key: cord- -t zbpyin authors: nan title: emerging pathogens: what are the sources and how can they be spotted quickly? date: - - journal: lab med doi: . /nm vcpd pgvgcl sha: doc_id: cord_uid: t zbpyin nan í í does it have a tendency for waterborne transmission? í í is it vector-borne with the ability to use humans as part of the life cycle? í í if it is directly transmitted, is it durable in the external environment? í í is it attendant-borne? í í is it needle-borne? í í if it is transmitted sexually, is it mutation-prone with a tropism for critical cell types or does it have invasive or oncogenic tendencies? looking at organisms that caused epidemics over the last centuries, they would have been identified as extremely dangerous pathogens using these criteria. yersinia pestis is durable in the external environment and is vector-borne. the partial eradication or the vectors-fleas and rats-have controlled plague so long as those vectors are absent. yellow fever, whose epidemic killed % of the population of memphis, tn, is vectorborne through human-mosquito cycles. the united states has seen the increase of west nile virus, also vector-borne through mosquito-wild bird cycles. in , the nihniaid research program was defined as directing research into broad areas, not necessarily targeting american problems alone. the interest of nihniaid, described as deriving from both humanitarian concerns and "enlightened selfinterest," focuses on the globalization of health problems. the announcement of the program identified broad goals for research: í í strengthening basic and applied research on the multiplehost pathogen and environmental factors that influence disease emergence. í í supporting the development of diagnostics, vaccines, and therapies needed to detect and control infectious diseases. í í maintaining and expanding the national and international scientific expertise required to respond to health needs. at almost precisely the same time, the department of health and human services (hhs) released an action plan to combat antimicrobial resistance. the action plan was put together by a task force that included the cdc, the nih, the fda, the agency for healthcare research and quality, the healthcare financing administration, health resources and services administration, the departments of agriculture, defense, veterans affairs, and the epa. the plan covered components and action steps, including priority steps. top priorities of the major sections include: í í surveillance: the cdc with state health departments and other task force members began planning a coordinated surveillance system, such that all entities would use similar methodology and develop patterns of use for antimicrobial drug used in human medicine, agriculture, and other consumer products. í í prevention and control: the hhs and others planned the launch of a national public education campaign to reduce overuse and misuse of antibiotics. pilot projects were already underway to identify effective strategies to promote appropriate antimicrobial drug use and reduce infection rates in clinical practice. í í research: the nih pledged to provide the research communities with genetic blueprints for various organisms to identify targets for needed new diagnostics, treatments, and vaccines. í í maintenance: the action plan pledged to maintain and expand the national and international scientific expertise that are required to respond to health threats. understanding the mechanisms that permit infectious diseases to move virtually unchecked is a key to defeating them. the human genome sequencing project, finished considerably before it was expected, has provided a head-start to this understanding. the function of the estimated , to , human genes will have an enormous impact on all areas of medicine, including our understanding of the host response to microbial pathogens. microbial pathogens are being sequenced; therefore, the interaction between microorganisms and the human genome can shortcut the development process for diagnostics, therapeutics, and vaccines. the first microbial sequencing project, haemophilus influenzae, was completed in july with extraordinary speed. the early use of a newly developed technique known as the shotgun approach sequenced thousands of fragments of the bacterium's genome. by comparing overlapping sequences, a complete dna sequence was designed that contained all of the genetic information of the bacterium. the nihniaid has funded projects to sequence the complete genomes of the bacteria that cause tuberculosis, gonorrhea, chlamydia, and cholera, as well as individual chromosomes of important organisms such as the malaria parasite plasmodium falciparum (http://www.niaid.nih.gov/dmid/genomes). many of these microbes have been completely sequenced and are now being annotated and analyzed. the nihniaid researchers and grantees are required to deposit the sequence data in specialized and public databases such as genbank, which is run by the national center for biotechnology information (http://www. ncbi.nlm.nih.gov/genbank/). access to the sequence data is available to anyone with internet connections, and the data is available even prior to publication in peer-reviewed journals. while there are advances in understanding and treating a variety of pathogens, the appearance of a well-understood pathogen that changes its characteristics can be disconcerting. unlike an outbreak of a completely unknown organism, an organism that fails to respond to recommended treatment triggers other problems. a case in point was published in the march issue of emerging infectious diseases as a letter to the editor, describing a multi-drug resistant shigella dysenteriae type organism, first seen in india in , where it was sensitive to nalidixic acid. a few cases of dysentery emerged in the years after , and a low-level resistance to fluoroquinolones was seen. then in the spring of , clusters of patients with acute bacillary dysentery were seen in eastern india. a total of , cases were seen and were found to be unresponsive to the newer fluoroquinolones but were found to be sensitive to ofloxacin. the authors of the letter, from the national institute of cholera and enteric diseases, kolkata, india and other institutions, noted that the drug-resistant shiga bacillus could spread further without an appropriate awareness programs, alternate drugs, and an effective vaccine. staphlococcus aureus and psuedomonas aeruginosa developed single mutations that caused multi-drug resistance to the broad use of fluoroquinolones. these agents were quickly followed by multi-drug resistance of campylobacter jejuni, escherichia coli, and neisseria gonorrhoeae. for these organisms, multiple mutations are required to generate clinically important resistance. according to david c. hooper (massachusetts general hospital, harvard medical school, boston, ma), writing in a special issue of emerging pathogens (mar-april, ), drug resistance in streptococcus pneumoniae, which is currently low, will require close monitoring as fluoroquinolones are used more extensively for treating respiratory tract infections. as consumers become more interest in healthy eating, a number of pathogens emerged quickly. fresh and minimally processed foods offer additional nutrients as well as fresh taste and a feeling of doing something good for your body. however, as a result, food-borne organisms have found it easier to survive and thrive. for instance, the addition of sprouts to everything from salads to sandwiches has increased produce-associated bacterial illness. although sprout-associated outbreaks have been reported since the early s, gastroenteritis with salmonella enterica serotypes newport and stanley in and , and the sakai city outbreak of enterohemorrhagic escherichia coli o :h in > , japanese schoolchildren have refocused attention on the public health hazard posed by seed sprouts. the risk for disease from sprouts is connected to seed production, distribution factors, and the sprouting process itself. seeds may be reared, harvested, milled, and sprouted locally or shipped globally to sprout growers; bacterial contamination may occur at any point in this chain. during germination, seeds are presoaked in water and then germinated in a warm, moist, aerated environment for to days. replication of pathogens by to orders of magnitude may occur during sprouting, resulting in high pathogen levels on mature sprouts, despite the fact that initial densities are low and the pathogens dispersed irregularly throughout the seeds. in an experimental model of seed contamination, salmonella serotype stanley added to alfalfa seeds increased from approximately x bacteria per gram of mature sprouts to bacteria per gram after to hours incubation, without affecting the appearance, smell, or taste of the sprouts. in the spring of , the outbreak of severe acute respiratory syndrome (sars) resulted in the issuing of a clinical description and preliminary information about diagnosis and cause. the incubation period is generally to days, although some isolated reports suggests that incubation may require days. illness begins with fever ( . °f) that may be associated with chills and rigors, and possibly accompanied by headache, malaise, and myalgia. after to days, a lower respiratory phase produces a dry, unproductive cough or dyspnea, and may progress to hypoxemia. the case fatality rate among patients that meet the world health organization (who) definition of sars is about %. initial diagnostic tests should include chest radiograph, pulse oximetry, blood cultures, sputum gram's stain and culture, and testing for viral respiratory organisms. as of march , , the case number of sars suspected in the united states stood at , while the who reported cases worldwide, including a number of deaths. recently, who reported finding a test that appears to identify the virus that may cause the disease. by isolating the organism from a patient and inoculating it with blood from a recovered patient, the virus was killed presumably because the recovered patient's blood included antigens to the virus. the who scientist who developed the test is not yet certain what type of virus he isolated. the paramyxovirus family-which includes measles, mumps, and canine distemper-remained a leading suspect. a new form of influenza, once the most feared scenario, is now low on who's suspect list. also recently, cdc announced that a previously unrecognized virus from the coronavirus family is the leading hypothesis for the cause of sars. two coronaviruses that are known to infect humans cause onethird of common colds and are also a common cause of health care-associated upper respiratory infections in premature infants. additional steps needed to confirm this hypothesis include further culturing of the virus from appropriate specimens, sequencing the viral genome, and examining specimens from patients at different stages of their illness. according to a cdc spokesperson, in one patient, a virus was seen in kidney specimens very clearly on the electron microscope. the cdc spokesperson said, "we also have done pcr, which is a way of probing for the genetic material of the virus, and we're finding virus pcr, very specific laboratorymedicine> may > number > volume evidence of that, in lung tissue, as well as the kidney in this individual patient. this patient, when tested with an antibody, in an antibody test to the virus, had a negative antibody test at the very beginning of the illness, and by the end of the illness that antibody test had become positive. it appears that, the patient seroconverted using a very specific assay for this new coronavirus. this is very strong evidence supporting coronavirus as an etiology, but we know from sequencing pieces of the virus dna that it is a virus unlike those that have been identified. . . . it's very premature to assign a cause or to make dogmatic statements about the etiology. we're still learning as we go, and we will maintain that spirit of collaboration." at the time that a distinct disease required identification, the disease outbreak was only about weeks old. it was first spotted in guangdong province, china. during the short time since the outbreak was first reported, the cdc acted decisively. the agency activated the emergency operations center; alerted public health partners in cities and states by issuing electronic messages. it also prepared and distributed more than , health alert cards to travelers returning from southeast asia; provided guidance to public health departments, health care facilities, and clinicians in monitoring and identifying potential cases; provided safe specimen-handling guidelines to laboratories; deployed more than a dozen cdc staff members, including medical officers, epidemiologists, infection control specialists, and patholo-gists to support the who in the global investigation; and provided regular media briefings to report on progress of the investigation. the new tools for identifying new viruses and bacteria have shortened the time needed for identification and genome sequencing. other tools make it possible to develop medications that may ease the problems with new pathogens. the difficulty is often recognizing that a new disease is present before it has become epidemic. guarding against the most dangerous emerging pathogens: insights from evolutionary biology more haemophilus and mycoplasma genes multi-drug resistant shigella dysenteriae type , forerunners of a new epidemic strain in eastern india? emerg infect dis an international outbreak of salmonella infections caused by alfalfa sprouts grown from contaminated seeds multinational outbreak of salmonella enterica serotype newport infections due to contaminated alfalfa sprouts clinical experiences in sakai city hospital during the massive outbreak of enterohemorrhagic escherichia coli o infections in sakai city available at key: cord- -qqm fbor authors: gulec, fatih; atakan, baris title: mobile human ad hoc networks: a communication engineering viewpoint on interhuman airborne pathogen transmission date: - - journal: nan doi: nan sha: doc_id: cord_uid: qqm fbor pathogens such as viruses and bacteria play a vital role in human life, since they cause infectious diseases which can lead to epidemics. recent coronavirus disease epidemic has shown that taking effective prevention measures such as wearing masks are important to reduce the human deaths and side effects of the epidemic. it is therefore requisite to accurately model the spread of infectious diseases whose one of the most crucial routes of transmission is airborne transmission. the transmission models in the literature are proposed independently from each other, at different scales and by the researchers from various disciplines. thus, there is a need to merge all these research attempts. to this end, we propose a communication engineering approach that melts different disciplines such as epidemiology, biology, medicine, and fluid dynamics in the same pot to model airborne pathogen transmission among humans. in this approach, we introduce the concept of mobile human ad hoc networks (mohanets). this concept exploits the similarity of airborne transmission-driven human groups with mobile ad hoc networks and uses molecular communication as the enabling paradigm. the aim of this article is to present a unified framework using communication engineering, and to highlight future research directions for modeling the spread of infectious diseases among humans through airborne pathogen transmission. in this article, we first review the airborne pathogen transmission mechanisms. then, the mohanet is given with a layered structure. in these layers, the infectious human emitting pathogen-laden droplets through air and the exposed human to these droplets are considered as the transmitter and receiver, respectively. moreover, the experimental methods for the proposed approach are reviewed and discussed. throughout the history, epidemics caused by infectious diseases have been a major threat to human life. epidemic diseases such as black plague, smallpox, spanish flu and recent coronavirus disease (covid- ) gave rise to millions of human deaths. in addition, epidemics can induce mental disorders in humans and recessions in the world economy due to prevention and control measures such as lockdown. owing to these facts, it is essential to understand and accurately model the spread of infectious diseases among humans. the interhuman spread of infectious diseases occur via direct contact and airborne transmission where pathogens are transferred from an infectious human to a susceptible one. the authors are with the department of electrical and electronics engineering, izmir institute of technology, , urla, izmir, turkey. here, transmission is employed synonymously with contagion rather than its usage in communication engineering. in airborne transmission, these pathogens (viruses, bacteria, fungi, and so on) are carried by large droplets and aerosols (droplet nuclei) which are emitted via breathing, speaking, coughing and sneezing [ ] . throughout this article, we use the term droplet to refer to both large droplets and aerosols together. as for the airborne pathogen transmission, it is not fully unraveled how its mechanisms operate between two humans, for example, it is still a matter of debate whether large droplets or aerosols are more infectious. in addition, the mobility and interplay of people during their daily life makes the problem of modeling infectious disease spread in an epidemic more chaotic. as people displace, there exist dynamic human groups exchanging pathogens among each other. due to their mobility, humans form different groups in an ad hoc fashion as their smart phones do in a mobile telecommunication network. actually, a human emitting expiratory droplets is an information source [ ] . when these emitted information carrying droplets are received by another human through sensory organs, we can consider there exists a communication path between them. hence, an analogy between human groups and mobile telecommunication networks can be established, since they both possess an intermittent connectivity which is detailed later. by utilizing this analogy, we propose an approach to modeling interhuman airborne pathogen transmission with communication engineering perspective where mobile humans forming a group are considered as a mobile human ad hoc network (mohanet). in a mohanet, the infectious human is the transmitter (tx), the susceptible human is the receiver (rx) and pathogen-laden droplets are information carriers propagating in the communication channel, that is, air. here, molecular communication (mc) employing chemical signals instead of electrical signals emerges as an enabler paradigm for the communication among humans due to its biocompatibility with the human body and multiscale applicability. on the other hand, researchers from many disciplines work separately in different scales to reveal the mechanisms of airborne pathogen transmission and model the behavior of epidemics. in fluid dynamics literature, researchers focus on the propagation of pathogen-laden droplets and their interactions with air [ ] . biologists deal with the survival of airborne pathogens in macroscale [ ] and their interactions with the human cells in microscale [ ] . furthermore, the medical literature conducts researches in cellular level to discover new drugs which cure the infectious diseases. in a larger scale, epidemiology literature focuses on the epidemic data to develop mathematical models for the spread of epidemics in time and space [ ] . however, there is a need to merge all of these research efforts in a unified framework. communication engineering approach can provide this framework by combining micro-and macroscale modeling issues. in this way, researchers will be able to utilize theoretical tools of communication theory in order to model the complicated nature of airborne pathogen transmission. in the remainder of this article, we first review the airborne pathogen transmission mechanisms and the motivation to use mc as the enabler communication paradigm. then, the communication engineering approach which merges different disciplines. in this approach, the layered architecture of mohanet is presented in detail and open research issues are discussed. finally, we give the existing and possible experimental techniques and conclude the article. this section provides a brief overview for the main issues of the airborne pathogen transmission mechanisms. then, the roles of molecular signals in the transfer of pathogens among humans are discussed. a. overview of main issues on airborne pathogen transmission ) respiratory activity and droplet size: pathogen-laden droplets are emitted to the air from an infected human via respiratory activities such as coughing, sneezing, speaking and breathing. these activities have different initial droplet velocities allowing different propagation distances. for instance, the initial velocities for coughing and breathing are about m/s [ ] and . m/s [ ] , respectively. therefore, a cough can infect people at a greater distance than breathing in still air. furthermore, the expiratory droplets are defined according to their diameters where aerosols and large droplets are assumed to have smaller and larger diameters than the size range - µm, respectively [ ] . while speaking, sneezing, and coughing release more large droplets into the air, breathing mostly contains aerosols. ) air distribution: in addition to the initial velocity, emitted droplets are influenced by the airflows, similar to a mc channel with drift. in outdoor environments, winds carry the droplets and dilute the concentration of pathogens via dispersion. therefore, it is less probable to get infected in outdoor environments. however, in indoor environments such as hospitals, offices or residential buildings, airflows generated by ventilation systems are critical for the spread of pathogens due to the circulation of air in bounded conditions. furthermore, personalized ventilation and exhaust systems are proposed as advanced ventilation systems to diminish the infection risk [ ] . these air distributions are required to be considered for realistic indoor airborne transmission models. ) posture, relative orientation, distance and movement of the human: for short distances, the posture, that is, standing, sitting or lying position, and the relative orientation of the infected and susceptible persons are important for the infection risk as shown in fig. . for instance, a doctor can reduce the exposure from an infected lying patient in a hospital ward via a standing posture and sideways orientation instead of face-to-face orientation [ ] . furthermore, a walking person can increase the infection risk in a closed and ventilated room by increasing the dispersion of the droplets [ ] . another important factor that influences the infection risk is the relative distance of the humans which is also referred as the social distance. surely, the infection risk decreases, as the relative distance between two people increases. the temperature difference between the human body surface and the surrounding air generates a thermal plume which is a buoyancy-driven upward flow of the surrounding air. as illustrated in fig. , this thermal plume leads to a convective boundary layer (cbl) around the human body, which should be taken into account for the movement of the droplets in the breathing zone [ ] . this upward flow can change the channel impulse response via generating an upward drift for the pathogens during the reception into the human body. ) survival of pathogens: subsequent to a respiratory activity, all of the emitted pathogens may not survive. in [ ] , it is shown that more than percent of the influenza viruses cannot survive within one minute. however, these survival rates are severely influenced by environmental factors such as temperature and relative humidity (rh). while increasing temperature decreases survival rates of the pathogens due to its effect at molecular levels, increasing rh results in decreasing evaporation of droplets [ ] . the decreasing number of pathogens results in a time-varying channel due to the dependence on the previous number of pathogens. via the aforementioned respiratory activities, a human can transfer pathogen-laden droplets to another human. this type of transfer (or communication) among humans is investigated in the medical literature where pheromone-based molecular signals are studied for the interaction of humans. in [ ] , it is proposed that pheromones secreted from the axillary apocrine glands of women living in close proximity provides a synchronization in their menstrual cycle. hence, molecular signals may give rise to some biological responses in human organism. as given in the next section, the transfer of the pathogen-laden droplets which cause infection can be considered in the context of mc. in this section, we present a framework with communication engineering perspective to model the spread of infectious diseases through airborne pathogen transmission. furthermore, open research issues are given. as shown in fig. , the proposed framework merges all of the multiscale research efforts in various disciplines such as fluid dynamics, biology, medicine, and epidemiology under the umbrella of communication engineering. mc emerges as the key paradigm that connects the studies among different disciplines in macro-and microscales. first, the mohanet is introduced through a layered architecture as depicted in fig. . layers are associated with different disciplines from µm to km scale in this architecture where each layer sends its output to a upper layer. the first layer is defined as the physical layer where the infectious human (tx) emits pathogen-laden droplets through the communication channel (air) as illustrated in fig. . the next layer is the reception layer which takes place at the susceptible human (rx) and includes two sublayers, that is, outer and inner reception sub-layers. the outer reception sub-layer comprises the interactions of the facial sensory organs with the droplets and inner reception sublayer provides the details about the interactions of pathogens with the biological cells in the human body. the networking layer where infectious diseases spread among different people is given at the top of the mohanet architecture. here, methods from mobile telecommunication networks literature are exploited and the outputs of the lower layers are employed rather. the details of this layered architecture are introduced as follows. a. physical layer ) transmitter: in a mohanet, an infected person is considered as a tx and her/his respiratory activities determine the tx parameters such as initial droplet velocities and droplet size distribution [ ] . the respiratory activities which are mentioned earlier can be classified as impulsive (sneezing and coughing) and continuous (breathing and speaking) emission signals. for continuous emissions, the frequency of the human exhalation is an influential factor for the transmission models. however, it is crucial to characterize speaking, since it is not always periodic and has more complex patterns than breathing. in addition, the respiratory organs such as nose or mouth affect the direction of the emitted signals. for example, the infection risk increases, when the tx uses the mouth instead of nose [ ] . furthermore, the convective boundary layer (cbl) of the human body, posture and relative orientation should be taken into account for accurate tx models. as mentioned earlier, the upward flow stemming from the cbl can affect the direction of the emitted pathogens in a tx model. ) channel: from the viewpoint of communication engineering, the channel is the physical medium between the tx and rx including the boundary conditions. as shown in fig. , channel modeling in the physical layer requires knowledge from fluid dynamics and biology due to the airdroplet interaction and survival of pathogens, respectively. the propagation dynamics of droplets can be examined under two subheadings depending on whether there is an external airflow or not. a) still air: in indoor environments such as residential buildings, it is generally assumed that there is no airflow, if there is not any ventilation system. after the emission of pathogen-laden droplets with an initial velocity, they are subject to newtonian mechanics during their interaction with the air. emitted droplets can be modeled as a cloud consisting of droplets and air particles. the movement of this cloud can be defined as a two-phase flow where these phases represent the gaseous state of air and liquid state of droplets [ ] . due to gravity, large droplets may fall earlier to the ground with respect to aerosols and evaporation can shrink the size of the droplets. as mentioned earlier, the temperature of the air and evaporation influence the survival rates of the pathogens. for continuous emissions, this fact can affect the channel memory, which is crucial for channel modeling. furthermore, initial velocities of droplets determined by respiratory activities can figure . two-layered receiver. give rise to short-term laminar and turbulent flows. these flows fade out as the distance between the tx and rx increases. b) windy air: for windy outdoor environments and indoor environments with airflows such as ventilation or wind arising from the open doors and windows, airflows dominate the propagation of droplets rather than other factors given for still air environments. the airflow which carries the pathogenladen droplets can be examined by advection and diffusion mechanisms. briefly, advection results from the airflow velocity and diffusion depends on the turbulent eddies during the mass transfer [ ] . it should be noted that molecular diffusion related with the thermal energy of molecules is negligible in macroscale. in order to calculate the concentration of droplets in time and space, deterministic and stochastic approaches which are based on differential navier-stokes and continuity equations are employed. for certain initial and boundary conditions, the solutions of these equations for deterministic concentration are known as gaussian plume for steady-state and gaussian puff model for transient analysis [ ] . actually, the concentration and velocity of droplets are random processes whose mean values are represented by these deterministic solutions. thus, stochastic differential equations are obtained which are non-trivial to solve as a closed form expression. therefore, these equations are mostly solved by numerical methods using eulerian and langrangian approaches [ ] . in addition, indoor ventilation types such as under floor air distribution, mixing, displacement, and downward ventilation should be incorporated into these airflow models. for example, downward ventilation can reduce the infection risk by diluting the dispersion of droplets [ ] . a human gets infected, when the transmitted pathogens are received into the body. as shown in fig. , the reception layer covers the issues related to biology and medicine in microscale where mc is utilized for the interactions of pathogens with the human body. the reception of these pathogens by the exposed human (rx) have not been well investigated, although there are myriads of theoretical, experimental and clinical studies for the propagation of pathogens. to this end, we propose a two-layered rx as shown in fig. and detailed below. the reception of pathogenladen droplets occur in the eyes [ ] , mouth and nose for many pathogens such as influenza virus [ ] . hence, we define the first step of reception as the outer layer sensing for the reception via facial sensory organs as illustrated in fig. . the whole surface of the human face is also important for the reception, since an infection may occur by touching the face contaminated with pathogens and these organs consecutively. pathogen-laden droplets emitted via a respiratory activity propagate as a mixture of droplets and air particles, which can be represented as a cloud [ ] . this cloud is affected by the momentum due to the initial velocity of droplets, gravity and buoyancy stemming from the temperature difference of the mouth and ambient air. according to this model, the trajectory of the cloud is given in fig. for a scenario that the tx emits pathogens by coughing. here, there is no airflow in the channel and, the tx and rx are standing toward each other at the same height as illustrated in fig. . fig. gives the change of the number of droplets in the cloud by taking settling and reception of droplets into account. the cross-section of the rx is assumed to cover a circular area including eyes, mouth and nose at the outer layer as illustrated in fig. . at this point, an analogy with the communication systems can be established by considering the infected state of the rx as symbol and no infection as symbol . this reception is accomplished by a detection according to a threshold value (γ = ) indicating the number of droplets required to become infected, as given in fig. . γ is a critical parameter in the airborne tranmission model, since it depends on the strength of human's immune system. to this end, biomedical data of humans such as body mass index, glucose level and whether or not having chronic diseases can be employed to estimate γ. in addition to these issues in the outer layer, the posture, relative orientation and cbl of the rx should be taken into account for an accurate receiver model as considered for the tx. furthermore, the reception of pathogen-laden droplets at the outer layer with different types of masks is an open issue to be investigated. ) inner reception layer: as shown in fig. , pathogens actually enter human body at the cellular level and increase their population. for example, viruses replicate themselves by inserting their genetic material (dna or rna) into human cells in two ways: they can bind their fusion (or spike) protein on specific receptor sites on the human cell or they can enter by using endosomes like a trojan horse [ ] . their binding sites can have different concentrations in different parts of the body. for instance, severe acute respiratory syndrome coronavirus- , which causes covid- , binds to angiotensin converting enzyme- receptors which are mostly found at upper respiratory tract [ ] . while large droplets are effective in upper respiratory tract, aerosols can reach down to alveoli in lower respiratory tract. hence, the droplet size can be effective to determine the infection risk according to the type of the disease. moreover, the viruses diffuse among human cells, bind to receptors and copy their genetic material in a random way. all of these issues at the inter-and intracellular level need to be modeled for an accurate transmission model for the spread of infectious diseases in mohanets. these modeling efforts can also contribute to drug and vaccine developments. what we examine up to here in lower layers of the mo-hanet architecture is about the transmission of infectious diseases between two humans. however, these transmissions occur many times in an epidemic, which requires a perspective to handle the population as a connected group, that is, a network. in the networking layer, the details of the mohanet architecture are presented in order to model the spread of infectious diseases in a large scale (km) within the communication engineering framework as shown in fig. . ) mobile human ad hoc networks: in epidemiology literature, each human, that is, a node, can be represented as susceptible (s), exposed (e), infectious (i) or recovered (r) according to the seir-based models in the infectious disease modeling approaches [ ] . according to the disease type, different combinations of these node types can be employed for the models such as sir or sirs. for example, covid- is suitable to use all the node types due to a non-infectious incubation period. in the literature, the number of these node types are modeled by ordinary differential equations where the number of the nodes can be deterministic or a stochastic process. the transition among different types of nodes (s,e,i,r) are defined with certain rates which are obtained by fitting statistical epidemic data. in experimental studies, these data are obtained by oral surveys or exploiting wireless sensor network technology [ ] . it is noteworthy that very few studies model the spatial change of the epidemic rather than its temporal change. by utilizing the widespread sir model, a mohanet is given in fig. which gives both the spatial and temporal changes. as the time elapses, the number of nodes may alter and the nodes can make transitions between states such as s, i or r. for example, a susceptible node can become infected, if it is in the transmission range of an infectious node or an infectious node can recover after a certain period. ) transmission types in mohanets: as illustrated in fig. , three transmission types are defined for the propagation of pathogen-laden droplets from the infectious nodes to the susceptible nodes as follows: • point-to-point transmission includes the communication between two nodes where the infectious and susceptible nodes are the tx and rx, respectively. • multicast transmission is the scheme that one infectious node spreads the disease to more than one node within its communication range. • multiple-access transmission comprises the scenario where a susceptible node is exposed to pathogen-laden droplets from multiple infectious nodes. humans are susceptible to infectious diseases in indoor places such as public transportation vehicles, shopping malls or offices. however, this is not the case that is encountered continuously. instead, the risk to get infected is intermittent due to the mobility of humans. as people displace, their smart phones can communicate opportunistically with each other as they are in the communication range. the same type of networking is also used in many applications such as wireless sensor, vehicular, and flying ad hoc networks. these dynamically changing structures defined as mobile ad hoc networks (manets) enable communication using the infrastructure at their location without a dedicated router. therefore, a mohanet can be resembled as a specific type of manet, that is, a delay tolerant network (dtn) in which an end-to-end link among the nodes may not always exist. the nodes in a dtn store their data and wait until they find a suitable connection. by considering this waiting delay, the routing algorithms in dtns provide the path to the desired user. similarly, an infected human can store its pathogens until finding a susceptible human to infect via airborne transmission. hence, we propose that opportunistic routing protocols such as epidemic or spray and wait can be adopted to model the spread of the infectious diseases. interestingly, epidemic routing protocol which is a reference method for routing in manets was already inspired by the mechanism of infectious disease spread during an epidemic [ ] . in order to observe and model the airborne transmission mechanisms among humans, experimental setups and computer simulations can be employed. in this section, we present and discuss how the performance of the proposed methods in different layers of the mohanet architecture can be evaluated. in physical and reception layers, the emulation of breathing, coughing and sneezing in experimental setups are realized by respiratory machines or thermal manikins which can be heated to change their temperature. these devices emit tracer gases including droplets. the concentration of droplets is measured by air samplers or via imaging techniques such as particle image velocimetry which gives the velocity and directions of droplets [ ] . moreover, sprayer-based mc systems can also be used instead of respiratory machines, manikins and air samplers. albeit reliable results can be obtained by the physical experiments regarding the consideration of droplet-air interaction and airflows, the collected data have a low-resolution in space and time and experimental devices are expensive. therefore, computational fluid dynamics (cfd) simulations are employed to evaluate the airborne transmission mechanisms with a high spatiotemporal resolution and less cost [ ] . however, the simulation software programs are based on navier-stokes equations which lack the capability to model all of the effects during the transmission realistically. these experimental techniques and cfd simulations can be employed to model the airborne pathogen transmission with communication engineering perspective for various scenarios between two humans. in a larger scale, for example, in a crowded city, it is essential to model the spread of infectious diseases with an approach that takes into account the interaction of people and their mobility in both time and space. the movement patterns of humans can be simulated by synthetic models such as random waypoint model or tracebased models which rely on real mobility data of mobile nodes as applied in manets. the adapted routing protocols for mohanets can also be evaluated in time and space by employing these mobility models according to the scenario via network simulation software. this article presents a framework to model airborne pathogen transmission with a communication engineering perspective. first, airborne pathogen transmission mechanisms are reviewed and mc is utilized to model the propagation and reception of this transmission. the concept of mohanet is proposed to handle the infectious disease spread modeling problem by using a layered structure in macro-and microscales. furthermore, simulation techniques and experimental methods to model airborne pathogen transmission are reviewed and discussed. throughout the article, open research issues possessing the potential for development opportunities are given. the efforts to model the infectious disease spread via airborne pathogen transmission with a novel approach given in this article has the potential for a holistic viewpoint. this communication engineering viewpoint can bring different disciplines such as fluid dynamics, medicine, biology and epidemiology together for accurate predictions about the spread of infectious diseases. hence, the most proper intervention method (lockdown, social distancing, wearing masks, and so on) can be chosen and how it will be applied can be determined to stop the epidemics in an effective way. airborne spread of expiratory droplet nuclei between the occupants of indoor environments: a review communication through breath: aerosol transmission survival of airborne influenza virus: effects of propagating host, relative humidity, and composition of spray fluids how viruses invade cells dynamics of infectious diseases close contact behavior in indoor environment and transmission of respiratory infection performance of "ductless" personalized ventilation in conjunction with displacement ventilation: impact of disturbances due to walking person (s) human convective boundary layer and its interaction with room ventilation flow mechanistic insights into the effect of humidity on airborne influenza virus survival, transmission and incidence regulation of ovulation by human pheromones a molecular communication perspective on airborne pathogen transmission and reception via droplets generated by coughing and sneezing air dispersion modeling: foundations and applications the severe acute respiratory syndrome a pneumonia outbreak associated with a new coronavirus of probable bat origin epidemic routing for partially connected ad hoc networks turkey as a research/teaching assistant under the supervision of assoc. prof. dr. barış atakan. his research interests include micro and macroscale molecular communications and molecular networks he is currently an associate professor with the department of key: cord- - i kktl authors: santra, hiran kanti; banerjee, debdulal title: natural products as fungicide and their role in crop protection date: - - journal: natural bioactive products in sustainable agriculture doi: . / - - - - _ sha: doc_id: cord_uid: i kktl seeking solutions from nature for solving one and all problems is the age-old practice for mankind, and natural products are proved to be the most effective one for keeping up the balance of development as well as the “healthy, wealthy, and well” condition of mother nature. fungal pathogens are proved to be a common and popular contaminant of agroecosystem that approximately causes – % of total microbial crop loss. to meet the proper global increasing need of food products as a result of population explosion, managing agricultural system in an eco-friendly and profitable manner is the prime target; thus the word “sustainable agriculture” plays it part, and this package is highly effective when coupled with nature-derived fungicidal products that can minimize the event of fungal infections in agrarian ecosystem. present study enlists the most common and effective natural products that might be of plant or microbial origin, their mode of action, day-by-day development of phytopathogenic resistance against the prevailing fungicides, and also their role in maintenance of sustainability of agricultural practices with special emphasis on their acceptance over the synthetic or chemical one. a large number of bioactive compounds ranging from direct plant (both cryptogams algae and moss and phanerogams)-derived natural extracts, essential oil of aromatic plants, and low-molecular-weight antimicrobial compounds known as phytoalexins to secondary metabolites that are both volatile and nonvolatile organic compounds of microbes (fungal and actinobacterial members) residing inside the host tissue, called endophyte, are widely used as agricultural bioweapons. the rhizospheric partners of plant, mycorrhizae, are also a prime agent of this chemical warfare and protect their green partners from fungal invaders and emphasize the concept of “sustainable agriculture.” natural products are the best weapon for the survival of any type of problems regarding infection, pathogenesis, or protection from diseases. due to their degradability in nature, they are the first options to be used by agriculturalists and plant biologist for combating fungal pathogenesis. the eukaryotic organism fungi have a separate kingdom in whittaker's five kingdom classification and are prime member of this ecosystem as a potent decomposer. in spite of their heavy and multidimensional applications in agricultural, medicinal, and industrial field ranging from the production of life-saving medicines to food supplements, they are the cause of huge global crop loss each year and lead to economic exhaustion. macro-and microscopic fungi-producing fruit bodies on different portions of a plant body (stem, leaf, fruit, root) lead to the death and decline of the crop species. several methods have already been tried since the start of civilization for crop protection but because of plant and fungal coevolution, fungi have dominated on the green eukaryotes and caused significant reduction in the crop yield. particularly in a country like india where the central gdp largely depends on agricultural output, the fungal pathogenesis has been a matter of grave concern for the agriculture department and policy makers. huge amount of money and manpower is invested to fight against the fungal diseases for ensuring higher and qualitative yield, but still it has been a burning problem of today's conditions. the problem with chemical and synthetic tools for combating the parasitic infections includes their toxicity leading to quality deterioration and environmental pollution accompanied with side effects on human health. in a case study, it has been reported that the extreme use of antibiotics in agricultural field and their direct consumption by humans through their daily meal have led to resistance of those antibiotics in human fungal pathogens. so we are in search of bioactive agents that will be of biological origin, selective to their host, and produce no secondary symptoms with least negative impact. the problem with fungi is their secretion of various types of mycotoxins (aflatoxins, ochratoxins, patulin, fumonisin, zearalenone, deoxynivalenol, etc.) in the stored food products, causing postharvest loss of cereals, pulses, dry fruits, and spices. mycotoxins are not only food spoilers but also potent disease-causing agents in humans leading to cancer, liver damage, kidney failure, and paralysis (miller ) . the severe effects of fungal crop loss are visible largely in tropical or subtropical regions where the temperature is moderately higher than the other parts of the world. fungal devastation occurs in two phases: firstly, when the crops are growing on the field and, secondly, when they are stored for further transportation, postharvest loss. the third type of contamination occurs when the microscopic airborne pathogens like molds grow on cooked foods and lead to food spoilage. at each and every level, scientists have developed techniques to minimize fungal food loss. on a gross annual estimate, almost % of agricultural food items are of no use due to fungal contamination (pittet ) . the major issues with fungi-related crop loss are deterioration as a result of increase of fatty acid conditions, change of color and texture of food items, poor nutritional conditions, and poor germinability of stored seeds (dhingra et al. ) . reports from asia and africa include death of humans and animals due to consumption of mycotoxin-contaminated foods (reddy and raghavender ) . fungal pathogens are sometimes dependent on more than one host for their successful completion of life cycle and disease development (puccinia graminis var. tritici, causal agent of black stem rust of wheat that requires berberis aristata for successful infection other than there main target wheat plant). so physical controls like eradication of secondary or collateral host and burning of the old livestocks and remnants of the field are the primary measures adopted by the farmers for disease-free crop production. so maintaining the sustainability along with less pathogenic infection is the deep ecological movement for crop maintenance. there are reports of resistance developed against the common and widely used antibiotics of agricultural importance. blasticidin s, an antibiotic obtained from streptomyces sp. (a type of actinobacteria predominantly present in soil samples), interacts with the protein synthesis and causes the death of the rice blast pathogens. development of resistance of this antibiotic is reported to be present in some fungal pathogens that detoxify it by deamination (dayan et al. ). compounds of bacterial and fungal origin from both soil and endophytic sources are used as an alternative source over the chemical ones. plant extracts especially essential oils from plant taxa of lamiaceae family are of immense importance and are used as fungicidal or fungistatic. most of the active ingredients act upon the fungal cell wall by either blocking the cellular processes like respiration, cell wall and cell membrane synthesis, ergosterol biosynthesis, protein synthesis, or dna replication. not only the secondary metabolites of plant and microbial origin but also the direct application of microorganisms in terms of biocontrol agent could be used as potent antifungals. other than these, plants' own defense molecules, known as the phytoalexins, could provide a strong line of defense against mycorrhizae; the root symbionts of higher plants can physically, biologically, and biochemically protect the plant root from pathogenic invasion and provide an enhanced resistance conditions to their hosts. this study includes the role of these compounds as natural agents of antifungal property and their role in disease prevention. mycorrhizae as a biocontrol agent mycorrhiza being the perfect example of symbiosis is known to be the oldest association between higher plant (both angiosperm and gymnosperm, monocot and dicot plants) and fungi and is an astonishing phenomenon of nature. the mycorrhizal association is one of nature's privileges for maintaining the sustainability of agriculture. in present day's changing environment, haphazard use of pesticides (fungicides) and chemicals poses a great risk to the existence and survival of mycorrhizal species in its complete biologically active form. there is a need to increase awareness in order to save mycorrhizal fungi from extinction. plants form beneficial association with other variants of life forms (animals, bacteria, or fungi) to complete their life processes, to fight against pathogenic microorganisms, and most importantly to thrive in adverse environmental situations. the plant root and its associated living microbial flora are together called "rhizosphere," particularly the area of mycorrhizal occurrence. the term mycorrhiza is derived from two greek words: mycos which means fungus and rhiza which means roots. in nature, more than % of angiosperms and almost all of gymnosperms are known to have mycorrhizal associations. the common two types of mycorrhizal associations that exist in nature are endomycorrhizae, also called arbuscular mycorrhizae (am), for example, endogone sp. and rhizophagus sp., and ectomycorrhizae (em), for instance amanita muscaria and laccaria bicolor. mycorrhizal associations support its host plants to survive in untimely soil conditions and drought situations by increasing the surface area of root and efficiency of mineral uptake. environmental threats including problems of temperature increase, climate changing, drought, and infertility of soil are some of the major challenges in agriculture and have to be mitigated to ensure global food security. in this context, mycorrhiza-based crop production is one of the key components of sustainable agriculture practices. in most of the cases, am fungi-mediated suppression of root pathogenic fungi is achieved by either morphological, physiological, or biochemical alterations of the host. several experiments on fungistatic activity of the mycorrhizal species have been done, and fruitful results are found against pathogenic fungi such as aphanomyces spp., botrytis fabae, chalara (thielaviopsis) basicola, dothiorella gregaria, fusarium oxysporum, gaeumannomyces graminis var. tritici, ganoderma pseudoferreum, pythium ultimum, p. splendens, phytophthora parasitica, p. cactorum, p. vignae, rhizoctonia solani, r. bataticola, and sclerotium rolfsii (lioussanne et al. ; bagyaraj ; bagyaraj and chawla ) . the most common outcome of am fungal colonization is seen as an increase in number of branches, resulting in a relatively larger proportion of higher-order roots in the root system. thickening of the cell walls due to lignification and production of polysaccharides in mycorrhizal plants are the common mode of prevention of penetration and growth of pathogens like fusarium oxysporum and phoma terrestris. a huge percentage of am-root pathogen interaction studies have been conducted in crop plants of agricultural and horticultural importance. but the information available on forest tree species is scanty. mycorrhizal technology can thus play an important role in production of low-cost quality seedlings and provide plant protection. like other methods of biological control, am fungi are not able to offer complete immunity against the infection caused by plant pathogens. they could only impart a degree of resistance against soilborne plant pathogens. however, the possibility of biologically controlling soilborne plant pathogens looks promising. am fungi play a protective role for plants by activating the defense mechanisms for the better resistance of crop plants and thus may protect the host plant from further fungal pathogenic attack, thus working as a potent biocontrol agent. researchers have proved that am symbiosis triggers the activation of several defense-related genes and also expression of pathogenesis-related proteins. evidences are drawn from modern techniques like molecular biology methods and immunological and histochemical analysis that strongly supports this concept. am fungi first act as a biotrophic agent, and before entering the host plant's root cell, they cause a sharp change in endogenous salicylic acid that is reflected in quick accumulation of reactive oxygen species (ros), a wide range of hydrolytic enzymes, and also the activation of phenylpropanoid biosynthetic pathway (güimil et al. , paszkowski , roman et al. . research findings have proved that the amount of defense-related compounds (essential enzymes like pal, phenylalanine ammonialyase, a product of phenylpropanoid pathway, enzymes needed for flavonoid or isoflavonoid biosynthesis like chalcone isomerase) that act for the protection of plant from fungal and bacterial pathogen is higher in the case of mycorrhiza-inoculated plant than in the uninoculated ones (volpin et al. (volpin et al. , . host's physiological and biochemical processes are greatly influenced by the mycorrhizal association in terms of decreased root exudation, higher concentration of phenylalanine and serine contents, ortho-dihydroxy phenols, increased membrane phospholipid content, etc. (smith et al. ) . when the phospholipid contents are high, it reduces the chances of root pathogenic attack, and higher concentrations of ortho-dihydroxy phenols show inhibitory activity against root rot pathogen sclerotium rolfsii (causal agent of southern blight), whereas the non-mycorrhizal plants are affected by the southern blight disease. tomato plants when inoculated with g. fasciculatum show inhibitory activity against root knot nematodes. host-am association leads to the formation of defense-related compounds like phytoalexins, chitinases (chi), β- , -glucanase (glu) (enzyme related to hydrolysis of fungal cell wall), peroxidases (pox), hydroxyproline-rich glycoproteins, and phenolics (st arnaud and vujanovic ) . synergistic effect of pgprs along with am fungi is proved to be a system of better protection than the use of am fungi alone (linderman ; bagyaraj and chawla ) . fungal wilt of common medicinal plant indian coleus (coleus forskohlii) caused by fusarium chlamydosporium could be minimized by the joint action of am fungus and trichoderma viride and cause a sharp increase in root yield and root forskolin concentration and may also reduce the severe disease conditions (singh et al. ). am causes a drastic change in the rhizospheric microbiota and intentionally either removes directly the pathogenic microorganisms or stimulates the accumulation of potent microbial partners especially fungus that are heavily antagonistic to the plant pathogenic ones. plants with mycorrhizal association harbor higher population of rhizospheric microorganisms, thus making it impossible for the pathogen to compete and invade the root. in the case of phytophthora cinnamomi, the numbers of sporangia and zoospores are found to be reduced when rhizospheric soil extracts of am plants are applied; it means the am fungi are able to alter the microbial population and particular functional groups of rhizospheric microorganisms (meyer and linderman ; larsen and bodker ) . they cause qualitative and quantitative changes in the fungal community by several factors like changed exudation patterns; altered root size and architecture; different physiological and biochemical parameters like sugar, organic acids, and amino acids; and also putative direct am fungal effects (toljander et al. ; ahmed et al. ; vigo et al. ) . fungistatic siderophore (low-molecular-weight chelating agents having higher affinity for ferric ion)-producing microorganisms are found to be crowded in mycorrhiza-infected roots and rhizospheric regions. mycorrhizal plants are to be reported with more actinomycetes and bacterial (gram-positive paenibacillus sp. against phytophthora parasitica) flora antagonistic to soilborne root pathogens (azcon-aguilar and barea ; budi et al. ). apart from providing biochemical and physiological defense strategies, arbuscular mycorrhizal species also exhibit physical barrier of defense by changing the root anatomy, morphology, and even architecture in terms of increased nutrient uptake, meristematic and nuclear activities of root cell, higher rate of growths, and branching patterns (atkinson et al. ; gamalero et al. ; gutjahr and paszkowski ) . thus responses of root morphology as a result from afm colonization seem to depend on plant characters, tap root system, etc. more benefits are seen in tap root system than fibrous root system in terms of gained biomass and nutrient acquisition. though there is a gap of knowledge in how increased root branching caused by mycorrhizal infection help the plant to defend fungal pathogenesis, synergism is seen as something that can balance the suppressed root growth caused by several root pathogens and restore the root health. mycorrhiza-mediated strengthening of the vascular system allows the higher rate of flow of nutrients, increased mechanical strength, and also inactivation of vascular pathogens. in conditions of limited resource such as carbon requirement and space for inoculation, a competition between the symbiotic partner (mycorrhizae) and pathogenic fungi is very common and expected (vos et al. ). in the direct warfare, mycorrhizae win over the pathogenic one and thus obtain higher amount of nutrients (almost - % of total assimilated carbon by host plant) and occupy large areas of available root cortical cells (jung et al. ; vierheilig et al. ) . defeating the pathogenic fungi in terms of nutrient uptake and providing a little or no room for infection are probably the mightiest cause of biocontrol ability of am fungus (hammer et al. ) . output of am and phytophthora interaction indicates that the pathogen does not penetrate cortical arbuscular cells, suggesting that localized competition for infection site does occur between the pathogenic fungi and the am fungus. not only fungi but also plant-invading nematodes are in the queue for colonization and nutrient uptake (smith ) . the infection of southern root-knot nematode (meloidogyne incognita, m. exigua) is reduced when the roots are priorly inoculated with symbiotic partners like in the case of coffee plants also (alban et al. ; dos et al. ) . reports have suggested that the number of infected sites is reduced within mycorrhizal root system than in the uninoculated one and thus strongly supports the mycorrhizal role as a biofungicide (vigo et al. ) . am fungi can help the plant uptake of nutrients like phosphate, nitrogen, minerals, microelements (zinc), and water at a higher rate than the uninfected one (baum et al. ; parniske ) , and as a result, they are provided with photosynthetic carbon (smith and smith ) . the plants capable of absorbing higher amount of nutrients in terms of am fungal association have the potential to tolerate pathogenic infections (karagiannidis et al. ) . though the improved nutrition and increased tolerance are not involved in a cause-effect relationship, proofs are there that higher uptake of phosphate results in remarkable reduction in pathogenic infection in mycorrhizal plant but not in non-mycorrhizal plant (bodker et al. ) . tomato plants already infected with rhizophagus irregularis are not colonized by the pathogen a. solani, whereas non-mycorrhizal plant is affected by the pathogen (fritz et al. ) . mixed action of arbuscular mycorrhizal fungi (amf) glomus intraradices and trichoderma harzianum as a biocontrol agent significantly reduces the damping off disease caused by rhizoctonia solani in the case of tomato seedlings (amer and seud ) . in order to combat parasitic (fungal, bacterial, viral, nematoidal, and insectal) infection like mammalian cells, plant cells also develop defense systems that mediate the release of low-molecular-weight and short-lived (generally - h of existence) antimicrobial compounds or molecules known as the phytoalexins (braga ; echiverri et al. ; paxton ) . these secondary metabolites help the plant to withstand biotic and abiotic stress (grayer and kukubun ) . most of them being lipophilic compounds can cross the plasma membrane and act inside the fungal cell causing cytoplasmic granulation of the infecting fungal cells, disorganization of the cellular components, rupture of the plasma membrane, and inhibition of the fungal enzymes and mycelial growth (cavalcanti ) . mode of action of phytoalexins against fungal pathogenesis varies from species to species (table . ). metabolism of phytoalexin mediated by fungus involves the tendency for its increased polarity by addition of hydroxyl group (oxygenation), removal of methyl group (demethylation), etc. (jeandet et al. ) . muller and borger first enlightened the concept of phytoalexins almost year ago (muller and borger ). the first reported case analyzed with the concept of phytoalexin was potato tuber infection by the different strains of causal organism of "late blight of potato," phytophthora infestans. this pathogenic fungus initiated the hypersensitive reactions that lead to the formation of some "plant secondary metabolite" that inhibited further infection of the same plant when infected with another strain of the same genus of phytophthora. muller and his coworker named this "principle" as "phytoalexins" that have protected the plant from secondary infection (deverall ) . accumulation of phytoalexins in the green plant tissue clearly indicates the presence of remarkable amount of fungal and bacterial infections in the host tissue (stoessl ) . phytoalexins are naturally occurring products secreted and accumulated temporarily by plants in response to pathogenic attack or abiotic stress and agents like heavy metal toxicity, uv radiation, and wounds on tissue (naoumkina et al. ). the inducer agent may be of two types, elicitor and elicitin. the elicitors are commonly the oligosaccharides from fungal cell origin (like hepatosaccharide from soja cell wall) (sharp et al. pezet and pont ( ) , adrian et al. ( ) , adrian and jeandet ( ) camalexin induction of the fungal programmed cell death (pcd) by apoptotic mechanisms et al. ( ) ). the elicitin types of molecules are generally a type of glycoproteins secreted by the fungal cells (cordelier et al. ) . reports on detailed investigations about phytoalexins have covered a very few families (leguminosae and solanaceae) of the green world (ingham ; kuc ) . though investigations on some selected number of species and genera are made from plant families including both monocotyledonous (amaryllidaceae, orchidaceae, poaceae) and dicotyledonous plants (apiaceae, asteraceae, convolvulaceae, chenopodiaceae, euphorbiaceae, linaceae, moraceae, piperaceae, rosaceae, rutaceae) and even gymnospermic taxa (ginkgoaceae) (coxon ) , cash crops like members of poaceae (focusing on maize and rice), vitaceae, and malvaceae (cotton) have been studied for their phytoalexin production (schmelz et al. ; langcake and pryce ; jeandet et al. ; sunilkumar et al. ) . though till date a lot of researches have already been performed regarding phytoalexins, a natural weapon against mycopathogens, but still to increase the fungitoxic effectivity of these stress metabolites, further advancement in design and genetic control is needed ). phytoalexin synthesis not only is dependent on pathogenic attack but also could be influenced by various abiotic factors such as temperature, humidity, and water availability ( fig. . ). there are evidences that different parts of the plant like leaves, flowers, stems, seeds, and root tubers are site of phytoalexin biosynthesis (mikkelsen et al. ) . different biochemical pathways are used for producing various types of phytoalexins. the three most common pathways include (i) the phenylpropanoic-polymalonic acid pathway, (ii) the methylerythritol phosphate (mep) and geranylgeranyl diphosphate (ggdp) route, and (iii) the indole phytoalexin (ip) pathway (jeandet et al. ). it is not always obvious that phytoalexins could be categorized not only by their chemical structure or biosynthetic pathway but also by their function and tissue specificity. examples include the occurrence of momilactone a on different plant parts of rice plant (lee et al. ; cartwright ) . momilactone a is known to be residing in rice husks and rice stems constitutively, but they are also a phytoalexin of rice leaves. further studies by toyomasu and his coworkers conclude that momilactone a is constitutively synthesized and oozed out from root of rice plants. still there is no sufficient data available to consider phytoalexins as ubiquitous throughout the whole plant kingdom. a lot of studies have revealed their complex biochemical synthetic machinery that involves their de novo synthesis, regulation, and mode of action (jeandet et al. ahuja et al. . regulatory mechanisms involve defense-related marker genes, calcium sensors, hormone signaling, phosphorylation cascades, and also their antipathogenic activity. there are reports on genetic engineering-mediated manipulation of phytoalexin production and increased disease resistance in the case of plants (delaunois et al. ; jeandet et al. jeandet et al. , . phytoalexins are secondary or stress metabolites that are produced when the host plant is infected with pathogenic fungus. phytoalexin-mediated defense response includes the expression of lytic enzymes such as chitinases and glucanases and a number of pathogenesis-related (pr) proteins, oxidizing agents, and lignification of cell walls (dixon and lamb ) . mode of action of phytoalexin involves the coordinated synergism between several defense factors for the effective inhibition of the fungal pathogen (purkayastha ; mansfield ) . in the case of sorghum plant, significant infection caused by fusarium proliferatum and fusarium thapsinum stimulates the production of -deoxyanthocyanidin, apigeninidin, and luteolinidin and also the concentration of defense-related proteins like peroxidases, β- , glucanases, and chitinases that help to fight the pathogenic infection (huang and backhouse (koga et al. ; fukuta et al. ). there are several ways of blocking the fungal infection in host plant tissues by phytoalexin-mediated response. that includes inhibition of fungal spore on the leaf surface and inhibition during and after penetration to host cell (usman et al. ) . the occurrence of fungal germ tube on the leaf surface and diffusion of fungal metabolites through the leaf cells cause the accumulation of phytoalexins by the underlying cells and provide the first line of induced chemical defense (vanwees et al. ) . phytoalexins may be located on papillae or cell walls, thereby producing a localized, fungitoxic barrier to penetration (friend ) . examples include occurrence of fungitoxic (against erysiphe graminis) flavonoid (thought to be phytoalexin) on papillae of resistant barley leaves. phytoalexins are known to be solely produced as a result of induction or stimulus by external agents. fruitful evidences could be drawn regarding this fact. induction of disease resistance in plants is developed through the direct and indirect involvement of elicitors. extracts of fungal basidiocarp, essential oils of aromatic plants (walters et al. ) , and also synthetic chemicals like aminobutyric acid, salicylic acid, jasmonic acid, and acibenzolar-s-methyl ( (matiello and bonaldo ) , hymenolobium petraeum, qualea albiflora, and corymbia citriodora (matiello et al. ) that act as the elicitors of deoxyanthocyanidins and glyceolin production. homeopathic preparations of species of calcarea (c. citriodora and calcarea carbonica), essential oils of eucalyptus globulus (telaxka et al. ; oliveira et al. ) , and mild concentrations of salicylic acid (durango et al. ) are major elicitins of pistain production and accumulation in cotyledons of common bean (phaseolus vulgaris). silicon-mediated enhancement of disease resistance by peroxidase (pox), polyphenol oxidase (ppo), chitinases (chi), β- , -glucanases (glu), and phenylalanine ammonia-lyase (pal) is found in the case of leaf spot of cotton plant caused by ramularia areola (curvêlo et al. ). southern amazonian amphibian family bufonidae represents the true toads, and their cutaneous secretions are of diverse source of bioactive compounds which can be fruitful as new chemical weapons for agrochemical development. use of elicitors in the case of crop protection nowadays is becoming a very popular method of inducing response which are proved to be durable and broad-spectrum disease control mechanism where the plant's own resistance is used. a group of seven brazilian scientists (deice et al. ) evaluated the possibilities of methanolic extracts of cutaneous secretions of two species of bufonidae, rhaebo guttatus and rhinella marina, on synthesis of phytoalexins named glyceolin (soybean plant), deoxyanthocyanidins (sorghum plants), and phaseolin (mung plant) in soybean cotyledons, sorghum mesocotyls, and bean hypocotyls, respectively. there is a direct relationship between the phytoalexins production and defense ability of the host plant against the fungal pathogenesis. studies reveal that when the phytoalexin glyceolin is produced in higher amount in the soybean plant (cultivar tmg rr) as a result of methanolic extracts of amphibian's (r. guttatus) cutaneous secretion (at a concentration of . mg/ml), stimulates the enzyme β- , -glucanase that can cause the hydrolysis of the fungal cell wall along with other defense-related enzymes (chitinase) is also produced in higher amount, but when suppression of glyceolin occurs, that particular enzyme is also not produced. there are evidences in the case of glycine max that the effectivity of phytoalexins varies from cultivar to cultivar. application of r. marina (amphibian) methanolic extracts induced glyceolin production in tmg rr and monsoy cultivars ipro but did not induce tmg rr cultivars to synthesize these defense-related compounds. less toxicity of phytoalexins than chemical fungicides is the reason for their universal acceptance. for over years, phytoalexins have been a detailed area of study for its antimicrobial activity, especially antifungal properties. several investigations include the in vivo bioeffectivity of the phytoalexins against serious plant pathogenic fungi (table . ). phytoalexin synthesizing genes have also been genetically modified to cope up with the pathogenic evolution. still reports are there that include examples of cruciferous phytoalexins detoxification by fungal enzymes (pedras and abdoli ) . modification of pathogen to overcome phytoalexinmediated damage includes curved germ tubes as a result of asymmetric growth of the germ tube. phytoalexins are natural products of diverse chemical nature, for example, alkaloids, coumarins, isoflavonoid (coumestans, isoflavans, isoflavones, isoflavanones, pterocarpans, pterocarpenes), lignans, polyacetylenes, pterocarpons (pisatin, phaseolin, glyceollin, medicarpin, and maackiain), terpenes, and non-isoflavonoid compounds (furanoacetylenes and stilbenes) ( fig. . ) (grayer and kokubun ) . both in vitro and in vivo fungicidal activity are shown by sakuranetin (rice phytoalexins) against the blast fungus (hasegawa et al. ). reduction of green mold (caused by penicillium digitatum) infections is achieved by the action of coumarin type of phytoalexin (scopoletin) of orange (sanzani et al. ). the loss of apples production caused by penicillium expansum and accumulation of patulin is minimized by the action of phenolic phytoalexins like resveratrol, scopoletin, scoparone, and umbelliferone (sanzani et al. ). in the case of medicago sativa (alfalfa), the isoflavonoid -o-methyltransferase provides increased resistance against phoma medicaginis by synthesizing maiackiain (he and dixon ) . for soybean plants, transformation of resveratrol to pterostilbene , langcake and pryce ( ) , coxon ( ) , and harding and heale ( ) (continued) (zernova et al. ) . scientists have proven that not only fungal infection acts as the stimulus for phytoalexin synthesis but also the hormone levels; phosphorylation cascades play a major role in this purpose. cytokinin overexpression in nicotiana tabacum is directly associated with its resistance against p. syringe by higher concentration of capsidiol and scopoletin (grosskinsky et al. .) the fungitoxicity of the phytoalexin could be enhanced by methylation or presence of electron-attracting groups on aromatic rings that is directly involved in affinity with membrane proteins being an uncoupler of ets system. endophytes are a type of hidden beneficial microorganisms that reside within the host plant causing no visible disease symptoms and syndrome and promote the plant to maintain its existence in typical harsh conditions. sometimes they could be latent pathogens at a very distant path of the host's life cycle but are simply a unique area of research where plant science and their microbial association get new definition. endophytes have been a constant and reliable source of exploration of bioactive compounds, but extensive search has not been performed till date, and that has given the endophyte biologists a great opportunity to search endophytic fungal and actinobacterial flora for the establishment of novel bioactive compounds. selection of plant for endophytic isolation is the most vital part of this study. exploitation of the proper isolates accelerates this search and opens up new angle of research. the search for uncommon products of agrochemical importance is a common demand of todays' world. the safer the antifungal agents become, the more it is well accepted in the scientific community as well as agricultural market. in general, the screening of thousands of natural products ends up giving only one commercial product. so indeed it's a tough job to end the search of new antibiotics with a hopeful result. a total of out of of the popular prescribed medicines are of fungal origin, and it is a fact that % of the fungi have been described till date (hawksworth (hawksworth , . so fungi serve as a continuous dependable source of new natural products. the intelligent screening procedure includes the selection of fungal flora of endophytic sources to open up the untapped potential of secondary metabolites synthesized by fungi. microorganisms grown in the petri plates or culture broth constitute minimal growth medium needed for their survival. any kind of stress or transfer of microorganisms on selective media acts as a stimulation for production of their secondary bioactive compounds. these secondary metabolites are produced for their survival in odd environments and strictly act as the selection force for the expression of their antimicrobial-producing genes. these crude by-products of microbial cultures are filtered and purified for their industrial, medicinal, and agricultural exploitation. soil microorganisms have been exploited for a long time for production of antibiotics, but microorganisms inhabiting plants are a new source in that respect. plants are selected usually with potent medicinal applications. here the knowledge of ethnobotany and folk taxonomy contributes a lot in this selection procedure. a strong literature survey supports the plant selection. the plants are surface sterilized and plated in nutrient-less solid plates. the fungi emerge out from different explants using the decaying plant parts as their primary growth substance. the isolates are identified by microscopic structures focusing on their conidial morphology, spore sculpturing, and colony characters. confirmatory identification includes s rrna analysis. endophytic fungi are tested for their antifungal activity against phytopathogenic fungi by one-to-one inhibition assay or antagonistic test ( fig. . ). two agar portions containing fungal hypha of endophyte and pathogen are placed on opposite sides of the plate. if the growth of pathogenic one is arrested partially or completely, that endophytic isolate is marked as antifungal agent and selected for further studies. another way of screening includes separating the agar plate into two equal halves, and two fungi are placed on two separate sides of the discontinuous plate. this test aims to screen the endophytes that produce volatile antifungals. if the isolate is potent enough to produce volatile organic compounds with fungi static or cidal activity, this will cease the growth of the pathogenic strain. then that isolate would be qualitatively and quantitatively measured for their volatile emissions using gc-ms as the master equipment ( fig. . ). liquid extracts of endophytic fungi are also tested for antifungal potentials by agar well-diffusion method. the fungal extract having antibiotic property shows clear zone of inhibition of growth of the pathogenic fungus surrounding the area of application of that fungal liquid. the potent isolate will be mass cultured, and the bioactive molecules will be extracted using organic solvents like ethyl acetate, ethyl ether, and n-hexane. steps include purification of that fungal extract by column chromatography, detection of the compound by thin-layer chromatography, and analysis of compounds by hplc and mass chromatography. field experiment includes the synergistic effect of a pure compound with mixture of natural compounds, and the effectivity of a newly applied antifungal agent strictly depends on the host plant and pathogenic microorganism's interaction, environmental condition, and development of drug resistance by that organism. application of pellets soaked in fungal extracts also is a method of determination of antifungal activity. secondary metabolites are itself diverse in nature. a variety of bioactive secondary metabolites are produced at significant concentrations by the endophytic microbial flora. the major components include quinones, phenols, phenolic esters, steroids, terpenoids, cytochalasins, benzopyranone, alkaloids, isocoumarins, and chromones. till date, a large number of plants have been studied for their endophytic flora as antifungal agents (table . ). alkaloid was the first ever reported insecticidal bioactive product. cryptocin was isolated from endophyte of tripterygium wilfordii, a plant of celastraceae family. the inner barks of the stem were used as explant, and cryptosporiopsis cf. quercina was isolated as a potent endophyte active against pyricularia oryzae and some other phytopathogenic fungi (li et al. ) . colletotrichum sp. produces -isoprenylindole- -corboxylic acid having inhibitory action against phytophthora capsici, a pathogen of cucurbitaceae, fabaceae, and solanaceae, and also other phytopathogens rhizoctonia cerealis and gaeumannomyces graminis var. tritici, a common pathogen of poaceae family . epoxycytochalasin h and cytochalasins n and h were isolated as chloroform and methanolic extracts of (fu et al. ) . a lot of endophytes have been explored for their antifungal production, but only a few of them were positive for antifungal metabolites categorizing in alkaloids. the common alkaloids acting as the antifungal agents of endophytic fungal origin are gliotoxin, cryptocanadin, tyrocidine a, fumigaclavine c, fumitremorgin c, -n-methyl albonoursin, and phomapsichalasin. the terpenoids, usually called isoprenoids, are large and diverse group of naturally occurring organic compounds derived from terpenes that are multicyclic. sixty percent of all the known natural products are terpenoids in nature. some endophytic fungicidal products are of terpenes by their native chemical structure. endophytic isolates (hormonema sp.) of gymnospermous plant juniperus communis were reported to be antifungal producers of a triterpene glycoside enfumafungin (pelaez et al. ) . known antifungal sterols of endophytic origin are β-hydroxyergosta- -ene, -oxoergosta- , , , -tetraene, etc. the sterols are strong inhibitors of helminthosporium sativum (present name: bipolaris sorokiniana), the asexual stage of cochliobolus sativus, a common root rot pathogen of wheat and barley crops which also infects leaf and stems of poaceae plants . sesquiterpenes are reported to be the growth inhibitors of cladosporium phlei (causal organism of leaf spot disease of timothy grass, phleum pratense). this is a unique example where the host plant (phleum pratense) itself harbors the endophyte (epichloe typhina) that inhibits the growth of its leaf spot pathogen (cladosporium phlei). from the point of view of organic chemistry, isocoumarins are defined as the isomer of coumarin where the orientation of the lactone is reversely arranged. zhang and his coworkers in the year isolated an endophytic fungus named microdochium bolleyi from fagonia cretica (also known as virgin's mantle of zygophyllaceae family), a herb of semiarid regions of gomera. isocoumarins were identified as the active compounds having antifungal activity against microbotryum violaceum (previously known as ustilago violacea), an obligate parasite of basidiomycete group and a common infectant of members of caryophyllaceae causing smut of anther. the four isolated and identified isocoumarins are monocerin, -oxo epimers of monocerin, and open-ring derivative compounds of monocerin. the compounds are obtained as mixtures by column chromatography followed by sephadex lh- chromatography techniques. preparative tlc further differentiated the four compounds. monocerin and its analogues were previously reported as antifungal compounds from fungal sources of drechslera monoceras, exserohilum monoceras, helminthosporium monoceras, exserohilum turcum, and fusarium larvarum (aldridge and turner ; robeson and strobel ; grove and pople ; claydon et al. ) . these secondary metabolites act on pathogens by interfering stages of divisional phases of cell cycle. the second isocoumarin was colorless oil. the third and fourth one are represented by the empirical formula of c h o and c h o . the fourth one is structurally correlated to fusarentin , -dimethyl ether. both the compounds are of heptaketide in their origin, and it is revealed that fusarentins are the probable precursors of the active compounds monocerins (scott et al. ; axford et al. ). dihydroisocoumarins, mellein (an isocoumarin derivative), (r)- -hydroxymellein, and fonsecinone were reported from species of xylaria (endophyte of piper aduncum), pezicula, penicillium (alibertia macrophylla), and aspergillus (cynodon dactylon), respectively (oliveira et al. ; schulz et al. ; song et al. ). phenols (popularly known as phenolics) represent a class of chemical compounds characterized with a hydroxyl group attached to an aromatic hydrocarbon group. phenol (or carbolic acid) is a colorless crystalline solid, aromatic compound having benzene rings. they are predominantly found in the plant kingdom as a response to stress and are of utmost importance. endophytic culture extracts are also known to be rich sources of phenolics; usually they are directly proportional to the antioxidative property of any fungal isolate, but in some particular cases, they are characterized with their antifungal potentials against phytopathogenic fungus. usually the liquid culture extracts of the endophytic isolates are subjected to solvent extraction using ethyl acetate, n-hexane, ethyl ether, etc. those organic solvents are believed to extract the phenolics from the water-based culture broth. those extracted compounds are further screened for their antifungal efficiency. ethyl acetate extracts of endophytic phoma sp. are reported to contain tetralone metabolites (derivatives of α-tetralone, , , -trihydroxy-α-tetralone) inhibiting the growth of two common broad phytopathogenic fungus fusarium oxysporum and rhizoctonia solani. griseofulvin is known to be the first antifungal compound isolated from penicillium griseofulvum. later it is isolated from several species of fungi including endophytic penicillium canescens and xylaria sp. (member of xylariaceae family). griseofulvin from endophytic p. canescens of popular chinese medicinal plant polygonatum cyrtonema (polygonaceae) showed strong inhibitory effectivity against phytopathogenic botrytis cinerea, sclerotinia sclerotiorum, colletotrichum orbiculare, and didymella bryoniae . other than penicillium, endophytic xylaria sp. isolated as an endophyte of abies holophylla yields griseofulvin and dechlorogriseofulvin for in vitro and in vivo effectivity against pathogenic magnaporthe grisea, corticium sasakii, blumeria graminis (park et al. ) . the ascomycete fungus pestalotiopsis is known to be a common plant pathogen but also has been reported many times because of their endophytic existence in the host plants. the two common species pestalotiopsis microspora (host: tropical plant terminalia morobensis) and p. fici are reported to be producing antifungal metabolites isopestacin and pestalofones d-e (harper et al. ; liu et al. a, b) . chlorogenic acid and colletotric acids are antifungal phenolics of colletotrichum gloeosporioides and sordariomycetes sp., respectively zou et al. ) . they were isolated from medicinal plants of china (artemisia mongolica and eucommia ulmoides) and effective against fungi imperfecti helminthosporium sativum. orcinol is used for the production of a dye called orcein used randomly for the staining of cells and chromosomes. orcinol is popularly known for its antifungal activity too and has been isolated as a product of endophytic origin of penicillium sp. from alibertia macrophylla (a plant of rubiaceae) showing bioactivity against cladosporium cladosporioides and cladosporium sphaerospermum (oliveira et al. ) . endophytic phomopsis sp., dothiorella sp., and diaporthe sp. have also been tested for their antifungal production and antifungal compounds that were detected (brady et al. ; xu et al. ; huang et al. ). volatile organic compounds (vocs) are said to be a type of organic low-molecularweight carbon-containing small compounds (up to c ) that have a high vapor pressure with low molecular mass ( - daltons) at room temperature. the high vapor pressure results from a low boiling point of that chemical compound, which causes a huge quantity of molecules to evaporate from the liquid, solid, or semisolid form of the compound and gets released into the surrounding environment. the endophytes are unique in their volatile emissions. the term mycofumigation that is very much popular with the treatment of agricultural phytopathogens is actually the output of vocs that originated from endophytic isolates. the first ever reported volatile antibiotic producer was muscodor albus (xylariaceae family), an endophyte of guazuma ulmifolia (a plant of sterculiaceae family collected from tropical forest of sw ecuador), isolated by gary strobel and his co-workers ). the major compounds isolated by gcms are known to be involved in antifungal, antibacterial activity. compounds include butanoic acid, -methyl-; butanoic acid, -methyl-; -butenal, -methyl-; butanoic acid, -methylbutyl ester; -buten- -ol, -methyl; guaiol; -octene, -ethyl-; formamide, n-( -methylpropyl); azulene and naphthalene derivatives; caryophyllene; phenylethyl alcohol; acetic acid, -phenylethyl ester; bulnesene; and various propanoic acid, -methyl-derivatives. these compounds were tested against a number of phytopathogenic fungi (botrytis cinerea, mycosphaerella fijiensis, pythium ultimum, phytophthora cinnamomi) showing partial or complete death or growth inhibition of those pathogens after or days of incubation. muscodor albus was reported from a diverse type of host plants, i.e., myristica fragrans, terminalia prostrata, cinnamomum zeylanicum, and ginkgo biloba, by several workers (worapong et al. ; sopalun et al. ; ezra and strobel ; mercier et al. ; ezra et al. a, b; atmosukarto et al. ; lacey and naven ; lacey et al. ; strobel et al. ; banerjee et al. a, b; corcuff et al. ; alpha et al. ) . the mycofumigants are effective against pathogen fusarium culmorum, causal agent of seedling blight, foot rot, ear blight, stalk rot, and common rot of cereals. sexual stage (teleomorph) of glomerella cingulata, a fungus of glomerellaceae, is a potent pathogen causing anthracnose-like symptoms of water-soaked, sunken spots and necrotic lesions on fruits of forest trees. this phytopathogen is strictly inhibited by the volatile emissions of this novel endophyte. banerjee et al. ( a, b) first reported muscodor albus strain gba from the usa as an isolate of ginkgo biloba (first isolate of m. albus from g. biloba) and tested the biological efficacy of its volatile mixtures against agricultural pathogens and also evaluated its promises to be used as a commercial mycofumigant agent for controlling the fungal diseases in storage fruits and vegetables, that is, agricultural productions and during food transportation. the strain gba in comparison to other strains of muscodor e and cz completely inhibits and potentially kills the member of phycomycetes, pythium ultimum after days of exposure of the mixture of volatiles. the organic compounds include alcohols, acids, esters, ketones, and lipids as their active components. -butanol, -methyl-, acetate was found in significant quantities. vitrine, a terpenoid, was first isolated from muscodor albus strain gab. the volatile mixture is artificially produced by the mixture of the pure compounds, and that mixture is again checked for antifungal activity. a positive mycocidal or mycostatic effect similar to the effect of endophyte's volatile emission will confirm establishment of the endophyte and its mixture as the biocontrol or antifungal agent. myrothecium inundatum, an endophyte of herbaceous acalypha indica (euphorbiacea member collected from northeastern part of india), produces unique mixture of volatile components having -octanone, -octanol, -octen- -ol, sesquiterpenes, organic acids, methyl esters, naphthalene, -octanoic acid, heptanoic acid, etc. this endophyte produces foam in its liquid culture predominant with long-chain carbon compounds like octane, , -cyclohexadiene, -methyl-and cyclohexane, and -ethylpropyl. (meshram et al. (meshram et al. , suwannarach et al. ; saxena et al. ; suwannarach et al. suwannarach et al. , suwannarach et al. , kudalkar et al. ; mitchell et al. ; worapong et al. ; daisy et al. ; siri-udom et al. postharvest fungal disease is one of the prime causes of agricultural loss of crops. use of biological agent to minimize this loss is one of the vital targets of agriculturalists, horticulturalists, and plant biologists. several chemical agents have been already tested for practical applications, but endophytes are less explored organisms in this arena. volatiles from endophytic source open up new scope of utilization of unique mixtures of chemicals to be used as mycofumigant agents. a large number of endophytes have already been screened for their postharvest disease management ability (table . ). the volatiles of the endophyte could be considered as the natural fungicides. muscodor vitigenus, an endophyte of hevea brasiliensis, was analyzed in gc-ms for their volatile production. the isolates produce a unique mixture of myroxylon balsamum , -cyclohexadiene, -methyl-; , -pentadiene and cyclohexene, -methyl- -( methylethenyl)-; alkyl alcohols starting with -butanol- -methyl, -propanol- -methyl, cinnamomum bejolghota. this isolate was known to produce azulene, a new compound detected first from any muscodor species. this species was tested in vitro and in vivo for antifungal activity against a common worldwide devastating pathogen rhizoctonia solani (causal agent of damping off). the vocs produced by this fungi include (s)-(+)- -methyl- -heptanol; ethyl acetate; propanoic acid, -methyl-, methyl ester; cis- , -dimethylthiane; s,s-dioxide; cyclopentane; butanoic acid, -methyl-, methyl ester; -butanol, -methyl-, acetate; β-humulene; azulene, , , , , , , , a-octahydro- , -dimethyl- -( -methylethenyl)-; s-( .α., .α., a.β); and eudosma- ( ), -diene , , , , , , -heptamethyl- , -bis(trimethylsiloxy) tetrasiloxane. rhizoctonia solani-infected seedlings were treated with volatile mixtures to assess the mycofumigation property. in vivo experiment was conducted on four seedlings of bird pepper, bush bean, garden pea, and tomato. it was concluded that gm of muscodor cinnamomi prepared on rye grain solid media is the minimum dose required for inhibition of rhizoctonia infection and total control and elimination of damping off symptoms. muscodor cinnamomi-infected soil does not show any seed germination inhibition in comparison to rhizoctonia solani-infected soil. so it is a type of pioneer study of using endophytic species as potent agents of fumigation and biocontrol. candida intermedia strain c (saccharomycetaceae) was isolated as an endophyte of strawberry (fragaria ananassa), and the volatile emission was known to be a mixture of organic compounds including esters, alcohols, alkenes, alkanes, alkynes, organic acids, ketones, and aldehydes of which , , , -cyclooctatetraene and -methyl- -butanol were the most dominant (huang et al. ). volatiles of strawberry endophyte were itself useful as postharvest control agent for the host plant against botrytis fruit rot. other compounds include , , , -cyclooctatetraene; -methyl- -butanol; -nonanone; pentanoic acid, -methyl-, ethyl ester; -methyl- -butanol, acetate; acetic acid, pentyl ester; and hexanoic acid, ethyl ester that were found to be extremely inhibitory to conidial germination (reproductive growth) and also vegetative (mycelial) proliferation of b. cinerea. when the fruits are exposed to c. intermedia synthetic volatiles or itself to the fungus, the incidence of botrytis fruit rot reduces significantly. strawberry fruits inoculated directly with the endophyte also remain disease-free. so mixtures of candida intermedia c , the unique natural products, are useful as mycofumigation technique or for postharvest disease management by biological control policies. tangerine fruit (citrus tangerine), the commercial citrus crop of northern thailand, faces huge postharvest losses due to pathogenesis of green mold (penicillium digitatum). the pathogen is the prime cause of worldwide deterioration of tangerine fruits by mycopathogenesis. out of detected compounds, the most predominant were -methylpropanoic acid and -methylbutan- -ol. other compounds include carbitol, octanoyl chloride, azulene, -methylhexane, -methylpropan- -ol, , -butanediol, caryophyllene, -methylbutyric acid, ethyl -hydroxyproponate, etc. the pathogen was treated both in vitro and in vivo for their inhibition by endophytic volatile components. in both cases, pathogen growth was restricted. during transportation of the fruits, fungus causes huge crop loss by infecting the fruits; when fruits were inoculated with gm of rye grain culture of m. suthepensis ( month old), the disease development is ceased. so it is a classic example of mycofumigation by the biocontrol agent of tangerine fruit for the control of rot lesions caused by p. digitatum infection. the in vivo application requires the proper surface sterilization (using sodium hypochlorite) of the targeted parts where the inoculation is going to be done, for example, fruit, stem, root, and leaf. usually infection on fruits for assessing the biocontrol potential is the most common and popular method. the seeds will be washed in distilled water, and using sterile needle, uniformly the whole area would be done, and the whole area would be infected or inoculated with the endophytic liquid extracts containing spore suspensions. muscodor albus vocs are potent enough to cause a significant reduction of in vitro spore germination of the tilletia species t. horrida, t. indica, and t. tritici. endophytic nodulisporium spp., trichoderma spp., phomopsis spp., and oxyporus latemarginatus are reported to produce vocs that inhibit mycelial growth of phytopathogenic fungi (lee et al. ; park et al. ; ajith and lakhsmidevi ; amin et al. ) . black sigatoka disease (also known as leaf spot or black leaf streak disease) of banana (musa paradisiaca) is caused by mycosphaerella fijiensis (ascomycete fungus). this phytopathogen is inhibited by the volatile emissions of muscodor sutura, an endophytic isolate of prestonia trifidi. the volatiles are effective also against ceratocystis ulmi, the causal agent of dutch elm disease of american elm (ulmus americana). the volatile mixtures include thujopsene, chamigrene, isocaryophyllene, and butanoic acid, -methyl-that are potent inhibitors of the common anthracnose pathogen of cucumber, muskmelon, and watermelon (members of cucurbits), colletotrichum lagenarium. so this unique endophyte and its chemical mixtures are potent mycofumigants and ensure crop protections against destructive pathogens like c. ulmi and c. lagenarium, sclerotinia sclerotiorum (causing white mold, cottony rot, water soft rot, stem rot, drop, crown rot, and blossom blight diseases of the host), and also phytophthora palmivora (oomycete fungi), the causal agent of bud root of palms and areca nut predominantly occurring in regions of south india (kudalkar et al. ) . liarzi and his coworkers tested the biological control efficacy of the endophytic daldinia cf. concentrica, isolated from olive tree (olea europaea l.) of israel against phytopathogens, and the unique mixtures of volatile were effective against the phytopathogenic mycelial growths. the mixtures include a variety of organic compounds: -methyl- -butanol, -methyl- -butanol, -methyl- , -cyclohexadiene, -methyl- , -cyclohexadiene, -heptanone, isoamyl acetate, -heptyn- -ol, -octenal, octanal, β-elemene, α-guaiene, β-selinene, α-selinene, α-bulnesene, germacrene a, etc. the unique mixtures having broad-spectrum antifungal property could be used for fumigation for eliminating the pathogenic infections of aspergillus niger (mold-causing organism on fruits of economic importance). so the endophytic d. cf. concentrica opens up opportunities for fungal disease control in food and agricultural industries (liarzi et al. ) . nodulisporium sp. strain gs d ii (hypoxylon anthochroum) and hypoxylon anthochroum strain blaci are potent enough to be used as biopesticide against fusarium oxysporum, a common contaminant of solanum lycopersicum var. cerasiforme (cherry tomato) causing a great percentage of crop loss globally. six vocs of alcohols' mixture, phenylethyl alcohol, -methyl- -butanol, -methyl- -butanol, eucalyptol, ocimene, and terpinolene, were detected and applied together with synergistic effect and individually both in vitro and in vivo. inoculation of pathogen on the cherry tomato fruits yields significant reduction in fusarium contamination. both agar dilution techniques and gas test were done to assess the in vitro antifungal activity, and the endophytic volatile mixtures were effective in both the cases. volatiles kill the pathogens probably by interfering cell membrane permeability, hyphal morphology, and respiratory activity of the pathogenic fusarium oxysporum. so it is a great opportunity to use the unique mixture of volatile organic compounds of the endophytic isolate to reduce the crop loss caused by the pathogenic infection on the commercially valuable plant of cherry tomato worldwide. endophytic phoma sp. (didymellaceae) and phomopsis sp. (valsaceae) were isolated from larrea tridentata and odontoglossum sp. singh et al. ) . the volatiles detected are effective against phytopathogens verticillum sp., ceratocystis sp., cercospora sp., sclerotinia sp. sclerotinia sp., and botrytis sp. algae are diverse group of autotrophs and the leading producers of o in the ecosystem. they range from prokaryotic unicellular to eukaryotic complex multicellular forms involved in the marine and terrestrial food chain. antifungal activity of the seaweed (members of phaeophyceae and rhodophyceae) is a major weapon for natural fungicides along with their antibacterial, anti-protozoan, and antiviral activities. algal seaweeds are potent holders of large number of secondary metabolites including phenolics, terpenes, alkaloids, and lectins which are not directly involved in photosynthesis and reproduction and thus fall under the category of secondary metabolites. they are common antimicrobial of algal origin that act on the target organisms by altering the microbial cell permeability accompanied with the loss of internal macromolecules or sometimes interfere with the membrane function causing cellular disintegrity ultimately leading to cell death (abu-ghannam and rajauria ). several studies include antifungal activity of algal members against human pathogens; a very few studies include their efficacy against plant pathogens (cheung et al. ; singh et al. ; stirk et al. ; padmakumar and ayyakkannu ; ismail et al. ; genovese et al. ; lopes et al. ). padmakumar and ayyakkannu tested species of algae against a variety of bacterial and fungal pathogens. out of the all screened organisms, % exhibited antibacterial efficiency, and only . % inhibited fungal growth. polysaccharides found in the cell wall and deposited in terms of storage food from red and brown algal sources include ulvans (obtained from ulva sp.), alginates and fucans (from fucus sp.), laminarin (laminaria sp.), and carrageenans that can induce defense responses in plants against phytopathogens by pathogen-associated molecular patterns (maps) and are capable of inducing plant resistance (vera et al. ) . polysaccharides stimulate regular cellular changes associated with pathogen perception and defense activation by change in ca + concentration and burst due to oxidative stress activation of salicylate, ethylene, and jasmonate biosynthetic pathways and by activating pathogenesis-related proteins (prps) (jaulneau et al. ; zhao et al. ) . as a result of the depolymerization of the polysaccharides, the obtained oligosaccharides induce protection against a variety of fungal, viral, and bacterial diseases by accumulation of the antimicrobial compounds in the cell. algal polysaccharides as an alternative weapon over the synthetic agricultural drugs for controlling plant disease have been widely studied (stadnik and freitas ; hahn et al. ). brown algae laminaria digitata, a genus of phaeophyceae, is commonly called seaweeds and known to be the potent producers of kelp, an iodine-rich substance needed for the normal functioning of thyroid gland. laminaria produces laminarin, glucan polysaccharide-containing , -linked β-d-glucose moiety, a reserve food material found on the vacuoles of the vegetative cells of this genus. β-glucans are involved as a major part of daily diet and obtained from the brands of common cereals. they are involved in the defense responses of agricultural crops like tomato (lycopersicon esculentum), eggplant (solanum melongena), pepper (piper nigrum), watermelon (citrullus lanatus), grape (vitis vinifera), apple (malus sp.), and pear (pyrus communis). elicitation of defense response by laminarin against causal agents of gray mold (botrytis cinerea) and downy mildew (plasmopara viticola) in grapevine plants remarkably suppresses their infection up to % and %, respectively (copping et al. ). so, natural product from brown algae laminaria sp. known as laminarin or laminaran can act as the biofungicide or biocontrol compounds. use of laminarin significantly reduces the mycelial growth and aflatoxin production in aspergillus flavus and ensures its use as a fungicide (liangbin et al. ) . the advantage of using laminarin over other products is that as it breaks down finally to glucose molecules, it has no maximum residue limit (mrl) on the plant treated with this product. so, there is no need of preharvest interval constraint. this has been a prime cause why laminarin has substituted five popular fungicides involved in the treatment of apple scab (venturia inaequalis) in france (mery et al. ). this phyto-pharmaceutical is used widely in france and some countries of europe in the name of vacciplant (major active constituent is laminarin). laminarin has broad-spectrum applicability on fire blight of apples and pears in greece, france, belgium, switzerland, portugal, and also morocco. it is effective for apple scab disease in france and belgium and for curing storage diseases of apples caused by gloeosporium sp. in belgium. laminarin comes out as a fungicide of natural origin after being eligible in tests between and in several parts of europe, for example, france, belgium, italy, and poland, on natural contamination of orchards on several sensitive strains of scab fungus including golden delicious, golden smoothie, read cheaf, galaxy, gala, and pink lady. laminarin is applied widely against secondary scab (to minimize secondary scab during summer and up to harvest) as a result of its uniqueness in its mode of action. it does not involve cell death of the host plant or hypersensitivity induction in the host organism but rather stimulates plants' natural resistance (klarzynski et al. ) . aziz et al. ( ) reported its effectiveness in tobacco plants, wheat, strawberries, apples, and vines. the application of laminarin and alginate reduced the development of wilt symptoms caused by verticillium dahliae on olive twigs, stimulating its phenolic metabolism (salah et al. ) . moreover, alginates reduced pathogen growth in vitro. laminarin induces the release of h o in cells of tobacco plants and leads to the increase in pal activity (phenylalanine ammonia-lyase) and causes the accumulation of pr- , pr- (glucanase), pr- (chitinase), and pr- . concerning red algae polysaccharides, carrageenans induced protection against a broad range of pathogens such as tobacco mosaic virus (tmv), b. cinerea, and e. carotovora on tobacco (vera et al. ) . again on tobacco, mercier et al. ( ) showed that carrageenan infiltrated the leaves and increased the expression of genes coding for a sesquiterpene cyclase involved in the synthesis of the antimicrobial terpenoid capsidiol, pr- proteins (basic chitinases), and proteinase inhibitor with antipathogenic activity. an adequate percentage of growth and spore germination inhibition of botrytis cinerea was mediated by the hexane extracts of laminaria digitata and undaria pinnatifida. porphyra umbilicalis, laverbread, is an edible seaweed (corato et al. ) . other than b-glucan polysaccharides (laminarin) of laminaria, other secondary metabolites (phenols, terpenes) of phaeophycean algae (sargassum sp.) showed effectivity against common pathogens fusarium solani, rhizoctonia solani, aspergillus spp., fusarium oxysporum, penicillium spp., and botrytis cinerea (khallil et al. ; ibraheem et al. ; mabrouk et al. ; liu et al. ). cyanophycean blue-green algae are abundant all over the world and ranging from pond ecosystem to oceanic system. though they have been reported to produce a large number of toxins and involved in death and disease of cattle and human being, they are of serious interest from the point of view of natural fungicidal products. drawing the similarities with bacteria, they are characterized with a mucilaginous or gelatinous sheath composed of polysaccharides which are the weapon against fungal pathogenesis. cyanobacterial polysaccharides (pol) show higher disease resistance against b. cinerea when they are applied on the intact fruit (preharvest conditions when fruit is attached to the plant) rather than the fruit detached (postharvest conditions) from the plant (zheng et al. ; feliziani et al. ; yao and tian ) . polysaccharides are involved in elicitation as elicitors for development of local and systemic disease resistance and expression of defense enzyme synthesis, for example, chitinases and glucanases that are involved directly in antifungal responses (paulert et al. ; reymond and farmer ; sharma et al. ) . water extracts of common bga anabaena sp., ecklonia sp. (common edible marine algae of japan and korea), and corallina sp. (hard seaweed of corallinaceae family) exhibit antifungal activity against podosphaera xanthii (causal agent of powdery mildew of cucurbits) on zucchini plant, cucurbita pepo, of cucurbitaceae (roberti et al. (roberti et al. , . in the recent past, fungi inhibitory ability of algal members has been reported by several workers (righini et al. ; corato et al. ; khallil et al. ; ibraheem et al. ) . in vitro growth inhibition of aspergillus oryzae and penicillium notatum has been seen by cyanophycean anabaena laxa (frankmölle et al. ) . devastating plant pathogens pythium sp., fusarium sp., and rhizoctonia sp. were restricted by extracts of anabaena sp. (moon et al. ; manjunath et al. ) . the use of bga extract as the growth inhibitor of pathogenic chaetomium globosum, cunninghamella blakesleeana, aspergillus oryzae, rhizoctonia solani, fusarium sp., pythium sp., and sclerotinia sclerotiorum is reported. the extracts of phormidium fragile and nostoc muscorum (rizk bryophytes, the simplest member of the broad umbrella of embryophyta, are situated between algae and pteridophytes, are known to be plant amphibians growing in the marshy or shady habitat, and require water for their fertilization and for the perfect swimming motility of their sperms. they have been evaluated for their antimicrobial activity for a long time. it has been proved that these cryptograms are rich source of bioactive secondary metabolites and can easily be exploited as an alternative source of fungicidal compounds. as they grow in marshy habitats and can protect themselves from biotic (ultraviolet rays, heat stress, and predation) and abiotic stress (fungal or bacterial attack), they are store house of diverse bioactive chemicals (xie and lou ) . members of hepaticopsida and mosses (the evolved members of bryophytes) are known to possess antifungal activity and are rich source of flavonoids, terpenoids, bibenzyls, and fatty acids of therapeutic importance (krzaczkowski et al. ) . bryophytes are known to possess antibiotic property (banerjee and sen ; banerjee ; singh et al. ; shirzadian et al. ; savaroglu et al. ) . their antibiosis has been evaluated against a large number of plant and human pathogenic fungus (mekuria et al. ) . antimicrobial compounds from bryophyte can cure the problems of conventional antibiotic resistance (vanden bossche et al. ) . the antifungal efficacy is tested by disc diffusion assay and microdilution method ( fig. . ). different concentrations of the extracts are prepared and checked for their antifungal efficacy against phytopathogenic fungi. they may be fungicidal or fungistatic in nature, interfering at cellular, genetic level and creating blockage at metabolic pathways. extracts are made on several organic solvents or water extractions and also mixture of one or two organic solvents. the solvents popularly used are ethanol, methanol, chloroform, ether, dimethyl sulfoxide (dmso), acetone, chloroform, and hexane (table . ). sporophytes and gametophytes of different bryophytes at different stages of growth and at a different amount are first surface sterilized and then crushed on the organic solvents and used as antifungals in vitro against the fungal pathogens (wolters (sabovljevic et al. ; pejin et al. ; veljic et al. ; gahotri and chaturvedi ; alam et al. ; deora and jain ; dey and de ; deora and suhalka ) . actinobacteria are a group of gram-positive filamentous bacteria that are called as the branched bacteria or ray fungi (from greek actis, ray beam, and mykes, fungus) and are characterized with the high g + c content occurring in mostly aerobic conditions but occasionally being anaerobes (ludwig and klenk ; olanrewaju and babalola ). their morphology varies from forming branching filaments or mycelial growth to external spores. they are ubiquitous in nature ranging their distribution from soil and human microbiota to plant and even animal kingdom. they are predominant in aquatic as well as terrestrial ecosystem playing a major part in mineralization and recycling of organic matters leading to soil formation (sharma et al. ). they are not only free-living members of the ecosystem but also a plant symbiont or endophyte, contributing to the plants' survival in extreme conditions and pursuing several bioactivities in vivo and in vitro. actinomycetes produce a diverse range of secondary metabolites, for example, antibiotics, antitumor, insectrepellent, and immunosuppressive agents, and plant growth-promoting regulators (pgprs) that are of immense pharmaceutical and agricultural importance. they are the prime producers of diverse antibiotics after the landmark discovery of penicillin in the year . the single genus of streptomyces sp. itself produces % of the total known bioactive ( , are produced by actinobacteria out of , produced by microorganisms, almost %) compounds from actinobacterial and riccia gangetica curvularia lunata deora and suhalka ( ) , guhil ( , ) bacterial source (berdy ) and is known to be the prime organism in the pharmaceutical world. they are equally profitable when isolated from plant source and designated as endophytic actinomycetes. so exploitation of the actinobacterial novel bioactive compounds both from endophytic and non-endophytic source is the ultimate way to fight against human and plant diseases. here we focus only on actinobacterial compounds' antifungal activity and role in plant protection from deadly diseases caused by severe phytopathogens leading to irreparable crop loss and economic breakdown of agricultural sectors. actinomycetes from soil source are selected based on the enrichment culture technique and are plated on selective media for isolation. antifungal agents, for example, nystatin and cycloheximide, are supplemented for the inhibition of fungal contamination. for isolation of endophytic actinobacteria from plant source, plants are first selected and surface sterilized for the elimination of the epiphytic contaminants and finally plated on the selective growth media like starch casein nitrate agar (scna), chitin-vitamin b, tap water yeast extract agar (twya), soybean, humic acid-vitamin b (hv), yeast extract casamino acid (yeca), modified gausse, and glycine-glycerol (ivantiskaya et al. ; küster ; küster and williams ; williams and davies ; hayakawa and nonomura ; crawford et al. ) . international streptomyces project (isp) medium is also popular media used for isolation, and they are supplemented with amino acids (l-asparagine for isp , tryptone for isp ), inorganic trace salts, starch or carbohydrate sources (malt extract for isp ), and agar as solidifying agent. ph set at near to optimum or slightly basic is mandatory for proper isolation techniques using isp medium. the actinomycete isolates are grown in solid or liquid medium for their antifungal bioactivity detection. antagonistic activities of the potent isolates are tested by growing them on both sides of the fungal hyphae, and isolate having anti-phytopathogenic activity will inhibit the growth of the pathogens. actinobacterial aqueous-or solvent-based extracts will be evaluated for either fungistatic or fungicidal activity by agar welldiffusion techniques. soluble bioactive compounds of antifungal importance will be extracted using wide range of organic solvents followed by purification by column and thin-layer chromatographic techniques. hplc analysis will be the most useful method for the detection of the purity of the compound, and further nmr studies are needed for the proper identification of the bioactive compound. cell line studies are made with the coupling of bioinformatics tools for the proper knowledge about their mode of action. actinobacteria can be a part of plant as endophyte, rhizospheric soil as symbiont for plant growth-promoting substance producer, and organisms' normal microbial flora as gut microorganism. so they are ubiquitous in their distribution. out of several biologically potent compound produced from the actinobacterial source, antibiotics are the major contribution of these microorganisms toward human civilization. all the known antibiotics (blasticidin, mildiomycin, natamycin, validamycin, kasugamycin) are of actinobacterial (most of them are the streptomyces sp.) source showing protective activity for the plants against agricultural fungal pathogens (tables . and . ). as human are dependent completely on nature and more particularly natural components of agricultural origin and importance, dependence on agricultural crops is of a known fact. but the problem arises when the crops are affected most by the fungal pathogens leading to huge crop loss, and thus the search for novel antibiotics is on, and the search has shifted to actinobacterial source, and endophyte plays an important role in this respect. there are significant reports of antifungal compounds from bacterial origin, but now the focus has shifted to microbes of endophytic origin (table . ). till date, a huge number of antibiotics are already reported and have minimized the crop loss to a notable amount ( fig. . ). antibiotics and other antifungal compounds include munumbicins a, b, c, d, e- , and e- , vanillin, saadamycin, , -dimethoxy- -p-methoxyphenyl coumarin, coronamycin, and fistupyrone isolated from different strains of streptomyces (shan et al. ; costa et al. ; igarashi et al. ; tian et al. ; zin et al. ) and are protecting a large number of cereals and other important cash crops from being affected by these common contaminants. endophytic actinobacteria directly counteract with fungal plant pathogens not only by producing bioactive compounds but also by enhancing the plant's growth through the production of plant growth promoters and making the plant less susceptible to pathogenic invasion. they are efficient agent of reducing the symptoms that arise due to exposure to environmental stress (shimizu ) . enhanced production of indole acetic acid (iaa) was mediated by streptomyces sp. (isolated from centella asiatica) and nocardiopsis sp. (dochhil et al. ; shutsrirung et al. ; gangwar et al. ) . experimental trials on cucumber indicate positive result as the isolates actinoplanes campanulatus, micromonospora chalcea, and streptomyces spiralis enhanced plant growth and improved yield conditions (el-tarabily et al. ) . other than auxin, auxin-like similarly functioning molecules named as pteridic acids a and b are found to be inducers of adventitious root proliferation in kidney bean plants at very minute concentrations of mm (igarashi et al. ) . chitin is a major fungal cell wall polysaccharide (the second most abundant polysaccharide in nature after cellulose) component and is the first line of defense of fungal cells. actinobacteria antagonize the fungal cell by producing chitinases (an enzyme capable of hydrolyzing fungal cell wall) and break the glycosidic bonds in chitin and lead to the death of the pathogenic cell. endophytic kitasatosporia sp. (isolate of catharanthus roseus) and kibdelosporangium sp. (isolate of achillea fragrantissima) are reported to be chitinase producers (el-shatoury et al. ; mini priya ) . actinoplanes missouriensis isolated from lupinus sp., a member of fabaceae family, produces chitinase causing hyphal cell lysis and reducing the conidial germination rate and protects the plant from pathogenic attack of plectosporium tabacinum, the causal agent of lupin root rot in egypt (el-tarabily ; el-tarabily and sivasithamparam ) . siderophores are soluble, small, high-affinity iron carriers produced by bacterial or fungal members and are involved in the transportation of iron (fe + ) across the cell membrane. they have caught sudden attention due to their involvement in plant growth promotion as well as antagonistic ability against phytopathogens (cao et al. ; tan et al. ; rungin et al. ) . endophytic actinobacteria from aloe vera, mentha arvensis, and ocimum sanctum are known to be producers of hydroxymate type and catechol type of siderophores, and the isolate saccharopolyspora o is known to be the potent inhibitor howell and stipanovic ( ) , homma et al. ( ) , thomashow et al. ( ) and smith et al. ( ) harpin proteins (erwinia amylovora), trade name: harpin αβ (proact) induction of systemic acquired resistance (sar) and less susceptibility to fungal and bacterial disease wei et al. ( ) strobilurin and oudemansin (members of basidiomycete grows on dead wood) commercial synthetic analogues: azoxystrobin and kresoxim-methyl of alternaria brassicicola, botrytis cinerea, and fusarium oxysporum (gangwar et al. ; el-shatoury et al. ). endophytic isolates of cucumis sativus (cucumber), identified as actinoplanes campanulatus, micromonospora chalcae, and streptomyces spiralis, are reported to control the growth and development of damping off, crown rot, and root rot pathogen pythium aphanidermatum. they are known to promote plant growth and to protect seedlings and mature plants. a novel bioactive compound identified as -prenylindole was isolated from endophytic streptomyces sp. showing strong antifungal activity against a broad range of phytopathogens: alternaria brassicicola and fusarium oxysporum (igarashi ) . another new prenylated indole derivative from endophytic actinobacterial source inhibited the growth of colletotrichum orbiculare, phytophthora capsici, corynespora cassiicola, and fusarium oxysporum (zhang et al. ) . naphthomycins a and k isolated from streptomyces sp. cs have antifungal activity against penicillium avellaneum shen , ) . biocontrol ability of fistupyrone has made it a useful tool to minimize the crop loss of brassica due to black leaf spot disease caused by alternaria brassicicola (igarashi ) . interest on actinomycetes of endophytic origin as an alternative tool for antifungal agent is increasing day by day (table . ). since the beginning of human civilization, whenever human race has faced any turbulence in its path of existence, they have rushed to their green friends, trees, for the ultimate solution. search for bioactive products of medical importance has been a thirst area from time immemorial. whether it is a concern of human or plant health, trees have given answers in all aspects. in the recent past, phytopathogenic infection has pushed the agricultural productive parameters to a real challenge, and plant extracts in its crude and purified form are applied as biocontrol methods (table . ). the existing synthetic chemicals are facing problem of immediate or delayed drug resistance and also issues of nephrotoxicity (the gold standard; amphotericin b), biomagnification, or quality assurance of the food products and thus are inconsistent in their business (goa and barradell ; cuenca-estrella et al. ) . so green plant extracts are the novel, safest, and the best effective treatment tool in this arena. plants are mysterious in their chemical nature and in respect to their secondary metabolite production. the faith is consistent on green plants due to the fact that plants protect themselves from fungal or bacterial diseases specially for the taxa that occur in marshy shady or water-logged or stress conditions (gurgel et al. ) . so the search is primarily made on the wild native taxa or invasive species that have higher potential of antimicrobial production. the knowledge of ethnobotany comes in this context, and tribal people are imitated for the gathering of crude knowledge. the problem of fungal pathogenesis is mainly faced by plants of economic importance, that is, cash crops. a single event of pathogenic attack can affect seriously the demand and supply ratio; thus the sustainability is lost, and restoring the good health of crops is a basic need of agricultural sectors but in an efficient way not hampering the soil health, ecosystem characters, and human health and also should be budget friendly. the search is strictly focused on plants of ethnomedicinal importance as history indicates the ability of medicinal plant extracts in human and animal mycoses and antifungal ability (mathias-mundy and mccorkle secondary metabolites are plants' best weapon against phytopathogenic invasion. several plant extracts have been assessed for their antifungal activity against a variety of phytopathogens of serious agricultural threats (table . ). the metabolites are divided into terpenoids, saponins, phenolic compounds, flavones, flavonoids, flavonols, alkaloids, and coumarins (table . ). plant extracts are primarily tested for antifungal efficacy and further are purified by solvent extraction and chromatographic procedures leading to discovery of new antifungal agents. terpenoids, also called as isoprenoids (under the chemical subclass of prenyllipids), are known to be the oldest group of widespread molecular compounds produced by plants. scher et al. ( ) reported a variety of six sesquiterpenes of antifungal importance against the causal organisms of bunch rot (botrytis cinerea) on grapes, scab of cucurbits (cladosporium cucumerinum), potato blight (phytophthora infestans), rice blast (pyricularia oryzae), and blotch of wheat (septoria tritici). sesquiterpene isolated from polygonum punctatum (dotted knotweed of knotweed family polygonaceae) named after the chemical polygodial is an effective control agent of zygosaccharomyces bailii (a common food spoilage yeast). scab of cucurbits is a common and devastating fungal pathogenic disease in agricultural fields, and this disease is to some extent prevented by the use of clerodane diterpenes extracted from detarium microcarpum, a plant of leguminosae family (cavin et al. ) . skaltsa ( ) reported fungi inhibitory (cunninghamella echinulata) activity of costunolide and eudesmane derivatives isolated from centaurea plants. other than terpenes, saponins (triterpene ad steroidal saponins) are also effective antifungals reported from plant sources. tea is one of the most vital cash crops in terms of foreign money earning and the most popular beverage having antioxidative properties. pathogenic infection by pestalotia longiseta causes a huge loss of tea production. nagata et al. in the year isolated triterpenoid saponins camelids i and ii from the leaves of camellia japonica (japanese camellia) that inhibited the tea pathogen p. longiseta. cucurbitacins i, a, b, q, and e isolated from cucurbitacins (ecballium elaterium) have antifungal activity against botrytis cinerea (har-nun and meyer ) . phenolics are odorous compounds having antifungal compounds and are also responsible for the plant pigment production. phenolics cover a large number of chemical compounds, for example, alkylated phenols, anthraquinones, coumarins, phenolic acid, phenols, phenylpropanoids, quinines, xanthones, hydroxycinnamic acid, p-coumaric acid, ferulic acid, and chlorogenic acid. phenol derivatives like crassinervic acid (p. crassinervium), aduncumene (p. aduncum), hostmaniane (p. hostamannianum), and gaudichaudanic acid (p. gaudichaudianum) are effective against strawberry blossom blight pathogen cladosporium cladosporioides (lago et al. ). -acetyl- -acetoxyacetophenone showed antifungal activity against spendley et al. ( ) , potterat et al. ( ) , martson et al. ( ) , marston et al. ( ) , viturro et al. ( ) , cavin et al. ( a) , and dhatwalia et al. ( ) pinocembrin from leaves of populus deltoides (salicaceae) shain and miller ( ) , hoof et al. ( ) cladosporium fruit and leaf rot and bitter root (cladosporium gloeosporioides) long-chain alcohol from peels of young fruit of persea americana from lauraceae, methylripariochromene a from roots of eupatorium riparium (asteraceae) prusky et al. ( ) , ratnayake bandara et al. ( ) pine needle pathogen (dothistroma pini) stearic acid from needles of pinus radiata (pinaceae) franich et al. ( ) pathogen of corn, sorghum, apple (helminthosporium carbonum) luteone and wighteone from leaf surface of lupinus albus (leguminosae) ingham et al. ( ) black and brown spot of banana (colletotrichum musae) dopamine from unripe banana fruit (musa sp.) muirhead and deverall ( ) powdery mildew of grains (erysiphe graminis) gramine from leaves of hordeum vulgare (poaceae) wippich and wink ( ) leaf spot, rots, and blights (alternaria alternata), disease of cereal (penicillium verrucosum) alizarin and emodin from root of rubia tinctorum of rubiaceae, alkylated phenols of peel and pulp of mangifera indica (anacardiaceae) cojocaru et al. ( ) , manojlovic et al. ( ) maize rot (fusarium moniliforme), epidemic outbreak of glume and kernel discoloration (curvularia lunata) flavan- -ols of root bark of sorghum cultivars of poaceae jambunathan et al. ( ) (continued) kobayashi et al. ( ) , endo et al. ( ) , cho et al. ( ) blue mold of tobacco (peronospora tabacina) diterpenoids from nicotiana tabacum of solanaceae reuveni et al. ( ) leaf and fruit pathogen (cladosporium cladosporioides) canaliculatol from bark of stemonoporus canaliculatus and long-chain alcohol from persea americana, phenylethanone from euodia lunuankenda, sinharine and methylsinharine from glycosmis cyanocarpa, illukumbin from glycosmis mauritiana (rutaceae), phenylethanone from euodia lunuankenda (lauraceae), benzoquinone from croton lacciferus (euphorbiaceae) bokel et al. ( ) , ratnayake bandara and wimalasiri ( ) , kumar et al. ( ) , greger et al. ( ) , pacher et al. ( ) , springob and kutchan ( ) miles et al. ( ) , miles et al. ( ) , lee et al. ( ) , deng and nicholson ( ) and yoganandam et al. ( ) (continued) sclerotinia sp. phenolic structures when contain a carbonyl group are known to be flavones, and the addition of an extra -hydroxyl group indicates flavonol. flavonoids are also known to hydroxylated phenolics but occurring as a c -c unit linked to aromatic ring. not only plant samples directly but also plant derivatives like porpolis (galangin isolated from the bee glue or resinous mixture produced as a result of the mixture of tree buds, sap, botanical extracts, and bee exudates) are shown to be antifungal against green rot or mold of tangerine (pathogens penicillium digitatum, p. italicum) and also control postharvest disease of cereal grains, legumes, and tree nuts caused by a. flavus (afolayan and meyer ) . flavones ( , , ′-trihydroxy- , ′dimethoxyflavone, , ′-dihydroxy- , ′, ′-trimethoxyflavone) from artemisia giraldi are effective against a. flavus infections (cowan ) . leaf wax of arrabidaea brachypoda (brazilian medicinal plant from bignoniaceae) contains herger et al. ( ) and abdu-allah and elyousr ( ) cassia tora (dealcoholized extract of leaves) mukherjee et al. ( ) thymol, carvacrol, citronellol, geraniol, citral, perillyl, menthol, eugenol, , - cirsiliol, cirsimaritin, and hispidulin and is showed to be effective against cladosporium sphaerospermum (alcerito et al. ) . galeotti et al. ( ) against fusarium oxysporum f. sp. dianthi (galeotti et al. ) . fusarium culmorum, a serous pathogen of seedling blight, foot rot, ear blight, stalk rot, and common rot of cereals and grasses, is found to be inhibited by six commercial coumarins: bergapten, herniarin, umbelliferone, xanthotoxin, and scopoletin. tithonia diversifolia, the source of tithoniamarin, is effective against the anther smut fungus microbotryum violaceum, earlier known as ustilago violacea (yemele-bouberte et al. ) . berberine and jatrorrhizine (alkaloids) are isolated from mahonia aquifolium (a plant of berberidaceae family commonly called as oregon grape and native to western north america) and are effective against human pathogenic candida species. pathogens of mango (c. gloeosporioides), anthracnose of lupin species, postbloom fruit drop of citrus, valencia and navel oranges in florida (caused by c. acutatum), and strawberry (caused by colletotrichum fragariae) are inhibited by findersine, anhydroevoxine, and haplamine (cantrell et al. ) . roots of cyathobasis fruticulosa are source of beta-carboline, tryptamine, and phenylethylamine-derived alkaloids and are antifungal in nature (bahceevli et al. ). essential oils (eos) of aromatic and medicinal plant origin are reported to possess antifungal properties and are of wide spectrum in their application for the control of agricultural pathogen (table . ). eos are mainly categorized under the plants' secondary metabolites and may fall under the category of terpenes, ketones, esters, aromatic phenols, ethers, alcohols, oxides, etc. (fig. . ) . they act by inhibiting the fungal hyphal growth either by accumulating in the fungal cell membrane or by crossing the cell membrane and entering into the eukaryotic cell. being lipophilic in their chemical nature, they can easily cross the cell and interrupt in sterol biosynthesis leading to growth retardation and finally cell death. as sterols are the maintenance, compounds of cellular integrity treatment with eo cause fungal cell death. metabolic processes like respiration, replication, transcription, and translation are inhibited. membrane permeability is drastically changed as they cause swelling and disruption of protein-lipid-protein membrane. leakage of useful ions like ca + and k + causes cell death. thymol, carvacrol, eugenol, and related phenolic compounds cause h + and k + leakage and water imbalance and deplete intracellular high-energy molecule (atp). essential oils are extracted from almost every parts of a plant, for example, roots, fruits, barks, twigs, leaves, seeds, and flowers, by several extraction procedures that include hydro and steam distillation, cold pressing, and zataria multiflora lamiaceae fermentation. the antifungal efficacy is checked by direct contact of the essential oil components and fungal hypha and poison food method, following micro or broth dilution techniques, or in vivo fumigation assay is also performed in case of field trials. essential oils from leaves of chenopodium ambrosioides, a member of amaranthaceae family, are effective against storage fungi aspergillus flavus, a. glaucus, a. niger, a. oryzae, colletotrichum gloeosporioides, c. musae, fusarium oxysporum, and fusarium semitectum (jardim et al. ) . lemongrass oil from cymbopogon citratus and cymbopogon martini are potent inhibitors of botrytis cinerea, rhizoctonia solani, aspergillus tamari, a. fumigatus, and a. conicus (tzortzakis and economakis ; mishra et al. ) . the members of lamiaceae family are well known for their pungent odor and are tested for their antifungal activity by agar and broth dilution methods (roby et al. ; omidbeygi et al. ). essential oils extracted from laurus nobilis, syzygium aromaticum, and origanum vulgare are effective antifungal compounds against two pathogens of rice, fusarium culmorum and fusarium verticillioides (rosello et al. ) . essential oils from cymbopogon exhibited antifungal activities against rot molds (soundharrajan et al. ) . antifungal activities of peppermint and sweet basil were tested against plant pathogenic fungi s. sclerotiorum, rhizopus stolonifer, and mucor sp. (edris and farrag ) . antifungal activity of β-dolabrin, γ-thujaplicin, and -acetyltropolone was tested against pythium aphanidermatum ifo (morita et al. ) . boyraz and ozcan ( ) tested the antifungal activity of the essential oils isolated from wild turkish summer savory (satureja hortensis). essential oils (carvacrol, thymol, p-cymene) extracted from origanum acutidens are effective against phytopathogens. growth of a. humicola, colletotrichum gloeosporioides, rhizoctonia solani, and phytophthora cactorum was inhibited by the essential oil of asarum heterotropoides var. mandshuricum (dan et al. ) . though there are several reports of essential oils being potent anti-phytopathogenic (penicillium purpurogenum, rhizopus stolonifer, spondylocladium austral, penicillium digitatum, penicillium luteum, monilinia laxa, curvularia lunata, etc.) in nature, still there are some problems regarding their maximum use and optimum effectivity. that includes their volatile natures, requirement of close systems, and degradation of eos by oxidation due to presence of extreme amount of hydrogenated compounds (kim et al. ). we are nourished by mother nature. so it is our prime duty to keep up the normal equilibrium of natural parameters. but in a way to seek solutions, some steps taken toward success may have negative impact on our environment. to fight against the fungal pathogens for the ensuring of better crop productivity, use of chemical fungicide is just another example of that fact. but we must emphasize on products from direct natural origin over the chemically synthesized one. natural products are the best weapon to fight fungal pathogenic diseases on economically important crop species. they are less toxic, stable, and of no side effects when used in crop fields. the crying need of modern era is obtaining pathogen-free crop species in one hand and assurance of environmental sustainability on the other. fungal and bacterial products are already used in large scales followed by the plants' secondary metabolites. phytoalexins as internal molecules are the plants' own defense system. the detailed biochemical analysis of the phytoalexins and study of their regulatory mechanisms are opening up new horizons for universal use of phytoalexin inducing elicitors as plant defense enhancers. mycorrhizae provide the basic line of physical barrier against pathogenic invasion, and reports include their ability to enhance plant growth, thus making the plant nonsusceptible to fungal attack. endophyte on the other hand can enhance the plants' defense system by direct incorporation and open up popular angles of green immunization or plant vaccination. researches on these fields are still scanty, but in the near future, they could lead to the ultimate solution of fungal pathogenic crop loss. effect of certain plant extracts and fungicides against powdery mildew disease of grapevines in upper egypt antimicrobial activity of compounds isolated from algae flavonoid and other constituents of bauhinia manca effects of resveratrol on the ultrastructure of botrytis cinerea conidia and biological significance in plant/pathogen interactions biological activity of resveratrol, a stilbenic compound from grapevines, against botrytis cinerea, the causal agent for gray mold the antimicrobial activity of , , -trihydroxyflavone isolated from the shoots of helichrysum aureonitens extraction and identification of bioactive compounds (eicosane and dibutyl phthalate) produced by streptomyces strain kx for the biological control of rhizoctonia solani ag- strain kx to control target spot disease in tobacco leaf - phytoalexins in defense against pathogens effect of volatile and non-volatile compounds from trichoderma spp. against colletotrichum capsici incitant of anthracnose on bell peppers in vitro antifungal efficacies of aqueous extract of dumortiera hirsuta (swaegr.) nees against sporulation and growth of postharvest phytopathogenic fungi evaluation of streptomyces griseorubens e g for the biocontrol of fusarium oxysporum f. sp. lycopersici: ultrastructural and cytochemical investigations interactions between a root-knot nematode (meloidogyne exigua) and arbuscular mycorrhizae in coffee plant development (coffea arabica) foliar epicuticular wax of arrabidaea brachypoda: flavonoids and antifungal activity metabolites of helminthosporium monoceras: structures of monocerin and related benzopyrans trans-trans- , -tridecadiene , , -triyne- , -diol, an antifungal polyacetylene from diseased safflower (carthamus tinctorius) mycofumigation by the volatile organic compound-producing fungus muscodor albus induces bacterial cell death through dna damage mycorrhizal fungi and trichoderma harzianum as biocontrol agents for suppression of rhizoctonia solani damping off disease of tomato effect of volatile metabolites of trichoderma species against seven fungal plant pathogens in vitro production of gliotoxin on natural substrates by trichoderma virens studies on antagonistic effect against plant pathogenic fungi from endophytic fungi isolated from houttuynia cordata thunb. and screening for siderophore and indole- -acetic acid production effect of mushroom extracts in the induction of phytoalexins and in the control of soy oidium in a greenhouse impact of mycorrhizal colonisation on root architecture, root longevity and the formation of growth regulators isolation and characterization of muscodor albus i- . s, a volatile antibiotic producing fungus influence of seed priming on the development of pearl millet downy mildew (sclerospora graminicola) synthesis and incorporation of the first polyketide synthase free intermediate in monocerin biosynthesis laminarin elicits defense responses in grapevine and induces protection against botrytis cinerea and plasmopara viticola streptomyces sanglieri which colonised and enhanced the growth of elaeis guineensis jacq. seedlings was antagonistic to ganoderma boninense in in vitro studies current status of biological control of plant diseases using antagonistic organisms in india status and prospects for enhancing the uptake of antagonistic organisms for nematode management in india alkaloids and aromatics of cyathobasis fruticulosa (bunge) aellen antimicrobial activities of bryophytes a review antibiotic activity of bryophytes an endophytic myrothecium inundatum producing volatile organic compounds muscodor albus strain gba, an endophytic fungus of ginkgo biloba from united states of america, produces volatile antimicrobials increasing the productivity and product quality of vegetable crops using arbuscular mycorrhizal fungi: a review seaweed polysaccharides as bio-elicitors of natural defenses in olive trees against verticillium wilt of olive thoughts and facts about antibiotics: where we are now and where we are heading in vitro screening of bryophytes for antimicrobial activity effect of phosphate and the arbuscular mycorrhizal fungus glomus intraradices on disease severity of root rot of peas (pisum sativum) caused by aphanomyces euteiches canaliculatol, an antifungal resveratrol trimer from stemonoporous canaliculatus induction and identification of sativan and vestitol as two phytoalexins from lotus corniculatus the cytosporones, new octaketide antibiotics isolated from an endophytic fungus phytoalexins induction in rubiaceae the camalexins: new phytoalexins produced in the leaves of camelina sativa (cruciferae) anti-fungal effects of cocoa tannin on the witches' broom pathogen crinipellis pernicious applied and environmental microbiology the chemical composition, antifungal, antioxidant and antimutagenicity properties of bioactive compounds from fungal endophytes associated with thai orchids isolation and identification of antifungal and antialgal alkaloids from haplophyllum sieversii isolation and characterization of endophytic streptomyces antagonists of fusarium wilt pathogen from surface sterilized banana roots isolation and characterization of two phytoalexins from rice as momilactones a and b munumbicins, wide-spectrum antibiotics produced by streptomyces nrrl , endophytic on kennedia nigriscans munumbicins e- and e- : novel broad-spectrum antibiotics from streptomyces nrrl aspectos bioquímicos e moleculares da resistência induzida bioactive diterpenes from the fruits of detarium microcarpum in vitro evaluation of fungicides, plant extracts and biocontrol agents against brown leaf spot of paddy bioactive diterpenes from the fruits of detarium microcarpum crop diseases and their management. phi learning private limited antifungal activity and action mechanism of ginger oleoresin against pestalotiopsis microspora isolated from chinese olive fruits studies on a chlorogenic acid-producing endophytic fungi isolated from eucommia ulmoides oliver antifungal activity of cinnamaldehyde and eugenol congeners against wood-rot fungi antifungal and antiviral products of marine organisms cytotoxic and antifungal triterpene glycosides from the patagonian sea cucumber hemoiedema spectabilis antimicrobial activity of -hydroxybenzoic acid and trans -hydroxycinnamic acid isolated and identified from rice hull diversity and antifungal activity of fungal endophytes of asparagus racemosus willd insecticidal secondary metabolic products from the entomogenous fungus fusarium larvarum -heptadecenyl)-resorcinol, the major component of the antifungal activity in the peel of mango fruit antifungal activity of neo-clerodane diterpenoids from scutellaria the manual of biocontrol agents effect of water activity on the production of volatile organic compounds by muscodor albus and their effect on three pathogens in stored potato biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by phytophthora megasperma glycoprotein elicitin biological control of phytopathogenic fungi by endophytic actinomycetes isolated from maize (zea mays l) plant products as antimicrobial agents identification of three hydroxyflavan phytoalexins from daffodil bulbs phytoalexins from other plant families isolation and characterization of actinomycete antagonists of a fungal root pathogen susceptibility of fluconazole-resistant clinical isolates of candida spp. to echinocandin ly , itraconazole and amphotericin b biochemical defense mechanisms in cotton plants against ramularia leaf spot mediated by silicon in vitro antifungal activity of -( , -dimethyl- , -dihydro- h-pyrrol- -yl)- -methylethyl pentanoate, a dihydro -pyrrole derivative phytoalexin accumulation in tissues of brassica napus inoculated with leptosphaeria maculans naphthalene, an insect repellent, is produced by muscodor vitigenus, a novel endophytic fungus activities of essential oils from asarum heterotropoides var. mandshuricum against five phytopathogens antagonist actinomycetes metabolites against plant pathogens fungi of agricultural importance induction of phytoalexins and proteins related to pathogenesis in plants treated with extracts of cutaneous secretions of southern amazonian bufonidae amphibians natural products in crop protection bioassay-guided isolation of allelochemicals from avena sativa l.: allelopathic potential of flavone c-glycosides antifungal activity of crude extracts from brown and red seaweeds by a supercritical carbon dioxide technique against fruit postharvest fungal diseases rhizospheric streptomycetes as potential biocontrol agents of fusarium and armillaria pine rot and as pgpr for pinus taeda geldanamycin, a new antibiotic induction of fusarium solani mutants insensitive to tomatine, their pathogenicity and aggressiveness to tomato fruits and pea plants molecular engineering of resveratrol in plants antifungal properties of surangin b, a coumarin from mammea longifolia phytochemical analysis and antifungal activity of moss bryum cellulare against some phytopathological fungi studies on antifungal potential of bryum cellulare against spore germination of fungus curvularia lunata in vitro antifungal activity of plagiochasma appendiculatum against alternaria solani evaluation of bryophyte for green fungicides as alternative treatment to control plant pathogen oxidative ring contraction of the phytoalexin cyclobrassinin: a way to brassilexin brassilexin, a novel sulphur-containing phytoalexin from brassica juncea l., (cruciferae) secondary metabolites production by actinomycetes and their antifungal activity antifungal bryophytes: a possible role against human pathogens and in plant protection isolation, characterization and antimicrobial activity at diverse dilution of wheat puroindoline protein free fatty acid accumulation and quality loss of stored soybean seeds invaded by aspergillus ruber molecular communication in interactions between plants and microbial pathogens seed germination enhancing activity of endophytic streptomyces isolated from indigenous ethno-medicinal plant centella asiatica interactions between an arbuscular mycorrhizal fungus (scutellospora heterogama) and the root-knot nematode (meloidogyne incognita) on sweet passion fruit (passiflora alata) application of plant extracts as inducers to challenge leaf rust of wheat inhibitory effect and mechanism of tagetes erecta l. fungicide on fusarium oxysporum f evaluating novel microbe amended composts as biocontrol agents in tomato biological activity summary for cocoa (theobroma cacao l.) effect of salicylic acid and structurally related compounds in the accumulation of phytoalexins in cotyledons of common bean phenylphenalenone phytoalexins, will they be a new type of fungicide? antifungal activity of peppermint and sweet basil essential oils and their major aroma constituents on some plant pathogenic fungi from the vapor phase production and genetic improvement of a novel antimycotic agent, saadamycin, against dermatophytes and other clinical fungi from endophytic streptomyces sp. hedaya antimicrobial activities of actinomycetes inhabiting achillea fragrantissima (family: compositae) an endophytic chitinase-producing isolate of actinoplanes missouriensis, with potential for biological control of root rot of lupine caused by plectosporium tabacinum performance of three endophytic actinomycetes in relation to plant growth promotion and biological control of pythium aphanidermatum, a pathogen of cucumber under commercial field production conditions in the united arab emirates nonstreptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters structures of antifungal diarylheptenones, gingerenones a, b, c and isogingerenone b, isolated from the rhizomes of zingiber officinale coronamycins, peptide antibiotics produced by a verticillate streptomyces sp. (msu- ) endophytic on monstera sp antifungal, anti-oomycete and phytotoxic effects of volatile organic compounds from the endophytic fungus xylaria sp. strain pb f isolated from haematoxylum brasiletto new endophytic isolates of muscodor albus, a volatileantibiotic-producing fungus effect of substrate on the bioactivity of volatile antimicrobials produced by muscodor albus antifungal volatile organic compounds from the endophyte nodulisporium sp. strain gs d ii a: a qualitative change in the intraspecific and interspecific interactions with pythium aphanidermatum preharvest treatments with chitosan and other alternatives to conventional fungicides to control postharvest decay of strawberry fungistatic effects of pinus radiata needle epicuticular fatty and resin acids on dothistroma pini blue-green alga anabaena laxa. i isolation and biological properties plant phenolics, lignification arbuscular mycorrhiza reduces susceptibility of tomato to alternaria solani antifungal metabolites from phomopsis sp. by , an endophytic fungus in gossypium hirsutum comparative efficacies in vitro of antibacterial, fungicidal, antioxidant, and herbicidal activities of momilactones a and b antifungal and antibacterial potential of methanol and chloroform extracts of marchantia polymorpha l flavonoids from carnation (dianthus caryophyllus) and their antifungal activity interactions between a fluorescent pseudomonad, an arbuscular mycorrhizal fungus and a hypo virulent isolate of rhizoctonia solani affect plant growth and root architecture of tomato plants diversity and biopotential of endophytic actinomycetes from three medicinal plants in india the diversity, plant growth promoting and antimicrobial activities of endophytic actinomycetes isolated from emblica officinalis gaertn agriculture and bioactives: achieving both crop yield and phytochemicals isolation and characterization of endophytic actinomycetes from mangrove plant for antimicrobial activity two phytoalexins from sugar beet (beta vulgaris) leaves lass-florl c ( ) the mediterranean red alga asparagopsis taxiformis has antifungal activity against aspergillus species introduction of some new endophytic bacteria from bacillus and streptomyces genera as successful biocontrol agents against sclerotium rolfsii fluconazole: an update of its pharmacodynamic and pharmacokinetic properties and therapeutic use in major superficial and systemic mycoses in immunocompromised patients plant-fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants sulfur containing cinnamides with antifungal activity from glycosmis cyanocarpa phytoalexin emit indolstruktur aus kohlrabi (brassica oleracea var. gongylodes) cytokinins mediate resistance against pseudomonas syringae in tobacco through increased antimicrobial phytoalexin synthesis independent of salicylic acid signaling metabolic products of fusarium larvarum fuckel. the fusarentins and the absolute configuration of monocerin potential of horsetail (equisetum sp.) preparations in the synthesis of defense metabolites in soy (glycine max l.) cotyledons and the effect on the growth of rhizoctonia solani kuhn, in vitro comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization chemical composition, antifungal and antitumor properties of ether extracts of scapania verrucosa heeg. and its endophytic fungus chaetomium fusiformis in vitro antifungal activity of dragon's blood from croton urucurana against dermatophytes multiple control levels of root system remodelling in arbuscular mycorrhizal symbiosis antifungal effect of five aqueous plant extracts on mycelial growth of penicillium expansum isolated from rotted yam tubers in storage alterations in root exudation of intercropped tomato mediated by the arbuscular mycorrhizal fungus glomus mosseae and the soil borne pathogen fusarium oxysporum f.sp. lycopersici host-pathogen interactions: xix. the endogenous elicitor, a fragment of a plant cell wall polysaccharide that elicits phytoalexin accumulation in soybeans tit for tat? a mycorrhizal fungus accumulates phosphorus under low plant carbon availability the accumulation of inhibitory compounds in the induced resistance response of carrot root slices to botrytis cinerea cucurbitacins protect cucumber tissue against infection by botrytis cinerea pestacin: a , -dihydro isobenzofuran from pestalotiopsis microspora possessing antioxidant and antimycotic activities the effect of post-infectional potato tuber metabolites and surfactants on zoospores of oomycetes the isolation of xanthoxylin from the bark of phytophthora and hendersonula-infected citrus lemon and its fungitoxic effect analysis on blast fungus-responsive characters of a flavonoid phytoalexin sakuranetin; accumulation in infected rice leaves, antifungal activity and detoxification by fungus the fungal dimension of biodiversity: magnitude, significance, and conservation the magnitude of fungal diversity: the ± million species estimate revisited efficacy of artificial humic acid is a selective nutrient in hv agar used for the isolation of actinomycetes genetic manipulation of isoflavone -o-methyltransferase enhances biosynthesis of ′-o-methylated isoflavonoid phytoalexins and disease resistance in alfalfa die wirkung von auszigen aus dem sachalin-staudenknoterich reynoutria sachalinensis (f. schmidt) nakai gegen plizkrankheiten, insbesondere echte mehltauplize production of antibiotics by pseudomonas cepacia as an agent for biological control of soilborne plant pathogens screening of poplar trees for antibacterial, antifungal and antiviral activity suppression of pythium ultimum induced damping -off of cotton seedlings by pseudomonas fluorescens and its antibiotic, pyoluterin effects of fusarium species on defence mechanisms in sorghum seedlings control of post harvest botrytis fruit rot of strawberry by volatile organic compounds of candida intermedia biodiversity of endophytic fungi associated with traditional chinese medicinal plants novel acidic sesquiterpenoids constitute a dominant class of pathogeninduced phytoalexins in maize antimicrobial activities of some brown macroalgae against some soil borne plant pathogens and in vivo management of solanum melongena root diseases antifungal and antiproliferative activities of endophytic fungi isolated from the leaves of markhamia tomentosa screening of novel bioactive compounds from plant-associated actinomycetes isolation of actinomycetes from live plants and evaluation of anti phytopathogenic activity of their metabolites isolation and identification of endophytic actinomycetes and their antifungal activity phytoalexins from the leguminosae fungitoxic isoflavones from lupinus albus and other lupinus species the polyoxins: pyrimidine nucleoside peptide antibiotics inhibiting fungal cell wall biosynthesis validoxylamines as trehalase inhibitors suppression of damping-off disease in host plants by the rhizoplane bacterium lysobacter sp. strain sb-k is linked to plant colonization and antibiosis against soilborne peronosporomycetes antibacterial and antifungal activities of brown alga zonaria tournefortii (jv lamouroux) direct isolation of micromonospora on selective media with gentamicin polyphenol concentrations in grain, leaf and callus tissues of mold-susceptible and mold-resistant sorghum cultivars composition and antifungal activity of the essential oil of the brazilian chenopodium ambrosioides l ulvan, a sulfated polysaccharide from green algae, activates plant immunity through the jasmonic acid signaling pathway modulation of phytoalexin biosynthesis in engineered plants for disease resistance metabolic engineering of yeast and plants for the production of the biologically active hydroxystilbene, resveratrol biosynthesis, metabolism, molecular engineering and biological functions of stilbene phytoalexins in plants deciphering the role of phytoalexins in plant-microorganism interactions and human health phytoalexins produced in the leaves of capsella bursapastoris (shepherd's purse) xanthotoxin: a phytoalexin of pastinaca sativa root mycorrhiza-induced resistance and priming of plant defenses isolation of endophytic actinomycetes from catharanthus roseus (l.) g. don leaves and their antimicrobial activity. iranian effect of verticillium wilt (verticillium dahliae kleb.) and mycorrhiza (glomus mosseae) on root colonization, growth and nutrient uptake in tomato and eggplant seedlings the possible association of phytoalexins with resistant gene expression in flax to melampsora lini antifungal potential in crude extracts of five selected brown seaweeds collected from the western libya coast in vitro antifungal, anti-elastase and anti-keratinase activity of essential oils of cinnamomum-, syzygium-and cymbopogon-species against aspergillus fumigatus and trichophyton rubrum insecticidal activities of aromatic plant extracts and essential oils against sitophilus oryzae and callosobruchus chinensis anthraquinones isolated from cassia tora (leguminosae) seed show an antifungal property against phytopathogenic fungi recent development in the use of blasticidin s, a microbial fungicide, as a useful reagent in molecular biology linear b- , glucans are elicitors of defense responses in tobacco-france induce systemic resistance and promotion of plant growth by bacillus spp antifungal activity of pisiferic acid derivatives against the rice blast fungus functional moiety for the antifungal activity of phytocassane e, a diterpene phytoalexin from rice phytoalexin induction in the sapwood of plants of the maloideae (rosaceae): biphenyls or dibenzofurans structural and functional characterization of gene clusters directing non-ribosomal synthesis of bioactive lipopeptides in bacillus amyloliquefaciens strain fzb evaluation of essential oils and their components for broad-spectrum antifungal activity and control of late leaf spot and crown rot diseases in peanut bryophytes, a potent source of drugs for tomorrow's medicine? a plant extract acts both as a resistance inducer and an oomycide against grapevine downy mildew outline of a comparative study of criteria used in characterization of the actinomycetes selection of media for isolation of streptomycetes phytoalexins from the solanaceae muscodor sutura, a novel endophytic fungus with volatile antibiotic activities endophytic fungi isolated from oil-seed crop jatropha curcas produces oil and exhibit antifungal activity identification of antifungal principle in the solvent extract of an endophytic fungus chaetomium globosum from withania somnifera isolation, characterization, and bioactivity of endophytic fungi of tylophora indica an endophytic nodulisporium sp. producing volatile organic compounds having bioactivity and fuel potential two fungicidal phenylethanones from euodia lunu-ankenda root bark antifungal and insect antifeedant -phenylethanol esters from the liverwort balantiopsis cancellata from chile efficacy of the biofumigant fungus muscodor albus (ascomycota: xylariales) for control of codling moth (lepidoptera: tortricidae) in simulated storage conditions the potential of the fungus, muscodor albus, as a microbial control agent of potato tuber moth (lepidoptera: gelechiidae) in stored potatoes benzoic acid derivatives from piper species and their fungitoxic activity against cladosporium cladosporioides and c. sphaerospermum the production of resveratrol by vitis vinifera and other members of the vitaceae as a response to infection or injury interactions between pea root-inhabiting fungi examined using signature fatty acids mycosubtilin overproduction by bacillus subtilis bbg enhances the organism's antagonistic and biocontrol activities momilactones a and b in rice straw harvested at different growth stages antibacterial activity of oriental medicinal plant extracts toward helicobacter pylori mycofumigation with oxyporus latemarginatus ef for control of postharvest apple decay and rhizoctonia root rot on moth orchid screening for endophytic fungi with antitumour and antifungal activities from chinese medicinal plants cryptocin, a potent tetramic acid antimycotic from the endophytic fungus cryptosporiopsis cf. quercina antifungal activity of camptothecin, trifolin, and hyperoside isolated from camptotheca acuminata effect of laminarin on aspergillus flavus growth and aflatoxin production use of the endophytic fungus daldinia cf. concentrica and its volatiles as bio-control agents mycorrhizae and plan health isoquinoline alkaloids from macleaya cordata active against plant microbial pathogens pestalofones a-e, bioactive cyclohexanone derivatives from the plant endophytic fungus pestalotiopsis fici bis( , -dibromo- , -dihydroxybenzyl) ether, a marine algae derived bromophenol, inhibits the growth of botrytis cinerea and interacts with dna molecules screening of a marine algal extract for antifungal activities a new macrolide antibiotic with antitumor activity produced by streptomyces sp. cs, a commensal microbe of maytenus hookeri a novel ansamycin, naphthomycin k from streptomyces sp new bioactive metabolites produced by colletotrichum sp., an endophytic fungus in artemisia annua overview: a phylogenetic backbone and taxonomic framework for prokaryotic systematics inhibitory activities of some marine algae on aflatoxin accumulation naphthoquinone spiroketal with allelochemical activity from the newly discovered endophytic fungus edenia gomezpompae plant disease control: understanding the roles of toxins and phytoalexins in host-pathogen interaction plant extracts, bau-biofungicide and fungicides in controlling some important diseases of rice cv. brri dhan biocontrol potential of cyanobacterial metabolites against damping off disease caused by pythium aphanidermatum in solanaceous vegetables bioprospecting for endophytes from australian flora with mycofumigation potential antifungal activity of rubia tinctorum, rhamnus frangula and caloplaca cerina antimicrobial compounds and resistance: the role of phytoalexins and antianticipins fungicidal and molluscicidal saponins from dolichos kilimandscharicus xanthones from polygala nyikensis ethnoveterinary medicine and development: a review of the literature elicitor activity of phytoalexins in soy and sorghum by extracts and tinctures of medicinal plant species synthesis of phytoalexins in soy and sorghum by extracts and tinctures from three forest species structures of ent-herbertane sesquiterpenoids displaying antifungal properties from the liverwort herberta adunca endophytic fungi associated with monarda citriodora, an aromatic and medicinal plant and their biocontrol potential bioactivity of bryophyte extracts against botrytis cinerea, alternaria solani and phytophthora infestans control of fungal decay of apples and peaches by the biofumigant fungus muscodor albus the algal polysaccharide carrageenans can act as an elicitor of plant defense laboratoires goëmar. parc technopolitain atalante muscodor ghoomensis and muscodor indica: new endophytic species based on morphological features, molecular and volatile organic analysis from northeast india muscodor camphora, a new record from cinnamomum camphora muscodor kashayum sp. nov. -a new volatile antimicrobial producing endophytic fungus muscodor strobelii, a new endophytic species from south india response of subterranean clover to dual inoculation with vesicular-arbuscular mycorrhizal fungi and a plant growth-promoting bacterium modulation oh cyp genes and glucosilate profiles in arabidopsis by defense pathways potential agrochemicals from leaves of wedelia biflora -trihydroxydihydrochalcone from psidium acutangulum fungi and mycotoxins in grain: implications for stored product research endophytic actinomycetes from indian medicinal plants as antagonists to some phytopathogenic fungi chemically characterized cymbopogon martinii essential oil for shelf life enhancer of herbal raw materials based on antifungal, antiaflatoxigenic, antioxidant activity and favorable safety profile volatile antimicrobials from muscodor crispans, a novel endophytic fungus volatile plant metabolites for postharvest crop protection -methoxybrassinin, a sulphur-containing phytoalexin from brassica oleracea brassicanal c and two dioxindoles from cabbage brassicanal a and b, novel sulfur-containing phytoalexins from the chinese cabbage brassica campestris l. ssp pekinensis dehydro- -methoxycyclobrassinin, a sulfur-containing phytoalexin isolated from turnip brassica campestris l. ssp. rapa calophycin, a fungicidal cyclic decapeptide from the terrestrial blue-green alga calothrix fusca biological activity of β-dolabrin, γ-thujaplicin, and -acetyltropolone, hinokitiol-related compounds bacillomycin d: an iturin with antifungal activity against aspergillus flavus chemical composition and fungitoxic properties to phytopathogenic fungi of essential oils of selected aromatic plants growing wild in turkey evaluation of , -dihydroxybenzaldehyde, dopamine and its oxidation products as inhibitors of colletotrichum musae (berk. and curt.) arx in green banana fruits antifungal activities of the leaf extract of cassia tora linn experimentelle untersuchungen über die phytophthora resistenz der kartoffel camellidins, antifungal saponins isolated from camellia japonica different mechanisms for phytoalexin induction by pathogen and wound signals in medicago truncatula glyceollin, a soybean phytoalexin with medicinal properties an endophytic actinomycete, streptomyces sp. aok- , isolated from mountain laurel and its antifungal activity antimicrobial activities of vernonia tenoreana streptomyces: implications and interactions in plant growth promotion dihydro isocoumarins produced by xylaria sp. and penicillium sp., endophytic fungi associated with piper aduncum and alibertia macrophylla activation of biochemical defense mechanisms in bean plants for homeopathic preparations inhibition of protein biosynthesis by mildiomycin, an antimildew substance antifungal activity of thyme, summer savory and clove essential oils against aspergillus flavus in liquid medium and tomato paste activity of fungal endophytes against four maize wilt pathogens efficacy of some agricultural wastes in controlling root rot of glycine max l. induced by rhizoctonia solani effect of seed inoculation with bacillus subtilis and streptomyces griseus on the growth of cereals and carrots stress induced carbazole phytoalexins in glycosmis species seasonal variation of antibacterial and antifungal activities of the extracts of marine algae from southern coasts of india griseofulvin from xylaria sp. strain f , and endophytic fungus of abies holophylla and its antifungal activity against plant pathogenic fungi arbuscular mycorrhiza: the mother of plant root endosymbiosis potential of the volatile producing fungus nodulisporium sp. cf for the control of postharvest diseases of apple isolation, abundance and phylogenetic affiliation of endophytic actinomycetes associated with medicinal plants and screening for their in vitro antimicrobial biosynthetic potential distribution and identification of endophytic streptomyces species from schima wallichii as potential biocontrol agents against fungal plant pathogens mutualism and parasitism: the yin and yang of plant symbioses effects of sulfated polysaccharide and alcoholic extracts from green seaweed ulva fasciata on anthracnose severity and growth of common bean (phaseolus vulgaris l.) biological control in greenhouse systems a new working definition of the term "phytoalexin biotransformation of the brassica phytoalexin brassicanal a by blackleg fungus phytoalexins from brassicas: overcoming plants' defenses phytoalexin accumulation and antifungal compounds from the crucifer wasabi pathogen inactivation of cruciferous phytoalexins: detoxification reactions, enzymes and inhibitors antimicrobial activity of rhodobryum ontariense. hemijska industrija the discovery of enfumafungin, a novel antifungal compound produced by an endophytic hormonema species biological activity and taxonomy of the producing organisms ultrastructural observations of pterostilbene fungitoxicity in dormant conidia of botrytis cinerea pers natural occurrence of mycotoxins in foods and feeds -an update review relation between the chemical structure and biological activity of hydroxystilbenes against botrytis cinerea two new antifungal naphthoxirene derivatives and their glucosides from sesamum angolense welw potential of plant extracts and fungicides for managing fusarium oxysporum f. sp lycopersici further evidence for the involvement of a pre-formed antifungal compound in the latency of colletotrichum gloeosporioides on unripe avocado fruits progress in phytoalexin research during the past years antifungal potential and defense gene induction in maize against rhizoctonia root rot by seed extract of ammi visnaga (l.) lam diterpene alcohols from croton lacciferus an antifungal chromene from eupatorium riparium outbreaks of aflatoxicoses in india removal of duvatrienediols from the surface of tobacco leaves increases their susceptibility to blue mold jasmonate and salicylate as global signals for defense gene expression use of algae in strawberry management antimicrobial activity of essential oils and ethanol natural products from plants and fungi as fungicides extract of phlomis fruticosa l. (lamiaceae) growth activities of the sugar beet pathogens sclerotium rolfsii sacc. rhizoctonia solani kühn. and fusarium verticillioides sacc. under cyanobacterial filtrates stress induction of defense responses in zucchini (cucurbita pepo) by anabaena sp. water extract activity of seaweed and cyanobacteria water extracts against podosphaera xanthii on zucchini antioxidant and antimicrobial activities of essential oil and extracts of fennel (foeniculum vulgare l.) and chamomile elicitation of foliar resistance mechanisms transiently impairs root association with arbuscular mycorrhizal fungi antifungal activity and potential use of essential oils against fusarium culmorum and fusarium verticillioides plant growth enhancing effects by a siderophore producing endophytic streptomycete isolated from a thai jasmine rice plant (oryza sativa l. cv. kdml ) bioactivities of extracts from some axenically farmed and naturally grown bryophytes antimicrobial activity of bryum argenteum screening of antimicrobial and antioxidant secondary metabolites from endophytic fungi isolated from wheat (triticum durum) structure biological activity relationships in triterpenic saponins: the relative activity of protobassic acid and its derivatives against plant pathogenic fungi pantoea agglomerans strain eh produces two antibiotics that inhibit erwinia amylovora in vitro effectiveness of phenolic compounds against citrus green mould control of penicillium expansum and patulin accumulation on apples by quercetin and umbelliferone determination of antimicrobial and antiproliferative activities of the aquatic moss fontinalis antipyretica hedw muscodor tigerii sp. nov.-volatile antibiotic producing endophytic fungus from the northeastern himalayas muscodor darjeelingensis, a new endophytic fungus of cinnamomum camphora collected from northeastern himalayas bioactivity guided isolation of antifungal compounds from the liverwort bazzania trilobata biosynthesis, elicitation and roles of monocot terpenoid phytoalexins biologically active secondary metabolites of endophytic pezicula sp pinocembrin: an antifungal compound secreted by leaf glands of eastern cottonwood endophytic actinomycetes from tea plants (camellia sinensis): isolation, abundance, antimicrobial, and plant-growth-promoting activities isolation of , -diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production plant bio-stimulants: a review on the processing of macroalgae and use of extracts for crop management to reduce abiotic and biotic stresses purification and partial characterization of a b-glucan fragment that elicits phytoalexin accumulation in soybean diversity and antimicrobial activity of culturable endophytic fungi isolated from moso bamboo seeds in: maheshwari dk (ed) bacteria in agrobiology: plant growth responses studies on endophytic actinomycetes (i) streptomyces sp. isolated from rhododendron and its antifungal activity 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polysaccharide on growth and flavonoid accumulation in fagopyrum tataricum sprout cultures actinobacteria associated with chinaberry tree are diverse and show antimicrobial activity the diversity and anti-microbial activity of endophytic actinomycetes isolated from medicinal plants in panxi plateau china preharvest l -arginine treatment induced postharvest disease resistance to botrytis cinerea in tomato fruits neoverataline a and b, two antifungal alkaloids with a novel carbon skeleton from veratrum taliense bioactive endophytic streptomycetes from the malay peninsula camalexin accumulation in arabis lyrata metabolites of colletotrichum gloeosporioides, an endophytic fungus in artemisia mongolica key: cord- -cnz qjy authors: pedersen, amy b.; davies, t. jonathan title: cross-species pathogen transmission and disease emergence in primates date: - - journal: ecohealth doi: . /s - - - sha: doc_id: cord_uid: cnz qjy many of the most virulent emerging infectious diseases in humans, e.g., aids and ebola, are zoonotic, having shifted from wildlife populations. critical questions for predicting disease emergence are: ( ) what determines when and where a disease will first cross from one species to another, and ( ) which factors facilitate emergence after a successful host shift. in wild primates, infectious diseases most often are shared between species that are closely related and inhabit the same geographic region. therefore, humans may be most vulnerable to diseases from the great apes, which include chimpanzees and gorillas, because these species represent our closest relatives. geographic overlap may provide the opportunity for cross-species transmission, but successful infection and establishment will be determined by the biology of both the host and pathogen. we extrapolate the evolutionary relationship between pathogen sharing and divergence time between primate species to generate “hotspot” maps, highlighting regions where the risk of disease transfer between wild primates and from wild primates to humans is greatest. we find that central africa and amazonia are hotspots for cross-species transmission events between wild primates, due to a high diversity of closely related primate species. hotspots of host shifts to humans will be most likely in the forests of central and west africa, where humans come into frequent contact with their wild primate relatives. these areas also are likely to sustain a novel epidemic due to their rapidly growing human populations, close proximity to apes, and population centers with high density and contact rates among individuals. emerging infectious diseases (eids) pose a serious and increasing threat to human health and welfare (daszak et al., ; king et al., ; jones et al., ) . diseases that have recently emerged in humans include sars, hiv, and swine flu. it is projected that . million people are currently infected with hiv/aids (who, ) , and in alone, it was estimated that billion dollars would be needed to prevent future hiv transmission and provide care for those already infected (who, ) . in wildlife populations, eids also have resulted in recent and dramatic declines in mammals (e.g., ebola in african apes; walsh et al., , canine distemper virus in wild dogs and lions; roelke-parker et al., ) , amphibians (e.g., chytridi-omycosis fungus in global amphibian populations; daszak et al., ) , insects (e.g., the mite varroa jacobsoni in honey bees; oldroyd, ) , and birds (e.g., conjunctivitis due to mycoplasma gallisepticum in passerines; williams et al., ) . human eids originate from multiple sources (e.g., host shifts from animal reservoirs [zoonotics] , evolution of existing organisms, and reemergence due to antimicrobial resistance), are increasing in number, and are globally distributed (jones et al., ) . although there is not sufficient data to evaluate whether there is a similar trend for increasing frequency of disease emergence in wildlife populations, if similar drivers influence the process of disease emergence we might expect this to be the case (daszak et al., ) . the ability to predict when, where, and within which species pathogens are most likely to emerge is therefore increasingly urgent. understanding the process of disease emergence will be critical for developing strategies to minimize risk and reduce the high cost of managing outbreaks once a disease has emerged. several review papers have explored the ecological and evolutionary drivers of disease emergence in human and wildlife populations and the conditions most likely to heavily impact new host species (daszak et al., ; osterhaus, ; jones et al., ; parrish et al., ) . ecological drivers that favor emergence are most frequently associated with changes in host or pathogen ecology, specifically ( ) an increase in host population density and contact rates, ( ) environmental changes that effect host quality and demography, and ( ) changes in host mobility and behavior. evolutionary drivers of emergence include high genetic variability in the pathogen population (morand et al., ; gupta et al., ) or a lack of variability in the host (daszak et al., ; altizer et al., b) . however, sustainable emergence of a pathogen on a host is only one outcome of a complex multistep process. we focus on disease emergence after host shifts, defined as the movement of a pathogen to a new host species irrespective of the long-term consequences (antonovics et al., ) . emergence after a host shift can result in rapid spread and high virulence because naïve hosts may lack appropriate immune responses (osterhaus, ; altizer et al., b) . the longer-term outcomes of a host shift can range from transient, unsustained ''spillover'' events (e.g., hantavirus; hughes et al., ) to persistent, self-sustaining epidemics (e.g., hiv; hahn et al., ; fenton and pedersen, ) . there are three key stages required for successful emergence of an infectious disease on a ''new'' target host: ( ) opportunity, ( ) infection and transmission, and ( ) establishment and sustainability ( fig. ) . close geographic proximity among hosts likely increases contact rates and, hence, opportunity for shifts (step , fig. ). although geographic overlap may be important for determining which pathogens a host species is exposed to, successful infection and then transmission of the pathogen in the new host will depend on evolutionary and ecological factors determined by the biology of both host and pathogen (step , fig. ). for example, recent comparative analyses have demonstrated that viruses are the most likely group to cross species boundaries (cleaveland et al., ; pedersen et al., ) , perhaps facilitated by high mutation rates and fast generation times providing the potential to rapidly overcome barriers to infection (parrish et al., ) . in contrast, within primates, half of the recorded helminth pathogens are specific to a single host species . pathogen taxonomy and, by implication, pathogen biology, may then serve as an indicator of potential for host . opportunity (biogeography of host & pathogen) . transmission (evolutionary & ecological barriers) (contact rate, demography, population density) figure . three key stages of disease emergence: ( ) opportunity, ( ) infection and transmission, and ( ) establishment. the biogeography of both hosts and pathogens will contribute to the opportunity for cross-species transmission. for natural populations, opportunity for new pathogens may be restricted to neighboring species; however, wildlife trade, invasive species, and domestic animals also may be a source of new pathogens. the ability for a pathogen to infect and be transmitted within the new host species is likely driven by ecological and evolutionary barriers, such as phylogenetic relatedness of the hosts and the evolutionary potential of the pathogen. lastly, multiple demographic factors will affect the ultimate sustainability and establishment of a pathogen following a host shift, including population density, contact rates, and the rate of spread of the pathogen. shifts. however, host characteristics also are important. close evolutionary relationships among host species might translate into similar immunological responses and lifehistory traits (pfennig, ; ricklefs and fallon, ; perlman and jaenike, ) , and hence increase the likelihood of successful cross-species infection. the rate of spread in the new host and the progression through the stages of host shift to sustainable disease emergence will be affected by host and pathogen demographics (step , fig. ), for example, population density, contact rates, and the rate of spread of the pathogen in the new host (r ; number of new cases per infected individual) (dobson and foufopoulos, ; fenton and pedersen, ; . mathematical models of hostpathogen interactions (e.g., traditional sir-susceptible, infected and recovered-models) indicate that for diseases transmitted in a density dependent fashion (i.e., by direct contact), pathogens will only persist and become established in populations above a critical threshold density (r > ; anderson and may, ) . disease emergence may be inhibited at any stage, providing the opportunity for alternative management strategies; unfortunately, this complexity contributes to the difficulty in generating accurate predictive models. in davies and pedersen ( ) , we demonstrated a significant relationship between the geographical distribution and evolutionary relatedness of primate host species, and the similarity of their pathogen communities. this relationship reflects both the opportunity for host-host contact (geographic overlap), and the barriers to infection (host biology) (steps and , fig. ). we expand on our previous analysis to explore the potential for future pathogen host shifts and disease emergence within and between wild primates and humans. first, we use the relationship between host relatedness, geographic overlap, and pathogen community similarity to quantify the risk of future host shifts that each species faces. second, we evaluate how past host shifts between primates might have shaped pathogen communities by analyzing patterns of host specificity. third, we explore whether the phylogenetic risk of host shifts may have shaped host geographic distributions over time. finally, we identify regions of the globe where we predict host shifts between nonhuman primates and humans may be most frequent. this analysis provides the first quantitative attempt to assess the risk of pathogens host-shifting to humans from wildlife populations, a critical step toward predicting disease emergence. pathogen species occurrences were obtained from the global mammal parasite database www.mammalparasites.org), comprising , records representing pathogen species (including viruses, bacteria, helminths, protozoa, arthropods, and fungi) across of the wild primate species recognized in the phylogenetic tree of bininda-emonds et al. ( ) . the human disease database from taylor et al. ( ) was used to measure pathogen sharing between primates and humans. we use the term ''pathogen'' broadly to include both microparasites (i.e., viruses, bacteria, and protozoans) and macroparasites (i.e., helminths, fungi, and arthropods). for all pair-wise primate-primate combinations we estimated pathogen community similarity as: a/(a + b + c), where a is the number of pathogen species found on two host species, x and y; b is the number of pathogen species on host x that are not found on host y; and c is the number of pathogen species on host y that are not found on host x (for further details see davies and pedersen, ) . critically, this metric is not biased by differences in sampling intensity or the relative sizes of the pathogen species pools of x and y. we focus on general patterns of pathogen sharing, thus our estimate of pathogen community similarity was calculated for all pathogen types combined (helminths, protozoa, and viruses). the relationship between community similarity and evolutionary divergence may differ across pathogen taxonomy; however, we currently lack comprehensive data on pathogen occurrences (and absences), which limits the scope of more taxon-specific analyses. in theory, our approach could be applied to any pathogen subset given sufficient data. following davies and pedersen ( ) , we derived the relationship between evolutionary divergence (representing time to most recent common ancestor from the dated phylogenetic tree of bininda-emonds et al., ) , and pathogen community similarity (as described above) between each primate pair using generalized linear modeling (glm) with binomial errors and a logit link function in the statistical package r (r: a programming environment for data analysis and graphics, v. . . ; http://www.r-project.org/). we used a glm approach because this enabled the shape of the rela-tionship between pathogen sharing with evolutionary relatedness to be characterized, and followed a similar protocol to that in previously published studies (gilbert and webb, ) . because we explore the combined risk of cross-species transmission between co-occurring primate hosts separately (see below), we did not include range overlap in the glm directly. for our model, we assume that pathogen community similarity (pathogen sharing) reflects the frequency of host shifts between any pair of primate species; the greater the frequency of host shifts, the more similar the pathogen communities. however, we note that pathogen community sharing will also reflect co-inheritance of similar pathogen communities from a common primate ancestor. although the relative importance of co-inheritance versus host-shifts in structuring pathogen communities remains unresolved, experimental cross-infection of fungal pathogens among tropical plant species (gilbert and webb, ) revealed a qualitatively similar relationship between host evolutionary relationships and cross-infection success, to that between host relatedness and pathogen sharing in primates (davies and pedersen, ) . we therefore suggest that, although co-inheritance will be important, the shape of the relationship may be determined by host shifts. nonetheless, disentangling co-inheritance from host shifts should be a focus of future studies but will require detailed information on pathogen phylogeny for many species. for each primate species, we calculated a metric encapsulating the summed risk of cross-species pathogen transmission (potential for host shifts) posed by all geographically co-occurring primates. this metric was calculated from the glm of pathogen community similarity against divergence times of each primate pair. we refer to this metric as the phylogenetic risk of host shifts (prhs). phylogenetic risk for species x (prhs x ) is then calculated as: where prhs x is the summation of the phylogenetic risk (prhs y ) for each s host species that overlaps in geographic range with the target species x. the phylogenetic risk from each overlapping species (y) to the target species x (prhs y ) is derived from the glm given the phylogenetic relatedness of species x and y. we hypothesize that primate species with high prhs will share a larger proportion of their pathogens with their geographic neighbors and close relatives. we therefore predict that: ( ) pathogen species richness will correlate positively with prhs, and ( ) the proportion of host-specific pathogens will decrease with increasing prhs. our metric (prhs) assumes that each overlapping species contributes additively to the total risk of a pathogen shifting to the target species. because the number of primate pathogens is finite, it is possible that the target species could accumulate all pathogens found in the local community of hosts. although we do not have complete data on the total pathogen community size of any one host or community, the large number of species-specific pathogens (> % in wild primates; pedersen et al., ) indicates that complete pathogen sharing within a community is unlikely. our model therefore predicts a positive relationship between the number of overlapping host species and within-host pathogen species richness. correlates of pathogen species richness in primates have been explored elsewhere (nunn et al., and may be confounded by variation in sampling intensities (altizer and pedersen, ) . we focused on testing our second prediction, relating to host range, which is the primary focus of this paper. if pathogen communities are shaped by host shifts, we predict that host species with high prhs would have fewer host-specific pathogens because they would be shared with other members of the host community. to test this prediction, we constructed a further glm to evaluate the relationship between prhs and the proportion of host-specific pathogens in each host's parasite community. because the number of recorded pathogens varies considerably between hosts (see above), some estimates of host specificity will be derived from very small sample sizes. to explore model sensitivity, we therefore repeated the glm weighting each datum by the logarithm of the total number of pathogens sampled for each species. previous studies found that viruses are the least likely to be specific to a single primate host . therefore, we ran a final glm of prhs against the proportion of host specific nonviral pathogens only. we hypothesize that phylogenetic risk of host shifts might limit geographic co-occurrence between closely related host species. this process would lead to the prediction that the observed prhs x should be lower than expected from a null model of drawing neighbors at random from the regional species pool. to evaluate whether prhs across wild primates varies nonrandomly or simply reflects the geographical distribution of host species within regional communities, we compared observed prhs x for each x primate host against a null distribution generated by randomly drawing ''neighbors'' from the phylogeny. for each primate host, we performed , random draws, with sample size of neighbors, s, matching the empirical number of hosts that overlaps with each x species' range. because of the large geographic distance separating the new and old worlds, new and old world primates will never have the opportunity to overlap, thus we performed our random draw of neighbors only from within the regional primate communities. to generate a geographical representation of the potential for host shifts within wild primates, we first constructed a weighted distribution map of primates, using our prhs metric as weights. primate species distributions were obtained from grenyer et al. ( ) . because the probability of pathogen sharing was influenced only by whether two species were found in sympatry across any part of their range, but not by the magnitude of range overlap (davies and pedersen, ) , we assumed prhs to be uniform across each target host species range. spatial data manipulation was performed in arcview (gis . , environmental systems research institute inc.) and arcmap (v . environmental systems research institute inc.) at a grid cell size of ° °. cell weights (cell w ) summed the phylogenetic risk of host shifts across co-occurring species and were calculated as: where k is the number of primate hosts with ranges intersecting with cell w . hotspots represent regions with high diversity of closely related primate species-where we predict host shifts will be most frequent. next, to provide an estimate of the cross-species pathogen transmission risk from wild primates to humans, we constructed a second hotspot map, weighting each primate distribution in proportion to its evolutionary distance from humans, using the nonlinear transformation determined from the glm model coefficients described above. here, cell weights (cell w ) are calculated as: where prhs h is the phylogenetic risk of host shifts from species x to homo sapiens. our hotspot maps provide a coarse estimate of the potential for host shifts, accounting for opportunity (geography) and biology (phylogeny). however, successful establishment of a pathogen in a novel host will depend on several additional factors, including those that influence contact rates among and within host species (fenton and pedersen, ; wolfe et al., ) . for example, habitat disturbance and transformation, resulting from human growth, may increase the probability of humans encountering new pathogens from wildlife. as a first approximation, we use estimates of human population growth ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ; sedac: http://sedac.ciesin.columbia.edu/gpw/) to represent human expansion into previously untransformed or isolated habitats, and hence potential for increased human-wildlife contact. we map the product of prhs to humans (cell weights cell w ) and an index of human population growth (increase in population density during years) to highlight areas of both high population growth and many closely related primates. here, cell weights (cell w ) are calculated as: last, we consider minimum threshold population densities required for sustained transmission and successful establishment of a disease after a host shift and map human population centers (cities > , ) on the weighted human risk map. population centers in close proximity to regions with high phylogenetic risk of host shifts and human population growth are likely to be foci of disease emergence. a third of the variation in pathogen communities can be explained by phylogenetic relatedness; more closely related primate hosts have more similar pathogen communities (glm of pathogen community similarity against divergence time: p value < . , z = - . , degrees of freedom (df) = , deviance explained = %; see also davies and pedersen, ) . phylogenetic risk of host shifts (prhs), our metric summarizing the likelihood of pathogens shifting between neighboring hosts, varied considerably across primates (fig. ) fig. a ), as expected, given that our metric, prhs, is a summation of all overlapping primates. the brown capuchin (cebus apella), with a geographic range extending from colombia and venezuela to paraguay and northern argentina, has the greatest prhs ( . ), and overlaps with more than other new world primate species across its large range. critically, however, phylogenetic risk of host shifts frequently differs even between primates overlapping with the same number of neighbors. for example, demidoff's bushbaby (galagoides demidoff) and the white-fronted capuchin (cebus albifrons) both have geographic distributions that overlap with other primate species, but the former has a prhs of . , and the latter . . across primates, our randomization procedure indicates a tendency for prhs to be greater than predicted from a random draw of neighbors (sign test of the number figure . a scatter plot of phylogenetic risk against the number of overlapping species for each primate host. risk correlated positively with overlap; however, there was still large variation in risk among species with the same number of neighbors. variation in phylogenetic risk is a product of both number of neighbors and phylogenetic relationships in the primate tree. b scatter plot of the proportion of host-specific pathogens against phylogenetic risk; symbol size is proportional to the total number of recorded pathogens for each host species respectively. hosts with high phylogenetic risk have significantly fewer host-specific pathogens, supporting our hypothesis that host shifts are more frequent among high-risk species. of times observed prhs is greater than predicted from random draws: p < . ), indicating frequent geographical overlap among close relatives. primates with higher phylogenetic risk had a significantly lower proportion of host specific pathogens (all pathogens combined), consistent with an increased frequency of host shifts in species with lots of closely related neighbors, but this was found only in the weighted glm (p value < . , z = - . , df = ; p = . for the unweighted glm). when excluding viruses, the relationship between prhs and the proportion of host-specific pathogens is significant with and without model weights (weighted glm: p < . , z = - . , df = ; unweighted glm: p < . , z = - . , df = ). unsurprisingly, there was large scatter in the relationship between prhs and the proportion of host-specific pathogens recorded for each primate host (fig. b) , suggesting other factors also are important in shaping pathogen communities. there are much fewer host-specific pathogens for primates with very high prhs (> ), although the sample size is relatively small. in contrast, primates with low prhs (< ) have varying proportions of host-specific and hostgeneralist pathogens (fig. b ). we show central africa and western amazonia to be hotspots of phylogenetic risk for host shifts between wild primates (fig. a) . these regions contain many closely related primate species that overlap in their geographical ranges. we interpret these hotspots to indicate regions where the frequency of pathogen transmission between wild primates is likely to be the highest, and thus we additionally predict increased pathogen community similarity, high within-primate pathogen species richness and a low frequency of host specific pathogens within these localities. centers of high phylogenetic risk of host shifts from wild primates to humans include central and western africa. areas of low risk include much of brazil, southern africa below °s, and northern india into nepal (fig. b) . therefore, despite the high frequency of predicted host shifts between wild primates within amazonia, it is not identified as a high risk region for shifts to humans. new world primates are more distantly related to humans than old world primates, and hence pose lower phylogenetic risk. the likelihood of successful establishment and sustained within-host transmission of a novel pathogen will be affected by several ecological and demographic factors. in humans, a critical factor is likely to be human-wildlife contact rates, population connectivity, and human population density. to stimulate discussion, we map one such factor-the intersection between human population growth (as a proxy for increasing human-wildlife contact; see methods), phylogenetic risk of host shifts to humans, and the distribution of human population centers of size > , (fig. c) . we do not have accurate data to model these terms directly; in particular, it is not clear how the likelihood of disease emergence decays with distance from population centers, or how human movement facilitates disease emergence. disease emergence is a complex, multistage process, but our results show that we can begin to make predictions about when and where diseases are likely to emerge. by understanding the factors contributing to each stage: opportunity, infection, and ultimately, establishment of a novel pathogen, we can identify which lineages and within which geographic areas disease emergence may be most likely. previous work has mapped where diseases have emerged in humans in the past (jones et al., ) and which pathogens likely pose the biggest risk of host shifts to humans (cleaveland et al., ) . our study is the first to make predictions about the likelihood of host shifts in the future. specifically, we use a robust evolutionary relationship between host relatedness and pathogen sharing to quantify the phylogenetic risk of host shifts within primates. . a hotspot map of the summed phylogenetic risk for host shifts across wild primates. central africa and west amazonia represent regions of high geographical overlap among many closely related primate species. we predict host shifts among primates to be frequent in these localities. b phylogenetic risk of pathogens host shifting to humans from wild primates. west central africa is a hotspot of high risk to humans, due to the overlapping ranges of many of our closest relatives. c the intersection between high phylogenetic risk and an index of human population growth (increase in density from [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , revealing regions where we expect high rates of human contact with primate species that pose the greatest risk of cross-species pathogen transmission to humans. open circles indicate human population centers (> , ) that may facilitate disease emergence following host shift events. we identify species between which host shifts are likely to be frequent, and map geographic areas in which they are distributed. in addition, we generate a hotspot map to highlight regions where humans may be most at risk of host shifts from wild primates. last, we explore recent demographic trends in human populations that might contribute to disease emergence following successful cross-species transmission. cross-species pathogen transmission events can heavily impact both human and wildlife populations (daszak et al., ; parrish et al., ) . however, limited data on disease in natural populations, in particular, historical information on when and where a pathogen first moved between host species, has made it difficult to generate predictive models (hopkins and nunn, ) . in a recent paper, we demonstrated that pathogen community similarity among primate species-pairs was correlated closely with their geographical proximity and phylogenetic relatedness (davies and pedersen, ) . close geographical proximity provides the opportunity for host-host contact and hence pathogen transfer, whereas phylogenetic distance might be indicative of the ease with which a pathogen can infect a novel host given the opportunity. in this paper, we extrapolate the observed relationship between pathogen sharing and host evolutionary relatedness to explore the likely frequency of host shifts within communities of primate hosts. we predict that host shifts will be most frequent among co-occurring and closely related hosts. therefore, the frequency of host shifts will reflect both the geographical distribution of host species across the landscape, as well as the shape of the phylogenetic tree that connects them (fig. ) . for example, the white fronted capuchin (cebus albifrons) overlaps in its distribution with many other new world primates and is nested within the recent new world radiation of capuchin monkeys (genus: cebus). we predict the risk of cross-species pathogen transmission to be high for this species. in contrast, demidoff's bushbaby (galagoides demidoff) is common across central and western africa, and also overlaps with many other primates in its distribution, but has only few close relatives. we predict intermediate risk of cross-species pathogen transmission for this species. the lowest risk of cross-species pathogen transmission is predicted for evolutionary distinct species in regions of low primate diversity (fig. ) . our model assumes that primate hosts will differ in their vulnerability to host shifts. we predict that frequent host shifts will increase pathogen species richness and de-crease the proportion of host-specific pathogens per host, as pathogens are shared with an increasing fraction of the host community. however, evaluating pathogen species richness is confounded by sampling artifacts (altizer and pedersen, ; but see nunn et al., ; lindenfors et al., ) , which may vary both taxonomically and geographically (hopkins and nunn, ) . we therefore focus on variation in host specificity. although it is possible that a systematic geographic bias in the distribution of pathogen species richness (e.g., as suggested for protozoan parasites; might additionally influence the frequency of host shifts, we do not believe that this trend would be sufficient to affect the strong geographical patterns found here. the results strongly support our hypothesis: primates with many close phylogenetic relatives and geographical neighbors have significantly fewer hostspecific pathogens. this relationship becomes stronger when we exclude viral pathogens, which tend to be less constrained by taxonomic barriers davies and pedersen, ) . to our knowledge, our study is the first to explore variation in host specificity among within-host pathogen communities across an entire mammalian order. in addition, we suggest that future studies figure . representation of the interaction between phylogeny and geography that may determine the frequency of host shifts within host communities. in the maps, warmer colors represent areas with many overlapping species ranges (geographically clumped distributions). in the phylogeny, red branches represent samples of closely related taxa (phylogenetically clumped). we predict the greatest frequency of host shifts will be observed among species which are both phylogenetically clumped (those with many close relatives) and geographically clumped (species with many overlapping neighbors) (top left). species that are neither geographically clumped nor phylogenetically clumped are predicted to have the lowest risk (bottom right). we expect intermediate risk of host shifts for species that are phylogenetically clumped but geographically dispersed, or geographically clumped but phylogenetically dispersed. investigating correlates of pathogen species richness should consider the frequency of host shifts. our results suggest that the frequency of host shifts between primates is likely highest in central africa and western amazonia-both centers of primate diversity. these findings are important because current estimates indicate that almost % of primates are considered threatened (iucn, ) , and infectious diseases, especially those that have resulted from a host shift, have been recognized recently as a significant driver of extinction risk in wild animals (pedersen et al., ; smith et al., smith et al., , . primates suffer from multiple threats, including habitat fragmentation and transformation, and direct exploitation (iucn, ) , which may increase susceptibility to disease-mediated declines (smith et al., ) . direct exploitation through hunting for bushmeat, necessitating frequent contact between humans and wild primates, is already associated with emerging infectious diseases moving to humans (i.e., siv, ebola, stlv- ; wolfe et al., ; nunn and altizer, ) . more recently, there has been increasing awareness of the risk for diseases moving from humans to wildlife populations. in tai national park, ivory coast endangered chimpanzee populations have suffered dramatic declines from pathogens shared with humans, including three outbreaks from pandemic human viruses (human respiratory syncytial virus (hrsv) and human metapneumoniovirus (hmpv); köngden et al., ) . significant declines in both chimpanzee and gorilla populations also have been documented across central africa due to infectious diseases (leroy et al., ) . ominously, it is likely that current risk scenarios are underestimates, based on limited pathogen sampling, especially in wild primates (hopkins and nunn, ) . geographic regions with high primate species richness, particularly, high hominidae diversity, such as west africa, pose a disproportionate risk of host shifts from wild primates to humans. notably, amazonia is not a hotspot for risks of shifts to humans, despite high primate diversity. new world monkeys diverged from old world monkeys and apes * mya, hence represent only distant evolutionary relatives to humans. although our model indicates host shifts from wild primates to humans to be most frequent within central and west africa, the likelihood of a host shift will be affected by additional factors influencing host-host contact rates. predicting successful host shifts will therefore require information on these additional factors. in this study, we used the rate at which humans are transforming natural habitat, indexed by human popula-tion growth, as a proxy for encounter rates between wildlife species and their associated pathogens. as human populations grow, encroach into new areas, and transform habitats, we may be at greater risk of contacting new pathogens (daszak et al., ; dobson and foufopoulos, ; weiss and mcmichael, ; wolfe et al., ) . this is of particular concern, given the positive correlation between human population growth and our modeled risk of host shifts (spearman's r = . , p = . , adjusting degrees of freedom to account for spatial autocorrelation), suggesting that in many parts of central africa, where host shifts are predicted to be frequent, human population growth also is high. global emergence of human infectious diseases frequently originate not from the point of first infection but from human population centers, where contact rates among individuals are high and where international travel and dissemination of the pathogen to other cities is facilitated (woolhouse, ; tatem et al., ; sharp and hahn, ) . in epidemiological models, directly transmitted pathogens are not likely to persist in populations below a critical size threshold (a function of both host and pathogen characteristics; anderson and may, ) . for example, measles is thought to go locally extinct in populations comprising less than , individuals (keeling and grenfell, ) . many host shift events may therefore never become established. however, large population centers in or near areas of rapid human expansion can provide the demographic component necessary to convert a host shift into a global emergence event. here, we map the distribution of the major human population centers across the globe. population centers that have the potential to facilitate the emergence of new diseases shifting from wild primates to humans are those in west and east central africa, including: kinshasa, democratic republic of congo; brazzaville, congo; and yaounde, cameroon to the west, and kampala, uganda; kigali, rwanda; and nairobi, kenya to the east. infectious disease detection and control, before emergence events, should focus on these regions. possibly the worlds most devastating pandemic, hiv- , provides a remarkable case study, closely matching our model predictions. resulting from a host shift from chimpanzees, one of our closest living relatives (korber et al., ) , hiv- , was first described in , but was introduced to human populations several times during the past years-two of which were from sivcpzptt (simian immunodeficiency virus) of chimpanzees (pan troglodytes troglodytes) from west central africa (korber et al., ) . one shift led to the global aids pandemic (hiv- group m), and the other resulted in a localized endemic in cameroon (hiv- group n) (keele et al., ) . although siv has been found in more than african primate species, chimpanzees are the only species that harbor the strain most closely linked to pandemic hiv- (sharp and hahn, ; keele et al., ) . recent analysis of hiv- group m suggests that several variants were circulating in the s, and molecular analysis now proposes that hiv- likely emerged from sivcpzptt at the turn of the th century ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) but had a period of relatively slow growth during the next years (worobey et al., ) . it is thought that hiv- group m was first transmitted to humans from wild chimpanzees in the southwest corner of cameroon (keele et al., ) , and its establishment and spread was likely facilitated by the rise of cities, specifically kinshasa, democratic republic of congo (formerly leopoldville, zaire). in , no population centers in the area of the cross-species transmission event were greater than , people; however, kinshasa grew rapidly as a trading center and had a population between , - , during the middle of the twentieth century when hiv- can be found in human samples (sharp and hahn, ; worobey et al., ) . kinshasa is km from the location of the original host shift event; however, it was a key transportation hub through river travel (sharp and hahn, ) . the successful emergence of hiv occurred in our predicted hot spot areas of high human risk. this example highlights the complex multistage process that drives disease emergence: a region with high risk of shifts to humans; a host shift from our closest relative; delayed emergence after the local growth of the human population at a transportation center. whereas human population density and encroachment into new habitats may affect the likelihood of the successful emergence of a primate pathogen into humans, the risk map of human disease emergence also highlights geographic locations where wild primates may be at the greatest risk of contracting human diseases. many pathogens from humans have been found in wild primate populations (e.g., polio, syphilis, influenza a, and measles) and pose a significant threat to their persistence (nunn and altizer, ) . almost half of wild primates face the risk of extinction (iucn, ) and pathogens have been linked to substantial population declines (formenty et al., , leroy et al., , köngden et al., ; understanding the patterns driving cross-species transmission and host shifts in wild populations will therefore be crucial for their conservation. within wildlife populations, ecological factors may be more important in determining rates of crossspecies transmission and host shifts within wild primates. host life history, ecological niche, feeding and activity patterns, sexual behavior, group size, and territoriality are strong determinants of pathogen transmission rates within a host population (altizer et al., a; nunn and altizer, ) , and therefore may be important for determining when and to whom cross-species transmission events are likely to be successful. although a comprehensive exploration of these ecological factors is beyond our current scope, knowledge of how host ecology is likely to interact with pathogen ecology, host phylogeny, and geography will be key for developing a predictive framework of wildlife disease emergence. in our analyses, we focused on primates because they are perhaps the best studied animal group and because of their close evolutionary relationship to humans. primates represent a significant disease reservoir for humans; approximately % of zoonotic human eids are shared with primate host species; however, other taxa (e.g., bats, rodents, birds, and domestic animals) also pose a threat of host shifts to humans (woolhouse and gowtage-sequeria, ) . nonetheless, we suspect that our results, specifically that phylogenetic tree shape and geographic dispersion are likely to affect the phylogenetic risk of a host shift, will likely extrapolate across taxa (see gilbert and webb, ) . last, we note that the relationship between pathogen taxonomy, host phylogeny, and host geography may be complex and vary across the different pathogen groups. for example, we might predict a greater frequency of host shifts among more distant relatives for viruses than for helminths, which suggests that the risk of viral emergence may be even greater than predicted in our framework. although data on pathogens from natural populations is incomplete, and there may be many factors that affect the likelihood of a host shift, our results suggest that a broad biogeographical approach can help predict cross-species pathogen transmission. by combining information across multiple species and communities, it is possible to detect general trends that are not apparent from single case studies. in contrast with traditional epidemiological studies, our analysis explores macroevolutionary and macroecological patterns in the distribution of pathogens across species and the 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emergence population biology of emerging and re-emerging pathogens emerging pathogens: the epidemiology and evolution of species jumps host range and emerging and re-emerging pathogens direct evidence of extensive diversity of hiv- in kinshasa by the authors thank k. smith key: cord- -w ogsk authors: perlin, david title: rapid detection of bioterrorism pathogens date: - - journal: beyond anthrax doi: . / - - - - _ sha: doc_id: cord_uid: w ogsk nan public health control is to identify rapidly the infecting pathogen and its source. some of these select agents are highly transmissible in the early stages of disease and it is critical to identify infected patients to limit the risk to the remainder of the population. in some cases, such as hemorrhagic fever with ebola virus, it is hypothesized that patients become infected through contact with an infected animal. yet, the natural reservoir of the virus is unknown, as is the manner in which the virus first appears in a human at the start of an outbreak [ ] . whether the initial source can be elucidated or not, rapid diagnostic procedures are critical to support infection control measures that monitor and limit the spread of infectious diseases agents [ , ] . finally, accurately defining the scope and progression of an infectious disease outbreak helps mobilize resources more efficiently and eases public anxiety that can lead to panic [ ] . rapid clinical diagnosis and aggressive preemptive therapy can limit the fatalities associated with a biological agent of mass destruction [ , ] . most clinical laboratories, however, still rely upon culture-based technology with phenotypic endpoints, approved by fda and/or cdc. these assays can take several days for definitive results. in addition to time delays, these techniques often lack adequate sensitivity and specificity and some organisms are difficult to isolate in culture. delays often translate into the initiation of empiric therapy in the absence of positive pathogen identification. the problem is not limited to a bioterrorism outbreak as hospital and public health laboratories, confounded by inadequate and slow methodology for pathogen detection, often have difficulty identifying pathogens. when occurring with very ill and/or immunocompromised patients, these delays can increase morbidity and mortality. in a bioterrorist event, like many naturally occurring disease outbreaks, there is a need to obtain rapid pathogen identification from clinical specimens but also from environmental specimens to minimize fomite-based transmission. time delays measured in days can prevent adequate public health measures from being instituted to contain an outbreak. such delays also subject exposed individuals to needless stress and anxiety. the inadequacy of phenotypic-based diagnostic assays is illustrated graphically by the ''gold standard'' public health laboratory-testing algorithm that was in place for positive identification of bacillus anthracis from environmental samples during the october anthrax outbreak (fig. . a) . a complicated matrix of phenotypic and biochemical assays required - days for a positive endpoint. what was needed was a streamlined approach that could progress from primary specimen to a positive or negative outcome in a matter of hours or less ( fig. . b) . rapid diagnostics have finally come of age and offer exceptional promise in this regard for accurate pathogen identification. rapid identification assays can be loosely divided into tests that either utilize antigen-antibody or other antibody-specific binding to identify a specific pathogen or genetic tests that identify pathogen-specific dna or rna sequences. serological techniques have been invaluable for detecting active infections with organisms that are difficult to culture and for documenting previous infection/ immunity. while conventional serodiagnosis has a limited ability to detect acute infections with select agents, it is invaluable for monitoring disease kinetics and dispersion of these agents within exposed or high-risk populations, especially where asymptomatic infections are frequent. such information is crucial to therapeutic intervention and prophylaxis, as well as to isolation protocols in the context of a biological attack. serodiagnosis is particularly useful for mass screening of infectious diseases because such techniques are generally simple to perform, inexpensive and amenable to high throughput technologies. the assay takes advantage of the exquisite sensitivity and specificity of antigen-antibody interactions. antigen capture assays represent a somewhat more recent addition to serological techniques. these methods have been useful for rapidly diagnosing acute infections where antigen levels are relatively high, especially in the urine where they may be concentrated. the enzyme linked immunosorbent assay (elisa) has become the workhorse of most clinical laboratories. it relies on specific antigen-antibody interactions to identify a pathogen [ ] . typically, a preliminary test can be performed quickly, usually within - h. a positive elisa test can be confirmed by performing a western blot with target-specific antibodies or by an immunofluorescent antibody (ifa). unfortunately, pathogen antigen production or a host antibody response is not always robust enough to permit reliable testing, especially in the early stages of an infection. in addition, a false positive result with an elisa test can occur due to interference from other antibodies. although the elisa test is highly specific, an antibody response may not be detected in either the early or late stages of a disease. improved sensitivity may be obtained by expanding the class of antibodies detected to igg, iga and/or igm classes of antibodies [ ] . elisa platforms are especially well suited for toxin detection and, when combined with procedures such as time-resolved fluorometry, sensitivities of - pg/ml can be obtained in a typical h assay for molecules such as botulinum type a or b neurotoxin and staphylococcus aureus enterotoxin b [ ] . a newer format for antigen-antibody detection where the detector antibody is labeled by chemiluminescence facilitates an even greater sensitivity [ ] . in one approach, target antigen is first complexed to paramagnetic beads coated with capture antibody and then identified using a detector antibody. an electrochemical flow cell with photon detector is used to detect the targetantibody interactions with detection limits approaching fmol/l and dynamic ranges of six orders of magnitude [ ] . more recently, a novel type of biosensor for rapid pathogen identification has been described using b cells as sensing elements. this system know as canary (cellular analysis and notification of antigen risks and yields) utilizes b lymphocytes genetically engineered to express both cytosolic aquorin, a calcium-sensitive bioluminescent protein, and membrane bound antibodies specific for a given pathogen or toxin [ ] . interactions of antigen with antibody elevate intracellular calcium levels resulting in light emission by the cytosolic aquorin molecules. the system responds in a fashion that is more rapid, sensitive, and specific than most antigen detection systems, and has been shown to detect yersinia pestis in less than min at levels of colony forming units [ ] . in recent years, the recognition that pathogens can evoke specific t-cell responses has been used to develop assays such those used in the enzyme-linked immunospot assay (elispot) t-spot tb and quantiferon-tb for the diagnosis of latent tuberculosis infection. the exquisite sensitivity of these assays in which signature tb peptides elicit specific interferon gamma release makes them applicable to immunosuppressed individuals, while the specificity of the assay overcomes problems such as prior vaccination with bacille calmette-gue´rin (bcg). these assays cannot distinguish between drug sensitive and resistant forms of tb, but are particularly valuable in following diseases transmission in settings where multidrug resistant (mdr) or extremely drug resistant (xdr) strains are prevalent. enzymatic activity has been used for some time in the detection of microorganisms. for example, the presence of significant levels of helicobacter pylori in gastric secretions has been made using the detection of urease activity of the bacillus. although bacterial urease production is not unique to h. pylori, the other urea producers are not found in the human stomach, the site of replication of h. pylori. an example of a bioterrorism antigen detection system utilizing a specific enzymatic interaction with a substrate rather than binding to an antibody is the micromechanosensor reported by liu, et al. [ ] . the functional nature of a toxin is detected utilizing microfabricated cantilevers [ ] . in this proof of concept model, botulinum neurotoxin b is detected by its activity as a endopeptidase, cleaving its neurotarget synaptobrevin (also referred to as vamp ). the reporting system detects the large change in resonance frequency of the vibrations of the cantilever following release of the agarose bead, which is bound to the cantilever by synaptobrevin. the cantalever system is most sensitive in gaseous or vacuum milieus and the fluid medium damps the vibration to some degree. it can detect botulinum toxin at nm concentration within min and is applicable to on-chip electronic technology that greatly increases sensitivity. genomic differences between microbes offer an alternative to culturing for detection and identification of pathogens by providing species-specific dna targets that can be accurately resolved by molecular methodology. nucleic acid-based molecular approaches for pathogen identification overcome many of the deficiencies associated with conventional methods by exploiting both large-and small-scale genomic differences between organisms. polymerase chain reaction (pcr)-based amplification of highly conserved ribosomal rna (rrna) genes, intergenic sequences, and specific toxin genes is currently the most reliable approach for identification of bacterial, fungal and many viral pathogenic agents. when combined with microarray or fluorescence-based oligonucleotide detection systems, these molecular approaches provide quantitative, high fidelity analysis [ , , ] . most importantly, these genetic probing systems offer rapid turn around time ( - h) and are suitable for high throughput, automated multiplex operations critical for use in clinical diagnostic laboratories. the need for rapid diagnostics was never more apparent than during the severe acute respiratory syndrome (sars) epidemic of the spring of . in the early stages of the epidemic, physicians and public health officials were relatively helpless to contain a fast moving, globally spreading epidemic of severe atypical pneumonia caused by an unknown respiratory agent. once the viral agent was identified, genomic sequences of the sars coronavirus (cov) were used to develop a diagnostic assay within days that rapidly (within - h) and reliably identified the sars cov in a range of clinical and environmental specimens [ ] . armed with molecular tools, physicians and public health officials in china and canada, hit hardest by the disease, could confirm the cause of rapidly spreading atypical pneumonias, monitor virus levels in patients and explore potential sources for the outbreak [ ] [ ] [ ] [ ] . this single event highlighted the importance of rapid molecular diagnostics in outbreak control and disease management, signaling the arrival of a new era in diagnostics. an effective diagnostic assay must be rapid, sensitive and specific as well as being simple and robust to facilitate use in clinical and public health laboratories. assays should detect a wide variety of pathogens and, where possible, have capacity to detect ''designer'' organisms (heterologously expressed toxin or virulence genes) created through recombinant technologies. genetic-based molecular assays are currently in development that not only include all category a through c pathogen and toxin genes but also include a wide range of common bacteria, viruses and fungi. with this approach, it is possible to recognize in clinical (respiratory secretions, blood, urine, tissue), environmental (including letters, nasal swabs and hair) and food or water samples the presence of a pathogenic organism that is present as a homogeneous population or is cloaked by dispersion within a large population of nonpathogenic organisms. it is also possible to detect unnatural events such as the expression of a lethal toxin gene (such as a botulinum toxin genes) in a recipient nonpathogenic organism such as escherichia coli or bacillus subtilis. similarly, a mixed powder containing anthrax-laden spores in a background of . % b. subtilis spores would be perceived as harmless, until the . % b. anthracis component began to cause disease. such complex mixtures can be easily resolved by molecular diagnostics. specificity and sensitivity are also key elements of the diagnostic assay. when specific probes are used, they must be shown to interact only with their designated targets to avoid false positive responses. the advent of real-time selfreporting probes capable of allele-specific sequence discrimination at the level of a single nucleotide allows these probes to react with exceptional fidelity [ , ] . sensitivity is a major requirement especially because in early stages of a disease few pathogens may be present for detection. some pathogens like francisella tularensis, the etiological agent of tularemia, need only a few organisms to cause lethal disease [ ] . microarrays of nucleic acids were developed to utilize the enormous amount of information provided by genome projects but have clear potential in mass screening and diagnostics [ , ] . a microarray allows thousands of targets to be analyzed simultaneously, being particularly useful for novel virus identification and characterization. microarrays consist of gene and genome-specific nucleic acid fragments, either cloned gene segments or long ( - mers) oligonucleotides, which are fixed to a glass slide or other solid matrix such as those used for computer chips [ ] . one such product is the genechip , a high density, oligonucleotide-based dna array developed at affymetrix. target dna or rna is labeled and hybridized to complementary dna sequences on the microarray. scanning lasers are used to detect high affinity interactions and each addressable position corresponds to a known target. the application of this maturing technology may be best illustrated during the outbreak of sars during early . to assist in trying to identity the pathogen, the cdc referred specimens containing the unknown agent to many laboratories, including that of dr. joseph derisi. derisi had developed a microarray chip called virochip containing nucleic acids specific for numerous viruses known to cause human disease. hybridization of the unknown virus genome segments to the chip revealed the presence of a previously uncharacterized coronavirus. subsequent molecular characterization and phylogenetic sequence comparisons confirmed that the virus was a new member of this family [ , ] . microarray technology is powerful but also expensive, technically demanding and labor intensive. amplifying the sample above background is critical to achieve dependable results. a highly purified nucleic acid sample is best for the assay as interfering substances may limit hybridization. false positive interactions can usually be minimized through careful microarray development with suitable redundancy of targets. it is best, however, to verify independently ''positive hits'' with a different technique. like any hybridization-based assay, target specificity is critical, although absolute fidelity can be fine tuned. allelespecificity at the level of single nucleotide changes can be desirable for subtyping of species [ ] . the ability to detect imperfect matches within a family is equally desirable for the discovery of new disease agents and variants of old ones (sars cov). of course, genetic microarrays like other genetic approaches can detect the presence of toxin genes, but they cannot be used to directly detect toxins. a trend toward fully automated microfluidic applications involving chipbased capillary electrophoresis that can perform in-line reagent dispensing, hybridization, and detection significantly reduces sample sizes and improves accuracy [ ] . the combination of array hybridization followed by direct viral sequence recovery provides a general strategy for the rapid identification and characterization of novel viruses and emerging infectious diseases. the ability of dna microarrays to identify either multiple gene targets from single or multiple pathogens in a single sample has the capacity to transform detection of emerging pathogens. it is particularly useful to evaluate rapidly changing disease agents such as influenza [ ] . new platforms such as the greenechippm, which is a panmicrobial microarray comprising , sixty-mer oligonucleotides, is suitable for comprehensive detection of a wide range of vertebrate viruses, bacteria, fungi, and parasites [ ] . this technology has the potential to transform blood testing by providing an integrated platform for comprehensive testing that replaces multiple individual assays [ , ] . the simplest pcr form involves the use of specific primers to amplify a known target fragment of dna or rna and detection the product with an intercalating dye. this approach relies upon the specificity of linear dna-dna or dna-rna hybridization probes in the amplification process. probe-target hybridization is highly temperature dependent, however, and, depending on the nucleotide composition of the probe, random annealing can pose a problem. sequences with high g þ c contents are especially vulnerable since the temperature profile for annealing is shifted and false priming may occur. in general, standard pcr-based amplification is insufficient for identification purposes due to relatively high levels of false positive results. a solution to this problem is the use of fluorescent probes such as the endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons that require high fidelity binding to target sequences for detection [ , [ ] [ ] [ ] . such high fidelity probes, especially self-reporting probes, have additional advantages in that both pcr amplification and detection can be done in a sealed tube, greatly reducing the possibility of contamination. they can also be used in real-time assays in which product formation is continuously monitored and validated. this is an important consideration for a clinical microbiology lab because pcr amplification has the potential to amplify small amounts of target dna from contaminating organisms or even human dna. real-time pcr does not require post-pcr sample handling, preventing potential pcr product carry-over contamination and facilitating high throughput assays. real-time pcr assays using high fidelity probes are also rapid ( . - h), quantitative and have a large dynamic range exceeding million-fold of starting target. probing systems including lightcycler tm [ , ] , taqman tm [ ] and molecular beacons [ , ] are widely used to identify pathogens in real-time assays. the lightcycler system measures the fluorescence resonance energy transfer (fret) between two linear oligonucleotide probes labeled with different fluorophores in a glass capillary tube format. the probes are hybridized to the target in a head-to-tail motif during the annealing stage of the pcr which bring the fluorophores in a close proximity, causing a transfer of energy resulting in an emission of a detectable fluorescent light. taqman probes are linear oligonucleotides that contain a reporter dye and acceptor with overlapping emission-absorption spectra. the reporter dye remains quenched by the acceptor, while hybridized to its target. cleavage of the reporter by nuclease activity of taq dna polymerase results in strong fluorescence signals. taqman can be utilized in a well format, amenable to high throughput screening. one limitation of fret-based systems is that multiplexing is more limited since several quenchers in the same reaction are required and spectral overlap can be a problem. molecular beacons are small, single stranded nucleic acid hairpin probes that brightly fluoresce when bound to their targets [ ] . the probes possess a stem and loop structure in which the loop contains a complementary target sequence. the stem forms by the annealing of short complementary nucleotide sequence arms adjacent to the target sequence. a fluorophore is covalently linked to one end of the stem sequence with a quencher covalently linked to the other end. in free solution, molecular beacons do not fluoresce because the stem structure keeps the fluorophore close to the quencher and fluorescence energy is absorbed and released as heat. in the presence of target dna, however, the loop sequence anneals to the target and a probe-target hybrid is formed forcing the stems containing the fluorophore and quencher apart, and fluorescence occurs. molecular beacons are better suited than most linear probes to monitor authentic amplicons in pcr reactions because a single nucleotide mismatch can prevent a molecular beacon from binding to its target and lighting up [ ] . both taqman and molecular beacons can detect single nucleotide changes and are highly suitable for allelic discrimination [ , ] . a single multiplex reaction assay that combines numerous probes and is capable of identifying multiple pathogens is a more efficient and cost-effective approach for a clinical microbiology lab, greatly expanding the capacity of pathogens being surveyed. multiplex assays require that probes representing different targets can be reliably resolved in the same reaction tube or well. typically, different probes are labeled with a range of fluorophores that have unique emission spectra that can be discerned with discrete optics or dispersed onto an array for detection. when dealing with fluorophores, the spectral properties of the probe-target hybrid must be significantly different from the unbound probes to permit unambiguous probe identification. lightcycler tm , taqman tm and molecular beacon probes are all suitable for multiplex assays. the ability to multiplex pcr by probe color and melting temperature (t(m)) greatly expands the power of real-time analysis. novel labeling techniques are evolving quickly that will allow more than targets to be simultaneously evaluated in a single reaction. for example, masstag pcr, which has been used detect viral hemorrhagic fever, is a multiplex assay in which microbial gene targets are coded by a library of distinct mass tags. nucleic acids are amplified by multiplex pcr using up to primers, each labeled by a photocleavable link with a different molecular weight tag. after separation of the amplification products from unincorporated primers and release of the mass tags from the amplicons by uv irradiation, tag identity is analyzed by mass spectrometry [ ] . a number of target sequences have been proposed for the identification of pathogens likely to be used in a bioterrorist event. some targets are specific to a single species, subspecies, toxin or virulence factor (table . ). other targets can be used more generally such as ribosomal rna (rrna) genes or heat shock genes. these genes contain highly conserved dna sequences (usually required for function) interspaced with variable regions that have been widely utilized in species-specific genetic assays. ribosomal genes in fungi and bacteria have conserved sequences that are ideal for universal primer targeting, contain variable sequence regions that are species-specific, and are present in high copynumber tandem repeats [ ] . the gene for the small-subunit ribosomal rna ( s-like) has been especially useful in evolutionary studies of distant phylogenetic relationships, remaining quite stable during evolution of all organisms [ ] . the s-like ribosomal genes can be amplified from total dna isolated from essentially any organism using a single set of primers recognizing the conserved regions of the gene. dna or rna from a wide variety of organisms can be amplified using a single set of ''universal'' pcr primers that bind to conserved regions of these genes. species determination may then be performed by analyzing the species-specific sequences contained in regions within the resulting amplicons [ ] . there are advantages to both types of targets. specific targets can be detected in samples that are heavily contaminated with nonpathogenic bacteria. they also make it possible to detect virulence factors inserted into normally innocuous bacteria. ''universal'' target amplification approaches have the advantage that they can be more easily multiplexed. a single set of primers serves to amplify multiple species, permitting the development of more general diagnostic assays. the same two approaches can be used to develop targets for viral detection assays, although ''universal primers'' are generally more restricted to bacteria, fungi and specific families of viruses. sample processing development is an integral component of a successful diagnostic program. during a bioterrorist event, depending on its size and duration, a public health lab may need to process tens of thousands of specimens. rapid nucleic acid extraction from clinical and environmental samples will be critical for downstream molecular evaluation. extraction of genetic materials must be automated, ultrasensitive and have high throughput capability to process and organize large sample populations. the detection of nucleic acids from infecting microorganisms or viruses in whole blood, tissues, respiratory secretions, urine, and other body fluids can be influenced by numerous factors. sample preparation depends on the type of biological material that vary in consistency and viscosity. highly viscous samples (such as mucous) can be difficult to handle and process. bacterial and fungal spores are encased in a heat and largely chemical resistant shell, making the isolation of nucleic acids troublesome. new cell and spore disruption techniques involving mechanical, chemical, enzymatic and thermal treatments have improved extraction efficiencies markedly [ ] [ ] [ ] [ ] . all of these procedures are suitable for robotic high throughput processing. when possible, liquid biological samples should be centrifuged to concentrate bacteria, spores and fungi prior to nucleic acid extraction, increasing purity and efficiency. anticoagulants such as edta, heparin or citrate can limit product formation by interfering with the pcr as can large excesses of free genomic dna. differential surface-based binding procedures have been developed to purify and concentrate target dna away from genomic dna and hostassociated inhibitors, improving sensitivity. magnetic bead technology is an ideal choice for nucleic acid isolation and purification because of its greater affinity for nucleic acids than other conventional methods. it reduces the risk of sample cross contamination found in other extraction methods by eliminating centrifugation and other manual steps during the extraction and purification process. magnetic bead based nucleic acid extraction can be performed from micro (< ml) volume samples, is completely automated for high throughput and can rapidly isolate purified nucleic acid in about h. commercial systems such as magna pure lc tm , kingfisher tm and nuclisens easymag tm are readily available in clinical laboratories for extraction of both dna and rna from a wide range of pathogenic bacteria, viruses and fungi present in clinical samples. these standardized products allow for highly efficient target capture and are scalable over a wide range of nucleic acid levels. the genexpert system is a bench-top sized fully self-contained microfluidic system for sample extraction and nucleic acid detection. the cepheid midas ii (microfluidic dna analysis system) provides rapid, on-site testing for bioterror pathogens from environmental samples. it automatically processes biological samples, extracts the nucleic acid, and prepares it for testing. the system then transfers the extracted nucleic acid and pcr reagents to a real-time thermal cycler with eight independently programmable reaction sites. all of the critical processes of the analysis are performed in a closed microfluidic system, including post-analysis clean-up and decontamination. this technique allows for continuous, automated operation over an extended time with an assay time is less than min for pathogen detection. one of the primary outcomes of rapid diagnostics development is to facilitate therapeutic intervention by detecting infectious agents early in infection. this goal requires that a new molecular detection technique be optimized for both sensitivity and specificity, and be validated. an important consideration is to be able to quantitatively compare culture and antigen-based detection in individuals with molecular probes. once molecular diagnostic approaches are validated, the new diagnostic tools can be refined and used to assess earlier predictors of disease such as elevated temperature or measurable immunological responses. ideally, validation of a new diagnostic should occur by statistically demonstrating its equivalence or superiority to conventional detection methodology on clinical samples. typically, such validations are best determined from clinical specimens obtained from patients in endemic areas of disease. for most select agents, human infections are rare and occur in remote regions outside the usa, making validation on human populations impractical. a partial solution is to use welldeveloped animal infection models to both optimize and provide initial validation for new diagnostic tools. the primary advantage of an animal infection model is that infection and progression of disease can be more precisely defined. the goal of optimization studies in animals is to achieve the highest possible level of detection while maintaining fidelity of identification in the absence of false positives. rapid advances in the genomic sequencing of bacteria and viruses over the past few years have made it possible to consider sequencing the genomes of all pathogens affecting humans as well as the crops and livestock upon which our lives depend. the chem-bio non-proliferation program of the us department of energy began a large-scale effort of pathogen detection in early in an effort to provide biosecurity at the winter olympic games in salt lake city, utah [ , ] . molecular assays were developed at the lawrence livermore national lab for likely bioterrorist agents by utilizing whole genome comparison methods to recognize unique regions of pathogen genomes suitable for identification. genetic-based rapid assays were developed for all major threat list agents for which adequate genomic sequence is available, as well as for other pathogens requested by various government agencies. the assays were validated by cdc and were used at the winter olympics [ , ] . the program continues to add new pathogens to expand the diversity of the detection platform. the olympic air monitoring utilized - monitor stations over an area centered on salt lake city. filters were removed on a h basis and tested for genome fragments of bioterrorism pathogens. during the screening period, a sample collected at the city airport was positive by initial screening. the airport was alerted regarding the potential for evacuation, but confirmatory tests were negative [ ] ; the cause of the false positive test result was not specified. in , the us department of homeland security expanded this program into biowatch, a multicity (initially ) program. from this ''early warning'' system, there has been one report of a positive assay. the incident originated in houston, texas where air filters detected genomic evidence of f. tularensis, the cause of tularemia, on air monitoring filters between october and , [ ] ; subsequent assays were negative. the source of the positive test was not clear but tularemia is endemic in the state. the $ million/year system was expanded to cities in late . yet, it has been criticized for being unable to detect small releases of pathogens [ ] . in july , the united states postal service employed at mail processing centers a high throughput bio-agent detection system (bds) developed by northrop grumman company. the automated system samples air from critical points around the mail sorting machines. the air is drawn through a spinning membrane of chemically enhanced water that removes contaminants. dna is then sampled by pcr probing methodology developed by cepheid, inc. the system is fully automated with run completion in min. if a biological agent is detected, a system alert is generated that that shuts down operations. bds was developed initially for anthrax, but it is being expanded to include other pathogens and will be adapted for toxins, as well. the science applications international corporation is developing a biosensor that combines advanced genomic and signal processing techniques to identify all known, newly emergent, and bioengineered pathogens (including all viruses, bacteria, fungi and protozoa). known as tiger (triangulation identification for genetic evaluation of risks), the biosensor uses mass spectrometry to determine the mass of core genetic material selectively extracted from a pathogen. tiger uses specialized algorithms to read a pathogen's genetic signature. the sensor then checks the pathogen's mass against the masses of known pathogens in its database. this system differs from most antibody-based biosensors that cannot detect unknown or bioengineered pathogens. an antibody-based microarray, which can be fabricated with a wide range of pathogen or toxin-specific antibodies, is a rapidly emerging approach that hold great promise for disease detection proteomics [ ] . similarly, mass spectrometry-based proteomics is emerging as an important tool, which can be used to study protein-protein interactions on a small and proteome-wide scale and generate quantitative protein profiles from diverse species [ , ] . the ability of mass spectrometry to identify accurately thousands of proteins from complex samples will continue to improve and impact biology and medicine [ ] . direct nucleic acid detection methods may not be sufficiently sensitive to detect pathogens that are either present at low levels in body fluids or tissues. a promising approach to assess host-pathogen interactions at a very early stage of infection is to develop a signature for the host's immunological response. both pcr and antigen capture assays require the presence of the causative agent. a number of recent studies suggest that there is a pathogen-specific difference in the innate host immune response and that these differences are detectible by transcriptional profiling with dna microarrays [ ] . in this approach, a molecular immunological signature or bar code-like response would be generated that corresponds to a given pathogen. in conclusion, rapid advances in diagnostic technology have facilitated realtime multiplex detection of a wide range of human pathogens. a range of platforms are now available in clinical and public health laboratories with the most advanced chip-based detection systems largely confined to academia. finally, as miniaturization of technology is a rising trend, it is likely that many of the diagnostic platforms will also emerge in deployment of handheld point-of-care devices suitable for rapid detection of 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rapid detection of point mutations by fluorescence resonance energy transfer and probe melting curves in candida species detection of bacillus anthracis dna by lightcycler pcr utility of random amplified polymorphic dna pcr and taqman automated detection in molecular identification of aspergillus fumigatus molecular beacons: probes that fluoresce upon hybridization homogeneous scoring of single-nucleotide polymorphisms: comparison of the -nuclease taqman assay and molecular beacon probes masstag polymerase chain reaction for differential diagnosis of viral hemorrhagic fever rapid diagnosis of bacteremia by universal amplification of s ribosomal dna followed by hybridization to an oligonucleotide array defining the unknown: molecular methods for finding new microbes pcr primers and probes for the s rrna gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid a method for simultaneous rna and dna isolation from dried blood and semen stains automated screening of blood donations for hepatitis c virus rna using the qiagen biorobot and the roche cobas hcv amplicor assay automated extraction of genomic dna from medically important yeast species and filamentous fungi by using the magna pure lc system recovery efficiences on nucleic acid extraction kits as measured by quantitative lightcycler pcr comparative genomics tools applied to bioterrorism defence biowatch program aims for nationwide detection of airborne pathogens air sensor detection -usa (texas) unveils bioterror sensor network high-throughput proteomics using antibody microarrays: an update mass spectrometry-based proteomics analysis of protein interaction and function with a -dimensional maldi-ms protein array patterns of host genome-wide gene transcript abundance in the peripheral blood of patients with acute dengue hemorrhagic fever key: cord- -x js vqe authors: callan, robert j; garry, franklyn b title: biosecurity and bovine respiratory disease date: - - journal: vet clin north am food anim pract doi: . /s - ( ) -x sha: doc_id: cord_uid: x js vqe although biosecurity practices play a role in minimizing respiratory disease in cattle, they must be used in combination with other management strategies that address the many other risk factors. because the pathogens involved in bovine respiratory disease are enzootic in the general cattle population, biosecurity practices aimed at the complete elimination of exposure are currently impractical. several animal husbandry and production management practices can be used to minimize pathogen shedding, exposure, and transmission within a given population, however. various combinations of these control measures can be applied to individual farms to help decrease the morbidity and mortality attributed to respiratory disease. and treatment, it seems that respiratory disease remains one of the foremost cattle health concerns. the challenge that the authors were presented with in writing this article was to consider the role that biosecurity could play in reducing the occurrence or effect of respiratory disease. it seems that little research has specifically evaluated the effects of biosecurity management practices on the occurrence of the problem in livestock operations. indeed, recognizing the multifactorial etiology of infectious respiratory disease and the ubiquitous presence of the pathogens involved leads to the conclusion that attempts to decrease disease prevalence must incorporate multiple management steps, of which biosecurity practices are only a single component. although biosecurity practices have equal potential to decrease respiratory disease losses in all food animal species, the authors focus this article primarily on bovine respiratory disease complex. this article addresses major areas of respiratory pathogen control and provides some suggestions for practical intervention. bovine respiratory disease is not a single entity, nor is it attributable to a single cause [ ] . one useful scheme for characterizing respiratory tract diseases in a practical manner distinguishes three different categories of problems [ ] . these include the bovine respiratory disease complex (brdc), epitomized by shipping fever pneumonia and enzootic calf pneumonia; acute interstitial pneumonias; and metastatic pneumonia. this scheme excludes many problems that involve only the upper respiratory tract, although these problems may predispose to lower tract infections. the interstitial pneumonias are most commonly attributed to toxicoses, and metastatic pneumonias are secondary complications of disease in other organ systems that spread hematogenously to the lung. although these disease problems are frequently fatal for affected cattle, they occur sporadically and are generally not considered to be contagious. the authors focus their attention for this discussion on brdc. this problem has an infectious origin, and it is by far the most frequently occurring form of cattle respiratory disease. cattle of all ages and in a variety of circumstances can be affected by brdc, but the disease most commonly manifests in young dairy calves (enzootic calf pneumonia) and in beef calves recently arrived at feedlots (shipping fever pneumonia). research over the past several decades has provided an increasingly clear picture of how brdc occurs and why it is so common. unfortunately, this knowledge has not led to a commensurate decrease in the morbidity and mortality associated with this problem, primarily because animals are commonly managed in ways that predispose to disease development. bovine respiratory disease complex refers to bacterial bronchopneumonia that may or may not be complicated by previous or concurrent viral or mycoplasma infection [ ] . numerous bacterial species can be isolated from the lungs of affected animals. in feedlot cattle and adult cattle, mannheimia (pasteurella) haemolytica is considered the most important pathogen, with lesser roles attributed to pasteurella multocida and hemophilus somnus. in younger calves, these same pathogens play a role, but mycoplasma spp. are also considered to be important. arcanobacterium pyogenes, fusobacterium spp., and bacteroides spp. are frequently isolated from animals with chronic, abscessing lung lesions but do not play a major role in acute bronchopneumonia. less common bacterial isolates, including streptococcus spp., staphylococcus spp., pseudomonas aeruginosa, and chlamydia spp., are also occasionally identified in young calves. all of the bacterial pathogens considered important in brdc can be isolated from the upper respiratory tract of healthy cattle and calves. these pathogens are considered ubiquitous in cattle populations, not because they can be found in each animal, but because they are readily identified in the nasopharynx of some animals in most populations. in the absence of other predisposing causes of disease, it seems that the simple presence of these bacterial agents is not of major significance. the disease complex is best characterized as being multifactorial, only occurring when a combination of factors involving the animal, environment, and infectious agents are present. viral pathogens are implicated in the development of brdc, although the final pulmonary pathology is primarily caused by bacterial pathogens [ ] . the principal viruses involved in brdc include bovine herpesvirus (infectious bovine rhinotracheitis), bovine parainfluenza virus type , bovine respiratory syncytial virus, and bovine viral diarrhea virus. lesser roles are attributed to bovine coronavirus, adenovirus, rhinovirus, reovirus, and enterovirus. these viral pathogens primarily infect the upper respiratory tract, resulting in rhinitis, tracheitis, and bronchitis. their ability to cause direct pulmonary disease is generally limited except for bovine respiratory syncytial virus, which can also cause severe lung damage as the primary agent. all of these viral pathogens predispose the lung to bacterial infection and bronchopneumonia. the primary role of these agents in brdc is to promote bacterial challenge to the lungs by compromising respiratory tract defense mechanisms. the predisposing causes of brdc act synergistically and are most commonly identified in combination rather than as single causative problems. the list of predisposing animal factors is long and includes animal age, decreased immune responsiveness due to animal stress, lack of previous viral exposure or vaccination, inadequate passive immunoglobulin transfer in young calves, nutritional deficiencies, and dehydration. environmental risk factors include high air humidity or dust content, rapidly changing environmental temperatures, extreme heat or cold, and high concentrations of noxious gases such as ammonia. several risk factors may increase pathogen density or pathogen exposure, although these risk factors probably act by other means as well. for example, commingling cattle from multiple sources may increase exposure to antigenically heterogeneous viral pathogens, while also increasing animal stress. poor ventilation and high humidity can increase pathogen density and survival time but also can increase noxious gas concentrations and adversely affect pulmonary function. animal crowding increases airborne pathogen exposure but also induces animal stress and reduces immune responsiveness. when evaluating the rate of brdc occurrence in cattle populations, it is clear that efforts to prevent this disease have not been effective on an industry-wide basis, although some individual producers have successfully used prevention strategies. the two biggest areas of brdc effect are in the form of enzootic calf pneumonia of dairy calves and shipping fever pneumonia of feedlot cattle. given our current understanding of this disease problem, it is clear that the animal management systems employed for these groups of animals (i.e., dairy calf-rearing systems and feedlot cattle-receiving systems) have failed to rigorously apply knowledge of disease pathogenesis and prevention into their processes. this situation may be changing currently, as the beef production industry increasingly uses quality-assurance principles in production systems and develops marketing procedures and animal-purchasing practices that reward improvements in animal health [ , , ] . similarly, the dairy industry has begun to recognize the economic benefit of improved calf health and increasingly uses specialized calf-rearing systems [ , ] . because the purpose of this article is to examine the role of biosecurity management in respiratory disease prevention, the authors do not attempt to provide a complete review of brdc preventive practices. many of the important means of preventing brdc do not employ biosecurity but are targeted toward enhancing animal immune preparedness and enhancing animal response to infectious challenge. effective respiratory disease preventive practices are those targeted at reducing identified risk factors for disease development [ , , , ] . these practices include management to improve animal nutrition with special emphasis on micronutrient nutrition, practices that reduce animal stress, reduced commingling of animals, improved animal transportation and feedlot receiving practices, improved preconditioning and vaccination programs that emphasize vaccination before shipment and during times of low calf stress, and improved ventilation with reduced crowding. it is important to consider the factors that drive the development and implementation of disease prevention and biosecurity programs. the most apparent of these factors is the effect on animal production and growth; however, all interventions have their cost, and these costs must always be considered relative to the potential economic returns. unfortunately, information regarding the financial impact of herd biosecurity programs is limited, and estimates based on clinical experience must often be applied. other issues, including herd pathogen status and its effect on livestock marketing, food product quality assurance, drug residues, injection site lesions, antimicrobial resistance, and animal welfare also contribute to the forces that drive the development of biosecurity programs. ultimately, a biosecurity program must be integrated into the overall herd management. it must be developed using a team approach that addresses the concerns of the producer, the economic effect on the production unit, the influence on product quality, and public health concerns. the veterinarian is best suited to effectively develop and implement such programs. the multifactorial nature of brdc and the ubiquitous presence of respiratory pathogens are important concepts when considering the role that biosecurity can play in decreasing the prevalence of disease. for infectious diseases in which point source pathogen exposure, high susceptibility, and high virulence are prominent features of disease transmission (e.g., anthrax, footand-mouth disease, rabies, and so forth), limiting animal contact with the pathogen is a key feature of disease prevention and may even provide the means of disease eradication. alternatively, when the causative pathogens are endemic in a population and individual susceptibility is dependent on numerous interrelated factors, the management of animal resistance and risk factors may be proportionally more important for disease prevention than biosecurity practices. it appears that brdc prevention requires a combination of management to enhance animal resistance plus management to reduce exposure to the pathogens. the important point is not to de-emphasize the value of reducing pathogen introduction, exposure, and transmission (i.e., biosecurity) but to also stress the importance of other management features that promote animal resistance. it is particularly important that preventive management practices be coordinated and used in combination, because no single management procedure will be successful without the complement of other practices. it is likely that our inability to reduce the prevalence of respiratory disease in cattle is, in part, attributable to our failure to integrate multiple aspects of respiratory disease prevention practices, including biosecurity. the fundamental concept of biosecurity is to decrease pathogen transmission between animals. transmission of respiratory pathogens occurs by close nose-to-nose contact, environmental or fomite exposure, and airborne exposure. increased contact between shedding and susceptible individuals increases pathogen spread. environmental exposure through common areas and equipment that involve oral or nasal contact such as feed bunks, water troughs, and salt blocks may be an even greater risk, however. total environmental pathogen load is extremely important in considering respiratory pathogen transmission. environmental contamination from animals in contact is the primary source of most respiratory pathogens. individual animal shedding is quite variable and depends on the etiologic agent, the time course of the disease, the clinical severity, and the immune response of the host. in general, clinically ill animals shed greater numbers of pathogens than normal or asymptomatic animals; however, it must be recognized that individuals periodically shed both viral and bacterial respiratory pathogens without evidence of disease. well-vaccinated animals may also periodically shed pathogens and should not necessarily be considered completely safe from disease transmission. the persistence of the pathogen in the environment also contributes to pathogen exposure. environmental pathogen survival times depend on many factors, including organic material, moisture, direct sunlight, and exposure to disinfectants. environmental survival times for most viral respiratory pathogens are probably on the order of minutes to several hours [ , ] . survival times for bacterial pathogens may be longer depending on the environmental conditions and the organism. airborne transmission is dependent on numerous factors, including ambient temperature, relative humidity, airborne particle (dust) density, ventilation, prevailing wind, and structural or geographic obstructions [ ] . airborne transmission of typical viral respiratory pathogens can occur over distances as far as meters and possibly further [ , ] . airborne transmission of other viruses such as footand-mouth disease virus or pseudorabies virus has been shown to occur over many miles, however [ , , , , ] . adding to the complexity of pathogen transmission, it seems that the efficiency of transmission is different between different strains of a given pathogen [ ] . understanding how management practices can reduce either pathogen shedding or exposure is the key to creating effective biosecurity programs. the term biosecurity is used for those management and hygiene practices that reduce introduction, exposure, and transmission of infectious agents. although biosecurity may not provide the single most important component of respiratory disease prevention, reducing pathogen exposure is a valuable part of any infectious disease management system. little information is available to specifically evaluate the effect of individual biosecurity practices in prevention of brdc, but there are some important respiratory disease prevention practices that limit pathogen exposure and good reason to more closely evaluate the role that biosecurity could play in the future. the authors emphasize five areas of biosecurity management that should be more rigorously applied for the reduction of respiratory disease prevalence in cattle, including ( ) strategic vaccination, ( ) calf biosecurity, ( ) housing ventilation, ( ) commingling and animal contact, and ( ) bovine viral diarrhea virus control. many improvements in vaccine technology have occurred over the past few decades, and practitioners have an array of improved bovine respiratory pathogen vaccines at their disposal [ ] . unfortunately, the current respiratory pathogen vaccines have not all been scrutinized for efficacy to the most desirable degree, and many do not protect against respiratory disease nearly as effectively as some veterinarians and producers would like to believe. although vaccines directed at specific conserved proteins, such as toxoid vaccines, may completely prevent a particular disease, vaccines against complex disease agents that have multiple antigenic strains are unlikely to be capable of such levels of protection. respiratory vaccines are better viewed as disease modifiers than absolute preventive agents. vaccines are usually used as a means to decrease the likelihood or severity of disease occurrence in the individual animal receiving the vaccination. indeed, vaccine efficacy may be evaluated in many ways, but the more rigorous evaluations involve the ability of a vaccinated animal to withstand a challenge of disease or pathogen exposure [ ] . practitioners tend to view vaccination as one of the management factors that enhance animal resistance to infection and thus augment the value of biosecurity management by working to reduce susceptibility to infectious disease rather than decrease exposure and transmission. for respiratory disease prevention, however, effective vaccination can also serve as part of a biosecurity management system. in addition to preventing disease, a vaccine's efficacy might also be considered for its ability to limit pathogen shedding when infection does occur. vaccineinduced immunity often results in decreased magnitude and duration of pathogen shedding [ , , ] . because exposure is directly related to pathogen concentration in the environment, it follows that vaccine-induced reductions in shedding should decrease transmission within a susceptible population. proper vaccine use and a well-managed vaccination program can be viewed as part of a complete biosecurity program. at a minimum, a good vaccination program should include the following: • proper storage and administration of the vaccine as indicated by the manufacturer's labeled recommendations. • vaccination of all susceptible animals, including both resident and incoming animals. • application of the vaccine to systemically healthy, well-nourished, minimally stressed, and immunocompetent cattle. • strategic timing of vaccination so that it precedes contact with new animals long enough to allow an appropriate immune response. • revaccination as recommended for the particular vaccine product. biosecurity management of calves is extremely important for development of healthy animals. many of the biosecurity recommendations for newborn calves focus on decreasing the transmission of enteric pathogens; however, these same principles can be important for minimizing respiratory disease problems. several details of calf biosecurity management deserve emphasis. environmental and housing factors significantly affect calf health and viability. differences in calf management for cow-calf herds versus dairies are related to the relative risk of respiratory disease between these two production groups. beef calves are generally raised in open-range situations that effectively dilute the exposure to respiratory pathogens. although beef calves are continually exposed to pathogens shed from adult cattle and other calves, the magnitude of pathogen exposure before weaning is generally low, resulting in relatively little respiratory disease. in contrast to many enteric pathogens, the environmental survival time of the respiratory pathogens is limited [ ] , and accumulation of pathogens in the environment is not considered a primary concern. dairy calf housing has a significant effect on the incidence of respiratory disease in neonatal calves. although the common viral respiratory pathogens can be transmitted over distances up to meters [ , ] , properly spaced calf hutches seem to effectively limit aerosol transmission of respiratory pathogens. the short survival of these pathogens in the environment limits the transmission between successive occupants of an individual hutch. disinfection procedures that are used for enteric diseases should be more than sufficient to decrease respiratory pathogen transmission (see article by barrington et al. in this issue). in contrast, there is a high risk of respiratory disease transmission in group-raised neonatal calves. factors including the number of animals, relative animal density, housing facilities, and ventilation conditions significantly contribute to transmission in grouped calves and are discussed in subsequent sections of this article. numerous management practices can decrease exposure and transmission of respiratory pathogens to calves in dairy operations. feeding pasteurized milk or milk replacer is a useful biosecurity practice for minimizing the spread of enteric agents such as salmonella spp. or mycobacterium avium subsp. paratuberculosis. these practices are also effective at limiting ingestion of potential respiratory pathogens. mycoplasma spp. bacteria are commonly implicated in newborn calf disease, including enzootic calf pneumonia [ , , , ] . although mycoplasma spp. may spread by the airborne route, it is also a common mastitis pathogen and can be shed from clinically or subclinically infected cows [ , , ] . nasopharyngeal colonization occurs after oral ingestion of contaminated milk, potentially resulting in clinical respiratory disease in calves [ ] . mycoplasma spp. and other pathogens can also spread hematogenously after ingestion by a susceptible calf [ , ] . similarly, other potential respiratory pathogens such as streptococcus spp., staphylococcus spp., salmonella spp., and escherichia coli can be recovered from milk and spread hematogenously to the lungs after oral ingestion. bovine viral diarrhea virus is shed in the milk of persistently infected cattle. ingestion of bovine viral diarrhea virus-contaminated milk can result in respiratory and systemic infections, possible immune suppression, and respiratory disease. proper cleaning and disinfection of calf feeding equipment, including nursing bottles, buckets, and mixing utensils, should be performed. equipment should be cleaned with a detergent and disinfected between uses. a common and economical disinfectant is standard household bleach used at a : dilution. bottles and equipment that are potentially shared between multiple animals should be soaked for to minutes in this solution. although bleach will not completely kill all potential pathogens, it is effective at significantly decreasing viable numbers and thus contributing to decreased exposure and transmission between feedings. prompt removal of dairy calves from the maternity pen environment, where they are exposed to numerous adult cow pathogens, can also decrease transmission of potential respiratory pathogens. newborn calves should not have direct contact with older calves and adults. calf hutch spacing should be evaluated, with a minimum of feet of separation between calves. worker hygiene can minimize contamination of calf feed and the calf environment. appropriate vaccination of dams before colostral production can increase passive transfer of effective antibodies, reducing the risk of exposure and potential shedding after infection. it has been demonstrated that good colostral transfer to beef calves was associated with decreased occurrence of disease episodes and improved calf performance all the way through the growing and finishing period in feedlot animals [ ] . it is unlikely that the passive transfer of immunoglobulins per se is specifically responsible for beneficial effects on the long-term health of animals, but profound effects may result from management that improves newborn health and disease resistance. this in turn provides for improved nutrition, growth, physiologic well-being, and decreased total pathogen load. numerous calfhood husbandry procedures should be considered as standard biosecurity protocols for all infectious diseases, including respiratory disease. sick animals should be identified and separated from healthy animals. a specific calf-isolation area should be established, with consideration to animal comfort and ease of cleaning and disinfection. where practical, individual equipment should be used for each separate calf. specific care and treatment personnel should be identified, and animals with suspected infectious diseases should be treated after handling healthy animals. additional personnel hygiene protocols include dedicated coveralls to be used in the sick pens, the use of rubber overboots, and disinfectant footbaths. personnel should be encouraged to wash their hands before and after entering the sick pens and between caring for animals with dissimilar disease conditions. in many cases, equipment and facilities need to be made available to help establish such procedures. similar biosecurity management practices can be used in cow-calf herds. although feeding pasteurized milk or milk replacer is obviously not a practical management practice, milk-borne exposure to pathogens can be minimized by proper attention to the adult cows. adult cattle must be appropriately vaccinated to provide optimal colostral immunity to the calves and to decrease adult cow infections and shedding. adult cow nutrition should be optimized to improve colostrum quality. adult cow nutrition can also have a dramatic effect on calving ease and decrease the incidence of dystocia. special attention should be placed on high-risk calves, including calves delivered with manual assistance, cesarean section, born in inclement weather, weak or premature calves, and multiple births. such calves often do not nurse colostrum in a timely fashion or have impaired absorption of immunoglobulin. cows should be evaluated for evidence of clinical mastitis and treated or culled as appropriate. decreased morbidity can be observed by minimizing the time that beef cow-calf pairs spend in a designated calving area, where pathogen loads tend to increase throughout the calving season. bovine viral diarrhea virus surveillance and eradication in cows and calves should also be used (see discussion in a following section). as can be seen from the preceding discussion, many of the management practices that contribute to biosecurity of respiratory disease are standard quality-assurance practices that are recommended for basic calf health. good ventilation is a critical aspect of animal management and can profoundly affect respiratory health. several discussions of ventilation and its effect on animal health are present in the literature [ ] [ ] [ ] [ ] , , , , , , , , , , ] . proper ventilation serves eight primary functions: . it decreases the airborne pathogen concentration . it eliminates noxious gases (ammonia, hydrogen sulfide, carbon dioxide, carbon monoxide, and methane) . it decreases airborne dust contamination . it decreases airborne endotoxin levels . it maintains optimum ambient temperature . it maintains optimum environmental humidity levels . it eliminates drafts . it eliminates areas of stagnant air with respect to biosecurity, one of the most important aspects of proper ventilation is the reduction in the concentration of airborne pathogens. all of the important viral and bacterial respiratory pathogens can spread aerogenously and can attain high concentrations in poorly ventilated housing areas. airborne pathogen concentration is a function of many factors, including animal type, housing system, stocking rate, bedding, humidity, dust particle density and size, and finally, elimination through ventilation. improved ventilation is one important means whereby airborne pathogen concentration can be readily decreased within the given constraints of an operation; however, pathogen removal is not a linear function, and practical and theoretical limits are often observed [ ] . studies of building ventilation for humans demonstrate potential reductions in airborne exposure of pathogens and disease incidence, although improved ventilation beyond that which provides comfort may not be practical or provide significant additional benefit [ ] . as the airborne pathogen load rises, ventilation provides progressively less protection against respiratory infections. it is important to realize that stocking rate has a more dramatic effect on airborne pathogen density than ventilation [ , ] . for example, a two-fold increase in stocking rate requires nearly a -fold increase in ventilation to maintain the same airborne pathogen density [ ] . ventilation cannot overcome grossly inadequate housing, management, or hygiene within a production unit. along with stocking density, there are other practical concerns that contribute to airborne pathogen density and transmission. one of these is related to animal handling and excitement. it is extremely important to handle grouped animals in a calm environment with minimal animal activity and stress. increased animal activity not only increases dust exposure (which contains airborne pathogens) but also increases ventilatory rate, ventilatory effort, and tidal volume, which in turn increases the amount of aerosolized pathogen shed by infected animals and the amount of pathogen inhaled by susceptible animals. the increased dust exposure will also adversely affect mucociliary clearance and respiratory defense mechanisms. part of the effect of ventilation is to minimize airborne contaminants that can impair respiratory function and defense mechanisms [ , , , ] . significant airborne contaminants include ammonia, hydrogen sulfide, carbon dioxide, carbon monoxide, methane, dust particles, and endotoxin. ammonia and hydrogen sulfide are toxic gases and can contribute to respiratory damage, decreased mucociliary clearance, decreased alveolar macrophage activity, and overall compromise to respiratory defense mechanisms. carbon dioxide, carbon monoxide, and methane contribute primarily as asphyxiative gases and generally do not contribute to significant impairment of the respiratory tract. dust particles also contribute to the impairment of respiratory defense mechanisms. dust particles can arise from both organic and inorganic sources. in general, particles greater than lm are filtered out by the nasal passages; most particles from to lm are removed by the mucociliary clearance of the trachea and bronchi, and particles less than lm can penetrate to the alveolar spaces [ , ] . organic and inorganic dust particles can impair mucociliary clearance and overload alveolar macrophage phagocytic clearance [ ] . organic dust particles are generally of more concern in confinement and intensive housing situations. in animal housing environments, most of the organic dust arises from fecal material, skin, and hair. organic dust is significant in that it often contains high endotoxin and pathogen levels [ , ] . inhaled endotoxin can contribute to pulmonary compromise by initiating inflammatory reactions within the alveoli and alveolar vascular endothelium. appropriate ventilation is also important in maintaining acceptable humidity and ambient temperature levels within confinement or semi-open housing. observed thermoneutral ranges (the range of air temperature that sustains optimal performance) for a variety of domestic livestock are available (table ) [ ] . in general, livestock can perform adequately within a fairly wide thermoneutral range. higher temperatures, especially when combined with high humidity, tend to be more problematic than low temperatures [ ] . depending on the given climate and temperature ranges of a geographic region, housing ventilation will need to be designed to provide either heating or cooling or both. cold temperatures and perhaps temperature fluctuations can decrease mucociliary clearance and predispose animals to respiratory disease [ ] . often, wide temperature fluctuations are more detrimental to animal health because they do not allow suitable adaptation over time. there is minimal information on how ambient temperature directly relates to airborne pathogen biosecurity. increased ambient temperature results in increased respiration and may increase pathogen shedding from infected animals. the direct effects of ambient temperature on pathogen survival are relatively unknown. some studies suggest that the concentration of airborne particles is increased at low temperatures, and airborne bacterial concentrations were higher in winter than in summer [ ] . there is slightly more information concerning the effects of relative humidity on pathogen survival and thus, airborne biosecurity [ , ] . in general, viruses with a hydrophobic lipid outer shell (i.e., enveloped viruses) survive better in lower humidity, and lipid-free viruses (i.e., foot-and-mouth disease virus) are more stable in moist air [ , ] . the four primary viral respiratory pathogens in cattle (bovine herpsevirus , bovine parainfluenza virus type , bovine respiratory syncytial virus, and bovine viral diarrhea virus) are all enveloped viruses and would be considered more stable in dry air, although the authors are unaware of specific studies documenting this conclusion. gram-negative bacteria have outer phospholipid membranes and are also expected to be more stable in dry air [ ] . mycoplasma are reported to be sensitive to relative humidity between % and % [ ] . extrapolation of these limited data suggests that typical airborne pathogens associated with respiratory disease in domestic animals survive better in cool, dry air such as is observed in the late fall, winter, and early spring months. although this correlates with clinical observations concerning the relative seasonal incidence of respiratory disease, a direct association has not been established. in beef cattle, seasonal increases in respiratory disease also correlate with seasonal management practices associated with movement of cattle to feedlots and increased animal density. it is likely that climate and management factors act together to dramatically increase pathogen exposure and transmission in feedlots. alternatively, the high humidity that can be observed with dairy confinement housing in cold weather probably contributes to increased respiratory disease because of the higher pathogen density associated with increased aerosolized particle concentrations. ventilation systems should be constructed to provide even airflow throughout the structure without areas of air stagnation or drafts. pockets of air stagnation have higher levels of airborne contaminants and contribute to the exposure and transmission of respiratory pathogens. air stagnation can often be remedied by appropriate use of inexpensive fans. correcting draft conditions can be more problematic and often requires complete evaluation of the housing structure for air leaks and evaluation of the ventilation system, especially air intake vents. guidelines for housing of livestock have been reported, including recommendations for ventilation (table ) [ , , , , , , ] . appropriate ventilation should flow from younger to older animals to minimize spread of pathogens to the more susceptible animals. the total air volume should be completely changed times per hour in winter, and it should be changed up to times per hour in summer [ , ]. the ventilation system should confinement housing • minimum of four air changes per hour (winter) • total exhaust capacity for up to air changes per hour (summer) • continuous (not intermittent) ventilation • single-speed fans, not variable-speed fans, should be used • fans must be able to sustain / -inch static pressure • one must allow for two to four different ventilation rates using multiple fans • enough inlet slot area should be provided to allow minimal inlet velocity of fpm (winter) and fpm (summer) • thermostats should be used to control ventilation fans • thermostats should be located at eye level near the center of the barn • the ventilation rate should be altered by stepping up the number of fans used for each level • wall fans should be mounted near the ceiling but collect air using ducts from within cm ( in) of the floor • the fresh air intake should be located near the ceiling but at least feet from any exhaust fan • adjustable eave slot inlets should be used to distribute incoming air uniformly provide constant rather than intermittent airflow. in the winter, the goal of ventilation is to minimize airborne pathogen density, remove excess moisture from animal respiration, and maintain adequate ambient temperature ( - °c, - °f). although higher ventilation rates improve air quality, they are inefficient because they require excessive heating costs. supplemental heating may be necessary as the outside temperature falls or stocking density decreases. at optimal stocking densities, livestock generally produce enough animal heat to maintain adequate ambient temperature in confined housing when outside temperatures remain above ÿ °c [ ] . winter ventilation is a compromise between the removal of airborne contaminants and the maintenance of ambient temperature. the primary goal of summer ventilation is to minimize ambient temperature and relative humidity. this requires high ventilation flow rates, which also enhance air quality. the goal is to maintain an ambient housing temperature to no more than °c above the outside temperature [ , ] . relative humidity levels should be maintained between % and %, and ammonia levels should not exceed ppm [ , , , , ] . maximum recommended stocking densities should not be exceeded (see table ) [ ] . separate age groups of cattle should be maintained in separate barns or be separated by barrier walls. calf hutches for individual dairy calves provide an ideal means of managing relative calf isolation and limiting airborne transmission if they are properly positioned and spaced. recommendations for calf hutches include one calf per hutch with a minimum separation of feet between hutches. hutches should be placed at least feet from oldercattle enclosures and feet from livestock building exhaust fans. many cattle management systems provide numerous opportunities for exchange of respiratory pathogens from animal to animal. assembling groups of beef calves for a feedlot often involves mixing calves from different origins, congregation of animals at sale barns or other holding pens, and movement in congested cattle transports. these activities are well known to increase the rate of respiratory disease occurrence by stressing the animals and providing circumstances that decrease disease resistance. these same animal contact and crowding circumstances can dramatically increase exposure to pathogens, often including pathogens to which the animal has not developed prior immunity. in a recent national survey, more than % of dairy producers housed sick animals in a manner that allowed direct nose-to-nose contact with healthy herdmates [ ] . many dairy producers expand their herds by purchasing animals from other sources, but less than % of them provide any quarantine time for the incoming animals. for producers who introduced % or more of their total animal inventory during an expansion, . % reported an increase in occurrence of respiratory disease during the year [ ] . during the early phases of respiratory disease, the shedding rates of pathogens via respiratory secretions increases dramatically. commingling, crowding, and the animal stresses that are involved in animal movement can precipitate respiratory problems. these same factors can increase spread of pathogens to other animals with close contact. although quarantine may not be effective against diseases with chronic carrier states such as johne's disease, it can substantially decrease the risk of spreading respiratory pathogens. furthermore, the duration of respiratory pathogen shedding has also been well characterized. in general, nasal shedding of viral respiratory pathogens is significantly reduced by days after infection but may persist longer in individual animals, which suggests that quarantine for approximately to days should significantly reduce the exposure and transmission of these pathogens within an operation. practical suggestions for limiting pathogen spread by contact include quarantine of incoming livestock, maintenance of hospital areas that do not allow contact with healthy animals, prevention of animal contact between different age groups of cattle, minimizing the time animals spend in market channels, and limiting the introduction of new animals to assembled herds or pens of cattle. the concepts of pathogen transmission within grouped housing can be effectively applied to weaned dairy calves. calves receive relatively low pathogen exposure while in calf hutches. on weaning and grouping in calf pens, the risk of exposure increases dramatically. it is important to appreciate that the risk of exposure rises with the number of calves housed together. for example, if one estimates that % of calves born in a herd with bovine viral diarrhea virus are persistently infected, then the probability of bovine viral diarrhea virus exposure in a group of calves is approximately . ( - . ). if the stocking rate increases to calves, the probability nearly doubles to . ( - . ). limiting the number of calves per pen to less than seven is associated with decreased respiratory disease mortality [ ] (also see article by smith in this issue). it must be emphasized that one animal can expose an entire pen of animals by simple close contact, airborne transmission, or environmental transmission at common housing areas such as feed bunks and water troughs. by dividing animals into smaller groups, the number of animals exposed is lowered significantly. using the same example of bovine viral diarrhea virus exposure, if one splits the calves into three separate pens, the probability of having all calves exposed to bovine viral diarrhea virus falls from . to the comparatively negligible level of . [( - . ) ]. simple segregation of animals is not sufficient unless physical barriers for fence line contact, separation of food and water troughs, segregation of likely fomites, and blocking airborne spread are used. these same principles can be applied to any group-housing situation. such management and housing decisions must be made based on a balance between the risk and cost of disease versus the availability and cost of facilities and labor. it was noted previously that the common bovine respiratory pathogens are considered to be ubiquitous in cattle populations in the united states and most other countries. although this does not suggest that every animal harbors each pathogen, these agents can be found routinely in the nasopharynx of healthy and diseased animals within most herds. in contrast, some european countries have successfully eradicated some viral respiratory pathogens such as bovine herpesvirus and bovine viral diarrhea virus (bvdv) from cattle populations. under the currently prevailing practices within the united states and many other countries, the authors do not suggest testing or identification of most respiratory pathogens as a viable means to identify carriers or to exclude the animals from introduction into a herd. the exception to this is bvdv. although bvdv is not considered a primary pneumopathogen, it is considered to have an important role in respiratory disease of cattle [ , ] . the immunosuppressive effects of the virus and the close association of bvdv infection and respiratory disease occurrence in some epidemiologic studies suggest that the virus plays a role by promoting secondary bacterial lung infection. although bvdv vaccines have been improved over the past several years, vaccination alone rarely eliminates bvdv from an infected herd. an effective bvdv biosecurity program must include the identification and removal of persistently infected animals, bvdv screening of incoming animals and their calves, and a comprehensive vaccination program [ , , , ] . persistently infected cattle do not mount an effective immune response against the virus and are capable of shedding large amounts of the virus into the environment through multiple routes. persistently infected animals have been implicated as the primary means by which bvdv infection is maintained in assembled dairy herds, and they are also considered a significant threat for transmission in cow-calf and feedlot operations [ ] [ ] [ ] [ ] , , ] . with the development of new tests over the past several years, our ability to accurately and expediently identify persistently infected animals has dramatically improved [ , ] . the serum immunoperoxidase monolayer assay (ipma) and antigen capture elisa tests and the immunohistochemistry test of skin biopsy material have appropriate sensitivity and specificity for detecting persistently infected cattle. the authors do not know of significant published research that evaluates the effect of test-and-cull strategies for bvdv on the occurrence of brdc; however, elimination of persistently infected animals from herds can have significant positive effects in decreasing other bvdv manifestations such as reproductive failure. implementing test-and-cull procedures for persistently infected animals may prove to be a powerful means of decreasing brdc prevalence. in general, all cattle introduced into a herd should be tested for bvdv before purchase or entry. acute bvdv infections of pregnant cattle can result in animals that are bvdv negative at the time of testing while the fetus is persistently infected. it is critical that all calves from newly introduced pregnant animals also be tested immediately after birth. to establish a bvdv-negative herd, it is generally more effective and economical to test calves as they are born rather than screen adult populations. a negative result for a calf indicates that not only the calf but also all of the calf's maternal ancestors are not persistently infected. a single positive test on a calf does not differentiate between acute and persistent infection. a confirmatory test may be performed in weeks, or the animal may be assumed to be persistently infected and euthanized or sold for slaughter. the dams of all persistently infected calves should be traced and tested as well to determine their status. in most cases, these animals will test negative, indicating fetal exposure due to acute infection during gestation. bulls should also be tested because they can contribute to animal exposure within a herd. although biosecurity practices play a role in minimizing respiratory disease in cattle, they must be used in combination with other management strategies that address the many other risk factors. because the pathogens involved in bovine respiratory disease are enzootic in the general cattle population, biosecurity practices aimed at the complete elimination of exposure are currently impractical. several animal husbandry and production 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respiratory syncytial virus vaccines in experimentally infected calves persistent bovine viral diarrhoea virus infection in us beef herds key: cord- - g dc z authors: mcintyre, k. marie; setzkorn, christian; hepworth, philip j.; morand, serge; morse, andrew p.; baylis, matthew title: a quantitative prioritisation of human and domestic animal pathogens in europe date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: g dc z disease or pathogen risk prioritisations aid understanding of infectious agent impact within surveillance or mitigation and biosecurity work, but take significant development. previous work has shown the h-(hirsch-)index as an alternative proxy. we present a weighted risk analysis describing infectious pathogen impact for human health (human pathogens) and well-being (domestic animal pathogens) using an objective, evidence-based, repeatable approach; the h-index. this study established the highest h-index european pathogens. commonalities amongst pathogens not included in previous surveillance or risk analyses were examined. differences between host types (humans/animals/zoonotic) in pathogen h-indices were explored as a one health impact indicator. finally, the acceptability of the h-index proxy for animal pathogen impact was examined by comparison with other measures. pathogens appeared solely in the top highest h-indices ( ) human or ( ) animal pathogens list, and occurred in both. of human pathogens, were zoonotic and were emerging, compared to and for animals. there were statistically significant differences between h-indices for host types (humans, animal, zoonotic), and there was limited evidence that h-indices are a reasonable proxy for animal pathogen impact. this work addresses measures outlined by the european commission to strengthen climate change resilience and biosecurity for infectious diseases. the results include a quantitative evaluation of infectious pathogen impact, and suggest greater impacts of human-only compared to zoonotic pathogens or scientific under-representation of zoonoses. the outputs separate high and low impact pathogens, and should be combined with other risk assessment methods relying on expert opinion or qualitative data for priority setting, or could be used to prioritise diseases for which formal risk assessments are not possible because of data gaps. disease or pathogen risk prioritisation exercises are used by organisations charged with providing surveillance and mitigation measures including disease management and control, and biosecurity measures. qualitative, semi-quantitative or quantitative approaches can be used, but most take significant time to develop, so their use is limited, and when research involving the study of multiple diseases or pathogens is planned, agents are rarely systematically selected. quantitative measures for risk prioritisation include the calculation of epidemiological parameters such as disease incidence, prevalence, mortality and morbidity rates, costs of prevention, treatment or control, and for human disease, years lived with disability (yld) and disability-adjusted-life-year estimates (daly). additional measures for animals include losses to production. for many diseases, robust estimates of these measures do not exist. semi-quantitative and qualitative risk assessments are less demanding of data than quantitative approaches. nevertheless, they require significant time and physical resources (for example, to obtain parameters and effect sizes from the scientific literature), need updating regularly, and they usually require expert-opinion, adding subjectivity [ , , , , ] . the h-index is an alternative approach to disease prioritisation. it objectively and rapidly provides a quantitative proxy of human disease or pathogen impact [ ] , (mcintyre, unpublished) . the hindex captures scientific interest in a disease by deriving a metric from the number of papers published and how many citations each receives. combining scientific impact (citations) with technical productivity (papers published) is useful as, individually, total papers does not account for the quality of publications, while citation count may be influenced by a small number of seminal papers or if a disease becomes 'fashionable' briefly. the h-index method is significantly correlated with more comprehensive measures of human infectious disease impact, including dalys [ ] , and deaths from disease (mcintyre, unpublished data). it can be rapidly obtained at low cost, attained automatically, and repeated regularly to reflect changes in impact, serving as a generic tool to assess the relative bearing of diseases or pathogens, in an easier, timelier manner than traditional risk assessments. while the h-index method undoubtedly has limitations, these tend to be different to those of other approaches; its use could be a step forward in separating high and low priority diseases or pathogens, in combination with other risk assessment methods. the enhanced infectious diseases (eid ) database integrates published data sources on pathogens, their hosts (including vectors) and geographic ranges [ ] . by coupling the h-index method with the eid , the primary aim of this study was to establish priority lists of human and domestic animal pathogens (including zoonoses) present in europe. we then consider reasons for the omission of some pathogens in our lists from those of other disease prioritisations: the global burden of disease (gbd) estimates [ ] , communicable human diseases reportable in the european community [ ] , the oie list of notifiable animal diseases, infections and infestations [ ] , and the eu fp discon-tools project [ ] . the gbd study was a large collaborative five year project which used all relevant published and unpublished evidence to create the strongest evidence-based epidemiological assessment of people's infectious and non-infectious health problems around the world [ ] . the discon-tools project, funded by the european commission over five years, investigated the impacts of domestic animal diseases, to focus and prioritise future research [ ] . as the zoonotic and emerging status of pathogens as well as their taxonomic division could affect the likelihood of their inclusion in surveillance and impact quantification work, these factors were also investigated as reasons for omission from the other disease prioritisations. the h-index can be obtained in the same way for both human and animal pathogens. it therefore has potential as a single metric for prioritising across both host groups. its potential as a quantitative one health indicator (i.e. a single measure applicable to both human and animal diseases) was investigated by comparing scores for human-only, zoonotic, and animal-only pathogen groups, including emerging status as this would likely drive research impact. previous work has shown that the h-index is a proxy for human disease impact [ ] , (mcintyre, unpublished) . we investigated its value as a proxy for animal disease impact by comparing domestic animal pathogen h-indices with other measures of impact including presence on the oie list [ ] , and inclusion in discontools [ ] . the eid database collates data on human and domestic animal pathogens: where, when, and in which hosts there is evidence of their occurrence. the database is built largely using automated procedures to interrogate publicly available databases. an eid background has been described previously [ ] ; here, we used similar criteria to define pathogens, including pathogenic status (frequently pathogenic: a pathogen which frequently causes a clinically pathogenic effect -morbidity or mortality -in humans or domestic animals; non-pathogenic: an organism which causes no clinical signs within any of its hosts; unknown pathogenicity: an organism for which there is insufficient evidence to decide), evidence of pathogens affecting hosts ('host-pathogen interactions': evidence from at least one piece of meta-data uploaded with dna or rna sequence information to [ ] , which describes where, when and from which host the pathogen came, or specific scientific publications [ ]), and evidence of pathogens occurring within countries (evidence from at least one piece of meta-data [ ] , or at least five publications in [ ] where pathogen name and a country mesh-term [ ] co-occurred in the title/abstract). information on host-pathogen interactions was collated when there was evidence of a pathogen occurring in at least one host of interest to the study (including humans and european domestic animals; see table ). further information about each organism, such as their taxonomic division for pathogens (bacteriaincluding rickettsia, fungi -including algal pathogens, helminths -including thorny-headed worms and pentastomids, protozoa, and viruses -including prion agents) or their taxonomic rank (genus, species, etc.) is stored using a series of statements. previously, we examined characteristics of pathogen species [ ] ; here, we include sub-species, to account for important strains e.g. escherichia coli o :h . information on whether pathogens were zoonotic, non-zoonotic, emerging and not emerging was examined based upon previously published information [ , ] . if not included in earlier work or if their status had changed due to more recent scientific evidence, updated pathogen information was based upon the previous definitions. zoonotic pathogens were classified as those naturally transmitted between vertebrate (non-human) animals and humans (as the definitive host), not including species which have recently evolved from animal pathogens but are no longer transmitted between animals and humans [ , ] . emerging pathogens are those that have appeared in a host population for the first time (including newly-evolved strains), or have occurred previously but are increasing in incidence or expanding into areas where they had not previously been reported [ , ] . pathogens needed to have emerged in several geographically distinct areas to be 'emerging'. information sources. h-index searches were undertaken in january using web of science (wos) [ ] . previous work established that results of h-index searches for pathogens undertaken using different bibliographic sources (e.g. wos, scopus, google scholar) are not identical but are highly correlated [ ] . eligibility criteria. searches were restricted to the years to , inclusive. english is used in wos, however searches also include foreign-language publication title translations. all literature in the wos database has been published. searches. searches were undertaken using search phrases specified in quotation marks (''''), the 'topic' search field and with no lemmatization. phrases were compiled including pathogen scientific name, alternative names, synonyms and alternative spellings according to ncbi taxonomy [ ] . h-indices for clinical diseases used clinical terms as well as pathogen phrases for the main pathogens of disease. virus searches also included synonyms and acronyms from the ncbi taxonomy database and international committee on taxonomy of viruses [ , ] , and the term 'virus', and excluded other entities (viral or non-viral) which shared acronyms. the boolean operators 'and', 'or', and 'not' linked multiple search phrases. example of a search phrase. (''mycobacterium tuberculosis'' or ''bacillus tuberculosis'' or ''bacterium tuberculosis'' or ''mycobacterium tuberculosis typus humanus'' or ''mycobacterium tuberculosis var. hominis''). a full list of human and domestic animal pathogens frequently causing pathogenic effects and for which there was evidence of european occurrence was created using eid information [ ] , and defined criteria, see figure . the relative impact of pathogens in this full list was assessed by calculating h-indices using the specified search protocol; high impact pathogens had the highest h-indices. the list was split according to host-pathogen interaction information, into two directories, one including pathogens with evidence of their occurrence in humans, and the second including domestic animal occurrence; zoonotic pathogens appeared in both lists. information was manually obtained on whether these pathogens cause diseases featuring in other prioritisation lists [ , , ] , by examining pathogens listed under each disease's details in the ncbi mesh library [ ] ; specific lists of diseases had been provided in other work [ , ] . these additional pieces of information are included in the results (tables and ). finally, information on the pathogenic status of each pathogen, whether they frequently occurred in the relevant hosts and in europe was verified by the study authors using manual literature searches of the scientific literature, for the pathogens with the highest h-indices. h-indices and previous prioritisations. pearson's chisquared tests with yates' continuity correction and fisher's exact tests (fet) were used to test for differences in counts of pathogens included in previous work [ , , ] , according to outcomes including their taxonomic division, zoonotic and emerging status. where appropriate, odds ratios (or) and % confidence intervals (ci) are presented. h-indices for one health. differences in h-indices for human-only, zoonotic, and animal-only pathogens were examined using a two-way analysis of variance (anova) with log transformation of the response, including emerging status as an explanatory covariate. post-hoc tukey multiple comparisons of treatments were undertaken using the hsd.test [ ] . h-indices for domestic animal pathogens. -oie list. homogeneity of variances of h-indices for animal-only (and not zoonotic) pathogens included or not within the oie list of notifiable animal diseases was examined using the fligner-killeen (median) test. one-way anova thereafter established differences in the (log -transformed) h-indices of pathogens included or not in the oie list. -discontools. h-indices and discon-tools scores were compared using spearman's rank correlations. if more than one pathogen had been included within disease information for the discontools rankings (for campylobacter, leishmaniasis, and salmonellosis), the higher h-index score was used for analyses. table . animal species including humans for which pathogens have been studied, including domestic animals we eat or companion animals we keep as pets, and exotic animals also used as food sources or as pets. all analyses were undertaken using the statistical software package r [ ] , with statistical significance determined by a pvalue of less than . . two lists each including the top human ( table ) and domestic animal pathogens (table ) which cause significant clinical disease and which therefore need consideration from a health and well-being perspective were short-listed using the hindex prioritisation method (for alternative names and synonyms see [ ] ). when combined, ( . %) pathogens appeared solely in the human or animal list, and ( . %) were in both lists. of the top human pathogens, were classed as zoonotic and were emerging, compared to and for domestic animal pathogens, respectively. of the top human pathogens identified, were either included in the gbd [ ] , or are reportable to the ec [ ] , or both. reasons for failure to include pathogens may be that pathogenic agents cause rarely diagnosed disease (e.g. human t-lymphotropic virus , lymphocytic choriomeningitis virus, and moraxella catarrhalis), or because disease agents are diverse, e.g. pneumonia or other lung infections (aspergillus niger, chlamydophila pneumonia, cryptococcus neoformans, klebsiella pneumonia, and mycoplasma pneumoniae) and gastro-intestinal (gi) symptoms or gi-tract infections (aeromonas hydrophila, bacillus cereus, bacteroides fragilis, clostridium species, vibrio parahaemolyticus, and yersinia enterocolitica). the impact of chronic disease or diseases causing low morbidity may be difficult to quantify or seen as less important (bartonella henselae, borrelia burgdorferi, human enterovirus c, human herpesvirus group, human papillomavirus, human parvovirus b , mycobacterium bovis, mycobacterium avium, and mycoplasma genitalium). in addition, some pathogens may generally be commensals or natural biota (aggregatibacter actinomycetemcomitans, candida species, enterobacter, enterococcus, staphylococcus species, candida tropicalis, helicobacter pylori, and porphyromonas gingivalis) or species existing in the environment (acinetobacter baumannii, burkholderia cepacia, candida glabrata, entamoeba histolytica, fusarium oxysporum, gibberella moniliformis, proteus mirabilis, pseudomonas species, rhizopus oryzae, serratia marcescens, staphylococcus aureus, and stenotro-phomonas maltophilia) causing opportunistic infections in immunecompromised individuals (including those young, old or pregnant); their impact upon the general population may not be quantified. of the top domestic animal pathogens described, were either notifiable according to the oie [ ] , or included in discontools [ ] , or both. reasons for failure to include may be similar to for human pathogens (only pathogens not previously mentioned are cited: multiple disease symptoms or lack of diagnosis -ascaris suum, feline immunodeficiency virus, feline leukemia virus, gallid herpesvirus , haemonchus contortus, and yersinia pseudotuberculosis; causes of specific disease being diverse, for respiratory infection -feline calicivirus, and gi symptoms -campylobacter fetus, cryptosporidium parvum, and listeria monocytogenes,; and existing in the environment and opportunistic -pseudomonas aeruginosa). in addition, some omitted pathogens may be production issues with impact difficult to quantify (streptococcus agalactiae causing mastitis in cattle and neospora caninum causing abortion in cattle and dogs) and some may be issues of pets (canine parvovirus, neospora caninum, and parainfluenza virus ). for human pathogens in our list (table ) , fungi and helminths are particularly under-represented in gbd assessments (percentage included in gbd: bacteria, %; fungi, %; helminths, %; protozoa, %; viruses, . %; fet, p = ? ). there was no difference between taxonomic divisions in the percentage reportable to the ec (fet, p = . ). human pathogens classed as emerging (compared to not emerging) were statistically more likely to have gbd estimates (fet, p, . , or = . , ci = . - . ) and be ec reportable (fet, p, . , or = . , ci = . - . ), but not more likely to have gbd estimates or be ec reportable if they were zoonotic compared to nonzoonotic (pearson's x , p = . and fet, p = . , respectively). for domestic animal pathogens in our list (table ) , fungi were particularly under-represented in the oie list (percentage included in oie: bacteria, %; fungi, %; helminths, . %, protozoa, . %, viruses, . %; fet, p = . ). by contrast, viruses are particularly under-represented in discontools (percentage included in discontools: bacteria, . %; fungi, %; helminths, . %; protozoa, . %; viruses, %; fet, p = . ). animal pathogens classed as zoonotic (compared to non-zoonotic) were (of borderline statistical significance) less likely to be included in the oie list (pearson's x , p = . , effect size(ø) = . , or = . ) but there was no difference in the percentage included in discontools, (pearson's x , p = . ) nor any effect of being emerging (versus not emerging) there was a statistically significant difference between the hindices of zoonotic, human-only or animal-only pathogens (twoway anova, f , = ? , p, . ); h-indices were significantly higher for human-only (untransformed mean = . . and lower for animal-only pathogens ( . . ) compared to zoonotic ( . . ). h-indices were higher (with borderline statistical significance) for emerging ( . . ) compared to not emerging ( . . ) pathogens (two-way anova, f , = . , p = . ). the interaction between zoonotic and emerging factors was not significant (p = . ). -discontools. there were significant correlations between h-indices and discontools estimates of public (human) health (zoonotic and animal pathogens) and impact on wider society (animal-only pathogens), and a further relationship of borderline significance between h-indices and the discon-tools overall result; no other correlations were significant (table ). the european commission has outlined measures to strengthen coordinated approaches to health security at eu level, including monitoring, early warning and combating specific threats of a cross-border nature. these measures could be for climate change resilience [ ] or for biosecurity, particularly for infectious diseases including communicable diseases, antimicrobial resistant and healthcare-associated infections related to communicable diseases, and biotoxins or other biological agents [ ] . in this study, we implement a number of previously defined actions [ ] , including presenting a quantitative evaluation for the impact of infectious pathogens affecting human health and well-being (via effects upon domestic animals) [ ] . the work is unique, starting with all known infectious pathogens, and then objectively and systematically deciding which occur in relevant hosts in europe using a transparent process. the study establishes priority lists of human and domestic animal pathogens (including zoonoses) present in europe, using the h-index as a proxy measure for impact. previous work suggests that higher h-indices indicate higher impact for a pathogen relative to lower h-indices [ ] , (mcintyre, unpublished material). the h-index method has both strengths and weaknesses. the strengths include that it is much more evidence-based and objective than semi-quantitative and qualitative approaches, and the results provide an easily understood quantitative estimate of impact. h-indices estimates can be simply and rapidly calculated, and they can therefore be repeatedly obtained to reflect changes in status, with the potential for automation of this process. the results are available for all pathogens at a global scale, and the scores reflect the wider scientific interest that would be expected to follow from a pathogen being either zoonotic or emerging [ ] . most importantly, within a study of human diseases, h-indices were correlated with daly estimates [ ] , (mcintyre, unpublished material). dalys are an accepted measure of true disease burden in humans which accounts for the years of healthy life lost as a result of poor health or disability as well as the potential years of life lost due to premature death [ ] . in further work, h-indices were also correlated with the number of human deaths (mcintyre, unpublished material). the weaknesses of the h-index method include that calculations need some manual oversight, as false positives can occur for instance when pathogens are used as model organisms; biases in results may happen because of trends in interest in specific pathogens, diseases or research fields or in certain regions; and estimates are subject to biases in funding (mcintyre, unpublished material) and research publication. h-indices are likely to underestimate the contribution of scientific literature published in non-english languages, although after translation some publications are included in wos and consequently in our calculations of h-indices. the literature searching method also doesn't account for the quality of publications in which pathogen names appear and the typical number of citations within different fields, and all bibliographic software packages incorporate newly published literature from different literature sources into their databases at different rates. finally, h-indices are only a proxy for impact, with the results susceptible to a lag in time-to-publication, and newly emerging pathogens likely to be under-represented. as the strengths and weaknesses of using the h-index method are different to those of other prioritisation methods, it is probably best used in combination with other approaches, for example, to shortlist a set of pathogens for more detailed risk assessment relying on expert opinion or qualitative data. it may also be used to prioritise diseases for which formal risk assessments are not possible because of data gaps. our priority lists of pathogens enabled investigation of why infectious pathogens are omitted from disease surveillance and impact quantification work [ , , , ] . we considered several reasons for exclusion, including lack of diagnosis or misdiagnosis [ ] , because the impact of particularly chronic infections is difficult to quantify or they are seen as less important, and because some pathogens are commensals or natural biota causing opportunistic infections in immune-compromised individuals; their pathogens include those which are zoonotic (z), non-zoonotic (nz), emerging (e) and not emerging (ne) [ , ] , or given a new status (ns) in this work. pathogens also included in the list of top animal pathogens are noted (a). the major pathogens causing diseases included within the global burden of disease (gbd) report are noted [ , ] , as are those reportable in the ec (ec) [ ] . doi: . /journal.pone. .t table . top domestic animal pathogens in europe, prioritised according to the h-index methodology [ ] with the same emerging and zoonotic definitions as for table . [ ] ; for some animal pathogens, this is the first time that emerging status has been examined. methods to assess disease impacts use metrics capturing either human or animal host effects; they neither measure the magnitude in all hosts nor take account of scientific knowledge and tools for control. it is hard to prioritise human and animal diseases, because of the different metrics used (health or societal impacts versus welfare or economic impacts). significant differences between hindices mean values for human-only, zoonotic, and animal-only pathogens provide evidence that this single measure may have some use as a one health metric accounting for such factors. for example values for zoonotic pathogens were higher than for animal-only, suggesting that they account for human as well as animal-impact. higher values for human-only compared to other pathogen groups suggests that zoonoses may be under-represented due to underestimation of their global burden [ , ] , or research impact [ ] , or because of biases in research impact and funding for chronic human pathogens [ ] . in addition, lower animal-only hindices may be due to funding biases. finally, there was limited evidence that the h-index method is a reasonable proxy for the impact of animal pathogens; animal pathogen h-indices were significantly positively correlated with subsections of discontools [ ] , including impact on public (human) health and overall results (borderline significance). if animal-only (not zoonotic) diseases were included, there was a significant positive relationship with impact on wider society. as the more animal-focussed subsections (disease knowledge, impact on animal health and welfare, impact on trade, and available control tools) were not correlated with h-indices, and h-indices were not affected by inclusion in the oie list [ ] , this suggests a human-centric bias in h-indices; for example, a pathogen causing little impact in animals may nevertheless have a high h-index if zoonotic. the priority lists presented in this work should be used by agencies and research organisations in combination with other risk assessment methods to identify gaps in working for priority setting. it has been suggested that zoonoses must be dealt with at the interface of human and animal health using all available information [ ] ; this work, combining the eid and h-index technique, demonstrates such 'big-data' approaches. pathogens also included in the list of top human pathogens are noted (h). the major pathogens causing diseases included within the oie list of notifiable terrestrial and aquatic animal diseases (oie) are noted [ ] , as are those included in the discontools project (disc) [ ] . doi: . /journal.pone. .t table . results of spearman's rank correlations between h-indices and the discontools prioritisation of major animal diseases [ ] . fungi ascaris suum, z, ne, ns helminths yersinia enterocolitica, z, e, h bacteria borna disease virus, z, e, ns, disc viruses bacillus anthracis evidence-based semiquantitative methodology for prioritization of foodborne zoonoses global change: impact, management, risk approach and health measures -the case of establishing priorities for national communicable disease surveillance prioritizing emerging zoonoses in the netherlands establishing priorities for european collaboration in communicable disease surveillance the h-index as a quantitative indicator of the relative impact of human diseases using open-access taxonomic and spatial information to create a comprehensive database for the study of mammalian and avian livestock and pet infections disability-adjusted life years (dalys) for diseases and injuries in regions, - : a systematic analysis for the global burden of disease study commission implementing decision of august amending decision / /ec laying down case definitions for reporting communicable diseases to the community network under decision no. / /ec of the european parliament and of the council oie-listed diseases, infections and infestations in force the ncbi nucleotide database homepage the ncbi medical subject headings (mesh) database homepage risk factors for human disease emergence host range and emerging and reemerging pathogens global trends in emerging infectious diseases overview -web of science the ncbi taxonomy database homepage agricolae package cran . - ed r: a language and environment for statistical computing adapting to climate change: towards a european framework for action decision no / /eu of the european parliament and of the council of october on serious cross-border threats to health and repealing decision no / /ec monitoring eu emerging infectious disease risk due to climate change quantifying the burden of disease -the technicial basis for diability-adjusted life years emerging infectious diseases: vulnerabilities, contributing factors and approaches neglected and endemic zoonoses the socioeconomic burden of parasitic zoonoses: global trends emerging zoonoses: the challenge for public health and biodefense years lived with disability (ylds) for sequelae of diseases and injuries - : a systematic analysis for the global burden of disease study thanks to helen roberts (uk department for environment, food and rural affairs) and john stephenson (institute of infection and global health, university of liverpool) for input during study development. key: cord- - kfi yvu authors: de graaf, miranda; beck, relja; caccio, simone m; duim, birgitta; fraaij, pieter la; le guyader, françoise s; lecuit, marc; le pendu, jacques; de wit, emmie; schultsz, constance title: sustained fecal-oral human-to-human transmission following a zoonotic event date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: kfi yvu bacterial, viral and parasitic zoonotic pathogens that transmit via the fecal-oral route have a major impact on global health. however, the mechanisms underlying the emergence of such pathogens from the animal reservoir and their persistence in the human population are poorly understood. here, we present a framework of human-to-human transmission of zoonotic pathogens that considers the factors relevant for fecal-oral human-to-human transmission route at the levels of host, pathogen, and environment. we discuss current data gaps and propose future research directions. in recent years there have been many examples of pathogens crossing the species barrier and infecting humans, although the vast majority of these zoonotic events did not result in sustained human-to-human transmission [ ] [ ] [ ] . nevertheless, the continuing emergence of zoonotic pathogens is a cause of concern globally, especially due to the high morbidity and mortality of pathogens like mers-cov and a/h n influenza virus [ , ] . humanto-human transmission of microorganisms generally occurs via one or multiple transmission routes, including the fecal-oral, airborne, direct contact, or vector-borne route. whilst pathogens including bacteria, parasites and viruses have very different biological properties, they can employ similar routes of transmission and emergence. identification of the mechanisms underlying the effective human-to-human transmission of emerging zoonotic pathogens and their commonalities across different pathogens, may help design of interventions aimed at reducing the risk of sustained human-to-human transmission after a zoonotic event. as part of the activities of the antigone consortium on the emergence of zoonotic pathogens, an expert opinion meeting was organized. using a comparative approach including parasites, bacteria and viruses that transmit via the fecal-oral route, the meeting aimed at identifying the key drivers of sustained human-to-human transmission after a zoonotic event, taking into account the host, the pathogen and the interface (transmission amplifiers). in addition, major knowledge gaps were identified that require future research in order to better control emerging zoonotic pathogens that potentially are transmitted through a fecal-oral route. the main conclusions of this meeting are presented in this perspective. enteric pathogens can be transmitted between humans by the fecal-oral route via direct contact or indirect contact via contaminated fluids, including surface water, food, and carriers such as fomites ( figure ). the risk of a zoonotic pathogen becoming human-to-human transmissible depends on its adaptation to the human host and the environment. to analyze this process, we considered fecal-oral transmission of a zoonotic pathogen between two human hosts as follows; the human host that is infected with a zoonotic pathogen after a zoonotic event is defined as the donor while the susceptible human host that is subsequently infected by the first human host is considered the recipient. the transmission interface is the environment that the pathogen encounters after release from the donor and before infecting the recipient. for sustained fecal-oral human-to-human transmission certain elements in this transmission cycle, which we will refer to as transmission amplifiers, appear essential whereas other elements are not an absolute requirement, but increase the likelihood of transmission. transmission amplifiers may interact and their presence may or may not depend on conditions under which transmission occurs, including for example socio-economic conditions and cultural and behavioral variation. we designed a framework of human-to-human transmission that includes the transmission amplifiers relevant for the fecal-oral transmission route at the levels of host, pathogen, and environment ( figure ). several key transmission amplifiers are specific for fecaloral transmission (figure ), such as the intestinal microbiomes of the donor and recipient hosts. individuals with a healthy intestine are less likely to become infected or colonized by opportunistic pathogens, although the resistance provided by a healthy colonization (microbiome) can, in principle, be disrupted by a pathogenic species depending on its pathogenic potential (virulence) [ , ] . the composition of the human intestinal microbiome is, amongst others dependent on the presence of a functional immune system [ ] [ ] [ ] . changes in microbiome composition, in addition to the impaired immunity itself, may impact the outcome of infection and subsequent transmission. clinical symptoms such as diarrhea and vomiting can increase the likelihood of fecal-oral transmission as they can facilitate the spread of a pathogen into the fecal-oral transmission between humans. after shedding from the host enteric pathogens can be transmitted between humans by the fecal-oral route via direct contact between humans, or via indirect contact via contaminated fluids, including surface water, food, and carriers such as fomites. environment and onto fomites [ ] . remarkably, most pathogens that transmit via the fecal-oral route are very stable and can survive under various conditions, which may be related to the fact that these pathogens have to pass the hostile conditions of the gastrointestinal tract. zoonotic pathogens need to adapt to factors specific to this niche, such as the acidic conditions in the stomach and low oxygen in the large intestine, the temperature and the availability of specific sugars and nutrients. for example, comparative genomics of cryptosporidium parvum genotype iic suggests that the ability to establish an infection in a particular host species may depend in part on the presence of transporters controlling the exchange of metabolites between the host cell and the pathogen [ ] . fecal shedding of a pathogen does not necessarily require replication in the intestine. for example, the hepatitis e virus (hev) is shed via the feces despite its liver tropism [ ] . however, the presence of receptors and the tissue distribution of these receptors is a crucial element for tropism of infection, the shedding of microorganisms in stool and subsequent human-to-human transmission. in addition, it should be noted that not all pathogens that are shed via the feces transmit via the fecal-oral route. several respiratory viruses of zoonotic origin, that are capable of human-to-human transmission, are shed in feces. during sars-cov, mers-cov and influenza a infection, viral rna can be detected in stool. however, for these pathogens there is currently no evidence of fecal-oral transmission resulting in disease [ ] [ ] [ ] [ ] [ ] [ ] . similarly, the enteric pathogen campylobacter subspecies jejuni was transmitted from human-to-human via sexual contact following a zoonotic event [ ] . once a donor is shedding the pathogen, environmental factors at the transmission interface can have a large impact on transmission efficiency. contamination of the surface water after flooding can magnify the size of an outbreak via waterborne and foodborne routes [ ] . food sources can be contaminated by irrigation with sewage-contaminated water or the use of manure that contains traces of human feces, or on site by food-handlers. anecdotally, even food preservation measures can impact transmission as some additives to preserve lettuce were shown to also increase the stability of hepatitis a virus [ ] . transmission via food can have a major impact on the global spread of pathogens. in nearly human-to-human transmission following a zoonotic event de graaf et al. framework for human-to-human transmission after a zoonotic event showing the key transmission amplifiers from the host (triangle), pathogen (blue) and environmental transmission amplifiers (green), respectively. the transmission amplifiers that are specific to the fecal-oral route are indicated with a red star. people were infected during an escherichia coli o :h outbreak in europe, resulting in deaths. epidemiological and trace-back investigations pointed to salad sprouts as the possible contaminated food source [ ] . transmission amplifiers not specific for fecaloral transmission route several factors that are important transmission amplifiers of the likelihood of fecal-oral transmission are generic to most human-to-human transmission routes. the immune system of the human host is an important factor that has to be confronted for sustained human-tohuman transmission. most pathogens that successfully transmit, have acquired genes that can counteract or evade the adaptive and or innate immune responses. pathogens may have adapted through altered domains that are recognized by the cellular or humoral immune system, to evade pre-existing immunity based on previously infecting pathogens. loss of genes or gene function may also be associated with adaptation to the human host. a salmonella enterica serotype typhimurium clone which causes bloodstream infection amongst children and hiv-infected adults in sub-saharan africa, has adapted to these immuno-compromised hosts through loss of gene functions enabling bacterial survival outside the host, whilst retaining the ability to cause enteritis in multiple host species [ ] . with the general population aging and technologies becoming more invasive, medical interventions can become an amplifier of human-to-human transmission. although medical interventions do not necessarily select for human-to-human transmissible pathogens, they can increase the duration of infection, and thereby the likelihood of evolution and adaptation [ ] . for instance, the application of extracorporeal membrane oxygenation prolongs and increases survival in patients with otherwise acute fatal infectious disease [ ] . the use of immune modulatory and suppressive drugs has created a human population that is more susceptible to prolonged pathogen proliferation and shedding [ ] [ ] [ ] [ ] [ ] . an immunodeficient individual was found to excrete a vaccine-derived poliovirus for twenty years. during this time the virus became virulent and changed antigenically [ ] . the unrestrained use of antimicrobial drugs in medical and veterinary care and in agriculture in low-income, middleincome, and high-income countries creates an unprecedented selective pressure that may select for pathogens that are more transmissible. salmonella enterica serotype typhimurium has the ability to develop a super shedder phenotype, that can be induced by antibiotic treatment in mice [ ] . a human reservoir for non-typhoid salmonella (nts) transmission of multiple serotypes was demonstrated in a study of nts-infected patients who continued to shed nts for months up to years, and strains of these patients acquired antimicrobial resistance genes and virulence genes that possibly affected host-pathogen interactions [ ] . the infectious dose, replication kinetics and the number of pathogens being shed can have a major impact on the efficiency of fecal-oral transmission. for example, norovirus and shigella spp. require a low infectious dose and can be transmitted via hands and fomites [ , ] , whereas listeria monocytogenes infections require a high infectious dose [ ] , making these transmission routes less likely. however, surprisingly little is known about the infectious dose for many fecal-orally transmitted human pathogens. as for shedding in the environment, several strategies can be successful, such as shedding of lower amounts of microorganisms over a long period (chronic/persistent infection) and thus a long period of transmission associated with mild clinical symptoms , or shedding of high loads of microorganisms for a relatively short period with significant clinical symptoms [ ] . receptor usage is a key element for successful human-tohuman transmission. not only are the site of the expression of these receptors in the host and the receptor specificity of the pathogen of importance, but also the prevalence of these receptors in the human population can potentially contribute to the likelihood of efficient transmission. for group a rotaviruses attachment to histobloodgroup antigens is an essential step for infection. interestingly, strains of the p [ ] , p[ ] and p [ ] subtypes that are generally found in cattle but can also infect humans, attach to the blood group a epitope [ ] . however, since the frequency of blood group a in human populations ranges from % to %, the majority of individuals is expected to be resistant to infection by these strains, which would constitute a barrier to transmission. sustained human-to-human transmission for a virus with a relatively low r would thus require an adaptation of the glycan binding capacity to the human hbga genetic polymorphism [ ] . hev genotype and exclusively infect humans while genotypes and infect pigs but occasionally infect and transmit between humans [ ] . the high conservation of hev attachment and entry factors may explain the observed cross-species transmission while factors limiting efficient human-to-human transmission are thought to include regulation of subgenomic translation and specific virus-host receptor interactions [ , ] . surprisingly few differences exist between the basic requirements of the different types of pathogens to become human-to-human transmissible. however, bacteria can replicate in the environment whereas viruses and parasites cannot. as the transmittable stages of parasites are environmentally very resistant, and can withstand water treatment processes, parasites are probably more likely to be transmitted via food and water compared to direct fecal-oral-transmission [ , [ _ t d $ d i f f ] ]. genome plasticity is an important factor for all pathogens but while parasites, viruses and bacteria can all adapt by mutations, recombination and lateral gene transfer, viruses can also acquire genes by genome rearrangements and bacteria can acquire mobile genetic elements that carry genes that may encode determinants that facilitate increased fitness in certain conditions. 'for a zoonotic pathogen the risk of becoming human-tohuman transmissible depends on further adaptation to the human host. for efficient fecal-oral transmission amplifiers in the transmission interface appear crucial.' the focus of the public health and emerging infectious disease communities on emerging viruses causing severe infections, has resulted in the discovery of several possible determinants of sustained human-to-human transmission of zoonotic viruses. however, the likelihood of sustained human-to-human fecal-oral transmission after a zoonotic event of any pathogen type is difficult to assess as these events are hardly described in current literature. one could conclude that zoonotic pathogens rarely become human-to-human transmissible through the fecal-oral route because there may be need for dual adaptation, i.e. to the harsh conditions in the environment in addition to the human host, and therefore these events are rare. however, we cannot exclude that we may be missing some of these events, because it can be difficult to distinguish between strictly human and zoonotic pathogens once the latter have established themselves in the human population or because they remain undistinguished with the use of current clinical microbiology tools. for example, recent results strongly suggest that a pig roundworm can act as an important source of human ascariasis [ ] [ ] [ ] but this can go unnoticed as the human and pig parasite population show minor phenotypic and genotypic differences. zoonotic pathogens that transmit via the fecal-oral route appear to cause similar clinical symptoms compared to other (related) enteric pathogens and further research is not pushed due the relative lack of (more) serious disease. we thus may neglect relevant pathogens and even if we do study these pathogens, the results may have undesired economic consequences for agriculture and food sectors. in addition, until recently we lacked tools for studying important zoonotic events; whilst the small genomes and rapid evolution of viruses allow identification of novel causative agents with limited sequencing effort, such analysis is much more complicated for bacterial and parasitic enteric pathogens which have relatively large genomes. some experimental models for fecal-oral transmission between hosts have been described, such as for norovirus [ ] , but there is a general lack of suitable animal models to study fecal-oral transmission. in fact, most research on host-pathogen interactions is focused on mechanisms of pathogenicity rather than on mechanisms of transmission, whilst the latter is crucial for the development of intervention strategies to prevent further spread and to prevent sustained human-to-human transmission of zoonotic pathogens. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest one health, multiple challenges: the inter-species transmission of influenza a virus zoonotic aspects of rotaviruses shiga toxin-producing escherichia coli o , england and wales sars and mers: recent insights into emerging coronaviruses intestinal microbial communities associated with acute enteric infections and disease recovery salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota the intestinal microbiota and susceptibility to infection in immunocompromised patients the intestinal microbiome in early life: health and disease gut microbiota composition correlates with diet and health in the elderly makison booth c: vomiting larry: a simulated vomiting system for assessing environmental contamination from projectile vomiting related to norovirus infection comparative genome analysis of two cryptosporidium parvum isolates with different host range hepatitis a: old and new viral shedding and antibody response in patients with middle east respiratory syndrome coronavirus infection influenza virus infection among pediatric patients reporting diarrhea and influenza-like illness enteric involvement of severe acute respiratory syndrome-associated coronavirus infection long-term sars coronavirus excretion from patient cohort extra-pulmonary viral shedding in h n avian influenza patients long-lasting outbreak of erythromycin-and ciprofloxacinresistant campylobacter jejuni subspecies jejuni from to in men who have sex with men quantitative assessment of infection risk from exposure to waterborne pathogens in urban floodwater survival of hepatitis a virus on modified atmosphere-packaged (map) lettuce the enemy within us: lessons from the european escherichia coli o :h outbreak loss of multicellular behavior in epidemic african nontyphoidal salmonella enterica serovar typhimurium st strain d the potential for respiratory droplet-transmissible a/ h n influenza virus to evolve in a mammalian host year in review : extracorporeal membrane oxygenation noroviruses as a cause of diarrhea in immunocompromised pediatric hematopoietic stem cell and solid organ transplant recipients high prevalence of prolonged norovirus shedding and illness among hospitalized patients: a model for in vivo molecular evolution persistent spiking fever in a child with acute myeloid leukemia and disseminated infection with enterovirus infectious complications and vaccinations in the posttransplant population prolonged influenza virus shedding and emergence of antiviral resistance in immunocompromised patients and ferrets twentyeight years of poliovirus replication in an immunodeficient individual: impact on the global polio eradication initiative host transmission of salmonella enterica serovar typhimurium is controlled by virulence factors and indigenous intestinal microbiota this study showed that nts strains of persistently infected humans acquired antimicrobial resistance and virulence genes inoculum size in shigellosis and implications for expected mode of transmission norwalk virus shedding after 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the past from the twig tips to the deeper branches: new insights into evolutionary history and phylogeography of ascaris prophylactic treatment with the nucleoside analogue -c-methylcytidine completely prevents transmission of norovirus the authors developed a murine fecal-oral transmission model the authors would like to thank all participants of the dahlem 'inter human barriers' workshop for their contributions and ryan kissinger (niaid, nih) for designing the figures. this work was supported by antigone (grant numbers ); edw is supported by the intramural research program of the national institute of allergy and infectious diseases, us national institutes of health; pf receives funding from the eu fp project prepare (grant number ); mdg is supported by the eu grant compare (grant number ) and the virgo consortium, funded by the dutch government (project number fes ). key: cord- -vp ydce authors: lanata, claudio f.; fischer-walker, christa l.; olascoaga, ana c.; torres, carla x.; aryee, martin j.; black, robert e. title: global causes of diarrheal disease mortality in children < years of age: a systematic review date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: vp ydce estimation of pathogen-specific causes of child diarrhea deaths is needed to guide vaccine development and other prevention strategies. we did a systematic review of articles published between and reporting at least one of pathogens in children < years of age hospitalized with diarrhea. we included rotavirus data from the rotavirus surveillance network coordinated by who. we excluded studies conducted during diarrhea outbreaks that did not discriminate between inpatient and outpatient cases, reporting nosocomial infections, those conducted in special populations, not done with adequate methods, and rotavirus studies in countries where the rotavirus vaccine was used. age-adjusted median proportions for each pathogen were calculated and applied to deaths due to diarrhea in children under years for , assuming that those observed among children hospitalized for diarrhea represent those causing child diarrhea deaths. articles and who studies done in countries were selected representing inpatient studies. studies seeking only one pathogen found higher proportions for some pathogens than studies seeking multiple pathogens (e.g. % rotavirus in single-pathogen studies vs. % in studies with – pathogens, p< · ). the percentage of episodes for which no pathogen could be identified was estimated to be %; the total of all age-adjusted percentages for pathogens and no-pathogen cases was %. adjusting all proportions, including unknowns, to add to %, we estimated that rotavirus caused [uncertainty range (ur) – ], enteropathogenic e. coli (ur – ), calicivirus (ur – ), and enterotoxigenic e. coli (ur – ) deaths. rotavirus, calicivirus, enteropathogenic and enterotoxigenic e. coli cause more than half of all diarrheal deaths in children < years in the world. despite global success in the reduction of all cause and diarrheaspecific mortality in the past years, diarrhea remains the second leading cause of death due to infections among children under five years of age worldwide [ , ] . it is estimated that diarrhea accounted for ? % of the ? million deaths among children under in [ , ] . several organisms have been implicated as important causes of these deaths [ , ] , yet there has not been a review using standardized methods to determine the importance of all of the common pathogens. the child health epidemiology reference group (cherg) has estimated the causes of child deaths from major causes since . we have undertaken this review to develop estimates of pathogen-specific diarrhea mortality among children under years of age. we present the results of a systematic literature review of studies of diarrhea etiology in hospitalized children and use these results to estimate the global burden of diarrhea mortality by pathogen for children under years of age for . we searched medline, lilacs, and medscape for studies published between and . we used the terms ''diarrhea'' (or ''diarrhoea''), ''gastroenteritis'', ''rotavirus'', ''e.coli'' (or ''escherichia coli''), ''salmonella'' (not ''typhi''), ''shigella'', ''campylobacter'', ''giardia lamblia'', ''vibrio'', ''cryptosporidium'', ''entamoeba'', ''norovirus'', ''calicivirus'', ''norwalk agent'', using ''and children'' as a search restriction. an example of one of the search instructions in medline-pubmed is: ''diarrhea'' [mesh] january , -december , . we also included data from the who rotavirus surveillance network for provided to us by who only from countries that had not introduced rotavirus vaccine as of december and had data covering the -month period. these studies used a standard protocol across the network [ ] . we included studies that sought at least one of the above listed pathogens and conducted or more months of surveillance among children less than years of age hospitalized with diarrhea. studies must have included all diarrhea patients at the selected study site or a systematic sampling of cases for the duration of the study. we did not require a minimal number of children evaluated to be included. laboratory tests were performed on rectal swabs or stools samples. we excluded studies conducted during reported diarrhea outbreaks, those that did not discriminate between inpatient and outpatient cases, those that included patients with nosocomial infections, and those conduced in special populations, such as hiv-positive patients. we also excluded studies that did not describe adequate surveillance methods or standard laboratory methods, according to the following criteria: a) salmonella and shigella isolation in salmonella/shigella agar, xylose-lysine-deoxycholate agar, hektoen enteric agar, and selenite enrichment for salmonella [ ] ; b) campylobacter isolation by use of transport media with antibiotics (skirrow's supplement or similar) and inoculation into % sheep blood with antibiotics (butzlers supplement or similar), cultivated at uc in micro-aerobic atmosphere [ ] ; c) vibrio cholerae isolation by alkaline peptone water enrichment and subculture at hrs into thiosulfate-citratebile salts -sucrose agar (tcbs) [ ] ; d) e. coli isolation from macconkey agar and identification of etec by dna probes or polymerase chain reaction (pcr) for heat-labile (lt) or heatstable (st) toxins, cell cultures (y , cho cells), ileal loop or mouse models [ ] ; e) epec isolation by the use of hep cell cultures or the presence of the plasmid for adherence (bfp) and the intimin gene (eae) identify in dna probes or by pcr [ ] ; f) rotavirus, calicivirus (or norovirus), astrovirus and enteric adenovirus identification with the use of enzyme-linked immunoassays (elisa), electronic microscopy, or pcr [ ] ; g) giardia lamblia identification by direct microscopic examination, or zinc-sulfate concentration from direct stools or by elisa [ ] ; h) cryptosporidium spp. identification by elisa, or the modified ziehl-neelsen stain for microscopy [ ] ; i) entamoeba histolytica identification by direct microscopic examination [ ] . we did not include studies in areas or countries where the rotavirus vaccine was used but included data from the placebo arm of rotavirus vaccine trials. articles published in languages other than english, spanish, portuguese, italian, german and french were not included. the following enteropathogens were considered: rotavirus, enteropathogenic escherichia coli (epec), enterotoxigenic escherichia coli (etec), salmonella spp. (excluding salmonella typhi), shigella spp., campylobacter spp., vibrio cholerae o and o , giardia lamblia, cryptosporidium spp., entamoeba hystolitica, human caliciviruses (genogroup i and ii norovirus and sapovirus) or astrovirus, coronavirus, and enteric adenovirus. we extracted data for all children less than five years of age for each pathogen. data from more than one hospital in a country were treated as separate studies if the presentation of data permitted. papers that published different etiological data from the same study site were grouped into one study. if co-infections were reported, they were not treated separately so each pathogen was counted as present if isolated alone or in combination. three reviewers (co, cxt, and cfl) did the primary extraction and all selected papers were reviewed by cfl and cfw independently. disagreements were resolved by cfl and/or reb. we calculated overall median proportions of positive diarrheal stool samples for each pathogen for children - months of age using the overall proportion for all children included in the study; studies enrolled children from a narrower age range so we calculated for these studies an age-adjusted proportion for the - months of age group by calculating a conversion factor for age group x as the median of - prevalence over age group x prevalence (median (prev - /prev x )) using studies that reported both - and the age group x for a given pathogen. to use this method we required at least studies, where each study reported both - months and age group x. in situations where less than studies were available we employed an alternative method where the conversion factor for age group x was taken as the ratio of the median prev - to median prev x (median (prev - )/median (prev x )). for this approach we required that or more studies contribute to each of the two medians, but dropped the method requirement that individual studies report both age groups. if neither of these sets of conditions were met, we borrowed the conversion factor for the age group x from a similar age group within the same pathogen (for instance, used the conversion factor calculated for studies including infants - months of age for studies that included infants - months of age) or from a similar pathogen (conversion factor for age group x for a study on epec borrowed from studies on etec). the - months prevalence proportion for each pathogen was estimated using the median individual study - months pathogen prevalence. we stratified studies by the number of pathogens sought and calculated the unadjusted and age-adjusted medians, as described above, separately for single pathogen studies and for studies that sought to pathogens. for estimating the proportion of diarrheal stools due to unknown pathogens, we included studies that sought or more pathogens. for the numbers of diarrheal deaths attributable to each pathogen, we assume that the distribution of pathogens observed among children hospitalized for diarrhea represents the pathogen prevalence among child diarrhea deaths. we applied the ageadjusted median proportion for each pathogen and for unknowns to the overall number of diarrhea deaths of estimated for the world in [ ] , adjusting all proportions equally to be constrained to add to %. we explored alternative estimates using all studies selected or only those that sought to pathogens, constraining or not all proportions to add to %. the uncertainty around each estimate was calculated using bootstrap confidence intervals [ ] . 'pseudo-data sets' were created by sampling studies with replacement from the real dataset. each of the pseudo-datasets was used in the estimation procedure described above to generate a corresponding prevalence proportions. the ? th and ? th percentile of these proportions gave the % confidence interval (ci). to estimate the uncertainty of the number of deaths for each pathogen, we paired each of the pseudo-datasets with random draws from the under total mortality envelope, the proportion of total deaths attributable to diarrhea [ , ] , and the proportion of diarrhea deaths due to unknown pathogens. the under year global total mortality envelope estimate and standard deviation were calculated by sampling and combining random draws from each of the countries in the world [ , ] . for each country, a normal mean and standard deviation was estimated from the point estimate and associated confidence interval. from citations identified in the electronic search, articles were selected for further evaluation (fig. ) ; articles were excluded because they had one or more of the exclusion criteria (about % because they were not longitudinal studies or inappropriate laboratory methods were used, % because no data was given for children , years of age, % for studies that lasted less than months of duration, and the rest because data were reported after rotavirus vaccine introduction, duplicate publications or reporting results on a pathogen not included in our list). a total articles and who rotavirus surveillance network sites were selected representing inpatient studies with data for at least one pathogen [list of the references can be found at www.cherg.org]. the geographical localization of the study sites is shown in figure . the median and age-adjusted median proportions (with % ci) of isolation of each enteropathogen in hospitalized diarrhea cases are shown in table . rotavirus, epec, calicivirus, and etec were the most frequently identified organisms. the sum of these age-adjusted median proportions, including unknowns was %, indicating a problem with many articles reporting mixed infections as separate causes. different isolation rates were observed in studies in which only one, versus at least enteropathogens were sought (table ) . rotavirus was more frequently isolated in single-pathogen inpatient studies in comparison with multiple-pathogen studies ( % vs. %, respectively, p, ? ). the same trend was observed between single-and multiple-pathogen studies for most pathogens, but mainly for giardia lamblia ( % vs. %, p, ? ), shigella ( % vs. %, p, ? ) and v. cholerae ( % vs. . %, p, ? ). very few studies sought a substantial number of pathogens. from the inpatient studies, only ( %) sought or more pathogens ( study with , studies with , studies with , and studies with pathogens). in these studies, ? % of cases had no pathogen identified. adjusting all proportions, including unknowns, to add to %, we estimated that rotavirus caused (uncertainty range ur - ), enteropathogenic e. coli (ur - ), calicivirus (ur - ), and enterotoxigenic e. coli (ur - ) deaths. these four pathogens were associated with % of all diarrhea deaths (table ) . these estimates varied substantially depending on the methods used. if the proportions were not made to add to %, rotavirus would be said to cause deaths or if only studies that sought . pathogens were selected and the proportions were adjusted to % rotavirus would be said to cause deaths (table ). when classifying studies by who region, most studies were done in the western pacific region ( studies) and less in the eastern mediterranean region ( studies) ( table ). rotavirus was more frequently isolated in the western pacific region ( %) and less in the american region ( %). other comparisons were limited by few or no studies in some regions (table ). in this review, we showed that more than half of the severe diarrhea episodes, most likely to result in death among children under the age of years in , could be attributed to rotavirus, epec, calicivirus, and etec. our estimates have been adjusted for age in studies that did not cover all children , years old, and campylobacter spp to add to %, including a fraction of episodes with unknown etiology. such adjustments have not been done in previously published estimates for single diarrhea etiologies [ , , [ ] [ ] [ ] . we identified a potential selection bias among studies that focus on a single pathogen. for example, the median proportion of diarrheal episodes with rotavirus identified varied from % in single-pathogen studies to % in studies that sought more than pathogens. it is possible that studies looking for a particular pathogen are more likely to be conducted in a study site with a high prevalence of that pathogen and/or a low prevalence of other pathogens. an urban hospital that treats children of higher socioeconomic status and living in more hygienic conditions than children in rural areas may find a higher proportion of cases with rotavirus. a study of cholera done in a hospital in an endemic area may not be representative of national or regional populations. because of the low number of studies that sought multiple pathogens, we have not restricted our analysis to only those studies, in an attempt to include as much global data as possible, but it should be recognized that the inclusion of single-etiology studies may result in a biased higher estimate for some pathogens. by including pathogens in this review we are able to address the problem of mixed infections, an important factor ignored in previously published single-pathogen estimates of deaths. no methodology has been developed to identify the true cause of an episode when more than one pathogen is identified in the stool. our adjustment of all percentages to fit % is done to correct for this problem, assuming that each pathogen is equally likely to cause the illness. this is probably not correct because some organisms are carried in the feces for a relatively long time after infection-causing illness, like norovirus [ ] , or may not cause illness, especially in older children who have acquired immunity that protects against disease, but not carriage of the organism, like some protozoa [ ] . this method of including all equally in the constraint to % of diarrhea deaths may result in an underestimate of the importance of some pathogens, such as rotavirus in young children, and overestimate the importance of others, such as giardia. we do not have data on the presence of these pathogens in the stools of asymptomatic children in the studies selected in this review so we cannot determine the attributable fraction related to each pathogen as done in other studies [ ] . however, controlling for pathogens found in non-ill children does not necessarily eliminate the problem because some pathogens with long excretion periods after illness, like norovirus, may be wrongly classified as not causing diarrhea. carefully conducted longitudinal studies are needed to separate long-term excretors after illness from asymptomatic infections, to reveal the true pathogenic role of these different organisms in developing countries. we estimated that the number of diarrhea episodes for which no pathogen can be identified is %, which is based on studies that sought at least pathogens, not necessarily all and thus may be an overestimate. these ''unknowns'' could be due either to the same pathogens not detected because insensitive methods were used to identify them (either the method itself or to using a rectal swab instead of a stool sample) [ ] , to the use of antibiotics prior to obtaining the stool sample, to other yet undiscovered infections, or to non-infectious causes of diarrhea. the proportion of samples with unknown causes was based on a selected group of studies that searched for or more pathogens. these studies do not represent the world as the rest of the studies did. the recently conducted studies called the global enterics multicenter study (gems) in countries in africa and asia were designed to fill this gap [ , , ] . however, they studied cases with moderate and severe diarrhea seen in health services (hospitals, emergency rooms and community clinics), not separating those being hospitalized from milder outpatient cases, therefore, those studies would not meet our inclusion criteria. given that we cannot distinguish among the reasons no pathogen was found during the episode, our estimates may represent an under-estimate, at least for some causes. we could not include some pathogens known to cause diarrhea in our review, such as organisms that cause food-borne outbreaks (i.e. clostridium perfringens [ ] , or staphylococcus aureus producing enterotoxins [ ] ), because there are very little data on their importance in developing countries. a recent review of rotavirus studies estimated that rotavirus caused deaths in children , in [ ] . if we would apply the median proportion of % rotavirus isolation found in the inpatient studies that sought it in our review, without any adjustment, to the million u diarrheal deaths in , we would estimate rotavirus deaths in . in it is estimated that diarrhea deaths have been reduced to [ ] . our estimate of deaths due to rotavirus, using our improved methods, still represents an important global public health problem, with children dying due to this condition every hour. this estimate does not account for any recent reduction in rotavirus-specific proportionate mortality due to the introduction of rotavirus vaccine, as seen in some latin america countries [ ] , but these countries account for a very small fraction of global diarrhea mortality. wide scale use of the rotavirus vaccine in high mortality countries will allow a more precise estimate of the true proportion of diarrhea deaths caused by rotavirus. our estimate of deaths for shigella is much lower than a previous estimate of deaths due to shigellosis in children under years in the world in published by kotloff et al [ ] . this initial estimate was not based on a systematic review of the literature; rather, it used a single study in latin america to estimate the proportion of shigella cases that were hospitalized and a bangladeshi study to estimate the case-fatality rate of children hospitalized with shigellosis to estimate the global burden due to this organism. using the same methodology of kotloff et al but with an updated review of the literature and current case fatality rates observed in bangladesh, bardhan p et al [ ] estimated that only children younger than years of age died due to shigellosis in asia in . our estimates are compatible with this asian estimate. the total number of deaths due to calicivirus of deaths has indicated to be the third most common cause of death due to diarrhea in children under years of age. few studies differentiated between gi and gii norovirus and other types of human caliciviruses, but in those few that did, most of calicivirus isolated in children with severe diarrhea have been due to norovirus gii [ , ] . patel et al [ ] estimated deaths due to norovirus among children under , but this was calculated using very different methods and assumptions: they used an attributable fraction due to norovirus when data on asymptomatic children was available, and applied their mean isolation rate of . % from inpatient studies (not much different from our median isolation rate of . %) to . million deaths due to diarrhea in the world; they did not adjust for mixed infections or unknowns. the deaths estimated to be caused by epec represent different sub-types of this type of pathogenic e. coli, a group that requires further epidemiological studies in different parts of the world to further characterize them since some sub-types are isolated with the same frequency in diarrhea and control children [ ] , new ''typical'' and ''atypical'' epec strains have been identified [ ] , and in some regions have been identified to cause more persistent than acute diarrhea [ ] . these estimates have several limitations. the studies included in this review were conducted in selected sites and in some cases in populations with increased risk of diarrheal diseases. thus, they may not be representative of the countries where they were conducted, nor of the world. for several regions, such as russia and the former soviet states or sub-saharan africa we have limited or no data ( fig. , table ). the gap of information from africa, for pathogens other than rotavirus, is most acute because of the number of diarrhea deaths in this region is very high [ ] [ ] [ ] . no study has been conducted to identify pathogens in children who died due to diarrheal diseases, so we assume that children in need of hospitalization are the best proxy of diarrhea deaths in low to middle income countries, but this may not be true for some pathogens. another limitation is the combination of laboratory methods with different sensitivities to identify a pathogen: from the culture-based identification of salmonella or shigella to the highly sensitive real-time pcr method for norovirus. this may have affected the relative importance of one vs another pathogen in our estimates. we excluded studies on nosocomial infections, on displaced populations and on diarrhea outbreaks, which may have caused us to under-represent deaths due to some pathogens like v. cholerae. we included in our estimates a total of pathogens ( viruses, bacteria and parasites) that have been incriminated as causes of severe diarrheal diseases. some viruses, like adenovirus, and parasites, like g. lamblia, have not been completely documented as a cause of severe diarrhea in developing countries [ , , ] . the subject of causality of diarrheal diseases is still not completely understood in settings where children are heavily exposed to many pathogens early in life. young infants may be protected by breast milk and trans-placental maternal immunity and very low doses of ingested pathogens early in life may result in subclinical infections and development of immunity. this immunity may not preclude, however, the excretion of these pathogens in the child's feces. practically all studies done in children who were studied when they were healthy as well as when they developed an acute diarrheal episode have found the same pathogens, although usually with lower frequency, in healthy states. thus, the assumption that any pathogen identified in a child with diarrhea is the cause of the episode is naive and additional methods are needed to determine the pathogenicity of microbes. with a better understanding of the pathogenicity of key organisms our estimates could be further adjusted. also, some studies suggest that children ill with a pathogen, as with epec, may excrete higher amounts in the stool, as compared with asymptomatic infections [ ] , so future studies may consider quantifying the amount of each pathogen in the stool to help identifying those ill with it. finally, the review period covering studies published between and (studies were conducted with a median mid-study period of , only ( %) studies were done prior to ). we have not identified a significant change of the proportions assigned to each pathogen over time, so this does not seem to affect our estimates, as shown in fig. for rotavirus. the global burden of disease study recently published cause of death estimates for countries in [ ] . for children , years of age, gbd estimated a total of deaths due to diarrheal diseases in while cherg estimated deaths for . gbd also estimated deaths due to etiologies and produced estimates for - , - , and - days and - years of age. cherg estimates for in children , years of age are slightly higher than gbd estimates for rotavirus ( vs rotavirus deaths, respectively), similar for epec and etec deaths ( vs , and vs , respectively), and lower for cholera, salmonella, shigella, campylobacter, entamoeba histolytica, and cryptosporidium spp. (table ). gbd did not estimate deaths due to norovirus, which was the third leading cause of death in our review. gbd used rates reported in diarrhea studies published between and done in outpatients, casecontrol, and community-based studies as a reference category to adjust the proportions seen in inpatient studies. cherg only used data from inpatient studies published between and . both gbd and cherg used modeling to obtain the total number of diarrheal deaths for children , , but unlike gbd, cherg has not used models for etiology-specific causes of deaths for each age group and for each country to produce its global estimate. age specific data and modeling may produce spurious results, more so if there are no data. for example, very few studies have been done describing causes of diarrhea in neonates in developing countries, but gbd has estimated deaths caused by each of the pathogens in neonates - and - days of age (table ). gbd only produced estimates for etiologies of diarrhea and by subtracting the total of these estimates from the total of diarrheal deaths; they estimated the proportion of other causes of diarrheal deaths. cherg estimated the proportion due to unknowns from studies that searched for - pathogens, which we feel realistically addresses the fact that a causative agent is not identified in every illness. this also explains why we estimated a higher number of deaths in this category ( ) than gbd for ''other causes'' which should include unknowns ( ). gbd and cherg recognized the problem of mixed infections, but the methods used to adjust for it was different: gbd only used proportions for each etiology from inpatient studies that searched for - etiologies and used that information to produce weights to adjust their estimates in the models. we choose to constrain all proportions, including unknowns, to % to correct for mixed infections, which we feel it is more appropriate until better data and analytical tools are available. we have done an extensive search of the literature to include the inpatient studies used in our estimates. gbd has not published the studies included, their search strategy, or modeling methods. until these are published we will not be able to completely compare these estimates. this is the first systematic review attempting to estimate the cause of deaths for these enteric pathogens. rotavirus, calicivirus, enteropathogenic and enterotoxigenic e. coli cause more than half of all diarrheal deaths in children , in the world. we have identified a potential selection bias in studies searching for only one enteropathogen, and the problem when mixed infections (more than one enteropathogen is identified in a stool sample taken from a child with severe diarrhea) are not taken into consideration when estimating causes of diarrheal deaths, factors that has affected previous published estimates. future studies should be done in hospital services dealing with all types of 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against treatment in developing world environments of high endemicity a systematic review and meta-analysis of the association between giardia lamblia and endemic pediatric diarrhea in developing countries quantitative real-time polymerase chain reaction for enteropathogenic escherichia coli: a tool for investigation of asymptomatic versus symptomatic infections global and regional mortality from causes of death for age groups in and : a systematic analysis for the global burden of disease study cynthia boschi-pinto of who and theresa diaz of unicef provided coordination of the involvement in cherg of their respective institutions. carolyn weidemann served as coordinator of the grant in support of cherg from the bill and melinda gates foundation. cherg provided advice on methods and interpretation of results. we thank walter mendoza for his initial literature review, cynthia boschi-pinto and laura lamberti for their support in searching for articles, and edda franco for editorial assistance. we thank the countries who provided data through the who-coordinated global rotavirus surveillance network of participating ministries of health, sentinel hospital sites, and the rotavirus laboratory network. we also thank john sanders and theresa j. ochoa for providing useful comments on early drafts of the manuscript. preliminary results of this study has been presented at cherg and food borne epidemiology reference key: cord- -k i wgs authors: woolhouse, mark e.j.; gowtage-sequeria, sonya title: host range and emerging and reemerging pathogens date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: k i wgs an updated literature survey identified , recognized species of human pathogen, % of which are zoonotic. of the total, are regarded as emerging or reemerging. zoonotic pathogens are twice as likely to be in this category as are nonzoonotic pathogens. emerging and reemerging pathogens are not strongly associated with particular types of nonhuman hosts, but they are most likely to have the broadest host ranges. emerging and reemerging zoonoses are associated with a wide range of drivers, but changes in land use and agriculture and demographic and societal changes are most commonly cited. however, although zoonotic pathogens do represent the most likely source of emerging and reemerging infectious disease, only a small minority have proved capable of causing major epidemics in the human population. a recent, comprehensive literature survey of human pathogens listed > , different species ( ), more than half known to be zoonotic, i.e., able to infect other host species ( , ) . the survey data showed that those pathogens regarded as emerging and reemerging were more likely to be zoonotic than those that are not ( , ) , confirming an association between these characteristics which had long been suspected ( , ) , but which could not be formally demonstrated without denominator data as well as numerator data. here, we revisit these calculations, using updated information on the biology and epidemiology of recognized human pathogens. we pay close attention to possible differences between the major pathogen groups-viruses, bacteria, fungi, protozoa, and helminths. we also examine in detail the relationship between host range and pathogen emergence or reemergence, considering both the type and diversity of nonhuman hosts. we catalog the kinds of proximate factors or drivers that have been linked with pathogen emergence and reemergence and ask whether these differ between the major pathogen groups or between zoonotic and nonzoonotic pathogens. we focus mainly on pathogen diversity (as numbers of species) rather than on the effects of disease that they impose, noting that many diseases, e.g., infant diarrhea, can be caused by more than one species of pathogen. however, we comment on the transmissibility of pathogens once they have been introduced into the human population because transmissibility is an important determinant of the potential public health problem. we obtained counts of pathogen species from an updated version of the previously published database ( ) . as before, we defined a human pathogen as "a species infectious to and capable of causing disease in humans under natural transmission conditions." we included pathogens that have only been reported as causing a single case of human disease and those that only cause disease in immunocompromised persons. we also included instances of accidental laboratory infection but excluded infections resulting from deliberate exposure in the laboratory. we added recently recognized pathogens listed online by the centers for disease control and prevention, the world health organization (who), promed, and elsewhere ( ) ( ) ( ) ( ) . we obtained taxonomic classifications online from the international committee on taxonomy of viruses, the national centre for biotechnology information, the cab international bioscience database of fungal names, and from standard texts ( ) ( ) ( ) ( ) ( ) ( ) . pathogen species were categorized as emerging or reemerging based on previously published reviews of the literature ( , ), again updated from online sources ( ) ( ) ( ) . a species was regarded as emerging or reemerging if any recognized variant fell into this category (e.g., escherichia coli o , h n influenza a). we considered the following pathogen groups: viruses (including prions), bacteria (including rickettsia), fungi (including microsporidia), protozoa, and helminths. we did not consider ectoparasites (ticks and lice). each group was further divided into subgroups (families) to test whether biases existed in numbers of emerging and reemerging species at this level. the viruses were also divided according to genome type (e.g., negative singlestranded rna viruses). we examined aspects of host range, both for all pathogens combined and separately for each of the viruses, bacteria, fungi, protozoa, and helminths. first, we distinguished pathogen species according to whether they were known to be zoonotic, using the who definition "diseases or infections which are naturally transmitted between vertebrate animals and humans" ( ) . note that this definition includes pathogens for which humans are the main host and other vertebrates are only occasional hosts, as well as the opposite, but excludes purely human pathogens that recently evolved from nonhuman pathogens, e.g., hiv. we then compared the fraction of emerging or reemerging species that were or were not zoonotic across the major pathogen groups and within each group by family. second, for all zoonotic species we identified the types of nonhuman vertebrate host they are known to infect, using the following broad categories: bats, carnivores, primates, rodents, ungulates, and other mammals and nonmammals (including birds, reptiles, amphibians, and fish). we excluded vertebrate intermediate hosts of parasites with complex life cycles. host types were ranked by the number of zoonotic pathogen species associated with them, and rankings were compared by using spearman rank correlation coefficient. third, we obtained a crude index of the breadth of host range by counting the number of the host types that each pathogen species is known to infect: (i.e., not zoonotic), , , and or more. we compared the fraction of emerging and reemerging species across these classes. for the emerging and reemerging pathogen species, we identified the main factors believed to drive their increased incidence, geographic range, or both, by conducting a systematic review of the emerging diseases literature. we allocated these drivers to or more broad categories (table) . note that although we chose categories that we considered to be useful and informative for our immediate purposes, and which were similar to those listed elsewhere ( ) , this is inevitably a subjective procedure and alternative categorizations may be equally valid. we then ranked the drivers (by number of emerging and reemerging pathogen species associated with each) and compared the ranking of drivers for the major pathogen groups and for zoonotic versus nonzoonotic pathogens. for the zoonotic species, we distinguished those known to be transmissible between humans, allowing that this might be through an indirect route (e.g., a vector or an intermediate host), from those for which humans can only acquire infection (directly or indirectly) from a nonhuman source. for the transmissible zoonotic species, we further distinguished those that are sufficiently transmissible to cause major epidemics in human populations from those that cause only relatively minor outbreaks. this classification was intended to distinguish between pathogens with r > in humans from those with r < , where r is the basic reproduction number, i.e., the average number of secondary infections produced by a single primary infection introduced into a large population of previously unexposed hosts. direct estimates of r are unavailable for most zoonotic pathogens. throughout the study, we quantified associations as the relative risk (rr) and tested for statistical significance using a standard χ test (with correction for small expected values). although these statistical analyses are susceptible to bias introduced by related species (e.g., several species of hantavirus exist, most of which are zoonotic and many of which are regarded as emerging or reemerging), the analysis at the family level is an indication of the extent of any such bias. the survey of human pathogens produced a count of , human pathogen species, with ( %) species regarded as emerging or reemerging (online appendix, available at www.cdc.gov/ncidod/eid/vol no / - _app.htm). of all pathogen species, are viruses or prions, including ( %) regarded as emerging or reemerging. for bacteria, the counts were and ( %), respectively; for fungi, and ( %), respectively; for protozoa, and ( %), respectively; and for helminths, and ( %), respectively. these numbers differ slightly from those previously published ( , ) as a result of adjustments to taxonomies and the discovery of previously unknown pathogen species. clear differences were found between the pathogen groups (χ = . , p<< . ), with viruses greatly overrepresented among emerging and reemerging pathogens and helminths underrepresented. more than virus families contain human pathogens, with just , the bunyaviridae, flaviviridae, togaviridae, and reoviridae, accounting for more than half of the species affecting humans and, likewise, more than half of the emerging and reemerging species. overall, no significant difference was found between the largest families (pooling the remainder) in the fraction of species regarded as emerging or reemerging (χ = . , p = . ). nor were any significant differences found according to genome type, e.g., between rna and dna viruses (χ = . , p = . ) or between positive and negative single-stranded rna viruses (χ = . , p = . ). more than bacteria families contain human pathogens; the enterobacteria and the mycobacteria account for the most species and for the most emerging and reemerging species. overall, no significant difference was found between the largest families (pooling the remainder) in the fraction of species regarded as emerging or reemerging (χ = . , p = . ). numbers of species of emerging and reemerging fungi, protozoa, and helminths were too small for meaningful comparisons between families, but no indication was found that emerging and reemerging species are concentrated in any particular taxa. of the , human pathogen species, ( %) are known to be zoonotic. in comparison, of the emerging or reemerging pathogens, ( %) are known to be zoonotic. this corresponds to an rr of . and confirms the expectation that zoonotic pathogens are disproportionately likely to be associated with emerging and reemerging infectious diseases. this pattern varies somewhat across the different pathogen groups: for bacteria and fungi the association is strongest with rrs of . and . , respectively; for viruses and protozoa, no obvious association was found, with rrs of . and . , respectively; and for helminths (which are almost all zoonotic but very rarely emerging or reemerging), rr is . . however, the numbers involved are small (particularly for protozoa and helminths), and these differences were not statistically significant (χ = . , p = . ). all the defined host types are potential sources of zoonotic infections, but differences occurred in their importance (ranked by number of pathogen species supported) across viruses, bacteria, fungi, protozoa, and helminths and no type consistently dominates ( figure a) , although ungulates are the most important overall, supporting over species of human pathogen. emerging and reemerging pathogens show similar trends ( figure b) , with ungulates again the most important overall, supporting over species. in general, ranking of host types in terms of numbers of species correlates well both overall (r s = . , n = , p< . ) and individually for each pathogen group. the general impression is that the emerging and reemerging zoonotic pathogens are not unusual in the types of nonhuman hosts they infect. however, when the fraction of emerging and reemerging species is compared with the breadth of host range (as the number of host types other than humans), a pattern becomes apparent (figure ) . overall, the fraction tends to increase with host range: > % of pathogens with the broadest host ranges ( or more types of nonhuman host) are emerging or reemerging (exact p = . ). however, this trend does not hold for the protozoa and helminths (although the numbers for these groups are small). we identified main categories of drivers of emergence and reemergence and ranked these by the total number of pathogen species associated with them ( ranking of drivers across different categories of pathogen showed poor concordance (e.g., spearman rank correlation for bacteria vs. viruses, r s = . , n = , p = . ). the most striking discrepancies were as follows: ) the marked association of emerging or reemerging fungi with hospitalization, poor population health, or both; ) the greater importance of pathogen evolution and contaminated food and water and the lesser importance of international travel and changes in land use and agriculture for bacteria in comparison with viruses; ) the greater importance of changing land use and agriculture for zoonoses than for nonzoonoses. overall, most zoonotic pathogens are either not transmissible (directly or indirectly) between humans at all (i.e., humans are a dead-end host) or are only minimally transmissible. examples include rabies virus, rift valley fever virus, and borrelia burgdorferi (the agent of lyme disease). a small minority (≈ %) of pathogen species that are technically zoonotic are, in fact, spread almost exclusively from person to person (e.g., mycobacterium tuberculosis or measles virus) or can do so once successfully introduced from a nonhuman source (e.g., some strains of influenza a, yersinia pestis, or severe acute respiratory syndrome (sars) coronavirus). however, a substantial minority of zoonotic pathogens (about %, i.e., species) are capable of some person-to-person transmission but do not persist without repeated reintroductions from a nonhuman reservoir (e.g., e. coli o , trypanosoma brucei rhodesiense, or ebola virus). this pattern is fairly consistent across the major pathogen groups. humans are affected by an impressive diversity of pathogens; , pathogenic species of viruses, bacteria, fungi, protozoa, and helminths are currently recognized. of this total, ( %) pathogen species are considered emerging or reemerging. this number must be viewed with some caution, given that these terms are still used somewhat subjectively. more rigorous definitions of emerging and reemerging have been proposed ( , , ) , but these are difficult to apply universally because they require long-term data on distributions and incidences which are available for only a small subset of infectious diseases (e.g., malaria [ ] and tuberculosis [ ] ). moreover, the counts of emerging and reemerging pathogen species reported here are subject to ascertainment bias. despite these caveats, our results suggest that pathogens associated with emerging and reemerging diseases share some common features. first, emerging and reemerging pathogens are disproportionately viruses, although they are not disproportionately different kinds of viruses. numerically, rna viruses dominate, comprising % of all emerging and reemerging pathogens. rna viruses are also prominent among the subset of emerging pathogens that have apparently entered the human population only in the past few decades, such as hiv or the sars coronavirus ( , ) . a possible explanation for this observation is that much higher nucleotide substitution rates for rna viruses permit more rapid adaptation, greatly increasing the chances of successfully invading a new host population ( , ) . second, emerging and reemerging pathogens are not strongly associated with particular nonhuman host types, although emerging and reemerging pathogens more often are those with broad host ranges that often encompass several mammalian orders and even nonmammals. this pattern is consistent across the major pathogen groups. the determinants of host range in general remain poorly understood, but among viruses for which the cell receptor is known, an association exists between host range and whether the receptor is phylogenetically conserved (as measured by the homology of the human and mouse amino acid sequences) ( ) . emerging and reemerging pathogens have been likened to weeds ( ) , and that the associations reported above are likely reflecting underlying "weediness," that is, a degree of biologic flexibility that makes certain pathogens adept at taking advantage of new epidemiologic opportunities. this characteristic seems to be reflected in the broad range of drivers of the emergence or reemergence of pathogens, ranging from changes in land use and agriculture, through hospitalization to international travel. although some drivers are numerically more important than others, the overall impression is that pathogens are exploiting almost any figure . relationship between breadth of host range (as number of nonhuman host types, as listed in figure ) and the fraction of pathogen species regarded as emerging or reemerging. a total of change in human ecology that provides new opportunities for transmission, either between humans or to humans from a nonhuman source. even if a pathogen is capable of infecting and causing disease in humans, most zoonotic pathogens are not highly transmissible within human populations and do not cause major epidemics. the possible magnitude of an infectious disease outbreak is related to the basic reproduction number, r ( figure ). for pathogens that are minimally transmissible within human populations (r close to ), outbreak size is determined largely by the number of introductions from the reservoir. for pathogens that are highly transmissible within human populations (r >> ), outbreak size is determined largely by the size of the susceptible population. for pathogens that are moderately transmissible within human populations (corresponding to r ≈ ), notable outbreaks are possible (especially if multiple introductions occur), but the scale of these outbreaks is very sensitive to small changes in r . in other words, small changes in the nature of the host-pathogen interaction can lead to large increases (or decreases) in the scale of the public health problem (figure ). such pathogens may be likely sources of emerging infectious disease problems in the future. however, we currently have no way of predicting whether a novel human pathogen will behave like rabies (frequently introduced into the human population, but not capable of causing major epidemics) or hiv (probably rarely introduced, but capable of causing a global pandemic). in conclusion, this study suggests that biologic and epidemiologic correlates of pathogen emergence or reemergence may be identified. however, the most striking feature of emerging and reemerging pathogens is their diversity (online appendix). for this reason, surveillance and monitoring of infectious disease trends may have to be broadly targeted to be most effective. given that threefourths of emerging and reemerging pathogens are zoonotic, in many cases this targeting might usefully be extended beyond at-risk human populations to include populations of potential animal reservoirs. emerging infectious diseases • www.cdc.gov/eid • vol. , no. , december figure . expected relationship between outbreak size (as fraction of the population affected) and key epidemiologic parameters: i is the number of primary cases of infection introduced into the human population from an external source such as a zoonotic reservoir (increasing in the direction indicated); r is the basic reproduction number, a measure of the transmissibility of the infection with the human population (see text). the curves are obtained from a modified version of the kermack-mckendrick equation and show that expected outbreak size is particularly sensitive to small changes in i or r when r is close to . examples of zoonotic pathogens with r > , r < and r close to are shown. rivf, rift valley fever virus. (reprinted with permission from [ ] ). risk factors for human disease emergence population biology of multi-host pathogens diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence factors in the emergence of infectious diseases microbial threats to health: emergence, detection, and response emerging infectious diseases world health organization. emerging diseases the promed-mail archives the microbial rosetta stone database: a compilation of global and emerging infectious microorganisms and bioterrorist threat agents international committee on the taxonomy of viruses. index virum topley & wilson's microbiology and microbial infection foundations of parasitology a summary of taxonomic recently approved by ictv zoonoses: second report of the joint who/fao expert committee. geneva: the organization population biology of emerging and reemerging pathogens -preface terrestrial animal health code- . general definitions the global distribution and population at risk of malaria: past, present and future the growing burden of tuberculosis: global trends and interactions with the hiv epidemic evolvability of emerging viruses emerging pathogens: the epidemiology and evolution of species jumps population biology of emerging and re-emerging pathogens emerging infectious pathogens of wildlife we thank louise taylor and sophie latham for their work on the original database and ben evans for his contribution to the updated database.dr woolhouse is professor of infectious disease epidemiology in the centre for infectious diseases at the university of edinburgh. his research interests include foot-and-mouth disease, e. coli o , scrapie, and sleeping sickness. he is an advisor to the uk government on issues relating to infectious disease epidemiology.dr gowtage-sequeira is a postdoctoral research assistant in the division of animal health and welfare at the university of edinburgh. her doctoral research, for the institute of zoology in london, was on the epidemiology of viral infections of canids in namibia. she is currently studying the ecology of wild dogs in eastern kenya. key: cord- -zd v b authors: kawashima, kent; matsumoto, tomotaka; akashi, hiroshi title: disease outbreaks: critical biological factors and control strategies date: - - journal: urban resilience doi: . / - - - - _ sha: doc_id: cord_uid: zd v b disease outbreaks remain a major threat to human health and welfare especially in urban areas in both developed and developing countries. a large body of theoretical work has been devoted to modeling disease emergence, and critical factors that predict outbreak occurrence and severity have been proposed. in this chapter, we focus on biological factors that underlie both theoretical models and urban planning. we describe the sars – pandemic as a case study of epidemic control of a human infectious disease. we then describe theoretical analyses of disease dynamics and control strategies. an important conclusion is that epidemic control will be strongly dependent on particular aspects of pathogen biology including host breadth, virulence, incubation time, and/or mutation rate. the probability, and potential cost, of future outbreaks, may be high and lessons from both past cases and theoretical work should inform urban design and policy. interdisciplinary collaboration in planning, swiftness of information dissemination and response, and willingness to forgo personal liberties during a crisis may be key factors in resilience to infectious disease outbreaks. infectious diseases pose an ever-present danger to human societies. despite tremendous advances in medical care, roughly one quarter of worldwide human deaths are attributed to infectious and parasitic disease (mathers et al. ) . several seemingly unalterable aspects of urban life, including long-distance travel and dense human contact networks, facilitate outbreaks from both known and newly evolved pathogens. epidemics are defined as widespread occurrences of infectious disease in a community at a particular time, and the th century bubonic plague, or "black death", was the most devastating epidemic in human history (benedictow ) . death rates were as high as - % in europe, africa, and asia from a disease caused by a bacterial infection (yersinia pestis) that persists in rodent populations and is transmitted by fleas to humans. close contact between humans and rats and worldwide travel contributed to the global impact of bubonic plague which appears to have originated in asia and traveled to europe via trade routes (especially rat-infested ships). the most destructive modern pandemic was the influenza that infected one third of the world's population (about million) and killed - million between january and december (taubenberger and morens ) . "spanish influenza", as the disease was named, is caused by the h n virus which is endemic in pigs and birds and often transitions into human populations. the lethality of the strain was high and showed an unusual relationship between lethality and patients' age: % of deaths were in the - age group which is the opposite pattern for milder flu strains (higher mortality among the very young and the aged). this partly reflected the impact of wwi where contagion was passed among troops both in training facilities as well as during warfare. however, the strain also had an unusual and lethal property; virulence was enhanced by a human immune over-reaction called a "cytokine storm" which causes the lungs to fill with liquid. some important aspects of the epidemic were: a deadly pathogen arose from a jump from animal to human (close between-species interactions were important in the origin of the virus), a few mutations were sufficient to confer strong lethality for the virus, and human travel allowed rapid spread (close quarters and massive troop movements helped to spread the virus and allowed new mutations to spread quickly). this chapter will focus on biological factors that are relevant for understanding and controlling epidemics. we will briefly describe some pathogens that cause human disease and their transmission mechanisms before analyzing the sars - epidemic as a case study of a modern urban epidemic. disease models will be discussed with a goal of determining how human societies can prepare to minimize the impact of future disease outbreaks. infectious diseases can be classified into two broad categories based on their pattern of transmission (table ) . "long-range" infectious diseases are infections that do not require close contact for transmission. for example, water-borne diseases, such as cholera, can rapidly spread throughout the community when the supply of drinking water becomes contaminated with the pathogen vibrio cholerae through poor sanitation or hygiene practices. food-borne infections follow a similar transmission pattern to water-borne diseases. transmission through contaminated food and water is also known as "fecal-oral transmission" because fecal matter is often the source of contamination while oral ingestion is the primary route for infection (mount sinai hospital ) . diseases transmitted by an animal vector, such as bubonic plague, are also considered long-range infections because a vector facilitates the spreading of the pathogen and direct contact is not necessary. one interesting aspect of some vector-borne infections is that direct contact with an infected individual cannot transmit the infection without the help of the vector. for example, dengue fever, caused by a mosquito-borne virus, can only be transmitted through the bite of an infected mosquito (us centers for disease control and prevention ). in contrast, plague, caused by bacteria living in fleas of rodents, is primarily transmitted through flea bites, but contact with contaminated body fluids like blood can also lead to plague bacterial infection (us centers for disease control and prevention a). in general, fecal-oral and vector-borne diseases are infections transmitted through an environmental (water, food) or a biological (animal) carrier that extends transmission range to large distances, but other routes are also possible depending on the specific pathogen. compared to long-range diseases, "short-range" infectious diseases are infections that transmit over limited distances and may require close or direct physical contact with an infectious individual. examples of short-range infections are pathogens that infect via contaminated airborne particles or expectorated droplets, and diseases that require contact with skin or bodily fluids such as blood or semen. infections capable of airborne transmission have the widest range among short-range infections and are caused by pathogens that spread through minute solid or liquid particles suspended in the air for an extended period of time (mount sinai hospital ) . in addition, the pathogen must be resistant to desiccation to remain viable for long periods of time outside its host. respiratory diseases are commonly believed to spread via airborne transmission of contaminated particles expectorated from coughing and sneezing. however, many respiratory pathogens do not have the capacity to withstand dry environments. instead, these pathogens transmit via "droplets"-expectorated moisture particles that are too big to indefinitely remain suspended in the air-to ensure ample moisture while outside the host. transmission occurs when contaminated droplets from an infected individual come in contact with surfaces of the eye, nose, or mouth. this mode of transmission is called "droplet contact". although diseases spreading via droplet contact have a more limited range than truly airborne infections, in the later sections, we will show how environmental factors can extend the range of droplet transmission. finally, diseases that transmit via direct contact generally have the most limited transmission range and some have stringent requirements for transmission. in the case of ebola, the disease is transmissible only via direct exposure of broken skin or mucous membranes with contaminated body fluids like blood, urine and semen, and excretions such as vomit and feces (us centers for disease control and prevention c). sexually transmitted diseases like hiv/aids are a special form of direct contact infection that requires sexual intercourse or sharing contaminated needles for exposure (us centers for disease control and prevention b). thus, short-range infections are characterized by some dependence on distance for infection and can be transmitted directly without a carrier. distinguishing between these two classes is important because measures to alleviate and control the spread of long-range infections are not applicable for short-range cases and vice versa. for instance, targeting the carrier or vector of the disease to control the spread of long-range infections (e.g., decontaminating or blocking off access to contaminated water or food) and reducing exposure to vectors of the disease, are irrelevant for mitigating the spread of short-range infections. in contrast, measures to control short-range diseases such as limiting person-to-person contact and imposing quarantine procedures do little to help alleviate the spread of water-borne or vector-borne illnesses. thus, identifying the mode of transmission is crucial to controlling the spread of any contagious infection. however, we will show that the distinction between long-and short-range transmissions is not always clear-cut. in this chapter, we focus on the emergence and spreading of severe acute respiratory syndrome or sars; the first worldwide pandemic in the age of globalized air travel and telecommunications. through theoretical analyses and data gathered from the epidemic, we examine how globalization exacerbates the problem of containing epidemics and show how urban environments can be especially prone to epidemics. the emergence and control of the sars epidemic is extensively documented. research on both the origin and epidemiology of the outbreak as well as the biological underpinnings of the disease making them excellent cases to determine methods to enhance urban resilience to epidemics. the history of the - global outbreak of severe acute respiratory syndrome (sars) provides key lessons on biological and policy factors that should be of general importance in designing resilient cities. we will summarize the history of the epidemic, with a focus on biological factors, before our discussion of disease models. according to the world health organization (who), over worldwide sars cases and over deaths occurred in different countries, mostly over a period of about four months (kamps and hoffmann ) . the severe, "atypical" pneumonia originated in guangdong province in southern china in mid-november . most of the early cases appear to have occurred among those who kill and sell animals and meat as well as food preparers and servers (breiman et al. ) . by mid to late january , the disease began to spread rapidly within the province, but a combination of symptoms difficult to distinguish from pneumonia (fever, dizziness, muscle soreness, coughing) and government policy to discourage coverage delayed the reporting of the epidemic until february . the initial communication reported cases (including > healthcare workers) and mortalities, but claimed that the epidemic was under control (enserink ) . the role of "superspreaders" and amplification in hospitals remained characteristics of sars as it spread to a worldwide epidemic. the first of several superspreading (generally defined as ten or more transmissions from a single infected individual) events occurred in hong kong on february , (braden et al. ). the index case was a physician from guangdong who stayed at the hotel metropole. the physician had treated sars patients in guangdong (although the disease was still unrecognized) and showed symptoms before his trip. he stayed only one night at the hotel before being hospitalized with severe symptoms but the short stay was sufficient to spread the infection to or more of the guests from the same floor of the hotel as well as a hong kong resident who visited one of the guests. eventually, over (almost half) of the documented sars cases could be traced to this "index" case. remarkably, there was no known direct contact in most of the transmissions among the hotel guests and visitors. the hong kong resident who visited a friend in the hotel subsequently infected over others at the prince of wales hospital in hong kong. others were business/holiday travelers who spread the pathogen to canada, vietnam, and singapore. as we will discuss below, this high transmission rate with little close contact in the metropole hotel remains mysterious. rapid recognition of a new epidemic was aided by a who disease expert, carlo urbani, who was asked to examine patients in a hanoi hospital. the affected included one of the metropole guests and roughly hospital staff who became affected not long after his admission. urbani recognized a severe, and possibly new, disease and warned who headquarters as well as the hospital and vietnam government before contracting, and eventually dying from, the disease (bourouiba et al. ) . response time is a critical parameter in epidemic control and his efforts played a large role in the effort to subdue the epidemic. who designated a new disease, "severe acute respiratory syndrome" (sars), on march and issued a global health alert on march followed by an emergency travel advisory on march . the etiological agent of sars was later discovered to be a novel coronavirus and was named sars-associated coronavirus (sars-cov). this discovery, in late march , came as a surprise to disease experts as previous human coronaviruses were only known to cause mild illness. in animals, related viruses were known to cause fatal respiratory as well as neurological diseases but coronaviruses are usually highly species-specific (kamps and hoffmann ) . forensic analysis of the metropole hotel in late april revealed physical components of sars in the common areas of the th floor including the corridor and elevator hall. however, no bacteria were found inside the guest rooms of the infected guests (the ventilation systems employed positive pressure within the guest rooms so that air was not shared among rooms). respiratory droplets, or suspended small particle aerosols generated by the index case-patient, are the most likely transmission mechanism (braden et al. ) . sars and other respiratory infections are considered to undergo short-range (approximately m) transmission via pathogen-infected droplets from host coughing or sneezing. such transmission requires "close contact", physical proximity between infected and susceptible individuals who can be infected when large droplets spray enter their bodies via air or touch. however, minute droplets or even solid residues that can arise via evaporation (droplet nuclei) may allow potential indirect and/or long-range transmission (bourouiba et al. ) . for example, contaminated gas clouds that form during coughing/sneezing may have carried the pathogen and extended its transmission range, removing the distinction between droplet contact and airborne modes of transmission. aerosol transmission probably caused high infection rates in an airline flight (air china ) from hong kong to beijing in which a single -year old individual infected at least others (olsen et al. ) . this feature of the disease may be highly relevant for medical and urban policy. long-range aerosol/nuclei transmission does not require direct contact between infected and uninfected individuals and can greatly elevate the number of "contacts" for a given infected individual. interestingly, genetic analysis showed that several sars strains entered hong kong, but only the hotel metropole index case was associated with the subsequent global outbreak (guan et al. ) . a related superspreading event occurred at a crowded high-rise residence, the amoy gardens, in hong kong. many of the infected individuals inhabited vertically placed apartments (in contrast to transmissions on a common floor at the metro hotel case). sanitary drainage fixtures that were malfunctioning and allowing air and sars-contaminated aerosols to flow back into resident bathrooms may have been the main driver of infection spread in the condominium (stein ) . the superspreader was likely a medical patient undergoing treatment for a kidney problem including hemodialysis, a medical treatment that inhibits immune capacity (stein ) . the index case carried a high viral load and suffered from diarrhea. an important feature of this event was again, a lack of direct contact between the spreader and the individuals he infected, and the "opportunity" for the pathogen to be exposed to a large number of individuals through airborne transmission (yu et al. ) . at the amoy gardens, more than individuals showed symptoms of sars almost simultaneously. high rates of hospital (nosocomial) transmission were an important and disturbing characteristic of the sars outbreak. the large fraction of infections among healthcare workers probably reflects a combination of contact from respiratory secretions from patients who were at a highly contagious stage (critically ill individuals also were the most infectious) as well as from medical procedures that inadvertently generated aerosol contamination. a single patient appears to have transmitted infections to over hospital staff in a span of two weeks at the prince of wales hospital in hong kong (see below). two other superspreader events occurred in hospitals in other countries (braden et al. ) . one infected patient (the son of one of the hotel metropole guests) infected over cases among patients, visitors, and healthcare workers at the acute care hospital in toronto, canada. finally, although taiwan instituted strict port entry screening and isolation of potentially exposed travelers entering the country, there was an outbreak in the ho ping hospital which spread into the community. in spite of a lock-down quarantine of over people in the hospital (included a large fraction of uninfected individuals), over cases emerged before the outbreak was contained. the initial rapid spread of sars caused widespread concern and panic and the epidemic seemed unstoppable. however, the disease was eventually contained within several months through efforts coordinated by the who. although advances in biomedical science and cooperative efforts among laboratories played key roles in isolating the infectious agent, "classic" epidemiological practices of patient isolation (separation of infected individuals from the general population), contact tracing, and large-scale quarantine (isolation of non-symptomatic individuals who have had contact with the infectious agent) were the main elements that halted the epidemic ). the - sars pandemic was caused by a moderately transmissible viral infection that produced . new cases for every infection (riley et al. ) and yet it spread to over countries across three continents potentially exposing tens of thousands of people in the span of only a few months. several studies have shown that the vast majority of infected cases had very low infectivity and that a few outliers were responsible for a disproportionate number of new infections riley et al. ; lipsitch et al. ; wong et al. ). in fact, riley et al. ( ) and lipsitch et al. ( ) found that early in the epidemic, an infected individual would only produce approximately three new infections when outliers are excluded. in singapore, % of the first probable sars cases showed no evidence of transmitting the infection yet cases appeared to have transmitted the disease to or more individuals (lipsitch et al. ) . shen et al. ( ) found a similar pattern in beijing where out of the confirmed cases did not infect others whereas four cases were responsible for infecting eight or more. the rapid spreading of sars despite only moderate average infectiousness has revived interest in the concept of superspreading events and heterogeneity in pathogen transmission. the transmission potential of an infectious disease is often described by the parameter r, the average number of new infections that infected cases produce over the course of their infection. r is the transmission potential of an infected individual within an otherwise completely susceptible population (dietz ) . however, population-based summary statistics may obscure individual variation of infectiousness and other types of heterogeneities. woolhouse et al. ( ) have shown that heterogeneities in infectiousness exist such that only % of the host population contributes at least % of a pathogen's transmission potential. these individuals who significantly transmit more than the average are called superspreaders. in hong kong, apart from the incident at hotel metropole, at least two large clusters of infection were attributed to superspreading events (riley et al. ) . data from the sars pandemic showed the effect that superspreaders and superspreading events could have on the trajectory of the epidemic. given their crucial role in intensifying an outbreak, we review the risk factors that facilitate superspreading events. co-infection and the presence of a comorbid disease could be risk factors for turning infected individuals into superspreaders (stein ) . studies on hiv/aids transmission showed that co-infection with another sexually transmitted pathogen increased the urethral shedding of hiv in infected individuals. moss et al. ( ) demonstrated that urethral hiv infection is associated with gonococcal infection and treatment for urethritis may reduce the risk of hiv transmission. in the case of sars, peiris et al. ( ) reported that other viral respiratory pathogens such as human metapneumovirus were detected in confirmed sars cases. in addition, the index case in the prince of wales hospital superspreading event was described to have a "runny nose" , an uncommon symptom for a lower-respiratory tract infection such as sars. these observations have led to the hypothesis that co-infection or presence of a comorbid condition could endow an infected individual with characteristics or behaviors that increases their infectiousness (bassetti et al. ) . for example, rhinovirus, the major cause of common colds, can cause swelling of nasal tissues that can elevate airflow speed and contribute to aerosol production (sherertz et al. ) . rhinovirus co-infection with more serious, but less transmissible respiratory ailments, such as sars, could be an important factor contributing to high infectivity. environmental factors also play an important role in facilitating superspreading events (stein ) . in the sars superspreading event at the prince of wales hospital, the index case was placed on a nebulized bronchodilator four times daily for one week (kamps and hoffmann ) . nebulized bronchodilators are often used to deliver drugs to the lungs of respiratory patients but may have inadvertently aerosolized the virus and left infected droplets in the immediate surroundings leading to extensive dissemination of the pathogen (tomlinson and cockram ) . tracheal intubation, which involves placing a flexible tube into a patient's windpipe to maintain an airway to deliver drugs, may also have inadvertently spread sars within hospitals. patients often emit respiratory secretions during the procedure. an outdated ventilation system and overcrowding likely also contributed to the spreading of the virus at the prince of wales hospital (riley et al. ; tomlinson and cockram ) . through a case-control study of hospitals treating sars patients, yu et al. ( ) confirmed overcrowding as one of the general risk factors of hospital-based sars superspreading events. the case-control study performed included wards in hospitals in guangzhou and wards in five hospitals in hong kong and showed that the main risk factors included closely arranged beds (less than m apart), a workload of more than two patients per healthcare worker, hospital staff that continued working despite experiencing symptoms of the disease, and lack of washing or changing facilities for staff. despite the explosive growth and global distribution of the sars outbreak, the pandemic was largely contained through isolation and quarantine, increasing social distance, and social behavioral adjustments (bell and world health organization working group on prevention of international and community transmission of sars ). isolation and quarantine were shown to significantly interrupt transmission of sars in several countries including hong kong (riley et al. ) , china (pang et al. ) , singapore (lipsitch et al. ) , taiwan (twu et al. ) , and canada (svoboda et al. ). in general, symptomatic cases were immediately placed in isolation while contacts of confirmed infected cases were placed in some form of quarantine. in some cases, contacts were not immediately confined but instead were monitored for the disease and isolated only when symptoms emerged. confinement was usually at home but designated facilities were available in countries like taiwan (twu et al. ) . in some cases, individuals under quarantine were allowed to travel with the permission from the local health authorities provided they wore masks and refrained from using public transportation or visiting crowded places. to further reduce the chance of transmission, hong kong and singapore also closed schools and public facilities, and canceled mass gatherings to "increase social distance". people were also required to wear masks when using public transport, entering hospitals, or in jobs where interacting with numerous people is unavoidable such as in restaurants (bell and world health organization working group on prevention of international and community transmission of sars ). the concerted effort has been marginally associated with the rapid reduction of new sars cases in several countries. however, because of the simultaneous introduction of these measures, it is difficult to evaluate the effectiveness of each. several characteristics of the infectious agent were important factors in controlling the sars epidemic. the incubation period from contact with the infectious agent to onset of symptoms was, on average, . days. importantly, peak infectivity coincided with clinical symptoms and often required an additional days or more ). thus, infectious individuals tended to be hospitalized before peak transmissibility. in addition, the two-week interval from exposure to high infectivity gave epidemiologists critical time to perform contact tracing to identify and quarantine potentially infected individuals before they reached high infectivity. this feature, in combination with moderate transmission rates (except in special cases), contributed to making sars a relatively controllable outbreak. in the next section, we present current theories on the emergence and spreading of epidemics and review the theoretical underpinnings behind control measures used to contain outbreaks. we briefly highlight different mathematical models used to describe epidemic dynamics in populations. we explain the factors that govern the emergence and transmission of diseases as well as the evolution of pathogens that cause them. finally, we examine how control measures such as isolation, quarantine, and vaccination mitigate the spread of infections. mathematical models have played an important role in our understanding of disease propagation. if biological factors can be accurately incorporated, such models may have predictive power to evaluate control strategies and guide policy. a key parameter in epidemic models is the total number of new infections that arise from a single affected host, the reproduction number, r. this value determines the outbreak potential of the infection; if r = , the infection will be maintained at a constant level (if we ignore random effects). r > leads to disease spread and r < predicts eventual extinction. however, r is not an intrinsic property of the pathogen. variability of the reproductive number across pathogens, hosts, and environments over time must be understood to accurately model disease. in the following three subsections, we discuss theoretical results on three important aspect of disease outbreak: ( ) the effect of "superspreaders" on the probability of outbreak, ( ) the impact of control strategies such as isolation and quarantine, and ( ) factors that affect the evolution of pathogen virulence. the - sars epidemic was characterized by the large impact of "superspreaders" on disease propagation. in theoretical models, superspreaders can be treated as individuals with large number of connections to other individuals. individual-based simulations incorporating network structures can efficiently address this topic and, in this subsection, we introduce three theoretical studies focused on the effect of network structure on disease outbreak. lipsitch et al. ( ) studied the effect of superspreaders on outbreak probability using the estimated parameters from the sars outbreak in singapore. the authors first estimated the distribution of the parameter r, which expresses the number of new infections from an infected host. probabilities of outbreak (persistence of initially introduced pathogen lineages) were determined for r distributions with a fixed mean but differing in variance. the authors found that large variance in r distribution greatly decreased the probability of outbreak (fig. ) . contrary to the expectation of the importance of superspreaders, their result showed that distributions strongly clustered around the mean had higher probabilities of outbreak than distributions that included superspreaders (right-hand tail outliers). one reason of this apparent inconsistency might be the assumption of a fixed mean r. under this assumption, increased variance in the r distribution increases both the numbers of individuals with extremely high r and low r. individuals with low r are essentially "dead ends" in disease infection and high numbers of such individuals will decrease outbreak risk. a similar result was obtained in meyers et al. ( ) . this study also focused on the case of sars outbreak in asian countries and used parameters estimated from the case study in individual-based simulations. meyers and co-workers examined differences in the probability of outbreak among three different networks among individuals. in the first network, termed "urban", many individuals have numerous contacts at public places including schools, hospitals, shopping centers and workplaces, and have more limited numbers of contacts at their home. the second network was a power law network, in which the distribution of the number of connections has a long right-hand tail. in such a distribution, a small fraction of people have large numbers of connections but most people have only a few connections. the third network was a poisson network, in which the majority of the people have numbers of connections close to the mean number. if the existence of superspreaders increases the probability of outbreak, then power law networks should show the highest outbreak probability. however, similar to lipsitch et al. ( ) , power law networks showed the lowest probabilities of outbreak. the reason might be similar to what we discussed above; in a power law network, the numbers of individuals with extremely small numbers of connection are elevated compared with the other two networks. pathogens cannot spread if they infect such individuals and will go extinct before they have a chance to infect superspreaders. fig. theoretically estimated probability that a single introduced pathogen persists after infinite time under a markov process with different mean (e) and variance (v) in the r distribution. in lipsitch et al. ( ) this persistence probability is considered as probability of an outbreak. modified from lipsitch et al. ( , fig. a) the two studies above indicated reduced probabilities of outbreak for populations that include superspreaders, but this conclusion may be strongly sensitive to model assumptions. networks with more total connections (including superspreaders) may realistically model urban environments (this relaxes the assumption of constant mean connectedness). fujie and odagaki ( ) modeled superspreaders as individuals with higher infection rates (strong infectiousness model) or more connections including connections with distant individuals (hub model). they calculated the probability of outbreak under different fractions of superspreaders in a population and showed that, as the fraction of superspreaders increases, the probability of outbreak increases greatly (fig. ) . they also analyzed several features of outbreaks like speed of disease spread and infection path between the two models and suggested that the hub model is consistent with data from the sars outbreak in singapore. these contrasting results highlight the need to validate model assumptions for applications to human society. higher outbreak probabilities with larger numbers of connections may seem obvious but this may be a realistic scenario for human society. a key issue is whether the number of connections of one person statistically affects that of others in human society. if not, the comparison between different fraction of number of superspreaders like fujie and odagaki ( ) would fig. theoretically estimated "percolation" probability of a single introduced pathogen under different fraction of superspreaders and population density in a hub model. in fujie and odagaki ( ) this percolation probability of percolation theory, in which a pathogen that has infected an individual in the bottom of × grid finally reaches an individual in the top of the grid, is considered the probability of an outbreak. as density becomes lower, distance between individuals becomes longer. the results for different fraction of superspreaders (λ) are shown in different markers. modified from fujie and odagaki ( , fig. ) more realistically predict the effect of superspreaders on the probability of outbreak. however, if higher number of connections of one person necessitates reduced numbers for others, the results in lipsitch et al. ( ) and meyers et al. ( ) could be more applicable for human society. in either case, models should focus on both outbreak probability as well as the nature (explosiveness) of disease spread. lloyd-smith et al. ( ) demonstrated that many previous human epidemics appear to have spread through superspreaders (although not to the same extent as sars). they showed that, although pathogen extinction probability increases with variance in reproductive number, populations with superspreaders experienced more rapid infection spread in cases of pathogen survival. under their model, host populations may suffer greatly from improbable epidemics. the first step to control the rise of any infectious disease is to understand how it transmits between hosts. often, we imagine these infections as readily communicable illnesses that can be caught by even the most fleeting contact. but as we have shown, exposure and transmission depends on the route the infectious disease pathogen takes. this means that some diseases can be transmitted even without direct or close contact with an infected individual. we have also shown how particular conditions can make a close-range disease transmit over extended distances, as is the case with sars transmission in the amoy gardens condominium complex. aside from mode of transmission, the timing between infectiousness and showing symptoms of the disease is another crucial factor to consider. an infectious disease is an illness caused by the presence of a pathogen within the host as well as the host's response to the invading pathogen. upon entry into the host, the pathogen begins to increase its numbers by redirecting resources to itself. after a certain time, its presence and the damage it has done to the host raises an internal host response to thwart the infection. it is at this stage of the infection that overt symptoms appear and the infection can be observed. the time elapsed between exposure to the pathogen and observing the initial signs and symptoms of the disease is called as the "incubation period" of the disease. the length of the incubation period varies among diseases and is affected by several factors such as dose and route of infection, and host susceptibility and ability to respond to the pathogen. because of these considerations, incubation period is described as a range of values depicting how short or how long it takes before an infection would show symptoms. during this period, the infected individual may or may not be contagious depending on the type of disease and the individual's health state. the disparity between the time we observe the symptoms of the infection and consider an individual ill and the time the individual is contagious are important aspects to consider in modeling as well as in prescribing infection control measures. the timings vary widely depending on the infectious disease (fig. ) . in the simplest scenario, the entire time an infected individual is contagious occurs after the first symptoms of the disease and ends well before the symptoms disappear. a completely overlapping timing where all symptomatic individuals are infectious would simplify identification and make control measures more effective. this timing pattern can be easily modeled by assuming that newly infected individuals simultaneously start to cause new infections to other individuals. and because the disease spreads specifically through a single class of individuals, control measures can simply identify symptomatic individuals to prevent new infections. in the case of sars, peak infectiousness occurs - days following the onset of disease symptoms and correlates with viral load over the course of the infection ). many believe this pattern helped contain the sars pandemic (chau and yip ; diamond ; fraser et al. ) despite exponential growth of the epidemic that quickly spread to multiple continents. in contrast, diseases such as hiv/aids have completely different infectious and symptomatic periods. the first signs of aids do not appear until the infecting pathogen has significantly damaged the host yet the infected individual is contagious throughout the asymptomatic phase and peak infectivity occurs before the onset of symptoms ). modeling diseases with disconnected infectious and symptomatic periods requires splitting the "infectious" class into "asymptomatic infectious" and "symptomatic infectious" classes to more accurately reflect the clinical characteristics of the disease. though sars and hiv/aids have significantly different timing patterns, the relationship between peak infectivity and symptomatic period is clear. however, some diseases exhibit partially overlapping contagious and symptomatic periods that make their outbreaks more difficult to stop. identifying the precise period that infected individuals are contagious is difficult because the values are affected by anderson et al. ( ) numerous factors such as susceptibility of the host, mechanism of infection, and immune response (baron ) . individual variation in incubation periods further complicates the problem. in dealing with diseases that exhibit partially overlapping periods such as pandemic influenza, it is best to rely on conservative measures that consider both exposed and likely infected individuals as targets of containment measures. note that it is possible to harbor an infection yet not show any signs or symptoms of the disease. called a "subclinical infection", this asymptomatic state may be a result of the pathogen infection strategy and the host's ability to tolerate an infection instead of purging it (baron ) . asymptomatic cases that are infectious can help spread the contagion despite strict control measures by being misclassified as uninfected individuals. asymptomatic cases are usually discovered by chance or by reviewing epidemiological data after an epidemic (baron ) . modeling asymptomatic cases requires adding an "asymptomatic infectious" class that is capable of exposing and transmitting the disease. containing the spread of an infectious disease suspected to have a high proportion of asymptomatic infected individuals is difficult but procedures such as contact tracing may reveal some of these asymptomatic carriers and quarantining of exposed and high-risk individuals can minimize their impact. most emerging infections have no available vaccine or treatment. thus the only way to control the spread of these diseases is to prevent exposure and further transmission. isolation and quarantine are two control measures that help block transmission by isolating the individuals who have, or may have, the contagious disease. "isolation" describes separating sick individuals (symptomatic) from people who are not sick (naïve) while quarantine pertains to the practice of separating and restricting the movement of asymptomatic individuals who may have been exposed to the disease to see whether they become sick. these control measures aim to progressively reduce the number of new secondary infections until the disease is eradicated from the population. formally, we can measure the effect of isolation and quarantine by taking a survey of new infected cases and deriving the basic reproduction number r of the infectious disease for each step of the outbreak. without any intervention, r is expected to eventually decrease as the number of susceptible individuals decreases in a finite population without migration. however, by the time the rate decreases to r < , a large proportion of the population has already been infected with the disease. by "removing" potentially infected individuals from the population, isolation and quarantine can more rapidly decrease r below by reducing the incidence of the disease, leading to fewer new infected cases capable of transmitting the infection. isolating symptomatic individuals prevents new cases by separating individuals spreading the pathogen from the host population. given a clearly defined set of symptoms to diagnose the disease, this strategy is intuitive and straightforward to implement from a public health point of view. a precise case definition also reduces misdiagnoses and prevents unnecessary isolation of non-target cases. however, many diseases share symptoms and may occur in combination with other infections so case definitions are not always precise. in the sars epidemic, infected individuals showing atypical symptoms were a major source of transmission, partly because co-infection may have elevated transmission rates (kamps and hoffmann ) . modern biomedical research may serve to quickly identify new pathogens and providing diagnostic tests may be the most important function of initial research (vaccines and treatments generally require months or years and may not be helpful for new diseases). isolating symptomatic individuals is most effective if peak infectivity occurs after observing the first symptoms of the disease and transmission only occurs in symptomatic cases ). while diseases like sars have shown such properties, other infections such as influenza appear to be transmissible even prior to showing overt symptoms. when peak infectivity occurs before the onset of symptoms, quarantine for symptomatic individuals may have little impact on dampening the spread of the infection . even for infectious diseases that transmit only after symptoms emerge, infected individuals may not immediately practice self-isolation or report to a healthcare facility. during the lag time between diagnosis and isolation, the pathogen can still spread to susceptible hosts undermining isolation as a way to control the spread of the infection. on the other hand, quarantining individuals that have been exposed to the disease addresses the shortcomings of isolation as a control measure. identifying exposure is dependent on how the pathogen spreads from one host to another. if the pathogen transmits via airborne droplets, then people present in the same room with an infected individual are considered "exposed". however, if the pathogen spreads only through sexual contact, then only individuals who have had sexual relations with the infected case are considered exposed. when the transmission mechanism is unknown, scenarios such as airborne transmission or via physical contact that lead to the most conservative outcome may be used instead. because the criteria to select individuals are independent of disease status, this strategy sacrifices sensitivity but works regardless of timing of infectivity and does not suffer from the lag time problem. such a conservative strategy is well suited for emerging infections, especially when the mechanisms of transmission and pathogenesis have yet to be revealed. in a perfect quarantine, all exposed individuals are expected to undergo quarantine regardless of whether they develop the disease or not, and during the quarantine period, exposed individuals do not transmit the disease. however, tracing all contacts is often problematic especially when an infected individual has traveled to numerous locations and when exposure occurred in public spaces and mass transit. compared to isolation, quarantine sometimes faces more resistance from expected participants especially from those who have been exposed but appear to be in a healthy condition. during the sars epidemic, mass quarantines were implemented in many countries. over , potentially exposed individuals were quarantined in taiwan, but in retrospect, the action may have spread panic among uninfected individuals and may not have been an effective strategy (university of louisville school of medicine ). in reality, quarantines are never perfect. compliance to the procedures is often problematic: quarantined individuals do not reduce their geographical movement or they only abide by the procedure for a short period. formal quarantines have good compliance rates but are costly and difficult to manage for a large number of cases. therefore a majority of quarantines are made voluntarily or with less monitoring than formal quarantines, but these suffer from reduced compliance and are less effective overall. knowledge about potential superspreaders to identify candidates for isolation can greatly enhance the efficacy of quarantines with much lower numbers of required isolations (diamond ) . although such knowledge may be rare at the beginning of an epidemic, rapid epidemiological analyses may play a critical role in reducing the costs of epidemic control. a critical aspect of human pathogens is their virulence or extent of damage to host. high virulence infectious disease such as hiv, plague or smallpox can be a great threat to human society, and the number of cases of pathogens that have been reported to have evolved virulence and/or resistance against drugs is alarming (altizer et al. ; holden et al. ). understanding the factors which affect the evolution of virulence in human society is an important issue. if it is possible to control these factors in urban design, human society can be more resilient against serious disease outbreaks. several classic theoretical studies on the evolution of virulence concluded that reduced virulence is generally adaptive and should evolve among pathogens. low virulence allows infected host individuals to survive, and pathogens can have more chance to spread to other host individuals. if pathogens have high virulence, they can propagate within an infected host individual, but risk killing the infected host and limiting their spread to other hosts. trade-offs between reproduction within a host and transmission among hosts is a well-studied explanation for the evolution of reduced virulence (anderson and may ; alizon et al. ). however, the balance (or equilibrium) of this trade-off can differ depending on biological characters of pathogens. ewald ( ) discussed how transmission mechanisms of pathogens can alter the predicted trajectory of virulence evolution. highly virulent diseases tend to immobilize hosts in early stage of infection. therefore, if pathogens are mainly transmitted by contacts between hosts, higher virulence would greatly decrease chances of new transmission. however, if pathogens can survive outside of the host and can be transmitted by air, water or vectors in which they are not virulent, host immobility should have less effect on the chances of new transmission. ewald ( ) noted that such pathogens, such as smallpox, tuberculosis or diphtheria, are often more virulent than pathogens that depend more directly on hosts for transmission. other factors can affect the balance of the trade-off and allow evolution of high virulence (galvani ) . for example, if multiple pathogen strains infect simultaneously and compete within individual hosts, high reproduction rate within a host (leading to high virulence) may be favored. in sexually-transmitted diseases, frequent exchange of sexual partners makes transmission of pathogens between hosts easier and as a result cause high virulence. this may be the case of hiv in human society (lipsitch and nowak ) . host population structure also affects the transmission of pathogens and therefore, has a large impact on the evolution of virulence. because urban planning and design can create or alter population structure by its use of the the environment, in the following paragraphs, we introduce two studies focused on the effect of host population structure on evolution of virulence. these studies are based on relatively simple models that may yield general insights. boots and sasaki ( ) incorporated a grid-like spatial structure of "sites" at which individuals can exist. each site can have one of three states: empty, occupied by susceptible individual, or occupied by infected individual. in the spatial structure, connections between individuals were divided into two types, those between neighbors and those between randomly chosen individuals. randomly chosen individuals can be in distant sites, and in such cases, pathogens can be transferred to distant locations. they found that pathogen virulence is favored as contact between hosts living in distant places becomes more common. in this model, a site becomes empty after death of an occupant. therefore, higher virulence is more likely to create a situation in which pathogens kill all susceptible hosts around them and can no longer spread. however, long-distance transfer allows pathogens to spread to new locations where they are surrounded by susceptible hosts. long distance transportation in human society allows contact between distant individuals and may be an important factor that facilitates the spread of outbreaks and favors pathogen virulence. boots and sasaki ( ) did not consider host immunity in their model. immune (infection-resistant) hosts can block pathogen spread and may have a large impact on the evolution of virulence. this question was theoretically addressed by the same authors. boots et al. ( ) incorporated the immune state after the recovery assuming a negative correlation between recovery rate and virulence and found that evolutionary trajectories could lead to low, or even extremely high, virulence depending on host population density. in host populations with high density, pathogens can easily find susceptible hosts and therefore, low virulence which increases the opportunity of infection to a new host evolves. on the other hand, in host populations with low density, immune hosts around a newly infected host efficiently block pathogen spread. in this case, highly lethal pathogens which kill infected hosts and make open spaces can spread more efficiently compared with pathogens with low virulence which induce immunity in hosts. even after killing some hosts, pathogens still have a chance to spread by infecting new susceptible hosts that emigrate to the open spaces. in boots and sasaki ( ) , infected hosts are assumed to be susceptible just after they recover and therefore, lower virulence pathogens spread more efficiently. however, in boots et al. ( ) , immune hosts block pathogen spread and create scenarios where highly lethal pathogens evolve. the results in boots and sasaki ( ) and boots et al. ( ) reveal scenarios in which low virulence can evolve to higher virulence depending on the structure of host populations. a key point is that outcomes are sensitive to the scenarios of population structure, transmission mechanisms, and host immunity. because of their short generation times and high genetic mutation rates, pathogens like rna viruses may evolve rapidly, even over the course of an outbreak. influenza virus, norovirus or dengue virus are well known examples of rna viruses that infect humans. because these viruses cause epidemics every year, controlling their impact is a very important aspect of urban resilience. as mentioned above, the models in boots and sasaki ( ) and boots et al. ( ) may be too simple to directly apply for particular diseases. theoretical studies under more realistic conditions based on structures that closely resemble actual human society and biological characteristics relevant to particular pathogens will be valuable to prevent and control outbreaks of high virulent diseases. important points to consider include parameters and assumption sensitivity for aspects of both host populations and pathogens. in addition, the definition of "connection" differs depending on the transmission mechanism of the pathogen. the concept of "network" must take the view of the pathogen and different networks may need to be considered for different diseases in the same human populations. many of the major human infectious diseases are zoonotic infections that have crossed over from animals into humans (wolfe et al. ). bubonic plague (schmid et al. ) , influenza (palese ) , hiv (gao et al. ) , ebola (marí saéz et al. ) , sars (lau et al. ; li et al. b ) and mers (memish et al. ; wang et al. ) have all been shown to have originated from animals before infecting humans. wolfe et al. ( ) surveyed major infectious diseases ranked by highest mortality and/or morbidity to identify patterns in their animal origins and geographical spreading. all the diseases they surveyed appeared to have originated from the old world (africa, asia, europe) and a remarkable proportion of causative pathogens arose from warm-blooded vertebrates while the remaining were attributed to birds. interestingly, the purported geographical origin of the disease was correlated with the type of animal to which the pathogen originally infected. for example, many diseases that trace back to tropical regions have come from wild non-human primates whereas diseases attributed to temperate regions often emerged from domestic animals. although the exact reason for this pattern is unknown, wolfe et al. ( ) suggested that, because livestock and pets were domesticated in the old world, ancestral pathogens had more opportunity to infect humans compared to more recently domesticated new world animals. for the disparity between old world and new world monkeys, they believe that closer genetic relatedness between human and old world monkeys may have aided in cross-species transmission. these results stress the importance of considering both environmental and biological factors as key determinants of cross-species transmission of infectious diseases. recent spreading of human population by urbanization exposes us to novel pathogens that were previously isolated from human society. the risk of zoonotic infections may be increasing and it is notable that many novel pathogens appear to have high virulence in human (reads ; schrag and wiener ) . during their long evolutionary history, pathogens and their original hosts may have been recurrently co-evolving by which hosts evolve to be resistant against the pathogens, and pathogens evolve to evade the resistant system (little ; woolhouse et al. ) . this means if hosts are exposed to a novel pathogen, it is highly possible that the hosts do not yet have immune resistance against the pathogen and are affected by high virulence (longdon et al. ) . there are also cases in which infections of novel pathogens cause inappropriate immune response and as a result, increase their virulence (graham and baric ). as introduced above, these highly virulent pathogens can spread in a host population depending on host spatial structure. however, it is important to note that not all novel pathogens have high virulence for human. highly virulent pathogens are more likely to be detected and studied and therefore, the patterns may result from ascertainment bias (alizon et al. ; longdon et al. ) . in any case, careful surveillance of both human and animal populations in regions of high human-animal contact may be an important component to defending against novel disease (woolhouse et al. ) . finding the original animal host of a new human pathogen requires scientific rigor but also guesswork and luck. the search for the animal reservoir of the sars pathogen first identified the himalayan palm civet (paguma larvata) after sars-like coronaviruses (sl-covs) were isolated from civets in live-animal markets in guangdong, china (guan et al. ) . however, tu et al. ( ) showed that while civets in live-animal markets were infected with sl-covs, civets on farms did not possess antibodies against the virus, which indicated that they have never been exposed to the pathogen. moreover, palm civets infected with sars-cov showed signs of illness contrary to the expectation that animal reservoirs should be clinically asymptomatic (calisher et al. ) . this observation and that other animals in the same live-animal markets were also infected by the virus (guan et al. ) indicated that the palm civets were infected in live-animal markets rather than being the ultimate source of the pathogen. surveillance of wild animals in the region later lead to the serendipitous discovery that chinese horseshoe bats (rhinolophus sinicus) are the original animal host of the coronavirus that became sars-cov (lau et al. ; li et al. b) . the focus on bats may have been inspired by outbreaks of nipah and hendra virus a decade before that were also traced back to these mammals (normile ). in addition, li et al. ( b) stated that the use of bat products in food and traditional medicine in southern china led them to investigate bats as a potential reservoir. interestingly, bats appear to harbor many human pathogens and have been implicated as the animal reservoir of nipah virus, hendra virus, ebola virus, and sars-cov. even mers-cov, initially transmitted from camels, have been traced back to bat through phylogenetic analysis and biochemical studies (wang et al. ; yang et al. ) . while sars-cov infection primarily affected the respiratory system, high concentrations of coronavirus were observed in bat feces and recovery from the small and large intestines indicate that replication is primarily through the excretory system (drexler et al. ) . lau et al. ( ) speculated that the use of bats in traditional medicine, especially bat feces, may have played a crucial role in the cross-species transmission of the virus. bat meat is also considered a delicacy and many chinese believe it possess therapeutic activity, which led to bat trade in live-animal markets such as those in guangdong, china. exposure between the pathogen reservoir and the new potential host species is a key factor dictating the probability of successful cross-species transmission. for example, hiv- and - seem to have transferred multiple times to humans since based on phylogenetic analysis, but only after was there a significant spreading of the infection (heeney et al. ) . one explanation suggests that the limited interactions between humans and primates created a barrier for the transference of the virus and insufficient interhuman encounters of infected cases delayed the rise of the epidemic (parrish et al. ) . to describe this phenomenon, let us model the underlying host contact network as a network of nodes (individuals) and connections (exposure). assuming a heterogeneously connected network such as human social network and contact networks (eubank et al. ) , we find that the probability of a new infection becoming extinct by chance is very high both because the pathogen may be poorly adapted to transmit in the new host (parrish et al. ; daszak et al. ; dobson and foufopoulos ) and because cross-species transmission events tend to occur in sparsely connected rural areas (tibayrenc ) . the limited connections inhibit emergence of the disease and only the few that avoid stochastic extinction proceed to produce an epidemic in the host population (lloyd-smith et al. ; eubank et al. ). this may explain why spillover events of animal infections, such as h n avian influenza, fail to take hold in the human population despite the hundreds of human cases and deaths that have been reported (parrish et al. ) . while distribution is skewed towards fewer connections in these networks, it is still possible that the cross-species transmission event occurs at a highly connected portion of the network. such an outcome will only make it more likely that the infection will take hold to produce an epidemic due to the presence of highly connected hubs that can spread the disease to a disproportionate number of hosts (rock et al. ) . lloyd-smith et al. ( ) expand this concept to show that any type of individual variance, for example infectiousness, produces the same effect. high individual variance increases the probability of extinction of an invading disease regardless of the strength of mean infectiousness. when the host population has a highly heterogeneously connected network, emergence of disease may be rare, but infections that survive stochastic extinction produce "explosive" epidemics similar to the case of sars in . these findings show that host population structure and demography significantly affects the probability of cross-species transmission as well as the subsequent epidemic that may follow. host factors also play a significant role in determining the success of new infections in novel hosts, especially for viruses. to infect a host, a virus must be able to interact with the host's cellular receptors to gain entry into cells and hijack the cell's machinery to replicate itself. at the same time, the pathogen must survive against the host's defense mechanisms. the initial interaction between the virus and host receptors is a critical step that determines host specificity and host range. for example, in sars as well as in other coronaviruses, the viral structure responsible for viral entry is the spike glycoprotein, which also appears to be the key determinant of host specificity (graham and baric ) . in humans, the receptor-binding domain on the spike glycoprotein interacts with a cell surface metalloproteinase called human angiotensin-converting enzyme (ace) to gain entry and infect lung epithelial cells (li et al. ) . however, ren et al. ( ) showed that sl-covs found in bats do not interact with palm civet or human ace receptors implying that changes must have occurred to gain this new interaction. in fact, there appears to be a sizeable difference between coronaviruses isolated from the putative bat reservoir and sars. sl-covs from bats were found to be at most only % similar compared to sars-cov (li et al. b) . later, ge et al. ( ) were able to isolate and characterize a sl-cov that utilizes the ace for cell entry in bats, palm civets and humans. this finding argues that ace utilization may have evolved prior to any cross-species transmission event. while gaining the ability to bind to a novel receptor appears to be a complicated process, in some instances, even a few amino acid changes may confer the ability to recognize a new species. initial studies comparing sars-cov isolated at different time points in the pandemic revealed the spike protein of viruses taken from palm civets and early human cases bind less efficiently than those from later on in the epidemic (yang et al. ) . further genetic and biophysical studies demonstrated that two amino acid changes had an enormous effect on the binding affinity of the sars spike protein to human lung epithelial cells (li et al. a, c; qu et al. ) . in most palm civet samples, lysine at position and serine at position of the spike protein were observed whereas asparagine and threonine were present in human samples. li et al. ( a) found that replacing lysine with asparagine removed the electrostatic interference with the histidine residue on the receptor while replacing serine with threonine provided a methyl group capable of filling in a hydrophobic pocket at the interface of the human ace receptor. although the structural changes appear to be subtle, these substitutions caused a thousand-fold increase in binding affinity to the human ace and lead to enhanced human transmission. however, li et al. ( a) also found that some civet specimens have asparagine instead of lysine at position yet this did not affect binding to the civet ace receptor. changes that are neutral to the original host but advantageous in the new host may have played a critical role in facilitating cross-species transmission between palm civets and humans. once a pathogen has evolved to reliably infect the new host's cells, the innate immune response is the host's first line of defense against the infection. when a virus successfully infects a cell, cytoplasmic enzymes that detect the production of double-stranded rna, a hallmark of virus replication, activate the expression and release of interferons from the cell. interferons act as an early-warning signal to other cells nearby by activating their intracellular antiviral response to combat viral infection and replication (roy and mocarski ) . because of the importance of this immune response against viral infection, many viruses have evolved features to subvert interferon signaling. for example, the influenza virus prevents the infected cell from detecting viral replication by using its ns protein to sequester double-stranded viral rna (lu et al. ) . another method to interfere with the innate immune response is to prevent interferons from activating antiviral mechanisms. nipah virus produces two proteins that prevent stat from translocating into the nucleus as well as another protein that sequesters stat present in the nucleus, obstructing the activation of interferon-stimulated genes (shaw et al. ). in the case of sars, kopecky-bromberg et al. ( ) found that sars-cov nucleocapsid and accessory proteins inhibit both the expression of interferon and associated transcription factors, as well as inhibiting cellular response to interferon by subverting the jak/stat activation of intracellular antiviral mechanisms. while infection with sars-cov did not induce production of interferons, coinfection with another virus did produce interferons. it appears that sars-cov does not induce interferon expression, yet does not shut down the whole pathway as interferon signaling continues to work when other stimuli are present (frieman et al. ) . by antagonizing the induction and response to interferons, the pathogen blocks the activation of more than interferon-stimulated genes which prevents the cell from going into an "antiviral state" (de lang et al. ). under the antiviral state, inhibitors are activated to prevent cell division, enzymes that digest proteins initiate programmed cell death, and proteins that present viral particles to activate the adaptive immune response are upregulated. blocking interferon signaling causes a general decrease of both innate and adaptive immune system response, allowing sars-cov to infect cells unimpeded and potentially cause a more serious disease. the rate at which a pathogen evolves is another biological factor that may determine risk of cross-species transmission. most recent emerging infections have been caused by rna viruses such as hiv (gao et al. ) , ebola virus (gire et al. ) , dengue virus (gubler ) , sars-cov (lee et al. ) and mers-cov (de groot et al. ) . rna viruses have an extremely high mutation rate because their rna polymerase, the enzyme that copies their genome, lacks proofreading activity which leads to error-prone replication. mutation rates for rna viruses range from − to − substitutions per nucleotide per cell infection, two orders of magnitude higher, on average, than dna viruses (sanjuán et al. ). at those rates, about out of , nucleotide changes every time an rna virus replicates itself. this may not seem high but note that hundreds of millions of viral particles may be produced during a single infection (haase ) , which gives the virus numerous opportunities to explore potentially advantageous mutations. although high mutation rates helps rna viruses to rapidly adopt advantageous changes and alter phenotype, deleterious mutations are also produced at an elevated rate. lauring et al. ( ) have demonstrated that viruses mitigate the effects of deleterious phenotypes by outcompeting and quickly purging these low-fitness variants. the ability of viruses to incorporate functional components made by other functional viruses within the same cell appears to also mitigate the negative effects of high mutation rates (makino et al. ). indeed, studies have shown that raising the mutation rate through mutagens can be used to create large numbers of dysfunctional mutants that rapidly leads to the extinction of the viral population (pathak and temin ; loeb et al. ; domingo ) . interestingly, in the case of sars, the coronavirus that caused the disease did not have a very high mutation rate relative to other rna viruses. coronaviruses have the largest genomes (approximately , nucleotides) among rna viruses and genome size and mutation rate appear to be negatively correlated (sanjuán et al. ) . one reason behind the relative stability of coronavirus genomes could be the presence of proofreading enzymes that guard against mutagenesis. in the case of sars-cov, smith et al. ( ) showed that the exoribonuclease domain in non-structural protein had proofreading activity and was responsible for protecting the viral genome against mutagenesis. although high mutation rates appear to facilitate adaptation of rna viruses to new environments, diseases of animal origin are not always caused by the fastest evolving rna viruses. the discussions above have introduced how several aspects of urban life, including high connectedness of individuals (including connections among distant individuals) and regions of high human/animal contact, are likely to elevate the risk of future epidemics. because these properties may be intrinsic to urban life and difficult to alter or control, monitoring and preparedness are critical for urban resilience to disease outbreak. opportunities for the evolution of new or variant human pathogens are difficult to limit and may, in fact, be increasing in modern societies. each contact between microbe and host can be considered a "trial" for a potential pathogen with random mutations in their genomes that may confer new functions or specificities. thousands of such trials occur daily in many regions and are likely spawning candidate emerging pathogens with the ability to reproduce within humans and possibly also to transmit from person to person. the trajectory of pathogen evolution depends strongly on the numbers of contacts (potential transmissions) among individuals in the host population as well as on chance. the vast majority of potential new pathogens are likely to be lost early in their histories. however, given continuous opportunities, the chance event of pathogen emergence is simply a matter of time. in guangdong province, several recent outbreaks of bird influenza (h n ) have led to limits on live poultry markets, but consumer preference for freshly slaughtered poultry and wild animals remain an impediment to regulating the high-risk concentration of multiple species (pig, poultry, dog, cat, rabbit, as well as reptiles, fish and numerous wild game) in close contact with one another (and often in poor health) as well as with humans. regions of recent human expansion where wild animal populations are in close proximity to high density human settlements must also be monitored carefully for new zoonotic diseases. new strains of swine and bird influenza are currently monitored as candidates for outbreaks but pathogen emergence is unpredictable and may come from completely unexpected sources. regardless of the source of new infectious agents, a major concern is that future pathogens may have properties that will make control much more difficult than sars. shorter incubation times and pre-symptomatic transmission strongly limit the efficacy of isolation and quarantine and may allow rapid disease spread. in this chapter, we have focused on biological factors that are central to disease emergence and control. policy prescriptions have been discussed extensively (university of louisville school of medicine ; beaglehole et al. ) and we highlight selected topics below. one of the important lessons from the sars crisis was the need for a rapid and organized response, even in the case of a relatively controllable disease. recognition of new epidemics through surveillance and global warnings and travel advisories are obvious critical factors but the necessary infrastructure has been difficult to implement, especially in developing regions. given the likely lag-time between the start of an outbreak and pathogen isolation and development of diagnostics, well-trained physicians and epidemiologists at the frontlines of the epidemic play a critical role in initial response. for an infected individual, the numbers of contacts and possible and actual transmissions increase rapidly with time so diagnosis and contact tracing are time-critical events. finally, communicating with, and educating the public and controlling panic are major concerns especially in the context of false reports and rumors. establishing trusted sources of information prior to emergencies should be a major objective for cities/regional governments. issues with coordination among government agencies or between medical and government agencies were strong obstacles in the response to sars in most affected regions (university of louisville school of medicine ). high transmission in medical care settings was one of the prominent features of both the sars and mers outbreaks. because infected individuals with weakened immune systems or co-infections may be the most difficult to diagnose and may show high infectiousness, proper training in pathogen containment is a critical element of epidemic preparation. similar basic techniques (proper use of gloves, gowns, masks, and goggles) were successful for sars and ebola suggesting that many practices will be of general value but specifics for particular transmission mechanisms (e.g. airborne versus vector transmission) are also critical. intervals between outbreak occurrences may be large, so regular confirmation of preparedness is important. low margins in health care are strongly linked to overcrowding and government and private organization incentives (e.g., increased funding for hospitals that rate highly on infection control training and preparedness) can greatly enhance hospital safety (committee on the future of emergency care in the united states health system ). the importance of patient isolation in limiting disease spread is clear from recent sars, ebola and mers outbreaks; hospitals must have containment facilities and "surge capacity" to limit superspreading events. although public health measures were sufficient to eventually control sars, additional measures including antiviral drugs and rapid vaccine development and production may be necessary for stronger pathogens. the economic impact of epidemics, roughly billion usd ( % of regional gdp) for east asia from sars and potentially over billion usd for pandemic influenza (the economist ) should help to justify the costs of outbreak preparation. informed sanitation (water purification, sewage treatment) and building regulations/inspections (e.g. airflow control) policies can play a key role in preventing disease emergence and spread. the sars example illustrates the need for extensive interdisciplinary efforts, combining expertise from physics (fluid mechanics), biology (especially understanding mechanisms of disease transmission), and building design for resilience to future outbreaks. isolation and quarantine were critical to controlling sars in hong kong, singapore, taiwan, china, vietnam and canada. compliance rates appeared to be high in all regions (university of louisville school of medicine ) perhaps partly because the "cultural" value placed on solidarity and cohesion was relatively high in these regions. more severe movement restrictions may be necessary for more transmissible and/or virulent pathogens. it is unclear whether similar measures can be employed with success in other regions where personal liberties are emphasized and/or government is less trusted. biological studies can help to determine the necessity and guide planning for future epidemics, but social and economic issues may be the more critical limiting factors in developing preparedness for disease outbreaks. understanding the social, psychological, and economic costs of previous and potential disease outbreaks among both citizens and government officials will be central to planning for resilient communities. the ability to overcome economic and psychological barriers (e.g., normalcy bias) to implementing such plans may require a fundamental transformation in human society. virulence evolution and the trade-off hypothesis: history, current state of affairs and the future rapid evolutionary dynamics and disease threats to biodiversity epidemiology, transmission dynamics and control of sars: the - epidemic. philosophical transactions of the royal society of 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co-evolution of pathogens and their hosts receptor usage and cell entry of bat coronavirus hku provide insight into bat-to-human transmission of mers coronavirus evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses evidence of airborne transmission of the severe acute respiratory syndrome virus why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others key: cord- -egwo oc authors: aw, tiong gim; rose, joan b title: detection of pathogens in water: from phylochips to qpcr to pyrosequencing date: - - journal: curr opin biotechnol doi: . /j.copbio. . . sha: doc_id: cord_uid: egwo oc waterborne pathogens pose a significant threat to human health and a proper assessment of microbial water quality is important for decision making regarding water infrastructure and treatment investments and eventually to provide early warning of disease, particularly given increasing global disasters associated with severe public health risks. microbial water quality monitoring has undergone tremendous transition in recent years, with novel molecular tools beginning to offer rapid, high-throughput, sensitive and specific detection of a wide spectrum of microbial pathogens that challenge traditional culture-based techniques. high-density microarrays, quantitative real-time pcr (qpcr) and pyrosequencing which are considered to be breakthrough technologies borne out of the ‘molecular revolution’ are at present emerging rapidly as tools of pathogen detection and discovery. future challenges lie in integrating these molecular tools with concentration techniques and bioinformatics platforms for unbiased guide of pathogen surveillance in water and developing standardized protocols. water is essential to human health as a means to reduce diseases and also as the vehicle through which diseasecausing microorganisms or pathogens may be transmitted. a growing number of enteric microorganisms, including bacteria, viruses and protozoa in sewage have been identified as important waterborne pathogens. most waterborne pathogens are introduced into drinking water supplies and recreational waters by human and animal wastes and poor wastewater infrastructure as well as inadequate treatment. this includes leaking sewers, combined sewer overflows, urban storm water runoff, agricultural land runoff, faulty septic tanks/drain fields and poorly treated wastewater discharges. the vital role of public health in minimizing community wide-spread acquisition of these waterborne pathogens and infectious disease control of the hundreds of pathogens has drawn attention to the need for detecting and identifying bacteria, parasites and viruses directly in water. until recently, scientists have had no accurate and comprehensive way to detect and quantify the presence of microbial pathogens in various environmental samples. culture-based methods have long been extensively used to detect microbial pathogens in environmental matrices and are considered to be 'gold standard', but inherent problems exist, such as lengthy time of analysis, labor intensive and the inability to detect uncultivated species. the term ''the great plate count anomaly'' was coined by staley and konopka [ ] over years ago to describe great discrepancy between direct microscopic counting and numbers of bacteria that can be enumerated from environmental samples by plating procedures. the ability to access dna sequences of microbial genomes offers an exciting alternative for exploring and improving the characterization and rapid detection of a diverse group of pathogens in water. the advent of molecular tools has resulted in tremendous paradigm shifts in microbial water quality monitoring: (i) direct detection of pathogens, including uncultivated pathogens, is now possible and it takes hours instead of days to weeks, (ii) the true positivity rate of some pathogens in waters is known to be much higher than previously thought based on culture-based detection alone, and (iii) traditional bacterial indicators often fail to predict or correlate with the presence of waterborne pathogens. by increasing the capacity to detect a wide range of microbial pathogens in waters, novel and potential pathogens could be recognized and associated with waterborne diseases and better characterization of known pathogens can be undertaken for waterborne diseases of unknown etiology. according to web of science, articles with qpcr and water as key words have been published since with over articles in and . citations of this work grew to over /year by . this article reviews some of the most recent and significant progress in molecular tools for the detection of pathogens in water. eliminated many iconic waterborne enteric diseases such as typhoid and cholera in the north america and europe. however, microbial pathogens continue to be a major cause of water-related disease outbreaks globally and remain a key public health challenge in providing safe drinking water and recreational water in the st century. in addition to these traditional waterborne pathogens, a significant number of emerging and re-emerging pathogens have now been recognized and water transmission has caused significant outbreaks; examples include escherichia coli o :h , helicobacter pylori, legionella pneumophila, campylobacter jejuni, mycobacterium avium complex, enteric viruses such as noroviruses and hepatitis e virus, and the parasites cryptosporidium parvum and microsporidia. according to the latest world health organization (who)'s guidelines for drinking-water quality, zoonotic pathogens which make up % of the emerging pathogens, pose the greatest challenges to ensuring the safety of drinking water and ambient water [ ]. the list of potential waterborne pathogens is extensive [ ] and table provides examples of emerging waterborne pathogens which are also listed on the latest us epa's contaminant candidate list (ccl- ). because of the nature of waterborne pathogens, what is perceived as small concentrations are associated with high levels of community risks [ ] . the use of quantitative microbial risk assessment allows for an improved understanding of how concentrations, loading and transport influence probability of infection associated with microbial contamination of water. the limitations in detecting and identifying pathogens directly from environmental water samples by culture or microscopy can now be addressed by integrating concentration techniques with molecular tools to provide sensitive, specific and quantitative data on any pathogens of interest. the tools have a number of advantages. multiple pathogens can be characterized by microarray technology, however often sensitivity is not adequate. qpcr is the most rapidly growing technique for use in the water environment for both microbial source tracking and rapid pathogen specific quantification; and finally the power of pyrosequencing for exploring the water environment has offered new insights into the microbial world. developed in the early to mid- s, dna microarrays are arrays containing a high density immobilized nucleic acids (genomic dna, cdna or oligonucleotides) in an ordered two-dimensional matrix that enable the simultaneous interrogation of thousands of genes in a single assay via nucleic acid hybridization [ ] . unfortunately, current limitations of microarrays including the lack of sequence information for many pathogens, non-specific binding and inability to offer the low detection levels available by qpcr, make the use of microarrays for the direct detection of very dilute spectrum of pathogens in environmental matrices impractical. until recently, microarrays have not been used to quantitatively detect detection of pathogens in water aw and rose table potential waterborne pathogens, diseases caused and recent qpcr assays for their detection major disease(s) qpcr detection limit (per reaction) ref. pathogens in environmental water samples and a previous pcr enrichment with gene-specific primers was usually needed to increase the detection resolution [ ] . multiple displacement amplification, an isothermal dna amplification reaction using phi dna polymerase and random hexamer primers, offers a promising unbiased approach for increasing the amount of target dna in the environmental sample before microarray detection [ ] . the rapid growth of s rrna gene databases has helped pave the way for developing high-density phylogenetic microarrays to simultaneously analyze many taxa in complex environmental samples. phylogenetic microarrays such as the phylochip have been proven to be useful in determining microbial community structure [ ] . the phy-lochip that targets the known diversity within the s rrna gene is able to simultaneously identify any of thousands of taxa present in an environmental sample. the current version (g ) of the phylochip allows simultaneous detection of up to , bacterial, archaeal and microalgal taxa [ ] . besides monitoring bacterial population during environmental bioremediation, phylochip technology has been used for monitoring airborne bacteria for bioterrorism surveillance [ ] , microbial community analysis in biological wastewater treatment systems [ ] and microbial source tracking of pathogens in coastal urban watershed [ ] . more recently, two methods of pcr-independent microbial community analysis using the microarray phylochip: direct rrna hybridization and doublestrand cdna generation and hybridization have been developed, providing cost-effective and pcr bias-free alternatives to pcr-amplified microbial community analysis [ ] . the virochip, a pan-virus dna microarray containing thousands of unique -mer oligonucleotide probes to target all viral families known to infect humans has been described by wang et al. [ , ] . the authors demonstrated that this approach coupled with a random pcr amplification strategy, was able to detect multiple viruses as well as novel members of known viral families. recently, several high-density microarray prototypes such as the pan-microbial detection array (mda) [ ] and the greenechip system [ ] have been developed for the molecular surveillance and detection of a panel of pathogens including bacteria, viruses and parasites in clinical samples. these methods have yet to be applied to realworld water samples for the detection of waterborne pathogens. to make this practical, these high-density microarrays will need to be further coupled with water sample concentration and purification techniques, perhaps other amplification techniques and then could be used to assess the diversity of pathogens in water and potentially discover the presence of previously unknown pathogens. polymerase chain reaction (pcr) developed in the early s by kary mullis, is now a common and often indispensable molecular technique in both clinical and biological research laboratories. the concept of pcr remains unchanged since its inception. however, the agarose gel-based detection of pcr amplification products has undergone dramatically changes that have led to a revolutionary testing platform, namely quantitative real-time pcr (qpcr), which enables quantification of dna targets by monitoring amplified products during cycling as indicated by increased fluorescence [ ] . in the past few years, the development of qpcr has provided significant improvements in pcr-based assays for the detection of waterborne bacterial, viral and protozoan pathogens (table ) . currently, qpcr offers numerous advantages over conventional, end-point pcr techniques such as higher sensitivity and specificity, faster rate of detection, no post-pcr analysis and thereby minimizing the risk of carryover contamination, and the capability to provide quantitative results. the dual-labeled fluorescent probe such as taqman probe has been the most popular technique of choice for detecting pathogens in environmental samples owing to its higher specificity. the advantage of this assay is that the target amplicon is verified by the probe which recognizes internal amplicon sequences. inclusion of an internal control in each sample is essential for the accuracy of quantitative results, particularly for complex environmental matrices, by monitoring the efficiency of nucleic acid extraction and reverse transcription (for rna target) as well as the presence of pcr inhibitors. detection of human pathogenic viruses has undergone a significant transition from conventional cell culture to novel molecular techniques, particularly quantitative reverse-transcriptase (qrt)-pcr for rna viruses, with higher sensitivity and specificity. detection of pathogenic viruses by qrt-pcr does not necessarily indicate their infectivity, but it has been an excellent methodology for studying virus pollution in the water environment. moreover, the use of qrt-pcr for the analysis of non-cultivable noroviruses [ ] and slow growing hepatitis a virus [ ] has been fundamental to advancing the understanding of the occurrence of these human pathogens in water environments. surveys around the world have begun exploring the occurrence of both dna and rna viruses in water by qpcr (see [ ] for a review). however, there were no consistent trends found in regard to the viral types or levels. the molecular characterization of environmental viral strains has revealed a great genetic diversity of human pathogenic viruses in polluted surface waters [ ] . yet no global survey of viruses has been undertaken by evaluating the same virus targets, using the same protocols. a temporal assessment is needed to compare the diversity and level of viral infections and water pollution to contrast developing to developed regions of the world, from tropical environments to temperate conditions and to contrast rural versus urban populations. it may be possible to take on global virus characterization in the future with evolution in technology that allows higher throughput and hundreds of targets. advances in microfluidics and nanobiotechnology allow for the construction of high-density and low-volume qpcr platforms, such as biotrove openarray system (applied biosystems) that are capable of accommodating reactions per array. with a novel internal amplification control approach, this next-generation qpcr platform offers high-throughput testing of environmental samples against a wide range of pathogens [ , ] . since the invention of the dideoxy chain termination technique, often referred to as 'sanger sequencing', was first described more than three decades ago, technological advances have led to the increasing development of massively parallel dna sequencing platforms, including pyrosequencing. based on the sequencing-by-synthesis principle, pyrosequencing is a dna sequencing technology that utilizes enzyme-coupled reactions and bioluminescence to monitor the pyrophosphate release accompanying nucleotide incorporation, in real-time ( figure ) [ ] . a major advance for pyrosequencing technology came with the introduction of the roche sequencing platform. this next generation sequencing technology holds several advantages over the more traditional sanger sequencing include higher throughput, simplified sample preparation and the miniaturization of sequencing chemistries, enabling massively parallel sequencing reactions to be carried out at a scale and cost not previously possible [ ] . the latest sequencing platform, gx flx+ is able to produce one million reads (with a read length of up to bases) per -hour sequencing run. pyrosequencing technology is revolutionizing the study of microbial ecology as well as direct metagenomic detection detection of pathogens in water aw and rose high levels of several classes of resistance genes in bacterial communities exposed to antibiotic were identified. [ ] reclaimed and potable waters viral dna and rna tangential flow filtration, dnase treatment, mda, pyrosequencing ( gs-flx and gs sequencer) over % of the viral sequences with no significant similarity to proteins in genbank. bacteriophages dominated the dna viral community. the rna metagenomes contained sequences related to plant viruses and invertebrate picornaviruses. [ ] wastewater biosolids viral dna and rna dnase and rnase treatment, reverse transcription for rna, pyrosequencing ( gs-flx sequencer) optimal annotation approach specific for viral pathogen identification is described. parechovirus, coronavirus, adenovirus, aichi virus and herpesvirus were identified. [ ] lake water viral rna tangential flow filtration, dnase and rnase treatment, random amplification (klenow dna polymerase), pyrosequencing ( gs-flx sequencer) % of the sequences with no significant similarity to known sequences. presence of viral sequences ( viral families) with significant homology to insect, human and plant pathogens. of human pathogens in both clinical and environmental samples. unlike pcr and microarray methods where investigators are limited by known sequence information and must select the range of pathogens to be considered in a given assay, high-throughput sequencing approach is unbiased and makes it possible to detect novel pathogens. figure shows the basic steps that are needed for the detection of microbial pathogens in environmental waters by pyrosequencing. besides the direct detection of pathogens in stool specimens [ ] , several recent papers describe the application of pyrosequencing to investigate the diversity of bacterial and viral pathogens in environmental samples (table ) . a general finding from these studies is that the majority of the sequences in environmental metagenomes, particularly viral sequences, have no similarities to known genes in the database, indicating high abundance of uncharacterized viruses which may be potential human pathogens [ , ] . the extremely high-throughput of pyrosequencing has significantly expanded the repertoire of complete bacterial pathogen genomes, particularly strains associated with outbreaks [ , ] , since whole bacterial genomes can be sequenced in a fraction of the time and at a much lower cost. as the majority of waterborne disease outbreaks had undetermined etiology agents [ ] , pyrosequencing and other high-throughput sequencing technologies are likely to identify novel pathogens associated with waterborne illnesses in the future and could address multiple etiologies. moreover, sequencing of outbreak isolates is of public health interest, particularly if rapid data about virulence markers can be obtained. most recently, a new whole genome amplification and sequencing approach called 'single virus genomics', which enables the isolation and complete genome sequencing by pyrosequencing of the single virus particle (bacteriophages lambda and t ), has been described [ ] . this proof-of-concept study is likely to enhance uncultivated virus discovery as well as provide relevant viral reference genomes for the assembly of metagenomic data and the design of qpcr primers and probes targeting uncultivated viruses. further study is needed to optimize this approach for human pathogenic viruses in environmental waters. although high-throughput sequencing has made remarkable advances in the past few years, the most critical challenge for its application in pathogen detection is the development of improved user-friendly bioinformatics and visualization platforms to facilitate rapid and robust analysis and interpretation of high-throughput sequencing data. challenges for pathogen detection in water using molecular tools: upstream sample processing adapting novel molecular tools to meet the needs of waterborne pathogen monitoring is a significant technical challenge. rapid detection and identification of waterborne pathogens by novel molecular tools are often hampered by the relatively low abundance of target organisms in water samples. much work has been carried out in the development of rapid, sensitive and specific analytical methods. however, less emphasis has been placed on the pre-analytical or upstream sample processing which must be able to selectively separate and concentrate all target pathogens in each water sample. the preconcentration step is important in improving the detection sensitivity of microbial contaminants, particularly by molecular detection tools which use extremely small sample volumes (a few microliters). recently, tangential-flow, hollow fiber ultrafiltration, which uses size-exclusion as the mechanism of concentration, has been shown to be a promising technique to concentrate diverse pathogens in a single process [ , ] . moreover, ultrafiltration is currently the method of choice to concentrate viruses from large volumes of water for metagenomic detection by pyrosequencing [ , ] . the relationship between detection by molecular approaches and viability or infectivity of waterborne pathogens still remains a concern, particularly for risk assessment to public health. one of the most recent approaches to determine viability or infectivity using molecular tools is propidium monoazide (pma) treatment before qpcr to reduce pcr signals from dna originating from dead cells [ , ] . although pma treatment has also shown promise in excluding signals from dead cells with microarrays [ ] and pyrosequencing [ ] , further validation is needed in environmental water samples, particularly after disinfection. disinfection mainly chlorination of water serves as the major process for inactivating pathogens. viable qpcr methods using pma for distinguishing live cells and dead cells (heat killed) has not been verified for water after disinfection. varma et al. [ ] and srinivasan et al. [ ] found that there was no observed reduction in the qpcr signals after disinfection of wastewater effluent samples. thus, currently, qpcr can be used as a tool to monitor pathogen loading and physical removal or dilution but cannot be used to address viability. this review shows that considerable progress has been made to achieve sensitive, specific and high-throughput detection of pathogens in water. overall, qpcr is among the most reliable and cost effective of the molecular tools owing to the shorten time to result as well as its potential to increase sensitive and specific, and it is therefore not surprising that qpcr is increasingly being used for the detection of waterborne pathogens. despite the potential for development of high-throughput analytical systems for the detection and differentiation of pathogens in water, further assay optimization is needed for high-density microarrays to demonstrate reliability and match the sensitivity of qpcr. roche pyrosequencing and other commercially available high-throughput sequencing platforms such as solexa/illumina genome analyzer, applied biosystem solid sequencing as well as the most recently released ion torrent system [ ] have revolutionized the study of microbial diversity. nevertheless, these high-throughput sequencing platforms are currently in their infancy for the direct detection of pathogens in water and are exploratory. whereas many technological and methodological challenges remain, high-throughput sequencing technology has the potential to provide an unbiased detection approach for waterborne pathogens with a single common protocol. by integrating with qpcr, concentration of key pathogen groups can be subsequently determined. a recent study by svraka et al. [ ] where metagenomic sequencing was used to identify viruses in samples that exhibited consistent cytopathogenic effects in cell culture but remained unexplained by established pcr, demonstrates the need for unbiased approach for pathogen detection in a public health setting. however, several questions arise when molecular tools are being introduced to microbial water quality monitoring. which systems offer the greatest promise for more routine water pollution diagnoses? can current laboratory personnel be trained to perform some of these complex assays on a routine basis? what changes are necessary in water sampling, transport and storage? are the improved sensitivity and decreased turnaround time worth the additional cost for water quality monitoring? currently, success is being achieved more in the downstream part of the detection process and strategic sampling as well as concentration techniques of pathogens in environmental waters should be exploited further. it is clear that the field of environmental pathogen detection is and will remain highly dynamic with tremendous potential for development of new tools and continuous improvement of existing concepts. however, the major challenge facing environmental microbiologists is to apply these tools for detecting pathogens in real environmental matrices on a routine basis. multi-laboratory round robin test should be performed to evaluate novel molecular tools and refine the prototype protocols into standardized methods with appropriate technology transfer. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats microbial health risks and water quality challenges of multiplexed detection: detection of pathogens in water and wastewater using microarrays a comprehensive review describing recent advances and challenges of microarrays for the detection of waterborne pathogens something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology high-density universal s rrna microarray analysis reveals broader diversity than typical clone library when sampling the environment deepsea oil plume enriches indigenous oil-degrading bacteria urban aerosols harbor diverse and dynamic bacterial populations bacterial community structure in geographically distributed biological wastewater treatment reactors characterization of coastal urban watershed bacterial communities leads to alternative community-based indicators pcr amplification-independent methods for detection of microbial communities by the high-density microarray phylochip microarray-based detection and genotyping of viral pathogens viral discovery and sequence recovery using dna microarrays a microbial detection array (mda) for viral and bacterial detection panmicrobial oligonucleotide array for diagnosis of infectious diseases why the need for qpcr publication guidelines?-the case for miqe the author provides guidelines to improve entire qpcr workflow, from experimental design to statistical analysis and reporting. general implementation of these guidelines is important for producing consistent and high quality data from qpcr assays. transcription-pcr assay for quantification of hepatitis a virus in clinical and shellfish samples molecular detection of pathogens in water -the pros and cons of molecular techniques prevalence and genetic diversity of waterborne pathogenic viruses in surface waters of tropical urban catchments accurate quantification of microorganisms in pcr-inhibiting environmental dna extracts by a novel internal amplification control approach using biotrove openarrays development and experimental validation of a predictive threshold cycle equation for quantification of virulence and marker genes by high-throughput nanoliter-volume pcr on the openarray platform pyrosequencing: history, biochemistry and future an informative introduction to the principle of pyrosequencing the development and impact of sequencing this article provides an overview of the sequencing technology direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach metagenomic analysis of viruses in reclaimed water metagenomic analysis of rna viruses in a fresh water lake these papers describe the metagenomic detection of viruses in environmental water samples by massively parallel pyrosequencing technology high-throughput genome sequencing of two listeria monocytogenes clinical isolates during a large foodborne outbreak variation in virulence among clades of escherichia coli o :h associated with disease outbreaks causes of outbreaks associated with drinking water in the united states from to single virus genomics: a new tool for virus discovery the authors describe a new approach that combines flow cytometry, whole genome amplification and pyrosequencing for the isolation and complete genome sequencing of a single virus particle. this approach has the potential to discover novel viruses and facilitate comparative genomic analyses by providing relevant reference genomes dead-end hollow-fiber ultrafiltration for recovery of diverse microbes from water tangential-flow ultrafiltration with integrated inhibition detection for recovery of surrogates and human pathogens from large-volume source water and finished drinking water quantification of viable legionella pneumophila cells using propidium monoazide combined with quantitative pcr use of propidium monoazide in reverse transcriptase pcr to distinguish between infectious and noninfectious enteric viruses in water samples selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology discrimination between live and dead cells in bacterial communities from environmental water samples analyzed by pyrosequencing quantitative real-time pcr analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater escherichia coli, enterococci, and bacteroides thetaiotaomicron qpcr signals through wastewater and septage treatment landscape of next-generation sequencing technologies the authors provide the current state of the art in next-generation sequencing technologies the authors demonstrate that metagenomic sequencing can provide an improved detection of unidentified pathogens in a public health setting rapid identification and quantification of campylobacter coli and campylobacter jejuni by real-time pcr in pure cultures and in complex samples quantification of viable but nonculturable escherichia coli o :h by targeting the rpos mrna failure to detect helicobacter pylori dna in drinking and environmental water in dhaka, bangladesh, using highly sensitive real-time pcr assays specific real-time pcr for simultaneous detection and identification of legionella pneumophila serogroup in water and clinical samples influence of environmental gradients on the abundance and distribution of mycobacterium spp. in a coastal lagoon estuary detection of live salmonella sp. cells in produce by a taqman-based quantitative reverse transcriptase real-time pcr targeting inva mrna development of a multiplex real-time pcr assay with internal amplification control for the detection of shigella species and enteroinvasive escherichia coli real-time pcr with an internal control for detection of all known human adenovirus serotypes a broadly reactive one-step real-time rt-pcr assay for rapid and sensitive detection of hepatitis e virus development and evaluation of a real-time pcr assay for quantification of giardia and cryptosporidium in sewage samples a duplex real-time pcr assay for the quantitative detection of naegleria fowleri in water samples pyrosequencing of the s rrna gene to reveal bacterial pathogen diversity in biosolids pathogenic bacteria in sewage treatment plants as revealed by pyrosequencing examples of how massively parallel pyrosequencing can be used to determine the diversity of bacterial pathogens in wastewater based on s rrna pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements using pyrosequencing, the authors demonstrate high levels of resistance genes in bacterial communities in river sediments exposed to antibiotics viral metagenome analysis to guide human pathogen monitoring in environmental samples another example of how pyrosequencing can be used to study the diversity of human pathogenic viruses in environmental samples. the authors also describe an improved bioinformatic approach for identifying pathogens in a virome data set key: cord- -xunyqlr authors: nan title: pathogen-inaktivierungssysteme für thrombozytenkonzentrate: stellungnahme date: - - journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: . /s - - - sha: doc_id: cord_uid: xunyqlr nan das auftreten neuer infektionskrankheiten oder die ausbreitung von identifizierten erregern außerhalb der bekannten endemiegebiete kann die transfusionssicherheit beeinträchtigen. bislang wurden in solchen fällen testmethoden adaptiert oder spenderauswahlkriterien verändert, um infizierte spender zu entdecken und von der spende auszuschließen. diese reaktive strategie erfolgte meistens mit einer zeitlichen verzögerung. die anwendung von inaktivierungsmethoden könnte die sicherheit von blutkomponenten hinsichtlich dieser risiken verbessern. die vorteile und grenzen dieser verfahren sollen gegen mögliche unerwünschte reaktionen und einschränkungen der produktqualität abgewogen werden. der begriff pathogeninaktivierung wird für methoden verwendet, die die infektiosität von pathogenen durch chemische reaktion oder durch physikalische einflüsse (z. b. uv-licht) zerstören. der begriff suggeriert, dass kein pathogen die behandlung überstehen kann. liegt jedoch eine hohe pathogen-kontamination vor oder ist das pathogen nicht empfindlich gegenüber dem spezifischen verfahren, kann es sein, dass die inaktivierungskapazität des verfahrens nicht ausreicht. es wäre deshalb besser von einer pathogenreduktion zu sprechen. im deutschen sprachgebrauch hat sich jedoch der begriff pathogeninaktivierung (pi) etabliert, so dass er auch durchgehend in dieser stellungnahme verwendet wird. in den vergangenen jahren wurden verschiedene methoden der pi für blutkomponenten entwickelt; einige systeme haben bereits ein ce-kennzeichen erhalten und sind damit innerhalb der eu verkehrsfähig. in deutschland ist darüber hinaus eine zulassung für die mit einem solchen system behandelten blutkomponenten erforderlich. die systeme wurden in klinischen studien getestet und werden mittlerweile in einigen ländern eingesetzt [ ] [ ] [ ] [ ] [ ] . es wird jedoch diskutiert, ob die angewandten inaktivierungsmethoden klinisch relevante veränderungen an den zellulären oder plasmatischen bestandteilen der blutkomponenten hervorrufen, die zu verminderter wirksamkeit oder unverträglichkeit führen bzw. immunreaktionen induzieren können. grundsätzlich zielen alle diese methoden auf die inaktivierung von nukleinsäuren von pathogenen. sie sind daher nur für die inaktivierung von dna-oder rna-haltigen pathogenen, nicht aber infektiösen proteinen wie prionen geeignet. sie können nur für blutkomponenten eingesetzt werden, die keine kernhaltigen zellen als wirkstoff enthalten, wie erythrozytenkonzentrate (ek), thrombozytenkonzentrate (tk) und therapeutisches plasma. in dieser stellungnahme werden nur systeme berücksichtigt, die eine ce-kennzeichnung erhalten haben [ ] und für die routinemäßige anwendung zur behandlung von thrombozytenkonzentraten zur verfügung stehen bzw. die gegenwärtig in klinischen prüfungen untersucht werden. die daten aus den hämovigilanzberichten / und [ , ] zeigen, dass in deutschland sowohl das risiko einer Übertragung von viralen erregern als auch das risiko einer transfusions-bedingten bakteriellen infektion durch tk [ ] sehr gering ist. in einem zeitraum von vier jahren ( - ) wurden , millionen tk-einheiten in deutschland transfundiert. die melderaten der einzelnen reaktionen ergeben sich aus der an- pathogen-inaktivierungssysteme für thrombozytenkonzentrate zahl bestätigter transfusionsreaktionen bezogen auf transfundierte tk-einheiten (. tab. ). allerdings ist hierbei zu berücksichtigen, dass einige spendeeinrichtungen bereits zusätzlich zu den angeordneten auflagen auf freiwilliger basis maßnahmen zur reduktion von bakteriellen infektionen durchführen (z. b. testung der produkte) und somit die häufigkeit dieser Übertragungen mutmaßlich bereits reduziert ist. die daten beziehen sich auf erreger, die bekannt sind und die durch spendertestung nachgewiesen werden bzw. nachgewiesen werden könnten. beim auf-treten neuer erreger ist ein mögliches Übertragungsrisiko anders einzuschätzen. vor allem für diesen fall könnte die pi die sicherheit von blutkomponenten weiter erhöhen, sofern der erreger für das angewandte pi-verfahren empfindlich ist. des weiteren führt die pi zu einer inaktivierung der noch im präparat befindlichen leukozyten, so dass nach pi eine bestrahlung der präparate mit gy zur vermeidung einer graft versus host (gvh) reaktion bei entsprechender indikation entfallen kann. aktuell haben in deutschland drei systeme zur pathogeninaktivierung von thrombozytenkonzentraten eine ce-kennzeichnung: intercept tm , mira-sol® und theraflex. aufgrund ihrer unterschiedlichen wirkmechanismen werden sie im folgenden zunächst separat beschrieben. die überwiegende mehrheit der heute zur verfügung stehenden daten zur inaktivierungskapazität und aus klinischen studien wurden von den herstellern oder in zusammenarbeit mit den herstellern veröffentlicht. das intercept tm blood system (in-tercept tm ) verwendet amotosalen-hcl ( -( -aminoethoxymethyl)- , , -trimethylfuro[ , -g]chromen- -one hydrochlorid, c h clno ). amotosalen, auch s- genannt, ist eine psoralen-verbindung mit hoher affinität zu nukleinsäuren [ ] . es durchdringt zellwände, kernmembranen und z. b. die lipidhülle von viren und lagert sich in die nukleinsäure ein [ ] . die reaktion, die letztendlich zur inaktivierung führt, erfolgt in drei stufen: ( ) die einlagerung von amotosalen in helikale regionen des genoms, ( ) die ausbildung einer kovalenten bindung von amotosalen mit einer pyrimidinbase im genom und ( ) die uva induzierte vernetzung (interstrang crosslinking) durch reaktion mit einer pyrimidinbase eines zweiten stranges [ ] . unter typischen reaktionsbedingungen liegen mehr als % der addukte strangvernetzt vor [ ] . durch die strangvernetzung wird eine transkription bzw. replikation der dna/rna verhindert. wegen der hohen vernetzungsdichte sind dna-reparaturmechanismen wirkungslos. die photoreaktion von amotosalen mit nukleinsäuren ist nicht sequenz-spezifisch. die reaktion erfolgt bevorzugt mit den pyrimidinbasen thymidin und cytidin; eine reaktion mit uracil ist sechs-bis achtmal langsamer [ ] . die bildung von psoralen-nukleotid-addukten ist hochspezifisch und erfolgt auch bei z. b. geringer konzentration von pathogenen und einem Überschuss von plasmaproteinen oder thrombozyten. interstrang-reaktionen sind möglich, so dass ein breites spektrum von nukleinsäuren einschließlich der von viren und anderen pathogenen inaktiviert werden kann [ ] in einem hämostase-globaltest (ro-tem®) war die gerinnungszeit nach tagen lagerung verglichen mit den kontrollen nur leicht verkürzt [ ] . trotz fehlens eines zellkerns enthalten thrombozyten mrna aus megakaryozyten. sie sind daher in begrenztem umfang zur proteinsynthese befähigt. ebenso verfügen thrombozyten über mirna, die post-transkriptional die genexpression beeinflussen und damit die proteinsynthese regulieren kann. eine reihe von untersuchungen zum einfluss von in-tercept tm auf mrna, mirna und das plättchenproteom zeigen veränderungen in unterschiedlichem ausmaß: in einer vergleichenden studie wurden nach pi mit intercept tm verringerte spiegel an von untersuchten mirna und an von anti-apoptotischen mrna gemessen [ ] . die autoren konnten keine quervernetzung der endogenen rna beobachten und sprechen von einer deregulation einzelner mrna. die verminderung der mirna-spiegel wurde auf deren vermehrte freisetzung in mikropartikeln zurückgeführt, was sowohl in tk mit additivlösung als auch in intercept tm -tk beobachtet werden konnte. die pi-technologie hatte in dieser untersuchung keinen einfluss auf synthese und funktion der mirna. dieser befund wird grundsätzlich gestützt durch untersuchungen von bakkour et al. [ unerwünschte effekte der pi von tk mit mirasol® betreffen mögliche toxische effekte der riboflavin-photoderivate, die bildung von neo-antigenen, veränderungen von in-vitro stoffwechsel-und funktionsparametern und einflüsse auf die mrna bzw. mi-rna sowie das proteom der thrombozyten. riboflavin ist nicht toxisch [ ] . die wichtigsten abbauprodukte des riboflavins als ergebnis des photochemischen prozesses sind lumichrom und lumiflavin. der riboflavinabbau ist zeitabhängig [ ] . umfangreiche toxikologische untersuchungen sind unternommen worden, um die sicherheit der mit mirasol® behandelten blutprodukte nachzuweisen (Übersicht in [ , ] ). die abbauprodukte von riboflavin treten auch als natürliche stoffwechselprodukte auf. experimentell wurde gezeigt, dass lumichrom und riboflavin nicht mutagen sind und auch keine chromosomenaberrationen durch bruch eines chromosoms induzieren. daher müssen überschüssiges riboflavin oder abbauprodukte nicht entfernt werden. untersuchungen im serum von patienten vor und tage nach transfusion von mirasol®-tk ergaben keine hinweise auf eine neo-antigen-bildung (capture p®technology) oder auf eine erhöhte rate an auto-und allo-antikörpern (pakauto und pak test) [ ] . die mirasol® behandlung bewirkt eine spontane thrombozytenaktivierung und damit verbunden einen signifikanten anstieg der anaeroben glykolyse für die energiegewinnung, gemessen als erhöhter glukoseverbrauch, gestiegene laktatproduktion und ph-erniedrigung [ ] [ ] [ ] [ ] [ ] [ ] . ausdruck der aktivierung sind u. a. die erhöhte p-selektin (cd p)-expression, annexin v-bindung sowie eine verstärkte freisetzung von thrombozytären zytokinen wie tgfβ und pf und die aktivierung des fibrinogenrezeptors gpiib/iiia. das ausmaß der beeinträchtigung von funktionsparametern wie die hypotone schockresistenz (hsr) und die mit unterschiedlichen agonisten stimulierte aggregation wird quantitativ unterschiedlich berichtet [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . abhängig von den versuchsbedingungen wurde sowohl über eine erhöhte spontan aggregation und verminderte thrombozytenadhäsion an kollagen oder subendothel [ , ] [ , , ] . dennoch finden sich nach einer pi mit mirasol® nur geringe veränderungen am gesamtproteom [ ] . veränderungen wurden gefunden bei proteinen, die für die zytoskelett-regulierung, also für adhäsion und "shape change" der thrombozyten, eine rolle spielen [ ] . der herstellungsprozess führte nur zu einem minimalen verlust an thrombozyten [ ] . von allen untersuchten in-vitro parametern zeigten nur laktatproduktion und ph bzw. glukoseverbrauch eine korrelation zur wiederfindung und Überlebensrate der thrombozyten in probanden [ , ] . der einfluss der mirasol® behandlung auf tk lässt sich wie folgt zusammenfassen: riboflavin und dessen abbauprodukte sind nicht toxisch und müssen daher nach der mirasol® behandlung nicht entfernt werden. während der entwicklung des uvc-verfahrens wurde die inaktivierung von viren, bakterien und protozoen in abhängigkeit von der uvc-dosis untersucht. dabei zeigte sich mit steigender uvc-dosis eine zunahme der inaktivierungseffizienz [ ] . das theraflex verfahren ist gegenwärtig für tk in additivlösung mit einer uv-dosis von , j/cm² ausgelegt [ ] . diese spezifikation wurde auch in der präklinischen entwicklung des theraflex uv-platelets verfahrens verwendet [ , ] . in den . tab. , und sind die inaktivierungskapazitäten mit dieser spezifikation zusammengefasst. auffällig sind die geringe wirksamkeit von uv zur inaktivierung von hiv und die relativ gute inaktivierung von nichtumhüllten viren wie hav und ppv [ , ] . andere umhüllte viren wie dengue virus (denv) - , chikungunyavirus (chikv), ross river virus (rrv) und zikavirus (zikv) wurden ebenfalls gut inaktiviert [ , ] . theraflex ist für die inaktivierung eines breiten spektrums von bakterien [ ] einschließlich der stämme des kürzlich etablierten referenzpanels der who [ ] geeignet (. tab. ). die wirkung von theraflex auf protozoen wurde mit trypanosoma cruzi, leishmania infantum, plasmodium falciparum und babesia divergens untersucht [ ] [ ] [ ] . in der inaktivierungsstudie mit babesien wurde in von kontaminierten tk eine vollständige inaktivierung erreicht (lrf ≥ , log ) und in den restlichen tk eine inaktivierung bis zur nachweisgrenze (lrf , log ). auch für die anderen protozoen (trypanosoma cruzi, leishmania unerwünschte effekte der pi von tk mit theraflex betreffen mögliche unverträglichkeiten durch die uv-bestrahlten tk, die bildung von neo-antigenen, veränderungen von in-vitro stoffwechselund funktionsparametern und einflüsse auf das rna-profil sowie das proteom der thrombozyten. in einem autologen hundemodell mit wiederholten gaben autologer uvc-tk wurden weder lokale noch systemische unverträglichkeiten beobachtet. ebenso wenig konnten antikörper gegen plasma-oder thrombozyten-neo-antigene nachgewiesen werden [ ] . in einer probandenstudie mit steigender dosis an autologen uvc-tk wurden zudem keine unerwünschten reaktionen beobachtet, und es konnten keine antikörper gegen neo-antigene gefunden werden [ ] . der einfluss der uvc-behandlung auf die in-vitro funktionalität der thrombozyten scheint gering zu sein. beschrieben sind eine erhöhte glykolyserate sowie ein moderater, aber signifikanter anstieg der annexin v-bindung und der gp iib/iiia-expression, während die werte für p-selektin oder zytokinfreisetzung (tgfβ, rantes) sich nicht von den kontrollen unterschieden; ebenso ppv , [ ] ist die aktivierbarkeit der thrombozyten nicht signifikant beeinträchtigt [ , ] . untersuchungen in der mikroflusskammer zeigten eine verminderte kinetik der gerinnselbildung an kollagen ab lagertag [ ] . es wurden nur geringere Änderungen im proteom der theraflex-behandelten thrombozyten gefunden. es sind vor allem proteine betroffen, die an der agonisten-induzierten veränderung der zell-morphologie (shape change) und der adhäsion beteiligt sind [ ] . der einfluss der theraflex-behandlung auf tk lässt sich wie folgt zusammenfassen: präklinische untersuchungen gaben keine hinweise auf unverträglichkeiten oder neo-antigen-bildung mit theraflex-tk. es kommt in vitro zu einem signifikanten anstieg der glykolyse. alle verfahren zeigen grundsätzlich eine gute inaktivierung von viren, haben aber auch limitierungen, die zu beachten sind. kwon und mitarbeiter verglichen z. b. die inaktivierung durch mirasol® und intercept tm . sie fanden eine bessere inaktivierung der umhüllten viren bvdv und prv durch intercept tm (bvdv , log vs. ≥ , log ; prv , log vs. ≥ , log ), während hiv durch intercept tm und mirasol® gleich gut inaktiviert wurde (≥ , log vs. ≥ , log ) [ ]. vergleichbar niedrige inaktivierung wurde in beiden systemen für die nicht-umhüllten viren berichtet (hav , log vs. , log und für ppv , log vs. , log ). jedoch konnte durch die behandlung mit mira-sol® eine reduktion von hev genotyp und eine reduktion von ≥ -≥ log erreicht werden [ ] . von anderen au-tab. inaktivierung von bakterien durch das theraflex uv-platelets system. die inaktivierungskapazität ist als logarithmischer faktor der abnahme der vermehrungsfähigkeit (log reduktionsfaktor, lrf) a dargestellt bakterien, gram-negativ enterobacter cloacae , ± , [ ] escherichia coli , ± , [ ] klebsiella pneumoniae , ± , [ ] morganella morganii , ± , [ ] proteus mirabilis , ± , [ ] pseudomonas aeruginosa ≥ , [ ] pseudomonas fluorescens , ± , [ ] serratia marcescens , ± , [ ] bakterien, gram-positiv bacillus cereus , ± , [ ] clostridium perfringens ≥ , [ ] propionibacterium acnes , ± , [ ] staphylococcus aureus , ± , [ ] staphylococcus epidermidis , ± , [ ] streptococcus bovis , ± , [ ] streptococcus dysgalactiae , ± , [ ] streptococcus pyogenes , ± , [ ] streptococcus pyogenes , ± , [ ] a lrf: ≥ bedeutet, dass im experiment eine vollständige inaktivierung erreicht wurde. die zahl gibt die nachweisgrenze an. aus den untersuchungen zur inaktivierung einer breiten palette von transfusionsrelevanten bakterienstämmen wurde geschlossen, dass alle drei verfahren das risiko bakterieller kontamination von tk reduzieren können. die inaktivierung sollte so früh wie möglich angewendet werden, um eine biofilmbildung [ , ] oder eine vermehrung kontaminierender bakterien auf eine hohe keimzahl zu verhindern. in beiden fällen könnte sonst die wirksamkeit der pi-systeme nicht ausreichend sein. durch die drei pi-verfahren werden auch protozoen inaktiviert, einschließlich t. cruzi, p. falciparum, l. infantum und b. microti. die höchsten lrf wurden nach inaktivierung mit intercept tm beschrieben. aufgrund ihres wirkmechanismus sind alle drei pi-systeme unwirksam zur inaktivierung von prionen. alle drei pi-systeme inhibieren die leukozytenproliferation und können die gammabestrahlung ersetzen. die transfusion markierter autologer pibehandelter tk zeigte eine signifikante verringerung sowohl der wiederfindungs-als auch der Überlebensrate verglichen mit unbehandelten tk bei allen drei verfahren [ , , [ ] [ ] [ ] . auch im klinischen einsatz kommt es nach der gabe von pathogeninaktivierten tk im vergleich zu unbehandelten tk im mittel zu einem geringeren anstieg der beim patienten gemessenen thrombozytenzahl. als messgröße wird hierzu der korrigierte anstieg der thrombozytenzahl stunde nach der tk-gabe verwendet (cci- h = -hour corrected count increment). tk ohne pi , ± , , ± , , ± , , ± , , ± , , ± , von klinisch größerer bedeutung ist jedoch die anzahl der (schwerwiegenden) blutungen bzw. der transfusionsbedarf. hierzu wurden in klinischen studien (s. . tab. ) und in der anwendung [ ] keine unterschiede gefunden. in systematischen reviews für inter-cept tm und mirasol® wurden schwerwiegende blutungen und refraktärität als endpunkte bewertet: der cochrane-report [ ] verglich studien mit insgesamt patienten, in denen pi-behandelte tk mit nicht behandelten verglichen wurden. neun von diesen betrafen in-tercept tm -tk. die untersuchten studien wiesen eine heterogenität hinsichtlich verschiedener aspekte wie endpunkt-definition, transfusionsprotokolle dauer der nachbeobachtung auf. die meta-analyse von fünf studien, die als endpunkt "blutung" definiert hatten, zeigte signifikant mehr blutungen über einen längeren betrachtungszeitraum mit einer auswertungsmethode (fixed effect model). dieses ergebnis zeigte sich nicht mehr in der metaanalyse mit einem anderen modell (random effects model). die patienten benötigten im mittel % mehr tk-transfusionen, und es fand sich ein signifikant höherer anteil an refraktärität. die autoren dieses reviews fanden zusammenfassend keine evidenz für unterschiede in mortalität, klinisch relevanter blutung, schwerer blutung oder unerwünschten ereignissen zwischen pi-tk und unbehandelten tk. für eine reihe von laborparametern zeigten sich unbehandelte tk überlegen gegenüber pi-behandelten. aufgrund der variabilität und der größe der einzelnen studien halten die autoren die daten jedoch nicht für ausreichend, um die schlussfolgerung zu ziehen, dass pi-behandelte tk gleichwertig sind. den vergleich erschweren qualitative (bis tage oder bis tage gelagert) und quantitative unterschiede (thrombozytenzahl/einheit) der verwendeten tk. bei einer aktualisierung des reviews konnten zwei weitere abgeschlossene studien mit zusammen teilnehmern einbezogen werden; drei weitere werden aktuell durchgeführt, diese wurden aber noch nicht in die analyse einbezogen [ ] . der erweiterte review bestätigte im wesentlichen die ergebnisse der ersten analyse: es fand sich weiterhin kei-ne oder wahrscheinlich keine evidenz für unterschiede in mortalität, klinisch relevanter blutung, schwerer blutung oder unerwünschten ereignissen zwischen pi-tk und unbehandelten tk. hinsichtlich des endpunkts "jede blutung" fanden die autoren erneut einen geringfügigen unterschied abhängig vom analysemodell zugunsten nicht-behandelter tk. die autoren fanden auch mit einschluss der neueren studien, dass die anwendung von pibehandelten tk signifikant häufiger zu refraktärität und auch zur entwicklung von anti-thrombozytären antikörpern führte. allerdings dominierte in dieser sub-analyse eine studie [ ] mit knapp % der daten. die autoren halten es für essentiell, dass unerwünschte effekte bei der anwendung von pi-behandelten tk sehr genau protokolliert werden. es gibt derzeit keine studien, welche inter-cept tm -tk und mirasol®-tk miteinander vergleichen. unterschiede in subgruppenanalysen weisen nach auffassung der autoren des aktualisierten cochrane-reviews auf einen vorteil von inter-cept tm hin bezüglich gesamtmortalität und transfusionsintervall. sämtliche eingeschlossenen studien betrafen ganz überwiegend (> %) hämatologisch-onkologische patienten, so dass hinsichtlich der untersuchten parameter keine aussage zur wirksamkeit und zu potenziellen unerwünschten reaktionen bei anderen patientengruppen getroffen werden kann. in einer der prospektiven, randomisierten open-label studie wurden tk in plasma, tk in additivlösung und in-tercept tm -tk mit dem primären studienziel verglichen, unterschiede im -h-cci zu finden. die autoren fanden verringerte -stunden und -stunden cci-werte und mehr blutungen (insbesondere auch fünf grad blutungen) im intercept tm -arm gegenüber einer bei plasma-tk und bei tk in additivlösung [ ] . der unterschied in der blutungshäufigkeit ist u. a. wegen des studiendesigns und der zu geringen anzahl der in die studie eingeschlossenen patienten nicht signifikant [ ] . eine publizierte, prospektive, randomisierte noninferiority studie an hämatologischen, thrombozytopenischen patienten hat ebenfalls die wirksamkeit von inter-cept tm -tk mit tk in plasma bzw. tk in additivlösung verglichen. unterschiede wurden nur für grad- -blutungen zwischen pi-tk ( , %) und plasma-tk ( , %), nicht aber zu tk in additivlösung ( , %) gefunden. die studienarme unterschieden sich nicht hinsichtlich grad- -und - -blutungen [ ] . es liegen noch keine ergebnisse aus klinischen phase-iii-studien mit the-raflex-tk vor. eine zusammenfassung der möglichen risiken bei der gabe von pathogenreduzierten thrombozytenkonzentraten hergestellt mit intercept tm , mira-sol® oder theraflex findet sich in . tab. . die wirksamkeit von intercept tm -tk bei einem routinemäßigen einsatz lässt sich auf der grundlage der schweizer hämovigilanzdaten relativ gut darstellen. seit der einführung der pathogeninaktivierung für thrombozytenkonzentrate im jahr wurden demnach der schweizer Überwachungsbehörde keine transfusionsassoziierten bakteriellen infektionen gemeldet. des weiteren kam es zu einer abnahme von nicht schwerwiegenden allergischen transfusionsreaktionen, was auf die geänderte zusammensetzung der lagerlösung zurückgeführt wurde. eine gleichzeitige landesweite zunahme des verbrauchs an tk oder ek als hinweise einer eventuell eingeschränkten hämostatischen wirkung der intercept tm -tk wurde nicht beobachtet [ ] . gleiches wurde von einem großen österreichischen klinikum berichtet [ ] . aufgrund der bisher vorliegenden sicherheitsdaten kann für alle drei pi-systeme ein verminderter thrombozytenanstieg gegenüber einer entsprechenden unbehandelten kontrollgruppe festgestellt werden. ein erhöhter ek-oder tk-verbrauch oder eine erhöhte blutungsneigung lassen sich mit den veröffentlichten klinischen studien nicht belegen. unerwartete reaktionen wie allergische reaktionen, immunologische refraktärität und phototoxizität bei neugeborenen wurden diskutiert, aber studien-wie auch die hämovigilanzdaten geben bislang keinen hinweis auf ein nachweisbares risiko [ ] . die auswertung der schweizer in-tercept tm -hämovigilanzdaten (spontanmeldungen) für die jahre / ergab keine zunahme der melderate von schwerwiegenden respiratorischen komplikationen [ ] . eine mögliche toxizität, kanzerogenität und genotoxizität bei dem empfänger von pathogen-inaktivierten tk wurde wiederholt thematisiert und im rahmen von pharmakologisch-toxikologischen studien untersucht. die ergebnisse der präklinischen pharmakokinetischen und toxikologischen studien sowie der vorliegenden klinischen studien (s. . tab auch bei einsatz von pi-verfahren sind Übertragungen von pathogenen aufgrund von limitierungen im wirkungsspektrum und in der kapazität der pi-methoden nicht auszuschließen. treten allerdings neue erreger auf oder ändert sich die epidemiologische situation, kann die einführung von pi-tk einen erheblichen sicherheitsgewinn erbringen, sofern die erreger durch das verfahren inaktiviert werden können. der sicherheitsgewinn tritt insbesondere dann ein, wenn andere präventive maßnahmen, wie spenderselektion und -testung, nicht praktikabel sind oder nicht den erfordernissen entsprechend schnell eingeführt werden können und nur dadurch die versorgung gewährleistet werden kann. diese voraussetzungen treffen aktuell nicht zu. dennoch sollten bereits jetzt strategien für die implementierung und finanzierung von pathogen-inaktivierten blutkomponenten erarbeitet werden. die bewertung, ob eine geänderte epidemiologische situation vorliegt, welche die einführung von pi-maßnahmen erforderlich macht, sollte situationsbezogen, aber mindestens alle zwei jahre durchgeführt werden. evaluation of the effectiveness of a pathogen inactivation technology against clinically relevant transfusion-transmitted bacterial strains. dieses papier wurde fertiggestellt am . . und vom arbeitskreis blut am . . verabschiedet. es wurde erarbeitet von den mitgliedern der untergruppe "bewertung blut-assoziierter krankheitserreger" des arbeitskreises blut: prof. dr. markus funk, dr. margarethe heiden listing of countries in which pathogen reduction technology systems and products are in use development of blood transfusion product pathogen reduction treatments: a review of methods, current applications and demands pathogen reduction technologies update on pathogen reduction technology pathogen inactivation technologies for cellular blood components: an update / of the european parliament and of the council of april on medical devices, amending directive / /ec, regulation (ec) no / and regulation (ec) no / and repealing council directives auswertung der meldungen von schwerwiegenden reaktionen und zwischenfällen nach § i amg monitoring bacterial contamination of blood components in germany: effect of contamination reduction measures pathogen inactivation of platelet and plasma blood components for transfusion using the intercept tm blood system cerus pressemitteilung vom . . . http:// www.cerus.com/investors/press-releases/ press-release-details/ /fda-approves-intercept-blood-system-for-plasma/default. aspx. zugegriffen cerus pressemitteilung vom . . . http:// www.cerus.com/investors/press-releases/ press-release-details/ /fda-approves-intercept-blood-system-for-platelets/default. aspx. zugegriffen the extent of amotosalen photodegradation during photochemical treatment of platelet components correlates with the level of pathogen inactivation photochemical inactivation of viruses with psoralens: an overview fundamentals of the psoralen-based helinx technology for inactivation of infectious pathogens and leukocytes in platelets and plasma isolation and characterization of pyrimidinepsoralen-pyrimidine photodiadducts from dna lc-ms/ms analysis and comparison of oxidative damages on peptides induced by pathogen reduction technologies for platelets redox proteomics and platelet activation: understanding the redox proteome to improve platelet quality for transfusion photochemical inactivation of pathogenic bacteria in human platelet concentrates photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light inactivation of cytomegalovirus in platelet concentrates using helinx technology photochemical treatment of platelet concentrates with amotosalen and long-wavelength ultraviolet in vitro assessment of buffy-coat derived platelet components suspended in ssp+ treated with the intercept blood system implementation of the intercept blood system for platelets into routine blood bank manufacturing procedures: evaluation of apheresis platelets functional characteristics of buffy-coat plts photochemically treated with amotosalen-hci for pathogen inactivation pathogen inactivation of double-dose buffy-coat platelet concentrates photochemically treated with amotosalen and uva light: preservation of in vitro function leukoreduced buffy coat-derived platelet concentrates photochemically treated with amotosalen hcl and ultraviolet a light stored up to days: assessment of hemostatic function under flow conditions platelet function assessed by shear-induced deposition of split triple-dose apheresis concentrates treated with pathogen reduction technologies preserved functional and biochemical characteristics of platelet components prepared with amotosalen and ultraviolet a for pathogen inactivation effects of intercept pathogen inactivation on platelet function as analysed by free oscillation rheometry effects of pathogen reduction systems on platelet micrornas, mrnas, activation, and function assessment of nucleic acid modification induced by amotosalen and ultraviolet a light treatment of platelets and plasma using real-time polymerase chain reaction amplification of variable length fragments of mitochondrial dna activated platelets can deliver mrna regulatory ago •microrna complexes to endothelial cells via microparticles platelets deliver small packages of genetic function differential expression analysis by rna-seq reveals perturbations in the platelet mrna transcriptome triggered by pathogen reduction systems proteomic analysis of intercept-treated platelets proteome changes in platelets after pathogen inactivation--an interlaboratory consensus pathogen reduction technology system pathogen inactivation of blood components: current status and introduction of an approach using riboflavin as a photosensitizer pathogen reduction technology treatment of platelets, plasma and whole blood using riboflavin and uv light establishment of culture systems for genotypes and hepatitis e virus (hev) obtained from human blood and application of hev inactivation using a pathogen reduction technology system photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light inactivation of viruses in platelet and plasma products using a riboflavin-and-uv-based photochemical treatment riboflavin and ultraviolet light: impact on dengue virus infectivity the effect of riboflavin and ultraviolet light on the infectivity of arboviruses photochemical inactivation of chikungunya virus in plasma and platelets using the mirasol pathogen reduction technology system riboflavin and ultraviolet light for pathogen reduction of murine cytomegalovirus in blood products the mirasol tm prt system for pathogen reduction of platelets and plasma: an overview of current status and future trends efficacy of the mirasol pathogen reduction technology system against severe fever with thrombocytopenia syndrome virus (sftsv) inactivation of orientia tsutsugamushi in red blood cells, plasma, and platelets with riboflavin and light, as demonstrated in an animal model evaluation of the mirasol pathogen reduction technology system against babesia microti in apheresis platelets and plasma riboflavin and ultraviolet light reduce the infectivity of babesia microti in whole blood reduction of leishmania donovani infectivity in whole blood using riboflavin and ultraviolet light inactivation of plasmodium falciparum in whole blood by riboflavin plus irradiation inactivation of plasmodium spp. in plasma and platelet concentrates using riboflavin and ultraviolet light evaluating pathogen reduction of trypanosoma cruzi with riboflavin and ultraviolet light for whole blood pathogen inactivation of trypanosoma cruzi in plasma and platelet concentrates using riboflavin and ultraviolet light bacterial contamination of blood components a laboratory comparison of pathogen reduction technology treatment and culture of platelet products for addressing bacterial contamination concerns functional inactivation of white blood cells by mirasol treatment mirasol prt treatment of donor white blood cells prevents the development of xenogeneic graft-versus-host disease in rag -/-γc-/-double knockout mice understanding loss of donor white blood cell immunogenicity after pathogen reduction: mechanisms of action in ultraviolet illumination and riboflavin treatment white blood cell inactivation after treatment with riboflavin and ultraviolet light inactivation of human white blood cells in platelet products after pathogen reduction technology treatment in comparison to gamma irradiation treatment of whole blood with riboflavin plus ultraviolet light, an alternative to gamma irradiation in the prevention of transfusion-associated graft-versus-host disease development of a riboflavin and ultraviolet light-based device to treat whole blood riboflavin's time-dependent degradation rate induced by ultraviolet a irradiation lack of antibody formation to platelet neoantigens after transfusion of riboflavin and ultraviolet light-treated platelet concentrates pathogen reduction technology (mirasol) treated single-donor platelets mitteilungen resuspended in a mixture of autologous plasma and pas platelet glycolytic flux increases stimulated by ultraviolet-induced stress is not the direct cause of platelet morphology and activation changes: possible implications for the role of glucose in platelet storage cell quality of apheresis-derived platelets treated with riboflavin-ultraviolet light after resuspension in platelet additive solution in vitro cell quality of buffy coat platelets in additive solution treated with pathogen reduction technology the effect of pathogen reduction technology (mira-sol) on platelet quality when treated in additive solution with low plasma carryover in vitro quality of single-donor platelets treated with riboflavin and ultraviolet light and stored in platelet storage medium for up to days effects of a new pathogen-reduction technology (mirasol prt) on functional aspects of platelet concentrates effects of mirasol prt treatment on storage lesion development in plasma-stored apheresis-derived platelets compared to untreated and irradiated units evaluation of white blood cell-and platelet-derived cytokine accumulation in mirasol-prt-treated platelets annexin v release and transmembrane mitochondrial potential during storage of apheresis-derived platelets treated for pathogen reduction treatment of buffy coat platelets in platelet additive solution with the mirasol® pathogen reduction technology system pathogen reduction treatment using riboflavin and ultraviolet light impairs platelet reactivity toward specific agonists in vitro impact of pathogen reduction technology and storage in platelet additive solutions on platelet function effects of riboflavin and ultraviolet light treatment on platelet thrombus formation on collagen via integrin αiibβ activation riboflavin and ultraviolet illumination affects selected platelet mrna transcript amounts differently oxygen removal during pathogen inactivation with riboflavin and uv light preserves protein function in plasma for transfusion treatment of platelet concentrates with the mirasol pathogen inactivation system modulates platelet oxidative stress and nf-κb activation riboflavin and ultraviolet light treatment potentiates vasodilator-stimulated phosphoprotein ser- phosphorylation in platelet concentrates during storage efficacy of apheresis platelets treated with riboflavin and ultraviolet light for pathogen reduction heyns adu p ( ) correlation of in vitro platelet quality measurements with in vivo platelet viability in human subjects macopharma theraflex uv-platelets uvc irradiation for pathogen reduction of platelet concentrates and plasma characteristics of the theraflex uv-platelets pathogen inactivation system -an update a novel approach to pathogen reduction in platelet concentrates using short-wave ultraviolet light tolerance of platelet concentrates treated with uvc-light only for pathogen reduction -a phase i clinical trial mitochondrial dna multiplex real-time polymerase chain reaction inhibition assay for quality control of pathogen inactivation by ultraviolet c light in platelet concentrates two pathogen reduction technologiesmethylene blue plus light and shortwave ultraviolet light -effectively inactivate hepatitis c virus in blood products effect of increased agitation speed on pathogen inactivation efficacy and in vitro quality in uvc-treated platelet concentrates inactivation of dengue, chikungunya, and ross river viruses in platelet concentrates after treatment with ultraviolet c light pathogenreduktion mittels uvc-licht: entwicklung des theraflex uv-plättchen-systems. dgti, freiburg influenza a virus h n is efficiently inactivated by the theraflex uv-platelets system reduction of zika virus infectivity in platelet concentrates after treatment with ultraviolet c light and in plasma after treatment with methylene blue and visible light hepatitis a virus is efficiently inactivated by the theraflex uv-platelets system enlargement of the who international repository for platelet transfusionrelevant bacteria reference strains the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates the effectiveness of uvc pathogen inactivation system on reducing the trypanosoma cruzi and leishmania infantum burden in platelets the efficacy of uvc pathogen inactivation on the reduction of plasmodium falciparum in buffy coat derived platelets the efficacy of the ultraviolet c pathogen inactivation system in the reduction of babesia divergens in pooled buffy coat platelets pathogen reduction by ultraviolet c light effectively inactivates human white blood cells in platelet 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ultraviolet a light radiolabeling of plts to assess viability: a proposal for a standard summary topic ii: new standards for platelet evaluation swiss medic haemovigilance annual report . www.swissmedic.ch/haemovigilancereport pathogen-reduced platelets for the prevention of bleeding pathogen-reduced platelets for the prevention of bleeding therapeutic efficacy and safety of platelets treated with a photochemical process for pathogen inactivation: the sprint trial clinical effectiveness of leucoreduced, pooled donor platelet concentrates, stored in plasma or additive solution with and without pathogen reduction therapeutic efficacy of platelet components treated with amotosalen and ultraviolet a pathogen inactivation method: results of a meta-analysis of randomized controlled trials comparison of the hemostatic efficacy of pathogen-reduced platelets vs untreated platelets in patients with thrombocytopenia and malignant hematologic diseases: a randomized clinical trial a randomized controlled clinical trial evaluating the performance and safety of platelets treated with mirasol pathogen reduction technology clinical safety of platelets photochemically treated with amotosalen hcl and ultraviolet a light for pathogen inactivation: the sprint trial a patient-oriented risk-benefit analysis of pathogen-inactivated blood components: application to apheresis platelets in the united states impact of platelet pathogen inactivation on blood component utilization and patient safety in a large austrian regional medical centre a prospective, active haemovigilance study with combined cohort analysis of , transfusions of platelet components prepared with amotosalen-uva photochemical treatment einführung der pathogeninaktivierung für thrombozytenkonzentrate in der schweiz key: cord- - twm hmc authors: vourc’h, gwenaël; plantard, olivier; morand, serge title: how does biodiversity influence the ecology of infectious disease? date: - - journal: new frontiers of molecular epidemiology of infectious diseases doi: . / - - - - _ sha: doc_id: cord_uid: twm hmc over the past years, biodiversity has been reduced on an unprecedented scale, while new infectious diseases are emerging at an increasing rate. greater overall biodiversity could lead to a greater diversity of hosts and thus of pathogens. yet disease regulation – due to the buffering role of host diversity – is considered to be one of the services provided by biodiversity. in this chapter, we ask how biodiversity is linked to infectious disease. first, we investigate the influence of the biodiversity of pathogens. we highlight that the number of pathogen species is not well known but that new findings are facilitated by the rapid expansion of molecular techniques. we show that, although there is a trend to find higher pathogen richness toward the equator, identifying a global pattern between the richness of all pathogen species and their latitudinal distribution is challenging. we emphasize that pathogen intraspecific diversity is a crucial factor in disease emergence and allows pathogens to adapt to the selective pressures they face. in addition, the selective pressure acting on hosts due to parasite, and reinforced by parasite diversity within hosts seems to be a major evolutionary and ecological force shaping hosts biodiversity. second, we investigate how the diversity of hosts influences infectious disease ecology. for multi-host diseases, a change in host species richness or abundance can modify the dynamics of local infectious diseases by either reducing (“dilution effect”) or increasing (“amplification effect”) the risk of transmission to the targeted host species. the underlying hypothesis is that, the competence of reservoirs varies according to the host species. the dilution effect has been demonstrated mainly through theoretical work and there have been only few case studies. regarding the genetic diversity of host, an important issue is: to what extent does a reduction of this diversity impact the ability of the host population to response to infectious diseases? third, we rapidly examine the role of biodiversity in the treatment of infectious diseases. to conclude, we consider that the consequences of the loss of species biodiversity on infectious diseases is still largely unknown, notably due to the lack of knowledge on the dynamics of host-pathogen relationships, especially at the population and at the community level.. we highlight that work on multi-host/ ulti-pathogen systems should be fostered and that new approaches, such as metagenomic investigations that does not require a priori assumptions, are promising to describe a community of pathogens and their interactions. investigate how the diversity of hosts influences infectious disease ecology. for multi-host diseases, a change in host species richness or abundance can modify the dynamics of local infectious diseases by either reducing ("dilution effect") or increasing ("amplification effect") the risk of transmission to the targeted host species. the underlying hypothesis is that, the competence of reservoirs varies according to the host species. the dilution effect has been demonstrated mainly through theoretical work and there have been only few case studies. regarding the genetic diversity of host, an important issue is: to what extent does a reduction of this diversity impact the ability of the host population to response to infectious diseases? third, we rapidly examine the role of biodiversity in the treatment of infectious diseases. to conclude, we consider that the consequences of the loss of species biodiversity on infectious diseases is still largely unknown, notably due to the lack of knowledge on the dynamics of host-pathogen relationships, especially at the population and at the community level.. we highlight that work on multi-host/ ulti-pathogen systems should be fostered and that new approaches, such as metagenomic investigations that does not require a priori assumptions, are promising to describe a community of pathogens and their interactions. over the past years, human activity has altered habitats and reduced biodiversity on an unprecedented scale that comes close to mass extinction (mea ) . at the same time, new infectious diseases seem to be emerging at an increasing rate (wilcox and gubler ) . during this period, there has been a dramatic spread of highly pathogenic diseases such as aids and multi-drug resistant bacterial infections, and in more recent years sars, west nile in north america, and highly pathogenic influenza viruses (jones et al. ) . habitat loss, largely a result of the conversion of forests and savannas into agricultural land, cities, and industrial sites, is the major cause of change in biodiversity. biodiversity represents the diversity of life at all levels of biological organization, from the genes within populations, the species that compose a community, to the communities that compose ecosystems. intuitively, one might assume that greater overall biodiversity would lead to a greater diversity of pathogens and hosts, and thereby increase the incidence of infectious diseases (dunn et al. ). yet disease regulation is said to be one of the services provided by biodiversity because a high level of species diversity creates a buffer that reduces the risk of transmission (mea ; walpole et al. ). scientific evidence supporting both of these views is beginning to emerge, but the core question remains: how is biodiversity linked to infectious disease? this is the question addressed in this chapter. pathogens are organisms that have a negative impact on the fitness of their host(s), often, if now always, by producing visible symptoms (e.g. a disease). such trophic interaction between two organisms, a host and a parasite, is just one of several interactions that take place within communities and ecosystems, others being those of prey-predator and plant-phytophagous for instance (begon et al. ) . to date, more attention has been paid to these other interactions, and their roles in ecosystem functioning (e.g. steffan and snyder ) , than to pathogen-host interactions, and food web studies only recently have begun to take parasites into consideration (arias-gonzález and morand ; lafferty et al. ) . studies incorporating pathogens are scarce (hudson et al. ) , probably due to the difficulties of surveying pathogens (using intrusive or even destructive sampling methods…). moreover, the systematics and even basic aspects of parasite biology often are unknown. however, although numerous species of pathogens still need to be described (dobson et al. ) , there is no doubt that pathogens represent a large part of biodiversity on earth. given that each free living species is host to numerous pathogens, and that pathogens of pathogens also exist (consider, for example, phages that are virus affecting bacteria), several authors believe that pathogens may be the most diverse living group on earth (windsor ) . the link between biodiversity and the ecology of infectious diseases is not simple. in this chapter, we investigate how biodiversity influences the ecology of infectious diseases at the intraspecific level (genetic variability of pathogens and hosts) and at the level of communities (species composition). although we mainly provide examples from human and animal diseases, we also use some illustrations from plants. we describe patterns of biodiversity and the consequences of changes in biodiversity on the ecology of infectious diseases. lastly, we rapidly examine the role of biodiversity in the treatment of infectious diseases. infectious disease ecology? we shall consider infectious diseases caused by bacteria, virus, fungi, protozoa and endo-parasites, and exclude from our analysis ecto-parasites that are considered here as disease vectors. in the light of the discussion above, the pathogen status of a given living organism clearly is not a straightforward question (consider, for example, the case of some rickettsia species that are considered to be not only blood vertebrate pathogens, but also tick symbionts, perlman et al. ). this status is related to the host, and varies with individual hosts and species as well as in space and in time (different hosts, for example, can have different resistance or susceptibility). when pathogens have complex life-cycles, some stages may have a different biology (such as biotrophic or necrotrophic plant pathogens, morris et al. ). furthermore, horizontal gene transfer is so extensive in bacteria that many microbio logists question the existence of species in bacteria, preferring to consider bacteria as populations that exchange genes. however, the existence of core genes responsible for the maintenance of species-specific phenotypic clusters is an argument supporting the identification of bacterial species (riley and lizotte-waniewski ) . for these reasons, combined with the limited knowledge available of the systematics of many pathogens (brooks and hoberg ) , it is difficult to accurately estimate the number of pathogen species. estimations of pathogen species richness vary from % to % of living beings (de meeûs et al. ; poulin and morand ) . in estuaries, the biomass of macro and micro-parasites has been estimated as exceeding that of top predators . although the existence of pathogens has been known for a long time, lists of species only were compiled recently for human and animals (ashford and crewe ; cleaveland et al. ; taylor et al. ) , with an update on human pathogens completed in (woolhouse and gaunt ) . approximately , human pathogens were reported, livestock pathogens (cattle, sheep, goats, pigs and horses), and domestic carnivore pathogens (dogs and cats). no clear figure was given for wildlife (but see the global mammal parasite database at http://www.mammalparasites.org/). on average, over two new species of human viruses also are discovered each year (woolhouse et al. ) . pathogens affecting humans have received more attention than those affecting other species. if one assumes that other animal species are affected in a proportional manner, huge numbers of pathogens remain to be discovered. altogether, , , , and , of fungi, viruses and bacteria respectively have been described, which represent only %, %, and - % of the total estimated number of species of fungal, viral and bacterial species. it is difficult to know the number of plant pathogens, but a significantly proportion of the fungal, viral and bacterial species are likely to be plant pathogens (ingram ) . until recently, many new pathogen discoveries relied on the investigation of atypical symptoms. today, new findings are facilitated by molecular techniques that render it possible to detect and characterise unculturable pathogens and to investigate the presence of genes and genomes independently of individuals (metagenomics). although multi-host pathogens are more numerous than single hosts, interactions between pathogens and hosts can evolve towards the specialisation of pathogens on a given host species (cleaveland et al. ; huyse et al. ; pedersen et al. ) . such a specialisation can lead to speciation, id est the birth of a new pathogen species. co-cladogenesis, a process of parallel diversification in hosts and pathogens, also can give birth to numerous pathogen species (page ) . until the development of molecular tools, these species were very difficult to distinguish (cryptic species). systematic investigations using molecular tools have made it possible, however, to reveal a high diversity of pathogens. for instance, in a systematic inventory of viruses in various vertebrate hosts conducted over a year period in the central african republic, different viruses were isolated, including new ones (saluzzo et al. ) . two species of plasmodium, p. falciparum, infecting humans, and p. reichenowi, infecting chimpanzees, were long considered to be within the clade that includes humans and the great apes. however, recent studies of apes in their natural habitat have revealed a much higher diversity of species infecting great apes; in addition, it has been found that p. falciparum also infect gorillas (liu et al. ; prugnolle et al. ) and are at the origin of human malaria. metagenomic studies in ecosystems such as human faeces (zhang et al. ) and marine sediments (breitbart et al. ) also have revealed that the majority of viral sequences found did not match in the databanks. finally, new investigations have been launched to monitor people, animals and animal die-offs in areas where people have a high exposure to wildlife. generic, broad screening tools will be used to detect pathogen species (wolfe et al. ). to our knowledge, a similar approach has not yet been implemented for pathogens of animals or plants. in addition to the inventories of pathogen biodiversity, scientists have investigated which part of the world holds the highest diversity of pathogen species. many studies on plants and animals have shown that species richness decreases the further one moves away from the equator. the reasons most likely are linked to the area, energy, time and habitat heterogeneity, and geometric constraints (gaston and blackburn ) . comparative studies exploring pathogen species richness in the tropics compared to temperate zones are scarce and have produced discrepant results. guernier et al ( ) studied the worldwide distribution of human pathogens (bacteria, virus, fungi, protozoa, and helminths) according to environmental, demographic and economical factors. they found that parasite species richness decreased with latitude and had a spatially nested organization; i.e. some species are widely distributed and occur in many communities while others have more restricted distributions and occur only in a subset of locations. such findings were confirmed by the analysis of dunn et al. ( ) , who showed that human pathogen diversity was strongly related to both mammal and bird species richness. diseases that occur in temperate zones also tend to occur in the tropics, while some tropical diseases are restricted only to the tropics. in primates, the number of protozoa species, which primarily are vector-borne transmitted, increase as one approaches the equator, however, the same trend was not found for viruses and helminths (nunn et al. ). lindenfors et al ( ), who examined the parasite richness of parasite species and terrestrial carnivore species, found that helminth parasite species richness increased the further away one moved from the equator. the reason for this finding is unknown and may be related to a bias in sampling because carnivores inhabiting areas of industrialized countries in the northern hemisphere may have been sampled more intensely. poulin ( ) and bordes et al ( ) did not find any correlation between helminth species richness at intra or interspecific levels and latitude. some studies have shown higher tick species richness at lower latitudes (cumming ) . however, this is not the case for flea species, which have been found to have low richness at lower latitudes (krasnov et al. ) . a final example is ichneumonid parasitoid hymenoptera. although a higher specific host diversity is found in the tropics, the number of species of this parasitoid group is similar in both tropical and temperate regions. it has been hypothesised that this is due to habitat fragmentation (leading to a lower density of hosts); lack of seasonality (and thus of a host population dynamics with peaks and high density of hosts), or the higher content in toxic compounds of tropical plants and thus in phytophagous insects (the "nasty hypothesis") (gauld et al. ) . a meta-analysis of parasite-associated host mortality (robar et al. ) revealed that host mortality risk declined as one moves away from the equator, indicating that, in addition to parasitic load, the effect of parasites on host mortality might be determined by some abiotic factors. thus, although there is a trend to find higher pathogen richness as we move toward the equator, it is thus challenging to identify a global pattern between the richness of all pathogen species and their latitudinal distribution. however, it should be noted that of the pathogens that have been discovered since , most have a global distribution (woolhouse and gaunt ) . pathogens generally are characterised as having higher mutation rates and generation times than those of their hosts (hamilton et al. ). genetic variability also results from recombination during sexual reproduction of eukaryotic pathogens, and any other genetic exchange mechanisms such as bacterial conjugation or viral recombination. in addition, many animal and plant pathogens use a vector to increase gene flow among populations and to reach a new individual host. this genetic diversity is a crucial factor in disease emergence (cleaveland et al. ) and allows pathogens to adapt to the main selective pressures they face: hosts' immune systems, the need to be transmitted, and treatments or vaccines used to counter infections. the capacity of some pathogens to genetically diversify facilitates their ability to evade host immune systems. one of the best examples is the human immunodeficiency virus (hiv), which is able to change its appearance faster than the time its takes for the immune system to reply (drosopoulos et al. ). another example is p. falciparum, which generates high levels of variability in genes involved in antigenic variability and virulence (var genes) by producing frequent recombination events between heterologous chromosomes (freitas-junior et al. ) . high genetic variation of pathogens also is involved in the interspecies infection process as it facilitates the infection of a broader range of host species, which is another characteristic of emerging pathogens (cleaveland et al. ; woolhouse and gowtage-sequeria ) . the evolutionary potential of pathogens allows them to respond quickly to the directional selective pressure provided by the massive use of drugs (palumbi ) . in areas where selective pressure is important, such as in hospitals, multiresistant bacteria are very frequent (levy and marshall ) . for bacteria, resistant genes probably originated from environmental organisms with which they shared their ecological niche (aminov and mackie ) . these genes can be transferred between different species of bacteria and even between species that colonize different hosts (nikolich et al. ) . although vaccination is a major advance of modern medicine, it thus far has contributed to the eradication of only one infectious disease in humans (small pox, www.who.int/mediacentre/factsheets/smallpox/en) and one in cattle, buffalo and wildebeest (rinderpest, normile ) . as many vaccines do not totally block transmission, vaccination modifies the selective pressure on pathogens. depending on how vaccines act on the pathogen, the epidemiology consequences can differ (gandon and day ) . for instance, theoretical work has shown that vaccines that reduce the growth rate or toxicity of pathogens also reduce selection pressure against virulent pathogens, leading to higher intrinsic virulence (gandon et al. ). in the poultry industry, an increase in virulence of avian tumour viruses has followed the use of vaccines that reduce virus growth rates (witter ) . although plants lack an adaptive immune system, through evolution they have developed various strategies to stop plant pathogen infections. an induced or acquired systemic resistance occurs following host recognition of a pathogen, which triggers the production of a hypersensitive reaction (jones and dangl ) . through this mechanism, the plant provides itself protection from secondary infection in distal tissues, even if the plant faces a pathogen for which it does not have the resistance gene (durrant and dong ). the immune system of vertebrates acquires its efficiency by being exposed to a diverse array of pathogens. the striking increase in hygiene standards that began in the early twentieth century has considerably lowered humans' exposure to pathogens, at least in developed countries. the immune response triggered by a pathogen can provide some cross protection against other pathogens (e.g. huang et al. ) . a low exposure to a diversity of pathogens has had immediate consequences in decreasing the risk of disease. but could this low exposure also induce evolutionary change in the ability of a host to respond to infection? due to a trade off between investment in disease resistance and other traits linked to fitness, low exposure could decrease the frequency of resistance down through the generations, setting the stage for a potentially devastating outbreak (altizer et al. ; graham et al. ) . domestic species that are bred to be protected from pathogens might be more susceptible to infectious diseases (lyles and dobson ) . furthermore, it has been suggested that on islands, where some pathogens may be absent, hosts may have lower immune response abilities (island syndrome) (lee and klasing ) . however, studies that have tested this hypothesis, both using experimental and theoretical approaches, have had contrasting results (beadell et al. ; hochberg and møller ; matson ) . infections by different species of pathogens or by different strains of the same species within the same individual host or vector are common (abbot et al. ; cox ) . in fact, parasite diversity in hosts seems to be a major evolutionary and ecological force for hosts (bordes and morand ). these concomitant infections can trigger cross-effective immune responses between pathogens that are antigenically similar, having thus an impact on the issue of the infection (lee et al. ). an infection also can enhance susceptibility to subsequent infection (cattadori et al. ). in particular, individuals with already are in poor physical condition may be more susceptible to multiple infections (beldomenico and begon ; telfer et al. ) . furthermore, concomitant infection may allow the exchange of genetic material between strains of a given pathogen species or even between species through horizontal gene transfer (see sect. . . above), allowing the emergence of new virulent strains. an extreme case is one in which a pathogen drives the extinction of a population or species. such scenarios are rare but do occur, generally due to a conjunction of pathogens and other causes. for instance, the decline of amphibian populations around the world is thought to be linked to a fungal pathogen batrachochytrium dendrobatidis causing chytridiomycosis (crawford et al. ). amphibians could have an increased susceptibility to the fungus due to changes in temperature variability (rohr and raffel ) . another example is the dramatic decline of the native red squirrel in the uk that has been attributed to a combination of direct competition with the grey squirrel and disease-mediated competition because the grey squirrel is a reservoir host of the squirrelpox virus that causes disease in the red squirrel (tompkins et al. ) . the local extinction of a host also may have tremendous consequences on an entire ecosystem (see for example the case of the wildebeest /rinderpest interactions in the serengeti, holdo et al. ). disease ecology? a change in species richness or abundance can modify the dynamics of local infectious diseases by either reducing or increasing the risk of transmission to the targeted species. the first outcome has been named, the "dilution effect", the second, the "amplification effect". the term "dilution effect" has conveyed different meanings since its first use in disease ecology literature (see box in the paper keesing et al. ) . the broad definition of the dilution effect refers to "the phenomenon -the net effect -when increased species diversity reduces disease risk" that is produced by a variety of mechanisms ("amplification effect" refers to the opposite phenomenon) (keesing et al. ) . this applies to vector-borne and directly transmitted diseases, although the concept of dilution has been developed most with regards to the tick-borne lyme disease (allan et al. ; logiudice et al. logiudice et al. , . the hypothesis underlying the amplification and dilution effect is that for many diseases, the competence of reservoirs, i.e. the ability to become infected and retransmit the pathogen, varies according to the host species (haydon et al. ) . the composition of the host community thus can influence the transmission dynamic of the disease. similarly, since vectors have different competence to transmit pathogens, the composition of the vector community likely influences transmission dynamics. different mechanisms are thought to be involved, but they are difficult to differentiate (begon ; keesing et al. ) . one is the modification of the encounter rate (when reduced, this corresponds to the "dilution effect" sensu stricto). in the presence of species that are poorly competent, the transmission event that should link an infectious individual to a susceptible individual instead links infectious individuals to non-competent individuals. for vector-borne diseases, the increased diversity of a poorly competent host species on which the vector feeds increases the proportion of vector bites that are wasted. for direct transmission, the addition of non competent hosts can decrease transmission if these hosts remove infectious particles (begon ) . a second mechanism at work is that a high diversity of host species regulates the abundance of the competent host population. this regulation can be mediated by interspecific competition for limiting resources or by predation upon competent hosts. this typically is the idea that underlies biological controls, where organisms prey upon reservoir hosts, vectors or intermediate hosts (straub and snyder ) . a third mechanism is based on the link between species richness and host mortality. this is the case when predators modify the mortality rate of a host and lower pathogen transmission by feeding on a heavily diseased individual (packer et al. ) . two other mechanisms are cited by keesing et al ( ) , but they are difficult to demonstrate: (i) the modification of recovery when species added to a community facilitate the recovery of infected individuals by, for instance, providing resources, and (ii) the modification of transmission once the contact has occurred, for instance, when adding a species modifies the pathogen load within the host. the dilution effect has been demonstrated mainly through theoretical work; there have been few case studies. one of the main examples is lyme disease in the usa that is caused by pathogenic bacteria transmitted by ticks. these ticks feed readily on many species of vertebrates and these species vary in their degrees of reservoir competence. the white-footed mouse (peromyscus leucopus) is thought to be the most competent host and dominates in fragmented forests. in native forests, which harbour a higher diversity of species than fragmented forests, ticks have a higher probability to dilute their bite by feeding on a less competent host (allan et al. ; logiudice et al. logiudice et al. , . however, such a dilution effect has not been demonstrated in europe, probably because of the complexity of the disease ecology which involves numerous reservoir host and bacteria species (halos et al. ) . another example is the west nile virus, where an increased diversity of non passerine birds, which are less competent reservoir hosts compared to passerines, was associated with decreased west nile virus infection in mosquitoes and humans (ezenwa et al. ; swaddle and calos ) . to date, there have been few examples of directly transmitted diseases, but studies on hantaviruses have shown that higher diversity of small mammals appears to regulate reservoir host populations through competition or predation. high small-mammal diversity also might inhibit intraspecific aggressive encounters between reservoir hosts that result in hantavirus transmission (suzán et al. ). in plants, crop diversity can reduce the total burden of disease in agricultural systems. this results from the combined effects of (i) the limitation of pathogen dispersal thanks to the physical barriers provided by the presence of non-host plants (burdon and chilvers ) , (ii) induced systemic resistance, and (iii) competition among pathogens. the efficiency of crop mixtures is linked to the size of the area on which this method is used: a high level of success has been observed in a field trial with susceptible and resistant varieties of rice conducted on a large scale ( , ha) in china (zhu et al. ) . illustrations of amplification effects are typically the consequences of species introduction that radically modifies encounter rates. the added species can introduce new pathogens that infect native hosts (spillover) (bruemmer et al. ) or amplify the circulation of local pathogens (spillback) (kelly et al. ). the introduction of additional species also can provide sources of vector meals and increase vector numbers or activity (saul ) . for instance, the introduction of siberian chipmunks (tamias sibiricus) in suburban forests could increase the risk of lyme disease because this host seems to be more competent than native hosts (vourc'h et al. ). the introduction of the mosquito aedes albopictus in many parts of the world has facilitated the transmission of the chikungunya virus (benedict et al. ; charrel et al. ) . theoretical works based on deterministic modelling have looked at the conditions in disease transmission dynamics that are needed for the amplification or the dilution effect to occur (begon ; dobson ) . when there is a relationship between the risk of a disease, the abundance of the reservoir host, and the abundance of an additional host, the addition of a species does not necessarily decrease the risk. in the case of lyme disease, for example, tick abundance mainly is determined by the abundance of deer, which are in fact a non competent reservoir. an increased abundance of deer may reduce infection prevalence when immature ticks are feeding on the deer. at the same time, however, the overall number of adult ticks increase proportionally with the number of deer (begon ) . further research in this field are relying on the modelling of the global community competence of hosts and vectors (roche ) . scientists and societies are increasingly interested in the dilution effect (mea ) due to the link between habitat disturbance, generalist host characteristics, and their competence in disease transmission. disturbance seems to favour generalist hosts (hosts that use different types of habitats) (devictor et al. ; marvier et al. ) , and these hosts often have a broad geographical distribution (mckinney and lockwood ; smart et al. ). crucially, these species also seem to have a higher competence reservoir or vector reservoir than species that are not favoured by disturbance (mills ; molyneux et al. ; vittor et al. ) . for example, many murid rodents that are recognized hosts of hemorrhagic fever viruses are opportunistic species that seem to be favoured in disturbed environments. the question is whether there is a causal link between a species' generalist and opportunist character and its disease competence. why are murid species associated with hemorrhagic fever more generalist than those which are not? could it be possible that specialist species also carry hemorrhagic fever viruses, only these viruses have not yet been identified? or is there something intrinsic in opportunistic species that makes them more likely to evolve and maintain hemorrhagic fever viruses (mills )? only a very small subset of plant and animal species have been domesticated (diamond ) . many species of that small subset, for example, cattle (in animals) and maize (in plants), have seen their genetic diversity considerably reduced for the purpose of intensive production (the bovine hapmap consortium, matsuoka et al. ) . in the wild, small populations of endangered species often have a very reduced genetic diversity (keller and waller ) . this then raises the following question: to what extent does a reduction of the genetic diversity in a host species impact the ability of the host population to response to infectious diseases (may ) ? genetic loci associated with the major histocompatibility complex (mhc) plays a key role in the acquired immune response of vertebrates (altizer et al. ) . mhc genes code for molecules that recognize foreign peptides (antigens) and display them on the cell surface. when the mhc-protein is displayed, it can be presented to immune cells, such as t lymphocytes or natural killer cells, which subsequently can trigger an adaptive immune response. because mhc genes are faced with an important diversity of antigens, they must themselves be diverse. the measure of the genetic diversity of mhc based on an analysis of polymorphism sequences of mhc among individuals in populations has been widely used in conservation biology as a proxy to estimate the fitness of populations confronted by pathogens (alcaide et al. ; bernatchez and landry ; sommer ) . however, the level of genetic variation at mhc loci results from different evolutionary forces (selection, gene flow, mutation) varying both in space and time in co-evolutionary systems involving both hosts and pathogens, making conservation genetics of non-model organism a challenging task (stockwell et al. ) . we already have many examples where low genetic diversity of species has favoured the diffusion of, and/or susceptibility to, pathogens. for example, the low genetic diversity of the tasmanian devil could be involved in its susceptibility to facial tumor disease (mccallum ) . the low genetic diversity found in commercially traded bee queens has been hypothesised as being one of the factors explaining colony collapse disorder (le conte and navajas ). the problem is even more critical in intensive crops in which disease resistance has relied on the use of a very small number of genes. this selection strategy has proven to be ineffective as pathogens manage to overcome the resistance. for example, the resistance of brassica napus (canola, oilseed rape and colza) to leptosphaeria maculans (causing the blackleg disease) due to a major resistance gene was overcome in an area covering approximately , ha in south australia in a period of years (sprague et al. ). even with advances in synthetic chemistry, which provides many biologically active molecules, pharmaceuticals derived from nature remain an important part of pharmaceutical practice today (newman et al. ). all organisms have developed compounds to protect themselves against infectious diseases and to interact with individuals of their own species or other species (e.g. rogerio et al. ). these molecules, coming from all organisms (bacteria, fungus, animals, plants) in terrestrial, marine and extreme ecosystems, represent an amazing diversity that has been tested in the field for millions of years by involving millions of individuals. however, only a very small subset of plants and marine organisms has been investigated for novel bioactive compounds. furthermore, it is estimated that less that % of bacterial species and only % of fungal species are known. those which have not yet been identified could be sources of novel molecules (cragg and newman ) . observations of natural medicine practices used by indigenous people have led to the discovery of many drugs. the most well known and widely used pharmaceuticals are quinine, used as a model to synthesize anti-malarial drugs (chloroquine and mefloquine), and artemisinin, indentified as a potent anti-malarial drug by chinese scientists (newman et al. ) . animals also are a source of inspiration for drugs against infectious diseases. for instance, compounds of the sponge cryptotethya crypta inspired the elaboration of antiviral medication such as azt used in the treatment of hiv/aids (cragg and newman ) . observing great apes medicate themselves through the plants they eat also could help to reveal new active compounds (krief et al. ). pathogens constitute a large part of biodiversity on earth and are present in all ecosystems and at all trophic levels, where they have a large impact on ecosystem functioning and on the population dynamics and evolution of their hosts. the recent acceleration of biodiversity loss due to human activities deeply impacts host-pathogen dynamics. pathogens and hosts form co-evolving systems exercising major selective pressures on each other. furthermore, the virulence or pathogenicity of a given species depends on its environment -which includes the hosts -that is highly variable in space and time. in such a context, human beings will never be able to completely control or eradicate every pathogen species; rather, we should accept that we must coexist with pathogens. to better understand and predict the evolution of pathogens and the impact of human activities on them, more in-depth studies are needed on how pathogens interact with host communities within different ecosystems. to understand human, animal, and plant epidemics, two-species systems involving only a single host and a single pathogen species are no longer appropriate. the approach considering multi-host/multi-pathogen systems in their environment is the framework that now needs to be used to deepen our understanding of disease dynamics woolhouse et al. ) (fig. . ) . however, these dynamics are very complex and difficult to study because precise knowledge regarding the diversity of pathogens and of interactions taking place on several scales is lacking (lloyd-smith et al. ). in addition to intensive fieldwork to collect adequate data and modeling to understand the main processes, the use of molecular tools in a multi-host/multi-pathogen framework will facilitate investigations into pathogen-host community interactions. in particular, new generation sequencing techniques render it easier to characterize the genetic diversity of pathogens and hosts. for instance, metagenomic investigations allow an approach that does not require a priori assumptions that is useful to describe a community of pathogens and their interactions. molecular techniques also may be used to clarify the taxonomic status of pathogens, revealing cryptic species or host races. with suitable molecular fig. . schematic representation of the link between biodiversity and the ecology of infectious diseases. diseases results from the complex interactions between the three compartments corresponding to pathogens, hosts and vectors (in the case of vector-borne diseases). the biodiversity of these three compartments can be considered at the community level (each circle corresponding to a species) or at the intraspecific level (each circle corresponding to a population or an individual within a population). the gray shading off of each unit considered (species, population or individual) illustrates its genetic or phenotypic variability in space and time, while variations in size illustrate frequency or density differences within the ecosystem. interactions within each compartment can be direct (competition) or indirect (apparent competition…), synergic or antagonistic as illustrated by the different arrows markers (producing a high level of polymorphism), the analysis of genetic variability within a spatially explicit framework renders it possible to identify the routes followed by a given pathogen. moreover, molecular techniques can be used to identify genes involved in important life history traits of a pathogen such as virulence and transmission. better knowledge of the mechanisms involved in host-pathogen interactions, and the extent 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biology of multihost pathogens temporal trends in the discovery of human viruses rna viral community in human feces: prevalence of plant pathogenic viruses genetic diversity and disease control in rice key: cord- -wmelyonk authors: roe, kevin title: a proposed treatment for pathogenic enveloped viruses having high rates of mutation or replication date: - - journal: scand j immunol doi: . /sji. sha: doc_id: cord_uid: wmelyonk several enveloped viruses, particularly some rna viruses, have high rates of mutation or replication, which can make them virulent pathogens in humans and other mammals. a proposed treatment could use synthesized proteins to mask pathogenic viral surface proteins to quickly induce an immune attack on specific enveloped viruses by using existing immune cells. one treatment could inject dual‐protein ligand masks into patients' blood streams to mask pathogenic surface proteins used to infect mammalian cells. the mammalian immune system already uses an analogous, more complex structure called a pentraxin to neutralize some pathogens by connecting their surface proteins to immune cells. and several types of antiviral peptides have already experimentally demonstrated effectiveness in blocking various viral pathogen infections. these treatments offer advantages, especially for currently untreatable viral pathogens. furthermore, using dual‐protein ligands and the antigenic memory of some subpopulations of nk cells would also allow the creation of defacto vaccines based on a host's nk cells, instead of vaccines utilizing cd and cd α:β t cells, which are limited by the requirement of mhc presentation of the target antigens to α:β t cells. targeted nk cell vaccines could attack host cells latently or actively infected by intracellular pathogens, even host cells having pathogen downregulated mhc antigen presentation. eight postulates concerning the effects of pathogen mutation, or change in phenotype from genetic recombination or rearrangement, and replication rates on pathogen versus host dominance are also listed, which should be applicable to viral and non‐viral pathogens. every human has barrier surfaces to suppress viral infections through the epithelium of the respiratory system and the gastrointestinal tract, the endothelium of blood vessels, placental barrier tropoblasts, and skin. the b.v report of the international committee on taxonomy of viruses (ictv) lists viral genera and viral species. but only a small percentage are pathogenic to humans, and presently viral genera are known to cause human viral diseases. yet despite anti-viral medicines and vaccines, pathogenic viruses infect millions of children and adults yearly world-wide. [ ] [ ] [ ] [ ] even though some viral infections can be treated or at least avoided by vaccination, for many of the untreatable viral infections, a large fraction of the survivors frequently suffer mild to severe life-long impairments. for example, the eastern equine encephalitis (eee) virus, endemic to the western hemisphere, has no human vaccine or even a treatment, and has been called the most deadly mosquito-borne pathogen in north america, because it can kill % to % of infected humans depending on the medical care received, and an estimated % to % of the survivors suffer severe and permanent neurological brain damage. [ ] [ ] this makes the need for a eee virus vaccine obvious, but the only vaccine currently available is for horses, not humans. another untreatable and lethal virus, transmissible by bodily secretions of humans and other mammals, and even considered fully capable of a world-wide pandemic spread after mutation, is the nipah virus, with a mortality rate ranging from % to % in the indian subcontinent. - peptide inhibitors against the nipah virus have been designed and modeled to target and inhibit interacting sites on the viral attachment g receptor, the f fusion protein trimer used to fuse the viral envelope with the host cell membrane, and the m matrix protein dimer used for initiating the budding of the virus; and the bonding stabilities of various antiviral peptide inhibitors were assessed with molecular dynamics (md) simulations. but at this time, there is still no vaccine and not even a treatment for humans. this article is protected by copyright. all rights reserved the two preceding viruses illustrate the virulence of some viral pathogens after transmittal to humans, but some extremely virulent viruses can have both high rates of mutation and high rates of replication. some dna viruses, and particularly several rna viruses, have high mutation rates, which can be expressed as nucleotide substitutions per nucleotide per cell infection (s/n/c); and these mutation rates typically range from - to - s/n/c for dna viruses to - to - s/n/c for rna viruses, although nucleotide insertions and deletions can also make a smaller contribution to the overall mutation rate. and independently from mutation rates, some viral pathogens can have a high replication rate among host cells, especially after transmission to secondary hosts of other species, such as viruses that originally evolved high replication rates while they infected animals such as bats and were thus selected by the fast immune responses of bats. [ ] [ ] and it will obviously be quite challenging to treat virulent viruses that have both high mutation rates and high replication rates. however, there are several known antiviral peptides that can inhibit or block viral infections, and some of them could potentially treat infections by virulent enveloped rna viruses having high rates of mutation and replication. [ ] [ ] there are even databases of experimentally verified antiviral peptides, typically derived from micro-organisms. these antiviral peptides are members of the larger group of antimicrobial peptides that contribute to the innate immune response of many species, and they are known to act either directly or by creating an immune response. several antiviral peptides have been experimentally demonstrated to be effective against different viruses, even in serum solutions with micro-molar concentrations, and their actions typically include blocking one or more stages of a virus's infection cycle; such as host cell attachment, host cell entry, replication inside a host cell, transcription, translation, maturation, or release from a host cell. [ ] [ ] this paper is focused on antiviral applications of custom designed dual-protein ligand masks that can be synthesized by conventional recombinant dna biotechnology, and these ligand masks are accepted article theoretical and functional analog extensions of pentraxins, which are more complex immune structures used by the mammalian immune system to neutralize some pathogens. in targeting specific viral pathogens, dual-protein ligand masks (for brevity, henceforth called dualprotein ligands) should be able to create a quick and powerful immune memory response with existing memory immune cells against some viral pathogens or virus infected cells, without some of the practical limitations of vaccines. which viral pathogens or virus infected cells are susceptible to memory t cell and memory b cells? enveloped viruses, and some non-enveloped viruses, typically have pathogenic surface proteins, such as glycoproteins, needed for viral infection of host cells. [ ] [ ] some of these surface proteins cannot mutate too much without losing their functionality for infection of a host cell, so critical sections of these essential surface proteins can be targeted to block viral pathogen infections. most t cell activations require that an antigen (i.e., a molecular pattern that a patient's immune system recognizes as foreign to the patient) be presented by another cell, such as a dendritic cell, on a specific surface protein known as a major histocompatibility complex (mhc), in humans this is also called a human-leukocyte-associated (hla) protein. t cells predominantly are α:β t cells with the mhc requirement for antigen presentation to activate α:β t cells; whereas a different subset of t cells called γ:δ t cells have no need for any mhc presentation. although activating γ:δ t cells is challenging, there are several distinct sub-populations of γ:δ t cells; while they are more abundant in mucosal tissues, sub-populations of γ:δ t cells are also found in several types of tissues and they are even found in blood. - furthermore, γ:δ t cells can be transformed into memory t cells like α:β t cells; and for γ:δ t cell sub-populations residing in various tissues, peptides of distinct proteins, such as an endothelial protein c receptor β sheet, in the presence of other co-stimulating peptides of proteins, such as icam- (known also as cd ) can activate their γ:δ t cell receptors. [ ] [ ] [ ] activating the γ:δ t cell receptor and this article is protected by copyright. all rights reserved one or two co-stimulatory receptors, can activate γ:δ t cells, which can also result in antibody production by activated b cells. so γ:δ t cells are the t cells referenced herein. dual-protein ligands could make specific viral pathogens targets for existing immune memory cells or innate immune cells. dual-protein ligands could induce an immune response by mimicking the key parts of antigens that activate existing immune memory cells or innate immune cells to attack tagged viral pathogens. there are significant benefits in using the immune memory system to neutralize viral pathogens. one benefit is that when memory immune cells are triggered by a dual-protein ligand antigen, the antibody response of the immune memory system will produce antibody numbers much larger than the antibody numbers released by a primary immune response, or released by long lived plasma cells resident in the bone marrow, or b cells in the respiratory and intestinal mucosal tissue. other potential benefits of using dual-protein ligands include avoiding the need to use many different preventative anti-viral vaccinations, and sufficient mass bondings to pathogenic viral surface proteins could potentially minimize t cell death (lymphopenia) from too many inflammatory stimulating signaling proteins (a "cytokine storm") caused by some viral pathogens, such as the ebola virus. in summary, surface proteins are used by viral pathogens to infect mammalian cells. however, dualprotein ligands could mask and block these surface proteins before the widespread viral pathogen infection of mammalian cells. a suggested treatment for humans uses a dual-protein ligand, that includes a first protein ligand that will mask (i.e., specifically bond to) a unique virus surface protein, or alternatively bond to a distinctive surface protein of a virus infected cell, and a second protein ligand that matches or mimics the section of an antigen that would activate memory immune cells, wherein the second protein ligand is connected to the first protein ligand. both the first protein and the second this article is protected by copyright. all rights reserved protein ligands can separately have their three dimensional conformations strengthened by covalent disulfide bonds (s-s bonds) formed between the sulfhydryl (-sh) groups of precisely placed cysteine amino acid residues. both the bonding between the first protein ligand to a viral surface protein, and the bonding between the second protein ligand to an immune cell, could be bondings utilizing non-covalent forces, such as electrostatic forces, van der waals forces, hydrophobic forces, cation-pi interactions and hydrogen bonds. some component amino acid residues can produce several bonding forces; the aromatic amino acid residue tyrosine is typical and can bond by hydrogen bonding, hydrophobic forces, and through it's side chain pi-electron system have cation-pi interactions with nearby cations. in the brain or central nervous system (cns) especially, synthesis of the second protein ligand to mimic an antigen to cause an attack on virus infected cells by memory γ:δ t cells must also take into consideration the risk of excessive inflammation. there are several sub-populations of γ:δ t cells, that can be distinguished by their t cell receptor variable regions (vγ), and they can be separated into bigger classifications, such as γ:δ t cells and γ:δ t cells. however, there are some classifications of γ:δ t cells, the pro-inflammatory interleukin- producing γ:δ t cells for example, that can stop certain infections, but they can also potentially create excessive inflammation and promote cancers and some auto-immune diseases. other alternatives for treatments involving the cns and brain are second protein ligands that utilize the less inflammatory γ:δ t cells, which are relatively scarce in blood, but more common in tissues and organs, that produce interferon-γ, which activates macrophages, but this can also cause some inflammatory interleukin- release. some potential ligands that could be mimicked by the second protein ligand to activate the γ:δ t cell receptors are listed later in this paper in the section that discusses the nk cell activating receptor nkg d. another alternative is to synthesize a second protein that utilizes specific innate immune cells. one option is the microglia, the main resident macrophages for neurons in the cns and brain, and they and cns border-associated macrophages in general are among the innate immune cells that could be used. microglia are essentially accepted article macrophages, so the second protein ligand could be synthesized to bond to one of the many distinct types of receptors expressed by macrophages -including the pattern recognition receptors (prr), such as the toll-like receptors, the fc antibody receptors, or the complement receptors such as cr , for example. the receptor structures of some macrophages and other immune cells, such as the nk cell nkg d activating receptor, are known to some extent; but any targeted receptor structure will probably require considerable research to determine enough detailed structure to enable the design of a second protein ligand having a strong bond to the targeted immune cell receptor. the nk cell nkg d activating receptor and the structure of its ligands are discussed in more detail later in this paper. there are several potential surface proteins on specific viral pathogens that can be targeted. for example, these surface proteins include the glycoproteins e and e on the surface of the hepatitis c virus of the hepacivirus genus. - another example is human immunodeficiency virus (hiv- ), an enveloped virus of the lentivirus genus with a surface protein, a trimeric glycoprotein, to induce membrane fusion for viral entry into a human host cell, and this glycoprotein is known to be targeted by antibodies. measles virus is an enveloped virus of the morbillivirus genus, with a morbillivirus surface protein complex with tetrameric attachment (h) and trimeric fusion (f) glycoproteins for viral entry and infection of human host cells. eastern equine encephalitis virus is an enveloped virus of the alphavirusgenus with two trans-membrane envelope glycoproteins e and e , where the surface protein e bonds to human cells for viral entry. [ ] [ ] in summary, there are several possible target viral surface proteins for bonding to dual-protein ligands for the immune system neutralization of viral pathogens. as previously discussed, the structures of some viral surface proteins are already known to some extent; but any targeted viral surface protein will probably require considerable research to determine enough detailed structure to enable the design of a first protein ligand having a strong bond to the targeted viral surface protein. this article is protected by copyright. all rights reserved one treatment option injects dual-protein ligands into the blood stream or localized regions to mask pathogenic surface proteins used by viruses to infect mammalian cells. the dosage of dual-protein ligands necessary to treat a viral pathogen infection will vary, depending on how long the dual-protein ligands will reside inside the patient before they are removed or inactivated, depending on the concentrations of the targeted virus and the immune cell utilized, and depending on how strongly each dual-protein ligand will bond with both the targeted viral surface protein and the immune cell utilized. the strength of bonding between ligands and proteins is quantified by an association constant (k a ), which is defined as the equilibrium molar concentration of a protein bound to a ligand, divided by the multiplication of the molar concentration of the unbound ligand and the molar concentration of the unbound protein, as seen in equation ( ). other papers use the dissociation constant (k d ), the reciprocal of the association constant k a , as seen in equation ( ) as mentioned before, a different option is utilizing cells of the innate immune system (e.g., innate lymphoid cells, macrophages or other phagocytes). as a specific example, the first protein ligand would bond to either a surface protein of a viral pathogen, or bond to a surface protein of a cell infected by the viral pathogen, and the second protein ligand would be designed to strongly bond to and activate phagocytes to consume the viral pathogen or the virus infected cell, by designing the second protein ligand to bond to a surface protein of human macrophages or neutrophils, for instance the fcαri receptor (cd ). this option would bypass some viral defenses against memory t cells or b cells, and would also quickly initiate an attack by innate immune system cells, in this instance by phagocytic cells. an injection of dual-protein ligands into the blood stream of patients will be easier than trying to introduce dual-protein ligands from inside the gastrointestinal tract, whether by using oral capsules or therapeutic bacteria. [ ] [ ] and injections into the blood stream, or injections into accepted article localized regions, can be used separately or in a combination. a combination of both treatment approaches is probably better for treating viral pathogen infections that also include the brain or cns. in such cases, a lumbar puncture through the patient's spine can safely inject the dual-protein ligands into the cerebrospinal fluid of the patient, and this is a standard procedure to deliver antibiotics directly into the brain and cns, while avoiding the blood brain barrier, a serious impediment to introducing most medicines into the brain by blood circulation. these proposed treatments raise the question of whether there is any risk of creating dangerous immune system reactions by injections of dual-protein ligands. this risk is small, because as discussed in an earlier paper, by themselves most pure proteins and peptides rarely induce an immune response. this is one motivation to combine a chemical adjuvant with many vaccinations to make their antigens immunogenic to induce a strong immune response, and influence the antibody titer and isotype and increase cell-mediated responses. however, since memory t cells and memory b cells already are activated effector cells, dual-protein ligand injections should be able to activate these targeted memory cells without requiring any adjuvants combined with the injections. but including certain cytokines can be helpful; cytokine therapies using type i interferons and interleukin- have been approved for some cancer treatments; and specific cytokines help nk cell receptor activation, which will be discussed in more detail later. it should be possible to synthesize dual-protein ligands in advance in the quantities needed to stop viral pathogens at relatively low cost. the modification of bacterial genomes, such as the genome of escherichia coli, using restriction enzymes can induce expression of specific and desirable peptides and proteins, and this is a very mature and long commercialized technology. thus, recombinant dna techniques, applied to one of the commercially available strains of bacteria, could make dualprotein ligands. protein ligands having an equivalent number of amino acid residues, this implies that entire dualprotein ligands could be synthesized with several hundred amino acid residues, in some cases with as little as amino acid residues, possibly including a short section between the first protein ligand to the second protein ligand to provide more flexibility. each protein ligand only needs to be long enough to provide a strong bond with its respective target, and each protein ligand may need conformal stabilization provided by disulfide bonds between precisely placed cysteine amino acid residues within each protein ligand. one last question concerns the therapeutic duration of the dual-protein ligands in the blood stream of a mammal. one significant factor determining the duration of a dual-protein ligand will be the presence and concentrations of pathogen and mammalian proteolytic enzymes, also known as proteases or peptidases, having nucleophilic active sites, that typically cleave peptide bonds by hydrolysis, such as at the carbonyl (c=o) of the peptide bond. any peptide segment of the dualprotein ligand is a potential peptidase target, but the peptide segment in the linkage of the two protein ligands of a dual-protein ligand will be particularly accessible to peptidases. fortunately, many peptidases also have known inhibitors, and carefully chosen specific inhibitors for the most inconvenient peptidases could also be injected with the dual-protein ligands to prevent or slow peptidase attacks, preferably without causing major disruption to the normal physiological functions of mammalian peptidases. the most appropriate inhibitor for a peptidase can be found in an extensive database of peptidases, their substrates and their this article is protected by copyright. all rights reserved inhibitors, such as the merops database. as of september , there were peptidase identifiers and inhibitor identifiers listed in the merops . database, along with the peptidase's substrate. eight postulates concerning the effects of pathogen mutation, pathogen change in phenotype by genetic recombination and pathogen replication rates on pathogen versus host dominance are listed below and should be applicable not only to viral pathogens, but also applicable to bacterial, fungal, and protozoan pathogens. the term "pathogen strain" as used below is defined as a sub-species or sub-type of a pathogen species. the term "dominate" as used below is defined as survive, or at least maintain an infection, where a pathogen strain possibly, but not necessarily, could kill its host; whereas when a host "dominates" a pathogen strain, it may, or may not, be able to eliminate the pathogen strain. the term "mutation" as used below is defined as random changes in one or more genetic nucleotides, by substitution, deletion or insertion of nucleotides, etc. the term "beneficial mutation" as used below is defined as a mutation that helps the pathogen strain, against the host or against other pathogen strains. a "beneficial change in phenotype" as used below is also defined as helping the pathogen. the term "genotype" as used below is defined as the entire genome of a pathogen strain, and the term "phenotype" as used below is defined as the characteristics of a pathogen strain, expressed as the interaction of a pathogen strain's genotype with its age, its various conditions of activity or latency, or its environment. it should be noted that a pathogen's phenotype can also change as a result of genetic recombination or rearrangement (called "genetic recombination" for brevity henceforth), either randomly or by programming, recombining or rearranging groups of nucleotides within a single pathogen strain or from multiple pathogen strains; such as a change in viral phenotype after random genetic recombination during co-infection of a host cell by two different viral strains. one specific example of a viral change in phenotype would be the antigenic shift in one or more antigenic determinants of an influenza virus resulting from the genetic recombination of various bird and mammalian influenza virus strains. a beneficial mutation for a pathogen strain could be a higher rate of replication, an improvement in its infectivity or the transmission of the pathogen strain, an improvement in reducing environmental, resource or metabolic requirements for the survival, replication or spread of the pathogen strain, an improvement in evading or overcoming the immune defense of a host by changing one or more antigenic determinants, and so forth. and as previously discussed, a beneficial change in phenotype by genetic recombination of a pathogen strain can possibly result in an antigenic shift in one or more antigenic determinants that were essential to the host's immune system for recognition and defense against the pathogen strain. examples of how the effectiveness of host defenses could be improved include an improved blocking of the infectivity of the pathogen, or an improvement in targeting, accessing or in neutralizing the pathogen, such as provided by adaptive immune system immunoglobulin antibody isotype switching or somatic hypermutation and affinity selection of antibodies against the pathogen, producing more effective cytokines, activating other more effective innate or adaptive immune cells, and so forth. , introduction of medicines or treatments are an alternative equivalent means to improve the effectiveness of the host defenses. a host can replicate effective defenses include increasing the number of effective antibodies, increasing the number of innate or adaptive immune cells, increasing the number of effective this article is protected by copyright. all rights reserved cytokines, increasing the number of activated immune cells, and so forth. introduction of medicines or treatments are an alternative equivalent means to replicate host defenses faster. recombination against a first host's immune defenses faster than its first host can improve the effectiveness of its immune defenses will dominate its first host, and after transmission to a second host it will be enabled to more virulently dominate a second host individual or species having a slower or weaker immune defense, which otherwise provides an equivalent host environment for the pathogen strain. host environment for the pathogen strain. can beneficially mutate or beneficially change its phenotype by genetic recombination against the host's immune defenses can eventually dominate the pathogen. replicate can eventually dominate the pathogen. this article is protected by copyright. all rights reserved these eight postulates summarize the effects of pathogen mutation, pathogen change in phenotype by genetic recombination, and pathogen replication rates on pathogen versus host dominance and their scope is limited to pathogen versus host dominance, but these postulates should be applicable to viral pathogens, and also applicable to bacterial, fungal, and protozoan pathogens. the host could be a mammal, but could possibly be non-mammalian, or even outside of the animal kingdom, since plants also struggle for dominance over several types of pathogens. targeted dual-protein ligands could mask viral surface proteins to quickly treat some untreatable virus infections by using already existing immune cells. one treatment uses injection of the dual-protein ligands into the blood of patients, and another treatment injects the dual-protein ligands into less accessible viral pathogen infections, such as the brain or central nervous system, to bond to a viral surface protein used to infect mammalian cells. these treatments could have advantages, especially for enveloped rna viruses having high rates of mutation or replication, although the initial development and implementation of these new treatment approaches will require substantial resources. furthermore, using dual-protein ligands and the antigenic memory of some subpopulations of nk cells would also allow the creation of defacto vaccines based on a host's nk cells, instead of vaccines based on α:β t cells, which are limited by the requirement of mhc presentation of the target antigens to α:β t cells. targeted nk cell vaccines could attack host cells latently or actively infected by intracellular pathogens, even host cells having less mhc antigen presentation capabilities, such as neurons. eight postulates concerning the effects of pathogen mutation, or change in phenotype from genetic recombination or rearrangement, and replication rates on pathogen versus host dominance have also been listed, which should be applicable to viral and non-viral pathogens. the author confirms that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. no ethical approval was required as this is an article with no original research data. data sharing is not applicable to this article as no new data were created or analyzed in this study. the author attests that he conceived the paper, wrote the paper, and approved the final version of the manuscript, and attests that he meets all of the icmje criteria for authorship. type iii interferons in antiviral defenses at barrier surfaces international committee on taxonomy of viruses (ictv) global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young childer in : a systematic review and modelling study eastern equine encephalitis virus -old enemy heparan sulfate binding by natural eastern equine encephalitis viruses promotes neuro-virulence morbidity and mortality due to nipah or nipah-like virus encephalitis in who south-east asia region world health organization nipah virus infection. world health organization 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trevor title: myalgic encephalomyelitis/chronic fatigue syndrome in the era of the human microbiome: persistent pathogens drive chronic symptoms by interfering with host metabolism, gene expression, and immunity date: - - journal: front pediatr doi: . /fped. . sha: doc_id: cord_uid: zim nond the illness me/cfs has been repeatedly tied to infectious agents such as epstein barr virus. expanding research on the human microbiome now allows me/cfs-associated pathogens to be studied as interacting members of human microbiome communities. humans harbor these vast ecosystems of bacteria, viruses and fungi in nearly all tissue and blood. most well-studied inflammatory conditions are tied to dysbiosis or imbalance of the human microbiome. while gut microbiome dysbiosis has been identified in me/cfs, microbes and viruses outside the gut can also contribute to the illness. pathobionts, and their associated proteins/metabolites, often control human metabolism and gene expression in a manner that pushes the body toward a state of illness. intracellular pathogens, including many associated with me/cfs, drive microbiome dysbiosis by directly interfering with human transcription, translation, and dna repair processes. molecular mimicry between host and pathogen proteins/metabolites further complicates this interference. other human pathogens disable mitochondria or dysregulate host nervous system signaling. antibodies and/or clonal t cells identified in patients with me/cfs are likely activated in response to these persistent microbiome pathogens. different human pathogens have evolved similar survival mechanisms to disable the host immune response and host metabolic pathways. the metabolic dysfunction driven by these organisms can result in similar clusters of inflammatory symptoms. me/cfs may be driven by this pathogen-induced dysfunction, with the nature of dysbiosis and symptom presentation varying based on a patient's unique infectious and environmental history. under such conditions, patients would benefit from treatments that support the human immune system in an effort to reverse the infectious disease process. toward the end of a career spent studying persistent bacteria in chronic disease, microbiologist gerald domingue wrote, "it is unwise to dismiss the pathogenic capacities of any microbe in a patient with a mysterious disease" ( ) . this thinking greatly applies to the illness me/cfs. me/cfs is characterized by neuroinflammation, severe fatigue, excessive post-exertional exhaustion, disturbed sleep, flu-like episodes, cognitive problems, sensory hypersensitivity, muscle and joint pain, headache, bowel symptoms, and severe impairment of daily functioning ( ) . severely ill individuals are often wheelchair dependent, bedridden, and unable to perform basic tasks of work or daily living. the history of me/cfs strongly suggests that infectious agents play a central role in driving the disease process. these include early associations with epstein barr virus (ebv)/human herpes virus (hhv ), the relapsing-remitting nature of me/cfs symptoms and antibodies/"autoantibodies" detected in patients with the disease ( , ) . a number of me/cfs outbreaks have also been reported, in which numerous people in the same geographical location developed the illness simultaneously ( ) . indeed, many me/cfs patients present with symptoms after suffering from a severe bacterial or viral infection. these infections often correlate with travel to a foreign country or exposure to pollutants or molds, suggesting that such pathogens take advantage of factors that compromise the host immune system. chronic inflammation is a hallmark of persistent infection. reports of cytokine activation in me/cfs clarify that the disease is associated with an inflammatory response ( ) . montoya et al. ( ) found me/cfs cytokine activation increased with disease severity, suggesting patients may struggle with a growing infectious burden over time. other me/cfs research teams have identified various forms of mitochondrial dysfunction in patients with the illness ( ) . there are now dozens of well-characterized mechanisms by which bacteria and viruses dysregulate mitochondrial metabolism ( , ) . many research teams have searched for well-characterized single pathogens in patients with me/cfs. these analyses often reveal elevated titers of igg and igm antibodies toward pathogens such as epstein barr virus, cytomegalovirus, parvovirus and m. pneumonia ( , , ) . several teams have also attempted to identify a single novel pathogen that might drive the entire me/cfs disease process. however, the discovery of the human microbiome now allows single microbes and viruses to be studied as members of complex communities. humans harbor these vast ecosystems of bacteria, viruses and fungi in nearly all tissue and blood ( ) ( ) ( ) ( ) . organisms in the microbiome continually interact with each other, and with the human genome, to regulate host metabolism and gene expression in both health and disease ( , ) . a growing number of inflammatory disease states, including neurological conditions and cancers, are tied to dysbiosis or imbalance of these human microbiome communities ( ) ( ) ( ) ( ) . gut microbiome dysbiosis has been identified in me/cfs ( ) . this dysbiosis is characterized by changes in microbe species composition and/or diversity. pathogens, or groups of pathogens, can promote dysbiosis by altering their gene expression in ways that promote virulence, immunosuppression and dysregulation of host genetic and metabolic pathways ( ) . when seemingly disparate biomedical findings on me/cfs are interpreted through the lens of these microbiome-based paradigms and platforms, a cohesive picture of the me/cfs disease process emerges. me/cfs may be driven by pathogeninduced dysfunction, with resulting microbiome dysbiosis varying based on a patient's unique infectious and environmental history. under such conditions, patients would benefit from treatments that, like those now being developed for cancer, support the human immune system in an effort to reverse the inflammatory disease process. in the usa, me/cfs cases were first formally reported to the cdc in the s ( ) . at the time, human microbes were typically only detected with culture-based laboratory methods. then, around the year , novel genome-based technologies began to revolutionize the field of microbiology ( , ) . these technologies identify microbes based on their dna or rna signatures rather than their ability to grow in the laboratory. the results of these genome-based analyses were remarkable: vast communities of microbes were identified in the human body that had been missed by the older culture-based techniques. these extensive ecosystems of bacteria, viruses, fungi, and archaea are collectively known as the human microbiome ( ) ( ) ( ) . today, so many novel microbes have been identified in homo sapiens that our human cells are equivalent to or even outnumbered by those of our microbial inhabitants ( ) . the tens of millions of unique genes harbored by this microbiome dwarf the ∼ , genes in the human genome ( , ) . for example, just one analysis of the human gut, skin, mouth, and vaginal microbiomes uncovered millions of previously unknown microbial genes ( ) . this has forced science to redefine the human condition. humans are best described as holobionts, in which the microbial genomes and the human genome continually interact to regulate metabolism and immunity ( , ) . early human microbiome studies characterized microbial ecosystems in the gut and on mucosal surfaces. however, the microbiome has now been shown to extend to nearly every human body site. these include the lungs, the bladder, the placenta, the testes, and the uterus [ ( , ( ) ( ) ( ) ( ) ]. jakobsen et al. ( ) found that previously sterile implants removed from joints, bones, pacemakers, and skulls of symptom-free patients were colonized by a range of bacterial and fungal organisms. another study demonstrated the presence of novel tissue specific bacterial dna profiles in a variety of mouse organs including the brain, heart, liver, muscle and adipose tissue ( ) . microbial communities also appear to persist in healthy human blood ( ) ( ) ( ) . a dna virome was recently identified in healthy human blood ( ) . another study reported both bacterial and fungal communities in the blood of healthy subjects. analysis of these organisms was performed by microbial resuscitation of blood culturing and microscopy in addition to next generation dna sequencing ( ) . whittle et al. ( ) recently characterized a human blood microbiome using a range of complementary molecular and classical molecular biology techniques. another study identified a larger amount of bacterial rdna in blood specimens from healthy individuals than in matched reagent controls ( ) . kowarsky et al. ( ) detected over , previously unidentified viruses, bacteria, and fungi in human blood samples obtained from immunocompromised patients. the study almost doubled the total number of anelloviruses found in humans. in order to classify many of these organisms the team was forced to add new branches to the "tree of life." they concluded that the newly discovered microbes "may prove to be the cause of acute or chronic diseases that, to date, have unknown etiology." the microbiome is inherited and evolves with the host. babies are seeded in the womb, during birth, and after birth by extensive microbiome communities in the placenta, the vaginal canal, and breast milk, among other body sites ( , ) . microbes/pathogens acquired from the external environment are further incorporated into the microbiome over time. for example, once acquired, cytomegalovirus persists as a member of the microbiomewith a significant impact on host immunity. brodin et al. ( ) found that the lifelong need for the body to control cmv causes approximately % of all t cells in cmv+ individuals to be directed against the virus. immune cells and associated microbes can travel between the human body and brain via several newly discovered pathways that bypass the classical blood-brain barrier ( ) . benias et al. ( ) documented a previously uncharacterized fluidfilled lattice of collagen bundles that appears to connect all human tissues. this human interstitium drains directly into the lymph nodes. two research teams have demonstrated the existence of a previously undiscovered meningeal lymphatic system ( , , ) . the network's fluid pathways connect the cerebrospinal fluid and cervical lymph nodes directly to the brain. while great progress has been made in characterizing the human microbiome, our understanding of the body's microbial ecosystems is still in its infancy. metagenomic analyses of the microbiome in all body sites regularly identify species or strains of bacteria, archaea, fungi, and/or viruses not previously understood to persist in homo sapiens ( ) . for example, just one study of the bladder microbiome identified previously unidentified viruses in subjects' urine samples ( ) . new strains of known microbes are also regularly identified. for example, as of august , the ncbi database contains ∼ , lactobacillus genomes, with newly characterized lactobacillus genomes added on a weekly basis ( ) . successful identification of these known or novel human organisms hinges on the careful choice of technology and methodology used for detection purposes ( ) . while the majority of human microbiome studies center on bacteria, awareness of viruses, fungi, and archaea has increased in recent years ( ) . for example, manuela et al. ( ) found an almost : ratio of archaeal to bacterial s rrna genes in human appendix and nose samples. identification of this archaeome abundance and diversity required use of a very specific archaea-targeting methodology. viruses are the most abundant life forms on the planet and in the human body, but have been relatively hard to detect until very recently ( ) . viruses that primarily infect bacteria, called bacteriophages, are particularly abundant in human microbiome communities. nguyen et al. ( ) estimate that ∼ billion bacteriophages traffic human tissue and blood on a daily basis. however, the human virome also harbors a plethora of human-associated viruses. newberry et al. ( ) are correct to assert that this human virome is understudied in me/cfs. paez-espino et al. ( ) at the joint genome institute have undertaken a large project called "uncovering the earth's virome." the project's goal is to better identify known and novel viruses in earth's ecosystems including the human body. viral identification requires the use of specific metagenomic tools, pipelines, and annotation platforms, with findings entered in the jgi img/vr database ( ) . the project is progressing at such a rapid pace that viral diversity in img/vr has more than tripled since august ( ), (figure ) . nevertheless, the vast majority of the gene content (over million genes in total) remains unknown or hypothetical. we must subsequently consider the possibility that asyet unidentified microorganisms may contribute to chronic inflammatory conditions like me/cfs. failure to do so would be akin to studying ∼ % of the animals in the rainforest and arriving at firm conclusions about the entire ecosystem based on that information alone. certain members of the human microbiome may also be difficult to detect based on their location and/or lifestyle. for example, chen et al. ( ) found that elevated cytokine expression in response to hsv-infected peripheral nerve ganglia persisted even when the virus entered a latent, non-replicating state. while it is important to pursue identification of novel organisms in me/cfs blood and tissue, ample data already exists on better-studied components of the microbiome. microbial and viral survival strategies, virulence mechanisms and collective behaviors are also characterized by a high degree of functional redundancy ( , ) . we must accept the complexity inherent to the human microbiome and further study these common mechanisms of survival and persistence. we must examine microbe and viral activity, microbe and viral gene expression, and the myriad ways in which the proteins and metabolites created by these organisms interact with the host immune system, the host genome, and each other. the genes of our microbial inhabitants greatly outnumber the ∼ , in the human genome. it follows that the majority of metabolites in homo sapiens are produced or modified by the microbiome. wikoff et al. ( ) found a large effect of the gut microbiome on murine blood metabolites including antioxidants, toxins and amino acids. for example, production of the metabolite indole- -propionic acid was completely dependent on the presence of gut microbes and could be established by colonization with the bacterium clostridium sporogenes. many microbes, viruses and their corresponding proteins/metabolites directly modulate the activity of host metabolic, immune, and neurological pathways. in other words, the human holobiont is controlled by the human genome, our microbial/viral genomes and their respective metabolites working in tandem. a growing number of studies provide examples of this metabolic overlap. while many such studies have been conducted in mice, their general trends carry over to humans. for example, me/cfs is associated with natural killer (nk) cell abnormalities, including reduced natural killer cell activity ( ) . these findings must be interpreted to account for the fact that nk activity is modulated by the bacterial microbiome. one study found that bile acids modified by the gut microbiome impacted liver cell gene expression in a manner that controlled nk cell accumulation and anti-tumor activity ( ) . similarly, a mixture of lactic acid bacteria from kefir increased the cytotoxicity of human nk khyg- cells to human chronic leukemia cells and colorectal tumor cells ( ) . the microbiome and its metabolites also impact the activity of related immune cells. rothhammer et al. ( ) found that tryptophan created by the gut microbiome interacted with the ahr receptor on microglia/astrocytes. subsequent changes in gene expression regulated communication between the two cell types. various forms of autonomic dysfunction are also common in me/cfs ( , ) . it is subsequently important to consider that the microbiome may contribute to host blood pressure regulation. pluznick et al. ( ) found that gut microbiomederived short chain fatty acids such as acetate and propionate travel to the kidneys and blood vessels. there they impacted activity of olfr and gpr , two host receptors that control circulation and blood flow. microbial modulation of host pathways can also drive inflammatory disease processes. pathogens and their associated proteins/metabolites control human metabolism and gene expression in a manner that can push the holobiont toward a state of imbalance and illness. for example, rizzo et al. ( ) found that human herpes viruses a/ b infected nk cells. this infection significantly modified expression of key host mirnas and transcription factors. mycobacterium leprae has been shown to alter human gene expression in a manner that allows it to hijack and reprogram adult schwann cells to a stem-like state ( ) . in a murine model of diabetes, liu et al. ( ) found that eltas, a protein created by s. aureus, prevented insulin from correctly binding its target receptor. this inhibited the phosphorylation of downstream signaling proteins and caused the mice in the study to develop impaired glucose tolerance. the ability of pathogens to interfere with host metabolism is tied to the dynamics of the communities in which they persist. like organisms in any ecosystem, human microbes constantly interact, both directly and indirectly. the proteins and metabolites they create are also in continual interplay. communities of microbes often exhibit synergistic interactions for improved nutrient acquisition, protection from host defenses, and survival in an inflammatory environment ( ) . these include biofilm formation and cooperative signaling via quorum sensing peptides. humphries et al. ( ) recently reported that biofilm bacteria can additionally communicate via ion channel-mediated electrical signaling. even viruses seldom act as single entities. diversity and equilibrium of the bacterial microbiome is regulated by bacteriophage predator-prey dynamics ( ) . pfeiffer and virgin ( ) found that enteric viral virulence is regulated by the activity of neighboring bacteria, fungi, and even helminths. these processes are called "transkingdom interactions." for example, human norovirus can bind carbohydrate histo-blood group antigens present on certain bacterial cells. this facilitates the ability of norovirus to infect human b cells ( ) . interacting microbes may contribute to dysbiosis or imbalance of microbiome communities ( ) . this dysbiosis is characterized by substantial shifts in community structure and diversity. in many cases, pathogens proliferate to inhabit niches once occupied by more innocuous microbes. most well-studied inflammatory disease states are tied to some form of microbiome dysbiosis. these include psoriatic arthritis, systemic lupus erythematosus, type and diabetes, parkinson's disease and a growing number of cancers ( , , ) . the gut microbiome can initiate and promote colorectal cancer at all stages of tumorigenesis by acting as an inducer of dna damage, generating epigenetic changes, regulating cell growth, and modulating host immune responses ( ) . the breast tissue microbiome of women with breast cancer has been shown to differ substantially in composition, virulence and diversity from that of healthy controls ( ) . species composition of the bronchoalveolar microbiome shifted toward a more pathogenic state in patients with sarcoidosis ( ) . alterations in the enteric virome were reported prior to disease onset in children susceptible to developing type diabetes ( ) . several research teams have tied me/cfs to bacterial gut microbiome dysbiosis. giloteaux et al. ( ) found that gut microbiome bacterial diversity was decreased in me/cfs subjects compared to heathy controls. the team also noted increases in certain bacterial species associated with either pro-inflammatory or anti-inflammatory activity. nagy-szakal et al. ( ) also analyzed the me/cfs gut microbiome. the study detected seven gut bacterial species whose relative abundance differed from that of control subjects and were strongly associated with me/cfs. while these findings are of interest, gut microbiome composition is additionally impacted by a host of environmental variables that cause large shifts in the region's microbial ecosystems. these include geographic location, food consumption, and even time of day ( ) . many research teams studying inflammatory conditions have struggled to isolate and/or replicate disease-induced gut microbiome dysbiosis in the face of this "noise." for example, frémont et al. ( ) found that intestinal microbiome species composition differed between patients with me/cfs and healthy controls. however, significant changes in intestinal microbiome composition were also identified between control subjects from norway and controls subjects from belgium. this variation was proposed to arise from differences in diet between the two cultures. studies of the me/cfs blood microbiome may be less subject to this environment-induced variability. furthermore, identification of microbial and/or viral communities in me/cfs blood would allow for a broader picture of possible infectious and inflammatory processes. for example, giloteaux et al. ( ) found that me/cfs subjects had higher levels of bacterial lipopolysaccharides (lps), lps-binding proteins and soluble cd in blood. the team suggested that these inflammatory markers may indicate translocation of gut bacteria into the blood. however, the markers could also reflect the presence of bacteria in the blood itself. the blood microbiome can be characterized if the microbial dna/rna in samples is first separated from that derived from the human genome. olde loohuis et al. ( ) used rna sequencing of reads from whole blood to analyze microbial communities in the blood of almost patients with three neurological conditions: bipolar disorder, schizophrenia, and amyoytrophic lateral sclerosis. the team identified a wide range of bacterial and archaeal phyla in subjects with all three disease states. they observed increased microbial diversity in schizophrenia subjects compared to the two other groups, and replicated the finding in an independent dataset. stephen quake's cell-free dna shotgun sequencing technologies can also characterize bacterial communities in human blood, and can be additionally extended to identify viruses and fungi ( ) . communities of microbes and viruses may also persist in me/cfs brain tissue. readhead et al. ( ) recently detected a range of persistent viruses in the alzheimer's brain. these included herpesviruses, torque teno viruses, adenoviruses, and coronaviruses. the alzheimer's brain has also been shown to harbor bacterial and fungal communities ( , ) . branton et al. ( ) identified hundreds of bacteria and bacteriophage-derived samples in brain tissue removed from patients with epilepsy, and in brain samples obtained from hiv/aids patients after autopsy. in fact, studies of the virome provide significantly extended context on microbiome community dynamics and disease processes. this is because bacteriophages (phages) infect, and subsequently modulate the activity of the bacterial microbiome ( ) . for example, duerkop et al. ( ) characterized the intestinal virome in a model of t-cell-mediated murine colitis. the intestinal phage population changed in colitis, and transitioned from an ordered state to a stochastic dysbiosis. phage populations that expanded during colitis were frequently connected to bacterial hosts that benefit from or are linked to intestinal inflammation. tetz et al. ( ) identified changes in the parkinson's gut bacteriophage community. these included shifts in the phage/bacteria ratio of bacteria known to produce dopamine. species-level studies of the microbiome are also greatly enhanced by analyses that provide further context on disease activity. this is an important consideration because, in theory, the microbiome of a patient with me/cfs could harbor the exact same microbial, viral and/or fungal species as that of a healthy subject. yet many of these organisms could be acting very differently in patients with the disease. species-level analyses of the me/cfs microbiome must subsequently be accompanied by studies that characterize microbial and viral gene expression and/or metabolism. since many of the proteins and metabolites in the human holobiont are microbial in origin, composition of the me/cfs proteome and metabolome change with microbiome species composition. proteome and metabolome analyses additionally reflect microbiome activity. this is because microbes and viruses frequently alter their gene expression in ways that cause them to express different proteins and metabolites over time. the human genome and related epigenetic changes also contribute to the metabolic diversity, although the high level of redundancy between human and microbial metabolites can make the origin of these associations hard to pinpoint. schutzer et al. ( ) demonstrated that the me/cfs cerebrospinal proteome differs substantially from that of healthy controls (figure ) . indeed, of , identified proteins ( . %) were unique to patients with me/cfs, providing strong evidence that me/cfs is indeed characterized by microbiome dysbiosis in tissue and blood. composition of the blood metabolome has also been shown to shift in me/cfs. one such study reported elevated plasma levels of choline, carnitine, and complex lipid metabolites in me/cfs patients ( ) . another analysis demonstrated a sustained hypo-metabolic response in patients with the disease ( ) . this dour-like state can be driven by exposure to adverse environmental conditions, as would be expected if the me/cfs immune system struggles to manage microbiome dysbiosis and associated pathogens. studies of the metabolome are often conducted in an attempt to identify disease-specific biomarkers. however, the metabolome can also be screened for metabolites that directly induce or suppress biological function in patients with a given illness. these studies help dissociate cause from effect and allow for possible modulation of disease phenotype. for example, johnson et al. ( ) investigated the metabolic influence of microbial biofilms on colon cancer tissue and related cancer occurrence. they found that up-regulation of a biofilm-derived polyamide metabolite enhanced both biofilm formation and cancer growth. studies of microbiome activity must account for the fact that pathogens detected in patients with me/cfs are also regularly identified in healthy subjects or in patients with related inflammatory conditions. this is particularly true of studies that have searched for ebv, hhv , cytomegalovirus, and other viruses able to be identified by pcr and/or antibody testing in me/cfs cohorts. this same trend is likely to hold for lessstudied or unidentified human microbes and viruses. while these "overlapping" results are often viewed as problematic, they make sense in light of research that clarifies how differently microbes act depending on host immune status, neighboring species, and a wide range of other variables. for example, susceptibility to hiv infection has been shown to vary based on the species composition and activity of the bacterial vaginal microbiome ( ) . indeed, most human microbes are pathobionts: they can change their gene expression to act as pathogens under conditions of imbalance and immunosuppression ( ) ( ) ( ) . for example, s. pneumoniae can persist as a highly adapted commensal or a virulent pathogen depending on its ability to evade the host immune response ( ) . s. aureus causes a range of illnesses, from skin infections to life-threatening diseases such as endocarditis and meningitis. however, ∼ % of the healthy human population harbors s. aureus as a member of the normal nasal microbiome ( ) . s. aureus virulence in these communities is determined by a number of factors, including the signaling and competitive strategies employed by neighboring microbes. the same is true of escherichia coli (e. coli), which also persists in numerous forms. one study found that "commensal" e. coli could evolve into virulent clones in fewer than generations ( ) . for most microbes, this evolution toward pathogenicity occurs via the acquisition of new genes or alteration of the current genome in a manner that induces gene loss ( ) . for example, loss of muca increases the ability of pseudomonas aeruginosa to evade phagocytosis and resist pulmonary clearance ( ) . it should also be noted that every microbial species represents many different strains, each of which may vary in the set of genes it encodes or in the copy number of such genes. this intra-species variation endows each strain with distinct functional capacities, including differences in virulence, motility, nutrient utilization, and drug resistance ( ). greenblum et al. ( ) identified extensive strainlevel copy-number variation across species in metagenomic samples obtained from patients with irritable bowel syndrome. this was especially true of genes tied to specific community functions, including functions related to community lifestyle. differences in gene copy-number also impacted adaptive functions linked to obesity. yao et al. ( ) found that deletion of a single bacteriodes gene-and the bile salt hydrolase it expresses-altered host metabolism in a manner that impacted weight management, circadian rhythm and immunity. a microbe or a virus' location can also influence its activity. for example, much of the human population harbors hhv- . however, in alzheimer's disease, hhv- a was recently identified in human brain tissue ( ) . there, its activity was shown capable of regulating host molecular, clinical, and neuropathological networks in a manner that can contribute to inflammation and neuronal loss. vanelzakker ( ) has proposed that hhv- may also infect the vagus nerve in me/cfs, resulting in altered gutbrain axis signaling in patients with the illness. the human immune system also plays a central role in determining microbe and viral activity. a robust immune response is often capable of controlling pathogen virulence. however, if pathogens overcome the immune response, or the immune system is suppressed by medications, chemicals, or other environmental factors, pathobionts are more likely to alter their gene expression in a manner that promotes disease. pathobionts can subvert the human immune response by collectively altering their gene expression. yost et al. ( ) performed an excellent gene ontology (go) enrichment analysis of the oral microbiome during periodontal progression. over the two-month study period, changes in the metagenome of non-progressing sites were minor. however, active sites that progressed to periodontitis were characterized by numerous functional genomic signatures. in fact, the team reported a complete rearrangement at the metagenome level between baseline sites that progressed to periodontitis and those that did not. go terms associated with processes including peptidoglycan biosynthesis and potassium transport were highly enriched at baseline sites that later progressed to periodontitis. genes controlling ciliary motility and crispr-associated proteins were also active during initial stages of disease progression. at the breakdown point, active sites expressed genes associated with ferrous iron transport and response to oxidative stress. progression to periodontitis was also correlated with increased expression of putative virulence factors associated with a range of bacterial species. mycoplasma, bacteriophage, and eukaryotic viral activity were higher in progressing sites compared to baseline samples. the team concluded that periodontitis progression is driven by the whole oral microbial community and not just a few select pathogens. in effect, under conditions of increasing inflammation and imbalance, the entire oral community appeared to act together as a pathogen. indeed, groups of bacteria not generally considered pathogens upregulated a large number of the putative virulence factors in active sites. these included veillonella parvula, a microbe almost always associated with dental health. community-wide shifts in microbiome virulence are often driven by dominant pathogens-organisms that become established as central components of the microbiome while suppressing commensal growth and activity ( ) . in other cases, keystone pathogens promote inflammation even when present as quantitatively minor members of the microbiome. for example, p. gingivalis often comprises just . % of periodontal biofilms, yet drives destructive changes in host-microbe interplay by profoundly impairing the innate immune response ( ) . pathogens able to persist inside the cells of the immune system are uniquely positioned to drive inflammatory disease ( ) . indeed, most well-characterized pathogens, including many connected to me/cfs, are capable of intracellular persistence ( ) . by surviving in this fashion, they can directly interfere with human transcription, translation, and dna repair processes (figure ) . pathogens in the cell cytoplasm may further dysregulate the epigenetic environment ( ) . for example, upon infecting a macrophage, mycobacterium tuberculosis alters the expression of human genes ( ) . h. pylori infection predisposes to genomic instability and dna damage, including double strand breaks ( ) . ebv infection of b cells can also promote persistent damage to human dna ( ) . the thousands of metabolites and proteins expressed by intracellular pathogens also interact with the host genome, further modifying human gene expression in a manner that promotes disease. even bacterial quorum sensing peptides can dysregulate human pathway activity. wynendaele et al. ( ) found that quorum sensing molecules created by gramnegative bacteria altered human gene expression in a manner that promoted in vitro angiogeneisis, tumor growth, and neovascularization in colon cancer. intracellular pathogens can also travel between cells via recently characterized tunneling nanotubules (tnts) ( , ) . these cytoplasmic extensions of dendritic cells, glial cells and related human cells allow for the intracellular transfer of micrornas, messenger rnas, prions, viruses, and even whole organelles such as mitochondria ( , ) . for example, hivinduced tunneling nanotubule formation appears to mediate approximately half of hiv virus spread among monocytederived macrophages ( ). dysfunction driven by intracellular infection is compounded by the fact that microbial proteins and metabolites are often identical or similar in structure to those created by their human hosts. the molecular mimicry or sequence homology between these proteins and metabolites makes it increasingly difficult for the human holobiont to recognize "foreign" from "self." for example, altindis et al. ( ) found that viruses carry sequences with significant homology to human insulin-like growth factors (vilps). these vilps can bind human and murine igf- receptors in vitro, resulting in autophosphorylation and downstream signaling. e. coli harbors a large, diverse network of proteins that actively promote endogenous dna damage in cells ( ) . however, at least of these dna-damaging proteins have human homologs that also promote dna damage and mutagenesis in the human host. indeed, redundancy between human and microbial metabolites, proteins, and pathways is so great that the potential for molecular mimicry to contribute to host immune and metabolic dysfunction is semi-infinite. for example, viral vesicles and human extracellular vesicles (evs) share considerable structural and functional similarity ( ) . these similarities are so extensive that it is difficult to distinguish evs from (noninfectious) viruses. many pathogens employ common survival mechanisms to persist in host cells, tissue and blood. the metabolic dysfunction driven by these different microbes and viruses can result in similar clusters of human inflammatory symptoms. the ability of various pathogens to dysregulate activity of the vitamin d nuclear receptor (vdr) is an excellent example of how different microbes can drive similar disease processes. the vdr regulates expression of hundreds of human genes, many of which regulate inflammatory and malignant processes ( , ) . the receptor also controls signaling of tlr and several families of antimicrobial peptides including cathelicidin (ll- ) ( ) . pathogens capable of slowing vdr activity can subsequently facilitate their survival by slowing the innate immune response. pathogens frequently linked to me/cfs or inflammatory disease have evolved to survive in this fashion. vdr activity is downregulated as much as times in epstein barr virus-infected lymphoblastoid cell lines ( ) (figure ) . hiv, m. tuberculosis cytomegalovirus, borrelia burgdorferi and mycobacterium leprae additionally dysregulate vdr activity to various degrees ( , ( ) ( ) ( ) ( ) . the fungus aspergillus fumigatus secretes a gliotoxin that significantly downregulates vdr expression ( ) . because disabling the innate immune system via the vdr pathway is such a logical survival mechanism, other uncharacterized bacteria, viruses or fungi may have also evolved to dysregulate receptor activity. it follows that me/cfs patients with similar symptoms may not always test positive for the same mix of pathogens and/or communities of pathobionts. similarly, composition of the me/cfs microbiome, proteome and metabolome should be expected to differ somewhat from study to study. instead of worrying about these inconsistencies, the me/cfs research community should strive to better characterize even more common mechanisms of pathogen survival and persistence. the ability of pathogens to disable the host immune response extends far beyond the vdr pathway. pushalkar et al. ( ) identified a distinct and abundant pancreatic microbiome associated with progressive pancreatic cancer. this dysbiotic microbiome drove oncogenesis by suppressing macrophage differentiation and t cell activity. another study found that candida albicans's transition from extracellular to intracellular pathogen was accompanied by a coordinated, time-dependent shift in gene expression for both host and fungus ( ) . these gene expression changes led to a gradual decline in proinflammatory cytokine activation by the host immune system. these findings shed light on a recent me/cfs study. hornig et al. ( ) reported distinct alterations in plasma immune signatures-including prominent activation of both pro-and anti-inflammatory cytokines-early in the course of me/cfs. however, these alterations were not observed in subjects with a longer duration of illness. there are a number of explanations for hornig's observations. me/cfs-associated pathogens may gradually alter their gene activity to persist in more latent, intracellular forms less recognized by the host immune system. or, in early-stage me/cfs, the immune system may actively attempt to target a growing infectious burden. over time however, pathogens in the microbiome may disable the immune response to a point where "immune exhaustion" occurs ( , ) . immunopathology and cytokine production would subsequently drop. the resulting disease state could be compared to a garden, in which healthy plants become progressively stifled by others, such as kudzu vine. many research teams are studying how "acute" pathogens (i.e., well-characterized agents associated with known infectious diseases) can cause chronic symptoms by persisting in latent forms. these include zika, borrelia burgdorferi, influenza, and other well-characterized viruses and bacteria ( ) . zika was shown capable of persisting in the cerebrospinal fluid (csf) and lymph nodes of infected rhesus monkeys for months after the virus had been cleared from mucosal secretions, peripheral blood and urine ( ) . viral persistence in both the lymph nodes and csf was correlated with upregulation of genes involved in pro-inflammatory and anti-apoptotic pathways. tens of thousands of ebola survivors have developed chronic symptoms months or years after initial infection ( ) . these "post-ebola syndrome" symptoms include extreme fatigue, severe pain, eye problems, and a host of neurological issues. while the virus is hard to identify in the blood of such patients, ebola has been detected in men's semen years after "recovery" ( ) . another study found that, up to a month after initial infection, influenza viruses regulated the long-term expression of inflammatory and neuron/glia-specific genes in mice ( ) . this was associated with chronic neuroinflammation characterized by an increase in the number of activated microglia and impairment of spatial memory formation. persistent measles virus is associated with conditions such as paget's disease, multiple sclerosis and crohn's disease ( , ) . measles virus rna has been detected in blood, respiratory secretions, urine, and lymphoid tissue for weeks to months after clearance of the "acute" infection ( ) . doi et al. ( ) found that, in vitro, measles virus persistence correlated with viral transition from a lytic to non-lytic mode that allowed the virus to evade the host innate immune response. hundreds of studies demonstrate that acute bacterial pathogens can transition into latent forms that lack a classical cell wall ( ) . these persistent variants are referred to as l-form bacteria. antibiotic use can induce persistent l-form growth. in fact, researchers create l-forms by deliberately culturing classical bacteria in conjunction with the beta-lactam antibiotics ( ) . one microarray analysis of l-form growth revealed upregulation of genes shared in common with persister cells and biofilms ( ) . l-form bacteria have been implicated in dozens of chronic conditions including rheumatoid arthritis, multiple sclerosis and sarcoidosis ( , , ) . a better understanding of these chronic sequelae would greatly benefit the me/cfs community, since a similar chronic progression of symptoms is frequently documented in me/cfs. at different points in history, me/cfs has been called "post-polio syndrome, " "chronic mononucleosis syndrome" and "post-viral syndrome" due to the fact that chronic symptoms are often noted after acute infection with polio virus, epstein barr virus, influenza or a range of other pathogens ( ) ( ) ( ) . chia et al. ( ) found that patients with acute enterovirus infection went on to develop a multitude of chronic symptoms consistent with an me/cfs diagnosis. years after the initial infection, enterovirus protein and rna were still present in these patients' stomach biopsies. it is clear that novel methodologies may be required to best identify pathogens in their latent forms. pathogens that persist inside human immune cells and associated tunneling nanotubuoles have been particularly hard to detect. when persistent zika virus was identified in rhesus monkeys, zikaspecific antibodies were not detected in the csf, despite prolonged and robust responses in peripheral blood. this suggested an additional mechanism for viral persistence in certain "anotomic sanctuaries." modern living has also increased the likelihood that "acute" infections may generate chronic sequelae. before the advent of antibiotics, steroid medications and childhood vaccines, almost half of all children under the age of five died from acute infectious disease ( ) . today, most individuals in first-world countries survive repeated acute infections over the course of decades. while certain dominant or keystone pathogens may be reliably identified in patients with me/cfs, composition of the me/cfs microbiome will likely differ between patients. even in hiv/aids, where an easily detected virus dysregulates immunity, disease symptoms reflect a mix of those driven by hiv, and those driven by "co-infectious" agents able to take advantage of the immunocompromised host ( ) . no two patients with hiv/aids are expected to harbor the same mix of these additional persistent bacteria, fungi, and viruses. the same is true of most cancers, an increasing number of which are now tied to severe microbiome dysbiosis ( ) . it is widely accepted that no two cancers are alike, and any tumor-associated microbiome is expected to differ somewhat among individual study subjects ( ) . this same pattern, in which unique infectious history impacts symptom presentation may also occur in me/cfs. a recent study by brodin et al. ( ) demonstrated the profound impact of infectious history on host immunity. the team performed a systems-level analysis of healthy twins between the ages of and . they measured immune parameters, including cell population frequencies, cytokine responses, and serum proteins, and found that % of these are dominated, and % almost completely determined, by non-heritable environmental influences. many of these parameters became more variable with age, emphasizing the cumulative influence of environmental exposure. the team also calculated how acquisition of just one chronic pathogen-cytomegalovirus (cmv)-conditions the immune response. identical twins discordant for cmv infection showed greatly reduced correlations for many immune cell frequencies, cell signaling responses, and cytokine concentrations. in general, the influence of cmv was so broad that it affected of the measurements dispersed throughout the immune network. these and related findings led brodin and team to conclude that the immune response is "very much shaped by the environment and most likely by the many different microbes an individual encounters in their lifetime." the above suggests that me/cfs may be driven by a process we have termed "successive infection." during successive infection, an "acute" infection or "initial immunosuppressive event" dysregulates the host immune system. this makes it easier for microbes to subvert the immune response by acting as polymicrobial entities. pathobionts alter their gene expression to better promote community-wide virulence. infected human cells fail to correctly express human metabolites in the presence of pathogen-generated proteins, metabolites, and enzymes. dysfunction driven by molecular mimicry increases. certain pathogens may infect mitochondria or central nervous system tissue. intracellular pathogens slow the human immune response, causing the host to more easily acquire other infectious agents. this creates a snowball effect in which the microbiome becomes increasingly dysbiotic as the strength of the immune response weakens over time. eventually, the human host may present with symptoms characteristic of me/cfs or a related inflammatory diagnosis. the unique symptoms any one patient develops are expected to vary based on the species, strain, virulence, and location of the pathogens driving dysbiosis, along with the myriad ways in which the metabolites and proteins created by these organisms cause dysfunction by interacting with those of the host. early childhood infections may predispose to dysbiosis at a later date ( ) . for example, measles depletes host b and t lymphocytes for up to - years after initial infection. this immunosuppression can reset previously acquired immunity and renders the host more susceptible to other pathogens ( ) . in other cases, a toxic environmental exposure, infection during surgery or the difficulty of enduring a traumatic event may weaken the immune response to a point where previously subclinical infections become active. me/cfs outbreaks, in which numerous patients developed the illness at relatively the same time, may well represent this phenomenon at work. the successive infectious process may even begin in the womb. depending on the health of the parent, founding microbiome communities in the placenta, vagina and breast milk may already be dysbiotic. cabrera-rubio et al. ( ) found that the breast milk microbiome of obese mothers tended to contain a different and less diverse bacterial community then that of normal-weight mothers. pathogens in the placental microbiome can alter methylation of human dna in a manner that may negatively impact the later life disease of premature babies ( ) . many aspects of modern living can additionally drive successive infection. antibiotic use disrupts the ecology of the human microbiome ( ) . antibiotic resistance genes from farm animals and produce are regularly transferred into the human food supply ( ) . electromagnetic radiation has been shown to lower immunity ( ) . the immunosuppressive biologics and supplements often prescribed for inflammatory disease further allow pathobionts in the microbiome to proliferate. for example, diaz et al. ( ) found that the salivary bacteriome of patients taking immunosuppressive biologics was more permissive to the growth of oral opportunistic pathogens. modern society also exposes the average person to pathogens our recent ancestors were unlikely to encounter. international airports harbor pathogens from across the globe ( ) . food products, and the microbes they contain, are frequently imported from foreign destinations ( ) . one study found a range of opportunistic pathogens enriched in showerhead biofilms ( ) . numerous pathogens were identified in commonly smoked cigarettes ( ) . many such pathogens are capable of persisting in human microbiome ecosystems, where the host may lack immunity toward their presence. me/cfs is a spectrum disorder with a diagnostic criterion that includes a range of physical and neurological symptoms ( ) . if successive infection contributes to me/cfs, this variability in symptom presentation is expected. furthermore, factoring "unique infectious history" into the disease process helps explain why patients with me/cfs often suffer from a multitude of symptoms not included in the official diagnostic criteria. because patients with me/cfs suffer from such diverse symptoms, it has been argued that they should be grouped into separately studied "subgroups" ( ) . in some cases this makes sense. for example, studies that distinguish earlystage/late-stage me/cfs patients may further elucidate how the immune response is modified by the microbiome over time. however, if successive infection contributes to me/cfs, future research should also focus on better understanding the common pathogenesis shared by all subjects. indeed, successive infection may contribute to other conditions tied to microbiome dysbiosis, persistent infection, and adverse environmental exposure. me/cfs should be studied in concert with these other conditions, which include gulf war syndrome, ehlers-danlos syndrome, chronic lyme disease, and fibromyalgia among others ( ) ( ) ( ) ( ) ( ) . the high levels of comorbidity and symptom overlap between patients with me/cfs and these related inflammatory diagnoses strengthens this assumption. a number of autoantibodies have been detected in patients with me/cfs ( ). this has led some research teams to postulate that me/cfs should be regarded as an "autoimmune" disorder ( ) . however, the classical "theory of autoimmunity" is in the process of being re-evaluated ( , ) . increasing evidence suggests that "autoantibodies" are actually created in response to chronic, persistent microbiome pathogens. under such conditions molecular mimicry or structural homology between pathogen and host proteins can result in "collateral damage" toward human tissue. for example, vojdani et al. ( ) found that different alzheimer's-associated pathogens or their molecules could react with antibodies against amyloid beta via molecular mimicry or the binding of bacterial toxins to amyloid beta. a growing body of research documents "autoantibody" production in response to a range of bacterial, viral and fungal pathogens/pathobionts. these pathogens are not shortterm "triggers" but persist as members of complex microbiome communities. for example, "autoantibody" production was recently tied to microbiome pathobiont enterococcus gallinarum. manfredo et al. ( ) detected e. galinarum in the mesenteric veins, lymph nodes, spleens and livers of mice made genetically prone to autoimmunity. in these mice, the bacterium initiated the production of "autoantibodies, " inflammation and activated t cells. however, this "autoantibody" production stopped when e. gallinarum's growth was suppressed with the antibiotic vancomycin or with an intramuscular vaccine. in addition, e. gallinarum-specific dna was recovered from liver biopsies of human autoimmune patients, and co-cultures with human hepatocytes replicated the murine findings. indeed, many pathogens can drive the activated or clonal t cell expansion often associated with "autoimmune" conditions. for example, tuffs et al. ( ) found that s. aureus endotoxins triggered uncontrolled activation of t cells. this led to a proinflammatory cytokine storm that accounted for both t cell activation and related inflammation. microbes and their metabolites control bidirectional signaling between the gut and the brain via pathways collectively known as the gut-brain axis ( ) . the gut-brain axis involves various afferent and efferent pathways including the vagus nerve, with signaling impacting neural, endocrine, and immune processes. the gut's enteric nervous system contains over million neurons-more than in either the peripheral nervous system or spinal cord ( ) . it follows that gut microbiome dysbiosis may modulate brain activity. for example, sampson et al. ( ) transplanted fecal samples from patients with parkinson's disease into germ-free mice. these mice, and not controls, exhibited physical symptoms associated with parkinson's. however, there is also growing evidence that humans may harbor a brain microbiome. researchers at harvard university are characterizing this ecosystem as part of the ongoing "brain microbiome project." in what marks a major paradigm shift, multiple research teams have shown that both amyloid beta and prion protein (prp) are potent antimicrobial peptides ( , ) . amyloid beta exhibited antimicrobial activity against a range of common microorganisms with a potency equivalent to, and in some cases greater than, cathelicidin (ll- ) ( ). these pathogens included s. typhimurium, candida albicans and, more recently, a number of herpesviruses capable of persisting in the alzheimer's brain ( , ) . the findings strongly suggest that amyloid beta and prp are not useless byproducts of abnormal brain catabolism. rather, they appear to form a mediated response of the innate immune system toward infectious agents in brain tissue ( ) . a number of brain abnormalities have been reported in patents with me/cfs ( ) . these findings must be interpreted in light of these novel infection-based paradigms and in concert with emerging data on the brain microbiome. in a study of aortic aneurysms, gottlieb et al. ( ) reported that bak snp-containing alleles were detected in aortic tissue but not in blood samples from the same patients. more recently ursini et al. ( ) found that schizophrenia gene risk loci that interact with early-life complications are highly expressed in the placenta. however, these loci were differentially expressed in placentas from women who suffered complications during pregnancy. they were also differentially upregulated in placentae from male compared with female offspring. these and related findings strongly suggest that the environment can select for human genome activity. for example, harley et al. ( ) found that in ebv infected cells, ebna and its transcription factors modulated the activity of human genes associated with risk for multiple sclerosis, rheumatoid arthritis, type diabetes and other conditions. in fact, nearly half of systemic lupus erythematosus risk loci were occupied by ebna and co-clustering human transcription factors. studies of the human genome must also account for the full extent of microbial dna and rna in human tissue and blood. if a genomic assembler fails to account for this contamination, chances of a false positive single nucleotide polymorphism (snp) increase significantly during analysis ( ) . contamination with even a small amount of microbial dna/rna-just one or two base pairs of difference-is enough to cause significant statistical errors in this fashion. if me/cfs is driven by successive infection, treatments that support or activate the human immune system could improve microbiome health by allowing patients to better target persistent pathogens. development of such therapies should be a priority for the me/cfs research community. however, most immunostimulative treatments that target pathogens are characterized by immunopathology-a cascade of reactions in which inflammation, cytokine release and endotoxin release are generated as part of the immune system's response to microbial death ( ) ( ) ( ) . the death of intracellular pathogens is particularly difficult for the host to manage, as the body must deal with debris generated from apoptosis. inflammation is also generated in response to bacterial cell wall components including the endotoxin lipopolysaccharide of gram-negative strains ( ) . luckily, immunopathology-generated symptoms are generally temporary in nature, and tend to subside as an increasing number of pathogens are eradicated. temporary immunopathology resulting from antimicrobial treatment has been documented for over a century, with symptoms varying depending on the nature of targeted pathogens. first referred to as the jarisch-herxheimer reaction, the phenomenon was originally observed during treatment of syphilis with mercury and penicillin ( , ) . the jarisch-herxheimer reaction/immunopathology has since been noted in a broad spectrum of chronic inflammatory conditions including tuberculosis and brucellosis ( ) . short-term immunopathology is also a central feature of acute infection. if a patient develops the flu, inflammatory symptoms increase as the immune system releases cytokines and chemokines in response to the infecting virus. hiv/aids patients undergo a form of immunopathology called immune reconstitution inflammatory syndrome (iris) following treatment with combination antiretroviral therapy (art) ( ) . iris occurs as art enables the host immune system to better target pathogens acquired during previous periods of hiv-driven immunosuppression. a range of well-characterized pathogens have been linked to iris including the herpesviruses and m. tuberculosis ( ) . however, the inflammatory reaction is also noted in culture-negative patients, suggesting iris may also involve novel or uncharacterized pathogens ( ) . over the past decade, in concert with our clinical collaborators, we developed an immunostimulative therapy used to treat patients with a range of chronic inflammatory conditions ( ) . treatment centers on the use of a putative vdr agonist in the form of olmesartan medoxomil, with the goal of reactivating components of innate immunity under vdr control. in , we published a series of case histories demonstrating improvement in me/cfs patients administered this treatment ( ) . however, all me/cfs subjects administered the therapy experienced immunopathology and associated inflammatory symptom increases that lasted for many years. as a general trend, patients administered the treatment during earlier stages of disease experienced less immunopathology, emphasizing the need for immunostimulative therapies to be used in a predictive and even preventative fashion. while some me/cfs physicians may feel uneasy about the suffering induced by immunopathology, other research communities have become accustomed to treatments that cause temporary discomfort. novel cancer immunotherapies also generate immunopathology by activating patient t cells. this allows the immune system to better target malignant tumors ( , ) . the resulting "cytokine release syndrome" leads to profound, temporary, symptom increases. however, these increased symptoms are considered acceptable, as patients who endure the reaction are more likely to enter a state of remission. since many forms of cancer are tied to severe microbiome dysbiosis, at least part of the "cytokine release syndrome" may result from the death of persistent pathogens. the history of me/cfs strongly suggests that infectious agents play a central role in driving the disease process. however, the discovery of the human microbiome has revolutionized the manner in which persistent infection and chronic inflammation are understood and studied. humans harbor extensive microbiome communities of bacteria, viruses, and fungi in nearly all tissue and blood. the hundreds of millions of unique genes harbored by this microbiome dwarf the ∼ , in the human genome. humans are best described as holobionts, in which these microbial genomes and the human genome continually interact to regulate host gene expression, metabolism and immunity. many inflammatory disease states, including neurological conditions and cancers, are tied to dysbiosis or imbalance of human microbiome communities in various body sites. while gut microbiome dysbiosis has already been identified in me/cfs, distinct microbial and viral communities may additionally persist in me/cfs blood and brain tissue. possible identification of these microbiomes should be a priority for the me/cfs research community, but analysis requires the use of very specific technologies and methodologies. pathogens and their associated proteins/metabolites control human metabolism and gene expression in a manner that can push the human holobiont toward a state of illness. studies of microbiome dysbiosis in me/cfs must consider this microbe and viral activity. most human microbes can alter their gene expression to act as pathogens under conditions of imbalance and immunosuppression. this pathobiont behavior is further determined by the activity and virulence of neighboring microbes. patients with me/cfs may harbor many of the same microbes and viruses as heathy individuals, yet these pathobionts may act with increased virulence in patients with the illness. intracellular pathogens, including several associated with me/cfs, have been shown to directly interfere with human transcription, translation, and dna repair processes. molecular mimicry between host and pathogen proteins/metabolites further exacerbates this interference. interacting microbes can also drive disease by changing their collective gene expression. other pathogens disable mitochondria, or may infect central nervous system tissue in ways that dysregulate signaling via the gutbrain axis. antibodies and/or clonal t cells identified in patients with me/cfs are likely activated in response to many of these persistent microbiome pathogens. different pathogens have evolved similar survival mechanisms to disable the host immune response and/or host metabolic pathways. the metabolic dysfunction driven by these different microbes can result in similar clusters of inflammatory symptoms. me/cfs may be driven by this pathogen-induced dysfunction, with the nature of dysbiosis and symptom presentation varying based on a patient's unique infectious and environmental history. an initial infection or environmental exposure weakens the host immune system. this makes it easier for pathobionts to subvert the immune response and interfere with host gene expression and metabolism. a snowball effect begins, in which the microbiome becomes increasingly dysbiotic as the strength of the immune response weakens over time. the unique symptoms any one me/cfs patient develops are expected to vary depending on the location, species, strain and virulence of the pathogens driving this dysbiosis. thus, while certain dominant or keystone pathogens may be identified in me/cfs, composition of the me/cfs microbiome, metabolome, and proteome should be expected to differ somewhat among individual patients. these common mechanisms suggest that me/cfs is best studied in concert with other chronic conditions tied to microbiome dysbiosis, persistent infection and adverse environmental exposure. these include fibromyalgia and gulf war syndrome, but also conditions like post-ebola syndrome in which severe chronic symptoms develop after infection with an "acute" infectious agent that is able to persist in latent forms. treatments that support or activate the human immune system could allow me/cfs patients to improve microbiome health by better targeting pathogens over time. like the novel immunotherapies being developed for cancers, these immunostimulative therapies would be expected to generate temporary immunopathology. institutional reviewers hesitant to approve immunopathology-based therapies should consider that me/cfs quality of life is typically very low, with patients demonstrating a substantial increase in mortality from suicide ( ) . it often takes patients years to receive a diagnosis of me/cfs. this delay wastes a valuable period during which the immune system is most responsive to immunostimulatory treatment. patients treated during earlier stage disease are also less likely to experience severe or long-lasting immunopathology. this suggests that immunostimulative therapies should be administered in a predictive and even preventative fashion. in addition, interventions or treatments that might help patients better manage the byproducts of immunopathology (bacterial lps etc.) should become a priority for the research 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chronic fatigue syndrome bak gene variation and abdominal aortic aneurysms convergence of placenta biology and genetic risk for schizophrenia transcription factors operate across disease loci, with ebna implicated in autoimmunity the human microbiome and autoimmunity on the translocation of bacteria and their lipopolysaccharides between blood and peripheral locations in chronic, inflammatory diseases: the central roles of lps and lps-induced cell death hiv & immune reconstitution inflammatory syndrome (iris) managing cytokine release syndrome associated with novel t cell-engaging therapies the jarisch-herxheimer reaction in syphilis: could molecular typing help to understand it better? jarisch-herxheimer reaction in early syphilis tuberculosis-associated immune reconstitution inflammatory syndrome and unmasking of tuberculosis by antiretroviral therapy iedea southern and central africa immune reconstitution inflammatory syndrome in patients starting antiretroviral therapy for hiv infection: a systematic review and meta-analysis immunostimulation in the treatment for chronic fatigue syndrome/myalgic encephalomyelitis immunostimulation in the era of the metagenome cytokine release syndrome mortality of people with chronic fatigue syndrome: a retrospective cohort study in england and wales from the south london and maudsley nhs foundation trust biomedical research centre (slam brc) clinical record interactive search (cris) register the authors would like to acknowledge michael eason kirkpatrick and janet raty for their help with graphic design. they also thank sara nicole azevedo for her help with technical editing of the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © proal and marshall. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -i pdngb authors: böhm, r. title: chapter pathogenic agents date: - - journal: waste management series doi: . /s - ( ) - sha: doc_id: cord_uid: i pdngb publisher summary this chapter describes the pathogens of humans, animals, and plants that may be present in organic wastes. the recycling of biological wastes by aerobic or anaerobic biotechnological treatment is necessary to protect the environment and to save natural resources. the recycling process may be conducted either in large-scale plants operated mostly in urban industrial areas, or in small plants operated primarily in the rural environment to improve the farmer's income. a relatively large number of pathogens are found in solid and liquid organic wastes and the most prevalent are bacteria, viruses, fungi, and parasites. fungi present in wastes and materials used for composting are mainly of interest from the point of view of occupational health and phytohygiene. the presence of parasites or their infective stages in wastes or residues of plant, animal, or human origin depends on the nature of the wastes and the level of pretreatment. parasites are of veterinary and medical importance if the raw materials used for composting are generated in wastewater treatment facilities or in slaughterhouses. plant pathogenic parasites must also be considered, even if some of them are highly specialized on certain plants, which limit their epidemiological importance. cyst-forming nematodes are the most relevant because these cysts may survive in the soil for several years. recycling of biological wastes by aerobic or anaerobic biotechnological treatment is necessary to protect the environment and to save natural resources. the recycling process may be conducted either: ( ) in large-scale plants operated mostly in urban industrial areas, or ( ) in small plants operated primarily in the rural environment to improve the farmer's income. municipal solid wastes, sewage sludge, and other organic sludges may contain different kinds of pathogens that are infectious to several species of animals and plants, as well as to humans. the origin and nature of organic wastes and the different types of sludges always cause hygienic risks in storage, collection, handling, processing, and utilization. these risks exist if the organic wastes are collected and processed via source separation (biowastes), if they are collected mixed with other wastes from households or relevant processing industries, if they are generated in the treatment of industrial or municipal wastewater, or if the sludge results from industrial processing of other organic materials. generally, three main types of risks are associated with recycling of organic wastes: . occupational health risks; . environmental risks; and . risks associated with product safety. therefore, hygienic principles must be followed in the collection, transportation, processing, storage, and distribution of such materials. occupational health risks exist in small as well as in large-scale plants; however, this is not the subject of this book and more details on occupational health risks may be found in other publications (hickey and reist, ; grüner, ; böhm et al., ) . environmental risks and risks concerning epidemiological pathways closed via contaminated products, as well as measures that can be adopted against such risks, will be dealt with in this chapter, along with relations to the pathogens involved and to the different epidemiological situations that can be expected under the given conditions. the information in table . summarizes the main epidemiological risks associated with recycling solid and liquid organic wastes. the risks related to the different raw materials are mainly defined by the presence of certain organisms and their properties. this will be covered in more detail in other sections of this chapter. generally, they can be divided into those causing phytohygienic risks and those related to human and animal health. the data in table . provide a survey concerning the hygienic relevance of some organic wastes originating from households. table . . epidemiological importance of processed wastes and residuals, and of the resulting products contamination of meadows introduction of pathogens by storage and processing close to susceptible animals aerogenic transmission by spreading the materials onto farm land handling of contaminated products in the household occupational exposure to contaminated products accidental transmission to immune-compromised persons c. indirect transmission to farm animals via feed from contaminated sites via living vectors d. indirect transmission to humans via introduction of zoonotic agents into the food chain via food contaminated by living vectors e. introduction into the environment generation of carriers in the fauna introduction of undesired properties into the microflora meat-leftovers (raw or insufficiently heated) -meat cuttings, tendons, rinds, etc. + − food of animal origin -egg shells + − -several meat and dairy products + − -raw milk products + − -leftovers from fish and shellfish + − other wastes (animal and man) -dirty packing material for meat and products of animal origin + − -used litter and wastes from pets + + -used paper handkerchiefs and sanitary pads the data in the table show that pathogens for humans and animals are not always limited to materials from warm-blooded individuals, but that they can originate from plant materials as well. a relatively large number of pathogens are found in solid and liquid organic wastes; the most prevalent are bacteria, viruses, fungi, and parasites. a brief discussion of each type follows. bacteria may propagate in the raw materials during collection, transport, and storage and they are involved in the composting process itself. in the latter case, especially in thermophilic processes, the bacterial pathogens are more or less reduced in number. it has to be considered then that they often propagate in the raw materials before being processed. this increases the risk and leads to a general contamination of the collected materials during transport. if sludges from wastewater treatment (biosolids) are composted, nearly all gut-related pathogens may be found, while some materials of industrial origin are nearly sterile if they were heated during processing. a compilation of bacterial pathogens of humans, animals, and plants that may be present in organic wastes is presented in tables . and . . presence of bacterial pathogens alone has nothing to do with the resulting risk of infection. transmission via the environment and the resulting route of infection must be a factor in the epidemiology of the resulting disease. the most important pathogens in this connection are salmonella spp.; others, like listeria or clostridia, may also be present in the material, but they are also present in the soil and therefore are of secondary importance if the product is used as a soil conditioner. several viruses of plant origin may be present in the raw materials (table . ), as well as gut-related viruses of animal and human origin. special risks are connected with viral causative agents of animal diseases such as foot and mouth disease (fmd), which may be present in meat and meat products if the animals had been slaughtered before showing clinical signs of illness. in sludges from wastewater treatment or in other wastes with fecal contamination, hepatitis a virus, rotaviruses, and caliciviruses have to be taken into account as emerging pathogens. fungi present in wastes and materials used for composting are mainly of interest from the point of view of occupational health and phytohygiene. from the species pathogenic to warm-blooded animals and humans in europe, mainly candida albicans and aspergillus fumigatus have to be mentioned in this connection. for plants, a variety of species are important pathogens; well known in this framework is plasmodiophora brassicae. a selection of relevant plant pathogenic fungi is given in table . . the presence of parasites or their infective stages in wastes or residues of plant, animal, or human origin depends on the nature of the wastes and the level of pretreatment. parasites are of veterinary and medical importance if the raw materials used for composting are generated in wastewater treatment facilities or in slaughterhouses (e.g., contents of the digestive tract) and, in general, if the wastes are of fecal origin or contaminated with fecal matter. some of the most important parasites of epidemiological relevance are listed in table . . from the protozoal ones, cryptosporidium parvum seems to be the most relevant, while eggs of ascaris species of humans and animals are the most important metazoic parasites. parasites at large are not only important as pathogens, they may also be a risk factor as vectors in transmission of diseases from wastes to susceptible populations (e.g., by flies and cockroaches). plant pathogenic parasites must also be considered, even if some of them are highly specialized on certain plants, which limit their epidemiological importance. cyst-forming nematodes are the most relevant because these cysts may survive in the soil for several years. roots and other soil-containing materials used in composting may contain such transmissible stages; more details can be obtained in the literature, such as in menke ( ) . a selection of relevant nematodes is listed in table . . most countries in the world, including most member states of the european union, do not have legal regulations defining the hygienic requirements for finished compost. in most cases, indirect parameters, e.g., maturity of the product, frequency of turning of windrows, temperature-time relationships, have to be kept voluntary or in the framework of quality networks. sometimes, only recommendations or legal demands are given by defining the particle size, minimum temperature, and exposure time, which are insufficient if they are not based on validation experiments or scientific investigations. a typical example is the requirement for composting in a reactor requiring a mm particle size combined with an exposure time of at least h and a temperature of at least • c, as set forth in the european animal by-product regulations (ec, ) , which is not covered by any validation experiment. there is no doubt that according to the state of science and technology, a composting process if properly applied can inactivate relevant pathogens and weeds. therefore, sometimes recommendations are given on how to operate the composting process in order to achieve hygienic safety of the product. such strict recommendations may be: "composting plants shall operate with the material at a moisture content of - % and a ph of about . if the facility uses windrows, the exposure time shall be at least for a period of weeks at • c. if the facility uses in-vessel technology, the exposure time will be for a period of week at • c." from a scientific point of view, other combinations of temperature and exposure time will lead to sufficient inactivation of the target organisms, since those are mainly vegetative bacteria, viruses, eggs of parasites, or similar transmission stages and seeds. even spores of bacillus anthracis can be inactivated under certain circumstances, as shown by miersch ( ) . until now it is not definitely clear if transmissible spongiform encephalopathies (tse) agents could be inactivated in composting; more research is needed in order to confirm or disprove the findings of brown and gajdusek ( ) , indicating that the scrapie agent may survive for more than years in the soil. however, their experimental setup does not appear to be representative of the situation in composting. nevertheless, inadequate technical design and improper management of the composting process may result in survival and transmission of the pathogens involved; therefore, only treatment in a validated process under steady supervision will lead to a hygienically safe product. this means that the following strategies may be combined in order to assure hygienically safe utilization of the processed materials: • validation of treatment (disinfection by chemical, physical, or biological means), • continuous recording of relevant process parameters (e.g., temperature, ph, exposure time), • microbiological monitoring of the final product (indicators), and • restrictions on the utilization of the final product. the capability of a process to inactivate pathogens causing risks that depend on the raw material cannot be judged simply by analysis of presence or absence of indicators (bacterial, viral, fungal, or parasitic) in the final product. when only product monitoring is used in order to validate a process, it can provide a false sense of security that the process is able to control the relevant hazards in the final product. absence of one or all of the mentioned pathogens or indicators in the final product may be caused by several other reasons: • they were not present in the raw material, • they were present in the raw material, but in a low concentration (less than log), • due to ineffective enrichment procedures (e.g., bacteria), re-isolation was insufficient, or • failure of isolation due to effects of the complicated matrix (e.g., viruses). therefore, the possibility of validating a process by input-output analysis of certain indicators is, in principle, possible but under practical conditions a rare event depending on the microbiological properties of the input materials processed and other strategies that must be followed, e.g., process validation with one or more representative testorganism. if either the thermophilic process itself or if an additional thermal treatment shall provide the inactivation of pathogens belonging to the indicated level of thermoand chemo-resistance, representative test-organisms must be exposed in a similar matrix as that being treated in a suitable test-body in a defined validation experiment. the relevant process parameters must be recorded during the exposure in order to define the technical conditions to be kept for safe inactivation according to the results of the survival experiments. the validation of the treatment with respect to hygienic safety for animals, humans, and, if necessary, plants may be done in several ways. for example, the "german laga m , " (laga-länderarbeitsgemeinschaft abfall, ) offered a relatively broad approach for solving this problem with respect to composting based on the "atv" (german association of wastewater experts) recommendations from for sewage sludge treatment (atv-abwassertechnische vereinigung, ) . process safety concerning the inactivation of relevant transmissible agents for humans and animals is validated in two steps. the first step is the validation of the process as designed by the producer of the technical equipment in the basic procedure. the second step involves putting into service validation of a treatment process at the plant with the input material used under practical conditions. in both validation procedures, salmonella senftenberg w (h s negative) is used as a test organism exposed in specially designed test carriers. in such a validation procedure, testing is always done twice, in summertime and in wintertime. this is a very complete and safe system; if due to economic considerations the system should be simplified and only a one-step procedure should be the aim, it must be the one that deals with putting validation into service. a scheme of how this validation could be organized in this case, taking into account the annual throughput of material in the plants, is given in table . from the "german biowaste ordinance" ( ) . the question of how validation should be performed under practical conditions and which test bodies can be applied is not easy to answer. in biogas plants, mainly two types of test bodies may be used, depending on the test organisms (rapp, ; böhm et al., ) . those test organisms like bacteria, fungi, and parasites type test bodies that can be retained by a membrane filter in a test body filled with liquid, as shown in fig. . , could be used. exposure of viruses to a process requires different test bodies. the virus material is adsorbed onto a special filter material and released after exposure by desorption, by washing with a special solution. such a type test body is shown in fig. . . in composting, different approaches are described for bacteria, because a representative amount of raw material can be contaminated directly with the test strain and put into textile sacks protected from mechanical destruction by a perforated metal basket, as shown in fig. . . this system may also be used for phytopathogenic fungi and viruses (contained in plant tissue), as well as for seeds. viruses not related to tissue may be exposed in the material, as described above in a type test body. year plus one more sample for every tons throughput) . > ( samples per year plus one more sample for every tons) total a at small plants half the number of samples (≤ tons/acre). b every statement concerning the hygienic safety of the product is always based on the result of the supervision of the final product together with the result of the validation of the process. c every sample is a "mixed sample" (about kg) based on five single samples of the final product. the choice of test organisms and the techniques applied depend on the origin of the raw materials and the intended utilization of the product (böhm, ) . several test organisms other than salmonella senftenberg, w (h s negative) are in discussion for different purposes and treatment processes, especially in the scandinavian countries (christensen et al., ) . some of the most important are: enterococci, better known as enterococcus faecalis, may be especially used for validating thermal treatment, since their heat resistance covers most pathogens in this epidemiological context, providing a high safety margin. the application of enterococci for the general purpose of validation of composting processes, on the other hand, has some disadvantages. first, the quantitative enrichment is less effective than in salmonella if contaminant flora shall be excluded. if the carriers are exposed to a composting process, it may happen that the test material is contaminated with indigenous enterococci. in this case, contamination from the substrate cannot be detected in an easy and reliable manner. finally, it must be kept in mind that enterococci are more chemo-and thermo-resistant than most relevant pathogens in this field, but do not have any epidemiological importance in this context. this means that the application of test organisms must be strictly related to the process to be judged. if only a process of thermal inactivation like a pasteurization unit is to be validated, enterococcus faecalis is the proper organism. if a thermophilic aerobic or anaerobic process shall be validated, it is more realistic to use the above characterized strain of salmonella senftenberg because enterococci would be too hard a criterion for this purpose since their chemo-resistance is different from the relevant mostly gram-negative pathogens. for practical considerations, the proposed serovar salmonella senftenberg has the advantage that it is rarely present in the raw material and can be easily identified by a natural marker (h s negative) from accidental contaminants. escherichia coli and campylobacter jejuni are not as resistant as the above-mentioned salmonella strain; the same applies to ecbo virus. it could be demonstrated that if dealing with a moderate epidemiological risk, e.g., given in composting source-separated biowastes, salmonella senftenberg w will cover the most relevant viral pathogens causing notifiable diseases in farm animals and which may be present in low concentrations in the raw material. the time necessary for the inactivation of viral pathogens is generally shorter or in the same range as for inactivating log of salmonella senftenberg w , h s negative as has been demonstrated by braumiller et al. ( ) , as well as moss and haas ( ) . the latter results are summarized in table . ; the results demonstrate the range of time necessary for reducing the infectivity of the tested viral pathogens for - log steps. since recently it was stated by emmoth et al. ( ) that heating of animal by-products to • c for min is not enough for the inactivation of circoviruses and it had been demonstrated by böhm et al. ( ) that the plant pathogenic tobacco mosaic virus withstands such treatment without any significant reduction, the consequence for the future will be to validate any kind of treatment if such viruses may be present in involved materials. it is obvious that the recommendations of regulation ec ( ) will not even lead to a safe inactivation of vegetative bacteria, as can be seen from fig. . , given the temperature curve measured in the reactor in relation to the survival of enterococci in the substrate and on exposed carriers (braumiller et al., ) . as a consequence, concerning composting of animal by-products and catering wastes, a validation with a test organism covering the higher epidemiological risk is necessary. this means that there is a clear indication for using bovine parvovirus in such a situation. it must be kept in mind that validation with a representative test-organism alone ("direct process validation") has, as every solitary procedure, advantages and disadvantages, which can be summarized as follows: • quickly provides the basic information regarding whether or not a technical process leads to a safe product. • validates that the producer or technical equipment is protecting the processing plant. • helps to define the technical requirement for a safe process. • gives a reproducible result which allows comparison of data. • high cost and labor intensive. • it is a rare event. • cannot detect accidental disturbances of the process. the validation with pathogens and seeds may be regarded as an "indirect process validation." however, this validation must be accompanied by continuous recording of measurable process data like temperature, ph, humidity, etc. in order to detect deviations and disturbances of the process over the entire year, which may result in an insufficient bactericidal effect. the advantages and disadvantages of an indirect process control applied alone are as follows: • easy to achieve and quick. • continuous control is possible. • no special expertise is necessary and laboratory work is limited. • limited representativity for the entire process (gradient formation in the material). • influences due to inhomogeneity of the material could not be detected. • valuable only in combination with the direct process validation. the system of process validation and control has to be completed by continuous monitoring of the final product, at least twice a year. as mentioned above, the investigation of the final product to detect every pathogen that may be present in the material is extremely difficult. therefore, representative indicator organisms have to be determined from the point of view of human and animal health, and if necessary for the purpose of safe plant breeding and production. several strategies have been followed in different countries and in several normative approaches; an example is given in table . . since the philosophies followed in this connection are very different, indicators will be discussed in another section. additionally, sample collection, storage, and transport have to take into account the special properties and behavior of the biological agents; simple adaptation of methods and recommendations common for chemical analysis is misleading. the advantages and disadvantages of end product monitoring alone as a single strategy to achieve hygienic safety in composting are as follows: • easy to achieve; limited expertise is necessary. • easy to administer and supervise. • inexpensive and independent from the site of production. • representative sampling of bulky material is difficult. • occurrence of pathogens and indicators depends on the type, origin, and microbiological properties of the raw material. • microbiological analysis is influenced by the different products (e.g., growth inhibition). • valuable only if applied on products from validated and supervised processes. restriction in the use of compost resulting from insufficient treatment should prevent introduction of undesired chemical residuals by contaminated crops into the food chain or direct transmission of pathogens to susceptible animals via feedstuff. this had been practiced in the past, especially with sewage sludge. such a strategy alone does not prevent the environmental risks or introduction of pathogens into vector populations, which will lead to indirect transmission cycles. (hellmann, ) . one of the sources of infection in seagulls has been found to be sewage treatment plants. other ways of introduction of a certain lysotype of salmonella enteritidis can be demonstrated by the work conducted by köhler ( ) . köhler identified the waste delivered from west berlin to a waste disposal site in the former german democratic republic and followed the introduction of this pathogen via birds into chicken populations and finally to humans via products containing eggs. williams et al. ( ) , as well as several other authors such as coulson et al. ( ) and mayr ( ) , described the importance of vectors in the transmission of salmonella to farm animals and humans. foster and spector ( ) described specific molecular mechanisms responsible for the ability of salmonella to survive the environmental stress, which is underlining the importance of this group of pathogens. this means that even if the fertilizers containing pathogens are immediately ploughed into the soil, they may generate carriers by attraction of certain species (seagulls) or prolonged survival in subsurface soil layers. thus, restrictions in use are a tool with limited effect from the point of view of epidemiology and should be avoided if possible and feasible. moreover, concerning plant pathogens and seeds, this strategy is ineffective if the products are to be used in agriculture. regrowth mainly concerns bacterial pathogens, since viruses except bacteriophages cannot propagate due to the lack of host cells in this environment. several bacterial pathogens like salmonella can grow at temperatures between and • c, as long as enough nutrients and moisture are available. this is not only a matter of material properties and storing conditions, but also of proper management of tools and equipment from a hygienic point of view. a strict division of the technical equipment involved must be provided. one type of equipment should be used only with the raw material and another one should be used only with the finished product (black and white principle). otherwise, a reliable disinfection measure has to be carried out in between if the strict division cannot be assured. there is sufficient evidence from practical experiments that moist and contaminated fresh material adhering to the equipment will lead to recontamination of the product even if the material seems to be stable and dry. since most bacteria will not propagate in a dry product even if nutrients are present, the product must be stored under dry conditions. the vapor pressure within the product should be in balance with a relative humidity of the air of at least %. xerophilic fungi may still grow if this balance is above a relative humidity of about %. in general, most bacteria and fungi do not grow in the material if the water activity (a w value) is below . . in order to achieve product safety by preventing regrowth, recontamination by living vectors like birds and rodents should be avoided by taking adequate measures. if a product must be evaluated in the framework of microbiological quality control, it is very difficult to investigate it for presence or absence of all pathogens that could be attended. since financial and technical limitations are given, one of the most practical means to come to a conclusion concerning hygienic safety of a product with a reasonable effort and limited costs is the application of a suitable indicator concept in combination with process validation and indirect process supervision. the indicator concept is only effective if the raw materials are relatively homogenous in their microbiological properties or certain (mostly fecal) contaminants can either be totally excluded in the raw material or have to be always present. this, in general, is not the case here, neither for biowastes nor for industrial wastes or organic sludges. this means that any indicator concept within this framework must be a compromise and the selection of a microbial indicator is extremely difficult and mainly influenced by underlying philosophies. nevertheless, those indicator organisms must fulfill several requirements: • they have to be present with a high probability in the raw materials involved, • the transmission via products must be a factor in epidemiology, • if a biotechnological process is used, the indicator should not be involved in the process itself, • the indicator should not be an organism that is generally present in soil and soil-related materials, and • the method for isolation and identification must be simple, definitive, and reliable if applied to a substrate with a complex microbiological matrix such as compost, sludge, or related materials. with respect to public health and veterinary requirements, several indicators and parameters are currently under discussion: since compost and related products are coming out of a microbial degradation process and the knowledge about the microbiological ecology of such materials is very limited, one must be careful to use isolation and identification techniques common in clinical microbiology without careful validation in combination with the involved sample materials. the variety of species to be present in environment, samples, and materials resulting from aerobic or anaerobic biological treatment far exceeds the limited number of species to be taken into account in excreta, as well as in body fluids and the variability of species is high and not yet fully understood. moreover, microbial parameters that are used in the field of water hygiene and food inspection are not applicable to substrates like manure, stabilized sludge, or compost because most of those indicators belong to the indigenous flora of agricultural soils (böhm, ) . if the limited reliability and applicability of methods used in clinical microbiology and in water inspection for the intended field of use are taken into account, as well as the fact that the exclusion of organisms that generally may be found in normal soils gives no sense for a substrate and fertilizer such as compost or sludge, the following microbial parameters are inappropriate: staphylococcus aureus, enterobacteriaceae, clostridium perfringens, sulphite-reducing clostridia, and listeria. especially enterobacteriaceae, for which threshold values are given in the ec regulations / , are an inappropriate parameter in the evaluation of products after aerobic or anaerobic biotechnological treatment. this is due to the fact that this parameter does not correlate with the presence or inactivation of pathogens as can be observed in fig. . . the data in fig. . show the results of input and output analyses in validated and non-validated composting plants. one parameter that seems to be very useful and reliable in this connection is the absence or presence of salmonella. salmonella are generally found in fresh biowastes or untreated sewage sludge with a high probability in various concentrations. since it is known that the probability of identifying a positive sample is basically related to the amount of material investigated, a compromise between feasibility and reliability has to be found. it is proposed to investigate or g ( × g) of compost for the presence or absence of salmonella with the method described in principle in the german biowaste ordinance using a pre-enrichment in buffered peptone water and an enrichment step (rappaport et al., ; edel and kampelmacher, ; vassiliadis, ) , or other validated method that may be developed within the framework of cen tc . some other parameters are still in discussion with respect to sewage sludge treatment and composting in the framework of eu directives. enterococci, for example, cannot be used as an indicator in the examination of compost and compost-related products, but they are valuable for the thermophilic anaerobic treatment in biogas plants and for pure thermal treatment (bendixen, ) . for e. coli, campylobacter, and yersinia, besides the lack of reliable re-isolation techniques, it must be stated that their thermo-resistance and, with minor exceptions, chemo-resistance are lower than those of salmonella. this means that it will make no sense if they are used as additional microbial parameters for describing a hygienically safe product. enteroviruses are generally present in sludge of fecal origin but not regularly in sludges coming from other sources. in principle, enteroviruses may be used as additional indicators but the re-isolation procedures, as for all viruses from environmental samples, are labor intensive and costly. their resistance in the involved treatment processes is not higher than that of salmonella; this means that the additional information resulting from using these indicator organisms is also low. the same applies for rotavirus, even it is of special environmental importance, according to metzler et al. ( ) and pesaro et al. ( ) . recent investigations have shown that coliphages do not correlate high with gut-related pathogens or other comparable fecal indicators even in substrates of fecal origin, as can be seen from fig. . (samhan, ) . the question of whether or not nematodes or nematode eggs are useful indicators in this connection is not easy to answer. with respect to nematodes pathogenic to men and/or animals, experience shows that even eggs of ascaris suum are less thermo-resistant than salmonella, but behave differently in chemical treatment. this means that if salmonella does not survive the composting process, ascaris eggs and all other nematodes eggs would not either. this does not apply for treatment with slaked lime or long-term storage, especially in connection with organic sludges. this means that ascaris eggs will not be a necessary indicator in all processes in which the thermal effect is the predominant one, but they will give valuable additional information if used in the monitoring of all other treatment processes. finally, the problem of indicator organisms from the phytohygienic point of view must be discussed. no virus, fungus, or bacterium pathogenic to plants has been found thus far that is of comparable importance as salmonellae are for the purpose mentioned above. the only indicator that is widely distributed in biological wastes from households is tomato seeds. even knowing that this indicator will not totally cover all requirements, it seems to be reasonable and feasible to define the term "phytohygienic safety" of the total coliform group (mpn index/ ml) coliphage count (pfu/ ml) r = − . fig. . . correlation between the parameters "coliform bacteria" and "coliphages" in sewage samples (samhan, ) . the final product should not contain more than two seeds capable to germinate and/or reproduce parts of plants in l. a suitable test method is described by entseuchung von klärschlamm, . arbeitsbericht der atv (vks-arbeitsgruppe . . ) hygienic safety -results of scientific investigations in denmark (sanitation requirements in danish biogas plants) keimemissionen bei der kompostierung hygienic safety in organic waste management. resource recovery and reuse in organic solid waste management abschlußbericht zum forschungsvorhaben -wa / veterinär-und seuchenhygienische untersuchungen zur Überprüfung von gülleaufbereitungsverfahren und der erzeugten gülleaufbereitungsprodukte aktuelle bewertung der luftkeimbelastung in abfallbehandlungsanlagen. m.c. baeza-verlag untersuchungen zur inaktivierung von indikatororganismen in anaeroben kofermentationsanlagen, final report in the framework of the research programme lebensgrundlage umwelt und ihre sicherung (bwplus) abschlußbericht forschungsvorhaben hs aerobe und anaerobe behandlung von bio-und küchenabfällen sowie anderen rest-und abfallstoffen tierischer herkunft unter den aspekten der seuchenhygiene, teil b: untersuchungen zur inaktivierung von indikator-bakterien und -viren im kompostierungsprozeß survival of scrapie agent after years internment methodenhandbuch zur analyse von kompost nr development of a nordic system for evaluating the sanitary quality of compost, nordic council of ministers the herring gull loras argentatos as a likely transmitting agent of salmonella montevideo to sheep and cattle laying down health rules concerning animal by-products not intended for human consumption salmonella isolation in nine european laboratories using a standarized technique heat inactivation of porcine circovirus type . abstracts of ramiran how salmonella survive against the odds verordnung über die verwertung von bioabfällen auf landwirtschaftlich und gärtnerisch genutzten böden (bioabfallverordnung -bio-abfv) gesundheitszustand und belastung von beschäftigten im abfallbereich latente salmonellen-infektionen der tiere und ihre ursachen health significance of airborne microorganisms from wastewater treatment processes beispiele für die anreicherung von salmonellen in der umwelt laga-merkblatt m verbreitung von infektionserregern über abfälle durch haus-, gemeinde und freilandungeziefer unter besonderer berücksichtigung der gesundheit des menschen hygienische aspekte der bioabfallkompostierung. teil : phytohygiene. proc. symp. des umweltministeriums baden-württemberg und der lg-stiftun viren und parasiten im trinkwasser: risiken und prävention. mitt hygienische untersuchungen zur bewertung von kompostierungsverfahren hinsichtlich der aufbereitung von abfällen aus dem kommunalen und landwirtschaftlichen bereich. dissertation abschlußbericht forschungsvorhaben hs aerobe und anaerobe behandlung von bio-und küchenabfällen sowie anderen rest-und abfallstoffen tierischer herkunft unter den aspekten der seuchenhygiene, teil a: tenazität viraler tierseuchenerreger in biogenen abfällen in biogasanlagen bei der kofermentation mit gülle sowie in kompostanlagen inactivation of animal viruses during thermophilic fermentation of source separated waste in a full scale biogas plant hygienisch-mikrobiologische untersuchungen zum verhalten von ausgewählten bakterien und viren während der längerfristigen speicherung von flüssigkeit in güllegemeinschaftsanlagen a new enrichment medium for certain salmonellae coliphages as indicators for hygienic safety of water, wastewater and liquid organic wastes the rappaport-vassiliadis (rv) enrichment medium for the isolation of salmonellae: an overview the transmission of s. livingstone to cattle by the herring gull (larus argentatus) key: cord- -i q bhs authors: makhanova, anastasia; plant, e. ashby; maner, jon k. title: capturing fluctuations in pathogen avoidance: the situational pathogen avoidance scale date: - - journal: evol psychol sci doi: . /s - - - sha: doc_id: cord_uid: i q bhs pathogen avoidance is an important motive underlying human behavior and is associated with numerous psychological processes—including biases against social groups heuristically associated with illness. although there are reliable measurement scales to assess chronic dispositional levels of pathogen avoidance, no measurement scale currently exists to directly assess moment-to-moment fluctuations in pathogen avoidance. this paper presents the situational pathogen avoidance (spa) scale, which assesses situational variability in pathogen avoidance, especially as it pertains to avoidance of social stimuli. across six studies, we demonstrate the reliability and validity of the spa scale, show that the scale is influenced by situational activation of pathogen avoidance motives, and demonstrate that it mediates the association between pathogen avoidance motives (both chronic and situational) and social biases against obese and foreign targets. the spa scale provides a valuable measurement tool for researchers who study pathogen avoidance and to those who study social biases more generally. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. avoiding pathogens is a key goal for most organisms, including people. throughout human evolutionary history, coming into contact with pathogens has led to a range of negative consequences, not the least of which is severe illness and death. from avoiding foods that have passed their expiration date to staying away from people with runny noses and coughs, people possess psychological adaptations aimed at helping them steer clear of pathogen threats. the psychology of pathogen avoidance comprises both reactive strategies that regulate behavior when a person perceives an acute pathogen threat and proactive strategies that regulate behavior in the absence of such situational cues (schaller ) . individual differences in pathogen avoidance are associated with a range of social processes and, as such, have been the focus of significant attention (e.g., murray and schaller ; park and isherwood ; van leeuwen et al. ) . indeed, scales intended to measure those individual differences have been developed and used with increasing frequency to test hypotheses pertaining to pathogen avoidance haidt et al. ; olatunji et al. ; tybur et al. ). over and above individual differences in pathogen avoidance, the motivation to avoid pathogens can be acutely activated in certain situations. finding your seat on an airplane only to discover that your neighbor has a nasty cough, for example, is likely to evoke an acute desire to lean away, cover your face, or switch seats. situationally activated pathogen avoidance motives, like individual differences in pathogen avoidance, can affect a range of social biases (faulkner et al. ; miller and maner ; park et al. ). despite the need for a measure that captures situational fluctuations in pathogen avoidance, no such measure currently exists. individual difference measures of pathogen avoidance typically are not affected by experimental manipulations that increase situational salience of pathogen threat (e.g., ainsworth and maner ; makhanova et al. ; makhanova et al. ; mortensen et al. ; however, see sacco et al. ) . and although researchers can readily measure feelings of disgust, such measures fail to capture the broader experience of pathogen avoidance. electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. the current research generates and tests a new scale-the situational pathogen avoidance (spa) scale-that captures moment-to-moment fluctuations in pathogen avoidance. the spa scale assesses affective, cognitive, and behavioral responses that comprise pathogen avoidance psychology, especially as it pertains to avoidance of potentially pathogenic social stimuli. across six studies, we provide evidence for the spa scale's reliability and construct validity. we show that the spa scale is responsive to situational cues indicating the presence of pathogen threat. furthermore, we link the spa scale to social psychological biases known to reflect activation of pathogen avoidance motives. our goal is to provide researchers with an essential tool that captures moment-tomoment state-level changes in the psychology of pathogen avoidance. throughout history, pathogens have posed a consistent threat to human health and well-being. falling ill has many negative consequences, such as experiencing pain, missing out on important opportunities or resources, and the most severedeath. from an evolutionary perspective, those who were better able to avoid illness would have had greater reproductive success compared to those who were less able to do so. consequently, humans and other species possess powerful psychological mechanisms that mitigate the threat of illness by helping them avoid potential sources of pathogens (ackerman et al. ; schaller and park ; tybur et al. ). early research suggested that disgust plays a key role in helping people avoid illness. implicated primarily in the realm of ingestive behavior and contamination fears, disgust was shown to promote avoidance of potentially harmful foods and other objects (e.g., rozin and fallen ) . as theories evolved, disgust was also implicated in antipathy toward contact with undesirable people, both those perceived as harboring contagious illness and those perceived as violating moral rules (haidt et al. ). thus, a long-standing literature suggests that disgust plays a crucial role in self-protection from social threats (for a review, see tybur et al. ). some theories suggest that acute disgust may be particularly important for in-the-moment, reactive responses to encountering a stimulus perceived as potentially infectious (schaller ) . a flurry of recent findings has built on this disgust literature to illuminate the larger psychology of pathogen avoidance. pathogen avoidance is a fundamental motive underlying human behavior and promoting a range of important social psychological processes. one of the most well-documented consequences of pathogen avoidance involves social prejudice. for example, people motivated to avoid pathogens tend to display negative biases against immigrants, people with obesity, the elderly, or people who have physical deformities-all groups people tend to heuristically associate with the presence of illness (ackerman et al. ; faulkner et al. ; miller and maner ; navarrete and fessler ; park et al. ) . such biases fit with the logic of error management theory (haselton and nettle ) . from this theoretical perspective, people display such biases because the error of assuming something is safe when it is not (a false negative) has more costly consequences than the error of assuming something is dangerous when it is not (a false positive). a fast-growing literature has now linked a range of social biases to pathogen avoidance motives. the consequences of pathogen avoidance do not end with prejudice. pathogen avoidance also has implications for morality. people motivated by pathogen avoidance tend to espouse moral world views that help bind the ingroup together to protect the group from internal instability, as well as outgroups who carry novel pathogens to which one's own group has not developed immunity or who may not follow group norms (murray et al. ; park and isherwood ; van leeuwen et al. ) . motivation to avoid pathogens also seems to affect juror decision-making (brown et al. ) . pathogen avoidance has also been linked with a range of individual differences (e.g., social conservatism, traditionalism, religiosity, conformity) reflecting group-based processes (murray and schaller ; terrizzi jr et al. tybur et al. , c.f. shook et al. tybur et al. a, b) as well as preferences for social interaction partners (brown and sacco ; young et al. ) . pathogen avoidance has even been tied to basic units of personality such as neuroticism (tybur et al. ; tybur and de vries , see also mortensen et al. ). a burgeoning literature has linked the aforementioned social phenomena to individual differences in pathogen avoidance. some people display higher chronic levels of pathogen avoidance than others and, for example, many of the prejudices mentioned earlier appear to be rooted in chronic levels of pathogen avoidance. individual differences in pathogen avoidance have been implicated in negative biases against people with obesity (e.g., park et al. ) , prejudice toward immigrants (e.g., faulkner et al. ) , and group-focused moral values (e.g., van leeuwen et al. ) . individual differences in pathogen avoidance are typically assessed using scales that measure affective, behavioral, and cognitive processes. the field predominantly relies on two such scales: the perceived vulnerability to disease questionnaire (pvd; ) and the three domain disgust scale (tdd; tybur et al. ). the pvd comprises two subscales: germ aversion and perceived infectability. germ aversion focuses on people's tendency to avoid possible sources of pathogens in the environment whereas perceived infectability focuses on chronic cognitions about vulnerability to illness. the tdd examines three social domains (pathogen disgust, sexual disgust, and moral disgust) in which the emotion of disgust tends to be elicited. pathogen disgust focuses on revulsion in response to stimuli that pose a contamination threat; sexual disgust focuses on revulsion in response to sexual partners and behaviors that deviate from those that would enhance reproductive success; and moral disgust focuses on revulsion in response to others who violate social norms. the pvd and the tdd are widely used and have greatly facilitated the development of the literature on pathogen avoidance. nevertheless, both scales reflect trait pathogen avoidance and are typically not affected by experimental manipulations of pathogen avoidance motives (e.g., ainsworth and maner ; makhanova et al. ; makhanova et al. ; mortensen et al. ) . notably, several other scales also tap into trait pathogen avoidance (e.g., burns et al. ; haidt et al. ; olatunji et al. ) but these scales are less frequently used in studies examining the links between pathogen avoidance and social outcomes. although much of the pathogen avoidance research has focused on individual differences, those individual differences are expressed through proximate psychological states and such states are affected by people's current environment. indeed, independent of their chronic motives, people sometimes find themselves in situations in which there is an immediate threat of pathogens. such situations can activate acute pathogen avoidance motives. signs of acute pathogen threat shift people's motivations toward pathogen avoidance in the moment and facilitate psychological processes that mitigate the threat. the acute activation of pathogen avoidance motives is consistent with models of functional flexibility, which suggest that motivational systems come "online" in situations that afford adaptively relevant threats or opportunities (neuberg et al. ) . the distinction between chronic and acute pathogen avoidance is also consistent with models of personality that differentiate between dispositional processes and processes that are activated in particular circumstances (e.g., mcconnell ). although some people are more chronically avoidant of pathogens than others, acute pathogen avoidance motives should come online in situations that afford an immediate threat of infection. in fact, situational activation of pathogen avoidance leads to some of the very same psychological processes that have been linked to chronic pathogen avoidance. situationally activated pathogen avoidance, for example, is associated with social biases against people who display heuristic cues to illness like people with obesity, the elderly, and people of foreign nationality (faulkner et al. ; miller and maner ) . moreover, situations that increase the salience of pathogen threat promote vigilance to violations of moral values associated with ingroup protection (murray et al. ) . a variety of experimental manipulations have been used to examine psychological processes that reflect situationally activated pathogen avoidance. some of these manipulations involve participants viewing slideshows of others who are ill (e.g., lund and miller ; park et al. ) , reading articles about the threat of a novel infectious disease (e.g., miller and maner ; makhanova et al. ) , and reading articles that use disease metaphors (e.g., brown et al. ) , as well as recalling and verbally describing a time they felt afraid of illness (e.g., murray et al. ) . despite a growing literature examining the effects of such manipulations on social psychological processes, the field lacks a measure that directly captures psychological fluctuations resulting from the situational activation of pathogen avoidance. if research does attempt to quantify situational activations of pathogen threat, this is typically measured by state feelings of disgust. although feelings of disgust reflect an affective component of pathogen avoidance, simple measures of disgust fail to capture the broader cognitive and motivational aspects of pathogen avoidance. one study attempted to make up for this limitation by altering the beginning of the pvd items to include phrases such as "currently" and "at this time" (sacco et al. ) . in this study, participants who were ostracized, compared to those who were not, reported lower state pvd scores. however, a measure specifically designed to assess situational fluctuations in pathogen avoidance does not exist and is greatly needed. the current research develops and validates a scale that captures situational fluctuations in pathogen avoidance: the situational pathogen avoidance (spa) scale. the spa scale assesses affective, cognitive, and behavioral responses to hypothetical social situations where pathogen transmission is likely (e.g., "right now, if someone coughed next to me without covering their mouth, i would move away from them"). to create items, we drew upon the interpersonally focused items from the pathogen disgust subscale of the tdd (e.g., how disgusting is "shaking hands with a stranger who has sweaty palms") and the germ aversion subscale of the pvd (e.g., "it really bothers me when people sneeze without covering their mouths"). the phrasing of the spa scale items highlights people's current feelings (rather than typical levels of disgust sensitivity) and behavioral responses to situations where contagion risk is high. thus, the spa scale homes in on fluctuations in people's pathogen avoidance motives. we did not include items that assess people's general beliefs about their susceptibility to illness. such items are assessed (at the trait level) with the perceived infectability subscale of the pvd. that subscale assesses cognitions about one's perceived likelihood of becoming ill. although perceived infectability is a relevant construct in the pathogen avoidance literature, it does not tend to predict social avoidance outcomes as consistently or powerfully as does germ aversion (e.g., brown et al. ; makhanova et al. ; wang and ackerman ) . moreover, similar to the construct of germ aversion, the focus of the spa scale is to assess people's affective and behavioral reactions to social situations that are associated with increased contagion risk. therefore, assessing global perceptions of illness risk is beyond the scope of what the spa scale is designed to measure. study is a preliminary study intended to establish the reliability and factor structure of the scale and examine predictive validity by assessing the association between the spa scale and aversive reactions toward an obese target (a heuristic cue associated with pathogen avoidance). next, in studies, we examined whether experimental manipulations of pathogen threat affected scores on the spa scale. in study , we also provide evidence for convergent and discriminant validity of the spa scale. finally, in studies and , we focus on how the spa scale functions as a mediator between pathogen avoidance (both chronic and situational) and social biases. study provides initial evidence for the reliability and the one-factor structure of the spa scale and examined whether spa scores were associated with bias against a target linked to heuristic pathogen threat (an obese target). as mentioned previously, past research has demonstrated that pathogen avoidance (both chronic and situationally activated) is associated with prejudice against targets who are obese (lieberman et al. ; miller and maner ; park et al. ) . evidence for such biases is in line with error management theory (haselton and nettle ) ; people display negative biases not just for others who are actually contagious but anyone who possesses cues that heuristically associated with the presence of illness (schaller and park ) . therefore, examining such biases allowed us to assess the predictive validity of the spa scale. participants participants (n = ) were students approached by research assistants in common campus locations at a large, public university in the southeastern us. research assistants asked participants to answer questions on a (provided) tablet or on their phone, in exchange for candy. one participant was excluded from analyses because they were only years old. the final sample thus included participants (m age = . , sd age = . ; % women; % heterosexual; % white/ caucasian; % not hispanic). participants reported being slightly liberal (m = . , sd = . ) on a (very conservative) to (very liberal) political orientation scale and moderately religious on average (m = . , sd = . ) on a (not religious at all) to (very religious) scale. correlations between spa scores and demographic variables for all studies can be found in the supplemental materials (table s ). some demographic variables were included in some studies but not others; we report all that were measured for each study. participants were randomly assigned to either complete the spa first or rate target photographs first (thus, order of presentation was counterbalanced). the spa scale consists of items (see table ) that focus on affective and behavioral responses to encountering potential pathogens. these items are grounded in past research on the emotion of disgust and draw upon concepts from items in existing scales such as the pvd questionnaire ) and the tdd scale (tybur et al. ). the spa scale uniquely highlights responses to encountering a potential social pathogen threat (e.g., "right now, i would want to wash my hands immediately, if i shook hands with a person with sweaty palms."). this focus on the immediate social context makes the spa scale valuable for studying social biases and social processes. furthermore, the focus on pathogen transmission during social interactions is particularly relevant to the study of pathogen avoidance responses because non-zoonotic pathogens (those that can be transmitted human-to-human) may have exerted more pressure throughout human evolutionary history than zoonotic pathogens (those that can infect humans but are not transmitted human-to-human) (see thornhill et al. ) . to assess avoidance of zoonotic pathogen sources would have required the addition of a substantial number of additional items, and that was beyond the scope of the current scale. the items in the spa scale focus on affective (e.g., "right now, if i was standing next to a person who sneezed i would feel disgusted") and behavioral responses (e.g., "right now, i would try to sit on the opposite side of the room if i walked into the room where there was a person blowing their nose") and participants are given the following instructions: for this task, you will answer a questionnaire about your attitudes. for the following questions, try to imagine yourself being placed in these situations right now, that is at this moment in time. please indicate how much you agree or disagree with each of the following statements on a scale of (strongly disagree) to (strongly agree). for the target perception task, participants looked at a photograph and imagined what it would be like to interact with the target in the photo-an obese man. participants indicated the degree to which they were happy, disturbed, disgusted, creeped out, and would feel uncomfortable if they were in the same room as the target. we averaged the items to create a composite of aversive reactions toward the obese target (α = . ; the happiness item was reversescored). the average score on the spa scale was . (sd = . ; range . to ) and the scale displayed adequate reliability (α = . ). a confirmatory factor analysis assessed the factor structure and model fit (see table for standardized factor loadings and item-level descriptive statistics). a one-factor solution resulted in a good-fitting model. the χ /df test, for which values lower than indicate good model fit, resulted in a value of . (χ = . ; df = ), srmr = . , rmsea = . . two items had relatively low factor loadings, but these items were reverse-scored (which can reduce reliability but are nevertheless valuable, barnette ; weems and onwuegbuzie ) . those items displayed higher inter-item correlations in subsequent studies, and we retained them based on theoretical rationale. a two-factor solution (separating affective and behavioral items into different factors) did not result in appreciably better model fit (χ = . ; df = ; srmr = . ; rmsea = . ). the chisquare difference test between the two models was not significant, χ ( ) = . , p = . ; thus, we retained the one-factor solution. although the rmsea indicates only moderate fit (maccallum et al. ) , the srmr is within the recommended . cutoff (hu and bentler ) . given recent caution against using fit indices as the only indicators of scale acceptability (e.g., mcneish et al. ; stanley and edwards ) , it is important to examine whether the spa scale functions as intended by responding to experimental manipulation of pathogen threat to judge the acceptability of this scale. we provide those tests in studies through . the combined column reports data from analyses that combine study spa data with those from the control conditions across all studies. these analyses are detailed after study next, we examined whether the spa scale had predictive validity by regressing aversive reactions toward the obese target onto spa scores. spa scores were significantly associated with aversive reactions toward the obese man, b = . , se = . , t( ) = . , p = . , % ci [ . , . ], semi-partial r (sr) = . . order of presentation did not affect this association, b = . , se = . , t( ) = . , p = . . this study provided preliminary evidence for the reliability of the spa scale. additionally, individuals who reported higher (relative to lower) spa scores also reported more aversive reactions toward an obese target, consistent with prior research linking pathogen avoidance to prejudice against targets heuristically associated with illness. our main goal in developing this measure was to create a scale that would be influenced by situational changes in the salience of pathogen threat. in study , we thus conducted an initial test of the hypothesis that spa scores would be higher when people were exposed to a pathogen threat compared to a nonpathogen threat. because different experimental manipulations are used in the literature to amplify pathogen threat, it is crucial that any measure of situational pathogen avoidance is generalizable across these manipulations. we thus used two common experimental procedures (an article manipulation and an image manipulation) to heighten pathogen threat. we predicted that regardless of the manipulation used, participants in the pathogen threat condition would score higher on the spa scale than participants in the matched control condition. participants participants (n = ) were recruited from amazon's mechanical turk and were compensated $ . . based on exclusion criteria established a-priori (see makhanova et al. ) , participants were excluded from analyses because they spent fewer than s viewing the manipulation article. our final sample included participants (m age = . , sd age = . ; % women; . % white/ caucasian). participants self-reported being fairly liberal (m = . , sd = . ) on a -point likert scale ( = "very conservative" and = "very liberal") and just slightly below the scale midpoint for religiosity (m = . , sd = . ) on a -point likert scale ( ="not religious at all" and = "very religious"). we used two different procedures to manipulate situational pathogen threat: an article manipulation and an image manipulation. in the article manipulation, participants were randomly assigned to one of two conditions: a pathogen threat article (n = ) or a weather threat article (n = ). because the study was conducted during the summer, both articles focused on threats that could be heightened during that season (see makhanova et al. ) . the pathogen threat article stated that methicillinresistant staphylococcus (mrsa) cases were on the rise and that people should be careful in public areas such as grocery stores, pools, and bars. the control article was about the threat of severe summer weather (e.g., flash floods and fire). in the image manipulation, participants were randomly assigned to view either images of others who were visibly ill (n = ) or of people pointing guns at the camera (n = ). four photographs for each condition were taken from stimuli used in previous research (see schaller et al. ) . each image appeared on its own page and participants were allowed to click through them at their own pace. we instructed participants to pay attention to the images as they may be asked about them later. in all conditions, immediately after the manipulation, participants completed the spa scale (α = . ). we conducted a sensitivity analyses using g*power for a one-tailed test of the difference between two groups; with our sample size, we would have the power to detect an effect of d = . . we examined whether the experimental conditions increased spa scores relative to the control conditions (see table for descriptive statistics). we regressed spa scores onto condition (pathogen threat vs. non-pathogen threat), method of manipulation (articles versus images), and the condition x method of manipulation interaction. manipulations of pathogen threat significantly increased spa scores, b = . , se = findings provide initial evidence that the spa scale responds to experimental manipulations of pathogen avoidance. this effect emerged across two different manipulations of pathogen threat: one using an article about the threat of an infection and one using photographs of people who were ill. importantly, the control conditions also represented threats, albeit ones unrelated to pathogens. thus, spa scores reflect responses to pathogen threat, specifically, as opposed to physical threats more generally. in study , we attempted to replicate the results of study using a slightly different manipulation. we again manipulated pathogen threat with articles, although these articles focused on winter threats (flu and winter weather) to be consistent with the time of year during which the study was conducted. participants were undergraduate students (n = ) recruited for an online study through the participant pool of a large, public university in the southeastern us. we excluded participants based on a-priori criteria. twenty-two participants spent fewer than s on the page with the manipulation article (same criterion as in study ) and participants answered the catch question embedded in the survey incorrectly ( participants failed both attention checks). our final sample included participants (m age = . , sd age = . ; % women; . % heterosexual; . % white/caucasian; . % not hispanic). participants self-reported being fairly moderate (m = . , sd = . ) on -point political orientation scale ( = "very conservative," = "very liberal"). a sensitivity analysis indicated we had % power to detect an effect size as small as d = . . participants were randomly assigned to read an article about either the threat of a new avian flu or the threat of severe winter weather (makhanova et al. ) . immediately following the manipulation, participants completed the spa scale (α = . ). participants also completed several tasks beyond the scope of the current analyses. an independent samples t test confirmed that participants in the pathogen threat condition scored higher on the spa scale (m = . , sd = . , n = ) compared to participants in the severe weather threat condition (m = . , sd = . , n = ), t( ) = − . , p < . , d = . . findings replicated those of study -spa scores were heightened for participants who read an article about an acute pathogen threat. these results provide further support for the hypothesis that the spa scale captures fluctuations resulting from the situational activation of pathogen avoidance. in study , we replicated and extended the findings from studies and in a laboratory study using a slideshow manipulation of pathogen threat. a third condition was included in which participants washed their hands with a disinfectant hand wipe after encountering a pathogen threat prime. we expected that, although the pathogen prime would increase spa scores, wiping their hands with the disinfectant wipe would reduce or eliminate that effect (for a similar effect see huang et al. ) . a second goal was to provide evidence for convergent and discriminant validity of the spa scale. for these purposes, we included a subset of measures used in the development of the pvd questionnaire and the three domain disgust scale (tdd). in the scale development paper for pvd ), germ aversion was negatively associated with agreeableness, conscientiousness, and positively associated with neuroticism. germ aversion was unassociated with extraversion, openness to experience, need for cognition, and social desirability. likewise, in the scale development paper for tdd (tybur et al. ), pathogen disgust was associated with neuroticism, but not the other factors of the big five. thus, in this study, we assessed the associations between spa scores and the big five. we additionally wanted to demonstrate the specificity of the spa scale for assessing situational fluctuations in pathogen avoidance, so we included state we also included the perceived vulnerability to disease questionnaire ). method and results are reported in the supplemental materials. participants read descriptions of two men and responded to questions about how much they would want to engage (or avoid interacting with) each target. these results are reported in the supplemental materials. participants also completed a measure to test a separate hypothesis that is beyond the scope of the current manuscript. and trait measures of anger. finally, because spa scores should reflect pathogen avoidance specifically, we assessed people's need for cognition and social desirability concerns to demonstrate discriminant validity. participants were undergraduate students (n = ) recruited through the participant pool of a large, public university in the southeastern us. data from participants were excluded due to issues with following the study protocol (e.g., participant continued to a task before they were instructed to). we excluded additional participants because they incorrectly answered the catch question imbedded in the survey. our final sample included participants (m age = . , sd age = . ; % women; . % heterosexual; . % white/caucasian; . % not hispanic). participants were fairly moderate in their political orientation (m = . , sd = . ) on a (very conservative) to (very liberal) scale and moderately religious (m = . , sd = . ) on a (not religious at all) to (very religious) scale. participants arrived at the lab and were randomly assigned to one of three conditions. in the control condition, participants watched a -min slideshow of household objects. in the pathogen threat condition, participants watched a slideshow of others who were visibly ill ). both slideshows included ten images shown in random order for s at a time, with repetition. in the pathogen threat plus wipe condition, participants watched the same pathogen threat slideshow then, as part of an ostensible product rating task, used a disinfectant hand wipe to clean their hands and keyboards (huang et al. ) . all participants performed the product rating task in which they rated a pen and a hand wipe on various characteristics. however, only in the pathogen threat plus wipe condition did participants actually use the hand wipe (in the control and pathogen threat conditions the participants only rated the packaging). participants completed the spa scale immediately after (α = . ). next, participants completed a series of questionnaires including measures of chronic pathogen avoidance. questionnaires were presented after the manipulation and main dependent measure because we did not want to make pathogens salient for participants in the control condition. moreover, in studies with similar designs, trait pathogen avoidance measures were not affected by experimental manipulations (e.g., ainsworth and maner ; makhanova et al. ; makhanova et al. ) . the pvd questionnaire includes items that fall into two subscales: germ aversion and perceived infectability. germ aversion (α = . ) is related to the desire to avoid potential pathogens in one's environment (e.g., "i don't like to write with a pencil someone else has obviously chewed on") and perceived infectability (α = . ) is related to one's general perception about the susceptibility of one's immune system to disease (e.g., "if an illness is going around, i will get it"). participants rated their agreement with each statement using a -point scale ( = "strongly disagree," = "strongly agree"). the tdd scale includes items that fall into three subscales: pathogen disgust, sexual disgust, and moral disgust. pathogen disgust (α = . ) is associated with feelings of disgust elicited by objects in the environment that pose contagion risk (e.g., "sitting next to someone who has red sores on their arm"). sexual disgust (α = . ) is associated with disgust elicited by undesirable sexual encounters (e.g., "hearing two strangers have sex."). and moral disgust (α = . ) is associated with disgust elicited by violations of social norms (e.g., "shoplifting a candy bar from a convenience store". participants rated their agreement with each statement using a -point scale ( = "strongly disagree," = "strongly agree"). participants also completed the ten item personality inventory (tipi; gosling et al. ) , which is a short measure of the big-five personality traits that includes two scale items each for neuroticism, extraversion, openness to experience, conscientiousness, and agreeableness. the need for cognition scale (α = . ; cacioppo et al. ) included items (e.g., "i appreciate opportunities to discover the strengths and weaknesses of my own reasoning") and assessed the trait enjoyment of thinking as an activity. we used the balanced inventory of desirable responding (α = . ; paulhus ) as a measure of social desirability that included items (e.g., "i don't care to know what other people really think of me"). for both of these scales, participants rated their agreement with each item on a -point scale ( = "strongly disagree," = "strongly agree"). we adapted state and trait anger questions from existing scales. for the state anger measure, participants answered questions (α = . ; e.g., "i feel angry") using a -point scale ( = "strongly disagree," = "strongly agree"). for the trait anger measure (α = . ), participants used the same scale to answer questions (e.g., "once in a while i can't control the urge to strike another person"). participants also completed two measures of bias against groups heuristically associated with illness. participants completed the ethnocentrism questionnaire (α = . ; the revised generalizable ethnocentrism scale, neuliep and mccroskey ) , which included items (e.g., "most other cultures are backward compared to my culture"). participants also completed an explicit anti-fat measure (α = . ; crandall ), which consisted of items (e.g., "i really don't like fat people much"). participants rated their agreement with each item on a -point scale ( = "strongly disagree," = "strongly agree"). at the end of the study participants provided demographic information and were debriefed. first, we examined effects of the manipulations on spa scores. we regressed spa scores onto two dummy coded condition variables (with the control condition serving as the reference group). the omnibus test confirmed the presence of differences in mean spa scores across the three conditions, participants in the pathogen threat plus wipe condition, however, did not differ significantly in spa scores from those in the no wipe pathogen threat condition, b = . , se = . , t( ) = . , p = . . thus, it appears that the experimental manipulation heightened people's situational pathogen avoidance, but cleaning ones' hands with a cleansing wipe did not reduce the effect of the manipulation. next, we examined whether spa scores were correlated with other scales that previously have been shown to be associated with pathogen avoidance. correlations among all variables are presented in table . spa scores were positively associated with all three scales of trait pathogen avoidance: germ aversion, perceived infectability, and pathogen disgust [b = . , se = . , t( ) = . , p < . , sr = . , when controlling for moral and sexual disgust]. spa scores were further marginally positively correlated with neuroticism (p = . ). spa scores were not associated with other factors of the big five. spa scores were also positively associated with trait, but not state, anger. moreover, as predicted, spa scores were not associated with the need for cognition or social desirability scales. next, we examined whether spa scores, as well as measures of trait pathogen avoidance, were associated with ethnocentrism and prejudice against obese people. as predicted, spa scores were positively associated with ethnocentrism, r = . , p = . , whereas the associations between ethnocentrism and germ aversion (r = . , p = . ), perceived infectability (r = − . , p = . ), and pathogen disgust (r = . , p = . ) were not significant. however, spa scores were not associated with prejudice against obese people, r = . , p = . . although neither subscale of the pvd was associated with prejudice against obese people (germ aversion: r = . , p = . ; perceived infectability: r = . , p = . ), a significant association emerged with pathogen disgust (r = . , p = . ). the experimental manipulation of pathogen avoidance did not affect ethnocentrism, f( , ) = . , p = . , or prejudice against obese people, f( , ) = . , p = . . study conceptually replicated studies and and demonstrated that spa scale scores are increased by an experimental pathogen threat prime. participants in the pathogen threat conditions reported higher spa scores than those in the control condition; however, the hand wipe manipulation did not reduce spa scores. we also observed evidence for convergent and discriminant validity of the spa scale. that is, the spa scale was correlated with all three assessments of trait pathogen avoidance (germ aversion, perceived infectability, and pathogen disgust), marginally correlated with neuroticism, and was not correlated with social desirability or need for cognition. in this study, we also assessed whether spa scores predicted measures of social bias. individuals who scored higher on the spa scale, compared to those who scored lower, reported higher levels of ethnocentrism. however, spa scores were not associated with an explicit measure of anti-fat prejudice. in study , we examined social bias against an obese target using a measure that focused on aversive reactions toward a target in a photograph. it could be that the spa scale is more closely associated with affective responses than the more cognitive responses captured by the explicit prejudice scale used in the present study, which assessed perceptions of ability, agency, anticipated social decision-making, and personal worries about own potential weight-gain. moreover, whereas the spa scale predicted responses to an image of an individual obese target, it did not predict responses to questions about the obese at the more abstract group level. thus, situational pathogen avoidance may be more closely tied to reactions to an individual obese target rather than fat people as a group more generally. consequently, we reverted back to the photographbased measure for the next two studies as we continued to examine the association between the spa scale and social biases. study examined whether the associations between pathogen avoidance-both trait levels of and situational activations of pathogen avoidance-and social biases are statistically mediated by situational pathogen concerns, as measured with the spa scale. we once again examined aversive reactions participants also completed a measure to test a separate hypothesis that is beyond the scope of the current manuscript. correlations between variables in study v a r i a b l e s . . *** toward an obese target (an example of a heuristic cue associated with pathogen avoidance). we also included a non-selfreport measure of ethnocentrism (oaten et al. ) in which participants saw faces of men of different races who held neutral facial expressions and were asked to identify how likely they thought they would be to catch an illness from each target. in previous work, white individuals who had a chronic health condition that decreases immune system function (rheumatoid arthritis), and thus increased susceptibility to pathogens, were more likely to perceive non-white targets to be likely to transmit illness than white targets (oaten et al. ) . theories have suggested that avoidance of groups perceived to be foreign may be adaptive for various reasons such as the threat of novel pathogens (fincher and thornhill ) , wariness of foreign norms (karinen et al. ) , lower trust of social partners , and as mentioned previously, heuristic association with disease (faulkner et al. ; van leeuwen and petersen ) . we predicted that spa scores would statistically mediate any effects of trait pathogen avoidance and experimental manipulation of pathogen threat on these two social biases. participants were undergraduate students (n = ) recruited through the participant pool of a large, public university in the southeastern us. we excluded participants based on apriori criteria. fifty-seven participants spent fewer than s on the page with the manipulation article and additional participants either did not correctly answer the catch question embedded in the survey or did not correctly identify the topic of the article they read. our final sample included participants (m age = . , sd age = . ; . % women; . % heterosexual; . % white/caucasian; . % not hispanic). participants were fairly moderate (m = . , sd = . ) in their political orientation on (very conservative) to (very liberal) scale. the study was conducted online. participants were randomly assigned to read an article about either the threat of a new avian flu or the threat of severe winter weather. this manipulation was identical to that used in study . following the manipulation, participants completed the spa scale, pvd questionnaire, and state anger scales. because pathogen disgust and germ aversion typically demonstrate similar associations with measures related to prejudice (e.g., makhanova et al. ) and because we switched from the anti-fat prejudice scale used in study , we only included the pvd questionnaire in order to limit the length of the online questionnaire. participants also performed two photo-rating tasks. first, participants completed the task described in study , in which participants responded with their gut feelings about an obese target. the second photo-rating task was adapted from oaten et al. ( ) . participants saw photos of men ( white, black, asian, and latino) who displayed neutral facial expressions. faces were selected from the chicago face database and were rated similarly on a variety of attributes. participants were given the following instructions: for the next task, you will see a variety of faces. these individuals were instructed to hold a neutral facial expression while being photographed and not show how they might be feeling. some of these individuals were ill at the time their photo was taken. we are interested if people are able to pick up on any subtle cues even when individuals are holding neutral facial expressions. please look at each photo and answer the question below. then, participants responded to the question "how likely are you to catch a disease from this person?" on a scale of (not likely at all) to (very likely). we hypothesized that non-white targets would be perceived as more contagious than white targets. the presentation of the state scales (spa and anger) and the photo-rating tasks was counterbalanced. all participants completed the pvd last. finally, participants reported demographics. first, we examined whether spa scores were responsive to both chronic pathogen avoidance and situationally activated pathogen avoidance. we regressed spa scores onto germ aversion and experimental condition. germ aversion significantly predicted spa scores, b = . , se = . , t( ) = . , p < . , % ci [ . , . ], sr = . . however, condition did not predict spa scores, b = − . , se = . , t( ) = − . , p = . , % ci [− . , . ], sr = − . . an independent samples t test confirmed that spa scores in the pathogen threat condition (m = . , sd = . ) did not differ from those in the control condition (m = . , sd = . ), t( ) = − . , p = . , cohen's d = . . we preregistered the sample size, exclusion criteria, and study design on the open science framework. because condition did not predict the spa scale in this study, we could not conduct some of the analyses we preregistered. analyses for state anger are reported in supplemental materials. participants also completed a measure to test a separate hypothesis that is beyond the scope of the current manuscript. notably, the photo-rating tasks required all participants to think about catching illnesses, and this could have affected spa scores for participants who performed that task before completing the spa. therefore, we conducted exploratory analyses focused only on participants who completed the spa immediately after the manipulation. results indicated that participants in the pathogen threat condition (m = . , sd = . ) tended to report higher spa scores than participants in the control condition (m = . , sd = . ) although this trend was not significant, t( ) = − . , p = . , cohen's d = . . because condition did not predict spa scores, the remainder of our analyses included experimental condition only as a covariate. first, we examined whether the effect found in study was replicated in this sample by regressing aversive reactions toward the obese target onto spa scores, controlling for condition and order of presentation. higher spa scores were associated with more aversive reactions toward the obese target, b = . , se = . , t( ) = . , p = . , % ci [ . , . ], sr = . . in a similar model, ga was not associated with aversive reactions toward the obese target, b = . , se = . , t( ) = . , p = . . although there was no direct effect, we examined whether spa served as a mediator of the indirect effect between ga and aversive reactions toward the obese target. we used process to explore this association, controlling for condition and counterbalancing order (see fig. a ). as presented above, ga scores significantly predicted spa scores. in addition, spa scores predicted aversive reactions toward the obese target, b = . , se = . , t( ) = . , p = . , % ci [ . , . ], controlling for ga, order of presentation, and condition. the indirect effect of chronic pathogen avoidance through spa scores was significant, b = . , se = . , p < . , % ci [ . , . ]. notably, condition also emerged as a significant predictor of aversive reactions toward an obese target, b = . , se = . , t( ) = . , p = . , % ci [ . , . ]. next, we examined whether spa scores were associated with thinking that neutral targets who were perceived to be foreign harbored a contagious illness. previous work using this measure found that white targets were rated as less likely to spread contagion than non-white targets but used the analytical strategy that first tested groups for differences within the non-white targets (oaten et al. ). thus, following those guidelines, we first examined whether there were any differences in the associations between the spa scale and the three non-white targets. a repeated measures general linear model indicated that there were significant differences in these associations, f( , ) = . , p = . . the association between spa scores and perceptions of contagion was significant for both latino targets (r = . , p = . ) and asian targets (r = . , p = . ) but not black targets (r = . , p = . ). thus, it may be that the cultural context for our american sample is different from that of the australian sample in the original study. all three non-white targets would likely be viewed as immigrants in australia. however, in the usa, it is possible that only asian and latino targets would be viewed as immigrants whereas black targets may be perceived as nonimmigrants. furthermore, this distinction is in line with prior research suggesting that pathogen avoidance biases target groups perceived as relatively more foreign and unfamiliar (faulkner et al. ). thus, we examined whether spa scores were associated with perceptions of contagion for immigrant (composite of asian and latino) versus nonimmigrant (composite of white and black) targets. we ran fig. the models depict mediational analyses from study . models control for experimental condition and counterbalancing order. the model in panel a uses the whole sample (n = ). the model in panel b uses a subsample of participants who self-reported being white or black (n = ). this model additionally controlled for contagion risk from non-immigrants (white and black targets) when estimating effects for contagion risk from immigrants (asian and latino). values are unstandardized coefficients analyses on a subsample of participants who self-reported being either white or black (n = ). we regressed perceptions of contagion from immigrants onto spa scores, controlling for condition, order of presentation, and perceptions of contagion from non-immigrant targets. spa scores were significantly associated with relative perceived contagion from immigrant targets, b = . , se = . , t( ) = . , p = . , % ci [ . , . ], sr = . . spa scores were not associated with perceived contagion from non-immigrant targets, b = − . , se = . , t( ) = − . , p = . , % ci [− . , . ], sr = − . . in a similar model that examined associations between ga and perceptions of contagion from immigrant targets, we did not find a direct effect, b = . , se = . , t( ) = . , p = . . nonetheless, we examined whether spa scores served as a mediator of the indirect effect between ga and perceptions of contagion from immigrant targets. we used process to conduct the analyses of the indirect effect of ga onto perceptions of immigrant targets through spa scores, controlling for condition, order of presentation, and perceptions of non-immigrant targets (see fig. b ). in path a, ga significantly predicted spa scores, b = . , se = . , t( ) = . , p < . . in path b, spa scores significantly predicted perceptions of contagion in immigrant targets, b = . , se = . , t( ) = . , p = . . there was a significant indirect effect through spa scores, b = . , se = . , p < . , % ci [ . , . ]. in this study, we found mixed support for our hypotheses. we replicated the association between chronic pathogen avoidance and spa scores. unlike studies - , however, in study , the experimental manipulation of pathogen avoidance did not affect spa scores. notably, the presence of non-significant results is expected in a set of studies (lakens and etz ) . thus, we reported these results to increase transparency and conducted a within-manuscript meta-analysis of the effects of experimental manipulations on spa scores across all studies (reported immediately following study ). nevertheless, we found support for our hypothesis that spa scores would mediate the association between trait pathogen avoidance and social bias. indeed, spa scores predicted biases against an obese target (replicating study ) and targets perceived as foreign, even when trait pathogen avoidance levels were not linked to these biases. for the bias against targets perceived as foreign, we found that white and black participants who demonstrated higher spa scores (compared to those who demonstrated lower scores) were more likely to perceive contagion in asian or latino faces compared to white or black faces. the latter finding may reflect increased desire to avoid immigrants (i.e., increased ethnocentrism). furthermore, spa scores statistically mediated the association between germ aversion and both social biases. taken together, these findings demonstrate the added value of measuring situational pathogen avoidance in addition to trait pathogen avoidance. in study , we again examined whether social biases associated with pathogen avoidance are accounted for by spa scores. we predicted that spa scores would statistically mediate the effects of both trait pathogen avoidance and situationally activated pathogen avoidance on aversive reactions toward obese targets. we wanted to examine the effect of experimental manipulations of pathogen avoidance again because although we found support for such an effect in studies - , it did not emerge in study . participants were undergraduate students (n = ) recruited through the participant pool of a large, public university in the southeastern us. we excluded participants because they spent fewer than s on the page with the manipulation article. one participant was , and their data were excluded. our final sample included participants (m age = . , sd age = . ; % women; % heterosexual; % white/ caucasian; % not hispanic). participants were fairly moderate in their political orientation (m = . , sd = . ) on a (very conservative) to (very liberal) scale and moderately religions (m = . , sd = . ) on a (not religious at all) to (very religious) scale. participants were randomly assigned to read a priming article about either the threat of an antibiotic-resistant infection or the threat of summer weather (as in study ). immediately following the manipulation, participants completed the spa scale (α = . ). next, participants rated a photo of an obese person and reported on their gut-level evaluations (as in studies and ). finally, participants completed the pvd and provided demographic information. first, we examined whether experimental manipulation of pathogen threat increased spa scores. as in studies - , participants who read an article about pathogen threat (m = . , sd = . ) demonstrated higher spa scores compared to participants who read an article about weather threat (m = . , sd = . ), t( ) = − . , p = . , d = . . ga was not affected by the experimental manipulation, t( ) = − . ; pi was marginally lower for participants in the pathogen threat condition (m = . , sd = . ) compared to the weather condition (m = . , sd = . ), t( ) = . , p = . . next, we examined the zero-order correlations between the key variables: spa, ga, pi, condition, and bias against the obese target. bias against the obese target was significantly associated with spa (r = . , p < . ), ga (r = . , p = . ), and pi (r = . , p = . ), but not condition (r = − . , p = . ). in multiple regression analyses, however, when bias against the obese target was regressed onto spa, ga, pi, and condition, only the association with spa scores was significant, b = . , se = . , t( ) = . , p = . , % ci [ . , . ], sr-= . (all other p's > . ). next, we turned to our main analyses. we first regressed spa scores onto both ga and the experimental manipulation of pathogen threat. both ga, b = . , se = . , t( ) = . , p < . , % ci [ . , . ], sr = . , and the experimental manipulation, b = . , se = . , t( ) = . , p = . , % ci [ . , . ], sr = . , were independently associated with higher spa scores. second, we estimated a mediational model using structural equation modeling in mplus. we utilized this approach (as opposed to using process like in study ) in order to simultaneously estimate the indirect effects of ga and experimental manipulation through spa scores. we report standardized effects (see fig. ). spa scores were positively associated with aversive reactions toward the obese target. the direct effects between measures of pathogen avoidance and aversive reactions toward the obese target were not significant (ga: b = . , se = . , p = . ; condition: b = − . , se = . , p = . ). however, as predicted, there was a significant indirect effect of ga through spa scores, b = . , se = . , p = . , and simultaneously a marginally significant indirect effect of condition through spa scores, b = . , se = . , p = . . study demonstrated that spa scores were linked to both trait levels of pathogen avoidance and situationally activated pathogen avoidance. indeed, over and above associations with trait pathogen avoidance, spa scores were positively associated with bias against an obese target. moreover, spa scores mediated the associations between pathogen avoidance (both trait and situationally activated) and bias against an obese target. thus, these findings and those from study highlight the benefit of using the spa scale alongside measures of trait pathogen avoidance. because one goal of these studies was to develop a scale that responded to experimental manipulations of pathogen avoidance, we conducted a meta-analysis across the studies to provide a summary of the effect of priming pathogen threat on spa scale scores. following recommended procedures (shadish and haddock ) , we first transformed the effect sizes from studies - using the fisher r-to-z transformation (borenstein ). notably, for study , we included the semi-partial r effect size from the model which collapsed across, but controlled for, the two different priming methods (r = . , n = ). for study , we included the semi-partial r effect size from the association between condition and spa scores (r = . , n = ). for study , we included the effect size for the difference between the object and pathogen threat conditions, excluding the hand wipe condition (r = . , n = ). in order to be consistent, for study , we included the effect size for the subsample of participants who completed the spa scale directly after the manipulation (r = . , n = ). finally, for study , we included the effect size for the experimental manipulation without controlling for ga (r = . , n = ). because experimental studies in the pathogen avoidance literature use many different types of manipulations, we used a random-effects model using the q-based estimate (q = . ) to obtain a summary effect size (transformed back into an r-based effect). across the five samples, the meta-analytic findings suggest a robust, mediumsized positive effect of experimental manipulation on spa scores, r = . , % ci [ . , . ]. this is equal to a cohen's d of . . to confirm the factor structure of the spa scale, we combined the data from study and the control conditions from the experimental studies - and performed a confirmatory factor analysis. a model with one latent factor had acceptable fit, χ ( ) = . , p < . , rmsea = . , srmr = . , cfi = . , and tli = . . factor loadings and descriptive statistics are presented in table . the two reverse-scored items have lower factor loadings, as is often the case with reverse-scored items (barnette ; weems and onwuegbuzie ) . consistently, a model with a twofactor structure (with the two reverse-scored items loading on to second latent factor) had better model fit, χ ( ) = . , p < . , rmsea = . , srmr = . , cfi = . , and tli = . . however, because from a theoretical standpoint both factors measure the same construct, we retained all items in the scale. research on pathogen avoidance has increased steadily in the past decade and has linked pathogen avoidance to myriad psychological processes from attraction to xenophobia (ackerman et al. ) . although reliable measurement scales exist for directly assessing trait pathogen avoidance, to date, the field has lacked a scale designed to directly assess situational fluctuations in pathogen avoidance, including those that follow experimental manipulations. in this paper, we developed and validated a scale-the situational pathogen avoidance (spa) scale-to address the need for such a measure. across five experiments (studies - ), we examined whether various experimental manipulations of pathogen threat increased people's spa scores. we also conducted a meta-analysis across the studies to provide a summary of the effect of pathogen threat on spa scale scores. this metaanalysis documented a reliable effect of pathogen priming on spa scores. moreover, we demonstrated convergent, discriminant, and predictive validity of the spa scale. spa scores were associated with social biases against targets heuristically linked with pathogen threat. indeed, associations between such biases and pathogen avoidance motives (both trait levels and situationally activated) were statistically mediated by the spa scale. overall, the spa scale was associated with both trait and situationally activated pathogen avoidance motives and with some of the key variables of interest in the pathogen avoidance literature. our results are in line with theoretical perspectives suggesting that environmental inputs affect the salience of chronic goals and individual differences, which consequently influence perceptions and behavior (e.g., mcconnell ; neuberg et al. ) . in study , we found that spa scores reflect both chronic pathogen avoidance levels and acute environmental cues of increased pathogen threat. furthermore, consistent with the hypothesis that pathogen avoidance processes influence perceptions and behavior through psychological states, trait pathogen avoidance, and environmental cues affected biases against an obese target through spa scores. that is, biases against social targets are affected by both people's chronic pathogen avoidance levels as well as current information about pathogen threat, which are both captured by spa scores. thus, the spa scale is a measurement tool that can complement the use of existing pathogen avoidance methodology. the spa scale contains specific features that make it uniquely valuable as a research tool. first, the scale emphasizes current, situational feelings. both the overarching instructions and each item in the scale bring people's focus to how they are feeling right now. second, the scale includes items that focus on affective (e.g., "right now, if i was standing next to a person who sneezed i would feel disgusted") and behavioral (e.g., "right now, i would try to sit on the opposite side of the room if i walked into the room where there was a person blowing their nose") responses to pathogens. such items additionally emphasize the important context of social interactions. the spa scale thus includes items that directly measure the desire for social distancing as a result of pathogen threat (e.g., "right now, if someone coughed next to me without covering their mouth, i would move away from them). because of these features, the spa scale could be a valuable tool for researchers interested in processes at the intersection of pathogen avoidance and social behavior. one way that researchers can use the spa scale is to assess whether situational pathogen avoidance motives are associated with social processes. in this work, spa scores were associated with two social biases: ethnocentrism and aversive reactions toward an obese target. one limitation of the obesity bias measure, however, was that it lacked a control target (i.e., someone of average weight) such as the non-immigrant group on the ethnocentrism measure in study . thus, rather than bias against a specific social target displaying a heuristic cue to illness, this association may reflect a general aversive reaction to social targets consistent with the link between pathogen avoidance and decreases in affiliative motivation (sacco et al. ) . future research could directly test this distinction and whether spa scores are negatively associated with affiliation. furthermore, the spa scale could also be associated with social processes beyond prejudice that reflect situationally we additionally performed a cfa in a larger dataset that included the spa scale (makhanova and shepherd ; study ) and found the fit indices somewhat improved: srmr = . , rmsea = . , tli = . , cfi = . . furthermore, a cfa of the pvd scale from the same study resulted in nearly identical fit indices: srmr = . , rmsea = . , tli = . , cfi = . . activated pathogen avoidance, such as risk-aversion (prokosch et al. ) , moral vigilance (murray et al. ) , and heightened desire for improving one's appearance (ackerman et al. ) . another way that researchers can use the spa scale is to examine whether and how situations influence pathogen avoidance motives. although the present research validated the scale's function by assessing how experimental manipulations of pathogen threat increase spa scores, pathogen avoidance is also affected by other situations such as being rejected (sacco et al. ) or imagining oneself in a crowded place (brown and sacco ) . thus, researchers could use the spa scale to assess how various situational factors and ecological variables (e.g., sng et al. ) affect peoples' pathogen avoidance processes. yet another application for the spa scale may be for research that tracks seasonal or longitudinal fluctuations in pathogen avoidance. for example, pathogen avoidance motives may increase during the flu season compared to other times of the year. indeed, the authors have more recently collected data consistent with this hypothesis (makhanova and shepherd ) . data collected at the start of the covid- pandemic demonstrated significantly higher spa scores (m = . , sd = . ) compared to the mean across the control conditions of samples reported in this manuscript (m = . ), one-sample t( ) = . , p < . . scores were not higher for ga or pi at the start of the pandemic as compared to the scores collected for this manuscript. furthermore, spa scores significantly increased as the threat of the pandemic grew whereas neither ga nor pi scores increased. another important longitudinal fluctuation in pathogen avoidance may occur around pregnancy and the post-partum period. consistent with past research (navarrete et al. ), spa scores may increase when women become pregnant because mothers' immune systems become more vulnerable to pathogens and illness can have negative effects on fetal development. however, research has yet to examine how these processes develop over time within women and what happens in the post-partum period. spa scores may remain elevated because mothers may be motivated to protect their newborns who do not have functional immune systems of their own. alternatively, spa scores may decrease because dealing with the newborns' bodily functions may desensitize mothers to potential sources of pathogens. one limitation of the spa scale is that it does not directly address two other domains linked with the emotion of disgust-sexual and moral disgust (tybur et al. ). sexual and moral disgust predict important social behavior. sexual and moral disgust, for example, are uniquely associated with agreeableness, conscientiousness, and lower psychopathy (tybur et al. ). we designed the spa scale to specifically focus on pathogen avoidance. thus, although the spa scale represents a useful tool to examine how state fluctuations in pathogen avoidance are associated with social perceptions, it is not intended to measure state fluctuations in avoidance based on sexual or moral disgust. the spa scale is also not meant to replace existing measures of chronic pathogen avoidance-the three domain disgust scale and the perceived vulnerability to disease questionnaire-but rather to be a complementary tool that specifically assesses moment-to-moment changes in pathogen avoidance. because the spa scale focuses on social avoidance and disgust at the moment of administration, it cannot be relied upon as an individual difference measure of pathogen avoidance and, consistently, is correlated only moderately with individual measures of pathogen avoidance. future research could examine whether individuals who perceive themselves to be more vulnerable to pathogen threat (i.e., report higher levels on the perceived infectability subscale of the pvd) may have stronger reactions to situations that connote increased contagion risk. overall, the use of complementary state and trait measurement tools may be especially useful for addressing several outstanding questions in the field of pathogen avoidance (see tybur et al. ) . one important area for future research is to examine when chronic versus situational changes in pathogen avoidance lead to similar social processes and when they lead to different social processes. for example, as mentioned previously, research has linked both chronic and situationally activated pathogen avoidance motives to social biases against targets heuristically linked with illness (e.g., faulkner et al. ; miller and maner ) . chronic versus situational motives, however, may also lead to differing social processes and responses. chronic pathogen avoidance, for example, has been linked with holding moral values that more strongly emphasize protecting one's ingroup from potential harm (van leeuwen et al. ; park and isherwood ) . situationally activated pathogen avoidance, however, may not be associated with changes in one's moral values (makhanova et al. ), but might instead facilitate stronger blame toward others who behave in ways that violate such group-protective values (murray et al. ) . future research can benefit from using the spa scale alongside existing pathogen avoidance scales to further examine parallel and divergent effects associated with chronic versus situational sources of pathogen avoidance. furthermore, because the spa scale focuses on threats from human interactions, it may be used in future research to examine whether situational pathogen avoidance is responsive to cues of nonzoonotic (pathogens that can be transmitted human-to-human) versus zoonotic (pathogens that can infect humans but are not transmitted human-to-human) pathogen presence (see thornhill et al. ). research on pathogen avoidance has grown exponentially in recent years. an impressive array of findings suggest 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sequence data date: - - journal: nat rev genet doi: . /nrg sha: doc_id: cord_uid: w de infections are one of the major selective pressures acting on humans, and host-pathogen interactions contribute to shaping the genetic diversity of both organisms. evolutionary genomic studies take advantage of experiments that natural selection has been performing over millennia. in particular, inter-species comparative genomic analyses can highlight the genetic determinants of infection susceptibility or severity. recent examples show how evolution-guided approaches can provide new insights into host–pathogen interactions, ultimately clarifying the basis of host range and explaining the emergence of different diseases. we describe the latest developments in comparative immunology and evolutionary genetics, showing their relevance for understanding the molecular determinants of infection susceptibility in mammals. supplementary information: the online version of this article (doi: . /nrg ) contains supplementary material, which is available to authorized users. the way in which these analyses have helped to clarify the genetic determinants of species-specific infection and disease, as well as the reasons behind pathogen emergence. although arms races involve both the host and the pathogen, in this review we only focus on genetic diversity in mammalian hosts. host-pathogen genetic conflicts are not confined to mammals (and their pathogens): they drive molecular evolution in most realms of life, including bacterial-bacteriophage systems , plants and their infectious agents , as well as invertebrates and their pests , . although we review studies and methods (boxes - ) that analyse genetic diversity at the inter-species level, the investigation of intra-species and intra-population signatures of pathogen-driven selection has also provided extremely valuable insight into infectious disease susceptibility, especially in our species. the interested reader is directed towards several recent reviews for more information [ ] [ ] [ ] [ ] [ ] . the dynamics of host-pathogen interactions a central tenet of the red queen hypothesis is that organisms must continually adapt to survive and thrive in the face of continually evolving opposing organisms. nonetheless, evolution is not all about biotic interactions. at a macroevolutionary level, mixed models of evolution are likely to operate; biotic factors mainly shape species diversity locally and over short time spans, comparisons among species take a snapshot of selective events that have been unfolding over long timescales. most of these approaches use extant genetic diversity and phylogenetic relationships among species to infer underlying evolutionary patterns. briefly, inter-species approaches rely on the alignment of orthologous coding sequences, analyse these alignments site-by-site, and at each site determine which, among all possible substitutions, would be non-synonymous (amino acid replacing) or synonymous (non-amino acid replacing) (see the figure) . the observed number of non-synonymous differences per non-synonymous site (dn) and the observed number of synonymous differences per synonymous site (ds) are then estimated. under neutral evolution, the rate at which amino acid replacements accumulate is expected to be comparable to the rate for silent changes and, therefore, dn/ds should be equal to (green codons in the figure). nonetheless, most amino acid replacements are deleterious and, as a consequence, are eliminated by selection; this results in a large preponderance of sites with dn/ds < , a situation referred to as purifying (or negative) selection (shown in blue in the figure) . conversely, the selective pressure exerted, for instance, by a pathogen, may favour amino acid replacements (for example, changes that modify the sequence and structure of a cellular receptor): in this case, dn/ds may reach values greater than , a hallmark of positive (or diversifying) selection (red in the figure) . the figure shows a hypothetical example whereby a virus uses a cellular receptor to infect the host. to prevent viral binding and infection, selection favours variants that modify the sequence and structure of the host receptors; on the other side, the virus adapts to such changes by gaining mutations that keep re-establishing receptor binding. this process fuels a genetic conflict, which is evident at the interaction surfaces. some lineages may be under stronger selective pressure than others and may display lineage-specific selected sites (episodic selection; cyan). in this case the branch of the phylogeny leading to these species may show significant evidence of positive selection . whereas shifts in the physical environment (for example, climate changes and oceanographic and tectonic events) drive evolution at a large scale, across much longer time periods . recently, a new interpretation of the red queen hypothesis was proposed ; the analysis of several phylogenies from different taxa indicated that speciation mostly occurs at a constant rate through rare stochastic events that cause reproductive isolation . this view curtails the role of biotic interactions as major determinants of species diversity . despite these observations, the red queen hypothesis has proven to be an extremely useful framework for the study of host-pathogen interactions. in this context, red queen dynamics can be divided into different types (see ref. for a recent review). frequency-dependent selection, for example, determines allele frequency fluctuations in both host and pathogen populations. in this scenario, rare alleles are favoured by selection (the pathogen, for instance, may be adapted to the most common host genotype and may fail to infect hosts carrying a rare allele), and diversity within populations is maintained. escalatory arms races are another form of selection that usually apply to quantitative or polygenic traits and proceed through recurrent selective sweeps. selection results in an escalation in the phenotypes of both the host (for example, resistance) and the pathogen (for example, virulence). finally, in chase red queen scenarios the host is under pressure to reduce the strength of the interaction through de novo evolution of novelty, whereas the pathogen evolves to tighten the interaction by reducing phenotypic distance. chase scenarios occur when host-pathogen interactions have a complex genetic basis (polygenic); they determine selective sweeps and tend to reduce genetic diversity within populations. over the years, the red queen hypothesis has been supported by the description of rapid rates of evolution in genes involved in genetic conflicts and, in a few instances, by the temporal reconstruction of host-pathogen co-evolution in natural settings . more recently, the development of experimental evolution approaches has allowed its formal testing , . although extremely valuable, laboratory-based studies often use an isogenic host population that is infected by one or a few pathogen strains, and such studies only partially recapitulate the complex nature of host-pathogen interactions that occur in real life. for instance, phenotypic plasticity (an environmentally based change in the phenotype) and multiway host-pathogen interactions are common in nature. a remarkable example of phenotypic plasticity is the vertebrate adaptive immune system: through rearrangement and somatic hypermutation, the same genetic arsenal is used to combat a wide array of pathogens and to develop lifelong resistance to some infections. despite the relevance of adaptive immunity for host defence, its action does not preclude pathogen-driven selection at several genes involved in innate immunity or, more generally, in the interaction with pathogens (these represent the focus of this review). as for multiway interactions, these represent the norm: the same host can be infected by multiple pathogens (or even by multiple strains of the same infectious agent) during its lifetime, whereas pathogens differ in their ability to infect one or more host species. thus, multiple host-pathogen interactions might drive the evolution of the same or different molecular systems, blurring the expectations of the red queen hypothesis. finally, hosts with long generation times (such as mammals, which are the focus of this review), evolve at lower rates compared with most of their pathogens and also display smaller population sizes, resulting in an asymmetry of the arms race (although parasites with life cycles involving two or more species may be constrained in their ability to adapt (reviewed in ref. ) ). even in the presence of a strong selective pressure (for example, a fatal infection), several generations may be required before the molecular signatures of the genetic conflict can be detected in mammalian host genomes . nevertheless, natural selection signatures have been described at several mammalian genes that interact with recently emerged human infectious agents (for example, hiv- ), possibly as a result of the pressure imposed by extinct pathogens or because these agents have established long-lasting interactions with non-human hosts. the 'site models' implemented in the phylogenetic analysis by maximum likelihood (paml) package are widely used to infer positive selection and to identify positively selected sites. these models allow dn/ds to vary from site to site, assuming a constant rate at synonymous sites. data (alignment and phylogenetic tree) are fitted to models that allow (selection models) or do not allow (neutral model) a class of codons to evolve with dn/ds > . likelihood ratio tests are then applied to determine whether the neutral model can be rejected in favour of the positive selection model. if so, the gene is declared to be positively selected. also, if (and only if) the null hypothesis of neutral selection is rejected, a bayes empirical bayes (beb) approach can be used to detect specific sites targeted by selection (beb calculates the posterior probability that each site belongs to the class with dn/ds > ) , . the paml approach implicitly assumes that the strength and direction of natural selection is uniform across all lineages. because this is often not the case, murrell and co-workers recently developed the mixed effects model of evolution (meme, hyphy package) . meme allows the distribution of dn/ds to vary from site to site and from branch to branch; thus, the method has greater power to detect episodic selection, especially if it is confined to a small subset of branches in the phylogeny. a major issue related to these approaches is their extreme sensitivity to errors in sequence (coverage), annotation and alignment. misalignments and incorrect sequence information may result in apparently fast evolutionary rates and thus inflate the false-positive rate [ ] [ ] [ ] . the use of specific alignment algorithms (for example, prank) and filtering procedures (for example, guidance) may partially overcome this problem . likewise, genetic variability that is generated by recombination can be mistaken for positive selection . thus, to limit false positives, alignments should be screened for recombination before running positive selection tests (and, if necessary, split on the basis of recombination breakpoints) or recombination should be incorporated into the model. the accumulation of favourable amino acid-replacing substitutions, which results in more non-synonymous changes than expected under neutrality (dn/ds > ). coronavirus (mers-cov) as a dangerous human pathogen. both ebov and mers-cov are thought to have originated in bats and spread to humans either directly or through an intermediate host. because eids are almost inevitably caused by an existing pathogen that adapts to infect a new host, comparative analyses of different species may help to unveil the genetic and immunological determinants underlying pathogen spillover and infection susceptibility. hiv- , for example, originated from the crossspecies transmission of the simian immunodeficiency virus siv cpz , which naturally infects chimpanzees . old world monkeys are resistant to hiv- infection owing to a post-entry viral block operated by cellular restriction factors. this differential susceptibility to infection was exploited to isolate tripartite motif-containing protein (trim ; also known as trim α), a major retrovirus restriction factor, from a rhesus macaque cdna library . the protein product of trim binds directly to the incoming viral capsid and targets it for disassembly. whereas macaque trim is highly efficient against hiv- , the human protein is not . most species-specific determinants of antiviral activity were mapped to a short amino acid stretch in the so-called b . (or spry) domain of trim (ref. ). in primates, this region has evolved under positive selection, and the human lineage shows some of the strongest selection signatures . why then is human trim so highly inefficient against hiv- ? possibly because the human gene evolved to fight another retrovirus. in a seminal paper, kaiser and co-workers resurrected an extinct pan troglodytes endogenous retrovirus (pterv ) and showed that the amino acid status of a single residue in the trim b . domain modulates its activity against pterv and hiv- , with the gain of restriction for one virus resulting in decreased control of the other one . human trim is very active against pterv , suggesting that our ancestors adapted to fight this virus or some related retrovirus, and this left them (us) unprepared against the hiv- epidemic. more recently, several genes identified as hiv- host factors were analysed in primates, and evidence emerged of positive selection at five of these (ankyrin repeat domain a (ankrd a), cd , microtubule-associated protein (map ), nucleoporin kda (nup ) and ran binding protein (ranbp )) . importantly, most of the positive selection targets in cd , map and nup are located in protein regions or domains that are responsible for direct interaction with the virus. the authors suggested that the selective pressure on these genes was exerted by ancient lentiviruses , . overall, a number of concepts can be taken from these studies: past infection events may leave a signature that affects the ability of extant species to fight emerging pathogens. evolution may act through trade-offs, whereby changes that are favourable in one specific environment (in this case, the presence of a specific pathogen) may be unfavourable when conditions change. protein regions at the host-pathogen interface are expected to be targeted by the strongest selective pressure. evolutionary studies based on inter-species comparisons allow the identification of molecular determinants of infection susceptibility at single amino acid resolution. mammals display different susceptibility to distinct pathogens, and infection with the same agent can have extremely different outcomes in diverse species (see ref. for a recent review). thus, domestic and wild mammalian (and non-mammalian) species represent natural reservoirs of human pathogens and/or may provide the adaptive environment for pathogen spillover. because host reservoir species and their pathogens often signatures of selection along specific branches can be detected through the so called 'branch-site' models implemented in the phylogenetic analysis by maximum likelihood (paml) package . in analogy to the site models described in box , alignment errors result in high false-positive rates when branch-site models are applied ; this issue can be partially mitigated by the use of specific aligners . branch-site models require the phylogeny to be divided into 'foreground' and 'background' branches. a likelihood ratio test is then applied to compare a model that allows positive selection on a class of codons for the foreground branches with a model that does not allow such selection . designation of the foreground branches needs a priori information, possibly based on biological evidence. if no clues are available as to which branches are more likely to have undergone selection, it is still possible to run the analysis by designating each branch of the tree as 'foreground'; this generates a multiple-hypothesis testing problem that must be appropriately corrected . two alternative methods can detect selection at specific lineages without a priori branch partition. the branch site-random effects likelihood (bs-rel) method considers three different evolutionary scenarios (purifying, neutral and diversifying selection) for all branches in a given tree, and each branch is considered independently from the others; the algorithm applies sequential likelihood ratio tests to identify branches with significant evidence of positive selection . the second method, the covarion-like codon model (fitmodel) , allows each site to switch between selective regimes at any time on the phylogeny. thus, switches are not necessarily associated with tree nodes. recently, this approach was shown to be more powerful than the branch-site tests if a priori information is available . both fitmodel and the paml branch-site methods envisage a bayesian approach to identify sites evolving under episodic positive selection. however, extensive simulations revealed that the branch-site approach is accurate but has limited power at detecting sites . this problem has been referred to as the 'selection inference uncertainty principle' -that is, it is difficult to simultaneously infer both the site and the branch that are subject to positive selection . co-evolve for millions of years, evolutionary analyses may help to explain host adaptive events associated with low susceptibility and mild disease outcomes. the most extensive body of knowledge on host-pathogen specificity focuses on viral infections, as the example of trim mentioned above testifies, but recent work has also shed new light on bacterial diseases. leptospirosis, one of the most prevalent human bacterial zoonoses worldwide, is caused by bacteria of the leptospira genus. wild rodents are considered to be the main reservoirs for human leptospirosis, but a study of malagasy small mammals indicated that several endemic species of tenrecs and bats are also infected with leptospira species that are markedly specific to their hosts, suggesting long-term adaptation of the bacterium to different hosts . a feature that pathogenic leptospira species share with other bacteria is complement evasion. indeed, these spirochetes have evolved different strategies to elude complementmediated killing; thus, leptospiral immunoglobulin-like (lig) proteins can bind complement factor h (cfh) and c b-binding protein (c bp) to mediate complement inactivation at the bacterial surface. a genome-wide analysis of positive selection in six mammalian species indicated that the complement system has been the target of extremely intense selective pressure . similar results were obtained by analysing positively selected genes in the bat myotis brandtii . thus, selection-driven speciesspecific differences at complement genes might explain differential susceptibility to infections. in line with this view, human-specific pathogens such as neisseria gonorrhoeae and neisseria meningitidis bind cfh of human origin, but not cfh from other primates, and a single amino acid change (n r) in the chimpanzee molecule restores cfh binding to sialylated gonococci and bacterial killing . several sequenced mammalian genomes are now available; it will be important to study the detailed pattern of molecular evolution at complement genes, with the aim of gaining insight into the determinants of species-specific complement evasion. yersinia pestis provides another remarkable example of differential susceptibility to a bacterial infection. again, rodents act as a natural reservoir for this human pathogen. as with other gramnegative bacteria, lipid a, the biologically active component of y. pestis lipopolysaccharide (lps), is recognized by toll-like receptor (tlr ) and its co-receptor lymphocyte antigen (ly ; also known as md ) (see below). recent data showed that, compared with mouse cells, human cells respond less efficiently to hypoacylated lipid a; this effect is almost entirely due to differences in tlr and ly sequences, as assessed by the generation of humanized mice . different responsiveness to variably acylated lps from other sources (for example, escherichia coli) had previously been described . starting from this premise, ohto and co-workers solved the crystal structure of the mouse tlr -ly -lps and tlr -ly -lipid iva (a synthetic tetra-acylated lipid a precursor) complexes and compared them to the human counterparts. structural differences were detected in the interaction of lipid iva with the two mammalian receptors, with some amino acid replacements in ly and tlr possibly being responsible for the observed differential binding . analysis of tlr in mammals revealed that the receptor has evolved adaptively . we mapped positively selected sites onto the structure of the human and mouse complexes and observed that some of these may indeed account for structural differences between humans and mice (fig. ) . rodents are the most established animal model for human disease, including for susceptibility to infection. in recent years, however, technological advances have made the sequencing of whole genomes a relatively quick and inexpensive process. the genome sequences of non-model mammals that serve as natural reservoirs of human infectious agents are now available, allowing the unprecedented opportunity to exploit these data for molecular evolution studies. bats, for example, are known to host a wide range of viruses that are highly pathogenic to humans . the genomes of six bat species have been sequenced so far, and three of these (m. brandtii, pteropus alecto and myotis davidii) were analysed in detail to unveil the evolutionary history of specific traits . results showed that different families of immune receptors -including killer cell immunoglobulin-like receptors (kirs), killer cell lectin-like receptors (klrs), sialic acid-binding immunoglobulin-like lectins (siglecs) and leukocyte immunoglobulin-like receptors (lilrs) -have expanded or contracted in distinct bat species. also, in these three bat species, as well as in the common ancestor of p. alecto and m. davidii, genes involved in immunity represented preferential targets of positive selection . this is not unexpected: immune-response genes have been shown to have evolved rapidly in most mammalian species analysed to date . thus, although these sequenced bat genomes have not yet provided an explanation as to why bats are tolerant to ebov, for instance, they pave the way for further analyses to test specific hypotheses and/or to address the molecular determinants of host-pathogen interactions. in a recent study, demogines and co-workers showed how this can be accomplished. the authors focused on angiotensinconverting enzyme (ace ), which serves as a receptor for severe acute respiratory syndrome coronavirus (sars-cov) cell entry. in particular, the receptorbinding domain of the viral spike protein is responsible for ace binding and is a major determinant of host range . although the human sars epidemic was suggested to have originated from the zoonotic transmission of sars-cov from bats to humans, possibly via an intermediate host (for example, palm civets) , , no ace -binding sars-cov-like virus had been identified in bats when demogines and collaborators started their work . the authors analysed ace genes in bat species, and results revealed that the gene evolved adaptively and that the positively selected residues of the bat genes map at the ace -sars-cov interaction surface (fig. ) . positive selection localized to a subset of sites or confined to a few species in a phylogeny. these data led to the conclusion that ace -binding coronaviruses originated in bats . this finding was confirmed in a subsequent study that isolated an ace -binding sars-like coronavirus from horseshoe bats in china , highlighting the power of evolutionary studies in predicting host range and disease emergence. similarly to sars-cov, mers-cov is thought to have originated in bats and to have spread to humans via an intermediate host, possibly dromedary camels . infection is initiated by binding of the mers-cov spike protein to human dipeptidyl peptidase (dpp ; also known as cd ) . recent data indicate that five amino acids in dpp that differ between humans (mers-cov susceptible) and hamsters (non-susceptible) are key determinants for host specificity (fig. ) . we extended a previous evolutionary analysis of mammalian dpp (ref. ): strong evidence of positive selection was found with episodic selection in the vespertilionidae bat family and the panda and ferret branches, as well as in the dog lineage (fig. ; see supplementary information s ,s (box, table)). as shown in fig. , most positively selected sites are located at the dpp -spike protein interaction surface , and one of these is among those described as binding determinants . thus, as observed for ace , mers-cov and related viruses (for example, coronavirus hku ) are likely to act as drivers of molecular evolution on mammalian dpp genes; it will be especially interesting to evaluate the contribution of positively selected sites in ferrets because these animals are resistant to mers-cov infection. immune responses in mammals are highly coordinated processes involving multiple systems that sense infection, activate antiviral and antimicrobial responses, and trigger adaptive immunity. the evolutionary history of several such systems has been analysed in detail, and below we describe the most recent findings. innate immune receptors. the mammalian immune system is endowed with a repertoire of molecular sensors called pattern-recognition receptors (prrs). these molecules detect pathogen-associated molecular patterns (pamps) and initiate a downstream signalling cascade that culminates in the production of cytokines and antimicrobial factors. the main families of prrs include tlrs, nod-like receptors (nlrs), rig-like receptors (rlrs) and aim -like receptors (alrs). in the host-pathogen arms race, these molecules represent one of the foremost detection-defence systems; consistently, several studies have reported adaptive evolution at genes encoding mammalian prrs. analyses in primates, rodents and representative mammalian species indicate that positive selection shaped nucleotide diversity at most tlrs, with the strongest pressure acting on tlr (refs , , ) . similarly to tlr (fig. ) , several positively selected sites in other tlrs are located in pamp-binding regions, raising questions as to whether species-specific host-pathogen co-evolution is occurring, and how these sequence changes translate into differential pamp recognition. in fact, as mentioned above for lps, species-specific differences in ligand binding by tlrs seem to be common and potentially affect the overall immune response to specific pathogens . integration of evolutionary, immunological and genetic studies will be instrumental in the future for medical applications, especially in light of nature reviews | genetics sites that are positively selected in mammals are mapped onto the tlr structure (red): several of these flank or correspond (orange) to residues that differ between humans and mice and that surround the phosphate groups of lipid iva (yellow) . if lys and arg are replaced with the human residues (glu and gln , respectively), the responsiveness of mouse tlr -ly to lipid iva is abolished. b | structures of human cd (white; transmembrane and juxtamembrane region) and mir (grey; encoded by kaposi sarcoma-associated herpesvirus). cd sites that are involved in the interaction and that are positively selected in mammals are shown in red. c | complex of transferrin receptor protein (tfr ) with the surface glycoprotein (gp ) of machupo virus (macv), a rodent arenavirus that can also infect humans through zoonotic transmission. tfr residues involved in the interaction with gp are in yellow, positively selected sites are in red and positively selected sites that directly interact with gp are in orange. the proposed use of tlr ligands as vaccine adjuvants, a step that may require tailoring to distinct species . compared with tlrs, mammalian alrs are much less conserved and more dynamic, with distinct species carrying different sets of functional genes (ranging from in mice to none in some bats) , . as a consequence, analysis of several mammals indicated that, with the exception of absent in melanoma (aim ), which is non-functional in several species, no unequivocal orthologues can be inferred for the remaining alr genes. this prevents the application of standard codon-based tests across the entire mammalian phylogeny, although closely related species can be analysed. thus, interferonγ-inducible protein (ifi ) and aim were shown to have evolved under positive selection in primates. positively selected sites were observed to mainly localize near to regions or domains involved in dna binding and protein-protein interaction, suggesting modulation of substrate specificity or genetic conflicts with viral inhibitors . positive selection was also described for the three mammalian rlrs (retinoic acid-inducible gene i (rigi; also known as ddx ), melanoma differentiation-associated (mda ; also known as ifih ) and lgp (also known as dhx )), the primate nlr family apoptosis inhibitory protein (naip) and rodent naip genes , . indeed, as is the case for alrs, rodents have multiple naip paralogues that show widespread evidence of inter-locus recombination. this led to the application of a dn//ds sliding window approach: the naip sites evolving with dn/ds > were found to be located in the bacterial ligand domain . studies on antiviral restriction factors have been extensive because these molecules represent obvious targets in hostpathogen arms races. specifically, genetic conflicts between host restriction factors and viral components often play out in terms of binding-seeking dynamics (the host factor adapts to bind the viral component) and binding-avoidance dynamics (the virus counter-adapts to avoid binding and restriction by the host factors). the evolutionary history of antiviral restriction factors has been comprehensively reviewed elsewhere [ ] [ ] [ ] , and we only highlight a few recent developments here. the first restriction factor to be identified was the product of the mouse gene friend virus susceptibility (fv ), a protein that protects against murine leukaemia virus (mlv) infection . the origin and evolution of fv is extremely interesting: early sequence analysis revealed that it derives from the gag gene of an ancient endogenous retrovirus that is not directly related to mlv . thus, fv exemplifies a paradoxical twist of the arms race scenario whereby a viral gene is co-opted by the host to serve an antiviral function (this is not the only instance, see ref. ) . recent results showed that the fv gene was inserted into the mouse genome between million and million years ago, long before the appearance of mlv. thus, the selective pressure exerted by other viruses must have maintained fv function and driven its evolution . indeed, analysis of fv from wild-type mice indicates that different fv products can recognize s (box, table) ). genes that evolved from a common ancestral gene through speciation. homologous genes created by a duplication event within the same genome. the observed number of non-synonymous substitutions per non-synonymous site. the observed number of synonymous substitutions per synonymous site. and block multiple genera of retroviruses (for example, equine infectious anaemia virus and feline foamy virus), and a number of positively selected sites in the carboxy-terminal region of fv are directly involved in restriction specificity . thus, in a similar way to trim , fv was identified for its ability to restrict an extant virus, but its evolution was driven by different waves of retroviral species, some of which are likely to be extinct. other restriction factors that have been the topic of recent investigation are encoded by two paralogous genes, myxovirus resistance (mx ; also known as mxa) and mx (also known as mxb). the protein products of the two genes display high sequence identity but different antiviral specificity. mx has broad activity against rna and dna viruses. recently, mitchell and collaborators showed the potential of evolutionary analyses to generate experimentally testable hypotheses on the nature of genetic changes that affect species-specific susceptibility to infection. the authors applied an evolution-guided approach and identified a cluster of positively selected residues in an unstructured surface-exposed mx loop (loop ), which confers antiviral specificity; genetic variation in loop is a major determinant of mx antiviral activity against thogoto and avian influenza a viruses, and replacements at a single positively selected site alter the ability of mx to restrict these pathogens . more recently, the selection pattern at the mx gene, which encodes an antiretroviral effector , was shown to parallel that of mx , with most selected sites located in loop (ref. ). in mx , sites selected in the primate lineage were detected outside loop , and mx also showed evidence of selection in other domains , ; these sites are promising candidates for being additional determinants of antiviral activity. antigen presentation and the ensuing t cell activation are central processes in mammalian cellmediated immune response (fig. ) . therefore, a convenient strategy for pathogens to elude immune surveillance is to hijack the molecular pathways responsible for these processes , . in line with the arms race scenario, there is evidence of positive selection at several mammalian genes involved in antigen presentation and the regulation of t cell activation , (fig. ) . the pathogen-driven mechanisms underlying evolution at these genes are likely to be manifold. one mechanism is genetic conflict with a pathogen-encoded component, evidence of which can be seen in the protein cd . positively selected sites in the transmembrane and juxtamembrane region of cd interact with mir (fig. ) , a kaposi sarcomaassociated herpesvirus (kshv) protein that downmodulates cd expression , . a second mechanism is the use of cell-surface molecules as viral receptors: some adenovirus strains, for example, have been reported to exploit cd and cd for cellular attachment , . a third mechanism is the broadening or tuning of the host's ability to process and present pathogen-derived components. for example, a positively selected site in the carbohydrate-recognition domain of cd (also known as langerin; a birbeck granule molecule) affects an amino acid position that is directly involved in the binding of pathogen-derived glycoconjugates . these mechanisms are not mutually exclusive. for example, a plethora of viral pathogens (such as herpes simplex virus , human papillomavirus, hiv- and kshv) interfere with cd d trafficking and recycling , . as a consequence, the cytoplasmic and transmembrane regions of cd d display positively selected sites, one of which is within a primate-specific trafficking signal. additional positively selected sites are located in the cd d extracellular region and flank the t cell receptor interaction surface and the lipid-binding pocket, which suggests that they exert an effect on antigen-binding specificity . finally, we draw attention to one of the few attempts at assessing the part that helminth infections have played as selective pressures for mammals and at integrating epidemiological information into molecular evolutionary approaches. machado and co-workers found evidence of positive selection at the mammalian gene fc fragment of igg, low affinity iiib, receptor (fcgr b), which is expressed by eosinophils and is involved in the binding of immunoglobulin g (igg)-coated parasites. notably, the authors also tested a specific hypothesis whereby mammalian lineages hosting a wider range of helminth species should show stronger evidence of selection compared with other species (this was accomplished by running the phylogenetic analysis by maximum likelihood (paml) branch-site models with helminth-rich lineages as foreground branches . their hypothesis was verified, providing a plausible explanation for the evolutionary pattern at fcgr b and suggesting that similar approaches may be used to detect other mammalian genes involved in genetic conflicts with helminth parasites. as exemplified by ace , host-pathogen interactions are not limited to immune system components. the reasons why genes with no specific defence function may be targeted by the selective pressure imposed by infectious agents are manifold. the best known instances probably refer to gene products that act as incidental receptors for pathogens, as is the case with ace . other host gene products that engage in genetic conflicts include those that participate in the coagulation cascade and the contact system, which are commonly hijacked by bacterial pathogens to promote tissue invasion or to elude detection by immune cells (see ref. for a review). an alternative possibility is that the host builds a line of defence based on the sequestration of essential micronutrients from the pathogen, a phenomenon known as 'nutritional immunity' . housekeeping genes. incidental receptors are often represented by the products of housekeeping genes, which are typically expressed at high levels by different cell types. among these, the transferrin receptor (tfrc) gene encodes a cell-surface molecule (transferrin receptor protein (tfr )) that is essential for iron uptake. tfr is used as a receptor by mouse mammary tumour virus, canine parvovirus and rodent new figure | genes involved in antigen processing and presentation and t cell regulation are common targets of positive selection in mammals. all pathway components are designated using official gene names (excluding the major histocompatibility complex (mhc) and t cell receptor (tcr)) and are highlighted in red if they are targets of positive selection in mammals or primates , , . the molecular components of different antigen processing and presentation pathways are shown (details from refs , ) to provide a general overview of the extent of positive selection and to highlight the function of positively selected genes, as most of their protein products directly interact with the antigen. thus, the figure is not meant to show all molecules involved in the process or to convey mechanistic insights. also, some genes may show tissue-specific expression or may be induced under specific circumstances: their products are nonetheless included for the sake of completeness. as for t cell regulatory molecules, the representation does not reflect the stoichiometry of binding (for example, cd functions as a dimer). notably, the same molecule may be expressed by different populations of t cells, although here each molecule is shown on one t cell type only (to avoid redundancy). the dashed arrows and '?' indicate steps that lack clear molecular definition or are only inferred. the orange circles, and red and blue shapes at the bottom of the figure represent proteolytic fragments. b m, β -microglobulin; blmh, bleomycin hydrolase; calr, calreticulin; cd lg, cd ligand; ctla , cytotoxic t lymphocyte protein ; cts, cathepsin; cyb, cytochrome b; erap, endoplasmic reticulum aminopeptidase; havcr , hepatitis a virus cellular receptor ; hla-dm, major histocompatibility complex, class ii, dm; icos, inducible t cell co-stimulator; icoslg, icos ligand; ifi , interferonγ-inducible protein ; inkt, invariant natural killer t; itcr, invariant tcr; lgmn, legumain; lnpep, leucyl-cystinyl aminopeptidase; ncf, neutrophil cytosol factor; npepps, puromycin-sensitive aminopeptidase (also known as psa); nrd , nardilysin; pdcd , programmed cell death ; pdcd lg , programmed cell death ligand ; pdia , protein disulfide-isomerase a ; ros, reactive oxygen species; tap, antigen peptide transporter; tapbp, tap-binding protein (also known as tapasin); thop , thimet oligopeptidase ; tpp , tripeptidyl-peptidase . the elimination of deleterious amino acid-replacing substitutions, which results in fewer non-synonymous changes than expected under neutrality (dn/ds < ). world arenaviruses. in line with the arms race scenario, tfrc evolved adaptively in rodents and caniforms, and positively selected sites are mainly located in the extracellular domain regions that interact with rodentinfecting arenaviruses (fig. ) and carnivore-infecting parvoviruses, respectively , . interestingly, positive selection at the primate transferrin (tf) gene, which encodes the tfr ligand, was also recently described ; in this case, selection is driven by bacteria, not viruses . transferrin is the major circulating iron transporter in mammals and is also thought to participate in nutritional immunity by sequestering iron from bacteria. consistently, most positively selected sites were found to have evolved to counteract binding by bacterial transferrin surface receptors that scavenge host iron . thus, different selective pressures exerted by distinct molecular mechanisms contributed to shaping the evolution of a central homeostatic process -in this case, iron transport in mammals. another housekeeping gene product that acts as a viral receptor is niemann-pick c protein (npc ), a sterol transporter located in the membrane of late endosomes and lysosomes. npc is expressed by most cell types and is used by filoviruses (such as ebov and marburg virus). evolutionary analysis of mammalian npc genes indicated that three positively selected residues are located in the amino-terminal portion of the second npc luminal loop; binding of this loop by the ebov glycoprotein (gp) is necessary and sufficient for the viral receptor activity of the sterol transporter , (fig. ) . the second luminal loop of npc is also bound with high affinity by the gp encoded by lloviu virus, a bat-derived, ebov-like filovirus . thus, npc may represent a universal receptor for filoviruses, and the constant selective pressure exerted by such viruses might have greatly contributed to shaping mammalian genetic diversity at loop . these data may have great and immediate practical values. in fact, small molecules that directly target npc and disrupt gp binding are regarded as possible therapeutic compounds against ebov [ ] [ ] [ ] (fig. ) . because mammalian npc diversity at the interaction surface is driven by selection, future efforts in this direction are likely to benefit from the incorporation of evolutionary analysis; this would be especially important when testing therapeutic molecules on model organisms and non-human mammals. in humans, mutations in npc cause niemann-pick disease type c , a progressive neurodegenerative condition. this is in line with the central role of this transporter in housekeeping functions; thus purifying selection. is expected to constrain variation in the gene. indeed, the human-mouse dn/ds calculated for the npc whole-gene region is definitely lower than , as is the case for most genes (fig. ) . in fact, mammalian npc genes show a large preponderance of codons evolving with dn/ds < , and positive selection is extremely localized in loop (fig. ) . this specific example illustrates a general concept, whereby molecules involved in central homeostatic processes may be engaged in genetic conflicts with pathogens, although in several instances the sequence space accessible for adaptive mutation without a high fitness cost is expected to be limited. the coagulation cascade and contact system. as anticipated above, several components of the coagulation cascade and contact system evolved adaptively in mammals, most likely as a result of genetic conflicts with bacterial pathogens , . for instance, staphylococcus aureus is endowed with an arsenal of proteins that target such systems, including two cysteine proteinases (scpa and sspb) that cleave plasma kininogen at each terminal side of the bradykinin domain to generate kinins, with a consequent increase of vascular leakage . these events are central for bacterial virulence and are linked to the pathogenesis of sepsis. in kininogen (kng ), positively selected sites are located in all domains, with the exception of the highly conserved bradykinin region . one of the positively selected sites defines the n-terminal cleavage site of scpa and sspb, suggesting that sites flanking the bradykinin sequence are evolving to avoid recognition and cleavage by bacterial-encoded proteases. in analogy to the strong purifying selection acting on the bradykinin region, analysis of calculation cascade genes indicated that disease-causing mutations are more likely to occur at sites targeted by purifying selection and are rarer at positively selected sites . again, these observations highlight the coexistence of distinct selective regimes at the same gene regions and exemplify the concept of evolutionary trade-offs. the advent of high-throughput sequencing technologies has allowed for the generation of an unprecedented wealth of genetic data, including the whole-genome sequences of host reservoir species for human pathogens, as well as genetic information for multiple microbial and viral species and strains. moreover, large-scale approaches such as rna interference and mass spectrometry are providing detailed pictures of host-pathogen interactomes , . finally, an increasing number of crystal structures of interacting host and pathogen proteins solved in complex are available, allowing the opportunity to determine the structural basis of these interactions to identify regions or amino acids that lie at the host-pathogen contact surface. integration of these data with evolutionary analysis will allow the testing of specific hypotheses, including which species have responded to the pressure exerted by one or more pathogens (see the sars-cov example), which molecules and residues have participated in the arms race and which host-pathogen interacting partners are expected to have co-evolved. these advances are also expected to progressively change evolutionary genetics from a hypothesis-driven to a hypothesis-generating discipline. in this respect, we note that although the arms race scenarios we have described in this review imply some form of host-pathogen co-evolution over time, the nature of the interaction and its dynamics have often been inferred from the observed pattern of variation. indeed, the fact that the same residues that affect specific host-pathogen interactions are targeted by positive selection does not necessarily imply a causal link, and in many instances the specific selective agents underlying molecular adaptations remain to be determined. as shown above, these may well be accounted for by extinct pathogens or by agents that had a major co-evolutionary role in the past but that are now fading away from the landscape of common infections. with a few exceptions , , evolutionary studies only investigate extant genetic variation and modern pathogens, with little reconstruction of past events. nevertheless, we do not necessarily need to go back in time: evolutionary analyses can be used as predictive tools to pinpoint which genes and residues are more likely to contribute to present-day host-pathogen interaction and help explain species-specific susceptibility nature reviews | genetics the values for some of the genes discussed in this review are indicated. data were derived from the ensembl biomart database (see further information). b | natural selection acting on mammalian niemann-pick c (npc ) genes. npc is shown with its predicted membrane topology and protein regions coloured in hues of blue that represent the percentage of negatively selected sites (as detected by the single-likelihood ancestor counting method using datamonkey); the darker the blue, the higher the percentage. the location of three positively selected residues (red) is indicated on the left, and an alignment of the corresponding region is shown on the protein to the right (with red and blue representing positively and negatively selected sites, respectively). the interaction with the glycoprotein (gp; green) of filoviruses (such as ebola virus, marburg virus or lloviu virus) is shown. gp binds npc after processing by cellular proteases. ace , angiotensin-converting enzyme ; darc, duffy blood group, atypical chemokine receptor; mx , myxovirus resistance ; ssd, sterol-sensing domain; tfrc, transferrin receptor; tlr , toll-like receptor . to infection. several studies mentioned above, including those investigating selection at mx (ref. ), tfrc (refs , ) , tf and other protein-coding genes , , , , used experimental analyses to show that evolutionary information can indeed be exploited to gain highresolution insight into the molecular determinants of binding affinities at host-pathogen interfaces. the studies of iron transporters hold particular value because the authors analysed the genetic variability of both the host and the pathogen and showed that both parties evolved in response to mutually exerted pressures, in line with the red queen principles. so far, few attempts have been made at integrating evolutionary analyses of host and pathogen interacting partners into a common framework. however, efforts in this direction hold the promise of improving our understanding of the strategies used by both hosts and pathogens to adapt and counter-adapt. in turn, this knowledge has possible biomedical and therapeutic implications, given the ability of different pathogens or distinct strains of the same infectious agent to elude not only natural host defences but also drugs and vaccination strategies. as a final note, we mention that we have exclusively focused on adaptive events involving coding gene regions. nevertheless, several recent studies (see ref. for a review) have highlighted the role of non-coding variants as important determinants of susceptibility to infection within species. thus, host-pathogen conflicts are more than likely to have contributed to adaptive evolution at regulatory elements during speciation. detection of these adaptive events will benefit from the availability of high-throughput techniques (for example, rna sequencing and chromatin immunoprecipitation followed by sequencing) and the development of methodological approaches for dissecting molecular evolution in non-coding regions; notably, recent data have shown the usefulness of a framework similar to dn/ds to analyse the evolutionary history of mammalian transcriptional enhancers . application of this methodology (or extensions thereof) to the study of host-pathogen interactions will provide valuable information on which non-coding sequence changes have been targeted by selection and thus modulate susceptibility to infection or related phenotypes. signatures of environmental genetic adaptation pinpoint pathogens as the main selective pressure through human evolution a new evolutionary law running with the red queen: the role of biotic conflicts in evolution the causes of evolution (longmans, green, & co, ) revenge of the phages: defeating bacterial defences genomic variability as a driver of plant-pathogen coevolution? mainstreaming caenorhabditis elegans in experimental evolution insights from natural host-parasite interactions: the drosophila model from evolutionary genetics to human immunology: how selection shapes host defence genes population genetic tools for dissecting innate immunity in humans the red queen's long race: human adaptation to pathogen pressure human genome variability, natural selection and infectious diseases natural selection and infectious disease in human populations the red queen and the court jester: species diversity and the role of biotic and abiotic factors through time phylogenies reveal new interpretation of speciation and the red queen host-parasite 'red queen' dynamics archived in pond sediment a matching-allele model explains host resistance to parasites antagonistic coevolution accelerates molecular evolution biological and biomedical implications of the co-evolution of pathogens and their hosts global trends in emerging infectious diseases origins of hiv and the aids pandemic. cold spring harb the cytoplasmic body component trim α restricts hiv- infection in old world monkeys positive selection of primate trim α identifies a critical species-specific retroviral restriction domain restriction of an extinct retrovirus by the human trim α antiviral protein positive selection of primate genes that promote hiv- replication hiv- capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency this is an excellent review highlighting the importance of non-model organisms in understanding zoonotic infections diversification of an emerging pathogen in a biodiversity hotspot: leptospira in endemic small mammals of madagascar patterns of positive selection in six mammalian genomes genome analysis reveals insights into physiology and longevity of the brandt's bat myotis brandtii this work helps to clarify the species specificity of n. gonorrhoeae infection by analysing the binding of sialylated gonococci to human and chimpanzee cfh humanized tlr /md- mice reveal lps recognition differentially impacts susceptibility to yersinia pestis and salmonella enterica lipid a modification systems in gram-negative bacteria structural basis of species-specific endotoxin sensing by innate immune receptor tlr /md- signatures of positive selection in toll-like receptor (tlr) genes in mammals mass extinctions, biodiversity and mitochondrial function: are bats 'special' as reservoirs for emerging viruses? an extremely interesting study providing an overview of the evolutionary history of three bat genomes, with possible implications for immunity-related evidence for ace -utilizing coronaviruses (covs) related to severe acute respiratory syndrome cov in bats a good example of how evolutionary studies can provide insight into host range and disease emergence retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation and characterization of a bat sars-like coronavirus that uses the ace receptor full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus a central work showing that dpp of human and bat origin acts as a functional receptor for mers-cov host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase adaptive evolution of bat dipeptidyl peptidase (dpp ): implications for the origin and emergence of middle east respiratory syndrome coronavirus molecular basis of binding between novel human coronavirus mers-cov and its receptor cd adaptation and constraint at toll-like receptors in primates contrasted evolutionary histories of two toll-like receptors (tlr and tlr ) in wild rodents (murinae) variation matters: tlr structure and species-specific pathogen recognition extensive evolutionary and functional diversity among mammalian aim -like receptors ancient and recent selective pressures shaped genetic diversity at aim -like nucleic acid sensors rig-i-like receptors evolved adaptively in mammals, with parallel evolution at lgp and rig-i molecular basis for specific recognition of bacterial ligands by naip/nlrc inflammasomes rules of engagement: molecular insights from host-virus arms races evolutionary conflicts between viruses and restriction factors shape immunity a cross-species view on viruses positional cloning of the mouse retrovirus restriction gene fv paleovirology and virally derived immunity evolution of the retroviral restriction gene fv : inhibition of non-mlv retroviruses a study in wild mice showing that fv antiviral activity is broader than previously thought. it identifies positively selected residues in the c terminus that contribute to antiviral specificity evolution-guided identification of antiviral specificity determinants in the broadly acting interferon-induced innate immunity factor mxa a seminal paper that applies an evolution-guided approach to detect mx residues that confer antiviral specificity human mx is an interferon-induced post-entry inhibitor of hiv- infection evolutionary analysis identifies an mx haplotype associated with natural resistance to hiv- infection manipulation of costimulatory molecules by intracellular pathogens: veni, vidi, vici!! plos pathog mhc class i antigen presentation: learning from viral evasion strategies an evolutionary analysis of antigen processing and presentation across different timescales reveals pervasive selection a million year history of t cell regulatory molecules reveals widespread selection, with adaptive evolution of disease alleles the intertransmembrane region of kaposi's sarcoma-associated herpesvirus modulator of immune recognition contributes to b - downregulation the nef protein of hiv- induces loss of cell surface costimulatory molecules cd and cd in apcs members of adenovirus species b utilize cd and cd as cellular attachment receptors structural basis for langerin recognition of diverse pathogen and mammalian glycans through a single binding site hiding lipid presentation: viral interference with cd d-restricted invariant natural killer t (inkt) cell activation a threonine-based targeting signal in the human cd d cytoplasmic tail controls its functional expression evolutionary history of copynumber-variable locus for the low-affinity fcγ receptor: mutation rate, autoimmune disease, and the legacy of helminth infection one of the few studies of helminth-driven selective pressure in mammals that also integrates evolutionary analysis with epidemiological information thrombosis as an intravascular effector of innate immunity dual host-virus arms races shape an essential housekeeping protein an extremely interesting study extending the arms race scenario to a housekeeping protein, the transferrin receptor evolutionary reconstructions of the transferrin receptor of caniforms supports canine parvovirus being a re-emerged and not a novel pathogen in dogs escape from bacterial iron piracy through rapid evolution of transferrin mammalian npc genes may undergo positive selection and human polymorphisms associate with type diabetes niemann-pick c (npc )/ npc -like chimeras define sequences critical for npc 's function as a flovirus entry receptor cell entry by a novel european filovirus requires host endosomal cysteine proteases and niemann-pick c multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection inhibition of ebola virus infection: identification of niemann-pick c as the target by optimization of a chemical probe small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection evolutionary analysis of the contact system indicates that kininogen evolved adaptively in mammals and in human populations positive selection during the evolution of the blood coagulation factors in the context of their disease-causing mutations induction of vascular leakage through release of bradykinin and a novel kinin by cysteine proteinases from staphylococcus aureus viral immune modulators perturb the human molecular network by common and unique strategies genome-wide rnai screen identifies human host factors crucial for influenza virus replication a novel test for selection on cis-regulatory elements reveals positive and negative selection acting on mammalian transcriptional enhancers paml : phylogenetic analysis by maximum likelihood accuracy and power of bayes prediction of amino acid sites under positive selection bayes empirical bayes inference of amino acid sites under positive selection detecting individual sites subject to episodic diversifying selection high sensitivity to aligner and high rate of false positives in the estimates of positive selection in the drosophila genomes class of multiple sequence alignment algorithm affects genomic analysis estimates of positive darwinian selection are inflated by errors in sequencing, annotation, and alignment the effects of alignment error and alignment filtering on the sitewise detection of positive selection effect of recombination on the accuracy of the likelihood method for detecting positive selection at amino acid sites evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level the effect of insertions, deletions, and alignment errors on the branch-site test of positive selection multiple hypothesis testing to detect lineages under positive selection that affects only a few sites a random effects branchsite model for detecting episodic diversifying selection modeling the site-specific variation of selection patterns along lineages performance of standard and stochastic branch-site models for detecting positive selection among coding sequences statistical properties of the branch-site test of positive selection towards a systems understanding of mhc class i and mhc class ii antigen presentation cd antigen presentation: how it works the authors declare no competing interests. key: cord- - jeexu authors: amalaradjou, mary anne roshni; bhunia, arun k. title: modern approaches in probiotics research to control foodborne pathogens date: - - journal: adv food nutr res doi: . /b - - - - . - sha: doc_id: cord_uid: jeexu foodborne illness is a serious public health concern. there are over known microbial, chemical, and physical agents that are known to cause foodborne illness. efforts are made for improved detection, control and prevention of foodborne pathogen in food, and pathogen associated diseases in the host. several commonly used approaches to control foodborne pathogens include antibiotics, natural antimicrobials, bacteriophages, bacteriocins, ionizing radiations, and heat. in addition, probiotics offer a potential intervention strategy for the prevention and control of foodborne infections. this review focuses on the use of probiotics and bioengineered probiotics to control foodborne pathogens, their antimicrobial actions, and their delivery strategies. although probiotics have been demonstrated to be effective in antagonizing foodborne pathogens, challenges exist in the characterization and elucidation of underlying molecular mechanisms of action and in the development of potential delivery strategies that could maintain the viability and functionality of the probiotic in the target organ. foodborne illness is a serious public health concern. the global burden of foodborne illness is currently unknown. however, the world health organization (who) reported that in , . million people died from diarrheal diseases, largely due to contaminated food and water (greig & ravel, ; newell et al., ) . in the united states, the centers for diseases control and prevention (cdc) estimates that each year there are about million cases of foodborne infections with , hospitalizations and deaths (scallan et al., ) . there are over known microbial, chemical, or physical agents that can result in illness when consumed (newell et al., ) . of these, microbial source comprising of bacterial, viral, and fungal is of major concern. cdc estimates that of all the foodborne infections, % of the hospitalizations and deaths are attributed to known pathogens (scallan et al., ) . in light of this serious public health crisis, efforts have been directed toward the detection, control, and prevention of well-recognized foodborne pathogens and diseases in the food chain. it is estimated that a reduction in foodborne illness by % would keep about million americans from getting sick each year (scallan et al., ) . with increasing trend in consumer preference for safe and wholesome food, probiotics offer an effective and alternative intervention strategy to control foodborne illnesses. pathogens, which include norovirus, nontyphoidal salmonella, clostridium perfringens, campylobacter spp., and staphylococcus aureus. these pathogens enter into the food system through contaminated raw materials, water, humans, meat animals, wild life, and insect vectors. there are several factors that affect the trends in the occurrence of foodborne illness: large-scale production and wide distribution of food, globalization of food supply, eating outside of the home, microbial genomic diversification yielding the emergence of new pathogens, and growing population of at-risk consumers. the diseases caused by these pathogens have different consequences and sequel (table . ). foodborne pathogens sicken more than million americans annually, that is, in in the u.s. population (scallan et al., ) . reducing foodborne illnesses by % would keep about million americans from getting sick every year. food safety concerns are also elevated because of consumers demand for high quality, low preservatives, and minimally processed convenient ready-to-eat meals. such foods are highly vulnerable to contamination and heighten public health safety concerns. this has led to the development of several control strategies both by the government and the industry. strategies to control foodborne infections can be classified as preharvest and postharvest interventions. traditionally, much of the research effort was aimed at improving the safety of meat products as postslaughter sanitation and product formulations. however, the continual incidence of outbreaks and increase in knowledge about the pathogens have led to the development of preharvest intervention strategies (doyle & erickson, ) . preharvest intervention step is a logical food safety approach that allows reduced levels of pathogen loads in the incoming raw materials (callaway, anderson, et al., ; soon, chadd, & baines, ) . antibiotics are known to alter the microbiological ecology of the intestinal tract . this led to the prophylactic use of antibiotics in animal agriculture to control disease and improve animal growth rate and efficiency. with emergence of antibiotic resistance among pathogens, research has focused on the use of naturally occurring antimicrobials as an alternative to control foodborne pathogens in live animals and foods. antimicrobials comprise organic acids, essential oils, plant extracts, bacteriocins, probiotics, and bacteriophages (baugher & klaenhammer, ; callaway, anderson, et al., ; hassan, kjos, nes, diep, & lotfipour, ; negi, ) . chemical rinses using organic acids that are generally recognized as safe such as acetic, lactic, and citric acids are commonly used in the meat industry to rinse animal carcasses and produce (fruits and vegetables) (sirsat, muthaiyan, & ricke, ). these acids reduce the ph of the food and hence control the growth of microorganisms. lactic acid is most effective when applied at higher temperatures and at a concentration of - % (sirsat et al., ). these organic acids are sometimes used in combination with oxidizing agents such as hydrogen peroxide to enhance their antimicrobial efficacies. essential oils extracted from clove, cinnamon, thyme, and oregano, and their components have been used in the control of foodborne pathogens such as nontyphoidal salmonella (johny, hoagland, & venkitanarayanan, ) , escherichia coli o :h (amalaradjou et al., ) , and listeria monocytogenes in microbiological growth media, on live animals or food systems (hyldgaard, mygind, & meyer, ) . unlike antibiotics, essential oils have multifaceted antimicrobial effects thereby making it difficult for the bacteria to develop resistance. microbial contamination can also be controlled by the use of microbicidal treatments, such as ionizing radiations, and heating. application of nonthermal methods such as high hydrostatic pressure, high-intensity pulsed electric fields, oscillating magnetic fields, intense light pulse, photosensitization, or a combination of above (hurdle approach) has also been shown to be effective (luksienė & zukauskas, ; morris, brody, & wicker, ) . one of the most common physical methods of decontamination is irradiation (radomyski, murano, olson, & murano, ; smith & pillai, ) . food irradiation destroys the indigenous flora and prolongs shelf life of products during storage. food is exposed to doses of ionizing radiation sufficient enough to create positive and negative charges to kill bacteria in the food. the type of physical method used and the dosage of the treatment depend on the type of food matrix to be decontaminated. biological methods of control include the use of bacteriophages, bacteriocins, and probiotics. in food matrix, these components can be present naturally or added extrinsically. these biological agents can be used at the pre-and postharvest phase to prevent bacterial contamination (hagens & loessner, ) . bacteriophages are viruses that can infect and kill bacteria and are considered alternatives to antimicrobials for use in the food industry (garcía, martínez, obeso, & rodríguez, ) and for therapeutic application to treat diseases (fischetti, (fischetti, , hanlon, ) . however, bacteriophages have narrow target spectra, and some can be used only against a particular strain. this high degree of specificity allows phages to be used against targeted microorganisms in a mixed population without affecting the microbial ecosystem (callaway, anderson, et al., ) . bacteriophages have been used to control foodborne pathogens in farm animals against specific pathogens (hagens & loessner, ; lejeune & wetzel, ; wall, zhang, rostagno, & ebner, ) . in addition, several phages or phage cocktails have been approved by the fda for use modern approaches in probiotics research to control foodborne pathogens as food additive in ready-to-eat meat or for application in cattle/poultry prior to slaughter (hagens & loessner, ) . the use of probiotics, prebiotics, and synbiotics (combination of prebiotics and probiotics) has also gained increased attention in recent years. the use of microflora to reduce pathogen load in the gut is termed as a probiotic strategy (callaway, anderson, et al., ) . probiotic techniques involve the introduction of a normal microbial population into the gut to provide a nutrient (prebiotic) that is limiting and allows the growth of a specific subset of the gut microflora. the goal of this approach is to fill all the niches available in the gut so as to exclude the establishment of pathogenic microbes (doyle & erickson, ; gaggia, mattarelli, & biavati, ; patterson & burkholder, ) . due to the increased concern about the emergence in antibiotic resistance, use of probiotics provides an effective alternative to combat foodborne illnesses (baugher & klaenhammer, ; dobson, cotter, ross, & hill, ; hassan et al., ) . in addition, vaccine and antibody therapy also offer a viable option to reduce the burden of foodborne illness. vaccine therapy involves the stimulation of the animal's immune system to limit pathogen colonization. two types of vaccines can be employed to immunize food animals: the use of killed or inactivated bacterial cells or live attenuated cells. this approach has been used in poultry to reduce the colonization of salmonella using salmonella-specific antibodies (de buck, van immerseel, haesebrouck, & ducatelle, ; tellez et al., ) . similar approach in cattle and swine has shown promising results by enhancing immunoglobulin, iga, igg, and igm in serum and by reducing pathogen carriage (house, bishop, parry, dougan, & wain, ; mastroeni, chabalgoity, dunstan, maskell, & dougan, ) . along with these intervention strategies, good animal management or good agriculture practices are equally crucial to the production of healthy animals and agricultural products to ensure food safety. antibiotics have been widely used in animal agriculture to control disease and to increase animal growth rate or efficiency. although antibiotics are used to target specific bacteria, the specificity can sometimes be too narrow to be highly effective. therefore, in several occasions, broad-spectrum antibiotics are often included in animal rations. such treatments can disrupt the intestinal microbial ecosystem and can lead to the establishment of opportunistic pathogens. this could also impose deleterious effects on animal health, performance, and food safety. in addition, the use of antibiotics in human and veterinary medicine to treat infectious diseases has led to the rise and spread of antibiotic resistance (callaway, anderson, et al., ; . increased antibiotic-resistant strains can pose a significant public health hazard yielding increased frequency of treatment failures, severity of infection, prolonged duration of sickness, increase in systemic infections, and increased hospitalizations and mortality (newell et al., ) . antibiotic use in plants, animals, and humans for healthpromoting purposes can lead to emergence and dissemination of resistant bacteria and resistance genes. since antibiotic resistance can spread horizontally, the use of antibiotics in one ecological compartment can have a consequence on the resistance status in another (kruse & sorum, ; newell et al., ) . food of plant or animal origin can be a source of both antibiotic-resistant bacteria and resistance genes. the presence of antibiotic-resistant bacteria in food presents a direct hazard to food handlers and consumers equally. additionally, resistant traits can be transferred from bacteria of food origin to human pathogens directly or via a commensal resulting in an indirect hazard (newell et al., ) . in addition, antimicrobial resistance can also arise due to continued exposure of bacteria to antimicrobial residues in food. furthermore, the different routes through which bacteria acquire resistance are complex. there is an increasing evidence of incidence of antibioticresistant bacteria in food, highlighting the importance of antibiotic-resistant foodborne pathogens and their infections. several antibiotic-resistant strains of salmonella (threlfall, ) , campylobacter (fao/who/oie, ) , shigella, vibrio, and s. aureus (de boer et al., ), e. coli (walsh et al., ) , and enterococci (fao/who/oie, ) have been reported for foods of plant and animal origin (newell et al., ) . several of these strains have also been reported to be multidrug resistant. infections caused by such strains are an important health problem. an important step in curbing antimicrobial resistance is the enforcement of prudent use of antimicrobial agents in all sectors of animal and food production (fao/who/oie, ) and to employ alternative strategies to control food pathogens. at present, the interrelationship between diet and health is well established. the traditional role of diet is to provide the nutrients essential for metabolism. however, over the last few decades, this idea has evolved. it is now established that besides meeting metabolic needs, the diet also helps promote health and the state of well-being of the individual. this change in concept of the dietary role of foods has led to the development of a new class of dietary products called functional foods. a food can be considered functional if it beneficially affects target function in the body besides its nutritional value (figueroa-gonzalez, quijano, ramirez, & cruz-guerrero, ) . probiotics fall into the category of functional foods (nagpal et al., ) . these are nonpathogenic microorganisms that confer a health benefit on the host and prevent some diseases when administered in adequate amounts (fric, ) . the widely used probiotics include lactobacilli, bifidobacter, bacilli, yeast, and some nonpathogenic species within the genera of escherichia (i.e., e. coli nissle ), enterococcus, and bacillus. however, the most common probiotics belong to the genera lactobacillus and bifidobacterium. probiotics in general exert beneficial effects in three ways: (i) provide end products of anaerobic fermentation of carbohydrates such as organic acids that can be utilized by the host. these end products once absorbed into the blood stream are able to influence human mood, energy level, and even cognitive abilities. (ii) effectively compete with pathogen colonization to exclude them from causing disease, and (iii) stimulate host immune system by producing specific polysaccharides. these health benefits are generally strain specific (not species-or genera specific); thus in most cases, a cocktail of probiotics is used to gain the most benefits. probiotics cocktail can also ensure health benefits, in the event, if one strain fails. to control foodborne pathogens, probiotics are either used in competitive exclusion or as defined cultures. competitive exclusion involves the extrinsic administration of probiotics to food animals for intestinal colonization. probiotic bacteria also affect the composition and function of intestinal microbial population (o'toole & cooney, ) . the predominant presence of the probiotic in the gut prevents the pathogens access to the ecological niche, interfering with the attachment of pathogens to the gut and subverting the eventual infection process (gaggia et al., ) . besides physical displacement of pathogens, several probiotics also produce bacteriocins, which are antimicrobial peptides that inactivate pathogens. additionally, probiotics stimulate the immune system and help in mounting protective response against pathogen interaction with host cells (gill & prasad, ) . probiotic-induced enhanced butyrate production can also increase resistance against diseases through activation of antimicrobial host defense peptide (floch, ; sunkara et al., ) . although the use of probiotics is promising, there are several challenges for the industrial production of probiotic-based functional foods: (i) improvement in production techniques for large-scale manufacturing; (ii) costeffective production strategy; (iii) enhancement of probiotic viability during storage, manufacturing, and transit in the gastrointestinal tract; and (iv) development of efficient delivery system. strategies to overcome these challenges must be multidisciplinary and will help make the process cost-effective and beneficial to both the producers and consumers. bacteria are deliberately added to food either to enhance product flavor (starter cultures) or to enhance health benefits (functional additives). probiotic bacteria are used for the prevention or treatment of various diseases; thus, the probiotic organisms must be nonpathogenic to the host prior to its consideration for use. commonly used probiotic bacteria include members of the genera lactococcus, lactobacillus, and bifidobacterium. lactic acid bacteria are generally considered safe; however, some species of lactobacillus and bacillus have been associated with opportunistic infections in patients with underlying conditions resulting in endocarditis, bacteremia, and liver abscess (boyle, robins-browne, & tang, ) . presumably, the pathogenic property is associated with a specific strain rather than with the species in general. thus, the strain-specific characterization is essential to prove the absence of pathogenicity. prior to the approval of a probiotic for human use, it is essential that the bacteria be screened for potential pathogenicity and virulence traits (sanders et al., ) . providing evidence for the absence of virulence properties is relatively straightforward in elucidating the pathogenic potential. besides phenotypic characterization, it is also essential to genetically screen potential candidates for use as probiotics. another critical consideration is the scope for antimicrobial resistance. in addition to being sensitive to antibiotics, it is also essential that the probiotic bacteria do not carry any transferrable antibiotic resistance genes, which can serve as genetic reservoirs for other potentially pathogenic bacteria. besides acquisition of antibiotic-resistant genes, there is also the risk for uptake of virulence genes from pathogens that coinhabit the intestinal tract at the same time. however, there is no evidence in the literature of such event taking place in the gut. this could partly be due to the transient colonization of the gut by probiotics. scientific committee on animal nutrition (scan) has established a guideline (a decision tree, fig. . ) for the approval of a probiotic strain based on antibiotic resistance (scan, ) . similar screening strategies are also employed for the approval of a probiotic strain for use as a feed additive. considering all the factors that are essential in the assessment of safety of probiotics, it is paramount that the general conclusion that "probiotics are safe" cannot be broadly made. prior to the use of a probiotic or probiotic cocktail in foods or dietary supplement, they need to be determined to be safe for the general population. when intended for use as drugs, the safety assessment must balance risk with benefit (sanders et al., ) . the fao has outlined a guideline for approval of a probiotic strain for use in food ( fig. . ). the term probiotic means "for life" and is associated with bacteria that exert beneficial effects on humans and animals. this was first observed by eli metchnikoff in who suggested that the dependence of intestinal microbes on food makes it possible for them to develop measures to modify the gut flora and to replace the harmful microbes with useful microbes. the initial works of e. metchnikoff and h. tissier in the early twentieth century set the stage for the elucidation of the beneficial effects of probiotics and their multitude of applications in human health as summarized in a recent review article (bron, van baarlen, & kleerebezem, ) . the increasing body of scientific evidence that demonstrates the beneficial effects of probiotics on health and disease prevention and treatment has made probiotics increasingly important as part of human nutrition and has led to a surge in the demand for probiotics in clinical applications (deshpande, rao, & patole, ; ng, hart, kamm, stagg, & knight, ; vanderpool, yan, & polk, ) and as functional foods (ly, litonjua, gold, & celedon, ; nagpal et al., ) . probiotics have been defined based on their intent of use. fuller ( ) defined probiotics as "a live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance." this definition genus, species, strain deposit in international culture collection in vitro tests in vitro and/or animal model animal studies phase highlighted the microbial nature of probiotics. similarly, huis in't veld, havenaar, and marteau ( ) defined probiotics as "a viable mono or mixed culture of bacteria which, when applied to animal or man, beneficially affects the host by improving the properties of the indigenous flora." a more recent definition accepted by the fao/who ( ) defines probiotics as "live microorganisms which, when administered in adequate amounts confer a health benefit on the host." these definitions tend to reiterate the basic definition that probiotics are live microorganisms that in adequate dose can be beneficial to humans. probiotics can be classified based on their ability to colonize the intestine as resident or transient. resident strains are those that are common inhabitants of the human digestive tract, and probiotic supplements containing these strains are able to re-establish in the intestinal tract. resident strains may have least antagonistic effect on other beneficial resident strains in the intestinal tract. transient strains pass through the system and do not re-establish themselves. certain transient strains may not be effective when used as monocultures and hence in most cases are combined with other resident strains to enhance their efficacy. taxonomically, probiotics must be identified by their genus, species, and strain as is done with other bacteria. the commonly used probiotics include the members of the genus lactobacillus, bifidobacteria, streptococcus, enterococcus, leuconostoc, and yeast (saccharomyces). among these, the resident strains include lactobacillus acidophilus, lactobacillus salivarius, bifidobacterium bifidum, bifidobacterium infantis, bifidobacterium longum, bifidobacterium animalis, streptococcus faecalis, and streptococcus faecium. transient strains include lactobacillus casei, lactobacillus rhamnosus gg, lactobacillus bulgaricus, lactobacillus yoghurti, lactobacillus brevis, lactobacillus kefir, lactobacillus delbrueckii, lactobacillus plantarum, streptococcus lactis, and streptococcus thermophilus. bacteria should possess certain characteristics to be identified as a probiotic. table . lists the criteria that are essential for a bacterium to be classified as a probiotic. to be able to produce desired beneficial effects, it has been established that a dose of billion colony forming unit/day has been recommended for at least days (gronlund, lehtonen, eerola, & kero, ; williams, ) . probiotics either as mono or mixed cultures mainly consisting of lactobacilli have been used for human consumption in a variety of foods such as fermented milks (yogurt), chesses, fruit juices, chocolates, wine, and sausages. mixed cultures are highly desirable because they may have synergistic effects, and moreover, if one fails, others still can exert beneficial effects. it is well established that probiotics have several beneficial attributes (dicks & botes, ; nagpal et al., ; williams, ) . those include lactose metabolism and food digestion, production of antimicrobial peptides and control of enteric infections, antimycotic effects, anticarcinogenic properties, immunologic enhancement, enhancement of short-chain fatty acid (scfa) production, antiatherogenic and cholesterol-lowering attributes, regulatory role in allergy (thomas et al., ) , protection against vaginal or urinary tract infections, increased nutritional value, maintenance of epithelial integrity and barrier, stimulation of repair mechanism in cells, and maintenance and reestablishment of a well-balanced indigenous intestinal and respiratory microbial communities. several mechanisms have been proposed regarding action of probiotics. some of the major attributes are discussed below (table . ). the intestinal barrier function is an important defensive mechanism of the intestinal epithelium to maintain its protective effects to protect against invading pathogens and other harmful agents (ohland & macnaughton, ) . the barrier function is maintained by several mechanisms that include mucus secretion, chloride and water secretion, and maintenance of cell-cell tight junctions (thomas & ockhuizen, ) . disruption in the barrier table . criteria of an ideal probiotic . accurate taxonomic identification . normal inhabitant of the targeted species . generally recognized as safe . resistant to bile, hydrochloric acid, and pancreatic juice . ability to survive in both acidic conditions of the stomach and the alkaline conditions of the intestine . ability to persist in the gut even if it does not colonize . adhesion to epithelium to prevent physical removal . immunostimulatory action . nonpathogenic . maintain high cell viability and metabolic activity at the target site . stability of desired characteristics during processing, storage, and delivery . genetic stability nagpal et al. ( ) function can lead to various conditions such as inflammatory bowel disease (ibd), coeliac disease, enteric infections, and other autoimmune diseases (ng et al., ). several probiotics have been shown to protect the epithelial barrier and prevent mucosal damage triggered by food antigens, enteric pathogens, drugs, and proinflammatory cytokines (o'hara & shanahan, ) . these protective effects are mediated through several mechanisms either directly or indirectly through alteration of gut microflora populations. the first barrier that the intestinal bacteria and pathogens meet is the mucus. the entire length of the intestinal tract is lined by goblet cells. the percent of goblet cells increase from duodenum ( %) to descending colon ( %) relative to the epithelial cells (goto & kiyono, ) . intestinal microflora also regulates the goblet cell populations in the gut. goblet cells secrete mucin that are resistant to proteolysis and form a protective gel layer over the epithelial surface. intestinal bacteria and pathogens have to penetrate the mucus layer to reach the epithelial cells during infection. several microorganisms have developed diverse methods to degrade mucus either to aid in invasion or for uptake of mucus-derived nutrients (aristoteli & willcox, ; ohland & macnaughton, ) . additionally, several studies have also reported that the mucus layer is significantly thinner in areas of inflammation thus compromising the barrier and allowing for increased bacterial adherence and infiltration (swidsinski et al., ) . probiotics enhance the barrier by promoting mucus secretion. several lactobacillus species have increased mucin expression in in vitro cell culture models and have blocked pathogenic e. coli adherence and invasion (mack, ahrne, hyde, wei, & hollingsworth, ) . similarly, in vivo experiments in rats fed with probiotic cocktail vsl# for days demonstrated a significant increase in mucin secretion (caballero-franco, keller, de simone, & chadee, ) . thus, enhanced mucus production by probiotics in vitro and in vivo could be used as a protective strategy to augment the intestinal barrier function. once the bacteria come across the mucus layer, they find binding sites on the epithelium for colonization/attachment. pathogenic bacteria, however, proceed to penetrate or damage the epithelium to cause disease. the enterocytes express pattern recognition receptors such as toll-like receptors (tlrs) that sense the presence of conserved bacterial motifs and initiate cascade of proinflammatory signals (franchi, wamer, viani, & nunez, ). these receptors are present intracellularly and basolaterally on enterocytes. therefore, only after a pathogen breaches the barrier, they can come in contact with the receptors, which can differentiate between commensal and modern approaches in probiotics research to control foodborne pathogens pathogenic bacteria in the gut (franchi et al., ; franklin & latz, ) . enterocyte cell-cell adhesion is an essential component of the intestinal barrier. several components make up the cell-cell junctional complexes such as tight junctions, adherens junctions, gap junctions, and desmosomes (turner, ). these intercellular junctional complexes help maintain the epithelial barrier permeability and its integrity (groschwitz & hogan, ; marchiando, graham, & turner, ; ohland & macnaughton, ) . regulation of tight junctions and the associated epithelial permeability is essential to maintain the epithelial barrier function. however, pathogens have evolved different mechanisms to cross the epithelial barrier demonstrating the critical role the barrier plays in maintenance of homeostasis and prevention of inflammation (goto & kiyono, ) . chronic inflammation has been shown to be associated with altered tight junction barrier function that can promote pathogen access to the basolateral side of the epithelial barrier. several studies have demonstrated that pretreatment with probiotic bacteria can inhibit the loss of permeability associated with tight junction alteration caused by stress, infection, or proinflammatory cytokines (ait-belgnaoui et al., ; dahan et al., ; ewaschuk et al., ; sherman et al., ) . it has been shown that probiotics can directly alter epithelial barrier function by altering the structure and function of tight junctions. resta-lenert and barrett ( barrett ( , found that pretreatment with s. thermophilus and l. acidophilus independently decreased the permeability of cell monolayers formed by intestinal cells of ht- and caco- . this study also demonstrated that probiotics altered the expression of several proteins that are structural components of tight junctions thereby decreasing permeability. another study by yan et al. ( ) demonstrated that certain proteins produced by probiotic bacteria can interact with mammalian cell signaling proteins and lead to alteration in tight junction function and permeability. two such proteins produced by l. rhamnosus (p and p ) inhibited apoptosis in enterocytes and promoted survival. in addition, these proteins also altered structural components of the cell junctional complexes and enhanced barrier function (table . ). the intestine is the first site for foreign antigen encounter. therefore, the intestine has developed a tightly regulated mechanism to protect against pathogen invasion. the intestinal immune system is made up of several t seth, yan, polk, and rao ( ) lymphoid organs collectively called as the gut-associated lymphoid tissue. this is the largest collection of lymphoid tissues in the body and consists of the mesenteric lymph nodes, peyer's patches, isolated lymphoid follicles, lymphocytes, and dendritic cells (forchielli & walker, ; hakansson & molin, ; newberry & lorenz, ) . microbial colonization of the gut affects the composition of immune cell populations. several studies have demonstrated that bacterial colonization of the gut led to an increase in the number of intraepithelial lymphocytes, germinal centers with antibody-producing cells, and serum antibody concentrations (hakansson & molin, ) . this demonstrates the complex relationship that exists between the intestinal immune system and the gut microbiota (bron et al., ) . several in vivo and in vitro studies have demonstrated the immunostimulatory effects that probiotics have on the intestinal immune system (gill & prasad, ) . probiotics and probiotics-derived products are detected by the specialized membranous cells (m cells). antigens taken up by the m cells are processed by the antigen-processing cells (apcs) and presented to naïve t cells. the type of cytokine secreted, phenotype, and activation of apcs determine the lineage of the t cell that is produced, namely t helper (th ), t helper (th ), or the t regulatory (treg) cells. activation of th cells leads to production of ifn-g, tnf-a, and il- which leads to the development of cell-mediated and cytotoxic immunity. th cells activated by apcs mainly secrete il- , il- , and il- which promote antibody production. treg cells secrete il- and tgf-b, which downregulate activities of both th and th cells and help maintain homeostasis in the intestine (gill & prasad, ) . probiotics have been demonstrated to modulate the innate and acquired immune responses. the innate immune system forms the first line of defense against pathogens. the major components of the innate immune system include epithelial cells, phagocytic cells (monocytes, macrophages, neutrophils), and natural killer cells. several clinical trials have demonstrated that probiotics enhance the phagocytic activity of peripheral blood leukocytes (gill, ) . healthy subjects administered with l. johnsonii, l. rhamnosus, or b. lactis demonstrated an enhanced phagocytic capacity of peripheral blood leukocytes. the increase in phagocytic ability was also found to be dose dependent and lasted for several weeks after cessation of probiotic intake (gill & rutherfurd, b) . probiotics also can activate neutrophils through increased expression of phagocytosis receptors (pelto, isolauri, lilius, nuutila, & salminen, ) and an increased oxidative burst or microbicidal capacity of leukocytes (parra, de morentin, cobo, mateos, & martinez, ) . similarly, consumption of probiotics by healthy subjects led to an increase in the number and activity of nk cells and increased phagocytic action in animals fed with probiotic supplements (cross, ) . probiotic-mediated gut health is attributed to the stimulation of epithelial innate immunity. administration of probiotic mixture vsl# exhibited anti-inflammatory effect by stimulation of epithelial-derived tnf-a production and activation of nf-kb (pagnini et al., ) . besides modulating the innate immune response, probiotics have also been shown to augment the acquired immune responses through induction of cell-mediated immunity. intake of probiotics has led to an increase in antibody responses to natural infections and immunizations. a randomized trial in children with rotavirus administered with l. rhamnosus gg demonstrated an increase in specific mucosal and serum antibody responses (kaila et al., ) . similarly, administration of probiotics following immunization with salmonella vaccine in subjects led to a significantly higher specific serum iga and iga-mediated cell responses (linkamster, rochat, saudan, mignot, & aeschlimann, ) . these observations indicate that probiotic strains exhibit adjuvant properties increasing the efficacy of antibody production and immune responses to immunizations. probiotics mediate these effects through increased transport of antigenic materials across the gut mucosa and upregulation of antigen-presenting molecules and costimulatory molecules in immune cells (gill & prasad, ; hakansson & molin, ). an important component of the immune response mediators are cytokines. they are the largest and the most pleiotropic group of mediators. they are responsible for initiation, maintenance, and resolution of innate and acquired immune responses. several studies have demonstrated that ability of specific probiotic strains to enhance cytokine production and influence both innate and acquired immune responses. probiotic administration has been reported to enhance levels of ifn-g, ifn-a, and il- in healthy subjects (arunachalam, gill, & chandra, ) . long-term consumption of probiotic containing yogurt has been shown to increase production of il- b, il- , il- , ifn-g, il- , tnf-a, il- , il- , il- , and tgf-b by mononuclear cells and dendritic cells (cross, ; gill & guarner, ; niers et al., ) . several studies have provided direct evidence that the administration of specific probiotic strains can help in the prevention and treatment of gastrointestinal infections. studies conducted in animals have also demonstrated the ability of probiotics to enhance serum and mucosal antibodies, phagocytic cell function and modern approaches in probiotics research to control foodborne pathogens nk cell activity, and resistance to infection with pathogens (gill & prasad, ; hakansson & molin, ) (table . ). probiotics exert their antimicrobial and cytoprotective effects on host intestinal epithelium directly through the production of antimicrobial factors and indirectly through the increase in expression of host cell antimicrobial peptides, enhancement of barrier function, and immunomodulation. this section specifically discusses the antimicrobials produced by probiotics and their antagonistic effect on pathogens. the intestinal cells produce two main classes of antimicrobial peptides, namely, defensins and cathelicidins. cathelicidins are constitutively produced by intestinal epithelial cells to aid in host defense against pathogens (kelsall, ) . the only stimulus that appears to induce cathelicidin production is butyrate that is produced by intestinal flora (schauber et al., ) and also by probiotics (floch, ; sunkara et al., ) . shigella infection (dysentery) in rabbits was significantly reduced by feeding butyrate to induce cathelicidin production (raqib et al., ) . defensins produced by the intestinal cells can be classified as a-defensins and b-defensins. a-defensins are produced by small bowel paneth cells (hd- and hd- ), and b-defensins are expressed by epithelial cells throughout the intestine (kbd- through ). these defensins exhibit antimicrobial activity against a wide variety of bacteria, fungi, and viruses. defensins are constitutively produced in the intestines to keep pathogens from reaching the epithelium (wehkamp et al., ) . in vitro studies with probiotics have demonstrated that caco- cells upon stimulation by e. coli nissle, e. coli strain dsm , and several lactobacilli in the cocktail vsl# led to an increased expression and secretion of human b defensin- (hbd- ) (schlee et al., ; wehkamp et al., ) . the underlying mechanism through which probiotics stimulated hbd- production was elucidated using specific inhibitors. it was observed that hbd- secretion was enhanced through activation of map kinases (schlee et al., ) . besides activation of map kinases, it was also observed that e. coli nissle flagellin also stimulated hbd- production (schlee et al., ) . in addition to stimulating the production of host antimicrobial peptides, probiotics by themselves produce several antimicrobial compounds such as bactericidal peptides and scfas that can directly inactivate pathogens (dobson et al., ; hassan et al., ) . these secreted factors can be considered an integral part of the intestinal barrier. scfas include acetic and lactic acid which reduce the luminal ph resulting in the growth inhibition of some pathogens including enterohemorrhagic e. coli (ehec) and salmonella enterica serovar typhimurium in vitro. additionally, scfas produced by probiotics have also been shown to decrease shiga toxin gene expression by e. coli o :h (carey, kostrzynska, ojha, & thompson, ; fayol-messaoudi, berger, coconnier-polter, lievin-le moal, & servin, ) . furthermore, scfas can also disrupt the outer membrane of gram-negative pathogens such as ehec, pseudomonas aeruginosa, and s. typhimurium thereby inhibiting pathogen growth. increased permeabilization of the outer membrane of pathogens also potentiates the activity of other antimicrobial modern approaches in probiotics research to control foodborne pathogens molecules by aiding in their penetration of the cell wall (alakomi et al., ) . besides lactic acid, the predominant antimicrobial activity in lactobacilli is through the production of antimicrobial peptides, bacteriocins, and microcins (fayol-messaoudi et al., ) . bacteriocins and microcins are peptides with bactericidal or bacteriostatic activity produced in a strain-specific manner by probiotics (lievin-le moal & servin, ) . bacteriocins are peptides produced by gram-positive bacteria while microcins are produced by gramnegative bacteria. bacteriocins permeabilize the cytoplasmic membrane of bacteria leading to disruption of cell wall synthesis and formation of pores eventually leading to cell death. microcins, on the other hand, target the enzymes that are involved in dna or rna structure and synthesis or protein synthesis enzymes (duquesne, petit, peduzzi, & rebuffat, ) . collectively, these antimicrobial compounds protect the intestinal barrier by rapidly eliminating pathogens from the gut. bacteriocin abp- produced by l. salivarius has been shown to inhibit the growth of bacillus, listeria, staphylococcus, and enterococcus species. however, this bacteriocin did not affect the growth of most lactobacillus species thereby providing a selective advantage for intestinal colonization of probiotic and commensal bacteria (flynn et al., ) . similarly, lacticin produced by l. lactis has been shown to clostridium difficile as a potential therapy; however, this bacteriocin inhibited other resident bacteria including lactobacillus and bifidobacteria (rea et al., ) . in a separate study, banerjee, merkel, and bhunia ( ) showed that the soluble factors produced by probiotic lactobacillus delbrueckii subsp. bulgaricus were able to neutralize c. difficile toxin, thereby preventing cytotoxicity in a cell culture model. l. delbrueckii has been demonstrated to produce hydrogen peroxide that can inactivate pathogens by oxidation. l. delbrueckii also produces lactic acid, heat-sensitive, and heat-resistant bacteriocins. the heat-resistant bacteriocin has been observed to inhibit the growth of s. thermophilus (van de guchte, ehrlich, & maguin, ) . similarly, certain bifidobacterium strains produce lipophilic molecules that have been shown to inhibit the viability of e. coli, klebsiella pneumonia, yersinia pseudotuberculosis, s. aureus, and s. typhimurium. additionally, this antimicrobial compound has also been demonstrated to prevent invasion of caco- cells by s. typhimurium and can also kill intracellular s. typhimurium in a therapeutic model (lievin et al., ) . probiotics also exert their antimicrobial and cytoprotective effect by inhibiting pathogen adherence to the intestinal epithelium (collado, isolauri, salminen, & sanz, ; sherman, ossa, & johnson-henry, ). probiotics inhibit pathogen adherence by competing for the binding sites on the epithelial cells. pretreatment of hep- and t cell lines with l. rhamnosus and l. acidophilus significantly reduced the binding of enteropathogenic e. coli (epec) and ehec to the monolayers. in addition, probiotic pretreatment also reduced ehec-induced increase in permeability and helped maintain monolayer integrity (johnson-henry, donato, shen-tu, gordanpour, & sherman, ; sherman et al., ) . probiotic bacteria or its cell surface components also inhibited e. coli o :h adhesion to caco- cells (johnson-henry, hagen, gordonpour, tompkins, & sherman, ; medellin-pena & griffiths, ) or virulence-associated gene expression (medellin-pena, wang, johnson, anand, & griffiths, ) . lactobacillus strains have also been shown to compete directly with pathogens such as salmonella species, for binding sites on human mucins or caco- cell surfaces (gueimonde, jalonen, he, hiramatsu, & salminen, ) . besides physical displacement of pathogens, e. coli nissle has been shown to secrete a nonbacteriocin component that may act either on the pathogen or on the host cell to inhibit adherence of several pathogens (altenhoefer et al., ) . similar studies performed in a rat model suffering from chronic psychological stress that were administered with l. rhamnosus and l. helveticus demonstrated reduced commensal bacterial adherence and translocation (zareie et al., ) . thus, inhibition of pathogen adherence is another mechanism through which probiotic bacteria prevent intestinal infection. altogether, probiotic exerts its cytoprotective effect in the intestinal tract through their ability to enhance intestinal barrier function, immune modulation, toxin binding and neutralization, and inactivation and prevention of pathogen attachment (table . ). when a probiotic is administered orally, it first encounters various harsh environments in the gut. therefore, it is essential that probiotics survive in the intestinal tract in significant numbers to produce beneficial effects. survival of the probiotic is dependent on multiple factors such as stress response, metabolism, ph homeogenesis, cell wall maintenance, and fatty acid synthesis (breton et al., ; sanders, ) . gram-positive probiotic bacteria use several mechanisms to help them survive in the gut. these include proton pumps, amino acid decarboxylation, and electrogenic transport systems that aid in acid resistance, changes in the structure of their cell envelop, chaperones involved in repair of damaged proteins, and incremental expression of regulators promoting global gene responses (cotter & hill, ) . in addition, several probiotics have also been shown to modulate their gene expression in vitro under simulated gastrointestinal tract environment or in vivo in mouse model (bron, grangette, mercenier, de vos, & kleerebezem, ; bron, molenaar, vos, & kleerebezem, ) . once the probiotics survive the transit through stomach and intestine, it interacts with the major component of the gastrointestinal tract that is the resident commensal flora consisting of - of different microbial species (collado et al., ) . one of the earliest investigations into the interaction between probiotics and the resident microbiota by tannock et al. ( ) revealed that administration of l. rhamnosus dr resulted in modest fluctuations in resident lactobacillus and bifidobacterium numbers. most subjects ceased shedding the probiotic strain once the administration was stopped. however, one subject continued to shed the probiotic strain for over months after the test period, indicating that there are interhost variables such as bacterium-host interactions. probiotics are commonly applied in companion and farm animals as growth enhancers (patterson & burkholder, ; swanson & fahey, ) . it has been demonstrated that administration of a cocktail of lactobacilli, bifidobacteria, enterococci, and pediococci improved weight gain in broiler chickens associated with an increase in bifidobacterium, lactobacilli, and gram-positive cocci populations (mountzouris et al., ) . a similar study in piglets administered with enterococcus faecium strain reduced enterococcus faecalis population in the intestine of weaning pigs, but the total numbers of e. faecium remained unchanged, suggesting that the e. faecium strain introduced had displaced part of the same species (vahjen, taras, & simon, ) . another study performed in mice demonstrated that administration of l. casei and l. plantarum affected the overall diversity of the murine intestinal lactobacilli but not the overall bacterial community structure (fuentes et al., ) . furthermore, an increase in the population of lactobacilli related to the acidophilus complex was observed. use of animal models provides us with an insight into the interaction between probiotics and the resident flora; however, studies in humans are essential if that species is the desired host. few studies have been done on humans. alterations in gut microflora have been reported in humans with irritable bowel syndrome (ibs) (kassinen et al., ; satokari et al., ) , and administration of multispecies probiotic supplement was able to alleviate ibs with a stabilization of the gut microbiota over time (kajander et al., ) . besides treating inflammatory conditions, probiotics have also been efficacious in the treatment of infectious diarrhea (benchimol & mack, ; guandalini, a guandalini, , b . there are several ways in which probiotics can alter the microbiota, which include competition for nutrients, changes in microenvironment, production of growth substrates, direct antagonism, competitive exclusion, barrier function, immune stimulation, and reduction of inflammation (o'toole & cooney, ) . studies using transcriptional microarrays have shown that introducing a probiotic into the mouse gut changed the metabolic pathway of the endogenous microbiota (sonnenburg, chen, & gordon, ) . gnotobiotic mice colonized by bacteroides thetaiotaomicron were challenged with b. animalis or l. casei, resulting in shifts in gene expression pattern of b. thetaiotaomicron. many of the altered genes were found to be involved in carbohydrate metabolism (sonnenburg et al., ) . this suggests that probiotics alter resident microbiota population or gene expression through competition for substrate availability and by altering the dynamics of carbohydrate utilization (keeney & finlay, ) . probiotics can also alter the microenvironment of microbiota through a diverse range of metabolic pathway outcomes. colonization of germfree mice by microbiota from human baby exposed to lactobacilli strains resulted in microbiome modification measured by selected culture regimes (claus et al., ) . this was also associated with changes in fecal levels of choline, acetate, ethanol, unconjugated bile acids, and cecal concentrations of scfas. besides changing the microenvironment, several probiotic strains are also known to produce vitamins and growth factors. the enhanced availability of such growth factors can also modulate the diversity of intestinal microbiota. probiotic bacteria also impact the resident microbiota by direct antagonism. natural competition between probiotics and opportunistic pathogens could also be mediated through the production of bacteriocins that are exploited to modulate microbiota. the indirect mechanisms through which probiotics modulate inherent microbiota include enhancement of intestinal barrier function that can alter release rates of host-derived micronutrients and suppression of proinflammatory cytokines resulting in a reduction of gut inflammation (zyrek et al., ) . reduction in inflammation can alter the gut environment sufficiently to impact on the microbiota. additionally, administration of probiotics can also bolster the innate and acquired immune responses leading to subtle changes in the overall composition of the gut microbiota (gill & rutherfurd, a , c . there is adequate information in literature to indicate that administration of probiotics in high dosage impacts the diversity of gut microflora (preidis et al., ) . however, detailed studies are required to understand the interplay of diet, microbiota, and host factors in determining the outcomes to allow its manipulation. foodborne pathogens are a major concern worldwide due to increased mortality and morbidity (flint et al., ) . thus, various affordable intervention strategies including improved food-processing methods along with probiotic-based natural and functional food systems must be developed to protect people against foodborne infections. probiotics are live nonpathogenic microorganisms that are administered to maintain and to improve intestinal microbial balance and also to protect the consumers from untoward infection from pathogens. among the various etiologic agents, bacterial, viral, and mycotoxins are of major concerns. several studies have demonstrated the efficacy of either wild-type or recombinant probiotics against foodborne pathogens thereby help improving animal health and preventing foodborne infections (bhunia, ; dobson et al., ; salminen et al., ) . this section discusses the various probiotic-based intervention strategies in controlling foodborne pathogens. mechanism of probiotic-mediated antimicrobial action in gastrointestinal tract is illustrated in fig. . . (ferens & hovde, ; soon et al., ) . among the many intervention strategies investigated, probiotics are found to be effective. lema, williams, and rao ( ) demonstrated the efficacy of several probiotics including l. acidophilus, l. casei, l. fermentum, l. plantarum, and e. faecium in reducing e. coli o :h shedding by sheep. the microbial supplements were fed with freeze-dried fermentation products of the probiotics for a period of weeks. the animal group fed with the probiotic supplement showed a significantly lower numbers of e. coli o :h shedding in the feces and an increase in average daily weight gain and feed conversion. in addition to reducing fecal shedding, administration of bifidobacteria in mice showed a decrease in shiga toxin production. mice that were fed with bifidobacterium breve had a significant reduction in body weight loss and mortality compared to the control group demonstrating that b. breve is capable of protecting mice from e. coli o :h infection (asahara et al., ) . a study conducted by stephens, loneragan, karunasena, and brashears ( ) also demonstrated that direct-fed probiotics consisting of l. acidophilus at different doses led to a dose-dependent decrease in fecal shedding and presence on the hide. in addition to the use of lactobacillus, studies using nonpathogenic e. coli such as e. coli and nissle strains also inhibited bacterial growth and shiga toxin production by stec (reissbrodt et al., ) . studies using in vitro cell culture models have also been used to elucidate the efficacy of probiotics in controlling e. coli o :h (sherman et al., ) . intestinal cell monolayers (hep- and t ) were exposed to l. acidophilus r and l. rhamnosus r followed by infection with ehec or epec (e. coli o :h or e. coli o :h ), respectively. following infection, the adherence and cytotoxicity induced by ehec and epec were examined. exposure to the probiotic strains significantly reduced pathogen attachment to the monolayers. in addition, the probiotics also protected the monolayers from pathogen-induced loss of transepithelial resistance and tight junction integrity . besides reducing the adherence and cell injury, l. acidophilus cell-free spent media resulted in downregulation of several virulence genes involved in the colonization of ehec (medellin-pena et al., ) . similar studies conducted using e. coli o :h -infected balb/c mice demonstrated that preexposure to l. paracasei resulted in an upregulation of dendritic cells and helper t cell, antibody production, and downregulation of proinflammatory cytokines yielding enhanced protection of the intestinal integrity (tsai, cheng, & pan, ) . in addition to using wild-type probiotics, paton et al. ( ) generated a recombinant nonpathogenic e. coli carrying chimeric lipopolysaccharide capable of binding enterotoxin produced by enterotoxigenic e. coli (etec). the recombinant probiotic was able to neutralize % of enterotoxin in culture lysates of diverse etec strains . similarly, a recombinant l. casei strain carrying the k fimbriae from etec was able to reduce the attachment of etec to porcine intestinal brush border in a dose-dependent manner and to reduce infection in a mice model (wen et al., ) . these studies demonstrate that bioengineered probiotics can be used in the targeted control of specific enteropathogens. salmonella infection is the leading cause of foodborne gastroenteritis in humans worldwide and is commonly associated with raw or uncooked poultry and eggs, and fruits and vegetables (foley, lynne, & nayak, ; weill et al., ) . in the united states, nontyphoidal salmonella is responsible for % of total foodborne illness and % of hospitalizations (scallan et al., ) . salmonella utilizes numerous virulence factors to initiate infection and colonizes effectively on the epithelial cells in the gut (ahmer & gunn, ) . therefore, control strategies have to be applied along the food production chain to prevent the entry into human food supply (vandeplas, dubois dauphin, beckers, thonart, & théwis, ) . several studies have demonstrated that inoculation of cultures of one or several probiotic strains into broiler chickens may inhibit salmonella contamination (audisio, oliver, & apella, ; higgins et al., higgins et al., , van coillie et al., ) . neonatal broiler chicks were challenged with salmonella enteritidis and then challenged with lactobacillus probiotic culture at different doses orally. lactobacillus significantly reduced salmonella incidence in chicks by % (higgins et al., ) . similar study conducted in grower pigs challenged with s. enterica serotype typhimurium and administered with l. plantarum resulted in a reduction in fecal shedding of the pathogen. in addition, probiotic feeding also improved the performance of the pigs (gebru et al., ) . l. rhamnosus was also able to reduce epithelial cells stress induced by heat or cytotoxicity induced by s. typhimurium infection in a cultured epithelial cell model (burkholder & bhunia, ). commercially available probiotic cocktails were evaluated for their ability to inhibit salmonella colonization in neonatal broiler chickens and turkey poults. administration of probiotic cultures (floramax, ivs-wynco llc, springdale, ar) significantly reduced salmonella counts in the tonsils and ceca of chickens and poults (menconi et al., ) . furthermore, administration of lactobacillus reuteri strain that produced reuterin (bacteriocin) significantly reduced salmonella populations and increased the survival rate in chicks (zhang, li, & li, ) . probiotic bacillus subtilis dsm was also able to significantly reduce cecal loads of salmonella (knap et al., ) . an in vitro gut fermentation cellular model was used to evaluate the protective effect of probiotics against salmonella. addition of bacillus thermophilus rbl to salmonella in the reactors of the colonic fermentation model revealed a protective effect on epithelial integrity and increased the transepithelial resistance by % (zihler, gagnon, chassard, & lacroix, ) . probiotic has also been effective against antibiotic-resistant s. typhimurium dt . administration of l. casei shirota in mice challenged with s. typhimurium significantly reduced the pathogen growth and subsequent extraintestinal dissemination. the increase in concentration of organic acids and lowering of ph in the intestine were thought to reduce probiotic colonization, which correlated with the antimicrobial activity . the mechanism underlying the antibacterial effect of lactobacillus is multifactorial and involves lowering of the ph, production of lactic acid, and production of bacteriocins, nonbacteriocins, and nonlactic compounds (dobson et al., ) . it was observed that l. johnsonii la , l. rhamnosus gg, l. casei shirota yit , l. casei dn- , and l. rhamnosus gr dramatically reduced the viability of s. enterica serovar typhimurium through the production of nonlactic acid molecules, while the complete inhibition of salmonella growth was observed to be due to a ph-lowering effect (fayol-messaoudi et al., ) . in another study, soluble factors produced by b. bifidum also suppressed gene expressions in s. typhimurium that are required for adhesion and invasion for systemic spread (bayoumi & griffiths, ) . in vivo study using a mouse model demonstrated that continued administration of l. casei crl diminished salmonella counts in the intestine as well as its spread outside this organ. the probiotic-associated immunomodulatory effect involved both the innate and adaptive immune responses. probiotic administration reduced neutrophil infiltration, activated phagocytic activity, increased igaþ cells, and released siga specific to the pathogen in the intestinal fluid (de leblanc, castillo, & perdigon, ) . besides antimicrobial actions, probiotics also increased feed conversion and the performance in chickens and turkey poults. in poultry, campylobacter is another predominant bacterial pathogen that is responsible for numerous outbreaks. several probiotic strains have been evaluated for their efficacy in controlling campylobacter. human colon t and embryonic int- epithelial cells were pretreated with lactobacillus strains and then infected with c. jejuni. it was observed that l. helveticus r reduced c. jejuni invasion into t cells and int- cells by - % and %, respectively. in addition, l. helveticus r adhered efficiently to the epithelial cells suggesting that the inhibition of pathogen invasion could be due to competitive exclusion (wine, gareau, johnson-henry, & sherman, ) . in in vivo experiments conducted using a defined human microbiota-associated balb/c mice were orally infected with either c. jejuni or salmonella and then subsequently challenged with probiotic lactobacilli and bifidobacteria. probiotics were able to enhance colonization resistance by successfully excluding both pathogens from mice and also increased proliferation of lymphocytes against salmonella antigens. this study indicates that the probiotic administration reversed the immunosuppressive activity of salmonella in balb/c mice (wagner, johnson, & rubin, ). baffoni et al. ( ) evaluated the use of synbiotics to control c. jejuni in poultry. prebiotic galacto-oligosaccharide was used with probiotic b. longum subsp. longum pcb , and this synbiotic significantly reduced c. jejuni population in poultry feces thereby highlighting the positive effect of employing the synbiotic approach to reduce pathogen loads. among the various pathogens causing foodborne illness, hospitalizations, and deaths, listeria is responsible for % of the associated deaths (scallan et al., ) . upon arrival in the gastrointestinal tract, l. monocytogenes invades the intestinal epithelium and disseminates from the mesenteric lymph nodes to the spleen and liver (vazquez-boland et al., ) . during bacteremia, the organism reaches to liver, spleen, gall bladder, brain, and placenta. in the placenta, it can cause villous necrosis and microabscesses resulting in preterm abortion and infection of the fetus leading to stillbirth or neonatal listeriosis (bakardjiev, theriot, & portnoy, ; jiao et al., ) . several probiotics have been evaluated for their ability to control l. monocytogenes infection. dos santos et al. ( ) evaluated the monoassociation of l. delbrueckii in gnotobiotic mice and their effect on listeria colonization. administration of l. delbrueckii was capable of protecting the mice against death caused by l. monocytogenes and also led to a faster clearance of the bacteria from various organs such as the liver, spleen, peritoneal cavity, and the gut. additionally, probiotic-fed mice also showed an increase in ifn-g and il- signifying the role for effector molecules in probiotic-induced cytoprotection. similar results were obtained in a study where rats were fed with l. casei shirota strain. in addition to reducing listeria populations in the gut, spleen, liver, and feces, the probiotic strain also increased cellular immunity as determined by the delayed-type hypersensitivity response against heat-killed l. monocytogenes (de waard, garssen, bokken, & vos, ) . puertollano et al. ( ) evaluated the immunomodulatory effects of l. plantarum against l. monocytogenes infections in mice. administration of l. plantarum in mice infected with l. monocytogenes resulted in a reduction in the production of proinflammatory cytokines which circumvented listeria-mediated cytotoxicity. several species of lactobacillus and bifidobacterium were also able to inhibit l. monocytogenes infection in a cell culture model . later, the anti-infective property of probiotic l. salivarius was shown to be due to the production of a bacteriocin that protected mice from listeriosis when challenged with l. monocytogenes (corr, li, et al., ) . in addition to the use of wild-type probiotics, koo, amalaradjou, and bhunia ( ) generated a recombinant l. paracasei strain to control l. monocytogenes infection in a cell culture model. the recombinant probiotic was designed to express listeria adhesion protein, an essential virulence factor (burkholder & bhunia, ; jagadeesan et al., ) aiding listeria in transepithelial translocation during intestinal phase of infection. preexposure of intestinal monolayers to the recombinant probiotic followed by listeria infection led to a reduction in adhesion and paracellular translocation by % and %, respectively. the recombinant probiotic also protected the monolayers from listeria-mediated cytotoxicity and tight junction compromise. the use of such recombinant probiotics can help in the targeted elimination of enteric pathogens. probiotics have been evaluated for the control of c. perfringens in turkey poults (rahimi, kathariou, grimes, & siletzky, ) . administration of primalac, a commercial probiotic cocktail to turkey poults, significantly reduced cecal c. perfringens counts compared to the control. in another study, a recombinant pichia pastoris containing c. perfringens alpha toxin gene significantly increased weight gain, feed efficiency, sero conversion, and an absence of adverse reactions in histopathological evaluation of broiler chicks (gil de los santos, storch, fernandes, & gil-turnes, ) . probiotic bacteria have also been used to control vibrio parahaemolyticus attachment in cell culture model. l. plantarum as was shown to attach efficiently to ht- cells and reduce v. parahaemolyticus attachment by competitive exclusion and displacement (satish kumar et al., ) . a recombinant probiotic expressing a chimeric lipopolysaccharide was able to bind to cholera toxin and protected infant mice challenged with virulent v. cholera (focareta, paton, morona, cook, & paton, ) . probiotics have also been shown to be effective against other pathogens such as shigella sonnei, s. aureus, e. faecalis, proteus mirabilis, and p. aeruginosa (varma, dinesh, menon, & biswas, ) . probiotics have also been used to control viral infections. gnotobiotic pigs fed with l. acidophilus and l. reuteri enhanced ifn-g and il- responses in serum and decreased human rotavirus infection (wen et al., ) . protection against rotavirus-induced diarrhea was also induced by administration of l. paracasei expressing variable domain of llama heavy-chain antibody fragments (pant et al., ) . another major enteric virus responsible for % of foodborne illness is norovirus (marshall & bruggink, ; mattison, ) . probiotic-fermented milk containing l. casei shirota strain has been evaluated for its efficacy in controlling norovirus gastroenteritis in a health service facility. a total of people were enrolled in the study. intake of probiotic-fermented milk by the treatment group resulted in a reduction in the mean duration of fever after the onset of gastroenteritis (nagata et al., ) . wang, yu, gao, and yang ( ) generated a recombinant lactobacillus strain expressing the hemagglutinin of the avian influenza virus h n . oral administration of this recombinant probiotic balb/c mice triggered both mucosal and systemic immune responses. there was an increase in anti-ha iga and anti-ha igg levels with an associated increase in il- production. recombinant l. casei expressing human lactoferrin was shown to exhibit antibacterial and antiviral activities in in vitro and in vivo models (chen et al., ) . receptor mimetics has been used as a strategy to control foodborne pathogens. many enteric pathogens recognize oligosaccharides expressed on host cells as receptors for toxins and adhesion factors. this interaction between the ligand and the receptor is essential for the initiation of infection process. this critical step in the infection process can be adapted to develop intervention strategies. this involves the expression of molecular mimics of host oligosaccharides in probiotic bacteria to control enteric pathogens (paton, morona, & paton, ) . such designer probiotics can bind bacterial toxins to competitively inhibit pathogen binding to its receptor in the gut. such receptor mimic probiotics have been developed against stec and etec (paton et al., , cholera (focareta et al., ) , and c. jejuni (yuki et al., ) . in addition to the enteric pathogens, probiotics have also been developed against many other pathogens and infectious agents summarized in table . . fungal pathogens could be controlled by inactivation of their toxins (mycotoxins) that cause foodborne infections. mycotoxins are of clinical importance due to their ability to induce acute and chronic toxicity in the host (kabak, dobson, & var, ) . these mycotoxins can be mutagenic, carcinogenic, teratogenic, and immunosuppressive in nature (salminen et al., ) . probiotics are known to bind and neutralize toxins. this attribute of the probiotic has been exploited in the reduction of dietary exposure to fungal toxins. specific strains of probiotics have proved to be highly effective in removing mycotoxins in model systems. armando et al. ( ) have demonstrated that saccharomyces cerevisiae strains can adsorb ochratoxin and zearalenone in a mycotoxin binding assay under simulated gastrointestinal conditions. it was also observed that toxin binding was a function of cell wall thickness and binding occurs through physical adsorption. similar studies conducted using a food system (grape juice) also demonstrated the ability of saccharomyces strains to adsorb ochratoxin (bejaoui, mathieu, taillandier, & lebrihi, ) . rats fed with aflatoxin-containing diet showed that administration of lactobacilli casei and l. reuteri significantly reduced the oxidative stress induced by aflatoxins in comparison to the control group that did not receive any probiotics (hathout et al., ) . similar experiment conducted in rats fed with different doses of aflatoxin (afb- ) followed by administration with l. reuteri demonstrated significantly lower levels of afb- in the intestine than in the control group. this study demonstrated that probiotic strains can bind to toxin in the intestine and act as a barrier to toxin-induced cell cytotoxicity (hernandez-mendoza, gonzález-córdova, vallejo-cordoba, & garcia, ). besides aflatoxin, ochratoxin, and zearalenone, other fungal toxins such as patulin and fumonisin can also be neutralized by probiotics. buccato et al. ( ) adapted from wells and mercenier ( ) . several probiotic strains such as propionibacterium, l. rhamnosus gg, l. plantarum, l. casei bacillus species, and e. faecium were demonstrated to bind and neutralize mycotoxins (danicke & doll, ; hernandez-mendoza, garcia, & steele, ; hernandez-mendoza et al., ; khanafari, soudi, miraboulfathi, & osboo, ; niderkorn, morgavi, aboab, lemaire, & boudra, ; topcu, bulat, wishah, & boyaci, ) . when using probiotics for control of infections, it is essential that they survive the acidic gastric ph, the intestinal enzymes, bile, and alkaline ph to be able to exert their protective effect. this is critical when using recombinant probiotic to ensure production of the foreign protein at the target site. in general, probiotic supplements have been delivered as capsules, tablets, or powders. the choice of delivery matrix is also important to ensure probiotics activity and viability (hajela, nair, abraham, & ganguly, ) . probiotic encapsulation technology (pet) has emerged as the principal method for delivery of probiotics. a wide range of microbes have been immobilized using semipermeable and biocompatible materials that modulate their delivery and release. probiotics are either entrapped or encapsulated. entrapment refers to the trapping of material within or throughout a matrix. encapsulation involves the formation of a continuous coating around the inner matrix that is present within the capsule wall as a core of the encapsulated material. encapsulation helps to stabilize cells, enhancing their viability and stability during production, storage, and handling. pet helps in the controlled and continuous delivery of probiotic cells in the gut. the biomaterials used in the encapsulation of probiotics include alginate, carrageenan, gelatin, chitosan, whey proteins, cellulose acetate phthalate, locust bean gum, and starches (anal & singh, ; chandramouli, kailasapathy, peiris, & jones, ; krasaekoopt, bhandari, & deeth, ) . alginate gels are insoluble in acidic media and hence protect probiotics from gastric acidity (gbassi, vandamme, ennahar, & marchioni, ) . carrageenan is used for its ability to form gels that can entrap the probiotics bacteria (doleyres, fliss, & lacroix, ; doleyres, paquin, leroy, & lacroix, ) . cellulose acetate phthalate is insoluble at ph below but soluble when ph is greater than . this is especially advantageous for the stability of encapsulated probiotic in the stomach and for controlled release in the intestine (anal & singh, ) . microencapsulation of probiotics is performed by freeze-drying, extrusion, or emulsification. freeze-drying results in the formation of concentrated dry powder that has longer shelf life but do not protect probiotics from gastric fluid or bile (rokka & rantamaki, ) . it is essential to provide probiotics with physical barriers to protect them from the adverse conditions encountered in the gut. for this purpose, extrusion and emulsification techniques intended to develop gel beads or capsules were applied (lacroix, paquin, & arnaud, ) . extrusion technique involves the mixture of probiotics with the hydrocolloid finally resulting in the formation of gelled droplets called beads (gouin, ) . emulsification involves the mixture of probiotics with a continuous phase resulting in a water-in-oil emulsion. this technique has been used to develop encapsulated probiotics (shima, morita, yamashita, & adachi, ) . emulsification results in the formation of oily or aqueous droplets called capsules (gbassi & vandamme, ) . alginate microcapsules coated with chitosan were evaluated for their ability to increase gastrointestinal viability of b. breve. chitosan stabilizes the alginate microcapsules at ph above . coating with chitosan also increased the survival of b. breve in simulated gastric fluid and prolonged its release upon exposure to intestinal ph (cook, tzortzis, charalampopoulos, & khutoryanskiy, ) . calinescu and mateescu ( ) developed a tablet dosage system based on carboxylated methyl high amylose starch (cm-has) and chitosan excipient for probiotic colon delivery. cm-has ensures protection in the gastric ph while chitosan helps with slow release of the probiotic in the intestine. this encapsulation of l. rhamnosus resulted in an improved percentage of delivered bacteria in simulated intestinal conditions. to enhance the slow and sustained release of probiotics in the gut, a coacervate extended-release microparticulate delivery system was developed (alli, ) . core mucoadhesive l. rhamnosus was prepared using hypermellose and subsequent enteric coating with hypermellose phthalate. hypermellose has good mucoadhesive and release rate controlling properties, which are preferred in mucoadhesive formulations. use of such microparticles in simulated intestinal conditions exhibited appreciable mucoadhesion compared to the freeze-dried cultures. in addition to the use of the above-mentioned matrices, dna-based gels have also been evaluated as a vehicle for oral delivery of probiotics. dnabased gels were initially prepared to protect lactic acid bacteria from extreme conditions in commercial yogurts (jonganurakkun, liu, nodasaka, nomizu, & nishi, ; jonganurakkun, nodasaka, sakairi, & nishi, ) . the dna forms complexes with gelling agents such as gelatin and k-carrageenan at low ph. this resulted in the formation of two kinds of gel: hydrogels and complex gels. in a hydrogel, the bacteria are fixed in the gel when the yogurt containing bacteria is added to the precooled mixture of dna and gelling agent. in a complex gel, a mixture of yogurt and cacao oil is added to the gel solution containing the dna and the gelling agent to form the encapsulated probiotic. it was seen that complex gel was able to protect the probiotic from adverse simulated gastric conditions. hydrogels in addition to protecting from acidic conditions were also more stable during c storage which corresponds to the storage temperature at which the product would be maintained for prolonged storage (jonganurakkun et al., ) . thus, dna-based hydrogels offer a potential delivery system for oral administration of probiotics. probiotics have been extensively studied in in vitro and in vivo models. ample evidence is documented to support the potential application of probiotics for the prevention and treatment of enteric infections. health benefits of probiotics include the prevention of diarrhea, atopic eczema, antibiotic-associated diarrhea, traveler's diarrhea, prevention of dental carries, colorectal cancers, and treatment of ibd. however, as with any potential intervention strategies, probiotics also have a safety concern. therefore, it is essential that toxic and metabolic effects of probiotics on humans are assessed for patient safety, especially for critically ill or immunocompromised patients. significant challenges still exist in the effective application of probiotics in pathogen control. future studies are essential to define optimal doses and their correct combinations of various probiotic species based on their molecular mode of antimicrobial action. there is also a need for improvement of production techniques to understand and develop better approaches to probiotic delivery and bioavailability in the gut as we move from theoretical benefits to clinical application. now with the possibility to express different molecules in probiotic bacteria, such as enzymes, cytokines, receptor mimics, adhesion molecules, antibodies, and host targeting molecules, future research can help optimize applications and develop biologically contained strains to support clinical trials. thus, probiotic bacteria can be used to control and prevent pathogen colonization in the food animals to improve food safety and a realistic therapeutic option in humans to control enteric pathogens. interaction of salmonella spp. with the intestinal microbiota lactobacillus farciminis treatment suppresses stress induced visceral 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campylobacter jejuni invasion of human intestinal epithelial cells soluble proteins produced by probiotic bacteria regulate intestinal epithelial cell survival and growth probiotics and immune health. current opinion in gastroenterology carbohydrate mimicry between human ganglioside gm and campylobacter jejuni lipooligosaccharide causes guillain-barré syndrome probiotics prevent bacterial translocation and improve intestinal barrier function in rats following chronic psychological stress lactobacillus reuteri atcc and l display probiotic potential in vitro and protect against salmonella-induced pullorum disease in a chick model of infection protective effect of probiotics on salmonella infectivity assessed with combined in vitro gut fermentation-cellular models molecular mechanisms underlying the probiotic effects of escherichia coli nissle involve zo- and pkc zeta redistribution resulting in tight junction and epithelial barrier repair key: cord- -uw cfc authors: peacock, sharon j; weinstock, george m title: microbial sequencing to improve individual and population health date: - - journal: genome med doi: . /s - - - sha: doc_id: cord_uid: uw cfc recent advances in sequencing technologies are changing the face of infectious disease investigation and control. personalized anti-infective therapies and surveillance of emergent pathogen outbreaks are just two examples of the potential benefits of merging the fields of genomics and infectious diseases. this genome biology and genome medicine collaborative special issue on the genomics of infectious diseases is very timely. vaccination, access to clean water, and antimicrobial drugs have all changed the relationship between humans and pathogens, resulting in a marked increase in life expectancy. yet, infectious diseases continue to take their toll on human health worldwide, and events such as the recent ebola outbreak in west africa serve as a sharp reminder of how fragile any success is in the control of pathogens. a more insidious but pervasive threat to human health is the emergence and dissemination of antimicrobial resistance among numerous pathogens, paralleled by a decline in antimicrobial drug discovery. advances in sequencing technologies have resulted in the availability of instruments that can be operated in a clinical environment, together with highthroughput platforms that can be used to define pathogens at the population level. these technologies have numerous potential applications for the control of infectious diseases. sequencing will bring improvements in the detection and control of outbreaks associated with multidrugresistant and other pathogens in hospitals and the community [ ] . confirmation of an outbreak could lead to earlier implementation of interventions that bring the outbreak to a close [ ] . conversely, excluding an outbreak the wellcome trust sanger institute, wellcome trust genome campus, hinxton, cambridge cb sa, uk full list of author information is available at the end of the article with confidence will reduce unnecessary infection control interventions [ ] . pathogen sequencing will be used to tailor individual patient prescribing. capillary sequencing of the human immunodeficiency virus (hiv) is already used to guide the treatment of patients who are hiv positive, but newer sequencing technologies will bring the added benefit of detecting resistant variants present as a minority of the hiv population in a given individual. in tuberculosis (tb), sequencing technologies will be used to predict antimicrobial resistance of the causative agent, mycobacterium tuberculosis [ ] . this will bring the greatest benefit to patients with multidrug-resistant and extensively drug-resistant tb (against which first-and second-line drugs are not effective), because conventional testing of second-line drugs is lengthy. accurate prescribing could lead to more rapid resolution of infection and reduced risk of onward transmission. genome sequencing also defines transmission of m. tuberculosis between individuals with greater resolution and certainty than was previously possible [ ] . passive surveillance using sequence data generated for clinical use would provide an overview of the emergence and spread of antimicrobial resistance. active genomic surveillance of key human pathogens would provide an early warning system for outbreaks, inform vaccine strategies through tracking of vaccine escape, and detect the emergence of new clones that harbor known or novel virulence determinants. sequencing is being used to identify reservoirs of antimicrobial-resistance genes in hospitals, other healthcare facilities, the community, and livestock farming, as well as common transmission pathways between them. finding pinch-points to stop transmission between reservoirs could limit the dissemination of antimicrobial resistance. sequencing also provides insights into the emergence of infectious diseases. for example, reconstruction of the early dynamics of the hiv pandemic using sequence data and statistical approaches identified kinshasa in the s as the focus of early transmission and the source of pre- pandemic viruses elsewhere [ ] . sequencing of the more recently emerged middle east respiratory syndrome coronavirus and comparison of sequence data for isolates from humans and dromedary camels has been cited as evidence for the role of camels as a reservoir [ ] . sequencing also has a role in drug discovery pathways, the laboratory evaluation of lead compounds, and the clinical phases of drug evaluation. for example, in , in the first published use of pyrosequencing, the f subunit of atp synthase was identified as the target of bedaquiline [ ] . bedaquiline subsequently became the first representative of the only novel class of anti-tb agents to be approved in years. sequencing of m. tuberculosis during clinical trials can be used to distinguish exogenous re-infection from a relapse of the primary infection, which is crucial to assess the efficacy of study drugs. sequencing technologies will also underpin clinical trials evaluating the effect of a therapeutic alteration of the microbiome in a range of conditions. the benefit derived from duodenal infusion of donor feces in patients with recurrent clostridium difficile infection provides proof-of-principle for clinical utility [ ] . extending this to other diseases will need to be supported by detailed genomic analyses of the human microbiota, together with a better understanding of the interactions between the native or medically altered microbiome and host immunity. several challenges remain before microbial sequencing becomes routine for diagnostic and public health microbiology laboratories. a suite of software tools will be required to convert sequence data into a format that is relevant and useful for clinicians and infection control teams. new methods to handle and process everexpanding pathogen-specific microbial genome databases will also be needed, including global and region-specific listings of gene mutations associated with drug resistance. it is also essential that existing mechanisms for the development of standard operating procedures and accreditation of laboratory methods be applied to microbial sequencing. working within a tightly controlled diagnostic laboratory will reduce errors (for example, through sample tracking) and allow data to be handled within an existing framework that protects patient confidentiality. further technological advances are also required to reduce the turnaround time between taking a clinical sample and generating sequence data. refinements such as extracting dna directly from a bacterial colony on a culture plate can reduce the processing time by up to a day [ ] . however, the need to culture the sample to obtain a pure growth of bacteria from which to purify dna prior to sequencing, rather than performing direct sequencing on the sample, means that timelines are still tied to bacteriology methods that were developed more than a hundred years ago. regardless, the enthusiasm for direct sequencing of clinical samples should be tempered by the probable reality of doing so. most samples sent to a diagnostic laboratory are currently reported out as 'no growth' or, through the use of selective culture media that target specific pathogens, 'no significant growth'. in a brave new world where all samples are sequenced as the primary method for pathogen detection, it may prove the case that the majority of samples will be sequence-positive. re-defining what data can be disregarded and what might represent new and important findings will take at least a generation of microbiologists to resolve. routine use of microbial whole genome sequencing in diagnostic and public health microbiology whole-genome sequencing for analysis of an outbreak of meticillin-resistant staphylococcus aureus: a descriptive study zero tolerance for healthcare-associated mrsa bacteraemia: is it realistic? whole-genome sequencing for rapid susceptibility testing of m. tuberculosis whole-genome sequencing and social-network analysis of a tuberculosis outbreak lemey p: hiv epidemiology. the early spread and epidemic ignition of hiv- in human populations middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation a diarylquinoline drug active on the atp synthase of mycobacterium tuberculosis duodenal infusion of donor feces for recurrent clostridium difficile rapid single-colony whole-genome sequencing of bacterial pathogens cite this article as: peacock and weinstock: microbial sequencing to improve individual and population health key: cord- -q w fb i authors: ewald, paul w. title: evolution of virulence date: - - journal: infect dis clin north am doi: . /s - ( ) - sha: doc_id: cord_uid: q w fb i at the close of the th century, the germ theory had generated a new understanding of the causes of acute infectious diseases and revealed new directions for study. this understanding contributed to the greatest improvements in health in the history of medicine. at the end of the th century, the second stage of this disciplinary development is occurring. the old germ theory is being expanded into a new germ theory, which, by integrated the full spectrum of biologic disciplines. this new germ theory is emphasizing how environments and human activities influence the characteristics of infectious agents and the broader role of infection as a cause of chronic diseases. human history cannot be understood well without understanding the causes and consequences of human disease. this fact has become amply apparent over the past few decades as the impacts of infectious diseases have been studied in the context of war, colonization, and competition [ ] [ ] [ ] [ ] [ ] . it is much less widely appreciated that the reverse is also true. historical studies of infectious diseases may help guide modern health sciences to recognize options for controlling diseases of the present and future. ecologic and evolutionary perspectives are enmeshed with the historical perspective of infectious diseases, because infectious agents spread and evolve over times scales that accord with historical events. they may influence historical events and may be influenced by such events. the influence of historical events on the evolution of pathogens largely has been neglected until the past quarter century. it has become clear that activities that were undertaken for one purpose can have unforeseen effects on the evolution of important characteristics of pathogens, such as virulence (which is defined broadly here to mean the degree of harm imposed on the host). an understanding of these evolutionary effects helps in understanding why some pathogens cause more harm than others, the environmental circumstances that permit this harm, and, most importantly for the future, the human activities that can ameliorate or prevent this harm. for most of the th century, the prevailing dogma was that disease organisms eventually should evolve toward benign coexistence with their hosts; harmful diseases were interpreted as a transitory state of maladaptation [ ] [ ] . this belief has not been useful for ameliorating the suffering caused by infectious diseases, because it suggests that the occurrence of severe disease is bad luck and that not much can be done to control this evolutionary process. this view arose more from assumptions about the harmony of nature than from rigorous application of evolutionary principles. specifically, it failed to cast the problem in the context of natural selection. rather than asking whether harmful or mild variants would win out in competition with each other over the short run, the focus was on what was stable over the long run. natural selection is powerless to favor long-term stability if the variants that win in the short term destabilize the system. natural selection may favor the evolution of extreme harmfulness if the host exploitation that causes this harm enhances the competitive success of the harmful variants over benign variants in the short run. if predator-like variants of a pathogen population out-produce and out-transmit benign variants, benign coexistence may be precluded. instead of generating longterm stability, the evolutionary conflict of interest between a predator-like pathogen and its host generates an evolutionary arms race in which pathogen and host each evolve characteristics that give them a leg up on the other, shifting the level of host exploitation closer to the optimum for the pathogen or that of the host. before the last decades of the th century, a few authors expressed reservations about the traditional dogma [ ] [ ] [ ] , but they largely were ignored. over the past quarter century, however, the evolution of virulence has been broadly investigated by a theoretical framework that is based on the principles of natural selection [ ] [ ] [ ] [ ] [ ] . this framework offers explanations for the broad range of virulence found among host parasite relationships and offers possibilities for virulence management (ie, the control of diseases by controlling the evolution of virulence) [ ] . the evolutionary framework also provides insight into the true scope of infectious causation of chronic disease and a sense of which diseases can be prevented or cured by developing disease-control strategies, such as vaccines and antibiotics. for acute infectious diseases, theory about the evolution of virulence focuses on the negative effects of virulence on transmission of pathogens between hosts. much of the variation in these negative effects of virulence depends on whether pathogens can be transmitted from hosts that have become immobilized by the infections. pathogens that can be transmitted readily from immobile hosts should be molded by natural selection to exploit hosts severely and to be highly virulent [ ] . the reason is simple. variants that exploit severely gain the competitive advantages of this exploitation while incurring little, if any, competitive cost from the illness that their exploitation generates, because they still can be transmitted from hosts that have the severe, immobilizing illness. specific applications of this idea are presented. parasites transmitted by biting arthropods can be transmitted effectively from immobilized hosts and therefore should evolve to a higher level of virulence than directly transmitted parasites. a comparison of viral, bacterial, and protozoal agents of human diseases showed that vector-borne pathogens are more lethal on a per-infection basis than are directly transmitted pathogens [ ] . the association between vector-borne transmission and virulence explains why diseases such as malaria, yellow fever, dengue, sleeping sickness, and visceral leishmaniasis are so severe, whereas most of the respiratory-tract pathogens of humans are relatively benign. a follow-up comparison of vector-borne pathogens indicates that this greater virulence of vector-borne pathogens is related to adaptation to the conditions of vector-borne transmission rather than to some spurious correlate of vector-borne transmission, such as injection of a pathogen below the surface of the skin. this follow-up comparison used historical data to assess the virulence of particular vector-borne pathogens in humans in relation to the degree to which the pathogen had evolved in response to vector-borne transmission between humans. specifically, it compared the virulence of vector-borne pathogens that had just been transmitted to humans with the virulence of the same kind of vector-borne pathogen that had been cycling extensively in humans and should be better adapted to vector-borne transmission between humans. as expected from evolutionary theory, the pathogens that had been cycling in humans were more severe in humans than those that recently had been introduced to humans from some other vertebrate host [ ] . the yellow fever virus, for example, was less deadly in humans just after it entered the human population than it was in outbreaks that involved extensive cycling of transmission between mosquitoes and humans. evolutionary management of the virulence of vector-borne diseases requires interventions that elevate the immobilization of hosts more costly to the infecting pathogens. logic dictates that this goal can be accomplished by mosquito proofing of dwellings. people who are immobilized by illness are more likely to be at home or in a hospital than people who do not feel ill; transmission from homes and hospitals therefore should tend to involve relatively virulent variants. when such dewllings are mosquito-proof, however, vectors cannot gain access to the severely ill people who are incapacitated inside these dewllings. the vectors will instead transmit pathogens from those infected people who feel healthy enough to get up out of bed and walk outdoors. these pathogens should tend to be relatively benign. the vector proofing of dwellings therefore should favor transmission of the benign strains from people in the outside environment instead of transmission of the more virulent strains that infect the bed-ridden. this favoring of benign strains through the mosquito proofing of dewllings would be manifested as an evolutionary decline in virulence. though this idea has not been tested directly, geographic variations in virulence and the demonstrated effect of vector proofing of houses on disease transmission suggests that it will work [ ] . strains of malaria are mild where the potential for vector-borne transmission is low and sporadic [ ] [ ] , and mosquito proofing of houses has had a strong inhibitory effect on transmission of plasmodia [ ] . as with vector-borne pathogens, evolutionary theory predicts that waterborne pathogens should evolve to relatively high levels of virulence, because they can be transmitted from immobilized people. reliance on the mobility of infected hosts is low for water-borne pathogens, because the wastedisposal activities of attendants and the movement of water can contaminate sources of drinking water. the lethality of diarrheal bacteria is correlated positively with the extent to which they are water borne [ ] . geographic comparisons also support the idea that the virulence of diarrheal diseases is linked to water-borne transmission; among shigella, for example, severe strains have been disproportionately common where the potential for waterborne transmission is high [ ] . changes in such ratios over time support the idea that the virulence of diarrheal pathogens couuld be managed evolutionary by blocking waterborne transmission. diarrheal pathogens, such as shigella and vibrio cholerae, evolved toward lower virulence as the water supplies were cleaned up in north america, south america, europe, and asia. [ , ] . like vector-borne and water-borne pathogens, pathogens acquired while in the hospital can be transmitted from immobile hosts. in this case, the transporting is done on the hands of doctors, nurses, and other attendants and the objects they touch. such attendant-borne transmission is the major route for most serious hospital-acquired pathogens, such as the staphylococci, streptococci, enterococci, pseudomonas, and clostridium difficile [ ] . attendants usually do not get infected, partly because they are less vulnerable than their patients, they wash their hands before leaving the environment, and they may have generated some immunity to the hospital organisms. although the evolution of virulence in hospitals has been studied only superficially, the available information supports the idea that cycling in hospitals makes pathogens more harmful. a review of all hospital outbreaks of escherichia coli-related infection that occurred in the united states and united kingdom before the effective use of antibiotics assessed whether increased attendant-borne transmission was associated with increased lethality [ ] . a statistically significant association was found; strains that had been circulating for a week rarely caused death, but strains that had circulated for many months killed about in infants [ ] . the implications for virulence management of attendant-borne transmission in hospitals mirror the implications for vector-borne and waterborne pathogens. if the attendant-borne transmission is blocked through proper hand washing and glove use, strains circulating in hospitals increasingly are represented by strains that are brought from the outside community; such community strains depend on host mobility for transmission and tend to be less virulent than strains that have been cycling in hospital environments. pathogens that are durable in the external environment also can be transmitted from very ill people, because such pathogens can reach susceptible individuals by relying on the mobility of susceptible, rather than infected, people. the high durability of smallpox, mycobacterium tuberculosis, and corynebacterium diphtheriae in the external environment helps explain why these pathogens have been scourges throughout history. the agent of plague, yersinia pestis, can be transmitted as a durable pathogen by the respiratory route and as a vector-borne pathogen. a re-analysis of plague from evolutionary and historical perspectives suggests that both routes were important in the black death of the th century [ ] . perhaps it was this combination that led this outbreak of y pestis to be so unusually destructive. as is the case with the preceding categories, virulence management of sitand-wait pathogens requires selective blocking of transmission from immobile individuals. in this case, however, the intervention requires selective inhibition of durable pathogens from the chain of transmission; this goal could be accomplished by requiring frequent air exchanges and decontamination of surfaces. vaccination programs generally do not eradicate target pathogens; at the global level, only the smallpox vaccine has eradicated its target from the human population. when eradication does not occur, policymakers must consider effects of vaccination not only on the frequency of infection but also on the virulence of the pathogens that are left in the wake of the vaccination program. vaccination programs that cause evolutionary reductions in the virulence tend to be successful because they leave behind mild variants that may circulate and protect unvaccinated individuals against virulent variants that might remain in the population, arise by mutation, or enter from other areas. the circulating, benign strains may protect unvaccinated individuals and the population as a whole against the spread of harmful strains. this process of virulence management can be accomplished by a virulence antigen strategy. this strategy dictates that vaccines should be based on virulence antigens (ie, antigens that make mild but transmissible organisms harmful) [ , ] . the virulence antigen strategy differs from the traditional approach to vaccine development, which selects antigens on the basis of the protection conferred to study subjects regardless of whether the antigens are virulence antigens. by selectively suppressing the virulent variants, virulence antigen vaccines force the target pathogens to evolve toward benignity. the virulence-antigen strategy is well illustrated by the diphtheria toxoid vaccine, which is based on a modified diphtheria toxin. the intact toxin liberates nutrients to the bacterium by killing nearby human cells. the immunologic response to the toxoid vaccine neutralizes the toxin and causes the toxin to be a net drain on the bacterium's nutrient budget. toxinless c diphtheriae still can infect and be transmitted from people [ ] ; the toxin therefore qualifies as a virulence antigen, because it makes viable benign pathogens harmful. when vaccination prevents the negative effects of the toxin, the toxinless strains should have a competitive advantage over the toxigenic strains, because the toxinless strains do not waste valuable resources by producing an ineffective toxin. toxinless strains therefore should increase in frequency relative to toxigenic-strains wherever toxoid vaccines have been administered extensively. this transition is confirmed by the historical data [ ] [ ] [ ] [ ] . the most detailed data set came from the vaccination program administered in romania from through . as the acquired immunity rose to %, the percentage of isolates that produced toxin dropped from % to %, and diphtheria vanished [ ] . if all of the costs of vaccine development and administration could be tallied and health benefits per dollar spent calculated, the control of diphtheria by the toxoid vaccine surely would be one of the most costeffective vaccine programs in history. only the smallpox vaccination program would rank higher, because it eradicated smallpox, which allowed for the abandonment of continuous vaccination. theory about the evolution of virulence is fundamentally different for chronic infectious diseases than for acute infectious diseases. this difference is well illustrated by the virulence of sexually transmitted diseases, which are intermediate between acute and chronic infectious diseases. syphilis has an acute phase that is characterized by a primary chancre, an early chronic phase that is characterized by a pervasive rash, and a late chronic phase (tertiary syphilis), which may involve mental illness, tumors, paralysis, meningitis, tremors, and cardiovascular disease. in other sexually transmitted diseases, the acute phase is inconspicuous or entirely lacking. hiv type (hiv- ) causes a mild flu-like illness within about a month of the onset of infection but is generally lethal in its chronic phase. the human tlymphotropic virus type causes asymptomatic acute infection soon after the onset of infection but causes paralysis, leukemia, or lymphoma decades later in a minority of infected people. because infected hosts generally must be mobile to engage in sexual activity, natural selection favors benignity of sexually transmitted pathogens over the short run. to be successful in the context of natural selection, however, sexually transmitted parasites must be infectious over relatively long periods of time, because options for sexual transmission of a given infection are generally less frequent than opportunities for transmission of typical agents of acute infectious diseases-a person generally has sex with many fewer people per week than he can sneeze on. natural selection therefore favors long-term persistence and contagiousness of sexually transmitted pathogens within each host. accordingly, most sexually transmitted pathogens are characterized by adaptations that allow the pathogen to evade the immune system to persist in and be transmitted from the body. although sexually transmitted pathogens are molded by natural selection to be benign over the short run, this long-term persistence within hosts raises the possibility of long-term damage, even though there is low probability of severe damage during any small period of time during the first years of infection. according to this framework, the evolution of virulence depends on the potential for sexual transmission in the host population. if the population is characterized by a high potential for sexual transmission (high rates of partner changes and unprotected intercourse), pathogen variants that replicate to relatively high levels soon after infection tend to have a greater chance of being transmitted to new partners. if the host population is characterized by a low potential for sexual transmission, the chance of a partner change occurring soon after infection is low, and the advantages of a high shedding of pathogens soon after infection is also low. if sexual partners remain together for a long period of time, a low probability of infecting the partner per act of sexual intercourse is of little consequence to the probability of pathogen transmission between the partners, because a large number of sexual contacts will occur during the long-term relationship. the low potential for sexual transmission thus favors low levels of exploitation and low virulence. a high potential for sexual transmission should favor elevated exploitation, which should increase the chances that negative side effects eventually will occur in the long run. this theoretical framework leads to two central predictions: the virulence of sexually transmitted pathogens ( ) should be greater in populations in which the potential for sexual transmission is greater and ( ) should increase within a population in response to an increase in the potential for sexual transmission. tests of these predictions uniformly have confirmed them whenever comparisons provide clear differences in the potential for sexual transmission and the virulence of infections. these comparisons involve hiv, human papillomavirus, human herpesvirus , and human t-lymphotropic viruses [ ] . the confirmations suggest that the virulence of sexually transmitted pathogens could be reduced by reducing the potential for sexual transmission through interventions designed to reduce partner changes and increase the use of barrier-type contraception. information on the virulence of sexually transmitted pathogens from restricted regions provides a sense of the potential effect of such interventions. the evidence from senegal is perhaps the most informative in this regard. the population in senegal has a low potential for sexual transmission relative to populations of other countries in sub-saharan africa. the relatively benign hiv type has not been replaced by the more virulent hiv- in senegal, as it has in other areas of west africa [ ] . the hiv- subtype that predominates in senegal is more benign than the hiv- subtypes that predominate in sub-saharan countries with a higher potential for sexual transmission [ ] . a similar difference occurs among the strains of the sexually transmitted bacterium chlamydia trachomatis. the strains of c trachomatis that predominate in senegal are more mild than the strains that predominate in sub-saharan countries with a higher potential for sexual transmission [ ] . these comparisons illustrate how a low potential for sexual transmission can favor benign sexually transmitted pathogens even in relatively small populations that are not isolated from surrounding populations. chronic infectious diseases may seem passe´relative to the acute emerging diseases that have monopolized the headlines, such as ebola virus and severe acute respiratory syndrome. chronic diseases, however, pose a much greater threat over the near term, and something important probably can be done to control them if their causes are examined. these two claims may seem presumptuous at first. the worst plagues of history have been acute infectious diseases that spread swiftly and lethally through human populations. the most damaging examples generally have been well adapted to transmission through human populations, either directly from person to person or indirectly through a biologic vector, such as a mosquito, or a nonbiologic vehicle, such as water. these diseases as a rule were longadapted to humans and caused their harm when they spread through previously unexposed human populations. measles and smallpox decimated native populations in the americas when they were introduced during the early colonial period [ ] [ ] [ ] [ ] . syphilis probably caused large amounts of death in previously unexposed populations in europe as a result of a reciprocal introduction into europe from the new world [ ] [ ] . these outbreaks were devastating largely because they were introduced from human populations with which they had been in evolutionary arms races into populations that had no acquired immunity and little if any evolved resistance. terrible new outbreaks of long-standing human diseases are unlikely to be a great threat in the future because the current high level of worldwide transportation is far greater than the level needed for global transport of well-adapted human pathogens. so far as is known, the only pathogens of humans that have not already been mixed globally by human travel are zoonotic (ie, newly introduced into humans from other species). zoonotic diseases generally have limited potential for spread in human populations and therefore have little potential for causing devastating epidemics. aids is the only exception; however, even aids causes minor damage compared with the decimation that was caused in new world populations on contact with old world pathogens. the diseases that are known to be a threat in the near future are those that currently are killing massive numbers of humans. in rich countries, these diseases are chronic diseases that have been and still widely are presumed to be caused by bad genes and harmful environments rather than by infectious agents. for most of the th century, the accepted wisdom has been that the scope of infectious chronic diseases is narrow, largely limited to the chronic phases of sexually transmitted diseases and a handful of other diseases, such as tuberculosis and shingles, diseases that were thought of as chronic sequelae to acute infectious diseases. evolutionary theory, however, suggests that many if not most of the major chronic diseases of humans are caused by infection [ ] . the logic leading to this conclusion involves a simple application of natural selection. there are three general categories of disease causation: genetic, parasitic, and nonparasitic environmental. (''parasitic'' is broadly defined to include infectious causes.) evolutionary considerations severely limit the feasibility of genetic causation for the most common severe chronic diseases, because such diseases tend to reduce any causal alleles down to a frequency that can be maintained by mutation [ ] . if an allele provides some compensating benefit (as is the case with the allele for sickle cell anemia), it can be maintained, but few of the common and harmful diseases with unknown causes have characteristics that are consistent with such a scenario. although a great amount of research effort has been spent on attempts to discover genetic causes of chronic diseases, this research generally has identified only genetic predispositions to common damaging diseases rather than direct causes. even in the case of cancer, where mutational causes have been identified, these causes are insufficient to explain any more than a minuscule portion of human cancer without invoking other categories of causation. in no case has the research on genetic causation of chronic disease led to a practical breakthrough that decisively controls or cures any common and damaging chronic disease. in contrast to this lack of success, infectious causes of chronic diseases have been documented (table ) , and preventive or curative interventions have been enacted (with vastly less funding). peptic ulcers, stomach cancer, and liver cancer are recognized as being caused by infection. peptic ulcers and some stomach cancer can be cured and prevented by antibiotic treatment [ ] , and many cases of liver cancer have been prevented by screening the blood supply for hepatitis b and c viruses. infectious agents have been associated with a large proportion of the most common severe chronic diseases of unknown cause, such as diabetes, alzheimer's disease, atherosclerosis, and schizophrenia ( table ) . because infectious causation of chronic diseases generally cannot be demonstrated with the same level of certainty as infectious causation of acute diseases (eg, koch's postulates generally cannot be satisfied), acceptance of infectious causation is more protracted for chronic diseases [ ] . the evidence for infectious causation of these diseases is steadily mounting and often is making sense of the evidence for genetic and noninfectious environmental causation. this coalescence of perspectives is well illustrated by the e alleleassociated diseases: atherosclerosis, stroke, alzheimer's disease, rheumatoid arthritis, and multiple sclerosis. the e allele is maintained at frequencies that range from about % to % in different human populations. its frequency is lowest in populations that have been living in high densities for the past few thousand years. the frequency is higher in populations that have been relatively small and isolated during this time, and it is highest in people who have been hunter-gatherers into the th century. even the lowest frequency of the e allele is too great to be maintained simply by mutation. the e allele is the primary allelic form of the apolipoprotein e gene in other primates and cannot be considered a defective allele. one possible explanation is that the e allele increases vulnerability to at least one infectious cause of the e allele-associated diseases. although many pathogens have been associated with these diseases (table ) , one pathogen, chlamydia pneumoniae, has been associated with all of them. this finding raises the possibility that the e allele increases vulnerability to c pneumoniae infection. as a respiratory-tract pathogen, c pneumoniae undoubtedly inflicts a heavier cost on dense human populations than on sparse populations. if so, the longer that a particular ethnic group has lived in high-density populations, the greater the cumulative selective pressure against the e allele. in accordance with this scenario, individuals who are infected with c pneumoniae are about four times as likely to have the e allele as are individuals from the general population [ ] . c pneumoniae apparently has evolved to take advantage of people who have the e allele and has driven down the frequency of e allele over time. the theoretical framework for understanding the evolution of virulence of sexually transmitted pathogens provides clues about which infectious agents are the most likely causes of these illnesses. the primary requirement for infectious causation of chronic disease is persistent infection, and this a question mark indicates transmission route is uncertain. abbreviations: ebv, epstein-barr virus; n, nonsexually transmitted; s-o, sexually transmitted by oral contact; s-g, sexually transmitted by genital contact. theoretical framework proposes that sexual transmission favors persistent infections more than any other mode of transmission. one caveat applies. pathogens transmitted by sexual oral contact should be selected to be persistent for the same reason that pathogens transmitted by sexual genital contact are selected to be persistent (ie, because sexual oral contact occurs rarely relative to contact through coughing or sneezing). if sexual transmission is defined broadly to include transmission through sexual oral and genital contact, sexually transmitted pathogens over the past quarter century have been responsible for a disproportionately large fraction of the chronic diseases that have been accepted as being caused by infection; about % of all human pathogens are sexually transmitted by this definition, but about half of the pathogens that cause these chronic diseases are sexually transmitted (see table ). they are also candidate causes of the chronic diseases for which infectious causation strongly is implicated but not yet accepted (see table ). to identify infectious causes of chronic diseases, one should look closely at the sexually transmitted pathogens. ecological imperialism. the biological expansion of europe plagues and peoples guns, germs and steel epidemics and 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evolutionary epidemiology vaccines as evolutionary tools: the virulence antigen strategy diphtheria immunization: effect upon carriers and the control of outbreaks mutation in the structural gene for diphtheria toxin carried by temperate phage b diptheria: studies on the biology of an infectious disease diphtheria in the united states, - evolutionary control of hiv and other sexually transmitted viruses identification of all hiv type group m subtypes in senegal, a country with low and stable seroprevalence human immunodeficiency virus type subtypes differ in disease progression molecular epidemiology of genital chlamydia trachomatis infection in high-risk women in senegal, west africa the european-american exchange first european exposure to syphilis: the dominican republic at the time of columbian contact infectious causation of disease: an evolutionary perspective frequency of apolipoprotein e (apoe) allele types in patients with chlamydia-associated arthritis and other arthritides sexual transmission and the natural history of human herpesvirus infection use of versant trade mark tma and bdna . assays to detect and quantify hepatitis c virus in semen sexual transmission of hepatitis c and early intervention detection and cellular localization of enterovirus rna sequences in spinal cord of patients with als risk of sporadic amyotrophic lateral sclerosis associated with seropositivity for herpesviruses and echovirus- identification and localization of chlamydia pneumoniae in the alzheimer's brain herpesviruses in brain and alzheimer's disease infectious agents and multiple sclerosis-are chlamydia pneumoniae and human herpes virus involved? the association between multiple sclerosis and infection with epstein-barr virus and retrovirus multiple sclerosis and epstein-barr virus potential applications of the national collaborative perinatal project for the study of toxoplasma infections and psychiatric disease genes, germs, and schizophrenia: an evolutionary perspective endogenous retroviruses and schizophrenia antibodies to toxoplasma gondii in individuals with first-episode schizophrenia toxoplasma gondii and schizophrenia borna disease virus antibodies and the deficit syndrome of schizophrenia positive and negative syndromes, and borna disease virus infection in schizophrenia a viro-psycho-immunological disease-model of a subtype affective disorder post-streptococcal autoimmune disorders of the central nervous system toxoplasma pericarditis mimicking systemic lupus erythematosus: diagnostic and treatment difficulties in one patient systemic lupus erythematosus in adults is associated with previous epstein-barr virus exposure an association between an antibody against chlamydia pneumoniae and ischemic stroke in elderly japanese inflammation and infections as risk factors for ischemic stroke evidence of an association between chlamydia pneumoniae and cerebrovascular accidents porphyromonas gingivalis infection is associated with carotid atherosclerosis in non-obese japanese type diabetic patients periodontal disease and cardiovascular disease: epidemiology and possible mechanisms maternal herpesvirus infections and risk of acute lymphoblastic leukemia in the offspring human papillomavirus type infection and squamous cell carcinoma of the head and neck in never-smokers: a matched pair analysis identification of a proviral structure in human breast cancer mouse mammary tumor virus-like gene sequences in breast tumors of australian and vietnamese women jc virus dna is present in the mucosa of the human colon and in colorectal cancers association of human polyomavirus jcv with colon cancer: evidence for interaction of viral t-antigen and beta-catenin hepatitis c virus infection and incident type diabetes is crohn's disease caused by a mycobacterium? comparisons with leprosy, tuberculosis, and johne's disease i thank gregory m. cochran and levi g. ledgerwood for contributing to the development of the ideas presented in this article. key: cord- - uoberfu authors: tiwari, bhagyashree; sellamuthu, balasubramanian; drogui, patrick; tyagi, r.d. title: future impacts and trends in treatment of hospital wastewater date: - - journal: current developments in biotechnology and bioengineering doi: . /b - - - - . - sha: doc_id: cord_uid: uoberfu the world’s population growth and economic development result in the increased requirement of land, water, and energy. this increased demand leads to the deforestation, loss in biodiversity, imbalance in agriculture and food supply, climate change, and increase in food and travel trade, which result in emergence and reemergence of infectious diseases. this chapter discussed various emerging infectious diseases and their causative agents (buruli ulcer and bunyvirus). furthermore, this chapter further illustrates the emergence of superbugs and the associated threat due to the presence of pharmaceutical compounds in the environment. the prevalence of pharmaceuticals in the environment exerts ecotoxic effects on living organisms and causes thousands of death every year. the threats associated with the pharmaceutical presence in the environment were briefly discussed in this chapter. finally, this chapter provides the alternative methods to avoid the use of antibiotics and to develop novel treatment technologies (such as phage therapy) to degrade and remove the pharmaceutical compounds. infectious diseases are the second leading cause of death annually across the globe. the causative agent of most emerging infectious diseases is viruses; every year approximately more than two novel viral pathogens are identified, which can cause illness in a human. since the s, an average of infectious diseases has been emerged and known to cause pandemics such as ebola, swine flu, chikungunya, and zika (table À ) [ ] . the emergence of infectious diseases has severe health and socioeconomic impacts; thus the following section discusses the various infectious disease that emerged within the last two decades and presenting few pandemic viral diseases as a suitable example [ , ] . factors for emergence include natural process (evolution of pathogen), infectious agents transfer from vertebrate to mammals, antimicrobial resistance (amr), and climate change. the factors responsible for the emergence of infectious diseases such as ( ) the evolution of new strain, ( ) the introduction of a host to enzootic, ( ) translocation of infected wildlife, ( ) farming practices, and ( ) others were provided. the viral evolution rate occurs through mutation and adaption, which are much faster than any other microscopic organisms that is why a large fraction of emerging and reemerging pathogens are viruses ( %). among these % of emerging viruses, rna viruses are prominent one because of their higher nucleotide substitution rate which enables them to invade and amplify in broad host range [ ] . the environment changes, social condition, and their interaction are the important factors which lead to the viral evolution and ultimately to disease emergence. the decrease in biological diversity due to deforestation, prevalence of micropollutant in natural environment, and climate change triggers the evolution of opportunistic pathogen [ ] . for instance, the outbreak of nipah virus diseases was occurred due anaplasma phagocytophilum [ ] buruli ulcers mycobacterium ulcerans [ ] to the extensive deforestation of a forest of southeast asia [ ] . the deforestation leads to migration of fruit bats for food quest to the agriculture land. these fruit bats are natural reservoir (host) of nipah virus and their migration to cultivable land lead to transmission of nipah virus disease in farm animals and subsequently in humans [ ] . the increase in temperature due to climate change affects the ecology, survival, and behavior of arthropod vectors, which subsequently changes population dynamic with increased disease transmission rate. for example, the rise in temperature from c to c decreases the proliferation time of plasmodium falciparum in anopheline mosquitoes vector from to days. usually, p. falciparum requires days to proliferate in the vector at c. however, the high temperature reduces its proliferation time by increasing egg production, by increasing metabolic rate, and by reducing the larval and pupal period duration [ ] . moreover, the proximity of natural host such as rodents and human is the ease and travel aids for the spread of emerging viruses. the viral infection begins from binding of virus to the receptor of host cell, and during the emergence of new disease, viruses develop either the ability to bind the new receptor or use homologue receptor in new host species. the spread of coronavirus (cov) which causes severe acute respiratory syndrome (sars) in human occurs due to cross-species transmission from raccoon dogs and chinese ferret badgers [ ] . the examination of wild species of cov did not have the sign of sarsÀcov infection but the cov viruses isolated from horseshoe bats have sarsÀcov infection. the cov binds to angiotensin-converting enzyme (ace ) receptor to infect humans. however, in bats, ace viral receptor is not use for infection by cov. later it was studied that the mutation in ace receptor aids the adaptation of cov in human cells. the spread of ebola, marburg, and measles viruses is some of the example of virus evolution and which contributes to the emergence of disease. the virus evolution and adaptation is difficult to predict and thus raises a question how the effect of emerging viral infection can be reduced. this requires a holistic approach to identify the drivers of emergence with effective surveillance. in an increased cases of severe fever with thrombocytopenia syndrome (sfts) were frequently reported in rural areas of china. the associated pathogens of thrombocytopenia and other similar diseases were not detected in majority of the pathogen samples which implies that the pathogen associated with sfts was newly evolved. the emergence of this unknown infection having average fatality rate of % due to organ failure leads to the implementation of enhanced surveillance to identify causative agent of sfts infection [ ] . the surveillance data revealed strain of viruses as causative of sfts infection and regarded as sfts viruses (sftsvs). sftsvs are rna virus with three single-stranded rna genomes. the complete sequencing of sftsv, rna genome reveals that they belong to genus phlebovirus, family bunyaviriade. the sftsv is transmitted in human through infected ticks and transmission of blood and other body fluid of infected person lead to human-to-human transmission of infection. the phlebovirus infection in human results in mild febrile illness. however, the major symptoms of sfts include thrombocytopenia, high fever, leukocytopenia, and lymphadenopathy. currently, it is believed that the vertical transmission of phlebovirus in arthropod vector (ticks such as haemaphysalis longicornis) helps in maintenance cycle of virus and amplification of virus occur in vertebrate host (human). the laboratory diagnosis of body fluid samples of sfts patient showed elevated level of serum and important enzymes and cofactor such as creatine kinase, alanine aminotransferase, lactate dehydrogenase, and aspartate aminotransferase. the sftsv replicates in the spleen of infected patient which increases the number of macrophages and platelets. the in vitro assay revealed that sftsv facilitates the phagocytosis of platelets by adhering on the surface of platelets and ultimately causing thrombocytopenia. the spread of sftsv in china and the detection of sftsv like viruses in other parts of world such as in the united states and europe emphasized on the urgent need of understanding of pathogenesis and transmission cycle of virus which help in the development of efficient vaccine. the changes in the hostÀenvironment lead to the evolution of opportunistic and novel pathogens due to which infectious diseases emerge. the forces which shape the emergence of disease in human are similar to the drivers found in wildlife and domestic animals. these drivers alter the interplay of hosts, environment, and pathogen thereby modulating disease ecology and developing or acclimatizing pathogen in hosts. the drivers alter interaction pattern of pathogenÀhost and environment which to either of three events ( ) altering the genetic trait of pathogen which causes severe disease in same host; ( ) emergence of pathogen in new hosts; and ( ) redistribution of pathogens that result in its establishment in new geographical area. frequent use of antimicrobial compounds and mass rearing of animals result in amr and eventually increase virulence pathogenicity of a pathogen. mass rearing of food animals to meet the demand of growing population results in intensification of genetically similar animals of same sex and age at confined place. this leads to transmission of pathogen with increases population turnover which supports emergence of novel trait. the transmission of highly pathogenic asian avian influenza a (h n hpai) is the well-known example of mass rearing [ ] . the drivers which are responsible for the disease emergence in new host include interspecies contact, wildlife migration, and increase contact between different hosts. the worldwide change in ecological landscape results in close contact between human and animals which eventually lead to transmission of microbial reservoir of animals (birds, rodents, and bats) to human. for instance, the emergence of nipah virus in human was due to transmission of virus from foraging fruit bat to pigs [ ] . the transport of food, live animals, plants with accompany insects for international trade, migration of birds, and wild animals results in geographical jump of pathogens. the bacterium ralstonia solanacearum hitchhikes to the united states via geranium plant which is imported from kenya [ ] . persisters are slow growing or nongrowing organisms which remain in stagnant during the presence of antimicrobial compound but have the capacity to resuscitate and grow under specific conditions. the persisters formation is mediated by variety of stress such as due to nutrient depletion, oxidative stress heat, acidic ph, and the presence of toxic compound (antibiotics). antibiotics such as tetracycline, rifampin, and ciprofloxacin were shown to enrich persisters formation by inhibiting protein synthesis, rna synthesis, or by antioxidative defense [ ] . persisters are the cause of biofilm infection, recurrent infection, and chronic infection and contribute toward prolongation of therapy time (e.g., tuberculosis, lyme disease). the repeated treatment of persistent infection may result in the development of drug-resistance microbes, often seen in the case of tuberculosis. persistent could be pre-or postantibiotic depending on its development in the host. the capacity of formation of persisters varies greatly among the bacterial species. for instance, persisters of mycobacterium tuberculosis are not removed from the host even after chemotherapy, while the single antibiotic treatment is sufficient for curing infection caused by streptococcus pneumoniae. the physical and psychological stress, host immune and hormonal factor, and coinfection also affect the persisters formation in the host. the mechanism behind the persisters formation is not well understood. however, it is believed that epigenetic changes, changes in dna modification, and posttranslational modification induce expression of persisters gene. the genes which involve in persisters formation are rela, sucb, hipa, ubif, and phou. the mutagenesis approach is used to study genes involved in persisters formation; however, the short antibiotic exposure, screening of partial mutant library, and aeration during antibiotic exposure are factors which result in failure of mutagenesis process for identifying persisters genes. zoonotic diseases are vector (mosquito, ticks, and bugs) aided or nonaided disease caused by bacteria, virus, parasites, prions and fungi, and transmitted from animals to human. various modes of transmission are direct contact between infected animal and human, via arthropod vector, consumption of contaminated animal food (meat and pork), and ingestion of aerosolized pathogens present in the environment. in last two decades, many vector borne pathogens spread in new geographical regions. the emergence and reemergence rate of zoonotic infection has increased inescapably due to urbanization, deforestation, climate change, international trade (frequent travel), population movement, and encroachment into animal habitats. the most emerging zoonotic vector borne diseases in the united states and canada include tick borne lyme disease, babesiosis, human granulocytic anaplasmosis and mosquito borne west nile virus (wnv), california serogroup viruses, and cache valley virus [ ] . the change in land use alters the abundance and interaction of vectors and hosts (wildlife and domestic animals) which results in emergence of vectors. for instance, deforestation in amazon and eastern africa enhances the breeding of anopheles' mosquito due to sunlight and standing water. similarly in north america, increased hunting changes the predator community and results in increased abundance of small animals such as mice, chipmunks, and shrew which are main host of spirochete borrelia burgdorferi, causative agent of lyme disease [ ] . socioeconomic changes and human activities are the another factor which governs spread and emergence of pathogen. the lyme disease was reported to occur more in high-income people in europe due to the more recreational activity and living in new homes in broad-leaf woodlands with cooccurrence of wildlife result in frequent exposure to vector. vector born zoonotic disease could be controlled by prompt identification of cause with subsequent action which requires integration of public health officials, researchers, and public [ ] . arbovirus is an acronym for arthropod-borne viruses. dengue virus, wnv, and chikungunya virus are recent examples of arboviruses which are transmitted from the arthropods (ticks, bugs, and mosquito) to vertebrate. arboviruses use arthropods as a vector (carrier) for transmission and do not causes any sickness in them. bunyaviridae, reoviridae, flaviviridae, and togaviridae are the most prevailing arboviruses families that cause diseases in human and animals [ ] . the emergence of arboviruses is governed by three factors, that is, high mutation frequency, varying anthropological behavior, and climate change. they easily adapt new host by altering receptor specificity, antigenicity, environmental conditions, and by efficient transmission. for instance, emergence of chikungunya virus in asia due to mutation in surface protein which increases its reproduction, transmission, and infection efficiency in aedes albopictus. they are able to maintain themselves for years in mosquito eggs or via attaining transstadial stages in ticks. domestic animals, livestock, and human are not important part of arbovirus life cycle because of nonviraemic transmission of arbovirus between ticks without infecting vertebrate host. this feature of arbovirus adds additional limitation for controlling disease emergence [ ] . mosquito eradication, development of live-attenuated vaccines, antiviral drugs, and molecule are few methods used to control and prevent arboviral infection. however, high mutational frequency will result in emergence of new pathogenic arboviruses. since it has been proven by genome sequencing of mosquitoes that they are the carrier of various known and unknown viruses, control of localized arthropod during endemic could be a possible solution for regulating the emergence of arbovirus [ ] . buruli ulcer (bu) is necrotizing skin disease caused by mycobacterium ulcerans recognized as one of most neglected tropical disease by world health organization (who). the bu lead to the formation of skin ulcers which results in osteomyelitis. it causes pandemic in humid tropical and subtopical region, often where humans are in proximity with slow moving or stagnant contaminated water. the transmission of m. ulcerans follows multihost transmission dynamics, that is, multiple hosts of aquatic environment such as scrapers, scavengers, and predators become contaminated with m. ulcerans and result in its passive dissemination via organism-to-organism contact. phylogenetic studies revealed that the m. ulcerans was emerged from species mycobacterium marinum which causes cutaneous disease in human and also shown to infect fish. m. ulcerans are widely distributed in africa; however, the bu cases reported in specific geographical villages. researchers postulates that a specific m. ulcerans strain might have pathogenicity or virulence to cause bu [ ] . the current knowledge lacks the understanding of spatiotemporal distribution of m. ulcerans and required detailed scenario of diversity of m. ulcerans strains existing in environment [ ] . superbug is a term coined for bacterial species which confers resistant toward majority of antibiotics. emergence of superbugs implies the frequent detection of novel bacterial pathogens which caused unrecognized life-threatening infections. the frequent emergence and spread of superbugs worldwide causes a concern that human life is heading back, toward the preantibiotic era, where the entire population of a society was wipe out due to the simple infection. united nation (un) general assembly of addresses the amr [ ] as a health emergency and acknowledge that amr has deleterious effect on human health and sustainable development. who identified a group of amr resistant "priority pathogens" that requires urgent strategic action (table À ) . who categorize these priority pathogens into three categories, that is, critical, high, and medium priority based on the need for new antibiotics. the criteria for prioritization of pathogens were developed using multicriteria decision analysis technique which includes amr pathogens which causes mortality, prevalence of resistance, transmissibility, treatability, healthcare and community burden, -year trend of resistance, preventability in hospital and community settings, and current pipeline [ ] . to fight against increasing prevalence of antibiotic-resistant superbugs and to limit the indiscriminate use of antibiotics, who prepared three groups of antibiotics namely access, watch, and reserve group to ensure appropriate prescription and use. access group comprises of antibiotics which are prescribe as in common infection as first and second choice. the first-choice antibiotics are narrow spectrum with low-resistant potential, whereas the second-choice antibiotics are broad spectrum having higher resistant potential. antibiotics such as β-lactam, chloramphenicol, and clindamycin come under this category. watch group identifies pharmacological antibiotic classes which prescribes as first or second choice but have a limited number of indication. the watch group has higher resistance potential compared with the access group. watch group comprises macrolides, quinolones and fluoroquinolones, and glycopeptides class of antibiotics [ ] . who created reserve group of antibiotics to reserve some antibiotics as the last resort in superbug infection. this group of antibiotics should be recommended as "last resort," in case of life-threatening infection when other alternatives are failed in treatment. reserve groups include eight antibiotic or antibiotic class which are aztreonam, fosfomycin, fourth-generation cephalosporins (cefepime), oxazolidinones (linezolid), fifth-generation cephalosporins (ceftaroline), tigecycline, polymyxins (polymyxin-b, colistin), and daptomycin [ ] . recently, among these eight antibiotic classes, resistant determinant against colistin was detected in people of rural vietnam. the prevalence of colistin resistant escherichia coli in intestine of resident of vietnam was extremely high, that is, approximately %. previously, mutation that causes colistin resistant was not transferable; thus it is not considered as pathogenic, as intestinal e. coli is nonpathogenic. finally, a transmissible colistin resistance gene (mcr) was detected in china, which has the potential to transfer colistin resistant to pathogenic microbes. the prevalence of mcr gene represents a serious concern regarding the emergence of superbugs that are resistant to last resort of antibiotic [ ] . currently, there are only fewer option (such as last resort antibiotics) to tackle antibiotic-resistant superbugs, and the finding of mcr gene indicates toward the needs of innovative research which results in better understanding of superbug emergence and also to research and development on quality, safe, efficacious, and affordable antimicrobial medicines, especially new antibiotics and alternative therapies, vaccines, and diagnostics. the various chapters of this book discussed about the occurrence and prevalence of human and veterinary pharmaceuticals (antibiotics, antidepressant, antidiabetic, radioactive agents, etc.) at different environmental sites. these pharmaceutical compounds are biologically active compounds that are known to have a specific mode of action (moa) in human and animals even at low concentration. due to genetic relatedness (presence of conserved gene) across species, these compounds interact with protein and cell lineage of nontarget organisms (conserved therapeutic drug targets) and elicit a response in their body (targets metabolic pathway, enzyme or mediators of cell signaling molecule). for instance, ibuprofen which is a cyclooxygenase inhibitor was found to interfere with arachidonic acid signaling pathway in marine clams ruditapes philippinarum [ ] . however, the effect of pharmaceuticals may vary from species to species due to difference in gene expression of the same gene across species or due to change in solubility or potency of drugs because of binding of pharmaceuticals with organic molecules present at environmental sites. therefore to understand the environmental risk posed by these contaminants, conceptual model of moa of pharmaceuticals was used. the moa [ ] approach involves the assessment of drug targets and its evolution between mammals and the model species. the conceptual model of moa approach in marine organisms reveals the presence of fluoxetine at environmental concentration was able to control the serotonin signaling pathway, and this alteration in physiological signaling pathway results in defects in reproduction, locomotion, and metabolism of aquatic organisms [ ] . many short-term toxicity studies reported that the drug molecules do not have an acute toxic effect on aquatic organisms because of their presence in low concentration (ng/l to low μg/l), but their constant release and exposure to aquatic biota have long-term chronic effects [ , ] . however, many laboratory studies at high concentration of pharmaceutical compounds report direct lethal effects. tetracycline concentration around À μg/l leads to low periphyton (nematode, bacteria, and algae) concentration in mesocosm stream [ ] . structure disruption in kidney and intestine of rainbow trout and brown trout due to diclofenac ( μg/l) was reported [ ] . prolonged exposure to pharmaceuticals in low concentration leads to the change in species trait and behavior of aquatic organisms. antidepressant and psychiatric drugs such as fluoxetine and oxazepam cause disruption in ecological interaction even in low concentration [ ] . indeed, even by considering that these drugs are diluted after their release, it is evident that they have a toxic effect on the aquatic ecosystem. the widespread occurrences of pharmaceuticals at various environmental sites such as in ocean, river, and groundwater raise a concern regarding the risk associated with human health. for instance, the birth control and growth stimulator agent like estrogen have the potential to cause prostate and breast cancer in humans (if the estrogen concentration is used above the threshold limit). the worldwide discharge of estrogen from livestock and human ranges approximately , and , kg/year, respectively. the us national toxicology program declared estrogen as carcinogen, the no adverse effect concentration of estrogen in human is . mg/day and frequent detection of estrogen in drinking water concentration ranging from . to . ng/l represent a health risk [ ] . estrogen was reported to cause abnormalities in animals such as permanent infertility (clover disease in sheep due to feeding on clover plant which has high phytoestrogen level) [ ] . however, human health risk assessment studies reported no appreciable risk to human due to exposure of pharmaceuticals mixture [ ] . due to emergence of antibiotic-resistant pathogens and unavoidable use of antibiotics, concomitant environmental perturbation caused by climate change might make the earth is not suitable for humans and other livings. thus researchers focus on finding alternative methods to avoid use of antibiotics and developing novel treatment technologies to degrade and remove the pharmaceutical compounds. prediction of emerging signals by theoretical and bioinformatics tool will highly help to alarm the researchers, hospitals, and public about the nearby occurrence of resistant bug. such predictions are underway by monitoring microbial community dynamics in different environmental samples; however, these studies facing enormous challenges in the developmental face [ , À ] . several research studies have been conducted to observe a change in microbial metabolic pathways for a while. such study results could predict microbial interactions and route of evolution, and might apply to precisely pinpoint the emerging organism in the mixed microbial community. these studies are still under research stage, such studies are using genomic, metabolomic, and trancsriptomic tools to predict the bug emergence. recently, geoghegan and holmes [ ] attempted predicting the virus emergence with an interest of biomedicine and preventing unpredicted incidence [ ] . they have monitored the evolution of viruses in short-term period to highlight the cross-species transmission and emergence. they have concluded that predicting emergence requires a new mechanistic and integrated approach, which might permit or stop the emerging viral spread into new hosts [ ] . with the fact that microbial resistant to pharmaceuticals, the treatment options are reduced; however, alternative methods have been explored to protect human and animal from microbial illness. the microbial drug resistance leads to direct and indirect consequences as described by who report [ ] . the severe direct consequences are prolonged illness, impossible to protect the patients undergoing surgery and augmented treatment cost. whereas the indirect impacts of amr are depleting the global economy (due to the loss of productivity by sickness) and higher costs of treatment. thus who proposed a global action plan to assure preventing the transmission of infectious disease and providing complete treatment with the help of safe and effective medicines [ ] . to achieve the global action plan successfully, five objectives have been proposed by who: ( ) to improve awareness and understanding of amr; ( ) to strengthen knowledge through surveillance and research; ( ) to reduce the incidence of infection; ( ) to optimize the use of antimicrobial agents; and ( ) to ensure sustainable investment in countering amr. these objectives are likely to be met through political leaders, member states, the secretariat, and international and national partners across multiple sectors. notably, individual responsibilities like recycling unused medicines, intake of only prescribed medicines, and keeping the environment clean would highly help to achieve the who's global action plan for the betterment of human and animal health. increasing resistance to antibiotics and the emergence of "superbugs" that are resistant to drugs of last resort have highlighted the great need for alternative treatments of bacterial disease. this has led to renewed interest in the potential of phage to treat bacterial pathogens. the term "phage therapy" usually refers to the treatment of bacterial infections with intact phage; however, there are other ways in which phage can be used as antibacterials. phage can be used as "lethal agent delivery systems" to introduce nonspecific or toxic antimicrobials [ , ] , or genes encoding antimicrobials [ ] into selected pathogenic bacteria. in another method (targeted gene transfer), filamentous phage can be modified to carry therapeutic genes and to target cell surface antigens or receptors on mammalian cells, transducing them by receptor-mediated endocytosis [ ] . although phages are not an antimicrobial tool in itself, this approach could be used to deliver antimicrobials to intracellular bacterial pathogens. phage secrets lysins enzymes which lyse the host bacteria. lysins are host specific and use cell wall components that are essential for viability as receptors; therefore bacterial resistance is rare [ ] . initial tests of lysins against clinically relevant gram-positive bacteria, such as methicillin-resistance staphylococcus aureus, look promising [ , ] . the use of multiple phage lysins with different cleavage sites could increase therapeutic effectiveness and further reduce the chance of bacteria developing resistance. lysins can also be used to make bacterial ghost vaccines. to produce better quality of treated sludge, wastewater treatment processes must achieve a minimum log reduction in e. coli loads and a final end product (sludge) with a maximum admissible concentration of e. coli cfu/g [dry solids (ds)] and zero salmonella in g (ds). similarly, united states environmental protection agency regulations are categorized into class a and class b sludges. class a pathogen reduction requirements are more stringent than those of class b sludges which are subject to application restrictions. class a sludges are required to reduce pathogens to below the detectable limit (, most probable number of salmonella, , plaque forming unit of enteric viruses, and , viable helminth ovum per g ds). treatment processes designed to achieve pathogen reduction to a required level can incur substantial capital and operating costs [ ] . development of phage treatment of sludge may provide long-term and cost-effective control of potentially pathogenic bacteria (e.g., e. coli and salmonella). successful phage treatment of wastewater bacterial pathogens would be dependent on the prevalence and diversity of pathogen species within wastewater. it would be virtually impossible to produce phage targeted at all pathogenic serotypes. for example, there is a high diversity of e. coli serotypes [ ] and around known salmonella serotypes [ ] . however, wastewater treatment conditions intrinsically reduce the numbers of some pathogenic bacteria. therefore there is potential for phage treatment to be used successfully in combination with biological sludge stabilization processes to reduce the abundance of specific pathogenic bacterial strains such as e. coli o . indeed, research on phage therapy for reduction of this pathogen in animal reservoirs is already underway [ ] . although phage-derived therapies have several advantages compared with antibiotics, the combined use of phage, or phage lysins, and antibiotics is an attractive treatment regime. indeed, such a product is available commercially in georgia: phagobioderm is a biodegradable polymer composite impregnated with a lytic phage cocktail and ciprofloxacin [ ] . using phage with antibiotics should increase the efficiency of treatment and reduce the emergence of resistant mutants because the surviving bacteria would need to acquire at least two separate resistance mechanisms. to kill the multidrug-resistant bacteria researchers from ibn (institute of bioengineering and nanotechnology), chin et al. [ ] developed a novel synthetic molecule and demonstrated selectively destroying five multidrug-resistant bacteria [ ] . recently polymer-based advanced drug designing to combat multidrug resistances is widely explored, which are described in detail [ , ] . due to climate change, a steady increase in temperature (about . c) was recorded in the past years, which was clearly observed on lands than the ocean in recent days [ ] . however, ice melting in north pole, increase in sea level, and alteration in raining patterns are clear and known effects of examples of global warming. climate change affects the water cycle in the following aspects: ( ) poor water quality, ( ) less availability (quantity for consumption and use), ( ) evaporation leads increases the microbial load (concentration) and toxic ions concentration, ( ) water depletion in animal-farming and agriculture (leads to food scarcity), and ( ) effects on freshwater biodiversity is still undetermined. ultimately, these changes will hugely affect wastewater microbiome (including beneficial and pathogenic microbes), the concentration of toxic pollutants and biological process performance, which was discussed in detail in chapter , treatment of wastewater containing pharmaceuticals: biological treatment. recently, who reported that climate change affects the infectious disease transmission pattern (fig. À ) . various infectious diseases and their agents (viruses, bacteria, protozoa, and multicellular parasites) have adapted (via evolution) humans as a primary host, which raises serious threat mankind to face in near future. due to the negligent, persistent and accidental activities of humans (industrial) raised the environmental pollution (chemicals including pharmaceuticals), which led to inseparable effect like climate change. furthermore, development of drug-resistant organisms and increased pathogen survival rate, only raising panic about the human, animal, and environmental health. thus researchers are continuously searching alternatives to these environmental problems. due to pharmaceuticals release in the environment and climate change, the survival of pathogens prolongs. high load of infectious bacterial strains dwelling in wastewater further favors the transfer of antibiotic-resistance gene. this could be a classical example for multidrug-resistant bacterial strains that are prevalence in tropical countries (like china and india). this draws immediate attention of public, health, and international sectors to fight against, pollution, climate change, and drug resistance. the advancement in medicinal research helps in control and elimination of various infectious diseases such as smallpox. however, emergence and reemergence of infectious diseases due to various factors, that is, natural evolution, climate change, amr, and many more, is a matter of concern. the knowledge about the evolution and life cycle of pathogens (bacteria, virus, or parasite) using omics study (proteome, metabolome, epigenome, and transcriptome) and via next-generation sequencing could help in the prevention and control of infectious diseases. the implantation of ecological approaches, network, and system biology approaches could provide a better understanding of the environmental factors of disease-emergence and drug-resistance mechanisms. the information gained from these approaches 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personal care products, and artificial sweeteners) in surface and groundwater (drinking water) in the ganges river basin environmental impact of estrogens on human, animal and plant life: a critical review temporal dynamics of periphyton exposed to tetracycline in stream mesocosms water-borne diclofenac affects kidney and gill integrity and selected immune parameters in brown trout (salmo trutta f. fario) ocean viruses and their effects on microbial communities and biogeochemical cycles what is flux balance analysis? fast automated reconstruction of genome-scale metabolic models for microbial species and communities evolution of bidirectional costly mutualism from byproduct consumption a shared limiting resource leads to competitive exclusion in a cross-feeding system: role of environment for cross-feeder coexistence community structure follows simple assembly rules in microbial microcosms species interactions differ in their genetic robustness the spatial and metabolic basis of colony 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microbiology and molecular biology antigenic formulas of the salmonella serovars bacteriophage-resistance systems in dairy starter strains: molecular analysis to application a macromolecular approach to eradicate multidrug resistant bacterial infections while mitigating drug resistance onset antibiotic-containing polymers for localized, sustained drug delivery recent advances in the treatment of pathogenic infections using antibiotics and nano-drug delivery vehicles, drug des van der valk, managing water resources under climate uncertainty key: cord- -f f h r authors: afrough, b.; dowall, s.; hewson, r. title: emerging viruses and current strategies for vaccine intervention date: - - journal: clin exp immunol doi: . /cei. sha: doc_id: cord_uid: f f h r during the past decade several notable viruses have suddenly emerged from obscurity or anonymity to become serious global health threats, provoking concern regarding their sustained epidemic transmission in immunologically naive human populations. with each new threat comes the call for rapid vaccine development. indeed, vaccines are considered a critical component of disease prevention for emerging viral infections because, in many cases, other medical options are limited or non‐existent, or that infections result in such a rapid clinical deterioration that the effectiveness of therapeutics is limited. while classic approaches to vaccine development are still amenable to emerging viruses, the application of molecular techniques in virology has profoundly influenced our understanding of virus biology, and vaccination methods based on replicating, attenuated and non‐replicating virus vector approaches have become useful vaccine platforms. together with a growing understanding of viral disease emergence, a range of vaccine strategies and international commitment to underpin development, vaccine intervention for new and emerging viruses may become a possibility. immunization is arguably the most appropriate way of preventing infectious disease. the control of many important viral pathogens by vaccination is perhaps one of the outstanding achievements of medical intervention. vaccine-induced immunity that is established in advance of virus infection relies primarily on adaptive immune responses for protective efficacy. critically, vaccination depends on the properties of antigen recognition, activation, expansion, memory, trafficking and the multitude of specialist functions of lymphocytes. the extent to which vaccine-induced immunity is successful also determines the spread and maintenance of a viral pathogen within a population. viral vaccines have had profound and enduring consequences for human and animal health; the worldwide eradication of smallpox and rinderpest are testament to their outstanding contribution to modern society. nevertheless, infectious diseases still pose one of the greatest threats to public health, and the past three decades have brought a constant barrage of new human pathogens. more than % of these infections are zoonotic [ , ] , entering either directly from wildlife reservoirs or indirectly via an intermediate domestic animal host [ , ] . hiv, avian influenza, hendra (hev) and nipah (niv) viruses, severe acute respiratory syndrome (sars) and middle east respiratory syndrome coronavirus (mers-cov), ebola and marburg filoviruses, lassa virus (lasv), rift valley fever virus (rvfv) and crimean-congo haemorrhagic fever (cchf) viruses are all examples of zoonoses currently emerging from wildlife. all these emerging zoonoses present a serious and increasing threat to health, biosecurity and economies worldwide. the mechanisms underlying disease transmission from animals to humans are becoming better understood [ ] with the emergence of pathogens from wildlife (which represents the greatest threat to global health) occurring in a non-uniform pattern, being localized to distinct geographic 'hotspots' in africa, asia and south america, and with each high-threat pathogen being weighted towards a key wildlife species (e.g. bats, rodents or non-human primates (nhps). it is clear that such diseases will continue to place a substantial burden on global health, especially in dense human populations where the pressures on environmental and economic resources are greatest. more than one billion cases of human zoonotic disease are estimated to occur annually, and emerging zoonoses result in enormous economic losses [ ] . increased urbanization, international travel, commerce and climate change increase the likelihood that emerging zoonosis will continue, if not worsen, in the future. when a zoonotic virus spills over into a susceptible new species, it often has the advantage that the new host has little or no pre-existing immunity, enabling attachment, entry and replication of the virus in receptive cells. the amplified virus may then evade clearance by host defences for long enough to be transmitted to another susceptible host, and the lack of herd immunity will result in a rapid dissemination of the virus, leading to disease in more virulent cases of infection. each step in the process represents an opportunity for vaccineinduced immunity and, through such intervention, transmitters and susceptible hosts are removed from a population by the pre-emptive development of protective immunity, so that the spread of infection becomes less likely. vaccination is therefore a powerful strategy for preventing and controlling emerging zoonotic infectious disease. the development of vaccines for such emerging infections, however, needs to contend with several key challenges associated with such viruses. an emerging infection may be a recently discovered virus and the result of a rare outbreak for which basic biological information such as correlates of protection, antigenic variability or immunodominance are unknown. there may be a lack of time to develop an appropriate animal model of disease in which to study viral immunology and evaluate vaccine candidates for preclinical assessment of protective efficacy and safety. additionally, many emerging viruses have high case fatality rates, spread easily and cannot be treated. these characteristics mandate that all experimental investigations with such infectious material be carried out at high levels of bio-safety, such as biosafety levels or . for such pathogens the availability of resource-heavy laboratory infrastructure is a bottleneck to basic research. moreover, the often standard vaccine approach of using attenuated strains or inactivated viral vaccines is not always a feasible option, because of the possibility of reversion to virulence or the requirement for large-scale culture and production in high containment facilities. in addition to these significant hurdles, the economic cost of novel human vaccine development for rare pathogens, which are unlikely to provide an effective payback on investment, has been a major impediment to progress. thus, basic research into many emerging pathogens has been neglected for years. in the unpredicted size, speed and reach of the ebola virus outbreak in west africa [ ] acted as a wakeup call for researchers, pharmaceutical communities and governments, emphasizing the importance of investment into the study of emerging pathogens. spurred on by this development and at the request of its member states in may , the world health organization (who) convened a broad coalition of experts to develop a research and development (r&d) blueprint for action to prevent epidemics. focusing on severe emerging diseases with the potential to generate public health emergencies, and for which no, or insufficient, preventive and curative solutions exist, the r&d blueprint specifies r&d needs, including vaccine research. through international governance, the programme aims to define r&d roadmaps for prioritized pathogens and to catalyse funding strategies [ ] . key to the development of successful and effective vaccines is the design of an antigen delivery system that optimizes antigen presentation and induces broad protective immune responses. recent advances in vector delivery technologies, immunology and basic virology have led to a deeper understanding of the molecular and cellular mechanisms by which vaccines should stimulate both arms of the adaptive immune response, thereby offering novel strategies of vaccination. here we discuss some current vaccine approaches for safe and effective vaccines encompassing recombinant virus technology, nucleic acid vaccines and self-disseminating vaccine approaches. advances in recombinant virology and virus reverse genetics have provided key insights into the replication and pathogenesis of a wide range of viruses. notably, these have facilitated the development of vectors for protein expression and vaccination. to date, several virus families have been exploited as vectors [ ] [ ] [ ] [ ] including many for vaccination [ ] [ ] [ ] [ ] fig . a basic advantage of viral vectored vaccines is that the choice antigen is expressed in the context of an active heterologous viral infection, which stimulates the full gamut of innate immune responses required for the development of adaptive humoral and t cell-mediated immunity [ ] . an important aspect of a virus-vectored vaccine for emerging viruses is that the characteristics, type and intensity of the immune response, as well as safety considerations and manufacturing techniques are determined predominantly by the vector and not the pathogen. therefore, developing and testing a vaccine against a newly discovered virus can be significantly shortened by the use of a viral vector platform with an extensive record of safety and efficacy. vaccines for emerging pathogens: from research to the clinic. part vesicular stomatitis virus (vsv), a negative sense rna virus of the rhabdoviridae family, has become a prominent replication-competent vaccine vector platform [ ] . vsv is non-pathogenic in humans and has an inherent ability to elicit strong cellular and humoral immune responses. one of the most useful aspects of this virus vector platform is its almost promiscuous ability to assemble recombinant vsv (rvsv), with many different types of heterologous viral glycoproteins. the platform is designed such that the vsv g protein is replaced with a heterologous envelope glycoprotein from, for example, an emerging virus; while this arrangement renders the rvsv replication competent, the recombinant viruses are generally highly attenuated [ ] . in , the attenuation of a yellow fever (yf) virus via successive rounds of serial passage led to the development of the yf vaccine termed d. the impact of this successful vaccine was recognized by the award of a nobel prize in [ ] and it has been widely adopted for human immunization for more than years [ ] . based on the utility of the vaccine, an infectious cdna clone of the yf d virus [ ] has enabled the development of a d platform that can be used to drive antigen delivery of pathogens of interest [ ] . the technology (licenced as chimerivax™) is well suited to similarly related flaviviruses and successful recombinants have been constructed, involving a simple swap of the prm/m-e genes of yf d for the same membrane envelope antigens of other emerging flaviviruses, such as japanese encephalitis, dengue and west nile. the resulting recombinant vaccines are efficiently delivered in a live-attenuated virus context with the safety profile afforded by the d non-structural genes [ ] . a licenced vaccine for japanese encephalitis (chimerivax™-je) using this technology has been developed by sanofi pasteur (lyon, france). other replication-competent platforms. the ease of direct manipulation of viral genomes together with a growing understanding of their biology has led to the development of attenuated virus vaccines with increased safety and immunogenicity. for example, a new vaccine candidate for rvfv has been developed in which a viral virulence factor has been deleted, resulting in a highly attenuated but immunogenic replicating virus [ ] . a similar recombinant approach has been used to attenuate the emerging and neglected pathogen mayaro virus (mayv) by swapping the subgenomic promoter of this alphavirus for an internal ribosome entry site [ , ] , which reduces the expression of mayv structural proteins. while this vaccines for emerging pathogens: from research to the clinic. part arrangement makes the virus unable to infect mosquito cells, replication in mammalian cells is still possible, resulting in a highly immunogenic profile. similar studies on infectious clones of other viruses [ ] have demonstrated that the genomewide de-optimization of codon usage dramatically reduces gene expression and can be used to attenuate otherwise pathogenic viruses. this strategy has been used for the prototype arenavirus lymphocytic choriomeningitis virus (lcmv) [ ] ; the study showed attenuation in an otherwise fatal mouse model of lcm disease and the ability to induce protective immunity. together with a maintained and robust ability to multiply in cell culture, this live attenuated approach may be suitable for similar viruses. however, work to underpin confidence in the genetic stability of such attenuated viruses is critical before further consideration can be given to using this approach for clinical intervention, especially considering the inherent high error rates of the arenavirus polymerase. for several years, recombinant adenoviruses have been adopted as promising tools for antigen delivery and vaccine efficacy [ ] . deleting the e gene from the adenoviral genome and supplying it in trans from a packaging cell line allows replication-deficient recombinant adenovirus to be produced, with the novel heterologous antigen gene of interest inserted at the e locus. early setbacks in this platform technology relating to pre-existing anti-adenovirus vector antibodies in humans [ ] have been resolved by adopting simian adenoviruses as vaccine vectors. a number of different replication-deficient vaccine vectors [ ] have recently been developed from simian adenoviruses and the platform has progressed work on emerging pathogens such as ebola virus, rvfv mers-cov and zika virus. modified vaccinia virus ankara (mva) is licensed as a third-generation vaccinia type vaccine against smallpox and serves as a potent vector system for the development of new candidate vaccines against a range of infectious diseases, including those caused by emerging pathogens. historically, mva was developed by serial tissue culture passage in primary chicken cells of vaccinia virus strain ankara, and clinically used to avoid the undesirable side effects of conventional vaccinia vaccines [ , ] . adapted to growth in avian cells, mva does not replicate in mammalian hosts and lacks many of the viral immune evasion genes [ ] . the features of mva, such as its capacity to accommodate large gene inserts [ ] , thermostability for application in remote regions without an established cold chain [ ] , ease of inexpensive manufacture to gmp standards and established regulatory package for development as an investigational new drug, make the recombinant mva platform [ ] highly versatile as a heterologous viral vector. in the context of emerging infections, the recombinant mva platform has shown encouraging preclinical efficacy against ebola, zika, chikungunya [ ] and cchf [ ] viruses. additionally, mva elicits a strong immunological response against a range of other orthopoxviruses (opxvs) (including variola), and vaccines based on this platform can be considered as providing added value, as human immunity to opxvs is low (after the cessation of the smallpox vaccination campaign) opening a gap for opxv emergence, as evidenced by the recent occurrence of monkeypox virus in west africa and onward cross boarder transmissions [ , ] . rna replicon systems are derived from either positive-or negative-strand rna virus genomes and embody disabled virus vectors that are non-pathogenic and unable to revert to virulence. driven by autonomous rna replication, rna replicons result in high-level, cytosolic expression of recombinant heterologous antigens stimulating both the humoral and cellular arms of the immune system. replicon vaccine approaches have closely followed technical developments to genetically manipulate viral genomes. for rna viruses, replicons based on positive-stranded picornaviruses were some of the first [ ] , and these were followed by those based on alphaviruses [ ] and negative-strand rna viruses [ ] . while a series of different vaccine replicon systems are available, new capabilities with negative-strand viruses have opened up opportunities against a wider range of emerging viruses. recent developments [ ] include the construction of lasv replicons packaged into lasv-like particles which allow a single round of replication and are able to confer protection against an otherwise lethal challenge of lasv in a guinea pig model of disease [ ] . while recombinant replicons are devoid of the viral glycoprotein gene, its incorporation into vlps is achieved by the use of a cell line that expresses the glycoprotein separately (in trans). this enables the scalable propagation of replicon particles in a way that aims to combine the safety of replicon-delivered lasv antigens with the convenience of simply and rapidly producing an attenuated virus. similar replicon approaches have been used to develop promising replicon-based vaccine candidates for ebola virus [ ] and rvfv [ ] . replicon approaches have the potency of live attenuated vaccines but are inherently safer, as their design ensures a single cycle of replication in contrast to a fully replicating, live attenuated vaccine virus. for live attenuated rna virus vaccines which incorporate error-prone polymerases, reversion to virulence is a distinct possibility after multiple rounds of replication. interest in the use of virus-like particles (vlps) as vaccines candidates stems from their ability to present ordered and highly antigenic structures to the immune system. at the same time, they lack a viral genome, potentially yielding safer vaccines, as there is no viral vaccines for emerging pathogens: from research to the clinic. part sequence that can revert to virulence. they induce strong b cell responses in the absence of adjuvants by efficiently cross-linking specific receptors on b cells [ ] , and they can also trigger t cell-mediated responses [ ] . the basis of ordered viral self-assembly from protein subunits that is noted in many different virus families provides the foundation for work with vlps, with more than different viruses that infect humans or other animals being identified as able to produce vlps. they are structurally diverse, having single or multiple capsid proteins, or a lipid envelope, although not all viruses are suitable vlp candidates. those which are can elicit a protective response without requiring multiple booster shoots, thus significantly reducing the vaccine costs. to date, vlp-based vaccines for human papilloma virus (hpv), hepatitis b virus (hbv) and hepatitis e virus (hev) have been licensed and are commercially available worldwide [ ] . several vlp-based vaccine candidates for human diseases are under clinical development, including those directed against norwalk virus, ebola and marburg viruses and hepatitis c virus. vlp vaccines combine many of the immunogenic advantages of whole-virus vaccines with the safety advantages of recombinant subunit vaccines. dna vaccines have emerged as a safer alternative to standard live and inactivated vaccines for treating human and animal infections [ ] . they exhibit several advantages over traditional strategies in terms of safety, stability, ease of manufacturing and immunogenicity [ ] . they offer potential advantages for vaccination against emerging viruses, in that plasmids expressing a viral antigen can be produced rapidly. furthermore, antigen is expressed in vivo and induces both humoral and cell-mediated immune responses. additionally, large quantities of dna can be produced in a short time at reduced cost, and dna preparations are more stable than other types of vaccines, which are desirable properties for a vaccine that may be used in remote areas. furthermore, dna vaccines are considered safe. however, the main limitation in the development of dna vaccines is their intrinsic low immunogenicity. work to improve this has focused on optimizing delivery approaches with the use of gene guns, or electroporation; targeting immune effector cells; and the use of potent adjuvants. dna vaccines are also frequently used in combination with other vaccine platforms in heterologous prime-boost strategies. a dna vaccine is currently licensed to immunize horses against west nile virus [ ] and has undergone phase i clinical trials in humans [ ] . dna vaccines have also been evaluated as candidates against many emerging viruses, including ebov [ ] , rvfv [ ] , dengue virus [ ] and chikv [ ] . synthetic peptide-based epitope-vaccines (evs) make use of short antigen-derived peptide fragments that can be presented either to t cells or b cells [ ] . evs offer several advantages over other forms of vaccines, particularly with regard to safety, ease of production, storage and distribution, without cold chain issues. they also offer the opportunity to vaccinate against several pathogens or multiple epitopes from the same pathogen. however, drawbacks include poor immunogenicity and the restriction of the approach to patients of a given tissue type [human leucocyte antigen (hla) haplotype] [ ] and, as such, they need to be tailored to accommodate the natural variation in hla genes. although initially this was thought to be a major impediment, new technologies have made this personalized-medicine approach feasible [ , ] . recently, bioinformatics tools have been developed to identify putative cd + t cell epitopes, mapped to the surface glycoproteins of the emerging viruses lasv, nipv and hendra [ ] . while these vaccine candidates still need to be experimentally tested, the approach represents an interesting and novel strategy that shows promise for vaccination and which could also address immunity in particular target populations. the induction of immune responses by the delivery of inactivated pathogens has been a standard and successful vaccination approach for many years, and licenced, inactivated vaccines for diseases such as poliomyelitis [ ] and rabies [ ] are commercially available. the long history of this approach is underpinned by a well-defined regulatory framework that can be readily applied to new disease targets [ ] . the major challenge for the inactivated virus approach is that infection is not established, and therefore a full adaptive immune response is generally not achieved. however, because of the absence of living pathogens, these types of vaccines are safe and a basic capability to prepare such vaccines, especially for emergency use, might be worthwhile as a stop-gap while alternative longer-term approaches are developed. in this regard, studies of virus inactivation with x-ray radiation (as a simple and cheap alternative to gamma irradiation by the use of radioactive isotopes), which maintain the tertiary antigenic structures of virus particles while destroying infectivity, have shown useful promise for a range of applications including vaccination (b. afrough, unpublished). vaccines play a pivotal role in host protection against infectious diseases and have significantly reduced mortality worldwide. however, many vaccine candidates for emerging diseases have failed to make it into clinical development. this is perhaps surprising, given the breadth of vaccine technology available and the nature of many of the diseases in question. a case in point is lassa fever, a viral haemorrhagic illness endemic to many parts of west africa responsible for more than cases of serious disease and approximately deaths each year since its discovery more than years ago. it is often in the headlines, being the most commonly exported vhf to other territories, including the united kingdom, which has received a disproportionate share of traveller-related cases (each incident placing substantial burden on public health resources). during , lasv caused an unusually large increase in cases in nigeria, which led the who to classify it as a grade public health emergency. however, despite the high burden of lassa fever and public attention, no vaccines have so far been approved. multiple approaches to develop a range of effective preclinical candidates have been made during the last three decades, including attenuated vaccines [ ] , replication competent vaccines, [ ] [ ] [ ] , non-replicating lasv vaccines [ ] , a rationally designed live attenuated vaccine [ ] and dna vaccines [ ] . additionally, many of these platforms have shown efficacy in animal models including nhps [ , , ] . these preclinical data illustrate that multiple vaccine technologies have the potential to yield protective lassa fever vaccines. therefore, the lack of a clinical vaccine after years since the disease was first described must be due to other factors, such as economic considerations or safety issues, perhaps connected with the growing burden of regulatory thresholds for human medical interventions. thus, bringing a lasv vaccine and, by analogy, other potential vaccines for emerging diseases to the clinic, may be very difficult -for non-technical reasons. although there is renewed interest from multiple international agencies to develop human vaccines for certain emerging pathogens [ , ] , it is prudent to consider other approaches to the control of emerging disease, including the feasibility of controlling infections at source. the pattern of disease emergence from viral pathogens into humans from wildlife reservoirs is a clear and present threat [ ] [ ] [ ] [ ] [ ] which will continue. this makes the task of identifying, controlling and preventing zoonoses a difficult and daunting goal, particularly when a new emerging pathogen may be completely unknown. while surveillance is an essential component of a successful control programme, effective containment of an emerging pathogen, before epizootics have the opportunity to spill over into human populations, has been achieved most effectively by large-scale culling or mass vaccination [ , ] of animals. nevertheless, the ability to contain even known emerging viruses such as ebola virus in wildlife is currently not possible. furthermore, the management of diseases that involve livestock, such as rvf and cchf, pose problems [ , ] in that conventional vaccines are not suited for use in these environments. a major limitation of conventional vaccination is the requirement for individual inoculation of each animal -a costly and impractical strategy for the target/reservoir species of those animals frequently involved in the emergence of high-risk pathogens [ ] . surveillance work focusing on epizootics that are, or may become, human pathogens is a useful goal. currently however, predicting which animal pathogens will become established as globally significant emerging human diseases is guesswork. nevertheless, in the early stages of a new zoonosis, pathogenic viruses are often poorly adapted to their new human host in terms of sustained human-tohuman transmission [ ] . this lag-phase in early zoonosis may, therefore, provide a window of opportunity to control the unrelenting zoonotic pressure of an emerging pathogen before it adapts further to humans. vaccine targeting of the pathogen within the animal transmission species could therefore bring useful advantages. self-disseminating vaccines, which aim to immunologically contain emerging viruses within their non-human reservoir hosts, offer an alternative to the conventional vaccine approach. they are designed to exploit the ability of replicating virus-based vectors to spread through animal host populations, so avoiding the need for direct inoculation of every animal. in this way, vaccination of a limited number of initiator animals is used for the introduction of the vaccine into a target population. the vaccine is engineered to express target antigens from the emerging pathogen of interest, so its transmission from vaccinated to non-vaccinated animals will result in the co-ordinated spread of specific immunity for the emerging pathogen throughout the targeted animal population. following early work to underpin a proof of principle for a disseminating vaccine against an animal pathogen [ , ] , a study targeting the human pathogen sin nombre orthohantavirus (snv) in its rodent reservoir -the deer mouse (peromyscus maniculatus) -proved effective. this approach used an engineered cytomegalovirus (cmv) vector (which causes a benign but transmissible infection in the host), expressing the snv envelope glycoprotein g [ ] . a similar approach is also being developed to interrupt zoonotic transmission of ebola virus [ ] , in this case disseminating cmv vaccines specific to great apes and expressing ebov antigens are being studied in african ape populations in the wild [ ] . interestingly, one of the goals of this work is to protect the great apes themselves from ebola virus disease, a major threat to the survival of these animals in the wild. also, as vaccines for emerging pathogens: from research to the clinic. part approximately % of human ebola virus outbreaks are known to have resulted from the direct handling of infected ape carcasses, the disseminating cmv-based strategy may be significant in protecting humans [ ] . clearly, further work needs to be conducted to assess the risks of live transmissible vaccines evolving into a pathogen with increased virulence. engineering such wild-life reservoir vaccines to be weakly transmitting, such that their reproduction number is below (r < ), making their transmission chains short so that they cannot be maintained, might be a way to address the justifiable safety risks. indeed, a mathematical model has recently demonstrated the value of such an approach [ ] . emerging pathogens represent one of the greatest risks to global health. there is already good evidence [ , ] that zoonotic pathogens will most probably be transmitted from a few key animal species in resource-poor areas of the world. based on recent history, it is probable that such pathogens have never been seen before. the global impact of the west african outbreak of ebola virus in underlines how stark differences in health-care infrastructure can impact upon human-to-human transmission of emerging pathogens. until basic health-care infrastructure in all countries can be raised to a level that enables early identification and control of high-risk pathogens at source, we will continue to respond to outbreaks of emerging disease long after epizootics have spilled over into human populations. innovative strategies are therefore urgently required to control such pathogens, vaccination is a proven approach. many novel vaccination strategies that have been developed during recent years have the potential to specifically address the growing threat of new and emerging disease. the use of well-defined vaccine vector platforms, with an extensive record of safety and efficacy against similar pathogens, can expedite the process of development, validation and production (table ) . accordingly, the design and licensure for particular platform vaccine technologies will help to accelerate the development of new vaccines, as only the simple substitution of a new antigen gene into the vector platform is required. this allows manufacturers to move to a new target disease with minimal changes in chemistry, manufacturing and controls. thus, new vaccine development can focus on the safety and efficacy of the inserted gene. in addition, the ability of platforms to target multiple pathogens helps to justify the investment required to build and maintain manufacturing infrastructure that specializes in one platform, because a single manufacturing facility can be ready to produce multiple vaccines at any time. in addition, further research into, and the development of, self-disseminating vaccines to control potential pathogens in their wild-life reservoirs should be encouraged. however, the progress of new vaccines through the necessary regulatory pathways to bring them to the clinic requires long-term investment by governments and international organizations. global trends in emerging infectious diseases emerging pathogens: the epidemiology and evolution of species jumps bats and emerging zoonoses: henipaviruses and sars spillover and pandemic properties of zoonotic viruses with high host plasticity ecology of zoonoses: natural and unnatural histories after ebola in west africa -unpredictable risks, preventable epidemics a research and development blueprint for action to prevent epidemics rna viruses: emerging vectors for vaccination and gene therapy immunologic basis of vaccine vectors viruses -from pathogens to vaccine carriers viral vectors as vaccine platforms: deployment in sight self-replicating alphavirus rna vaccines rapid development of vaccines against emerging pathogens: the replication-deficient simian adenovirus platform technology the evolution of poxvirus vaccines live virus vaccines based on a vesicular stomatitis virus (vsv) backbone: standardized template with key considerations for a risk/benefit assessment rhabdoviruses as vaccine vectors: from initial development to clinical trials. biology, pathogenesis of rhabdo-, filoviruses attenuated vesicular stomatitis viruses as vaccine vectors yellow fever and max theiler: the only nobel prize for a virus vaccine pathogenesis and pathophysiology of yellow fever transcription of infectious yellow fever rna from full-length cdna templates produced by in vitro ligation the yellow fever d virus as a platform for new live attenuated vaccines from research to hase iii: preclinical, industrial and clinical development of the sanofi pasteur tetravalent dengue vaccine creation of a recombinant rift valley fever virus with a two-segmented genome ires dependent replication of venezuelan equine encephalitis virus makes it highly attenuated and incapable of replicating in mosquito cells ires-based venezuelan equine encephalitis vaccine candidate elicits protective immunity in mice modulation of poliovirus replicative fitness in hela cells by deoptimization of synonymous codon usage in the capsid region development of live attenuated arenavirus vaccines based on codon deoptimization adenoviruses as vaccine vectors pre-existing immunity against ad vectors: humoral, cellular, and innate response, what's important? a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity recombinant vaccinia viruses as vaccines mva vaccination against smallpox: clinical tests with an attenuated live vaccinia virus strain (mva) [author's translation] environmental risk assessment of clinical trials involving modified vaccinia virus ankara (mva)-based vectors infectious poxvirus vectors have capacity for at least base pairs of foreign dna long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass modified vaccinia virus ankara: history, value in basic research, and current perspectives for vaccine development vaccinia-based vaccines to biothreat and emerging viruses understanding orthopoxvirus host range and evolution: from the enigmatic to the usual suspects emergence of monkeypox as the most important orthopoxvirus infection in humans engineering poliovirus as a vaccine vector for the expression of diverse antigens selection of rna replicons capable of persistent noncytopathic replication in mammalian cells replicon rna viral vectors as vaccines use of a scalable replicon-particle vaccine to protect against lethal lassa virus infection in the guinea pig model replication-deficient ebolavirus as a vaccine candidate single-dose immunization with virus replicon particles confers rapid robust protection against rift valley fever virus challenge virus-like particles in vaccine development virus-like particles as particulate vaccines formulation and stabilization of recombinant protein based virus-like particle vaccines vaccines for the st century dna vaccines: recent developments and future possibilities dna vaccines -back in the saddle again? a west nile virus dna vaccine utilizing a modified promoter induces neutralizing antibody in younger and older healthy adults in a phase i clinical trial a dna vaccine for ebola virus is safe and immunogenic in a phase i clinical trial a dna vaccine encoding ubiquitinated rift valley fever virus nucleoprotein provides consistent immunity and protects ifnar(s/s) mice upon lethal virus challenge immunogenicity and protective efficacy of a vaxfectin-adjuvanted tetravalent dengue dna vaccine a dna vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates more than one reason to rethink the use of peptides in vaccine design hla class ii restriction of hiv- clade-specific neutralizing antibody responses in ethnic thai recipients of the rv prime-boost vaccine combination of alvac-hiv and aidsvax((r)) b/e the tubingen approach: identification, selection, and validation of tumorassociated hla peptides for cancer therapy innovative bioinformatic approaches for developing peptidebased vaccines against hypervariable viruses a bioinformatics tool for epitope-based vaccine design that accounts for human ethnic diversity: application to emerging infectious diseases polio vaccines: who position paper an overview of the immunogenicity and effectiveness of current human rabies vaccines administered by intradermal route a global regulatory science agenda for vaccines a live attenuated vaccine for lassa fever made by reassortment of lassa and mopeia viruses development of a new vaccine for the prevention of lassa fever yellow fever d-vectored vaccines expressing lassa virus gp and gp glycoproteins provide protection against fatal disease in guinea pigs vaccinia recombinant expressing lassa-virus internal nucleocapsid protein protects guinea pigs against lassafever development of live-attenuated arenavirus vaccines based on codon deoptimization of the viral glycoprotein enhanced efficacy of a codon-optimized dna vaccine encoding the glycoprotein precursor gene of lassa virus in a guinea pig disease model when delivered by dermal electroporation safety, immunogenicity, and efficacy of the ml reassortant vaccine for lassa fever in small non-human primates a dna vaccine delivered by dermal electroporation fully protects cynomolgus macaques against lassa fever who publishes list of top emerging diseases likely to cause major epidemics global partnership launched to prevent epidemics with new vaccines zoonosis emergence linked to agricultural intensification and environmental change impact of vaccines and vaccination on global control of avian influenza novel approaches to develop rift valley fever vaccines serological and virological evidence of crimean-congo haemorrhagic fever virus circulation in the human population of borno state, northeastern nigeria cross-species virus transmission and the emergence of new epidemic diseases isolation of an attenuated myxoma virus field strain that can confer protection against myxomatosis on contacts of vaccinates the current status and future directions of myxoma virus, a master in immune evasion replication and immunoactivity of the recombinant peromyscus maniculatus cytomegalovirus expressing hantavirus g glycoprotein in vivo and in vitro a cytomegalovirus-based vaccine provides long-lasting protection against lethal ebola virus challenge after a single dose ebola: outbreaks cause crisis for great apes and humans. toronto: the jane goodall institute of canada self-disseminating vaccines for emerging infectious diseases eradicating infectious disease using weakly transmissible vaccines the authors confirm that they have no competing interests in this work. key: cord- - obomty authors: pardon, bart; buczinski, sébastien title: bovine respiratory disease diagnosis: what progress has been made in infectious diagnosis? date: - - journal: vet clin north am food anim pract doi: . /j.cvfa. . . sha: doc_id: cord_uid: obomty when it is desired to identify infectious agents involved in an outbreak of bovine respiratory disease, a variety of possible sampling methods may be used. for field use, the deep nasopharyngeal swab, transtracheal wash, and nonendoscopic bronchoalveolar lavage are most feasible. at present, bacterial culture and polymerase chain reaction testing are most commonly used to identify infectious agents. interpretation of test results can be challenging, particularly for opportunistic pathogens. evidence-based guidelines for precise interpretation of microbiologic tests results are lacking; however, approaches that have been practically useful for the management of bovine respiratory disease outbreaks are presented. increasing pressure on antimicrobial use and the need for veterinarians and farmers to use antimicrobials more rationally. the use of diagnostic support by laboratory analysis is one of the frequently mentioned cornerstones of antimicrobial stewardship programs. however, scientifically reasoned, the evidence that systematic use of laboratory diagnostics, especially the antibiogram, would result in selection of a different first-choice therapy compared with an empiric decision preferably following (evidence-based) guidelines, is limited, especially in cattle. antimicrobial resistance in respiratory tract bacteria from cattle is present and varies highly between systems. resistance levels are generally lower in closed dairy and beef herds, substantially higher in feedlots, and most worrisome in veal calf operations, where oral mass medication is frequently used. , although there is no doubt of the presence of resistance, and multiresistance, in respiratory bacteria from cattle, to what extent this results in therapy failure when following guidelines for antimicrobial therapy is poorly documented. in recent years, guidelines specifying first-line, second-line, and third-line antimicrobial choices for the different cattle diseases have been initiated in several european union countries, including the netherlands, belgium, denmark, sweden, and germany. , , however, the amount of literature reporting the clinical benefit of every antimicrobial-bacteria combination in highly variable field settings is currently very limited. therefore, these guidelines mainly include the spectrum of the antimicrobial, pharmaceutical leaflet recommendations and follow classification of the importance of antimicrobials for human medicine of the world health organization. in contrast with human medicine, to the authors' knowledge there are no extensive, sufficiently detailed, and large-scale studies available on therapy failure caused by antimicrobial resistance in cattle. regardless of the limitations mentioned earlier, diagnostics are more and more frequently used when addressing bovine respiratory disease (brd). this increased use is understandable, because antimicrobial decision making for brd still often involves a decision to use group therapy, and, in the current climate, mass medicating without any evidence of the need for this therapy will increasingly be criticized. the authors fully acknowledge the complexity of advising on the implementation and interpretation of diagnostic tests for brd, given the huge gaps in the current knowledge. however, the need is urgent, and therefore this article provides a framework to assist practitioners and clinicians in their everyday decision-making process. this reasoning may not withstand time; this article does not represent a consensus of all leading experts, nor is the objective to provide a complete literature overview. this discussion reflects on the body site sampled, the test used, and the pathogen detected. the selected sampling site of the respiratory tract is of great importance for interpretation of the test result. and endoscopic bronchoalveolar lavage [bal] ; fig. ). nasal swabs, predominantly sampling the cutaneous part of the nose, are generally considered of limited value for infectious diagnostics. in contrast, dnss sample the respiratory and associated lymphoid epithelium of the nasopharynx and return more meaningful samples. however, the biggest issue with nasopharyngeal swabs is the large number of polymicrobial samples recovered (> %), which heavily compromises clinical interpretation when only opportunistic pathogens are retrieved. contamination can be reduced by rinsing the nares (with a single-use paper towel or a gauze with alcohol) or by using a guarded dns. however, studies specifically focusing on the effect of guarded swabs to reduce nasal contamination are, to the authors' knowledge, not available in cattle. recent reports on the respiratory microbiome in cattle also put the idea of contamination at that sampling site into another perspective, given the large variation in bacterial species normally present. , the largest disadvantage of dnss is that they do not directly sample the lower respiratory tract. despite some conflicting results, previous studies overall showed that, for most pathogens, an association between dns results and ttw or bal is present. , [ ] [ ] [ ] in addition to cotton swabs, brush swabs also exist, which cause more intensive swabbing of the mucosa (although possibly also blood staining of the sample), presumably with higher detection rates. no evidence on their benefit for use in cattle is currently available. complications of dns are rare and included nasal hemorrhage and fracture of the shaft of the swab. the latter is without any harmful consequence because the animal evacuates the remaining part of the swab either by sneezing or swallowing. to overcome the issue of nasal contamination, transtracheal sampling techniques relying on perforation of the trachea with a needle or catheter after surgical preparation of the skin have been developed. historically, transtracheal swabs have been used, but the transtracheal aspirate and wash are now common. although an aspirate (tta) only involves aspiration of mucus present in the respiratory tract, a wash (ttw) requires fluid instilment and immediate aspiration. despite the terminology tta being frequently used in the field, the technique usually used is a ttw. most frequently, for ttw in cattle, ttw kits (large animal trans-tracheal wash kit, mila international, inc, florence, ky), or human central venous catheters (eg. centracath , vygon, ecouen, france) are used, which are commercially available and sterile packed for single use. alternatively, a male dog urinary catheter can be used in combination with a -g catheter/needle to perforate the trachea in between tracheal rings. in veterinary medicine, the common thinking is that the ttw is preferred for bacteriology and bal to study inflammation (cytology). however, this recommendation generally comes from horse medicine, and seems to be expert opinion rather than supported by substantial peer-reviewed studies. in humans, ttw is generally not used for ethical reasons. the general idea is that the bronchial bifurcation is the site where the efflux of the mucociliary system of the whole of the lung comes together. hence sampling there would be representative for the whole of the lung. however, there are some counterarguments for this reasoning. first, the mucociliary system can be heavily impaired by pneumonia. second, microbial aspiration from the nasopharynx into the upper trachea is likely frequent. third, normal pathogenesis involves gradual descent of bacteria down the respiratory tree toward the lung. taking the second and third arguments into account, a positive ttw culture might equally represent a bacterial tracheitis or even an insignificant colonization or upper airway contamination, resulting in false positive diagnosis of infectious bronchopneumonia. advantages are that a new disposable catheter can easily be used for each animal, and sampling is theoretically achievable within a predictable time frame given that no active cooperation of the animal is required, in contrast with bal. however, sedation of the animal and local anesthesia of the puncture site can be done to improve animal comfort during the procedure. in a bal procedure, a bal catheter or flexible endoscope is introduced through the nose and trachea into the lower airways until it wedges into a larger (or smaller depending on catheter diameter) bronchus. next, while holding in this wedged position, a volume (usually ml in calves, if necessary followed by a second or third injection) of sterile saline is injected and immediately aspirated. classically, as in human medicine, a bal is performed by endoscopy. the major advantage is that a specific lung lobe, previously shown to be affected on radiology or ultrasonography, can be sampled. also, protective sheets or agar plugs can be used to reduce the risk of nasal contamination. the major disadvantage of the endoscope is the high operating costs and risk for equipment damage in the farm setting. also, sampling multiple animals becomes difficult because time to resterilize the endoscope between animals is needed ( - minutes minimum). to overcome the cost and risks of endoscopic bal, nbal techniques have been developed. in nbal, a bal catheter is blindly introduced through the nose, larynx, and trachea until the wedged position in a large bronchus is reached. next, a volume of saline is injected and gently aspirated. the volume used varies substantially between studies ( - ml , ), but a trend to reduce the volume for welfare/comfort reasons is present. on average, . % of the volume ( . %- . %) can be recovered in nbal, which is substantially larger than in a ttw procedure. it is important to realize that sedation not only suppresses the required responses (coughing, curving of the nose, and extroversion of the tongue) to ensure an intratracheal position but also causes systematic sampling of the diaphragmatic lung lobes, which are less likely to be affected. good restraint of the calf with the head fixed with the nose pointing upward as much as possible is advisable to ease blind introduction of the tube into the trachea. alternatively, the calf might be surprised into allowing the tube to be advanced into the airways by placing the head in a horizontal position, and introducing the catheter on inspiration visible by the opening of the nostrils. overall, in % of animals, nbal sampling can be completed within minutes. for the remaining %, the practical advice is to select another animal to sample when undertaking group diagnosis, rather than spending excessive time and causing prolonged irritation to a reluctant animal. alternatively, a technique where a double-guarded bal catheter is orally introduced into the larynx through a pvc (polyvinyl chloride) speculum has been described for calves that are at least to months old, where the guarded catheter is inserted through the larynx under visual control. the use of bal samples (especially nbal) for bacteriology is still highly controversial, mainly because of the risk of nasal contamination. although contamination is far less than with dns, . % of nbal samples were still polymicrobial. a large influence of the sampler seems to be present, likely depending not only on differences in hygienic sample handling but also on skills to swiftly introduce the catheter without touching too much of the nasopharynx. however, it is important to realize that hard evidence on substantial nasal contamination by using nbal catheters is currently not available in any species. the only available study on this matter showed pure culture and negative results in . % and . % of the nbal samples, even though dns samples of the same animals were polymicrobial. further, the currently most extensive study on sample method comparison showed very good agreement for bacteriology between dns, ttw, and nbal. interestingly, in human medicine, there are growing efforts toward the use of a mini-bal procedure for bacterial diagnosis in ventilator-assisted pneumonia. overall, sample contamination should be avoided and, in the case of nbal, this can be done by adequate training or visualization of the larynx by a video speculum (ivetscope, dairymac limited, hampshire, united kingdom) or endoscopic cameras intended for plumbers or auto mechanics. these devices are available at much lower prices than traditional endoscopes. next to the site of the respiratory tract sampled (upper or lower airway), the cultural perception of the effect of the sampling technique on animal welfare also plays an important role in what technique is currently preferred in a given country/region. no studies on the effect of respiratory tract sampling on stress or pain have yet been conducted in calves. a master of science thesis showed that both animals sampled by dns or nbal spent less time walking compared with the unsampled control group, whereas lying or eating were unaffected. for ttw as well as nbal, the required volume of saline to be instilled is unclear; it is also unclear whether the volume instilled influences bacteriology results, as it does for cytology. in summary, sampling techniques for the field need to be economically feasible both in terms of equipment/disposables cost and also invested time. the dns, ttw, and nbal best suit this profile and are currently most frequently used in the field. differences in use exist between countries, which mainly originate from historical or cultural preference. an overview of available diagnostic tests for pathogen identification in respiratory diseases in cattle is shown in table . it is beyond the scope of this article to provide a complete overview of all tests possible. the focus is on the most frequently used tests and the most promising future tests likely to become widely available for practice within the next years. in the current international context, the pressure to reduce antimicrobial use has become the main driver of diagnostic test performance for causal diagnosis of brd. a crucial aspect for field efficacy is a short turnaround time (tat), the time between sampling and availability of the test result. in order to be able to use the diagnostic test result to target therapy or initiate control measures tat needs to be as short as possible, ideally less than a day. however, having test results the next morning might also be workable for most outbreaks. also, the use of cow-side testing for a causal diagnosis of brd has great potential to reduce tat. however, to the authors' knowledge, no such tests are currently commercially available. hence, attention should be given to ensuring proper and timely transport to the laboratory. at refrigerator temperature ( c- c), the isolation rate of mannheimia haemolytica and pasteurella multocida was not reduced for hours, whereas a transport temperature of more than c resulted in reduced isolation as soon as hours later. serology serologic tests are useful to target vaccination programs, to determine protective status, and to evaluate infection dynamics at larger scale. however, they are not suitable to direct immediate therapy because they have a tat of weeks (required time for seroconversion) and only provide indirect evidence of infection. also, for the opportunistic pasteurellaceae family, maternal immunity smoothly shifts to acquired immunity, without any signs of disease or seroconversion. another important issue is that sensitivity and specificity can be highly variable between different antibody enzymelinked immunosorbent assays (elisas), hampering clinical interpretation and their use for individual animal decisions (eg, culling or purchase). for targeting therapy, direct identification of the pathogen is needed, and this can be achieved by microbial culture, matrix-assisted laser desorption/ionization time of flight (maldi-tof) mass spectrometry (ms), pcr, or ngs/third-generation sequencing. microbial culture is most frequently used for identification of bacteria. next to low operating costs, the possibility of antimicrobial susceptibility testing is an important advantage of culture. for mycoplasmata, specific media are required, and fastidious growers, especially histophilus somni, are easily overgrown, resulting in false-negative results. , sensitivity and specificity of microbial culture have not been determined for most of the bacteria involved in brd. a recent study using bayesian latent class analysis showed that mycoplasma bovis culture on solid medium containing tween is . % ( % bayesian credible intervals [bci], . to . ) sensitive and . % ( % bci, . - . ) specific. in the last decade, maldi-tof ms, which identifies bacteria by their unique protein profiles, has revolutionized routine diagnostics. it is primarily used for identification of bacteria after culturing, including mycoplasma species. however, maldi-tof ms can also be applied directly on the sample after a very short period of enhanced growth in a liquid medium. relative to classic microbial culture, these culture-enriched direct maldi-tof ms techniques allow correct bacterial identification in % of the samples (sensitivity . %; % confidence interval [ci], . - . ; specificity % [ - ]) within hours. the technique performed less well in polymicrobial samples and in samples with mixed infection. also for m bovis, a culture-enriched direct maldi-tof ms technique was developed, which was . % ( % bci, - ) sensitive and . % ( % bci, - ) specific in a bayesian latent class model including pcr and microbial culture on solid agar. tat was reduced from more than days to less than days. in addition, different maldi-tof ms methods are available for antimicrobial susceptibility testing. by means of mbt-astra (maldi biotyper antibiotic susceptibility test rapid assay), oxytetracycline resistance in p multocida could be identified with high accuracy (se . %; % ci, . - . ; sp %; [ % ci, - ]) in as little as hours, outperforming the disc diffusion antibiogram. the mbt-astra technique can be designed for every bacterium-antibiotic combination, but logistical changes are needed to create a good intralaboratory workflow. the costs of maldi-tof procedures are generally low, in line with microbial culture. pcr for the causal diagnosis of brd is now very popular. the main reasons are that multiplex pcr or multiple single pcrs allow detection of multiple bacteria and viruses, providing practitioners with a more extended view of the pathogens involved and hence more options to better target therapy, control, and prevention. fastidious and metabolically active viable but unculturable viruses and bacteria can be detected, in contrast with standard microbial culture. however, in contrast with sequencing techniques, specific primers are needed, and the pathogen of interest needs to be determined beforehand. in this way, the diagnostics are potentially biased and possibly lead to false-negative results. another problem is that viral genomes evolve rapidly and primers might become outdated, limiting the efficient detection of the pathogens of interest. pcr is generally not cheap, but, by pooling samples (dns, ttw, or bals), a group diagnosis can be reached and costs are decreased. in available studies, pools of samples from animals were shown to improve diagnostic accuracy at the group level. , the largest disadvantage of pcr is interpretative difficulty, because pcr can identify dead pathogens, opportunists currently not involved in infection, and contaminants, none of which signify a clinically meaningful test result. this disadvantage was shown, for example, for h somni. the use of quantitative pcr is more informative because the pathogen load, especially in the respiratory disease complex, is important to consider. for this reason, quantitative pcr is increasingly used in veterinary laboratories, although interpretative questions remain. especially, when multiple pathogens are detected, determining the attributable fraction of each pathogen to the clinical presentation remains very difficult. next-generation and third-generation sequencing ngs technologies are now becoming more widely available because of the democratization of the technologies, and because platforms such as minion (oxford nanopore technologies, oxford, uk) allow decentralized sequencing experiments. the first studies using ngs (metagenomics) to detect viruses involved in brd in feedlots are already reported; these detected known pathogenic viruses as well as previously unknown or incompletely understood viruses (eg, influenza d virus). , hence, the advantage of ngs is that all pathogens can be simultaneously detected, without prior selection of which pathogens to test for. also, semiquantitative results can be reported because, for most viruses, the number of reads corresponds to the initial load of the pathogen present. not only viruses but also whole genomes can be recovered at a scale that is constantly increasing. eventually, direct sequencing of bacteria will allow detection of virulence genes, phylogenetic clustering of strains during outbreaks, and ultimately prediction of antimicrobial resistance based on single nucleotide polymorphisms or resistance genes. a high total bacterial burden and low bacterial community diversity were associated with positive culture results in classic microbial culture. ngs is the basis for microbiome studies, which are discussed elsewhere in this issue. disadvantages are that ngs is costly and requires a long tat under the current conditions using the most accurate devices (eg, illumina, san diego, ca). however, with nanopore sequencing platforms (eg, minion) a higher throughput and shorter tat can be achieved. this long-read technology has been commercially available since and has made tremendous improvements in output and accuracy. in humans and pigs, minion has been used to characterize pathogens in different types of samples, even at the site of disease outbreaks, because data analysis can be done in the field on portable hardware. , on human lower respiratory tract samples, within hours of sampling, a result was given at a sensitivity of . %. however, in order to achieve wide implementation in veterinary practice, the cost, the ability to correctly interpret, and setup of an actionable logistic chain will be essential. therefore, this technology is another case in which analysis of pooled samples from multiple animals to obtain a group diagnosis of primary pathogens will be the most likely application. clinical interpretation of a diagnostic test result to determine the infectious cause of a respiratory tract disease requires information on the pathogen identified, the site of the respiratory tract sampled, the diagnostic test used, the clinical condition of the animal, and whether the sample originates from a single animal or is pooled. there is no current consensus on the way to sample the respiratory tract or to interpret diagnostic test results in humans and many other species. based on the available research, it is unlikely that an evidence-based consensus on respiratory tract sampling method, diagnostic testing, and interpretation of results in cattle can be reached. hence, this article assists readers to properly interpret results of testing by providing information not only on current recommendations but especially on the drawbacks and research gaps. the first point to consider is the nature of the pathogen retrieved, whether it is a primary or secondary pathogen. a primary or obligate pathogen (when present), per definition, induces damage to the respiratory tract, mostly followed by an inflammatory response. however, depending on the infectious dose and host immunity, infection might result in clinical disease or not. also, certain primary pathogens can chronically and even asymptomatically infect animals, resulting in carriers: for example, salmonella spp or m bovis. most primary pathogens weaken innate immunity of the airways, facilitating superinfection by opportunistic bacteria. some, such as bovine respiratory syncytial virus, bovine herpesvirus type (bhv- ), and potentially also others ( table ) , are able to induce life-threatening disease without bacterial superinfection. despite still being controversial in some scientific communities or countries, m bovis is generally considered a primary pathogen. detection of a primary pathogen can, with some caution, be interpreted straightforwardly. the primary pathogen should normally not be present, and, depending on its virulence, it can, either as a sole agent or in combination with other agents, be held responsible for the clinical picture. also, detection from any site of the respiratory tract is meaningful. for animal welfare reasons and following the pathogenesis, which starts with nasal infection, dns samples might be sufficient and even most appropriate to detect primary pathogens. however, detection rates at the different sites of the respiratory tract differ between pathogens. for bovine coronavirus, dns was more frequently positive than samples from the lower respiratory tract, whereas the inverse was true for bovine respiratory syncytial virus. for bhv- , dns is recommended given that the infection most frequently remains limited to the upper airways. the true interest of any diagnostic effort lies in extrapolation of test results from the sampled animals to the whole group. detection of primary pathogens in some animals makes involvement of the same pathogen in the part of the resident flora, differences in strain virulence described possibly resulting in some primary pathogenic strains. other studies show cattle to become ill from their own resident strain on exposure to other pathogens and/or risk factors , , chlamydia psittaci controversial, likely primary natural infections result in mild or subclinical disease (continued on next page) moraxella bovis/ovis secondary primary eye pathogen, occasionally isolated in pure culture from animals with bronchopneumonia pasteurella multocida secondary part of the resident flora. strain virulence differences exist, and some disease presentations (eg, septicemia or peritonitis) have been linked to certain strains , salmonella spp primary primary site of infection of most salmonella spp is the gastrointestinal tract. localization in the respiratory tract is possible, most likely after septicemic spread trueperella pyogenes secondary involved in purulent processes. often regarded as characteristic for chronicity. however, naturally resistant to fluoroquinolones escherichia coli, gallibacterium anatis, enterobacter hormaechei, staphylococci, streptococci, fungi secondary single reports on cattle-specific strains isolated in pure culture in an outbreak of pneumonia in calves , [ ] [ ] [ ] multiple other bacterial species can be detected in the bovine respiratory tract. this table is limited to either known primary pathogens or frequently isolated pathogens, currently assumed to have a pathogenic significance. data from refs. , , , , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] pardon & buczinski cohoused animals very likely. hence, to improve sensitivity of the group diagnosis, the use of pcr on a pooled sample (up to animals) can be considered. , a pitfall when working with pcr to detect primary pathogens is that vaccine antigen can be detected up to days after intranasal vaccination with a live vaccine, resulting in false-positives. a secondary or opportunistic pathogen can be part of the normal respiratory microbiome, without inducing inflammation. in general, breaching of innate immunity, either by another pathogen or a noninfectious cause, is needed before the opportunistic pathogen invades tissues and induces inflammation. interpretation of detection of an opportunistic pathogen is more difficult, given that they can be present in healthy animals. , , therefore, simply detecting the pathogen cannot be seen as evidence of its involvement. the pasteurellaceae family and a range of other bacteria (eg, streptococcus spp and trueperella pyogenes) are generally considered secondary pathogens. although there seems to be little discussion of p multocida, scientific opinions on the potential primary role and differences in strain virulence of m haemolytica and h somni vary greatly. , it is outside the scope of this article to review or take a position on this matter. similarly, in other species, including humans, this issue of opportunistic pathogens exists. when interpreting a positive culture result, a differentiation between contamination, colonization, and infection needs to be made. contamination is defined as the presence, usually in low numbers, of bacteria in a sample that are not expected to be present in the sampled site. colonization can be defined as the presence of a micro-organism in a host, with growth and multiplication of the organism, but without interaction between host and organisms, hence no inflammatory reaction, immune response, or clinical expression occurs. similarly, infection is isolation of a high number of bacteria from a site of the respiratory tract, but in the presence of inflammation of the mucosa, presenting either clinically or subclinically. hence, simply picking a suspected colony from an agar plate, to confirm the cause of the respiratory disease, and subsequently using an antibiogram based on this single colony may be misleading. more information can be derived from culture results if quantitative descriptions and at least the degree of contamination are described. a possible way to better describe culture results, previously used for research purposes, , is presented in table . it is also important to realize that using selective media for pasteurellaceae, as, for example, by adding bacitracin, ensures better growth and detection of these opportunistic pathogens, but information on the amount of pathogens and degree of contamination of the sample will be lost. , pasteurellaceae are part of the normal respiratory flora, and can even be abundantly present in the nasal cavity of healthy animals. an association between the presence of a pasteurella species in the nose and its presence in the lower respiratory tract is described. , , however, interpretation of dns results for opportunistic pathogens remains very difficult, especially because the composition of the nasopharyngeal microbiota seems to be heavily influenced by bioaerosols from the agricultural environment. loss of biodiversity and overgrowth of opportunistic pathogens occurs in the pathogenesis of brd, resulting in higher odds that pasteurellaceae can be cultured from nasal swabs in larger quantities in ill animals. , , however, with current knowledge on the interpretation of dns results at the individual or group level, samples of the lower respiratory tract are likely a better option to evaluate potential involvement of opportunistic pathogens. interpretation of detection of opportunists in lower respiratory tract samples remains difficult, even in ill animals, because, even with very strict clinical case definitions, pasteurellaceae can also be cultured from the lower airways in healthy animals. , previously explored ways to overcome the issue of interpreting detection of opportunistic pathogens in humans and other species are the use of quantitative cultures or cytologic evidence of inflammation. quantitative culture is derived from the assumption that, in case of a severe infection, the opportunistic pathogen will be present in larger numbers. cutoffs such as greater than colony-forming units per milliliter of bal fluid have been suggested in dogs and horses. , however, pathogen burden builds up, and sampling early in the disease process could mean that much lower numbers are detected despite the pathogen being involved in the inflammatory process. another option is derived from the assumption that a bacterial infection will result in a massive airway neutrophilia. however, clear cutoff percentages for neutrophilia differentiating a bacterial infection from a viral one or a strictly noninfectious airway inflammation have not been determined in calves. given that some bal techniques result in a larger contribution of the bronchial component in the total bal fluid, the neutrophil percentage is increased compared with a larger volume of mainly alveolar lavage fluid. primary insights in calf bal fluid analysis show that cytologic parameters coincide poorly with clinical or ultrasonographical findings or culture results for opportunistic pathogens, at least when using nbal. the presence of phagocytosis by bacteria in neutrophils or macrophages may be helpful to differentiate active infection versus simple presence of the bacteria. although interpreting culture results for opportunistic pathogens is already difficult, interpretation of pcr results is even more so. not only can insignificant quantities or even dead bacteria result in a positive pcr, the nasal passages also pick up bacterial dna, making the lower respiratory tract sample positive. quantitative pcr might overcome this issue, but, to the authors' knowledge, no guidelines on how to interpret these results are currently available in the bovine species. although laboratory costs are fixed, the return on investment of an analysis greatly depends on selection of appropriate animals to sample and on the technical sampling skills of the veterinarian. an animal in the first days of the disease, not previously treated with antimicrobials and not displaying severe respiratory signs, is first choice. by sampling in the acute phase of the disease, the odds of detecting the viral component are higher. by avoiding previous antimicrobial treatment and by sampling early in the disease course, the probability that the antibiogram derived is useful for empiric therapy increases. by avoiding sampling animals in heavy respiratory distress, the odds of aggravation of disease or even mortality can be decreased. in addition, in spite of all reasoning made earlier, veterinarians still make decisions at an individual animal level. when sampling an individual, the main interest is usually to make a decision representing the group, and to judge the utility of the diagnostic test to support this group decision. different epidemiologic approaches are possible to determine an appropriate sample size. the goal is more a detection of disease approach (being % confident that the pathogen was detected when present) rather than determining the prevalence of the pathogen in a group of animals. in the field, sample size is currently more driven by practical reasons such as available time to sample or maximum samples allowed to pool for economic reasons. for example, performing pcr on pools of animals increases sensitivity without diluting the sample too much. fig. provides an overview on the risk of not finding a positive animal in scenarios, related to % prevalence of the pathogen in the diseased population and scenario with a pretest probability that % of sick calves are affected by the pathogen (ie, where multiple pathogens are involved and can cause the same clinical disease). results of a test with % sensitivity (ie, detects the pathogen in of infected calves) and % specificity (no false-positive calves) are presented. using this test in a scenario with % of the affected animals being positive for the pathogen, after calves not finding a positive, will misclassify only . % of the herds (the scenarios assume that the pooled tests' accuracy is the same as the individual test). in the case of opportunistic pathogens, given that they can be found in healthy or subclinical animals, at this time it might be most prudent to only sample animals with evidence of clinical bronchopneumonia by using a combination of clinical scoring and thoracic ultrasonography. focusing on bacterial isolation to direct intervention strategies, without taking the clinical status into account, holds great danger for overtreatment with antimicrobials. knowledge of respiratory health is rapidly evolving in animals, following new developments in humans. in particular, better insights into the role of the respiratory microbiome and the interaction of the airway inflammatory response with different organisms and air pollutants are likely to change how the diagnostic tests discussed in this article are interpreted. the authors hope that the information, tools, and provisional advice provided can aid the large group of cattle veterinarians, already having to make rational treatment decisions today. b. pardon has received honoraria for acting as speaker or consultant for pharmaceutical (zoetis, msd, vetoquinol, dopharma, boehringer ingelheim, dechra, hipra, ceva, merial, and elanco), agricultural (algoet nutrition), and chemical (proviron) companies and nonprofit organizations (boerenbond, amcra, dgz-vlaanderen). s. buczinski has received honoraria for acting as speaker or consultant as well as research grants for pharmaceutical companies (zoetis, msd, hipra, and ceva) and companies involved in commercialization of ancillary tests used in respiratory diseases (ei medical imaging, geissler corp.). fig. . risk (probability ranging between and ) of not finding a positive animal for a given pathogen according to sample size (x axis) in a scenario where % (solid line) and % (dashed line) of affected animals are positive for a given pathogen. the graph represents a test with % sensitivity and % specificity. it assumes that there are no falsepositive results (ie, when the test indicates that the pathogen is present, this is a truepositive result). in the example where the pathogen is causing the disease in % of affected calves, the risk of not finding an infected animal after sampling n cases is ( -se)n , where se is the test sensitivity. in the alternative scenario where only % of cases are caused by the pathogen (ie, in % of cases, this is another cause), the probability of not finding a case is ( - . *se)^n. laboratory test descriptions for bovine respiratory disease diagnosis and their strengths and weaknesses: gold standards for diagnosis, do they exist? fve (federation 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medicated veal calves bovine adenovirus type pneumonia in dexamethasone-treated calves apoptosis in calf pneumonia induced by endobronchial inoculation with bovine adenovirus type (bav- ) structured literature review of responses of cattle to viral and bacterial pathogens causing bovine respiratory disease complex what is the evidence that bovine coronavirus is a biologically significant respiratory pathogen in cattle? bovine viral diarrhea virus-associated disease in feedlot cattle pathogenesis of influenza d virus in cattle randomized clinical trial to evaluate the pathogenicity of bibersteinia trehalosi in respiratory disease among calves evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to days after feedlot arrival recovery of moraxella ovis from the bovine respiratory tract and differentiation of moraxella species by tdna-intergenic spacer pcr fatal peritonitis caused by pasteurella multocida capsular type f in calves salmonellosis in calves influence of systemic fluoroquinolone administration on the presence of pasteurella multocida in the upper respiratory tract of clinically healthy calves first report of enterobacter hormaechei with respiratory disease in calves septicaemic escherichia coli and experimental infection of calves isolation of drug-resistant gallibacterium anatis from calves with unresponsive bronchopneumonia infection, disease, and transmission dynamics in calves after experimental and natural challenge with a bovine chlamydia psitacci isolate key: cord- -rlvk bcx authors: balloux, francois; van dorp, lucy title: q&a: what are pathogens, and what have they done to and for us? date: - - journal: bmc biol doi: . /s - - -z sha: doc_id: cord_uid: rlvk bcx microbes are found on us, within us and around us. they inhabit virtually every environment on the planet and the bacteria carried by an average human, mostly in their gut, outnumber human cells. the vast majority of microbes are harmless to us, and many play essential roles in plant, animal and human health. others, however, are either obligate or facultative pathogens exerting a spectrum of deleterious effects on their hosts. infectious diseases have historically represented the most common cause of death in humans until recently, exceeding by far the toll taken by wars or famines. from the dawn of humanity and throughout history, infectious diseases have shaped human evolution, demography, migrations and history. a pathogen is defined as an organism causing disease to its host, with the severity of the disease symptoms referred to as virulence. pathogens are taxonomically widely diverse and comprise viruses and bacteria as well as unicellular and multicellular eukaryotes. every living organism is affected by pathogens, including bacteria, which are targeted by specialized viruses called phages. the number of viruses and bacteria on earth is staggering and they occupy essentially every environment. a liter of surface seawater typically contains in excess of ten billion bacteria and billion viruses. the number of viruses on earth is estimated to be around , which corresponds to roughly ten billion times the number of stars in the universe [ ] . an average human is made up of about trillion cells but carries a similar number of bacteria, mostly in the gut [ ] . the vast majority of viruses and bacteria we are exposed to have no negative effect and some can even * correspondence: f.balloux@ucl.ac.uk ucl genetics institute (ugi), darwin building, gower street, london wc e bt, uk be beneficial, though a tiny fraction of these can severely affect our health. specifically, about one in a billion microbial species is a human pathogen. indeed, approximately human pathogens have been described, whereas it has been estimated that there are one trillion microbial species on earth, the vast majority of which remain uncharacterized [ ] . pathogens can be divided into two main categories, namely facultative and obligate pathogens, reflecting how intimately their life cycle is tied to their host. facultative pathogens are organisms for which the host is only one of the niches they can exploit to reproduce. facultative pathogens are primarily environmental bacteria and fungi that can occasionally cause infection. they include many of the most problematic hospital-acquired bacteria involved in the antimicrobial resistance pandemic. a distinction is sometimes made between facultative and accidental pathogens, with the latter representing those which only occasionally infect weakened or immunocompromised hosts. typical examples of 'accidental' pathogens include neisseria meningitidis or escherichia coli. obligate pathogens require a host to fulfil their life cycle. all viruses are obligate pathogens as they are dependent on the cellular machinery of their host for their reproduction. obligate pathogens are found among bacteria, including the agents of tuberculosis and syphilis, as well as protozoans (such as those causing malaria) and macroparasites. some obligate pathogens require multiple different hosts to fulfil their life cycle. the definite host, which supports the adult form of the pathogen, is often a vertebrate and the intermediate host (referred to as a vector) is generally an arthropod or a mollusc. this alternation of vertebrate and invertebrate hosts is found in viruses (for example the zika virus), bacteria (for example lyme disease) and protozoa (malaria). trematodes (parasitic flatworms) go even further and some exhibit among the most baroque life cycles. digenetic trematodes have a basic three-host life cycle, and for some species a fourhost life cycle. for instance, halipegus occidualis sequentially has to infect a freshwater snail, an ostracod, a dragonfly nymph and ends its cycle after the dragonfly is eaten by the green frog rana clamitans, where it resides under its tongue [ ] . what is the host range of pathogens? some pathogens are limited to infecting a single host species, whereas others can infect a multitude of host species. host ranges can feel highly idiosyncratic if not outright puzzling. for example, leprosy in humans is caused by two related intracellular bacteria mycobacterium leprae and mycobacterium lepromatosis, which are essentially restricted in the wild to humans, as well as armadillos in the americas and red squirrels in scotland [ ] . conversely, yersinia pestis, another intracellular obligate bacterium and the agent of plague, has a natural life cycle involving alternating infections of rodents and fleas, but can infect essentially any mammalian host. an interesting twist in the case of plague is that y. pestis is not well adapted to the human host. with the exception of uncommon occurrences of human-to-human transmissions, referred to as pneumonic plague, plague epidemics (bubonic plague) are caused by plagueinfected fleas biting humans. somewhat ironically for a pathogen that is possibly the biggest killer in human history, bubonic plague is a complete evolutionary disaster. the human host is at a very high risk of dying, the flea cannot reproduce on a meal of human blood and the bacterium is stuck in an evolutionary dead-end as it cannot transmit to another host. there is no obvious predictor for the host range of different pathogens. intuitively, it may be tempting to predict that pathogens with a more intimate relationship with their host are more closely adapted to their host, and thus have a more restricted host range. however, there is no obvious pattern suggesting that viruses (that rely on the host cells' machinery for reproduction) have a narrower host range than bacteria. also, intracellular bacteria do not seem to have a markedly narrower host range than extracellular ones, despite being more intimately tied to their host. we know relatively little about the underlying genetic changes required for a pathogen to infect a new host, though, interestingly, only a few mutations can be required for a host jump. for example, avian influenza is only around five mutations away from being able to transmit in mammals [ ] , and a single amino acid change was sufficient for the humanadapted bacterium staphylococcus aureus to become a pathogen of rabbits [ ] . obligate pathogens tend to be highly adapted to their hosts, with sophisticated mechanisms to synchronise their life cycles with that of the host, and the ability to manipulate the host's immune system, metabolism and sometimes even behaviour. genes encoding proteins specific to pathogenicity are referred to as virulence factors, which include a variety of molecules required for colonization of the host, immunoevasion and immunosuppression, scavenging nutrients within the host, and entry into and exit out of cells for intracellular pathogens. in bacteria, virulence factors are often found in groups of genes on pathogenicity islands, which can be transferred horizontally by plasmids or other transposable elements. for example, one of the defining features of the plague bacterium y. pestis from its less virulent closest relative yersinia pseudotuberculosis, is the inclusion, early in its evolution, of two plasmids carrying genes involved in pathogenicity [ ] . while acquisition of novel genes and repurposing of existing ones is essential in the evolution towards pathogenicity, a general feature during the evolution towards pathogenicity is genome reduction through the inactivation and loss of genes. this can be primarily explained by the fact that a host represents a fairly stable and resource-rich environment where some metabolic pathways required in the environment are not necessary. genome reduction is a general trend accompanying the evolution towards pathogenicity and is observed in mycobacterium tuberculosis, pathogenic e. coli strains and in the ongoing adaptation of klebsiella pneumoniae lineages to cystic fibrosis patients. the most extreme example is leprosy (m. leprae and m. lepromatosis), which has shed nearly half the genes found in their environmental relatives [ ] . another interesting tendency of many bacterial pathogens is the secondary loss of the ability to undergo genetic recombination [ ] . pathogens cause illness to their hosts through a variety of ways. the most obvious means is through direct damage of tissues or cells during replication, generally through the production of toxins, which allows the pathogen to reach new tissues or exit the cells inside which it replicated. bacterial toxins are among the deadliest poisons known and include famous examples such as tetanus, anthrax or botulinum toxin, known as botox in its commercial application. however, the damage to the host is often self-inflicted through a strong or sometimes excessive immune response that indiscriminately kills infected and uninfected cells and damages host tissues. typical examples of maladaptive over-reaction of the immune system include cirrhosis and liver cancer in hepatitis b [ ] , or the - influenza epidemic, where the toll was highest amongst the young and healthy possibly because they mounted the strongest immune response and as such died from a 'cytokine storm' in the lungs leaving patients literally drowning in their own body fluids [ ] . some pathogens benefit from the hosts' immune reaction to spread within an infected host or increase their transmission to uninfected hosts. influenza transmits mainly through aerosols created through the sneezing and coughing it causes. vibrio cholerae triggers a strong inflammatory response in the gut mucosa, leading to watery diarrhoea and ensuring its release in the environment and thus infection of further hosts. pathogens greatly vary in the severity of their symptoms from a mild inconvenience to assured death. it is sometimes assumed that the deadliest pathogens represent recent host jumps where the pathogens' virulence is maladapted to the new host, and that co-evolution between host and pathogen will lead to more benign symptoms over time. however, this is only true in the case of strict vertical transmission (such as from mother to child), where survival and transmission of host and pathogen are intimately linked. in the case of horizontal transmission, the situation is more complex and there is no straightforward way to predict the evolution of future virulence, as it will depend on a variety of factors, including the population structure of the host and the correlation between virulence and transmission [ ] . a textbook example for reduction in virulence is the introduction of myxomatosis into the european rabbit population in australia and france in and , respectively. upon introduction, the virus initially killed about % of infected rabbits but over a few years mortality went down to %, following the emergence of attenuated virus strains and genetic resistance in the rabbit population. while the virulence went down, which translated into higher transmission rates, the current % mortality rate remains exceedingly high and there is no evidence for further reduction in the short term [ ] . bdgpl, the global lineage of the amphibian fungal pathogen batrachochytrium dendrobatidis (bd), is the only known pathogen to have extirpated entire host species. yet, over the three decades since its discovery, it shows no sign of evolving lower virulence. the primary reason there is little selective pressure on bd for virulence attenuation is that it can infect a very vast host range so that the extinction of any particular host species has limited impact on its fitness. even worse, some host species, such as the widely introduced african clawed frog, xenopus laevis, and the american bullfrog, rana catesbeiana, carry the disease asymptomatically, fuelling the global bd pandemics and limiting any shortterm prospect for significant decrease in virulence [ ] . these are just examples of the evolution of virulence but both illustrate that there is no simple pattern of decrease in pathogenicity with time. how old are the major human pathogens? apart from a few putative ancestral pathogens, including helicobacter pylori [ ] , that might have co-speciated with their human host, the infectious diseases afflicting us were acquired through host jumps from other wild or domesticated animal hosts or sometimes from the wider environment. the timing of these events and the original source remains unclear in many cases. the traditional view has been that many human pathogens emerged during the neolithic revolution. the main arguments for an origin of human pathogens linked to agriculture are based on the proximity between traditional farmers with their livestock and the emergence of higher human population densities in stable settlements enabled by agricultural subsistence. high population density is indeed required by some epidemic diseases which could not have maintained themselves on scattered groups of hunter-gatherers [ ] . this argument, however, neglects the fact that pathogens can evolve fast. also, while the proximity of humans and livestock is conducive to host jumps, humans transmitted more diseases to domestic animals than they acquired, with tuberculosis in particular having probably jumped from humans to cattle rather than the other way around [ ] . finally, this argument also neglects the high burden of pathogens in wild populations, including in the great apes. ancient direct evidence is scant for pathogens, and historical records rarely allow unambiguous attribution of described symptoms to a disease. that being said, recent progress in sequencing technology and in particular the ability to generate sequences, if not complete genomes, from ancient samples has greatly improved our understanding of the age of the major human pathogens, often leading to unexpected results. figure summarises the current knowledge on the age of the seven current 'major killers' , as well as plague, which was included due to its major impact in the past. while some of these estimates may need to be updated in the future after the emergence of new evidence, it is unlikely the general pattern will change much. some human diseases are old (for example plasmodium falciparum malaria) and others recent, such as hiv or, more surprisingly, measles. there is also no obvious pattern pointing to the neolithic revolution as a strong driver for the emergence of human pathogens. where are the resistance-conferring genes in the human genome? infectious diseases have killed well over half of all humans who have ever lived on earth. pathogens, such as childhood diseases, that affect their host prior to reproduction, through death or reduced fertility, have exerted enormous selective pressures. yet, scans of the genome for signatures of pathogen-driven selection have identified only a few variants with clear effects. similarly, genome-wide association studies (gwas) for infectious disease resistance/susceptibility have identified only few loci impacting on infectious disease susceptibility [ ] , despite their success in identifying thousands of variants involved in chronic diseases and phenotypic traits such as height. even for diseases that have affected us for a long time, for example tuberculosis, we know of no obvious protective genetic variant. given the high selective pressure pathogens must have exerted, it is reasonable to ask where all the resistance genes are. strongly protective variants may have reached fixation, rendering them undetectable unless the pathogen has a highly heterogeneous distribution range. an interesting case for regional selective pressure is the duffy negative antigen mutation protective against plasmodium vivax that is found at close to % in sub-saharan africa but virtually absent anywhere else. another situation where a resistance gene does not reach fixation arises when the protective variant is deleterious when homozygous, as in sickle cell anaemia. we might also speculate that the evolutionary potential and high genetic diversity of most pathogens limits our ability to detect protective variants in the human genome, particularly so if these were only effective against a subset of lineages within a pathogenic species. in addition to the few variants protective against specific pathogens, we also know of genomic regions involved in immunity against a wide spectrum of pathogens, such as interleukin genes or the major histocompatibility complex (mhc) system. the very high genetic diversity of the mhc is believed to have been shaped by exposure to different pathogen species [ ] . also, following the recent development of techniques to sequence ancient dna, it has been suggested that immunity genes such as those encoding toll-like receptors have been acquired following hybridization with archaic humans and are over-represented in the current gene pool of anatomically modern humans relative to genes not involved in immunity [ , ] . infectious diseases had a massive impact on our history, leading to the rise and fall of civilizations, both through the toll they took on human life but also through economic and societal collapse following epidemics. more military campaigns have probably been lost or won due to infectious diseases than due to the tactical acumen of the armies' commanders. thucydides reports in his history of the peloponnesian war, written in the th century bc, how the plague of athens devastated the citystate of athens in ancient greece during the second year [ ] ; influenza [ ] ; hiv [ ] ; tuberculosis [ ] [ ] [ ] [ ] ; p. falciparum malaria [ , ] ; hepatitis b [ ] ; measles [ ] ; plague [ , ] of the peloponnesian war ( bc) when it was on the cusp of victory against sparta, ending the golden age of pericles and athenian predominance in the ancient world. the eventual fall of the roman empire was also largely down to another epidemic, the justinian plague in - ce, which precluded the emperor justinian recovering lost territories in the western part of the empire [ ] . infectious disease played an equally important role in past human migrations. the conquistadores' sweeping conquests of large swaths of the americas in the th century was greatly aided by the diseases they brought with them, such as measles and smallpox, to which the indigenous populations had limited immunity [ ] . conversely, one of the possible reasons europeans managed to colonize africa was that they used quinine, an antimalarial drug derived from the bark of the cinchona tree [ ] . history has been shaped not only by pathogens infecting humans, but also those affecting domestic animals and crops. for example, it has been suggested that the islamic conquest of the th and th centuries did not extend to sub-saharan africa because the horses and camels of the islamic armies were dying from trypanosma spread by tsetse flies [ ] . conversely, pathogens were at other times the drivers of large migration. around one million irish people died and another million migrated to the us to escape the famine caused by phytophthora infestans destroying potato harvests between and [ ] . at least in the developed world, the leading causes of human mortality are no longer infectious diseases but instead age-associated disorders such as cancer, heart disease and diabetes. numerous countries have undergone an epidemiological transition, starting some years ago in some developed countries and less than years ago for developing countries. diseases that once devastated human populations, such as smallpox, are now eradicated. others, such as the plague or leprosy, are largely under control with the exception of a few hotspots. the current situation is, however, one of new challenges. globalization and increased mobility, particularly air travel, have facilitated the transmission of diseases not just locally but between continents. the recent outbreak of zika in the americas, for example, has been attributed in part to an increase in air travel from infected areas into brazilian airports, extending both the incidence and geographic range of the virus [ ] . the outbreak of severe acute respiratory syndrome (sars) and recurrent ebola crises in central africa highlight the ability of new and existing diseases rapidly to become significant international health threats. in addition, our ability to combat infectious diseases is also challenged by the widespread emergence of pathogen drug resistance. the global antimicrobial resistance (amr) crisis is increasingly limiting our resources to combat disease through antimicrobial therapy. thus, in spite of the global health narrative supporting a decline in the number of deaths caused by infectious disease, the complexity of our interactions with diseasecausing agents are as significant now as through history. infectious diseases continue to be a major cause of mortality globally, responsible for between a quarter to a third of all deaths and nearly half of all deaths in people under the age of , with most of these in principle avoidable. microbiology by numbers revised estimates for the number of human and bacteria cells in the body the role of damselflies (odonata: ztgoptera) as paratenic hosts in the transmission of halipegus eccentricus (digenea: hemioridae) to anurans red squirrels in the british isles are infected with leprosy bacilli airborne transmission of influenza a/h n virus between ferrets a single natural nucleotide mutation alters bacterial pathogen host tropism yersinia pestis, the cause of plague, is a recently emerged clone of yersinia pseudotuberculosis insight into the evolution and origin of leprosy bacilli from the genome sequence of mycobacterium lepromatosis microbial diversity and the genetic nature of microbial species immunobiology and pathogenesis of viral hepatitis the influenza pandemic: insights for the st century infectious diseases of humans myxoma virus and the leporipox viruses: an evolutionary paradigm emerging fungal threats to animal, plant and ecosystem health age of the association between helicobacter pylori and man myths and misconceptions: the origin and evolution of mycobacterium tuberculosis evolution, revolution and heresy in the genetics of infectious disease susceptibility pathogendriven selection and worldwide hla class i diversity genomic signatures of selective pressures and introgression from archaic hominins at human innate immunity genes introgression of neandertal-and denisovan-like haplotypes contributes to adaptive variation in human tolllike receptors cooling and societal change during the late antique little ice age from to around ad insights from paleomicrobiology into the indigenous peoples of pre-colonial america -a review selected papers from the first international conference 'camel cultures: historical traditions, present threats and future prospects'. london: rn books after the famine: plant pathology, phytophthora infestans, and the late blight of potatoes zika virus in the americas: early epidemiological and genetic findings th century variola virus reveals the recent history of smallpox pandemic influenza -including a risk assessment of h n the early spread and epidemic ignition of hiv- in human populations detection and molecular characterization of -year-old mycobacterium tuberculosis from a neolithic settlement in the eastern mediterranean armed conflict and population displacement as drivers of the evolution and dispersal of mycobacterium tuberculosis pre-columbian mycobacterial genomes reveal seals as a source of new world human tuberculosis eighteenth-century genomes show that mixed infections were common at time of peak tuberculosis in europe plasmodium falciparum accompanied the human expansion out of africa genomes of cryptic chimpanzee plasmodium species reveal key evolutionary events leading to human malaria dating the origin and dispersal of hepatitis b virus infection in humans and primates origin of measles virus: divergence from rinderpest virus between the (th) and (th) centuries early divergent strains of yersinia pestis in eurasia , years ago historical variations in mutation rate in an epidemic pathogen, yersinia pestis the authors are grateful to liam shaw for discussions on the manuscript and for financial support from the mrc, bbsrc, nerc and esrc (grants ne/m / and mr/p / ). lvd and fb wrote the manuscript. both authors read and approve the final version. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -gvq uyfk authors: rosenberg, ronald title: detecting the emergence of novel, zoonotic viruses pathogenic to humans date: - - journal: cell mol life sci doi: . /s - - -y sha: doc_id: cord_uid: gvq uyfk rna viruses, with their high potential for mutation and epidemic spread, are the most common class of pathogens found as new causes of human illness. despite great advances made in diagnostic technology since the s, the annual rate at which novel virulent viruses have been found has remained at – . most emerging viruses are zoonoses; they have jumped from mammal or bird hosts to humans. an analysis of virus discovery indicates that the small number of novel viruses discovered annually is an artifact of inadequate surveillance in tropical and subtropical countries, where even established endemic pathogens are often misdiagnosed. many of the emerging viruses of the future are already infecting humans but remain to be uncovered by a strategy of disease surveillance in selected populations. in common with all organisms, pathogens evolve. every year brings reports of previously unrecognized human pathogens or of pathogens extending their geographic range, becoming less susceptible to treatment or prevention, or displaying unprecedented epidemic tendencies. as i write this an unprecedented ebola virus epidemic threatens west africa [ ] and chikungunya, a mosquito-borne virus, which first appeared in the western hemisphere in november , has already infected nearly , , people there [ ] . for those of us with responsibility for preventing or controlling infectious diseases, the speed with which new battles must be fought can be disconcerting. zaire ebola virus was first identified as a human pathogen only in and chikungunya in but neither reached pandemic magnitude until decades later. how can we be better prepared to identify emerging pathogens early? i will try to briefly examine some of the factors that influence our success in finding and characterizing previously unrecognized human viruses. the concept of emerging diseases is relatively recent [ ] , even if the phenomenon is not. the definition used by the world health organization [ ] is representative: ''an emerging disease is one that has appeared in a population for the first time or that may have existed previously but is rapidly increasing in incidence or geographic range''. in practice, determining if a disease is increasing in incidence or geographic range sometimes requires interpretation that might be considered arbitrary. for example, using this broad definition a recent paper [ ] claimed to have identified about emergent ''events'' between and , most of which were examples of antimicrobial resistance. a more limited and less ambiguous subset of emerging pathogens will be described here: virus species first recognized to cause human illness. before proceeding, it might be worth describing how three recently described pathogens were discovered and their disease characteristics. all three were first reported during the last six years and all three are generally accepted as distinct pathogenic entities causing serious human illness. in early september , a -year-old female resident of lusaka, zambia developed fulminant symptoms of an acute infection, beginning with headache and myalgia, and progressing over the next days to extensive rash, facial swelling and severe sore throat [ , ] . by the time she was airlifted to a hospital at johannesburg, south africa, she had developed cerebral edema, acute respiratory distress, and renal failure. despite intensive care, including hemodialysis, she died days after her initial symptoms. five of those who cared for her during transport to south africa or at the johannesburg hospital-a paramedic, two nurses and a cleaner -subsequently developed symptoms and four of these died. a previously undescribed arenavirus, lujo virus (a conflation of lusaka and johannesburg) was isolated from the index case and all four secondary cases [ ] . the arenaviruses, which have bisegmented, single-stranded, negative-sense rna genomes, are broadly divided phylogenetically into new world and old world groups. lujo belongs to the old world group, as does lassa virus. typically arenaviruses have rodent reservoirs but the specific host for lujo virus has yet to be determined and how the index case was infected is unknown [ ] . there have been no further cases reported. during summer, , two men, aged and years, were admitted within a few weeks of each other to heartland regional medical center, st. joseph, missouri, usa, with similar symptoms of fever, fatigue, anorexia, nausea and non-bloody diarrhea. the two men were farmers who lived approximately km distant from each other in northwestern missouri. both men had histories of frequent tick bite and were initially suspected to be infected with ehrlichia chaffeensis, a tick-borne rickettsia endemic to the area. serological and molecular testing of both, however, were negative for ehrlichia and neither responded to antibiotics. while in hospital both men developed precipitous thrombocytopenia and leukopenia. symptoms resolved with supportive care and both men were released from hospital and days after admission. culture of specimens indicated the presence of virus, which was confirmed by electron microscopy, and subsequently a unique bunyavirus, in the group phlebovirus, was sequenced from both patients [ ] . phleboviruses are singlestranded, negative-sense rna viruses with tripartite genomes, all of which appear to be transmitted by biting arthropods. heartland virus is the first pathogenic phlebovirus described from the western hemisphere and has a % nucleotide homology with the severe fever with thrombocytopenia syndrome (sfts) virus, reported from china in [ ] . heartland virus has since been isolated from ticks and antibodies to it have been found in a variety of wild animals, including white-tailed deer (odocoileus virginianus) and raccoons (procyon lotor) [ ] . there is no evidence for direct human to human transmission of heartland although a number of mostly nosocomial cases have been reported for sfts. between april, and late july, , middle east respiratory syndrome coronavirus (mers-cov) was definitively diagnosed in people, of whom died [ ] . the focus of cases has been in saudi arabia, united arab emirates and other middle eastern countries; the few cases detected in europe and north africa appear to be travelers from the middle east. the index case was a -year-old male admitted to hospital at jeddah, saudi arabia in june with a recent history of fever, cough and shortness of breath [ ] . at the time of admission his laboratory blood results were generally unexceptional but by days post-admission his white blood cell count had increased to , /cu mm and his platelets fallen to , . antibiotic-sensitive strains of klebsiella pneumoniae and staphylococcus aureus were cultured from his respiratory tract but he did not respond to antibiotic therapy. despite being on intensive support from the second day of admission, the patient died days post-admission of respiratory deterioration and renal failure. coronaviruses, which include severe acute respiratory syndrome coronavirus (sars-cov) and some agents of the common cold, have positive-sense, single-stranded rna genomes and are predominately transmitted between humans by fomites. many of the mers-cov cases have been nosocomial or appear to have been transmitted within families. neutralizing antibodies to mers-cov have been widely found in dromedary camels [camelus dromedarius] from the arabian peninsula and africa [ ] and virus has been isolated from them, strengthening the evidence that they are the immediate link to emergence in humans. despite the differences in clinical presentation and geographical location, these three pathogens share three characteristics: all were unknown before found infecting humans, all are rna viruses, and all have proven or putative non-human, animal sources. animal rna viruses are the most common source of emerging pathogens in a seminal study, woolhouse et al. [ ] tabulated pathogens first reported to be pathogenic to humans during - . two-thirds of these were viruses, % of which had single-stranded rna (ssrna) genomes. the predominance of rna viruses mostly owes to two characteristics. first, the rate of error during rna replication (* - ) is an order of magnitude greater than that of dna (* - ). rna replication does not benefit from the proofreading capabilities of dna polymerase or post-replication mismatch repair; consequently the potential for mutation per replication cycle is high [ ] and the lack of fidelity may have limited the size of rna genomes, many of which are in the range of , - , nucleotides. second, most rna viruses are zoonoses, that is, they were transmitted, at least initially, to humans from non-human mammal or avian hosts. examples of rna viruses retaining the capacity to be directly transmitted from animals to humans include influenza, nipa, and sars viruses, but even some viruses commonly transmitted exclusively between humans, such as hiv and hepatitis c, have likely animal origins [ ] . all arthropod-borne viruses (arboviruses) are zoonoses, although some, like dengue, yellow fever, and chikungunya, have adapted to efficient vectorborne transmission between humans. humans have been in contact with infectious animals since prehistory but their exposure accelerated with the development of livestock husbandry beginning about , years ago [ ] . the growing global population has not only increased the demand for domesticated meat in the st c but has increased encroachment on areas once wild, both are trends that increase human exposure to animals and animal products [ ] . by the end of , there had been, by one tabulation [ ] , virus species from virus families incriminated as causes of human disease. more than two-thirds of these viruses ( %) are known or presumed zoonoses. more than a quarter ( %) were first described from non-human mammals, birds or blood-feeding arthropods - years before being recognized as human pathogens. indeed, the first vertebrate virus described, the cause of foot and mouth disease, was isolated from a cow in [ ] but conclusively shown to cause human disease only in [ ] . the dates of discovery, regardless of host, are plotted in fig. a . the rate at which virulent viruses have been discovered has been governed by two equally important factors: the ability of existing technology to detect and discriminate between viruses, and the ability to collect specimens potentially containing novel viruses. initially, the lack of methods for the laboratory cultivation of viruses, which require cells for replication, prevented their isolation for study. early characterization as a virus depended mainly on demonstration that a filterable agent smaller than bacteria was responsible for transmissible disease. until the late- s, when embryonated chicken eggs and suckling mice began to be used commonly to culture animal viruses, only of the viruses now known to be pathogenic had been described. the rate of discovery again accelerated after the introduction of in vitro cell culture in (fig. a) . the mean annual rate of virus discovery during - was . . during this period methods for antigentically typing viruses using panels of antibodies were refined and came into wide use. there was a striking increase in the number of novel viruses described during - to . /year. this was followed, however, by a sudden deceleration in the rate of discovery to only about /year, which persisted through , despite the availability of increasingly powerful methods for genomic characterization, such as polymerase chain reaction (pcr) from the mid- s and, more recently, high-throughput, parallelized (''next-generation'') sequencing. vertebrate viruses can be sorted into two broad categories: those directly transmissible between humans or between animals and humans, and the arboviruses, which require the mediation of blood-feeding arthropod vectors, such as mosquitoes or ticks; % ( ) of pathogenic viruses are transmitted to humans only by arthropod vectors. the sudden, transitory increase in rate during - has been shown [ ] to be because the rates of discovery of these two classes differed (fig. b) . before the rates for the two were the same and each comprised about half the pathogenic viruses. after , however, the trends of the two classes of virus diverged. while about two non-arboviruses were discovered each year between and , the arboviruses dramatically increased during - , only to fall equally dramatically to nearly zero by . during - twice as many arboviruses ( ) were discovered than non-arboviruses ( ); by contrast, during - , only arboviruses were discovered compared to non-arboviruses. the difference in the rates for the two classes highlights the important role that strategies for specimen collection play in the recognition of novel pathogens [ ] . [ ] . all the field stations were located in tropical or sub-tropical countries and all carried out an integrated strategy that attempted to discover viruses from humans, vertebrate animals and biting arthropods. of the arboviruses discovered by the end of , ( %) were discovered by rf staff, of those during - . in comparison, the single most successful institutional discoverer of non-arboviruses, the united states national institutes of health, described of ( %). the rf protocol, which was the model for several other institutions, including the institut pasteur, was directly responsible for two additional characteristics differentiating arbovirus from non-arbovirus discovery. although % of all non-arboviruses were discovered in europe or the usa, % of all arboviruses were discovered in sub-saharan africa, latin america/caribbean, or egypt/india/ near east (fig. ) . second, % of arboviruses were first isolated from arthropods, a consequence of systematic vector collections. the predetermined cessation, by , of most rf support for international arbovirus researchincluding sponsorship of reference collections, conferences, new technology and reagents-was soon followed by a rapid, worldwide decline in arbovirus discovery (fig. b) . the east african virus research institute, for example, which was founded by rf in , isolated arboviruses after direct rf administration ended in , but none after the general rf program closed in . the disproportionate productivity of rf resembles that of especially effective individual discoverers of plant species [ ] . those botanical ''big hitters'' combined technical expertise and persistence over many years with concentration in a limited geographic area where they had gained deep knowledge. the rf was committed to a long-term strategy founded on five components. first, it chose study sites where it had evidence that arbovirus diversity would be high. these were mostly tropical and contained forested and rural areas. they chose countries where a professional work force could be recruited and where it was hoped the work would be sustained after the rf departed. second, the program concentrated only on one subclass of pathogens, the arboviruses. despite the wide competence of the professional staff, few reports were published dealing with local diseases other than arboviral. third, the research strategy called for long-term commitment. rf staff, including expatriates, typically lived on site and implemented projects for years, allowing for continuity of not only research but training. fourth, the program was integrative. human, animal and vector investigations were simultaneously pursued. because all scientists worked in a single unit connections between human virus isolates and those from animals or vectors were readily made. on the other hand, for many viruses isolated from animals or arthropods there has yet to be a link to human infection [ ] . and fifth, each unit was self-contained. each was capable of conducting both specimen collection and sophisticated laboratory analyses. how many pathogenic viruses remain to be discovered? mathematical methods for extrapolating from historical rates of discovery to estimate the pool of yet to be discovered organisms in a given taxon tend to be accurate only after most species have already been discovered [ ] . the fundamental weakness of these computations, which generally rely on analysis of cumulative frequency curves, is the assumption that the numbers of organisms known at a given time are the result of methods of discovery that have been consistent everyplace and throughout time. the increased rate of virus discovery during - was largely due to the temporary efforts of the rf, a ''big hitter'' [ ] , whose combination of active surveillance, geographic specialization, and integrated approach remains atypical. in contrast to sophisticated computations, a recently published prediction that a minimum of , mammalian viruses of families remain to be discovered was based on a simple arithmetical calculation using data from a single study [ ] . considering how few new, virulent viruses are found every year, the potential for any of , viruses jumping to humans and being discovered would then be very low ( . - ). the authors used degenerate, virus-family-level primers to amplify genomic segments from specimens of feces, urine, and throat swabs collected from the bat species pteropus giganteus in bangladesh. amplicons were as short as bp and no biological information was obtained. they found viruses (and statistically surmised an additional three existed), some of which might be novel, belonging to seven virus families. in calculating a number for the universe of viruses yet to be found they speculated that each of the known , mammal species will host an average unique viruses, unshared with other species. the authors concede that there is little evidence to support these presumptions. a single subtropical bat species hardly represents all mammal species and indeed many viruses are known to infect more than one species; they tested for only of the virus families pathogenic to humans. ultimately, the number of viruses remaining to be discovered is irrelevant if, as expected, there are many and they continue to rapidly evolve. the discovery of a virus can long predate its emergence as a recognized public health threat. the discovery of zika virus in a monkey in uganda preceded its first incrimination as the cause of a human epidemic- , km distant in micronesia-by years [ ] . as noted above, the availability of ever more powerful molecular techniques is substantially increasing the catalog of distinct viruses found in nature but the number found annually to be pathogenic to humans rarely exceeds a few each year. is it feasible to predict which animal viruses have the potential to cause disease in humans? steps to emergence the steps by which a virus might emerge from exclusively animal hosts are schematically depicted in fig. . at the most preliminary level (tier ) viruses circulate within mammals and birds, not necessarily causing disease, before some opportunistically infect humans (tier ). typical human-animal contact includes husbandry, capture of wild animals for food, and exposure to animal fomites or waste, as may happen in bat infested environments. indirect exposure via arthropods must be frequent, as evidenced by the large proportion of pathogenic viruses that are vectorborne [ ] . in most instances these tier , opportunistic infections are dead ends or remain rare events because the pathogen is not well adapted to transmission between humans (e.g., crimean-congo hemorrhagic fever virus) or because the type of contact between infected animals and humans is uncommon (e.g., sealpox virus). a small but fig. comparison of regions in which arboviruses and nonarboviruses were discovered fig. schematic of the emergence of zoonotic viruses as human pathogens. in tier , viruses are only transmitted among sub-human animals. in tier , viruses infect humans, but only directly from animals. some animal viruses (solid arrows), like west nile, can fuel zoonotic epidemics. others, like hantaviruses, are frequent but subepidemic causes of human illness (dashed black arrows), while many, like sealpox, are rare (dashed red arrows). in tier , zoonotic viruses have acquired the ability to be transmitted between humans without the contribution of the animal host. in some cases (w) a virus might leap directly to tier or transition through tier significant group of zoonotic viruses, including rift valley fever, nipah and west nile viruses, are capable of instigating human epidemics without ever adapting to humanhuman transmission. theoretically even sub-epidemic (r o \ ) transmission can favor mutations that will enhance future transmission [ ] . in rare instances (tier ) pathogens do evolve to allow human-human transmission (e.g., hiv) or appear to already posses that capacity (e.g., mers-cov, lujo). some viruses maintain animal-animal and animal-human cycles but are mostly propagated by human-human transmission (e.g., chikungunya, zika). the geographically limited sylvatic cycles of dengue in west africa and southeast asia account for a tiny percentage of the estimated million infections annually [ ] . human contact with an animal virus does not ensure infection. among the biological barriers for the virus are finding a route of entry, evading general immune defenses, invading host cells, replicating sufficient numbers before specific immune responses are mounted, and finding a route to the next host. aerosol delivery, for example, greatly enhances transmissibility but efficiency depends on the anatomical site within the respiratory system of the invaded cells [ , ] . arbovirus transmission, in which the vector amplifies, transports and inoculates the virus into humans, can be enhanced by viral mutations that increase the potential for successfully infecting the vector [ , ] or animal host [ ] without altering its virulence to humans. much recent research has focused on identifying determinants essential for viral invasion of host cells and how modification of the viral ligands might increase their ability for interspecies infectivity [ ] . it is not yet possible on the evidence of sequence alone to predict with confidence the probability of an animal virus transitioning to humans. in general, vertebrate specificity greatly limits the ability of viruses adapted to one species to invade similar cells in another, distant species. influenza a is the most studied and best understood of the few viruses that frequently jump from animals to humans. a number of mutations have been identified that enhance infectivity. these include substitutions in the hemagglutinin (ha) protein receptor binding sites that enable the virus to exploit sialylated glycan receptors on respiratory cells belonging to other species [ ] . for example, two nonsynonymous base changes in the ha receptor binding sites of avian h and h viruses, which converted their specificity from the avian a , -sa to human a , -sa, led to the pandemic of h n and the pandemic of h n [ ] . much current research on the determinants of influenza specificity is experimental and its extension to complex natural transmission of other virus families remains to be tested. understanding how such adaptability works could focus our attention on those virus families or species with the greatest chance of infecting humans but how this knowledge could be used more specifically to identify potential threats to humans among animal viruses, as has recently been proposed [ ] , is unclear. geographical bias, human behavior and likelihood of contact species richness of mammals and birds is greatest at the equator, thinning toward the poles [ ] ; mosquitoes [ ] and ticks [ ] appear to follow a similar latitudinal species diversity gradient. pathogen species are also richer in the tropics than in temperate zones [ ] although, as has been ruefully pointed out, ''the fact that warbler species distributions are better understood than the distribution of human pathogens is a gap that clearly deserves research attention'' [ ] . there is a strong association between mammal and pathogen richness but mammal diversity appears to be an indicator rather than the cause of pathogen diversity [ ] . pathogens that maintain external life cycles, for example vector-borne and helminthic, which are directly susceptible to variability in precipitation, tend to be more geographically restricted to the tropics than those directly transmissible between people, such as influenza [ ] . environmental barriers to dispersion can be circumvented. monkeypox virus, whose natural transmission is largely restricted to parts of equatorial africa by the range of its natural rodent hosts, has demonstrated the ability to make use of new hosts in temperate zones [ ] and a number of arboviruses, such as chikungunya, dengue and zika viruses, have widely expanded their natural ranges as their principal vectors have [ ] . the rf selection of field sites in was based on the relatively greater diversity of arboviruses found during the preceding years in tropical countries and it can be argued that their success in discovering new viruses owed as much to this factor as their choice of integrated, active surveillance. as would be expected, there appears to be a correlation between zones of species richness and frequency of reported vector-borne and zoonotic emerging disease events [ ] . the species richness of the tropics suggests that human populations there are exposed to greater risk and that they are fertile grounds for virus mutation. certain behaviors common to some regions, such as the harvesting of wild animals for food, aggravate that risk. the lack of housing barriers to rodents, bats and arthropod vectors are major vulnerabilities to pathogens carried by them. environmental and sanitation deficiencies also increase risk to enteric and vector-borne viruses; in the absence of dependable water supply, many people, for example, are forced to store containers of water that provide breeding for the virus-carrying mosquitoes. most importantly, as dunn et al. [ ] have shown, countries with the highest pathogen richness spend the least per capita on health care, and there is an inverse correlation between investment in public health and pathogen prevalence, independent of species richness. because of the obvious link between the abundance of novel viruses and the tropics, geospatial modeling could help target areas for surveillance. there is a tradition of developing and using models to identify those areas of the world whose species richness is most in need of conservation [ , ] and recently attempts have been made to use models to identify areas most liable to spawn emerging diseases [ , ] . considering the association between mammal and pathogen species richness there can be expected to be some overlap between the two sets of ''hot spots''. the predictive robustness of a model depends not only on the algorithms used but in the choice and weighting of variables, and the representativeness and validity of the data. for example, a widely cited model [ , ] , which chooses ''the original case or cluster of cases representing an infectious disease emerging (during - ) in human populations for the first time'', lists only of viruses first described as infecting humans during that period [ ] . on the other hand it credits the first occurrence of a number of viruses described years earlier to the period - ; these include measles, influenza a, and rabies, assigning to each a single, arbitrary origination location (us, hong kong, and costa rica, respectively) for modeling geospatial associations. because this model also lumps a variety of emerging disease types, including many examples of antimicrobial resistance, population density is a major factor, which might explain the higher likelihood for emergent events it assigns to india and java than to the amazon or equatorial africa, where so many novel viruses have been discovered. modeling has been more successful for single pathogens for which large amounts of specific data have been collected, such as for dengue [ ] or malaria [ ] . associating disease prevalence in a limited area with well-characterized environmental attributes, as has been done for plague bacteria (yersinia pestis) in uganda, can be used to predict areas potentially at risk that would be difficult to collect data from, such as large plague-prone tracts of the neighboring democratic republic of congo [ ] . because many of the arbovirus species are known only from places where long-term field operations were established by the rf, institut pasteur, and others, and because those sites were selected for logistical and political realities as well as scientific interest, their usefulness in modeling is still limited. surveillance for emerging pathogens: the problem of knowing the unknown successful surveillance depends on how and where one looks. ideally, an emerging virus will be detected at its source and contained before spreading. this ideal requires, however, extensive networks of alert health care providers, adequate laboratory resources, and an effective method for communicating results to an authority capable of responding. it also assumes that a zoonotic virus will not be spread by animal hosts impossible to control, as was the case with the avian arbovirus, west nile. in practice, human disease surveillance raises an alarm only after an arbitrary number of seemingly related, serious cases are reported and arouse attention. generally, number of cases and length of time to detection are least for anticipated pathogens with distinct presentations, such as poliovirus presenting with acute flaccid paralysis. in countries with rudimentary public health systems that threshold might be reached for unexpected pathogens only after the number of cases reaches epidemic proportions impossible to ignore, as the recent epidemic of ebola virus in west africa demonstrates. many viral disease cases present as clinically indistinguishable acute febrile illnesses (afi) or with symptoms so mild the patient does not seek attention. cases with neurological involvement or systemic bleeding can also be difficult to diagnose clinically without adequate laboratory support. in those tropical areas most likely to spawn emerging viruses, afi will be misdiagnosed or undiagnosed in at least % of patients [ ] [ ] [ ] [ ] . overlooking novel pathogens as the cause of nonspecific symptoms is not confined to developing countries: it is likely heartland virus was a cause of illness in the usa long before it was characterized in . nevertheless, the probability that more undescribed viruses infect humans in the tropics seems to be greater. it is likely, therefore, that many emerging diseases due to novel viruses will be overlooked, especially at tier , until they become epidemic, are transported to countries with more sensitive surveillance, or are discovered by chance. laboratory support for clinicians is critically deficient nearly everywhere in the rural tropics. the first step in determining if an illness might be caused by a rare or unknown virus is to eliminate the possibility of pathogens known to be endemic. simple, relatively accurate rapid diagnostic tests (rdts) are available for a few common causes of afi, notably malaria, but tests for most viruses require not only equipment, such as elisa readers, but modest, dependable infrastructural support-electric power, clean water, cold storage-rarely available outside major cities. poor roads often make the timely, proper transport of specimens to centers with laboratory capacity impractical. even in cities, many hospitals and government laboratories do not have the basic equipment, fresh reagents or accurate testing protocols to assay for most common endemic pathogens. should laboratory capability be available to eliminate most known etiologies for the disease observed, description of a novel pathogen typically requires both biological and molecular characterization [ ] . recovery of viable virus for culture and histological evidence of pathology remain fundamental steps establishing causality. the increasing power and availability of rapid, next-generation sequencing has made whole-genome analysis an increasingly routine and important part of describing novel viruses but because of the large number of commensal species found in the human virome [ ] , linking a novel genome with virulence will be tentative without supporting biological evidence. for example, wu and ki polyomaviruses, isolated in the mid- s from children suffering from acute respiratory infections and tentatively included in the list of human pathogens [ ] , have yet to be proven causal of illness [ ] . our ability to detect and characterize novel pathogenic viruses in hot spot locations lags behind global systems that have arisen to report and respond to unusual occurrences. the international health regulations (ihr) of the world health organization (who) binds countries to plans to improve their ability to detect and respond to outbreaks, and the global outbreak alert and response network (goarn) of who helps organize international response to public health emergencies. open source networks for reporting outbreaks include the program for monitoring emerging diseases (promed-mail), a free, internet-based system for disseminating information posted by , professional contributors in countries, and healthmap, which collects and continuously updates disease outbreak data from a variety of public sources, including news services. many of these reports seem never to be investigated or resolved. the full value of these systems can only be attained if provided with accurate information. considering the barriers to obtaining human surveillance data, it has been proposed [ ] that monitoring animal populations at sentinel locations could alert us to risk from viruses with pandemic potential. for such a plan to be feasible for emerging viruses it would be necessary to judge the potential risk posed by a virus not yet known to infect humans. epizootic disease in livestock or wild animals is used as a threat indicator for some known zoonotic viruses, such as influenza, rift valley fever virus, and west nile virus, but there is no assurance that an agent potentially pathogenic to humans will cause noticeable disease in animal hosts. coronaviruses closely related and putatively ancestral to sars virus, for example, seem not to cause disease in host bats [ ] . the extent of animal disease surveillance is also far less in the tropics than even the poorest human clinical networks so the likelihood of recognizing an unusual event is less. periodic sampling of animals can discover novel viruses but is too infrequent and limited to be surveillance. considerable attention has been given to human contact with bush meat, or animals captured for food [ ] . while harvesting and slaughtering wild animals appear to have provided the mechanism by which some important pathogens have emerged, such as hiv and sars, it has not played a role in the emergence of many others, including the three examples discussed in this paper: lujo, heartland and mers-cov. vectors obviate the need of direct human-mammal or human-bird contact and can move viruses across ecological zones. sequencing and cataloging the viruses of animals in selected areas can provide valuable insight to transmission dynamics and phylogenetics but, as discussed, cannot yet be used to predict. one must wonder if the us $ . billion proposed to catalog mammalian viruses not yet known to be pernicious [ ] would not be better spent on developing more suitable diagnostic tests for humans in remote areas most liable to emerging pathogen risk or on conducting sentinel human surveillance. ultimately, the best indication that a pathogen has the ability to jump to humans is finding it in humans [ ] . although there is not a strong rationale for conducting autonomous searches for potentially pathogenic viruses in animal populations, there is much value in animal investigations in support of surveillance for infectious diseases in selected, sentinel human populations. the discovery of lujo virus in a human, for example, should direct our attention to its epidemiology and ecology in the area where we suspect exposure occurred. had lujo been discovered in an animal instead, its significance as a pathogen would have been speculative until the detection of the first human case. the integrative, long-term approach of the rf can serve as a model but with primary focus on conducting population-based surveillance for acute illness. concomitant ecological profiling and virological studies in arthropods, mammals and birds can more quickly clarify the epidemiology of any novel viruses discovered in the human population. the zoonotic viruses pathogenic to humans represent a small but unknown proportion of those infecting mammals and birds. from this constantly evolving universe of vertebrate viruses two or three are recognized every year to have broken the species barrier, a remarkably small number considering the frequent contact between humans and animals, and the high adaptability of rna viruses. while most of these novel, emergent viruses have inconsequential public health significance, some, such as mers-cov or lujo, have obvious destructive potential. what is the best strategy for identifying and limiting the menace from novel, zoonotic viruses? identifying potential pathogens before they leap to humans (tier ) would seem ideal but is impractical. the determinants of pathogenicity are complex and poorly understood, while a system for wildlife or livestock surveillance in those areas with conditions most conducive to emergence cannot anytime soon reach a scale or effectiveness to be pragmatic. it is likely that yet to be recognized viruses already infecting humans will be sources of disease outbreaks of varying magnitude in the future. these tier infections can be uncovered as part of comprehensive, investigative surveillance in human populations at risk. in the near term this would be best accomplished in most places through specially designed sentinel surveillance sites. modeling might at some point provide guidance for site selection but too narrow a definition for target sites (e.g., bush meat markets) will be self-defeating. by identifying and eliminating poorly appreciated endemic agents, investigations can then focus on illnesses with unresolved etiologies. unlike investigations directed at animal populations there would be a tangible, immediate improvement in the health of the subject communities. complimentary studies of vectors and animals, as pioneered by the rockefeller foundation, would prepare for epidemiological investigations of those zoonoses uncovered but the primary focus must be on humans. ultimately, one hopes, surveillance for emerging zoonoses will be a part of improved health care systems throughout the world. global alert and response: ebola virus disease 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rate of human viruses berichte der kommission zur erforschung der maul-und klauenseuche bei dem institut für infektionskrankheiten in berlin das klinische bild der maul-undklauenseuche beim menschen, aufgestelt aus den bisher experimentell gesicherten erkrankungen the rockefeller foundation virus program: - with update to big hitting collectors make massive and disproportionate contribution to the discovery of plant species centers for disease control and prevention, international catalog of arboviruses including certain other viruses of vertebrates predicting unknown species numbers using discovery curves a strategy to estimate unknown viral diversity in mammals zika virus outbreak on yap island, federated states of micronesia the role of evolution in the emergence of infectious diseases the global distribution and burden of dengue airborne transmission of influenza a/h n virus between ferrets role of receptor binding specificity in influenza a virus transmission and pathogenesis venezuelan equine encephalitis emergence: enhanced vector infection from a single amino acid substitution in the envelope glycoprotein chikungunya virus adapts to tiger mosquito via evolutionary convergence: a sign of things to come? a single positively selected west nile viral mutation confers increased virogenesis in american crows investigating virus-glycan interactions using glycan microarrays early alterations of the receptor-binding properties of h , h , and h avian influenza virus hemagglutinins after their introduction into mammals global patterns of terrestrial vertebrate diversity and conservation insight into global mosquito biogeography from country species records using habitat models to map diversity: pan-african species richness of ticks (acari: ixodida) ecology drives the worldwide distribution of human diseases global drivers of human pathogen richness and prevalence spectrum of infection and risk factors for human monkeypox movement of chikungunya virus into the western hemisphere global trends in emerging infectious diseases threatened biotas: 'hot-spots' in tropical forests theory and data for simulating fine-scale human movement in an urban environment hot spot or not: a comparison of spatial statistical methods to predict prospective malaria infections improvement of disease prediction and modeling through the use of meteorological ensembles: human plague in uganda etiology of acute, non-malaria, febrile illnesses in jayapura, northeastern papua. indonesia etiology of severe non-malaria febrile illness in northern tanzania: a prospective cohort study undiagnosed acute viral febrile illnesses evidence of a major reservoir of non-malarial febrile diseases in malaria-endemic regions of recommendations for publication of viral genetic data and sample access for novel viruses and strains the human virome: new tools and concepts human polyomaviruses in disease and cancer bats and their virome: an important source of emerging viruses capable of infecting humans overviews of pathogen emergence: which pathogens emerge, when and why? key: cord- - m bsrw authors: shaw, alan r.; feinberg, mark b. title: vaccines date: - - journal: clinical immunology doi: . /b - - - - . - sha: doc_id: cord_uid: m bsrw nan vaccines represent one of the most effective and cost-effective medical and public health achievements of all time. worldwide, vaccination programs are currently estimated to save over million lives each year. in addition to having such a major beneficial impact on vaccine-preventable disease morbidity and mortality, the direct and indirect impacts of vaccination programs translate into economic savings of many billions of dollars each year. in what is considered to be one of the most significant medical successes of all time, a collaborative and comprehensive vaccination campaign against smallpox resulted in the global eradication of the disease in . similarly, efforts to eradicate poliomyelitis have made tremendous progress in reducing the global disease burden, and will hopefully soon overcome certain residual societal and programmatic obstacles to provide the second successful example of elimination of a major health threat by vaccination. concerted global efforts to provide measles vaccine have resulted in the control and elimination of the disease in many countries, including substantial reductions in mortality in a number of developing countries where the residual disease burden is greatest. these and other examples provide clear evidence of the power of vaccines in favorably manipulating host immunity to confer dramatic public health benefits, at both the individual and population level. as vaccines are administered to healthy individuals (often to entire age cohorts or populations), to prevent diseases caused by infectious agents to which they might be exposed in the future, they differ in important ways from pharmacologic agents that are used to treat individuals in whom a disease process is already manifest (or who display predispositions to disease). for this reason, vaccines are unique in the way that they impact on societies and in the way that societal commitment to vaccination determines their ultimate impact. as a result, vaccination efforts provide an informative window on challenges that need to be successfully navigated at the interface between scientific opportunity and societal capacity and commitment. indeed, current limitations in realizing the full global potential of available vaccines relate more to existing inadequacies in health care financing and infrastructure (especially as they are manifest in developing countries), and the relative value that societies place on disease prevention, than they do to any inherent biological limitations of vaccines themselves. fortunately, recent acceleration of new vaccine introductions in developing countries through public and private initiatives to build immunization infrastructure and provide funding of vaccine purchase offers hope that vaccines will one day be equitably available to all who need them. the importance of vaccines extends beyond their use as public health tools to include their role as drivers of immunologic discovery. the history of vaccine development is rich with immunologic insights that emerged from careful observations of how diseases spread in populations and how such spread differs in disease-naïve and experienced populations, as well as of how innovative experimental approaches revealed fundamental aspects of immune system function. the general concept of immunity induced by prior exposure to a disease (including its specificity and potential lifelong duration) was appreciated by the ancient greeks. use of the word 'immunity' itself dates to the th century when it was applied to describe the relative susceptibility and resistance of populations to plague. the subsequent successes of edward jenner and louis pasteur in the development of effective smallpox and fowl cholera immunization strategies, respectively, provided a foundation for modern immunology; pasteur himself coined the term 'vaccine' in recognition of jenner's use of vaccinia virus. jenner's smallpox immunization studies also provided early experimental support for the concept of immune memory. pasteur's efforts provided the first demonstration of the attenuation of pathogens by their propagation in culture (or by passage in nonnatural animal hosts), while robert koch demonstrated that killed pathogens could also engender immunity. the discovery of bacterial exotoxins by emile roux and alexandre yersin facilitated the discovery of antibodies and their potential use in passive immunotherapy with antitoxin antibodies by emil von behring and shibasaburo kitasato. these discoveries enabled the development of active immunization against diphtheria and tetanus using toxin-antitoxin mixtures. paul ehrlich's development of accurate methods for antibody quantitation made passive immunotherapy and active toxin-antitoxin immunization far more reliable and effective, and provided a stimulus for significant advances in immunologic theory. in each of these instances, vaccine development illuminated central mechanisms of immune system biology. vaccine development today has transitioned from an approach that was once largely empirical to one that is based on the hypothesis-driven application of techniques in molecular biology and immunology. evidence for this synergy can be seen in recent studies of vaccine-elicited immune responses to illuminate primary and memory t-and b-cell responses in humans, as well as the strong discovery stimulus provided by ongoing efforts to develop new vaccines for major infectious diseases for which vaccines are not currently available. vaccine development today faces a number of significant challenges. there exist tremendous public health needs to address major well-known pandemic diseases, including acquired immunodeficiency syndrome (aids), tuberculosis, and malaria, for which no vaccines currently exist and for which natural immunity does not provide a helpful guide for vaccine development. furthermore, there exists a need to confront effectively newly emerging and re-emerging diseases, ranging from the well-known, but constantly changing, threats from influenza pandemics to the appearance of previously unknown zoonotic infections such as the coronavirus that causes severe acute respiratory syndrome (sars). with changes in population density, mobility, and social constructs, along with alterations in the global climate, ecological circumstances, and the proximity of humans to animal reservoirs for previously confined infectious agents, the concept of new infectious agents entering human populations and spreading rapidly around the world is no longer novel. in confronting prevalent or newly emerging diseases, vaccines are looked to as the most promising line of defense. however, the speed at which new infectious disease threats have been shown to emerge and spread, and the fact that the pathogens that now need to be confronted may display tremendous genetic variability (e.g., human immunodeficiency virus (hiv)) or an identity that cannot be predicted in advance (e.g., avian influenza or agents like sars) places unprecedented demands on the vaccine development process. in addition to these new challenges, there remain unmet needs in the derivation of vaccines that can achieve the greatest public health benefit. these needs include the development of new ways to achieve more effective vaccine-elicited immune responses in neonates whose immune systems are immature (or are impacted by maternal antibodies) (chapter ) and in the elderly whose immune system function may be waning as a result of immune senescence (chapter ). fortunately, the scientific foundation provided by basic and applied immunology and the use of new methods for pathogen identification, antigen discovery, vaccine production, adjuvant development, and novel vector derivation afford important opportunities for vaccine development and additionally present the possibility of improving on natural immunity. success in vaccine development will be predicated on continuing the historical synergy between advances in vaccine technology and basic immunologic discovery. toward that end, this chapter focuses on preventive vaccines for infectious diseases and how they are developed. although current routine vaccine recommendations are reviewed, given the active state of new vaccine introduction and evolving vaccine recommendations, as well as differences in recommendations in different countries, readers are encouraged to refer to up-to-date national resources for the most current information. while vaccine approaches are being actively explored to modify beneficially malignant and immunologic diseases (autoimmunity and allergy), these are beyond the scope of the current discussion. n impact of vaccination programs n unlike other medical interventions, vaccines confer benefits to both individuals and populations. , while individuals may be protected from infection or disease by vaccine-induced immune responses, decreasing the number of susceptible hosts in a population also helps break the chain of transmission that pathogens require to spread and persist in human populations by induction of 'herd immunity. ' the benefits of herd immunity depend on achieving sufficiently high immunization rates in a population to impact pathogen transmission dynamics (including the potential for extinction of ongoing interhost transmission). the requisite level of vaccination coverage of a population needed to compromise pathogen spread significantly varies between pathogens, and is influenced both by vaccine efficacy (and its duration) and by the reproductive characteristics and infectiousness of the pathogen. analysis of the impact of vaccination programs in the usa provides an example of the beneficial impact of vaccines when used routinely and when high coverage levels are achieved. as shown in tables . and . , vaccination programs in the usa dramatically decreased the annual morbidity of many vaccine-preventable diseases. in many instances, the disease burden from several vaccine-preventable diseases of childhood has been reduced by over % since vaccine introduction (e.g., diphtheria, tetanus, measles, mumps, rubella, and polio). the somewhat lower rate of decline of pertussis (the annual morbidity of which has been reduced by a nonetheless impressive %) relates to the limited duration of vaccine-induced immunity, which is estimated to wane within - years after childhood vaccination. it is anticipated that recent availability of pertussis booster vaccines for use in adolescents and adults will lead to significant further declines in pertussis morbidity. even for diseases targeted by vaccines that have been in widespread use for less time (< years), impressive decreases in disease morbidity have been seen (e.g., varicella, hepatitis a, and pneumococcal disease). in a notable recent demonstration of the population benefits of vaccines, introduction of the -valent pneumococcal conjugate vaccine resulted in a decrease of % in disease morbidity in children under years of age within the first years of its introduction. interestingly, the rate of meningitis and bloodstream infections caused by antibiotic-resistant streptococcus pneumoniae also fell by % in this age group. in a striking related finding illustrating how vaccines can impact pathogen transmission dynamics, rates of antibiotic-resistant pneumococcal infections also declined by % in individuals over the age of who had not received the vaccine. thus, direct protection by vaccination of children who represent a reservoir of infection provided, via herd immunity, significant indirect benefits to those who did not themselves receive the vaccine. in addition to their benefits in preventing disease morbidity and mortality, routine vaccination programs are also impressively cost-effective. evaluation in the usa of the impact of ten vaccines routinely given as part of the childhood immunization schedule (diphtheria, tetanus, pertussis, haemophilus influenzae b (hib), polio; measles, mumps, rubella, hepatitis b and varicella) found that more than million cases of disease and more than deaths were averted over the lifetime of the immunized birth cohort of children. when the cost of the vaccination program was compared to the economic impact of diseases prevented, these vaccines alone are estimated to save nearly $ billion each year. when including indirect economic benefits (such as the time parents take off from work to care for sick children), the annual savings to society exceed $ billion. when preventive services were ranked based on clinically preventable disease burden and cost-effectiveness, childhood immunization received the highest score. progress in the development of new vaccines accelerated significantly towards the end of the th century, with the development of vaccines against diseases that were not previously preventable by vaccination, but also with the development of improved versions of existing vaccines. thus, the number of diseases that can be prevented by vaccines included in the us centers for disease control and prevention's (cdc) routine childhood and adolescent immunization schedules grew from seven in to in (table . and fig. . ) . moreover, in the past several years, new vaccines have been introduced for adolescents and young adults (e.g., pertussis booster (tdap), meningococcal conjugate, and human papillomavirus (hpv) vaccines), and older adults (e.g., tdap and zoster vaccines) have shown that the value of vaccines extends across the human lifespan (figs . and . ). new combination vaccines have been developed to increase the simplicity and acceptability of vaccination regimens, as well as to improve overall compliance with the recommended series of vaccines. such combinations include either those that contain multiple inactivated or recombinant antigens (such as a combination diphtheria, pertussis, tetanus, hib, and hepatitis b vaccine) or multiple live attenuated viruses (such as a combination measles, mumps, rubella, and varicella vaccine (mmrv)). the development of a combination vaccine is often more complicated than simply combining individual antigens, for when antigens are administered in combination, immunologic interference is sometimes seen. this necessitates titration of antigen combinations (and in the case of combinations of inactivated and/or recombinant antigens, adjuvant selection) to achieve immune responses that are not inferior to each of the antigens administered individually. despite their readily demonstrable public health impact, the value of vaccines is often not appreciated, for when vaccine programs are successful the diseases that they cause become less prevalent and may disappear. however, to prevent resurgence of an infectious disease that has been brought under control, vaccination programs need to be continued. the difficulties facing current efforts to eradicate poliomyelitis have demonstrated that failure to maintain high immunization coverage rates can lead to prompt re-emergence and spread of the disease. even in developed countries, maintenance of strong immunization programs with high degree of coverage is needed where infectious diseases can travel with remarkable speed -and do so even before the extent of spread is evident. n principles of immunization n the terms vaccination and immunization are often used interchangeably. however, vaccination specifically refers to efforts to induce protective immune responses by administration of a vaccine, whereas immunization more generically refers to interventions -either active or passive -that seek to confer immune protection. active immunization describes the induction of immune responses by administration of a specific antigen or antigens, while passive immunization involves the administration of exogenous immunologically active substances (historically, antibodies present in sera obtained from immune individuals or animals) to confer temporary protection from an infectious pathogen or toxin. although the approaches for passive immunization waned in the later half of the th century, the advent and increasing robustness of monoclonal antibody technology have led to a resurgence of interest in passive immunization. vaccines seek to engender immune responses similar to those that confer immunity to re-infection in individuals who experience (and survive) natural infection with a given pathogen. in lieu of formal demonstration of a specific type of antibody or cellular immune response that contributes to prevention or accelerated clearance of an infection, most often vaccine efficacy is demonstrated first in the course of a placebo-controlled trial. in some instances, specific immune effector mechanisms, such as a specific level or type of antibody response, can be identified that correlate with immune protection. in this case, the 'correlate of immunity' provides a benchmark against which similar vaccines can be compared. in the case of most inactivated vaccines, subunit vaccines, and recombinant vaccines that produce antibody responses, but generally meager cd t-cell responses, it is likely that humoral immune responses are the primary or sole protective immune mechanism. in the case of live attenuated vaccines that induce both cellular and humoral immune responses against the pathogen, it is likely that both arms of the immune system act in concert to confer immunity. however, the actual mechanisms of immune protection induced by either a natural infection or a vaccine are generally not understood in detail for many infectious diseases. similarly, although vaccines depend on the induction of immunologic memory, the magnitude, character, and duration of immune memory differ between vaccines, as can the actual mechanism of immune protection. for certain vaccines, such as those that protect against bacterial diseases induced via production of toxins (e.g., diphtheria or tetanus), protection induced by toxoid-based vaccines is clearly dependent on persistent antibody (igg) and memory b-cell responses, ensuring that sufficient antitoxin antibodies are present at the time of toxin exposure to inactivate and clear the toxin. in other cases, such as long-lived protection against hepatitis b, if sufficient levels of antibodies are achieved in the initial immunization period, even hosts who may with time lose detectable levels of antibody responses remain protected. in this instance, given the relatively long incubation period of hepatitis b, memory antiviral b-cell responses induced by the vaccine can be activated, facilitating neutralization and clearance of the infection before clinical disease is manifest. although it is popularly believed that vaccines confer protection by inducing 'sterilizing immunity' -wherein an infectious agent is blocked from even infecting one cell in an exposed host -this is clearly not the case for a number of vaccines. for example, the inactivated poliovirus and live attenuated rotavirus vaccines do not prevent some degree of local replication of their pathogenic counterparts in the gastrointestinal tract of exposed hosts. however, they are both effective in preventing clinical disease. in the case of poliovirus vaccine, this is mediated by elicitation of antibody responses that block dissemination of the infection to the central nervous system; while in the case of rotavirus, as yet unidentified immune effectors limit local virus replication so that significant gastrointestinal damage does not occur following infection. , the major types of vaccines licensed for use include live attenuated organisms, killed or inactivated organisms, subunit vaccines consisting of purified (or partially purified) components of an organism, and subunit vaccines produced by recombinant dna technologies. the use of live attenuated vaccines dates back to the early work of jenner and pasteur on smallpox and fowl cholera vaccines, respectively. , the fundamental concept of live attenuated vaccines is to mimic the effective host immune responses that follow natural infections. most live attenuated vaccines currently in use were derived by propagation of initially pathogenic organisms in culture on cells from different (nonhuman) species, or at nonphysiologic temperatures, for prolonged periods. driving pathogen evolution in culture to select for variants adapted to growth in heterologous cell types ex vivo often leads to the derivation of pathogen variants that grow poorly in vivo in humans and are unable to cause clinical symptoms. vaccines developed via this approach include those used to prevent a number of viral and bacterial infections, including yellow fever, measles, mumps, rubella, polio (the 'sabin vaccine'), varicella-zoster (used both for the prevention of chickenpox and shingles) and rotavirus (one version of the available vaccines), tuberculosis, and cholera. more recent technologies being applied to live attenuated vaccine development include the application of reverse genetic strategies ( fig. . ) and those involving genetic reassortment with attenuated viral variants, as have been used to develop polyvalent live attenuated vaccines against influenza and rotavirus ( fig. . ) . , the live attenuated vaccines currently in use are highly efficacious (> %) and protection is frequently durable. the efficacy of many live attenuated vaccines likely reflects the ability of the attenuated vaccine to replicate within vaccinated hosts, and to expose the immune system to pathogen-derived antigens in a manner that closely resembles the nature, location, and effects of natural infection. because live attenuated vaccines replicate within immunized individuals, they can induce both cellular (cd and cd ) and humoral (b-cell) effector responses and immunologic memory. in addition, as the live attenuated vaccines likely activate the host innate system in a manner similar to their pathogenic parents, they provide inherent adjuvant effects in augmenting adaptive immune responses. a key consideration in the development of any live attenuated vaccine relates to the relative balance between the ability to induce sufficient immune responses in vivo to confer protection (often associated with level of preserved replicative ability in vivo), and the ability to cause symptoms (which may also relate to the extent of in vivo replication). as such, an effective but also safe and well-tolerated vaccine needs to strike a specific balance between level of attenuation and level of immunogenicity. in addition, depending on the nature and number of genetic mutations responsible for the attenuated phenotype, a potential risk of reversion to a pathogenic form exists for certain vaccines. for most live attenuated vaccines, this has not been observed to be a problem in clinical practice -likely because the attenuating mutations are sufficiently numerous or genetically stable. one vaccine where reversion to pathogenic form was seen involved specific components of the live attenuated oral poliovirus vaccine (opv; the 'sabin vaccine'). in this instance, vaccine reversion to wild-type was shown to lead rarely to cases of paralytic polio (approximately one case per million doses administered). based on these observations and the elimination of endogenous polio transmission in many developed countries, the inactivated polio vaccine (ipv; the 'salk vaccine') was substituted for opv. however, in light of a favorable cost-benefit ratio, high degree of efficacy, and ease of administration, opv continues to be the mainstay of polio vaccination efforts in developing countries. the use of physical or chemical methods to kill or otherwise inactivate a pathogenic organism represents a second major approach to vaccine production. , in most cases, treatment with chemical agents such as β-propiolactone and formaldehyde is used to eliminate pathogen infectivity. while this approach has the benefit of presenting most of a pathogen's antigenic repertoire to the immune system of the immunized host, it can only be used in instances where the inactivated pathogen does not possess constituents that would confer significant toxicity. vaccines based on killed pathogens are believed to exert their protective effects via elicitation of pathogen-neutralizing antibodies and the induction of memory b-cell responses (likely in concert with cd t-cell memory). however, because inactivated pathogens cannot accomplish de novo synthesis of pathogen-derived gene products in antigen-presenting cells (apcs), they do not typically induce cd t-cell responses (chapter ). in addition, killed vaccines are generally less immunogenic than live attenuated vaccines. as a result, they are commonly administered with an adjuvant (most often alum: see section on adjuvants, below) to augment their immunogenicity. a number of viral and bacterial vaccines currently in use are killed/inactivated vaccines, including whole-cell bordetella pertussis vaccine and the influenza virus, rabies virus, and hepatitis a virus vaccines. a number of bacteria produce toxins that represent the major pathogenic components responsible for disease in infected humans. examples include corynebacterium diphtheriae and clostridium tetani. detoxified versions of these toxins are referred to as 'toxoids,' and represent the purified components of vaccines preventing diphtheria and tetanus, respectively. toxoids have historically been produced by chemical inactivation of toxins, but more recently, genetic inactivation via targeted mutagenesis has been employed. the acellular pertussis vaccine is also a purified subunit vaccine composed of a defined set of protein constituents prepared from cultured bordetella pertussis. the mechanism of immune protection conferred by purified subunit vaccines is the antibody response elicited by vaccination. antibodies directed against the capsular polysaccharides present on encapsulated bacteria also confer protective immunity in a number of important instances by inducing antibodies that exert opsonophagocytic effects (promoting phagocytosis of antibody-coated bacteria) and, in some instances, bactericidal effects. initial successful vaccine efforts against streptococcus pneumoniae and neisseria meningitidis utilized purified preparations of capsular polysaccharides. although such purified polysaccharides can induce protective levels of antibody responses in adults, they are poorly immunogenic in children under years of age (as a function of the relative immaturity of their immune systems). in addition, t-independent antibody responses elicited by purified capsular polysaccharides are less durable than those that are produced in the presence of cd t-cell help. as a means of both augmenting antibody responses against polysaccharide antigens in young children and facilitating their persistence, the development of conjugate vaccines represented an important advance. in this approach, purified polysaccharides are chemically conjugated to a carrier protein (such as diphtheria toxoid or an outer-membrane protein complex (ompc) derived from n. meningitidis). the carrier protein augments cd t-cell helper responses to the polysaccharide antigens, and enables elicitation of durable protective antibody responses even in young children. polysaccharideconjugate vaccines have been produced that protect against haemophilus influenzae b, streptococcus pneumoniae, and n. meningitidis infections. if two such segmented viruses with different genetic characteristics are used to infect one cell, the progeny viruses from this mixed infection will carry a range of mixtures of the genes of the two parent viruses. using either genetic or immunologic screening methods, reassorted viruses carrying the precise gene composition of interest can be selected. this approach has recently been employed to generate live attenuated vaccines against rotavirus and influenza virus. the strategy for generation of the pentavalent bovine-human reassortment rotavirus vaccine is shown above. rotaviruses have a segmented double-stranded rna genome comprising independent rna elements. the outer shell of the virus comprises two proteins vp and vp that are involved in cell binding and entry and that specify the viral serotype (p type for vp and g type for vp ). vp and vp also represent the targets of virus-neutralizing antibodies. the pentavalent bovine-human rotavirus vaccine was generated by a 'modified jennerian' approach in which the bovine rotavirus wc (which is attenuated in humans as a result of host range restriction) serves as the gene donor for the backbone on to which gene segments encoding four common human rotavirus g types (g - ) as well as one very common p type (p ) (derived from individual rotavirus isolates) were reassorted via a process of cell co-infection and subsequent selection of the recombinant viruses with the desired composition of bovine and human gene segments. an analogous genetic reassortment approach has also been used to generate live attenuated influenza vaccines. in this instance, three attenuated 'cold-adapted' viral strains (two a types and one type b) are used in co-infections in tissue culture with recent circulating wild-type influenza strains to derive vaccine strains that include the two relevant hemagglutinin (ha) and neuraminidase (na)-encoding gene segments admixed with the six 'backbone' genes from the attenuated master donor virus for use in annual influenza vaccines. the first recombinant vaccine developed, the recombinant hepatitis b surface antigen (hbsag) prepared in yeast, was developed in hopes of avoiding safety concerns related to the plasma-derived hbsag vaccine. the knowledge that immune sera could provide protection by passive immunization of naïve hosts, and that purified inactivated plasmaderived hbsag vaccine could elicit protective antibodies, laid the groundwork for development of this recombinant vaccine. the recombinant vaccine, when combined with adjuvant (alum), elicits favorable immune responses, is highly efficacious and is well tolerated -all features that recombinant vaccines are now expected to deliver. the second recombinant vaccine developed targeted prevention of borrelia burgdorferi infection (the cause of lyme disease), and was based on a purified recombinant version of the ospa protein. this vaccine, although conferring some degree of efficacy, faced implementation challenges, and was not widely embraced. as a result, it was withdrawn from the market. more recently, recombinant technology-derived purified subunit vaccines have been developed that consist of virus-like particles (vlps) that self-assemble when the l protein of hpv is produced in isolation of other viral proteins ( fig. . ). the l protein is the target of virusneutralizing antibodies and vaccines consisting of a mixture of types and (the cause of ~ % of cases of cervical cancer) and and (the cause of ~ % of cases of genital warts) or of hpv types and alone have been shown to be highly efficacious and well tolerated. interestingly, hpv vlps induce antibody responses that exceed those that follow natural hpv infections. in light of these successes, and the power and versatility of recombinant antigen production methods, a major proportion of new vaccine development efforts involves the use of protein subunit vaccines produced by recombinant technologies. vaccines produced by this method are those that depend largely or exclusively on the induction of antibodies against individual or a selected subset of pathogen proteins. because a number of proteins produced in isolation by recombinant methods have been observed to elicit lower immune responses than do natural infections or live attenuated vaccines, the development and use of adjuvants to optimize recombinant vaccine immunogenicity represent an important parallel area for future exploration. n vaccine development and evaluation n as a necessary prelude to clinical evaluation of candidate vaccines in humans, extensive preclinical research and development activities are undertaken to establish that the vaccine candidate has the desired properties. toward this end, a number of key issues need to be addressed. first, animal studies must show that the vaccine candidate raises the desired type and magnitude of immune response against the infectious agent. second, the vaccine needs to protect animals against death or disease in an appropriate challenge model, when feasible. ideally, in the course of these studies, a specific type or level of immune response, referred to as a correlate of immune protection, can be identified. third, the vaccine should be relatively free of serious discernible toxicities and side effects in animals when administered by the route intended for humans. fourth, it is necessary to demonstrate that the vaccine can be produced in a consistent manner by a process that is consistent with the current good manufacturing practices (cgmp) process by which the first clinical trial materials will be produced (www.fda.gov/cber/gdlns/indcgmp.pdf ). bioengineered l proteins ( ) l pentamer self-assembled virus-like particle in specific instances, vlps can be produced via a process of self-assembly of individual viral capsid proteins produced by recombinant dna methods in cell culture systems. this approach has a number of attractive aspects, including the ability to produce vlps that accurately display conformationally correct epitopes recognized by neutralizing antibodies and the absence of pathogen-derived nucleic acids. in addition, recombinant vlps have been employed to derive safe and effective vaccines for pathogens, such as hepatitis b virus (hbv) and human papillomavirus (hpv), that cannot be grown in culture (and are thus refractory to standard vaccine approaches of attenuation or inactivation). the generation of the vlps that comprises newly developed hpv vaccines is shown. the hpv l proteins (which represent the major capsid protein and target of virus-neutralizing, protective antibodies), derived from hpv types of interest (e.g., types , , , and ) are produced via recombinant methods. under appropriate conditions, individual bioengineered l proteins first self-assemble into pentamers, and then into vlps that are comprised of pentamers and that are almost identical, both morphologically and antigenically, to infectious hpv virus particles. vlps prepared from individual hpv types are then combined with specific adjuvants to prepare the final vaccine products. even before preclinical studies are completed, vaccine developers typically begin an initial dialog with regulatory authorities (such as the food and drug administration (fda) or the european medicines agency (emea)) to set expectations about what will be necessary and sufficient for advancement to clinical studies in humans (www.fda.gov/cber/ genetherapy/isct sh.pdf ). phase i studies primarily focus on detailed assessment of the safety and tolerability of a vaccine, but evaluation of its immunogenicity is also frequently conducted. generally, a phase i study includes fewer than healthy volunteers divided unequally between those who receive vaccine or placebo ( or vaccinees per placebo recipient). phase i studies typically employ escalating doses of the candidate vaccine, with a dose range progressively increasing in steps of three-to fivefold often being used. blood samples are taken at prescribed intervals and analyzed for laboratory evidence of potential toxicity, as well as for evidence of vaccine-elicited immune responses. a phase i study is considered successful if it demonstrates that the candidate vaccine is well tolerated or identifies any immediate safety concerns that will need to be closely monitored in potential future clinical studies. ideally, phase i studies also provide an initial indication of the optimal dose level and number of doses required. a phase ii study typically includes several hundred to a few thousand volunteers (randomized between vaccine and placebo) and can assume two general design types. phase iia studies provide additional safety data on a larger number of individuals of the intended age who receive the intended vaccine dose (who are more representative of the general population intended for vaccine use than the very healthy individuals included in the phase i study), as well as provide additional data on vaccine immunogenicity. even larger phase iib studies can provide additional data on vaccine safety and immunogenicity in subjects generally representative of those for whom the vaccine might be recommended, but importantly, also provide the first opportunity to address to answer the question, 'does this vaccine work in humans?' the size of a phase iib study needed to detect a signal of vaccine efficacy depends on the attack rate of the infection being targeted by the vaccine. the development of new vaccines depends on the convergence of public health need, biological plausibility, and practical feasibility. vaccine development programs are influenced by multiple considerations, including: >> what are the major unmet medical and public health needs today? >> what is known about the natural history and pathogenic mechanisms of the infection of interest? >> is immunity to a given antigen associated with protection against disease following re-exposure in the context of natural infection? >> if natural immunity capable of preventing re-infection follows an initial infection with the pathogen, can a specific host immune effector mechanism (e.g., antibody, cd t-cell) be identified as the likely agent (or 'correlate') of immune protection? if so, can a threshold level of this specific immune correlate needed for protection from re-infection be defined? >> can the pathogen be grown in culture? if so, does the pathogen cause such a life-threatening disease that an attenuated version of the virus would face an impossible barrier for demonstration of safety? >> can a specific antigen (or antigens) be identified that represents the target of protective host immune responses? >> if the protective immune response is mediated by antibodies, can the target antigen (be it a protein or polysaccharide) be produced in scalable quantities in a form that mimics its native structure so that it can effectively elicit antibody responses that can block the key functional role(s) of the target molecule in the pathogen lifecycle or otherwise lead to the clearance of an incipient pathogen infection? >> having chosen an antigen and presentation system, what is the best way to produce it on a large scale? choices will be limited by the nature of the antigen and delivery system, but definition of an optimal system for producing the vaccine (prokaryotes like escherichia coli, or diverse eukaryotic hosts including yeast, insect cells, plants, or cultured plant cells, mammalian cells) is a central consideration. >> what is the most effective way to present the antigens of the pathogen of interest to the immune system? modern molecular biology and biochemistry have provided numerous options for vaccine immunogen presentation, including recombinant proteins (and recombinant virus-like particles (vlps)), synthetic proteins, protein-polysaccharide conjugates, and gene delivery systems (recombinant viral vectors, or dna vaccines) >> is the antigen of interest sufficiently immunogenic on its own, or is augmentation of the desired immune response by conjugation to a specific carrier or addition of an adjuvant necessary to elicit a sufficient and sufficiently durable immune response in individuals in the target population for vaccination? >> what types of potential safety concerns can be anticipated for the vaccine in question? >> what is the attack rate of the infection in the general population? if the infection occurs relatively rarely in an overall population, can a subset of the population be identified that has a higher risk of infection so as to accelerate the achievement of statistically significant protection? is this subset sufficiently similar to the rest of the population to enable extrapolation of the clinical results to the broader target population as a whole? >> what tests to evaluate vaccine immunogenicity will need to carried out on clinical samples obtained from participants in the clinical trials? will measurement of antibody titers, t-cell responses, pathogen presence and quantity, pathogen serotype, and any other parameter peculiar to the disease in question represent the primary criteria for vaccine effect? development and validation of theses tests represent an essential component for the feasibility and success of a vaccine clinical study phase ii studies also present the first opportunity to identify a potential laboratory immunological correlate of protection from disease -if nature and prior experience have not already done so. in order to do so, the placebo recipients in the phase ii trial must experience a sufficient number of cases of disease while vaccine recipients need to exhibit significant evidence of decreased risk of infection or disease. in addition, immunological measurements in the vaccinees need to capture the relevant protective immune responses (e.g., the type and level of antibody and/or cellular immune response that predict protection) and measure them with sufficient precision and reliability. if laboratory measurements of immunity correlate with vaccine protection, subsequent refinements of the vaccine, its adjuvant, its manufacturing process, or its regimen may be assessed by simple immunogenicity studies, rather than repeating efficacy studies. once efficacy is established for a vaccine, it is very difficult to carry out a double-blinded, placebo-controlled efficacy study. vaccines that have been shown to be immunogenic and well tolerated in phase ii studies can then advance to pivotal phase iii studies required for vaccine licensure by regulatory authorities. phase iii studies are intended to expand further the safety database in a larger number of individuals (who are representative of the specific populations for which the vaccine will ultimately be used), establish definitive evidence of protective efficacy, and to establish clinical consistency of the vaccine made by the process run in the facility intended for licensure and commercialization (www.fda.gov/cber/genetherapy/ isct jcr.htm). typically, phase iii studies include or more subjects in a blinded, placebo-controlled design. this size trial allows the identification of less frequent safety events. it also provides an opportunity to capture data on health care utilization, cost, and impact of the vaccine on these parameters. as a new vaccine will ultimately be included in a vaccine program where multiple vaccines may be administered at the same time, it is also necessary to conduct concomitantuse studies. the developer of the new vaccine must show that the new vaccine does not impact on the immunogenicity of the existing vaccines, and that the existing vaccines do not impact on the immunogenicity of the new vaccine. in contrast to drugs, where licensure by the fda is the primary determinant of how a new product is implemented in medical practice, vaccine use in the usa includes an additional process that evaluates how best to employ a new vaccine to optimize its implementation and public health impact. the us cdc has responsibility for making recommendations about the use of licensed vaccines, and it relies on its advisory committee on immunization practices (acip) for guidance. the acip considers several aspects in addition to a vaccine's safety and efficacy, including the anticipated costeffectiveness and practical feasibility of potential alternative vaccine deployment strategies and consideration of how a new vaccine may be successfully implemented in clinical practice to achieve the greatest public health impact. once the cdc has received, reviewed, and accepted the recommendation of the acip, the recommendation is published in its final official form in morbidity and mortality weekly report (mmwr; www.cdc.gov/nip/publications/acip-list.htm). the recommended immunization schedule for children and adolescents ( fig. . ) is updated on an annual basis and can be accessed at www.cdc. gov/nip/recs/child-schedule.htm. the recommended adult immunization schedule (fig. . ) is also updated on an annual basis and can be accessed at www.cdc.gov/nip/recs/adult-schedule.htm. the recommended adult immunization schedule includes information concerning use in special populations (such as health care workers and pregnant women) and individuals with specific conditions associated with altered or impaired immune function (such as individuals with congenital and acquired immunodeficiency syndromes, recipients of immunosuppressive therapies, malignancies, asplenia, liver disease, and renal disease). readers are encouraged to check to ensure that they are following current recommendations. pregnancy registries currently exist for four vaccines in the usa. health care professionals are encouraged to report exposures of pregnant women to the appropriate registry: hbv vaccine ( - - ), hpv vaccine ( - - ), meningococcal vaccine ( - - ), and varicella vaccine ( - - ). n vaccine safety n unlike drugs that are utilized to treat individuals suffering from a given disease state, vaccines are administered to normal, healthy infants, adolescents, and adults. consequently, standards for the safety and tolerability of vaccines are set at a very high level. when developing a new vaccine, a graded process of clinical studies is employed that involves increasingly larger numbers of volunteers and that typically progresses from individuals who are selected to be free of any identifiable health problems to those who are selected to be representative of the overall population for whom the vaccine is being developed. if phase i studies reveal no evidence of safety concerns and the desired evidence of immunogenicity, a major focus of the series of larger randomized double-blind, phase ii placebo-controlled studies that are then conducted is to explore the safety and tolerability of a vaccine in increasingly vulnerable populations (such as those who may have identified pre-existing health problems or asymptomatic abnormalities detected on screening laboratory studies). reflecting the importance of documenting the safety of a new vaccine, phase iii studies to assess the safety and efficacy of a new vaccine now typically involve large numbers of volunteers. indeed, as a result of needing to provide evidence for safety, it is now common to have the size of the phase iii trial be significantly larger than would be necessary to document vaccine efficacy. the ability of a study to identify an increased risk of any given adverse event with sufficient statistical power is directly related to the size of the population in the study. as a general rule, a study of - subjects is needed to measure the risk of an event that happens in one out of individuals. for one in , - subjects are needed. even in studies of this size, very rare events may not be identified, and if a specific safety concern exists substantially larger trials may be needed. the recent experience with the development of rotavirus vaccines provides an illustrative example of the importance placed on documenting vaccine safety. rotavirus is an important cause of serious gastroenteritis in infants and young children, and the associated diarrhea and vomiting can lead to life-threatening dehydration. in developing countries where health care resources and effective rehydration options are limited, over infants die of rotavirus gastroenteritis each year. given the global importance of rotavirus gastroenteritis, the first licensure of an orally administered rotavirus vaccine in was a very welcome advance. however, as the vaccine entered routine pediatric practice, it was recognized that a low, but increased incidence of intestinal intussusception was seen after the first and second doses (with about one case of intussusception seen per vaccinees.) upon recognition of this association, the vaccine was withdrawn from the market. with the evident public health need for a safe and effective rotavirus vaccine, it was hoped that alternative rotavirus vaccines then in development (both oral vaccines based either on a combination of bovine-human reassortant viruses (fig. . ) or an attenuated human rotavirus strain) might differ from the first licensed rotavirus vaccine and not result in an increased rate of intussusception. however, to demonstrate that these alternative rotavirus vaccines were safe, and that an increased risk of intussusception was not inherent to rotavirus vaccines as a class, very large-scale safety studies were required. toward this end, the safety of each of these vaccines was evaluated in studies involving about infants -just to evaluate whether the rate of intussusception in vaccinees was discernibly increased compared to the normal background rates seen in the placebo recipients. , fortunately, both vaccines were found to be well tolerated and no increase in intussusception was observed in vaccine as compared to placebo recipients. in light of the documented efficacy of these vaccines determined in earlier and significantly smaller phase iii trials, both have now been licensed in a number of countries. however, even with the large phase iii studies conducted for these newer rotavirus vaccines, they will still be studied in large postlicensure active surveillance safety studies and closely monitored in active and passive vaccine safety surveillance systems (see below). following vaccine licensure, safety is tracked via a number of means, including both active and passive surveillance studies of adverse events. active surveillance includes phase iv postmarketing studies of vaccine safety in larger populations in real-world use. formal postmarketing studies can include tens of thousands of individuals or more. an alternative type of postmarketing safety study is carried out by the us fda and the cdc within the context of the vaccine adverse event reporting system (vaers) database (www.vaers.hhs.gov or by telephone: - - ). the vaers database accepts spontaneous reports of adverse experiences from health care providers, patients, parents, vaccine manufacturers, and other sources. the best use of the vaers database is to identify signals in a population that may appear following the introduction of a new vaccine. a newer vaccine safety surveillance system, known as the vaccine safety database (vsd), has been developed by the cdc in cooperation with seven large health maintenance organizations (hmos) around the usa. the vsd contains the complete medical records of all the members from the participating hmos, and the information used to populate the database is entered by health care professionals using relatively consistent terminology, improving the quality, uniformity, and usefulness of the data. particularly important is that the vsd construct allows comprehensive epidemiological analyses to determine if the incidence rate of a specific adverse event is higher among vaccinees than nonvaccinees. in addition to vaers and the vsd, the cdc has also created a clinical immunization safety assessment network that reviews patterns of clinical syndromes that may follow vaccination. while the safety profile of a vaccine can be relatively well defined through the efforts described above, confidence in vaccination programs has often been challenged by public perceptions, either real or unsubstantiated, about vaccine safety. in some instances, specific vaccines have been associated with increased incidence of a specific adverse experience, such as the association between the first-generation rotavirus vaccine and an increased risk of intussusception following vaccination. however, a number of other safety concerns that have emerged are not supported by scientific evidence. an example of this can be found in the case of concerns about the association of whole-cell pertussis vaccines with permanent brain damage -concerns that were later shown to be unfounded. nevertheless, public concerns about the safety of the whole-cell pertussis vaccine resulted in decreased levels of pertussis vaccination coverage that were soon followed by epidemics of whooping cough in the uk and japan. another example is the allegation that certain vaccines, such as the combination measles, mumps, rubella (mmr) vaccine, are associated with autism. highlighting how perceptions of temporal association can give rise to public concerns, mmr vaccines are generally given around year of age, and autism is generally diagnosed in the second year of life. although the alleged causal association between mmr and autism has been refuted by thorough scientific analyses, reports in the popular media in the uk resulted in a dramatic drop in vaccination rates, followed by an increased rate of new infections. , n vaccines not yet available n although an impressive armamentarium of vaccines is now available, safe and effective vaccines have yet to be developed for a number of very important infectious diseases. the reasons underlying the lack of effective vaccines for an array of important pathogens include biological considerations, safety concerns, and practical constraints. of these, the biological considerations are often the most important barrier. as discussed above, vaccines have been successfully developed for pathogens whose natural infections give rise to natural immunity wherein the infected host (at least those who survive initial infection) is no longer susceptible to re-infection (such as measles, yellow fever virus, or smallpox) or who experiences significantly less severe clinical sequelae upon re-infection (such as rotavirus). in instances where natural immunity follows natural infection, not only is a precedent for immune protection established, but the nature of protective host responses can be studied, providing a correlate of protection to guide vaccine development efforts. however, for many of the pathogens for which vaccines remain elusive, natural immunity does not follow natural infection. in the absence of natural immunity, not only is a precedent for successful immune containment lacking, but no potential correlates of protection are available to inform vaccine development. in some instances where natural immunity does not follow natural infection, persistent infections are established and maintained by active virus replication that cannot be controlled or cleared by host immune responses (such as hiv and hepatitis c). alternatively, other pathogens are able to persist in the host through establishment, via diverse mechanisms, of latent infections that are resistant to host immune clearance (such as tuberculosis or herpes viruses (such as herpes simplex virus (hsv) or epstein-barr virus (ebv))). in other instances, even when the host is cleared of an infection via drug vaccines not yet available treatment, the host remains susceptible to re-infection and disease in the future (such as malaria). although different pathogens have evolved diverse strategies for evasion of host immune responses -ranging from manifestation of tremendous genetic diversity and propensity for immune escape; to sequestration of critical structural domains that might be susceptible to antibody neutralization; to the utilization of specific mechanisms to evade host innate and adaptive immune effectors -the common end result is frustration of vaccine development. while failure of host clearance of an infection is a common theme underlying the lack of vaccines, additional obstacles to vaccine development include other immunologically related considerations as well as both practical and safety considerations. examples of immunologically related obstacles include instances where prior exposure to a given pathogen predisposes the host to more severe disease manifestations upon re-infection (as has been proposed in the case of dengue virus) or where earlier vaccine development efforts inadvertently lead to severe adverse events following infection with the targeted pathogen (such as respiratory syncytial virus (rsv )). in each of these cases, the adverse events that follow a secondary immune exposure are believed to be the result of immunopathologic responses that result from the nature of the immune response elicited by the initial exposure to pathogen-derived antigens (by either infection or vaccination). given that the mechanisms underlying these immunopathologic processes are incompletely understood, the development of vaccines that are highly immunogenic but not similarly inclined to elicit immunemediated adverse consequences represents a substantial challenge (especially given the very high expectations for vaccine safety). an additional immunologically related challenge relates to the observation that certain organisms encode antigens that resemble constituents of the human host. for example, in the case of neisseria meningitidis group b, the bacterial polysaccharide resembles those found on certain human cell lineages, thus raising concerns about whether polysaccharide-based vaccines successfully developed for group b n. meningitidis might yield undesirable autoimmune responses. an additional distinct, but important, practical barrier to new vaccine development relates to the prevention of diseases that are threats to pregnant women or their offspring (where immunization of the pregnant woman might be able to protect the neonate). although a number of inactivated vaccines are either routinely recommended for use in pregnant women (e.g., inactivated influenza vaccine) or can be used in pregnant women for pre-or postexposure prophylaxis for those at risk of infection (e.g., inactivated hepatitis a vaccine and recombinant hbsag vaccine), the development of new vaccines specifically for use in pregnant women or the study of new vaccines in pregnant women has been impeded by concerns arising from potential litigation that might follow the appearance of a congenital abnormality in a child born to a mother who was vaccinated while pregnant. given the - % prevalence of congenital abnormalities, the practical difficulties in proving the safety of a new vaccine specifically administered to pregnant women, and the current litigious environment surrounding vaccines, the development of new vaccines to address important infections of pregnant women and their neonates (e.g., group b streptococcus: gbs) faces significant challenges. there remain a number of important infectious diseases for which no effective preventive vaccines exist. below, we list the major 'missing' vaccines, comment on why they are not yet available, and highlight the major approaches currently being explored to develop them. at the end of , an estimated million people were living with hiv infection, and in the preceding year approximately . million people became newly infected, and approximately million individuals died of aids. as the most promising biomedical intervention to contain the aids pandemic, the development of an hiv vaccine is a top global health priority. yet, hiv infection represents a vexing challenge to vaccine development. , hiv infection does not result in clearance of the virus due to a host immune response. following infection of target cells, the genome of hiv -a retrovirus -is transcribed into a dna copy via the action of reverse transcriptase. the newly formed dna copy of the hiv genome then integrates into the host cell chromosomes (referred to as a provirus) as a requisite step in the viral lifecycle. once integrated into the chromosome of an infected cell, the hiv provirus can alternatively be actively transcribed, leading to the synthesis of viral mrnas and subsequently to production of new virus particles, or it can remain in a transcriptionally silent, functionally latent state in a small percentage of infected cells. as infected cells harboring latent hiv proviruses do not produce hiv protein antigens, they cannot be recognized by host antiviral immune responses and can thereby persist undetected. upon subsequent activation of latently infected cells at some later time, viral rna transcription can be coincidently activated leading to production of progeny virions. as hiv targets activated cd t cells for infection and consequent depletion, the host's ability to mount both hiv-specific and non-hivspecific immune responses is progressively impaired. the ability of the host to clear hiv infection is further complicated by the extensive genetic diversity of virus populations that emerge, and progressively diverge, within infected individuals as a function of a replicative cycle that is accomplished by the inherently error-prone reverse transcriptase and the numerous cycles of replication that occur in infected individuals. as a result of these influences, genetically diverse populations of hiv variants are established in infected persons that facilitate the outgrowth of genetic variants that can escape from selective pressures -be they effective host cellular or humoral immune responses, or the inhibitory effects of antiretroviral drugs. an extraordinary degree of genetic diversity is also manifest in the hiv variants seen in different individuals and in different geographic regions. as successful vaccines for other infectious agents have historically had to protect against pathogens exhibiting only limited genetic diversity, hiv represents an unprecedented challenge. as many successful vaccines protecting against viral infections are predicated on the induction of neutralizing antibody responses against the viral surface proteins that mediate attachment to and entry into target cells, significant efforts have focused on the potential of the hiv surface envelope (env) glycoprotein, gp , to elicit infectionneutralizing antibodies. unfortunately, hiv gp is highly resistant to the action of antibodies by virtue of its heavy glycosylation and its native conformation that shields functionally critical structural domains from antibody binding. as a result, candidate gp -based vaccines have failed to elicit meaningful levels of neutralizing antibodies in immunized human volunteers and have not protected from hiv infection in two large phase iii studies. given the inability, to date, of candidate hiv env-based vaccines to elicit appreciable levels of neutralizing antibodies, current vaccine strategies are largely focused on the induction of cd cytotoxic t-cell responses against the more constrained and conserved antigens, such as vaccines not yet available gag, pol, and nef. it has been hypothesized that induction of high levels of hiv-specific cd t-cell responses prior to infection may not prevent infection, but may enable infected individuals to control virus replication better. should this hypothesis be valid, individuals immunized with such vaccines may exhibit lower levels of ongoing hiv replication, progress to aids more slowly, and potentially be less likely to transmit hiv infection to others. much of this work involves vectored gene delivery systems (such as adenoviral vectors, described below). however, the recently announced results of a phase iib 'test of concept study' failed to demonstrate a beneficial effect on either prevention of infection or reduction of viral load among volunteers who received the vaccine despite the induction of appreciable levels of hiv-specific ctl responses by the recombinant adenovirus-based vaccine employed. while this study result does not, in and of itself, refute the 'ctl hypothesis', it represents a significant disappointment for the aids vaccine research effort, and raises important questions about the ability of vaccine-elicited cell mediated immune responses to favorably alter the outcome of hiv infection. a there are also efforts under way to utilize the recently solved three-dimensional structure of the hiv env glycoprotein to guide the derivation of nonnative structures that might serve as better immunogens to elicit broadly cross-reactive neutralizing antibodies. in addition, relatively conserved and functionally essential sequences of the extracellular domain of the hiv transmembrane env protein, gp , are being explored as immunogens to elicit broadly neutralizing antibodies. malaria is the world's most common vector-borne disease -estimated to cause approximately million clinical cases and million deaths annually. , the disease hits hardest in africa, and is especially severe in children under years of age. in addition to direct morbidity and mortality, malaria is responsible for debilitating illness with enormous social and economic consequences. of the four malaria-associated protozoal species, plasmodium flaciparum and p. vivax represent the two major agents. these parasites have a three-stage lifecycle taking place both within the mosquito, and in the liver and blood of the infected host, and each cycle is largely distinct from the others from an immunological perspective. as a result of the multiple strategies for evasion of host immune response that the parasite has evolved, parasite replication proceeds at high levels despite active host immune responses. , either as a result of these specific immune evasion strategies or the inability of the infected human host to mount immune responses that clear the parasite, prior infection does not protect an individual from repeated subsequent infections. although the severity of disease is often attenuated following repeated infection, the mechanism of disease modulation is incompletely understood, and the limited relative immunity engendered by prior infection is easily lost if an individual leaves a malaria-endemic region. as such, the limited impact and duration of host immune responses to malaria parasites suggest that any successful vaccine strategy will need to do far better than natural immune responses -a high bar for efforts to develop an effective vaccine. roughly two dozen antigens have been cloned and tested as potential vaccine immunogens, and with a few exceptions the results have been disappointing. one antigen, the circumsporozoite antigen, presented as a fusion with hbsag (rts,s), has shown modest promise in human studies. this vaccine is now undergoing larger-scale clinical efficacy testing to determine if the magnitude of protection would justify largescale implementation efforts. should 'proof of concept' be supported in these studies, but the absolute magnitude of efficacy be insufficient, future efforts will likely focus on the identification of an appropriate adjuvant to improve the magnitude and duration of immune responses. alternative approaches include immunization with irradiated or genetically attenuated sporozoites. definition of novel antigens expressed at specific stages of the parasite lifecycle, and evaluation of combinations of multiple parasite antigens. mycobacterium tuberculosis is an intracellular mycobacterial pathogen that represents one of the world's most common and most serious infectious diseases. over billion people are believed to harbor latent m. tuberculosis infections, and approximately million active cases of tuberculosis and over million deaths occur each year. furthermore, the interface of hiv infection and its attendant immune system damage both increases the severity of m. tuberculosis infection and increases the infectiousness of infected individuals. the emergence and dissemination of m. tuberculosis isolates that are resistant to multiple antimicrobial drugs represent a growing public health threat. however, while the need for a vaccine to prevent tuberculosis is clear, a significant number of challenges face vaccine development efforts. most individuals infected with m. tuberculosis can control the acute phase of mycobacterial replication, and mount vigorous innate and adaptive immune responses to the infection. however, the infection is often not cleared by the host's immune response, and the mycobacteria are able to persist and multiply within vacuoles inside macrophages. longterm latency is established in fibrotic cysts in the lung. the recrudescence and dissemination of m. tuberculosis occur at a later time in a number of infected individuals, likely as a result of waning host immune control. although the ability of m. tuberculosis to persist despite active innate and adaptive immune responses represents a major challenge to vaccine development, the fact that most individuals can contain (if not clear) m. tuberculosis infection suggests that a vaccine that can alter the course of the natural infection by limiting early dissemination and decreasing the risk of later recrudescence could provide major public health benefits. efforts to develop a vaccine against tuberculosis date back many decades. bacille calmette-guérin (better known as bcg), based on mycobacterium bovis, was first introduced in . currently, bcg is provided as a component of the routine expanded programme for immunization (epi) schedule and is administered to a significant majority of the world's children. although some protective efficacy ( - %) has been reported against miliary infection and m. tuberculosis meningitis in children, conflicting results have been obtained in different studies regarding the ability of bcg to protect against pulmonary tuberculosis in adults. one explanation for the overall limited efficacy of bcg emerges from formal genome sequencing studies that have disclosed significant differences between m. tuberculosis and of the vaccine strain of bcg. the variability in the results of bcg efficacy studies in different populations and geographies may derive from variations in the geographic prevalence of cross-reactive mycobacterial species (that may themselves confer partial protection), or the fact that bcg vaccines used throughout the world do not represent a homogenous preparation -with the root strain of bcg having been widely distributed and passaged extensively under diverse conditions. vaccine efforts against tuberculosis have primarily focused on the evaluation of specific mycobacterial antigens (e.g., esat , ag , and hsp ) that have been tested as vaccines in animal models with variable success. , some of these strategies are now being advanced into human clinical trials. an alternative strategy is based on improving the performance of the bcg vaccine by insertion of genes encoding specific potential protective antigens that it normally lacks. in addition, the development of auxotrophic mutants of m. tuberculosis is being explored as a potential immunogenic and specifically attenuated live vaccine. the determination of the sequence of the m. tuberculosis genome nearly a decade ago helped identify numerous previously unknown gene products, and increased the repertoire of antigens to be evaluated for their ability to induce protective immune responses. the pathogen sequence is also being used to elucidate virulence determinants and thereby help guide efforts to attenuate m. tuberculosis rationally. together with influenza virus, rsv and piv account for a substantial majority of pediatric upper respiratory illness and consequent acute otitis media. a variety of influenza vaccines are licensed for pediatric use, but vaccines to prevent infection with the paromyxoviruses rsv and piv remain elusive. a significant impediment to vaccine development for rsv and piv traces back to unanticipated untoward results obtained in clinical studies of inactivated rsv vaccines in the early s. these early-generation rsv vaccines -based on cultured virus that had been inactivated with formalin -raised a potent antibody response in immunized children. however, on subsequent natural exposure to rsv, vaccine recipients exhibited more frequent and significantly more severe lower respiratory tract rsv infections than did unimmunized children. as a similar phenomenon was also seen with a formalin-inactivated measles vaccine in the same era, a common immunopathologic mechanism may be operative. while the mechanism of exacerbation of rsv disease by the early inactivated vaccines is incompletely understood, it has been suggested that chemical inactivation of rsv and measles resulted in modification of a critical neutralizing structure on the surfaces of these viruses, thereby limiting the induction of the most potent neutralizing antibodies and favoring nonneutralizing and potentially immunopathologic antibody responses. (passive protection against rsv is available for premature infants in the form of monoclonal antibodies that target the rsv f protein (one of the viral envelope glycoproteins); certain anti-rsv antibody responses can clearly mediate protective as opposed to deleterious effects. ) alternatively, or in addition, it has been proposed that inactivated rsv vaccines may have preferentially induced a th -type immune response when a th -type response may be needed to effect protection of the lower respiratory tract from rsv infection and damage. while excellent live attenuated measles vaccines have been developed, rsv and piv have so far resisted the approach used for measles and mumps (these are all members of the paramyxoviridae family of viruses). based on the successful precedent provided by the live attenuated measles vaccine, an attenuated or reverse genetics-engineered rsv is considered the most promising approach. however, stable attenuation of rsv has been difficult to achieve and vaccine safety concerns result in their cautious advancement through clinical evaluation. effective vaccines for meningococcus types a, c, y, and w are available as straight capsular polysaccharides and as conjugated polysaccharides. the group b polysaccharide shares chemical similarity with a shorter sugar found on the surface of neuronal tissue. while it is possible to make highly immunogenic conjugates with the group b polysaccharide, theoretical concerns about cross-reactivity with self antigens has impeded the development of this type of vaccine. current work centers on a handful of relatively well-conserved surface proteins of meningococcus. gbs is a common component of the flora of the female genital tract, and transfer to the neonate is the cause of severe infections that are fatal or have serious sequelae. short-course intrapartum antibiotics are recommended for culture-positive women, and this approach has cut the incidence of neonatal infections by about two-thirds, thus reducing somewhat the urgency of vaccine development. however, short-course antibiotics could ultimately drive the emergence of antibiotic-resistant gbs. candidate vaccines have been shown to elicit a protective response. however, aside from a reduced market, the main impediment to development of a gbs vaccine is concern over vaccination of pregnant women or women of childbearing age. any birth defect might be attributed to the vaccine, and in a litiginous society, this would be problematic for a vaccine producer. prior to the advent of effective polymerase chain reaction methods for screening blood donations, hcv was a significant cause of transfusionrelated hepatitis. currently, transmission of hcv among the normal population is quite low; transmission among injection drug users remains high. hcv is another pathogen where infection does not typically result in an immune response that clears the infection. however, a minority of hcv patients do spontaneously clear their infection, suggesting that an appropriate immune response could do the job. current vaccine work is concentrated on vectored gene delivery vaccines, primarily adenoviruses, intended to raise antiviral cytotoxic t-cell responses. with the exception of the live attenuated varicella-zoster virus (vzv) vaccine used for the primary prevention of chickenpox and reactivation of latent vzv infections (the cause of shingles and postherpetic neuralgia in older individuals), there are no other vaccines available for use in humans to prevent infection with members of the herpes virus family. hsv types and cause recurrent vesicular eruptions "above or below the belt," respectively. like other herpes viruses, hsv infections are not cleared by the immune system and the virus can persist, remaining in a latent state that is functionally inaccessible to immune recognition and clearance. in addition, like other herpes viruses, hsv encodes a number of gene products that promote evasion of host immune responses. recent attempts to make hsv vaccines have used virus glycoproteins produced by recombinant dna methods. a recent clinical efficacy trial of this vaccine approach showed partial protection of women, but not men, who were seronegative for hsv . the reasons for this curious result are not clear, but efforts to develop this type of vaccine continue. in addition, a number of preclinical studies are exploring the ability of cell-mediated immune responses to hsv antigens induced by recombinant vaccine vectors (e.g., adenoviruses: see novel vaccine vectors, below) to prevent or ameliorate hsv infections. genetically engineered attenuated hsv variants have also been studied in experimental animal models. it is not clear when these new strategies may advance to clinical evaluation in humans. another herpes virus, cmv is a very common infection in humans, with - % of individuals being infected by adulthood. cmv is a cause of severe infections in neonates, causing debilitating neurological sequelae. following initial infection, cmv persists in infected humans, despite the fact that anti-cmv antibodies are present and that a very sizeable proportion of the overall host cd and cd immune responses are specific for cmv antigens. ongoing virus persistence and replication in the face of active host immune responses are likely explained by cmv's sophisticated repertoire of host immune evasion functions (including those that inhibit antigen presentation mechanisms and immune effector responses). for these reasons, to be successful, vaccine development efforts will need to elicit immune responses that are significantly more effective than the quantitatively impressive, but functionally limited, immune responses that are generated in the course of natural cmv infections. live attenuated vaccines have been investigated sporadically since the s. an attenuated strain, the towne strain, showed some effect, but was judged to be insufficiently immunogenic. hybrids of the attenuated towne strain and the virulent toledo strain remain in development. recent work has included recombinant dna (rdna)-derived proteins (via either dna vaccine approaches or recombinant viral vectors, such as attenuated poxviral vectors). , ebv is a herpes virus that represents the causative agent of infectious mononucleosis and is widespread among the human population. in concert with incompletely understood environmental (and perhaps additional host) factors, ebv is also etiologically associated with burkitt's lymphoma. the ability of ebv to establish persistent infections in humans (along with latent infections at the cellular level) despite readily detectable antiviral immune responses suggests that, like other herpes viruses, the development of effective ebv vaccine will likely be challenging. ebv vaccines have been in development since the s with the coat protein, gp / , as the most common vaccine antigen studied. dengue fever virus is a mosquito-borne flavivirus (the virus family that includes japanese encephalitis virus and yellow fever virus -for which successful vaccines exist). dengue virus is endemic in a substantial portion of tropical and subtropical areas and causes febrile disease as well as hemorrhagic fever. there are four distinct serotypes of dengue fever virus. prior infection with one serotype has been implicated in predisposing for more severe disease following infection with a second dengue fever virus serotype, although the evidence supporting this concept has been questioned and the underlying pathogenic mechanisms are incompletely understood. the processes of vaccine development have changed significantly in recent years -a process facilitated by substantial improvements in understanding of human immune system function, as well as the advent of powerful new technologies for vaccine development. as a result of these advances, vaccine development is now commonly pursued in a hypothesis-driven manner and is a far less empiric pursuit than in the past. however, at the same time, the infectious diseases for which no effective vaccines currently exist represent more challenging targets than those diseases that have yielded to vaccine development efforts in the past. furthermore, global changes that influence the emergence and rate of spread of infectious diseases place unprecedented challenges on the productivity and pace of new vaccine development efforts. vaccines not yet available >> the challenge of responding rapidly and effectively, with powerful new technologies, to newly emerging infectionsincluding those that haven't been seen in humans before (e.g., severe acute respiratory syndrome (sars)) or for which novel antigenic variants are anticipated but cannot be predicted (e.g., pandemic influenza) >> maximizing the value of innovative new approaches while ensuring the safety of new vaccines so derived one hypothesis proposes that antibodies against the initial infecting serotype bind to the surface of virus particles of the novel infecting serotype, but do not neutralize the infection. in a process referred to as "immune enhancement" of infection, still infectious complexes of antibody virus particles are then envisioned to be preferentially taken up by cells of the reticuloendothelial system that represent primary target cells for virus replication. although the veracity of this hypothesis in not established, it does present certain theoretical concerns about what type of antibody responses will need to be induced by vaccines to exert beneficial rather than detrimental effects. the general belief is that a vaccine providing equivalent immunity against all four serotypes will be required. vaccines based on inactivated virus, engineered chimeric viruses based on the yellow fever virus vaccine platform, engineered deletion mutant viruses, and rdna-derived proteins are in various stages of development. n new antigen discovery methods n historically, vaccine antigens were not discovered in the literal sense. rather, whole organisms were inactivated by either heat or chemistry or organisms were attenuated by forcing growth in nonphysiological conditions. the entire antigenic repertoire of the organism was delivered to the immune system. the isolation of tetanus, diphtheria, and pertussis toxins, along with chemical detoxification schemes, allowed the production of more refined vaccines. the isolation and purification of polysaccharide capsules from a range of important bacterial pathogens enabled the development of additional vaccines. with the advent of molecular biology in the late s, a new set of tools allowed a more directed approach for the discovery of pathogen virulence factors, vaccine antigen discovery, and vaccine development. the tools of molecular biology enabled for the first time the development of vaccines against pathogens that could not be propagated in culture, including the successful development of recombinant hbv and hpv vaccines. development of these vaccines was enabled by clinical and animal model studies showing that antibodies directed against a specific viral target antigen (e.g., the hbv surface antigen or the hpv l protein) were implicated in protection. in addition, molecular biologic approaches enabled the derivation of fully recombinant vaccine antigens (such as those developed using a limited set of defined antigens of bordetella pertussis), including genetically modified versions of bacterial toxins that maintain their proper antigenic structures but are no longer toxic. however, for many of the pathogens for which vaccines do not currently exist, application of these recombinant dna technology-enabled strategies are insufficient due to incomplete understanding of the pathogen antigens that would elicit a protective host immune response. as such, the development of additional techniques to discover protective antigens was needed. fortunately, several important technological advances that facilitate discovery of previously unknown protective antigens from even very complex microorganisms have opened a new era in vaccine development. the earliest rdna technology-enabled methods of antigen discovery involved the expression of individual pathogen-derived gene products (or fragments thereof ) in bacterial hosts (typically escherichia coli) using rdna expression vectors. here, the genome of a pathogen is broken up, and the fragments are inserted into a plasmid or a viral vector, typically a lambda bacteriophage. colonies, or plaques, are spread on a membrane, allowed to grow, and hopefully express the cloned gene fragments. the ability of these recombinant gene products (now isolated in individual colonies) to react with antibodies present in the serum of individuals who had recovered from infection with that pathogen could then be directly assessed. antibodies present in the immune sera are assessed for their ability to identify antigens by immunochemical reactivity. in this way, the entire genome of a given pathogen could be scanned for potential immunoreactivity. such reactivity would both indicate the in vivo expression of that gene product, as well as document its antigenicity. however, additional studies are needed to demonstrate whether antibody responses against a newly defined antigen have any protective potential. to document the ability of an antigen to elicit protective immune responses, it is necessary to immunize an experimental animal (most commonly, mice) and then, following experimental pathogen challenge, evaluate infection outcomes in immunized versus nonimmunized animals. as this approach has most often been used to identify antigens recognized by host humoral responses, sera from animals immunized with a candidate antigen can then be transferred to a naïve host to provide evidence that the antibody response to the antigen represents the relevant agent of immune protection. more recently, as dna sequencing became more efficient and scaleable, determining the entire sequence of the genomes of viruses, bacteria, and parasites has become routine, , allowing identification of previously unknown genes (and predicted gene products) that can be evaluated as vaccine immunogens. scanning the entire pathogen genome via specific computer analysis programs, genes that exhibit specific characteristics can be identified (e.g., predicted expression on the cell surface by virtue of possession of a leader sequence for secretion or membrane anchor sequences). in addition, the relative conservation of the gene within the pathogen population can be determined by assessment of gene sequences from multiple distinct isolates. once potential vaccine antigens are identified, each candidate gene is expressed in an appropriate rdna system, and the protein product is tested in an animal model. the first bacterial genome sequenced in its entirety was that of haemophilus influenzae, marking the beginning of a new approach to vaccine antigen discovery. since this initial bacterial genome sequence determination, genomic sequencing of pathogens has advanced exponentially. over bacterial genomes have now been sequenced, and hundreds more are currently in process. genome-based antigen discovery is being applied to a wide range of bacteria, including streptococci, pneumococci, staphylococci and chlamydia, as well as nonbacterial pathogens such as plasmodium falciparum. an alternate, promising approach to novel antigen discovery has been built on technological advances in proteomics. , these advances include development of high-resolution two-dimensional gel electrophoresis techniques and mass spectrometry methods that enable separation, identification, and purification of individual proteins from the complex mixture of proteins expressed by a pathogen. in proteomic analyses, a small culture of bacteria, preferably taken directly from an infected person, or otherwise grown in physiologically similar conditions, is subjected to physical or enzymatic treatment with specific proteases to generate peptide fragments that are then fractionated by a micro-high-performance liquid chromatography method and sequenced by molecular mass-by-mass spectrometry. an overlapping set of peptides of approximately - amino acids is sufficient to identify an antigen and provides the means to find the gene. although proteomic analysis is, in some ways, more involved than genomic analysis, it provides an important new approach to antigen identification, and offers a direct way to document that the specific protein identified is actually expressed by the pathogen (including, for example, demonstration that a protein of interest is expressed on the external surface of the pathogen). , a combination of proteomic and serologic methods to select potential novel vaccine immunogens, called serological proteome analysis, or serpa, , can be used to screen the pathogen proteome for expressed proteins that are recognized by antibodies present in sera obtained from individuals who have recovered from an infection with the pathogen. the proteomic approach to antigen discovery has been applied to identify novel vaccine candidates for a number of human pathogens, including helicobacter pylori, chlamydia pneumoniae, staphylococcus aureus, bacillus anthracis, haemophilus influenzae, and plasmodium falciparum. as in new genomic methods for antigen discovery, having identified a gene encoding a candidate antigen by proteomic methods, it is then necessary to show that an immune response of the desired type can be raised against the protein. in addition, it is necessary to show that immune responses elicited following immunization with the candidate antigen engender some degree of protection. often, pure protein antigens produced by recombinant methods are not very immunogenic. as such, many of the emerging recombinant vaccines so produced will likely require enhancement of their immunogenicity by means of an adjuvant. the term 'adjuvant' (derived from the latin adjuvare, to help) refers to any substance added as a component of a vaccine preparation -in addition to the vaccine antigens themselves -that improves the immunological response to the antigen. as such, 'adjuvant' is a catch-all term including a broad range of molecular entities that act via diverse -and, in a number of instances, yet to be elucidated -pathways. until recently, most adjuvants were derived empirically and the mechanisms by which they augmented immune responses were unknown . as a result, there were few, if any, principles available to guide the improvement of known adjuvants or the development of new ones. however, recent advances in understanding of the mechanisms by which dendritic cells sense the presence of pathogens and their constituents, and translate this information to shape the quantity, quality, and durability of host cellular and humoral adaptive immune responses, have transformed adjuvant discovery and optimization. what was once a process of trial and error now represents an area of hypothesis-driven research and mechanism-based discovery. a particularly promising advance emerged from the discovery that pathogen sensing by the innate immune system is mediated by recognition of specific pathogen-associated molecular patterns (pamps) by pathogen recognition receptors (prrs) such as the toll-like receptors (tlrs) that are expressed on dendritic cells (dcs) and other hematolymphoid and some epithelial cells (chapter ). the pathogen-derived pamps recognized by tlrs consist of structures that are found only in or on pathogens (including bacteria, viruses, and parasites) and are not part of normal vertebrate biology. following binding of a specific pamp to a specific prr, a specific cellular activation and response cascade is triggered that can directly confront an intruding pathogen and/or lead to the activation of specific host adaptive immune response mechanisms. these breakthroughs in basic immunology have been readily translated into what can now be considered the science of adjuvant biology. , such progress has occurred at an especially opportune time as new vaccine development strategies have transitioned from traditional approaches using attenuated or killed pathogens to highly defined and purified recombinant proteins (so-called "subunit" vaccines) or nonreplicating vectored antigens. although these newer approaches are promis-ing from the perspective of vaccine safety and the opportunity they afford to design the structures of vaccine immunogens, recombinant or synthetic vaccines are often inherently less immunogenic than traditional vaccines based on attenuated live viruses or intact killed organisms. in the context of current vaccine development and regulatory approval processes, an adjuvant is developed as part of a vaccine, not as an independent product. consequently, there are currently no adjuvants licensed by regulatory authorities as stand-alone products. most contemporary efforts to develop novel adjuvants are focused on the targeted activation of tlrs that are expressed on specific cells critical for the generation of innate and adaptive host responses to specific pathogens. a family consisting of distinct tlrs has been identified to date in humans (chapter ). tlrs are expressed in a number of innate immune cells, including dcs, macrophages, neutrophils, endothelial cells, and fibroblasts. given the importance of dcs as critical antigen-presenting cells, most studies of the biology of tlr signaling have focused on these cells. different tlrs are expressed on distinct subpopulations of dcs, and, depending on the tlr, in distinct cellular compartments. tlrs expressed on the surface of human myeloid dcs include tlr (which is heterodimerized with tlr or ), as well as tlrs , , , and ( fig. . ), while these same cells express tlrs and within endoplasmic reticulum (er) and phagolysosomes. plasmacytoid dcs (pdcs) express tlr and within er/phagolysosomes. the tlrs expressed on the cell surface are primarily activated by pamps encountered in the extracellular environment, while tlrs expressed in the er/phagolysosomes are activated by pamps (including viral pathogen-derived rna or dna) that tend to be routed through these endosomal compartments. activation of tlrs on innate immune cells leads to their production of specific cytokines, as well as their expression of co-stimulatory molecules, leading to induction of adaptive immune responses. given that different dcs express different tlrs, and that signaling via different tlrs results in the expression of a distinct pattern of cytokines, it is believed that activation of specific tlrs can variously favor the induction of th -or th -biased immune responses, or can differentially augment either direct or cross-presentation pathways for antigen presentation ( fig. . ). although most data on induction of specific types of immune responses by engagement of specific tlrs have emerged from murine studies (and have not yet been validated in humans), the ability to tailor an adjuvant preparation to achieve a desired type of immune response with a specific vaccine immunogen is a promising notion. naturally occurring ligands for tlrs include lipopolysaccharide (lps) from bacterial cell walls (recognized by tlr ), triacyl lipopeptides (recognized by tlrs + ), diacyl lipopeptides (recognized by tlrs + ), peptidoglycan (recognized by tlr ), flagellin (the monomer that makes up flagella, recognized by tlr ), singlestranded rna (recognized by tlr ), double-stranded rna (recognized by tlr ), and unmethylated dna containing the dinucleotide pair cpg (recognized by tlr ). based on these insights, a variety of approaches to develop adjuvants predicated to activation of specific tlr pathways are being actively pursued. one interesting aspect of adjuvant development is how it is revealing the mechanisms of action of adjuvants that were originally identified via a process of trial and error, as well as delineating important aspects by which certain empirically derived vaccines are able to induce high-level, long-lasting immune responses. one illustrative example can be found in the case of complete freund's adjuvant (cfa), which has long served as the benchmark for laboratory studies of adjuvants. cfa is a mixed emulsion of mineral oil, mannide monooleate, and killed mycobacteria. however, it is far too reactogenic for use in humans, causing significant pain and abcesses at the site of injection -reactions that would be exacerbated if cfa were to be used repeatedly. an alternative preparation termed incomplete freund's adjuvant (ifa) lacks the mycobacterial component, but it too is associated with injection site reactions that are severe enough to limit its use to experimental therapeutic cancer vaccines. although cfa's toxicity precludes its use as a vaccine adjuvant in humans, many of its constituents (including liposaccharides, dna, and specific bacterial cell wall components) are now understood to exert their adjuvant effects on vaccine-induced immune responses via engagement of specific tlrs. similarly, the live attenuated mycobacterium bovis strain, bcg, long widely employed as a vaccine for the prevention of tuberculosis, includes cell wall, peptidoglycan, and dna components that activate specific tlrs. interestingly, the highly effective yellow fever vaccine d has been shown to activate multiple tlrs as part of its induction of antiviral immune responses. it is quite likely that other live attenuated viruses that transiently replicate in immunized hosts also activate innate immune responses via engagement of tlrs. in yet another example involving a nonreplicating vaccine immunogen, one version of the hib polysaccharide conjugate vaccines now licensed for use in children for the prevention of invasive hib disease includes the meningococcal outer-membrane protein complex (ompc) as its protein carrier. ompc conjugates have favorable immunogenic properties that correlate with the ability of ompc to activate dcs via tlr . hundreds of different adjuvant formulations have been tested in animal models, and a few have been advanced into human studies. with a few α α ( ) toll-receptor agonists. alum, the classical adjuvant most often used in vaccines in humans, includes a range of salts of aluminum precipitated under basic conditions, usually aluminum sulfate mixed with sodium or potassium hydroxide plus a variable amount of phosphate. the relative proportions will determine the size, charge, and solubility of alum. the composition of alum used as an adjuvant varies in currently available vaccines and may influence vaccine immunogenicity. alum is utilized as an adjuvant in many of the currently available vaccines composed of inactivated toxins or recombinant proteins (live attenuated vaccines do not include alum or other adjuvants). alum serves two main purposes as an adjuvant. first, it acts as an antigen depot. vaccine antigens adsorb to alum and elute from it following injection into the host. second, alum acts a mild irritant, causing the recruitment of leukocytes necessary for generation of an immune response to the site of injection. adsorption of antigens on to alum routinely improves immunogenicity, particularly the antibody response. alum does not typically enhance cd t-cell responses. alum has been a component of many vaccines for decades and has an excellent safety record. as new adjuvants are developed, alum may remain as a component of combination adjuvant mixtures (as is the case with some newer adjuvants now approaching clinical use), or it may eventually be supplanted by other agents that more effectively provide favorable depot and local inflammatory responses to accentuate host immune responses. using lipids with polar head groups (e.g., triglycerides) and differing types of hydrophobic tails, one can form either micelles (spheres) or multilamellar sheets in aqueous environments. under the right conditions, antigens can be incorporated into the spheres or between layers of the sheets, providing a potential slow-release depot system. immunopotentiators such as qs or detoxified lps derivatives (such as monophosphoryl lipid a (mpl)) may be added to the lipid mix. iscoms are a proprietary form of liposomes made of cholesterol, saponins from quillaia bark (various members of the qs-x family of triterpene glycosides), and phospholipids that form cage-like structures into which antigens can be entrapped or intercalated. iscom complexes may provide a depot function, as well as facilitate the delivery, uptake, and processing of vaccine immunogens by apcs. purified influenza virus hemagglutinin (ha) and neuraminidase mixed with phosphatidyl choline and phosphatidyl ethanolamine (polar lipids) will form empty particles that have the surface properties of influenza virus. adding an antigen in solution before mixing the lipids results in the incorporation of the antigen inside the particle. this provides a vehicle for delivering antigens to the interior of a cell, via the influenza ha membrane fusion process, thereby enabling antigen processing and presentation via both major histocompatibility complex (mhc) class and pathways. emulsions numerous oil-in-water and water-in-oil emulsions have been tested as adjuvants. one such emulsion, mf , is used in a licensed influenza vaccine. mf consists of squalane, a metabolizable shark oil and two surfactants, polyoxyethylene sorbitan monooleate and sorbitan trioleate, in an oil-in-water emulsion. cytokines are host-produced immunomodulators that regulate immune cell action (chapter ). several cytokines are being tested as potential vaccine adjuvants, including granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin- (il- ), and il- . of the defined tlr agonists being explored as vaccine adjuvants, lps and its partially detoxified form, mpl, which activate tlr , have been most thoroughly explored in clinical trials. with evidence of enhanced ability to increase the percentage of individuals responding with protective antibody levels to hepatitis b as compared to a standard hepatitis b vaccine, one hepatitis b vaccine that employs an adjuvant formulation (termed as ) consisting of a combination of alum and mpl has been licensed for use in high-risk individuals. in addition, a vaccine against hpv that uses the same adjuvant formulation may be licensed soon, and candidate hsv- and malaria vaccines currently being studied in latestage clinical trials also include this alum-mpl combination adjuvant. a wide variety of tlr -specific agonists consisting of oligodeoxynucleotides containing unmethylated cpg motifs (cpg-odn) are being evaluated in preclinical studies. these cpg-odns resemble bacterial dna, modified to include a phosphorothioate backbone to increase their stability. two cpg-odn adjuvants have been evaluated in recent phase i and ii trials and shown to increase the timing and magnitude of induction of protective antibody levels, as well as the proportion of responding individuals, to recombinant hbsag vaccine as compared with the current commercially available version of the vaccine. one of these cpg-odn adjuvants also elicits protective antibody responses in immunized hiv-infected individuals who had previously failed to respond to the hepatitis b vaccine. this approach is now being studied as a way of inducing protective immune responses to hepatitis b earlier after initiation of the vaccination regimen or with fewer doses of the vaccine. in addition to the cpg-odn-based tlr adjuvants described above, small chemical compounds with structures that resemble nucleic acid bases have been identified that activate tlr (e.g., imiquimod) or both tlr and (e.g., resiquimod). these compounds are being evaluated as vaccine adjuvants in preclinical studies. flagellin, a tlr agonist, is also being explored as an adjuvant. recently, attention has also been focused on coupling, rather than mixing, tlr agonists to antigens. cpg oligonucleotides conjugated to antigens have been tested in preclinical studies of hepatitis b vaccines and in human clinical trials for treatment of allergy. ligands for tlr / have been coupled to hiv antigens, and the ligand for tlr (flagellin) has been fused to a variety of antigens. , in some instances, coupling a tlr ligand to an antigen resulted in a substantial improvement of the immune response compared to mixtures -potentially the result of enabling the antigen and the tlr ligand to co-locate in the same dc compartments. numerous preclinical studies have confirmed that many natural and synthetic tlr agonists possess adjuvant activity. importantly, early human clinical trials of tlr-predicated adjuvants have supported the promise of this approach to mechanism-based strategies to augment vaccine immunogenicity. an important challenge is to define the most potent and best-tolerated variants, and to define rules by which activation of specific tlr pathways might translate into predictable augmentation of desired types of immune responses. it is hoped that general rules will emerge to suggest which of an increasing number of novel adjuvants in development performs best with which type of vaccine immunogen, and if results obtained with a specific type of immunogen-adjuvant combination can be extrapolated to predict the likelihood of enhanced immunogenicity with other vaccines. although beyond the reach of available experimental results, the ability to tailor, titrate, and otherwise optimize immune responses to vaccines by manipulation of specific tlr pathways appears a realistic future possibility. however, important challenges remain. in particular, a primary challenge for nextgeneration adjuvant development is finding a combination that retains immunopotentiating action while minimizing vaccine-associated adverse experiences. short-term adverse experiences, such as local injection site reactions, represent undesirable side effects that may disqualify candidate adjuvants early in clinical development. however, given that vaccines are administered to healthy people to prevent potential future infectious diseases, the potential for rarer adverse experiences (such as autoimmunity) that may only be manifest with much longer latency from the time of vaccine adjuvant administration will undoubtedly be important considerations for use in prophylactic vaccines. as induction of cell-mediated immune responses is considered an important component of vaccine strategies for many diseases for which no vaccines are currently available (many of which are caused by intracellular pathogens), there is a need to develop safe and readily scalable approaches to elicit durable cd t-cell responses in immunized humans. further, given the critical role that cd t cells play in induction, differentiation, and maintenance of cd t-cell responses, any such novel vaccine strategy will likely also require appropriate cd tcell responses. as elicitation of cd t-cell responses against a foreign antigen usually depends on the de novo expression of the antigen within a host cell and its subsequent processing and presentation via class i mhc pathways (chapter ), most novel vaccine strategies are predicated on the need to achieve synthesis of pathogen-derived antigens within apcs of immunized human hosts. with an increasing appreciation of the role that cross-presentation pathways can play in elicitation of class i-restricted cd t-cell responses, such de novo antigen synthesis may not need to occur within apcs themselves (which may be an advantage for potential vaccine delivery strategies that do not directly target apcs). one of the many attractive attributes of effective live attenuated vaccines is their ability to recapitulate (to various degrees) many of the processes that lead to the generation of potent immune responses following natural infection. these processes include the fact that replication of all viruses depends on gaining access to host cells for genome replication and for the synthesis of essential components of virus particles that permit further propagation of the infection within and between hosts. one immunologic benefit of this requirement is that de novo synthesis of viral gene products within infected cells provides a key opportunity for viral antigen presentation (via mhc class i pathways) and elicitation of antiviral cellular immune responses. along with the processing and presentation of intact virus proteins via mhc class ii pathways leading to production of antiviral antibody responses, live attenuated viral vaccines have a strong track record for induction of broad cellular and humoral immune responses that likely both contribute to conferring protective immunity. however, despite their track record of success, it is likely that few, if any, new live attenuated viral vaccines will be derived in a manner that resembles previous successful efforts (e.g., the empiric derivation of live attenuated polio, yellow fever, or varicella-zoster vaccines). important reasons for this change include the desire for safe and well-characterized vaccines whose mechanisms of attenuation are defined and that can be monitored in the course of vaccine production and use. indeed, most of the recently developed live attenuated vaccines were derived using new approaches for genetic reassortment (fig. . ) wherein genome segments encoding pathogen-derived antigens of interest are recombined with a common set of genome segments that carry attenuating mutations (derived either by use of attenuating viral passage under specific conditions in cell culture (e.g., the cold-adapted influenza vaccine or use of a virus obtained from a nonhuman host that is itself inherently unable to replicate to high levels in humans, e.g., the reassortant rotavirus vaccine prepared via genetic reassortment between human and bovine rotavirus strains (fig. . ) ). although such approaches have proven successful, they are limited in that they can only be applied to homologous viruses (e.g., those derived from the same virus type) whose genomes are segmented and capable of ready genetic reassortment in culture, or to viruses that can be manipulated by reverse genetics. in response to the desire to produce vaccines that can safely and reliably elicit desired immune responses, especially t-cell responses, several approaches are being explored to develop novel vector systems that permit the expression of pathogen-derived antigens. as many of these approaches are based on viruses distinct from the viral pathogen targeted for induction of host immune responses, the inserted pathogen-derived gene products are expressed via recombinant methods as heterologous antigens. alternatively, in nonviral expression systems, such as dna vaccines, the pathogen-derived antigen is expressed in isolation and does not depend on virus-mediated antigen delivery to apcs following host inoculation. collectively, such recombinant heterologous expression systems are commonly referred to as 'vaccine vectors.' in some instances, such recombinant vectors express only a specific antigen (in the case of dna vaccines or certain viral vectors, e.g., adenovirus), while in others both the inserted pathogen-derived antigens and antigens encoded by the viral vector 'backbone' are expressed (e.g., poxvirus vectors). most new approaches employ expression systems that are inherently nonreplicating (e.g., dna vaccines) or that employ viral vectors that can replicate at high levels in tissue culture but not in vivo (e.g., complemented adenovirus deletion variants or host range-restricted poxviruses). while numerous approaches are being pursued to develop novel vaccine vectors, they will all need to meet certain common criteria to emerge as vaccine approaches applicable for widespread use. in particular, any successful approach must be safe in healthy and immunodeficient humans (given their increased representation in the population as a result of hiv infection and therapeutic immunosuppression), desirably immunogenic (including in individuals who may have been previously exposed to the virus from which the vector was derived, e.g., vaccinia or adenovirus), and able to be produced in large quantities and in a stable manner. a limited number of vaccine vector approaches now being pursued are likely to meet these criteria. should successful approaches emerge, there will likely be interest in applying them for use in vaccines targeting diverse pathogens. thus, while definition of promising, broadly applicable, vaccine vector approaches may help simplify certain aspects of vaccine regulatory review and manufacture, they may also present challenges to prioritize use for specific applications should administration of a given vaccine vector on one occasion compromise or preclude successful administration at a later time. nevertheless, the development of novel vaccine vectors is laying essential groundwork for the development of next-generation vaccines. several novel vaccine vectors currently being studied in preclinical studies and human clinical trials are described below, all of which depend on the delivery and expression of a candidate pathogen-derived gene sequence. in a number of ways, dna vaccines represent the simplest approach to deliver pathogen-derived genes. viral vectors similarly serve to deliver pathogen gene sequences to host apcs, either directly or indirectly, but do so in a manner that depends on and takes advantage of the lifecycle and tropism of the virus that is being adapted to express the exogenous pathogen gene products. the ability of purified plasmid dna containing heterologous antigens expressed under the control of eukaryotic transcriptional regulatory and rna processing signals to elicit immune responses when injected into experimental animals was discovered serendipitously. however, since the initial, quite surprising, description, the development of so-called dna vaccines has become an active area of preclinical and clinical vaccine development. reasons for this enthusiasm include the attractive simplicity and facile preparation of vectors that encode only the defined antigen of interest (which can itself be manipulated via recombinant methods to assume a desired configuration), a reasonably straightforward method for vaccine production, and the inherent stability to temperature (which is much greater than most currently live or subunit vaccines). although the dna vector is most commonly injected intramuscularly, the generation of specific immune responses depends on the uptake of the vector dna by apcs followed by the expression, processing, and presentation of vector-encoded antigens. as tissue and tissue fluids present a hostile environment for purified dna, and the process of dna uptake by apcs appears to be relatively inefficient, much of the dose of injected dna is degraded before it can be reached by an apc that can initiate the desired immune response. most dna vaccine research has been pursued in mice, although studies have now been performed in numerous animal species. studies have usually utilized intramuscular injection of vaccine vector dna, but various intradermal and transdermal approaches have also been explored. murine studies have shown that administration of antigen-encoding plasmid dna can elicit appreciable cellular and humoral immune responses that may confer protection against experimental challenge. however, translation of these promising results in animal models to humans has proven frustrating. while dna vaccines have been generally well tolerated in immunized volunteers, in most human studies of dna vaccines, administration of even substantial quantities of dna vaccine vectors has elicited relatively low-level immune responses. it is not yet known whether these disappointing results reflects fundamental differences in the immunogenic behavior of dna vaccines in humans and mice, or the fact that the dna doses administered to humans do not match those administered to mice (dna per weight of the immunized host). given the substantial size differences between humans and mice, it would likely be impractical (for reasons of both vaccine supply and the actual process of administration of sufficiently high doses of dna) to administer the relative murine dose to humans. as such, a variety of approaches are being explored to prolong dna survival in tissue, promote more efficient targeting of dna to apcs, or to develop novel adjuvants that might specifically amplify immune responses to dna vaccines. , dna vaccines are currently being used as candidate preventive vaccines for a wide variety of infectious diseases, including hiv, tuberculosis, malaria, and cmv. poxviruses represent the family of viruses that are physically the largest viruses and that possess the largest genomes. much of the poxvirus genome encodes gene products that serve to evade host immune responses, and that are not required for virus replication in tissue culture. further, facile techniques for the insertion and deletion of specific viral genes have been developed. the ability to accommodate sizeable foreign gene inserts is, in part, a function of the large size of the poxvirus genome (and the large packaging capacity of poxvirus virions). as a result of these favorable attributes, poxviruses have been utilized extensively in laboratory studies of virus biology, recombinant protein production, and host immune responses. although poxviruses encode multiple gene products that help the virus evade host immune responses, they are, nevertheless, potent immunogens. studies of individuals immunized decades ago with vaccinia virus (in the course of smallpox eradication efforts) have shown that this virus induces long-lasting memory t-and b-cell immune responses. in contrast to most of the other viral vectors currently being developed, poxviruses can replicate readily in culture and do not require an engineered host cell to support propagation ex vivo. one important limitation of all poxvirus vectors developed to date is that, given the large size of the poxvirus genomes and the multitude of gene products they naturally express, even large inserts derived from foreign pathogens of interest will present only a minority of the vaccine vector antigens delivered to and recognized by the host immune system. to be effective, approaches to focus immune responses on the antigen of interest will need to be developed. toward this end, a variety of so-called 'prime-boost' approaches are being explored where the host immune response is primed with one type of recombinant vaccine vector (such as a dna vaccine or adenovirus vector) and then boosted with subsequent delivery of poxvirus vectors encoding the same antigen. in this manner, immune responses to antigens of interest have been significantly augmented in a number of preclinical studies. vaccinia virus represents the prototypic vaccine vector. this virus is the same one that was employed in the successful smallpox eradication campaign, and has been used as a laboratory tool for decades. however, given current high expectations for vaccine safety, and the increased number of immunodeficient individuals present in the population (as a result of the new antigen discovery methods emergence of the hiv pandemic and the increased use of immunosuppressive therapies in clinical medicine) at high risk of serious adverse events, and potentially fatal consequences, from vaccinia immunization, the original vaccinia strains used in smallpox eradication efforts are not considered safe for general use. however, studies of vaccinia-based vaccine vectors have provided a strong basic foundation for research on other more highly attenuated poxvirus variants. modified vaccinia ankara (mva) is an attenuated vaccinia virus that was originally derived by prolonged passage of a vaccinia virus isolate on chicken embryo fibroblasts in culture. in the course of extensive passage in culture, a viral variant emerged that had fortuitously deleted large sections of the viral genome, including those that encode important poxvirus immune evasion genes and those that determine the ability of the virus to replicate on cells obtained from different animal species. specifically, while mva grows well on chicken cells, it cannot replicate in human cells in culture or in vivo, conferring an inherent safety feature. mva was safely administered to over individuals at high risk of adverse consequence for vaccinia immunization toward the end of the smallpox eradication effort. more recently, it has garnered renewed interest as a potential safer smallpox vaccine in the wake of concerns about bioterrorism threats. even though mva cannot replicate in mammalian cells, the virus demonstrates favorable immunogenic properties. mva has been used as a vector expressing genes for a wide variety of genes, including hiv and malaria antigens either alone or, as described above, in 'prime-boost' regimens, where mva has been administered following initial priming immunizations with other vaccine vectors. a concerted effort is under way to improve further the performance of mva by manipulating a series of poxvirus genes that dampen the human immune response to the virus (and to any antigens inserted in it). avipox is a family of poxviruses that infect birds and cause respiratory diseases in poultry. canarypox, a member of the avipox group, has been adapted as a vaccine vector. canarypox replicates well on avian cells in culture but cannot replicate on human cells in culture or in humans in vivo. as a result, canarypox, like mva, provides an interesting vector system with inherent safety features. canarypox vectors carrying hiv genes have been tested in several clinical studies, either alone, or in 'prime-boost' regimens following priming with adenovirus vectors and recombinant protein antigens. in a large ongoing phase iii hiv vaccine trial, a recombinant canarypox vector is being used as a priming vector, followed by boosting with a recombinant version of the hiv gp surface env protein. to date, the results from human clinical trials of canarypox vectors have been disappointing, with only low-level specific immune responses generated in human volunteers. adenoviruses, one of the common causes of upper respiratory and gastrointestinal infections, have seen extensive use in clinical trials and were one of the first gene therapy vectors. most adenovirus vectors currently being studied in preclinical and clinical settings are disabled by deletion of the early e genes that are necessary for replication in an immunized host. most adenovirus vaccine vectors developed have used the well-characterized and readily produced adenovirus serotype (ad ) as the vector 'backbone.' disabled adenovirus vectors are grown in cells that express the e genes artificially inserted into the cell's genome. once these disabled vectors, encoding a heterologous pathogen-derived antigen of interest, enter a cell, the pathogen gene product is expressed, processed, and presented by host apcs. as adenoviruses can directly infect dendritic cells, they promise to provide efficient vaccine vectors. robust antibody and cd t-cell responses to heterologous antigen genes expressed by adenovirus vectors have been observed in preclinical animal models. furthermore, in early-phase human clinical trials, adenovirus vectors have been generally well tolerated, and proven to be the most effective of any recombinant vector system studied to date in eliciting high-level cd t-cell responses. the main potential drawback to widespread use of adenovirus vectors in humans is that, depending on the adenovirus type and the geographic location, variable levels of pre-existing immunity are found in humans as a result of prior naturally acquired adenovirus infections. high levels of antibody against the adenovirus vector might blunt the immunogenicity and efficacy of an adenovirus vector-based vaccine, but it remains to be seen if this will be a significant limitation. should pre-existing immunity to adenovirus vectors derived from epidemiologically prevalent serotypes (e.g., ad ) limit vaccine immunogenicity, current efforts to develop vaccine vectors based on serotypes that are rare in human populations or novel adenovirus vectors specifically designed to avoid preexisting antibody responses may yield effective alternative approaches. adenovirus vectors are currently used in clinical trials for vaccines against hiv, malaria, influenza, and a range of other pathogens. alphaviruses are rna viruses that cause zoonotic diseases, such as venezuelan equine encephalitis. these viruses do not normally circulate in humans, so immunity to these viruses is quite rare in humans. alphaviruses have a strategy for overexpressing the proteins that make up the virion by making a separate subgenomic rna specifically encoding these gene products. current recombinant alphavirus vaccine vector strategies take advantage of this subgenomic transcript, replacing the viral genes with selected genes for other antigens, but maintaining the signals for translation and protein production. in addition, through use of genetic complementation, it is possible to generate virus particles that only contain this heterologous antigen-encoding expression cassette. such virus particles can efficiently mediate infection of host cells, but because they lack other alphavirus genes needed for virus replication cannot spread beyond the initial target cell infected. , alphavirus vectors rival the adenoviruses in efficiency of protein production in tissue culture and have induced robust antibody and t-cell responses in preclinical studies. one current limitation of the alphavirus vector system is the difficulty of scaling the production system; however, this is a technical matter that should be addressable. in addition, ample safety data will be needed before widespread use of alphavirus vaccines achieves endorsement by regulatory authorities for use in healthy populations. adeno-associated viruses (aav ) belong to a family of single-stranded dna viruses (parvoviruses) that include the b parvovirus that causes a rash in children known as 'fifth disease' (measles, mumps, rubella, and varicella make up the first four). aav is transmitted in conjunction with adenovirus infection, and is not known to cause any significant disease. it is poorly immunogenic in the course of natural infections. aav can integrate into the genome of the infected cell, usually in a particular place on chromosome , although integration does not appear to be efficient or site-specific when replicationdefective adenoviruses of the type being developed as vaccine vectors are used. the propensity for chromosomal integration and poor immune response to the virus made aav a good candidate for gene therapy; cells with an integrated viral genome could deliver a gene product for a long time without the immune system killing the infected cell. recently, efforts have been made to adapt replicationdefective aav as a vaccine vector. although encouraging results have been reported in preclinical studies, phase i studies in humans have demonstrated disappointing immunogenicity. n summary n the challenges to optimizing the full public health potential of existing vaccines largely relate to programmatic considerations. in contrast, the terrible impact of infectious diseases that cannot now be prevented by vaccines (such as the 'big three' killers of hiv, tuberculosis, and malaria) pose direct challenges to the scientific community to develop new generations of vaccines that overcome the largely biological obstacles to control and elimination of these diseases. the nature of the challenges posed by such pathogens necessitates that future vaccine efforts will not simply recapitulate the immune responses engendered by natural infection (as has been the premise of traditional vaccine development efforts), but rather, substantially improve upon them. as the development of vaccines to prevent infections with the so-far refractory pathogens is pursued, improved understanding of the immune response to natural infection, as well as delineation of the reasons why host immune responses fail either to clear incipient infections or prevent future new ones, will be essential. fortunately, early empiric approaches have now been replaced with hypothesis-driven strategies enabled by improved insight into the functioning of the human immune system, as well as new technologies, including higher-resolution tools to describe and quantitate pathogen-specific immune responses; novel methods for antigen discovery and targeted optimization of immunogenicity; the development of new, mechanism-based adjuvants; and the advent of innovative methods for vaccine vector-mediated antigen delivery. thus, although the challenges may be vexing, the scientific and technical foundations on which vaccine development efforts rest have never been stronger. n references n for many important infectious diseases for which no vaccines are currently available, successful derivation of effective vaccines will depend on improving upon natural immunity, especially in those instances where natural immunity does not follow natural infection (such as human immunodeficiency virus (hiv) and malaria) or where safety concerns limit the development of specific protective antigens (neisseria meningitidis group b). in addition, for other pathogens that typically manifest significant genetic (and antigenic) diversity (such as influenza, hiv, and bacteria such as streptococcus pneumoniae), a need exists to develop novel vaccines that can protect against a wide range of variants with a limited number of vaccine immunogens. towards these ends, a number of new approaches, enabled by new vaccine technologies, are being pursued, including: >> targeted alteration of protective antigens to increase their ability to elicit protective immune responses (e.g., efforts to alter the structure of the hiv env glycoproteins gp and gp so that they elicit higher-level, more potent, neutralizing antibody responses than their native counterparts) >> the development of synthetic consensus antigens able to elicit broader immune responses than would sequences obtained from individual pathogen isolates (e.g., efforts to develop consensus immunogens able to elicit cytotoxic t-lymphocyte (ctl) responses against genetically diverse hiv- variants) >> techniques for new antigen discovery to identify novel conserved antigens within otherwise genetically diverse pathogens (e.g., streptococcus pneumoniae) or those for which currently known protective antigens cannot be developed as vaccines (e.g., neisseria meningitidis group b) >> the use of novel adjuvants or vaccine vectors to enable 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- - sha: doc_id: cord_uid: oreg rnj over the past decade, a genomics revolution, made possible through the development of high-throughput sequencing, has triggered considerable progress in the study of ancient dna, enabling complete genomes of past organisms to be reconstructed. a newly established branch of this field, ancient pathogen genomics, affords an in-depth view of microbial evolution by providing a molecular fossil record for a number of human-associated pathogens. recent accomplishments include the confident identification of causative agents from past pandemics, the discovery of microbial lineages that are now extinct, the extrapolation of past emergence events on a chronological scale and the characterization of long-term evolutionary history of microorganisms that remain relevant to public health today. in this review, we discuss methodological advancements, persistent challenges and novel revelations gained through the study of ancient pathogen genomes. the long shared history between humans and infectious disease places ancient pathogen genomics within the inter est of several fields such as microbiology, evolutionary biology, history and anthropology. research on this topic aims to better understand the interactions between pathogens and their hosts on an evolutionary timescale, to uncover the origins of pathogens and to disentangle the genetic processes involved in their epidemic emer gence among human populations. over the past , years, major transitions in human subsistence strategies, such as those that accompanied the neolithic revolution , likely exposed our species to a novel range of infectious agents . closer contact with domesticated animals would have increased the frequency of zoonotic transmission events, and higher human population densities would have enhanced the potential of pathogens to propagate within and between groups. throughout human his tory, a number of epidemics and pandemics have been recorded or are hypothesized to have occurred (fig. ) . although most of their causative agents still remain speculative, robust molecular methods coupled with archaeological and historical data can confidently demonstrate the involvement of certain pathogens in these episodes. the investigation of past infectious diseases has tra ditionally been conducted through palaeopathological assessment of ancient skeletal assemblages , , although this approach is limited by the fact that most acute infec tions do not leave visible traces on bone. since the s, the field of ancient dna (adna) has brought molecular techniques to this study, providing a diachronic genetic perspective to infectious disease research. initial attempts relied on pcr technology [ ] [ ] [ ] [ ] [ ] , which restricted the study of ancient microbial dna to targeted, short genomic fragments that were amplified from ancient human remains. this method made infectious disease detection possible but gave limited information on the evolution ary history of the patho gen. in addition, complications associated with the study of adna, which is typically present at low quantities, is heavily fragmented and har bours chemical modifications [ ] [ ] [ ] , hampered efforts to reproduce and authenticate early findings [ ] [ ] [ ] . over the past decade, major advancements in geno mics, in particular, the development of high throughput sequencing, also called next generation sequencing (ngs) , radically increased the amount of data that can be retrieved from ancient remains. this techno logy has assisted the development of quantitative meth ods for adna authentication , , [ ] [ ] [ ] and has enabled the retrieval of whole ancient pathogen genomes from archaeological specimens. the first such genome, pub lished in (ref. ), was that of the notorious bacterial pathogen yersinia pestis, the causative agent of plague. since then, the field has expanded its directions to the in depth study of infectious disease evolution, providing a unique resource for understanding human history. here, we review the latest methodological innovations that have aided the whole genome retrieval and evolu tionary analysis of various ancient pathogens (table ) , most of which are still relevant to public health today. a scientific field focused on the study of whole pathogen genomes retrieved from ancient human, animal or plant remains. the cultural transition associated with the adoption of farming, animal husbandry and domestication as well as the practice of a sedentary lifestyle among human populations. the infectious disease transmission from animals to humans. in the second half of this review, we highlight the util ity of this approach by discussing evolutionary events in the history of y. pestis that have been uniquely revealed through the study of ancient genomes. methods for isolating ancient microbial dna the sweet spot for ancient pathogen dna. the retrieval of dna from ancient human, animal or plant remains carries with it a number of challenges, namely, its limited preservation and hence low abundance, its highly fragmented and damaged state and the perva sive modern dna contamination that necessitates a confident evaluation of its authenticity , . efficient adna recovery is best accomplished via sampling of the anatomical element that contains the highest quantity of dna from the target organism. for human adna analysis, bone and teeth have been the preferred study material, given their abundance in the archaeological record. recent studies suggest that the inner ear por tion of the petrous bone and the cementum layer of teeth have the greatest potential for successful human dna retrieval. however, petrous bone sampling and shotgun ngs sequencing of adna from five bronze age skeletons previously shown to be carrying y. pestis failed to detect the bacterium in this source material, suggesting that its preservation potential for pathogen dna is low . direct sampling from skeletal lesions, where present, has proved a rich source of adna for some chronic disease causing bacteria, such as mycobacterium tuberculosis, which was isolated from vertebrae ; mycobacterium leprae, which could be isolated from portions of the maxilla and various long bones , ; and treponema pallidum subsp. pallidum and t. pallidum subsp. pertenue, which have been isolated from long bones . of note, the sampling methods for recovering pathogen dna do not generally follow a standardized procedure, in part because of the great diversity in tissue tropism and resulting disease progression. in addition, acute blood borne infections do not typically produce diagnostic bone changes as opposed to those that affect their hosts chronically . therefore, if infections have caused mortal ity in the acute phase, as is the case for individuals from epidemic contexts who do not display skeletal evidence of infection, the preferred study material has been the inner cavities of teeth. pathogen adna is thought to be preserved within the remnants of the pulp chamber, likely as part of desiccated blood , . consequently, tooth sampling has proved successful in the retrieval of whole genomes or genome wide data (that is, low coverage genomes that have provided limited analytical resolution) from ancient bacteria such as y. pestis , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , borrelia recurrentis and salmonella enterica ; ancient eukaryotic pathogens such as plasmodium falciparum ; and ancient viruses such as hepatitis b virus (hbv) , and human parvovirus b (b v) . even m. leprae, which commonly manifests in the chronic form, has been retrieved from ancient teeth , . other types of specimen have also shown potential for adna retrieval. examples are dental calculus as a source of oral pathogens, such as tannerella forsythia pandemics refers to increased, often sudden, disease occurrence within populations across more than one region or continent, whereas epidemics refers to increased disease occurrences within a confined region or country. the evaluation of the health status of ancient individuals or populations, usually through the analysis of disease marker presence on skeletal assemblages. (adna). the dna that has been retrieved from historical, archaeological or palaeontological remains. tropism refers to the type of tissue or cell in which infection is established and supported. segregating the metagenomic soup: methods for pathogen detection. regardless of the source of genetic material, most ancient specimens yield complex metagenomic data sets. poorly preserved adna usually makes up a miniscule fraction of the total genetic mate rial extracted from a sample (< %), and the majority of dna usually stems from organisms residing in the envi ronment . hence, specialized protocols are necessary for the detection and isolation of ancient pathogen dna and its confident segregation from a rich environmental dna background (fig. ) . in this context, laboratory based techniques are sep arated into those that target a specific microorganism and those that screen for several pathogenic micro organisms simultaneously (fig. ). methods that screen for a single microorganism have used species specific assays of conventional or quantitative pcr (also known as real time pcr) , - , as well as hybridization based enrichment techniques , , (fig. ). these methods are particularly useful when the target microorganism is known, for example, in the presence of diagnostic skeletal lesions among the studied individuals , , or when a hypothesis exists for the causative agent of an epidemic . by contrast, broad laboratory based patho gen screening in adna research has used microarrays for both targeted enrichment and hbv that were sequenced using capillary sequencing (sanger method). a term used to describe a specimen or data set that includes nucleic acid sequences from all organisms within the sampled proportion. the diagram provides an overview of techniques used for pathogen dna detection in ancient remains by distinguishing between laboratory and computational methods. in both cases, processing begins with the extraction of dna from ancient specimens . as part of the laboratory pipeline, direct screening of extracts can be performed by pcr (quantitative (qpcr) or conventional) against species-specific genes, as done previously , , , . pcr techniques alone, however, can suffer from frequent false-positive results and should therefore always be coupled with further verification methods such as downstream genome enrichment and/or next-generation sequencing (ngs) in order to ensure ancient dna (adna) authentication of putatively positive samples. alternatively , construction of ngs libraries , has enabled pathogen screening via fluorescence-based detection on microarrays and via dna enrichment approaches . the latter has been achieved, through single locus in-solution capture , or through simultaneous screening for multiple pathogens using microarray-based enrichment of species-specific loci and enables post-ngs adna authentication. in addition, data produced by direct (shotgun) sequencing of ngs libraries before enrichment can also be used for pathogen screening using computational tools. after pre-processing, reads can be directly mapped against a target reference genome (in cases for which contextual information is suggestive of a causative organism) or against a multigenome reference composed of closely related species to achieve increased mapping specificity of ancient reads. alternatively , ancient pathogen dna can also be detected using metagenomic profiling methods, as presented elsewhere , , , through taxonomic assignment of shotgun ngs reads. both approaches allow for subsequent assessment of adna authenticity and can be followed by whole pathogen genome retrieval through targeted enrichment or direct sequencing of positive sample libraries. detection , whereby probes are designed to represent unique or conserved regions from a range of pathogenic bacteria, parasites or viruses. although amplification based or fluorescence based approaches can be fast and cost effective for screening large sample collections , , enrichment based techniques are usually coupled with ngs and therefore provide data that can be used to assess adna authenticity. when shotgun sequencing data are generated, com putational screening approaches can be used to detect the presence of pathogen dna as well as for meta genomic profiling of ancient specimens (fig. ). in cases for which a causative agent is suspected, ngs reads can be directly mapped (for example, using the read align ment software burrows-wheeler aligner ) against a specific reference genome or against a multigenome reference that includes several species of a certain genus with the purpose of achieving a higher mapping speci ficity to the target organism (fig. ). in addition, broad approaches involve the use of metagenomic techniques for pathogen screening. examples of tools that have shown their effectiveness with ancient metagenomic dna include the widely used basic local alignment search tool (blast) ; the megan alignment tool (malt) , which involves a taxonomic binning algorithm that can use whole genome databases (such as the national center for biotechnical information (ncbi) reference sequence (refseq) database ); metagenomic phylogenetic analysis (metaphlan) , which is also integrated into the metagenomic pipeline metabit and uses thousands (or millions) of marker genes for the distinction of specific microbial clades; or kraken , an alignment free sequence classifier that is based on k-mer matching of a query to a constructed database. taxonomic sequence assignments from the above methods, however, should be interpreted with caution, mainly because some pathogenic microorganisms have close environmental relatives that are often insuffi ciently represented in public databases. for example, a > % sequence identity was shown between environ mental taxa and human associated pathogens such as m. tuberculosis and y. pestis according to an analysis of s ribosomal rna genes . as such, given that envi ronmental dna often dominates ancient remains that stem from burial contexts , analyses should always ensure a qualitative assessment of assigned reads, that is, an evaluation of their mapping specificity and their genetic distance (also called edit distance) to the puta tively detected organism. in addition, one should con sider the known adna damage characteristics as criteria for data authenticity. although several types of chemical damage can affect post mortem dna survival, certain characteristics have been more extensively quantified. the first, termed depurination, is a hydrolytic mecha nism under which purine bases become excised from dna strands. this process results in the formation of abasic sites and is a known contributor to the fragmen tation patterns observed in adna. as such, an increased base frequency of a and g compared with c and t immediately preceding the ʹ ends of adna fragments is often considered a criterion for authenticity . a sec ond type of damage commonly identified among adna data sets is the hydrolytic deamination of c, whereby a c base is converted into u (and detected as its dna analogue, t) , . this base modification usually occurs at single stranded dna overhangs that are most acces sible to environmental insults, resulting in an increased frequency of miscoding lesions at the terminal ends of adna fragments , . consequently, the evaluation of dna damage profiles (for instance, by using map damage . (ref. )) is a prerequisite for authenticating ancient pathogen dna and is necessary for ensuring adna data integrity in general. more detailed overviews of authentication criteria in ancient pathogen research have been reviewed elsewhere , . targeted enrichment approaches to isolate whole ancient pathogen genomes. evolutionary relationships between past and present infectious agents are best determined through the use of whole genome sequences of pathogens. however, the recovery of high quality data is often challenging owing to the aforementioned char acteristics of adna and therefore requires specialized sample processing. for example, in cases in which adna authenticity has already been achieved in the detection step, u residues resulting from post mortem c deami nation can be entirely or partially excised from adna molecules using the enzyme uracil dna glycosylase (udg) to avoid their interference with downstream read mapping and variant calling. in addition, given the low proportion of patho gen dna in ancient remains, a common and cost effective approach for whole genome retrieval involves microarray based or in solutionbased hybridization capture. both methods constitute a form of genomic selec tion of continuous or discontinuous genomic regions through the design and use of single stranded dna or rna probes that are complementary to the desired tar get. microarray based capture utilizes densely packed probes that are immobilized on a glass slide . it is cost effective in that it permits the parallel enrichment of molecules from several libraries that can be subsequently recovered through deep sequencing, although competi tion over the probes can impair enrichment efficiencies in specimens with comparatively lower target dna con tents. nevertheless, this type of capture has shown its effectiveness in the recovery of both ancient pathogen and human dna , , , , , . more recently, in solutionbased capture approaches have gained popularity owing to their capacity for greater sample throughput without compromising capture effi ciency [ ] [ ] [ ] ; every sample library can be captured indi vidually, thus providing, in principle, an equal probe density per specimen. this technique has contributed to the increased number of specimens from which human genome wide single nucleotide polymorphism (snp) data could be retrieved , , even from climate zones that pose challenges to adna preservation (pre sented elsewhere [ ] [ ] [ ] ). in addition, in solutionbased capture has recently become the preferred method for microbial pathogen genome recovery for both bacteria and dna viruses (for examples, see refs , , , , , , ). nevertheless, deep shotgun sequencing alone has also been used for human [ ] [ ] [ ] and pathogen , , high quality an algorithm that assigns metagenomic dna reads to a species or a higher taxonomic rank (for example, genus or family) based on the sequence specificity. the matching, for each read, of multiple subsequences of length k without mismatches to a database. a hydrolytic reaction in which the β-n-glycosidic bond of a purine (adenine or guanine) is cleaved, causing its excision from a dna strand. the hydrolytic removal of an amine group (nh ) from a molecule. in ancient dna studies, the term deamination most often refers to the deamination of cytosine residues into uracils. the identification of polymorphisms (nucleotide differences) in sequenced data by comparison to a reference. www.nature.com/nrg | june | volume genome reconstruction, especially for specimens with fairly high endogenous dna yields, although this frequently carries with it a greater production cost. in the absence of ancient pathogen genomes, the tim ings of infectious disease emergence and early spread are inferred mainly through comparative genomics of modern pathogen diversity , , palaeopathological eval uation of ancient skeletal remains or analysis of his torical records , . such approaches are highly valuable and, when combined, can be used to build an inter disciplinary picture of infectious disease history; however, limitations also exist. for example, the analysis of con temporary pathogen genetic diversity considers only a short time depth of available data and cannot predict evolutionary scenarios that derive from lineages that are now extinct. in addition, skeletal markers of specific infections in past populations only exist for a few con ditions and, when present, can rarely be considered as definitive, as numerous differential diagnoses can exist for a given skeletal pathology . similarly, historically recorded symptoms can often be misinterpreted given that past descriptions may be unspecific and do not always conform to modern medical terminology . in the past decade, the reconstruction of ancient pathogen genomes has complemented such analyses with direct molecular evidence, often revealing aspects of past infections that were unexpected on the basis of existing data. the recent identification of hbv dna in a mummified individual showing a vesicopustular rash , which is usually considered characteristic of infection with varv, highlights the importance of molecular methods in evaluating differential diagnoses. the oldest recovered genomic evidence of hbv to date was from a , year old individual from present day germany , which shows that this pathogen has affected human populations since the neolithic period. in addition, the virus was identified recently in human remains from the bronze age, iron age and up until the th century of the current era (ce) in eurasia , , , . regarding bacterial pathogens, the identification of b. recurrentis in a th century individual from norway showed that -aside from y. pestis -other vector borne pathogens were also circulating in medieval europe. furthermore, the causative agents of syphilis and yaws, t. pallidum subsp. pallidum and t. pallidum subsp. pertenue, respectively, were recently identified in different individuals from colonial mexico who exhib ited similar skeletal lesions. this study demonstrates the power of ancient pathogen genomics in distinguishing past infectious disease agents that are genetically and phenotypically similar but that differ greatly in their public health significance. finally, the identification of g. vaginalis and s. saprophyticus in calcified nodules from a woman's remains ( th century troy) directly implicates these bacteria in pregnancy related com plications in the past. these findings, as well as other insights gained from analyses of ancient pathogen genomes (table ) , demonstrate the ability of adna to contribute aspects of infectious disease history beyond those accessible by the palaeopathological, historical and modern genetic records. the reconstruction of whole pathogen genomes has not only been a tool for demonstrating infectious disease presence in the past but also aided in the robust infer ence of microbial phylogeography, which is important for understanding the processes that influence pathogen distribution and diversity over time. the evaluation of genetic relationships between ancient and modern pathogens is often conducted by direct whole genome or genome wide snp compari sons of bacteria , , , , , viruses , , , or mito chondrial genomes and nuclear genome data from eukaryotic microorganisms , , . hence, accurate variant calling is critical for drawing reliable evolutionary inferences, although this process is often a challenge when handling data sets derived from samples with high rates of dna fragmentation (resulting in ultrashort read data), low endogenous dna content and high levels of dna dam age. in these cases, increased accuracy is best achieved through stringent ngs read mapping parameters and through visual inspection of the sequences overlap ping the studied snps . in addition, histograms of snp allele frequencies -used to estimate the frequency of heterozygous calls in haploid organisms , -can often demonstrate the effects of environmental contamination on ancient microbial data sets . once variant calls are authenticated, one of the most common types of evolutionary inference in patho gen research is through phylogenetic analysis, which is a powerful means of resolving the genetic history of clonal microorganisms (fig. ) . among the most commonly used tools in ancient microbial genomics are mega , which comprises several phylogenetic methods; phyml , raxml and iq tree , which implement maximum likelihood approaches; mrbayes , which uses a bayesian approach; and programs used for phylo genetic network inference, such as splitstree . two notable studies that examined phylogenetic relationships among ancient m. leprae genomes revealed a high strain diversity in europe between the th and th centuries ce , . considered alongside the oldest palaeopatholog ical cases of leprosy dating to as early as the copper and bronze age in eurasia , and the high frequency of protective immune variants against the disease identi fied in modern day europeans , these results may sug gest a long history of m. leprae presence in this region. moreover, the phylogenetic analysis of a th century s. enterica subsp. enterica genome from europe showed its placement within the paratyphi c lineage . further identification of the bacterium in th century colonial mexico revealed it as a previously unknown candidate pathogen that was likely introduced to the americas through european contact. given the low frequency of paratyphi c today, these results may be indicative of a higher prevalence in past populations. finally, an example from viral genomics is the recovery of hiv rna from degraded serum specimens , which high lighted the importance of archival collections in reconcil ing the expansion of recent pandemics. specifically, these data were able to dispute a long standing hypothesis regarding the initiation of hiv spread in the usa. when the evolutionary histories of pathogens are influenced equally by mutation and recombination, additional tools have been used to identify recombining loci and to determine genetic relationships within and between microbial populations (fig. ) . for example, the programs clonalframeml and recombination detection program (rdp ) have been used to infer potential recombination regions within ancient , , , respectively. in addition, principal component analysis (pca) and ancient admix ture component estimation using the bayesian modelling frameworks structure and finestructure on both multilocus sequence typing (mlst) and whole genome data were recently used for population assignment of a , year old h. pylori genome . these analyses revealed key information on changes of the bacterial population structure that occurred in europe over time. furthermore, the recent study of ancient t. pallidum subsp. pallidum and t. pallidum subsp. pertenue used the program tree puzzle , a maximum likelihoodbased phylogenetic algorithm, to gain a more robust phylogenetic resolution of ambiguous branching patterns among bacterial lineages. such whole genome analyses of both clonal and recombining pathogens have helped to elucidate not only past infectious disease phylogeography but also possible zoonotic or anthroponotic transmission events that reveal disease interaction networks through time. among others (table ) , a notable example is that of , year old pre columbian m. tuberculosis genomes isolated from human remains, which showed a phylo genetic placement among animal adapted lineages, being most closely related to a strain circulating in modern day seals and sea lions . although the extent to which these strains were capable of human to human transmission is unclear, this study supports the existence of tuberculosis in pre columbian south america and is helping to delineate the genomic and adaptive history of m. tuberculosis in the region before european contact . another example of intriguing evolutionary relationships revealed uniquely through the study of ancient pathogen genomes includes analy ses of neolithic and bronze age hbv. these genomes grouped in extinct lineages that are most closely related to modern strains identified exclusively among african non human p ri ma te s , , a result that raises further questions regarding past transmission events in hbv history. finally, the phylogenetic analysis of medieval m. leprae genomes suggested a european source for lep rosy in the americas , reinforcing the hypothesis that humans passed the disease to the nine banded arma dillo, the most common reservoir for this disease in the new world . importantly, the resolution of evolutionary analyses will depend on the quality, size and evenness of spatial sampling in the comparative data set. therefore, the incomplete and often biased sampling of ancient and modern microbial strains can introduce challenges for discerning true biological relationships and past evo lutionary events. nevertheless, in recent years, marked reductions in ngs costs have aided the increased pro duction of large whole genome microbial data sets from present day strains. current efforts for centralized data repositories that are continuously curated (such as the pathosystems resource integration center (patric) database and the recently introduced enterobase ) and the development of robust phylogenetic frameworks that can accommodate genome wide data from > , strains (for example, grapetree ) are becoming valua ble for integrating large sample sizes into microbial evo lutionary analyses. in combination with the increasing number of ancient microbial data sets, these tools will aid in the evaluation of genetic relationships by offering higher resolution. inferring divergence times through molecular dating. apart from providing a molecular fossil record and revealing diachronic evolutionary relationships, a third analytical advantage gained from the retrieval of ancient pathogen genomes is that their ages can be directly used for calibration of a molecular clock. the ages of ancient specimens can be determined through contextual information, through archaeological artefacts or directly through radiocarbon dating, predominantly of bone or tooth collagen. such temporal calibrations are required for high accuracy estimations of micro bial nucleotide substitution rates and in turn lineage divergence dates (fig. ) , particularly because both esti mations seem to be highly influenced by the time depth covered by the genomic data set . for such analyses, the most widely used program is the bayesian statistical framework beast , . a characteristic example of how ancient calibration points can considerably affect divergence date estimates is that of m. tuberculosis. according to modern genetic data and human demographic events, the m. tuberculosis complex (mtbc) evolution was suggested to have fol lowed human migrations out of africa, with its emer gence estimated at more than , years ago . recently, its emergence was re estimated to a maximum of , years ago on the basis of the , year old myco bacterial genomes from peru , a result that was further the diagram is an overview of whole-genome analysis applied to date for ancient microbial data sets and distinguishes the methods used for clonal and recombining pathogens; of note, the depicted summary is not meant to represent an exhaustive pipeline of all possible analyses that could be undertaken. ancient genome reconstruction is usually initiated through reference-based mapping or through de novo assembly of the data, although the latter has only been possible in exceptional cases of ancient dna (adna) preservation , . subsequently , the genomes are assessed for their coverage depth and gene content for evaluation of their quality , which is also relevant for the comparative identification of virulence genes over their evolutionary time frames. here, we show an example of virulence factor presence-or-absence analysis in the form of a heat map, as done previously , , , . in addition, a comparison of the ancient genome or genomes with modern genomes can be carried out for single-nucleotide polymorphism (snp) identification and for assessment of snp effects (using snpeff ), which is particularly relevant for variants that seem to be unique to the ancient genome or genomes. initial evolutionary inference can often be carried out through phylogenetic analysis and by testing for possible evidence of recombination in the analysed data set, for example, by comparing the support of different phylogenetic topologies and by identifying potential recombination regions and homoplasies , . if the data support clonal evolution, robust phylogenetic inference (for example, through a maximumlikelihood approach) is followed by assessment of the temporal signal in the data , . if the data set shows a sufficient phylogenetic signal, molecular dating analysis and demographic modelling are considered possible, although the size of the data set will determine whether such analyses will be feasible and meaningful. alternatively , if recombination is confirmed, genetic relationships between microbial clades or populations can be determined through phylogenetic network analysis or through the use of population genetic methods such as principal component analysis (pca) and identification of ancestral admixture components , . in this case, the assessment of the temporal signal and proceeding with molecular dating analysis is cautioned and likely best performed after exclusion of recombination regions from all genomes in the data set. mrca , most recent common ancestor. ngs, next-generation sequencing. a term used to describe that genome evolution occurs as a function of time and, therefore, the genetic distance between two living forms is proportional to the time of their divergence. a technique to estimate the age of a specimen on the basis of the amount of incorporated radiocarbon ( c) that after the death of an organism gradually becomes lost over time. denotes the frequency of substitution accumulation in an organism within a given time; usually represented as substitutions per site per year. the dates of separation between two phylogenetic lineages, for example, the split between two species. corroborated by the incorporation of th century european mtbc genomes in the dating analysis , , . in molecular phylogenies, the length of each individ ual branch usually reflects the number of substitutions acquired by an organism within a given period of time and, as such, varying branch lengths should represent heterochronous sequences. therefore, an important pre requisite for a robust dating analysis is that the nucleo tide substitution rate of the species whose phylogeny is to be dated behaves in a 'clock like' manner, meaning that phylogenetic branch lengths correlate with archaeological dates or sampling times. such relationships can be assessed through date randomization and root-to-tip regression tests (fig. ) . the former is used to assess the effect of arbi trary exchange of phylogenetic tip dates on the nucleo tide substitution rate and divergence date estimates , whereas the latter is used for estimation of a correlation coefficient (r) and coefficient of determination (r ) by relating the tip date of each taxon to its snp distance from the tree root (using, for example, the program temp est ). the resulting values determine whether there is a temporal signal in the data and suggest whether branches within a phylogeny evolve at a constant rate, in which case a strict molecular clock can be statistically tested, for example, using mega or marginal likelihood estimations , , and applied. if branches are affected by differences in their evolutionary rates, a relaxed clock would be more appropriate. in general, a constant mole cular clock will rarely reliably describe the history of a microbial species, even more so for infectious pathogens whose replication rates vary between active and latent or between epidemic and dormant phases , . in certain cases, neither of the two models may fit the data, such as when extensive rate variation weakens the temporal signal. this challenge was encountered in initial attempts to date the y. pestis phylogeny using too few ancient cali bration points , . similar limitations can arise when the evolutionary history of a microorganism is vastly affected by recombination, as observed for hbv , , although hbv molecular dating was recently attempted using a different genomic data set and suggested that the currently explored diversity of old and new world pri mate lineages (including all human genotypes) may have emerged within the last , years . molecular dating analysis requires the use of an appro priate demographic model for the available data, which can be determined through model testing approaches (for example, through marginal likelihood estimations , ). currently, the most widely used models for estimating dates of divergence are the coalescent constant size , which assumes a continuous population size history -and is unrealistic for epidemic pathogens -and the coalescent skyline , which can estimate effective population size (n e ) changes over time. moreover, the birth-death demographic model , , which is cur rently unexplored within adna frameworks, may prove an insightful analysis tool in the future. this model has shown its applicability on comprehensive pathogen data sets from modern day epidemic contexts . it has the ability to incorporate prior knowledge on incom plete sampling proportions and sampling biases within a data set, a frequent caveat of adna studies that is currently unaccounted for within molecular dating analy ses. finally, recently developed fast dating algorithms should also be noted, for example, the least squared dating (lsd) program, which does not use constrained demographic models but can handle uncorrelated rate variation among phylogenetic branches and has shown potential for analysing large genomic data sets . the pathogen best studied using adna analysis so far is y. pestis, the causative agent of plague. to date, ancient genomes of this bacterium have been published , - (fig. ) , and their analyses have yielded valuable infor mation on past pandemic emergence as well as in depth microbial evolution. integration of such knowledge into human population frameworks has provided key insights into the association of human migrations and infectious disease transmission in the past , . this sec tion describes the evolutionary history of y. pestis with the aim of demonstrating aspects of its emergence and spread as revealed through adna research. plague is a well defined infectious disease caused by the gram negative bacterium y. pestis, which belongs to the fam ily enterobacteriaceae. it evolved from a close relative, yersinia pseudotuberculosis, which is an environmental enteric diseasecausing bacterium . although the two species are clearly distinguishable in terms of their vir ulence potential and transmission mechanisms, their nucleotide genomic identity reaches % among chromo somal protein coding genes . in addition, they share the virulence plasmid pcd , which encodes a type iii secretion system common to three known pathogenic yersinia: y. pestis, y. pseudotuberculosis and yersinia enterocolitica. the distinct transmission mechanism and pathogenicity of y. pestis are conferred by the unique acquisition of two plasmids, ppcp , which contributes to the invasive potential of the bacterium , and pmt , which is involved in flea colonization , , as well as by chromosomal gene pseudogenization or loss throughout its evolutionary history . y. pestis is not human adapted. its primary hosts are sylvatic rodents such as marmots, mice, great gerbils, voles and prairie dogs, among others, in which it is continuously or intermittently maintained in so called reservoirs or foci [ ] [ ] [ ] . its global distribution includes numerous rodent species , and encompasses regions in eastern europe, asia, africa and the americas (fig. ) , where the bacterium persists in active foci, some of which have existed for centuries or even millennia , , , , . y. pestis transmission among hosts is facilitated by a flea vector (fig. ) . the best yet characterized is the oriental rat flea, xenopsylla cheopis, although others are also known to play important roles in y. pestis transmission , , . notably, recent modelling infer ences suggest important roles for ectoparasites such as body lice and human fleas in its propagation during human epidemics . landmark studies investigating the classical model of transmission have shown that y. pestis has the unique ability to colonize and form a biofilm within the flea, which blocks a portion of its a test that involves random shuffling of calibration points (tip dates) across a molecular phylogeny to evaluate the effect of randomizations compared to true data on the nucleotide substitution rate estimates. a test that uses a linear correlation to determine the relationship between branch lengths and sampling times within a time-dependent phylogeny. a mathematical model that aims to explain the size and density of a population over time. www.nature.com/nrg | june | volume foregut, the proventriculus (fig. ). this phenotype is determined by the unique acquisition and activity of certain genomic loci in y. pestis, namely, the yersinia murine toxin (ymt) gene, which is present on the pmt plasmid , and facilitates colonization of the arthropod midgut . in addition, it is dependent on the pseudogenization of certain genes, namely, the biofilm downregulators rcsa, pde (also known as rtn), pde (also known as y ) and the ure ase gene ured , , which are, by contrast, active in y. pseudotuberculosis. the biofilm prevents a blood meal from entering the flea's digestive tract, leaving it starving; as a result, the insect intensifies its feeding behaviour and promotes bacterial transmission to un infected hosts [ ] [ ] [ ] . this continuous transmission cycle among fleas and rodents, also called the enzootic phase of maintenance (fig. ) , is thought to drive the preser vation of plague foci around the world and is depen dent on environmental and climatic factors as well as on host population densities , [ ] [ ] [ ] . disruption of this equilibrium for reasons that are not well understood can cause disease eruption among susceptible rodent species, leading to so called plague epizootics (fig. ) . during that time, marked reductions in the rodent pop ulations force fleas to seek alternative hosts, which can lead to infections in humans and, as a result, trigger the initiation of epidemics or pandemics. plague manifestation in humans has three disease forms, namely, bubonic, pneumonic and septicaemic . bubonic plague is the most common form of the disease and can cause up to % mortality when left untreated . subsequent to the bite of an infected flea, bacteria travel to the closest lymph node, where excessive replica tion occurs, giving rise to large swellings, the so called buboes. in addition, following primary bubonic plague, bacteria can disseminate into the bloodstream to cause septicaemia (secondary septicaemic plague) and to the lungs, causing secondary pneumonic disease. both forms are highly lethal disease presentations and cause nearly % mortality when left untreated. only the pneumonic form can result in direct human tohuman transmission. early evolution: plague in prehistory. the time of divergence between y. pestis and y. pseudotuberculosis has been difficult to determine given the wide temporal interval produced by recent molecular dating attempts , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , are shown as grey circles within their geographical country or region of isolation, and the size of each circle is proportional to the number of strains sequenced from each location (number indicated when more than one genome is shown). the areas highlighted in brown are regions that contain active plague foci as determined by contemporary or historical data. ybp, years before present. adapted with permission from the 'global distribution of natural plague foci as of march ' from https://www.who.int/csr/disease/plague/plague-map- .pdf. based on adna data ( , - , years before present (ybp)) , . nevertheless, y. pestis identification in human remains from neolithic and bronze age eurasia suggests that it caused human infections during these periods and originated more than , years ago , , . these data have revealed important details about the early evolution of the bacterium. genomic and phylogenetic analyses have shown that strains from the late neolithic and bronze age (lnba) occupy a basal lineage in the y. pestis phylogeny, and a recent study suggests the presence of even more basal variants in neolithic europe (fig. ). such analyses have demonstrated that, during its early evolution, the bacterium had not yet acquired impor tant virulence factors consistent with the complex trans mission cycle common to historical and extant strains. one of these genes is ymt, whose absence has been asso ciated with an inability for flea midgut colonization in y. pestis . in addition, these strains possess the active forms of the rcsa, pde , pde and ured genes, which suggests an impaired ability towards biofilm formation and blockage of the flea's proventriculus , . finally, they possess an active flagellin gene (flhd), which is pres ent as a pseudogene in all other y. pestis, as it is a potent inducer of the innate immune response of the host . as a result, during its initial evolutionary stages, y. pestis may have been unable to efficiently transmit via a flea vector. flea borne transmission of y. pestis is a known prerequisite for bubonic plague development ; hence, it has been suggested that this disease phenotype was not present during prehistoric times , . in addition, these results have raised uncertainty regarding the pos sible vector and host mammalian species of the bacte rium. the bronze age in eurasia was a period of intense human migrations, which shaped the genomic landscape of modern day europe , . remarkably, the y. pestis lnba lineage was shown to mirror human movements during that time and was found in regions that do not host wild reservoir populations today (fig. ) . the wide geographical distribution of these strains, their supposed limited bubonic disease potential and their relationship with human migration routes might together be indica tive of a different reservoir host species compared to wild rodents that have a central role in plague transmission in areas such as central and east asia, where the disease is endemic today. nevertheless, an alternative mode of flea transmis sion, termed the early phase transmission, which occurs during the initial phases of infection and was suggested to be biofilm independent , should also be considered as a possible way of y. pestis propagation during its early evolution . although this transmission mechanism is currently not well understood, its comparative mode and efficiency in different rodent species have recently started to be assessed evidence showing the full capacity for flea colonization similar to modern and historic strains was identified in two , year old skeletons from the samara region of modern day russia . although this strain was shown to occupy a phylogenetic position among modern y. pestis lineages (fig. ) , molecular dating analysis indicated that it originated ~ , years ago, suggesting that it over lapped temporally with the other bronze age strains that lacked the genetic prerequisites for arthropod transmis sion. similar characteristics were previously identified in a low coverage , year old isolate from modern day armenia , which suggests that multiple forms of the bacterium were circulating in eurasia between , and , years ago that may have had different transmission cycles and produced different disease phenotypes. as the propagation mechanisms of those strains are still uncer tain, and the exact timing of flea adaptation in y. pestis is unknown, additional metagenomic screening from human and animal remains may provide relevant infor mation on disease reservoirs and hosts across neolithic and bronze age eurasia. it is becoming increasingly apparent that, aside from plague, other infectious diseases, such as those caused by hbv , and b v (table ) , were circulating dur ing the same time periods. further pathogen screening coupled with a temporal assessment of human immune associated genomic variants may reveal key aspects of disease prevalence and susceptibility during this pivotal period of human history. after the bronze age, bubonic plague has been associated with three historically recorded pandem ics. the earliest accounts of the so called first plague pandemic, which began with the plague of justinian ( ce), suggest that it erupted in northern africa in the mid th century ce , and subsequently spread through europe and the vicinity until ~ ce. the sec ond historically recorded plague pandemic began with the infamous black death ( - ce) and con tinued with outbreaks in europe until the th century ce. the most recent third plague pandemic began in the mid th century in the yunnan province of china, and it was during that time that alexandre e. j. yersin first described the bacterium in hong kong, in (fig. ) and has persisted until today in active foci in africa, asia and the americas. although the majority of mod ern plague cases derive from strains disseminated in this global dispersal, the pandemic is considered to have largely subsided since the s . the association of y. pestis with the two earlier pan demics has, until recent years, been contentious. on the basis of their serological characterization, modern strains were traditionally grouped into three distinct biovars, namely, 'antiqua' , 'medievalis' and 'orientalis' , according to their ability to ferment glycerol and reduce nitrate , . in addition, historical accounts of the dis ease seemed to correlate with the supposed distinct geographical distributions of these biovars , and their phylogenetic relationships, as inferred from mlst data, reinforced the hypothesis that each was responsible for a single pandemic . by contrast, later studies identified additional, atypical biovars , and more robust phylo genetic analysis suggested that phylogeography does not correlate clearly with the phenotypic distinctions described between these bacterial populations , , . recent genomic analyses have revealed high genetic diversity of the bacterium in east asia, which invaria bly led to the assumption that y. pestis emerged there . however, a strong research focus on the diversity of the bacterium in these endemic regions, mainly china, has contributed to a profound sampling bias in the available modern data (fig. ) . more recent investigations have revealed previously uncharacterized genetic diversity in the caucasus region and in the central asian steppe that ought to be further explored [ ] [ ] [ ] [ ] (fig. ) . currently, the evolutionary tree of the bacterium is characterized by five main phylogenetic branches (fig. ) . the most ances tral, branch , includes strains distributed across china, mongolia and the areas encompassing the former soviet union. the more phylogenetically derived branches - were formed through a rapid population expan sion event and are today found in asia, africa and the americas . their wide distribution mainly reflects the geographical breadth of branch , which is associated with the third plague pandemic that spread worldwide during the th and th centuries and is still respon sible for more confined epidemics such as those reported in madagascar . the analysis of adna from historical epidemic contexts has generated important information regard ing the evolutionary history of plague. the recovery of y. pestis dna via pcr from remnants of human den tal pulp suggested the involvement of the bacterium in both the first and second pandemics; however, these results were difficult to authenticate , , . subsequent pcr based snp typing of ancient specimens offered some phylogenetic resolution and revealed an expected ancestral placement of medieval strains in the y. pestis phylogeny [ ] [ ] [ ] . more recently, full characterization and authentication of the bacterium were achieved using plasmid and whole genome enrichment coupled with ngs , , , . historical accounts of the first plague pandemic ( th to th centuries ce) suggest that the disease expanded mainly across the mediterranean basin; however, its exact breadth and impact have been difficult to assess given the limited availability of historical and archaeo logical data, with the latter being currently under revision . two recent studies have reconstructed th century y. pestis genomes from southern germany , (fig. ) , a region that lacked historical documentation of the pandemic. phylogenetic analysis showed that both genomes belong to a lineage that is today extinct and is closely related to strains from modern day china , , which suggests the possibility of an east asian origin of the first pandemic. this hypothesis was recently reinforced by the publication of a nd century to rd century y. pestis genome from the tian shan mountains of modern day kyrgyzstan , which shares a common ancestor with the justinianic plague lineage (figs , ) . however, given the > year age difference between these strains , , , as well as the aforementioned east asian sampling bias of modern y. pestis data , the geographical origin of the pandemic remains hypothet ical. retrieval of additional y. pestis strain diversity from that time period, particularly from areas known to have played an important role in the entry of this bacterium into europe, that is, the eastern mediterranean region, may hold clues about its putative source. the beginning of the second plague pandemic, years later, was marked by the notorious black death of europe ( - ce), estimated to have caused an up to % reduction of the continental pop ulation in only years . historical records suggest that the first outbreaks occurred in the lower volga region of russia, and the disease then spread into southern europe through the crimean peninsula . initial analy sis of y. pestis via pcr from victims of the black death revealed a distinct phylogenetic positioning of two mid tolate th century strains and led to the proposal that the disease entered the continent through independent pulses . by contrast, whole genome analysis of ancient strains from western, northern and southern europe demonstrated a lack of y. pestis diversity during the black death, which suggests its fast spread through the continent and favours a single wave entry model of the bacterium into europe , , , although the possible presence of additional strain diversity during that time has recently been explored . intriguingly, the phylo genetic positioning of the black death y. pestis genomes places them on branch , only two nucleotide substitu tions away from the 'star like' diversification of branches - (fig. ) , which gave rise to most of the strain diversity identified around the world today , . after the black death, plague epidemics continued to affect europe until the th century , . inferred climatic data from tree ring records in central asia and europe have recently suggested that such epidem ics were likely caused by multiple introductions of the bacterium into europe as a result of climate driven disruptions of pre existing asian reservoirs . by con trast, ancient genetic and genomic evidence supports the persistence of the disease in europe for years after the black death , , . analysis of y. pestis strains spanning from the late th to the th century ce has revealed the formation of at least two european lin eages that were responsible for the ensuing medieval epidemics (fig. ). both lineages derive from the black death y. pestis strain identified in th century west ern, northern and southern europe , , , suggesting that they likely arose locally. the first lineage survives today and gave rise to modern branch strains , (which are associated with the third plague pandemic), suggesting the european black death as a source for modern day epidemics . the second lineage has not been identified among present day diversity and currently encompasses strains from th century germany and th century france (great plague of marseille, - ce) (fig. ) . these phylogenetic patterns are consistent with a con tinuous persistence of the bacterium in europe dur ing the second plague pandemic. in addition, they are supported by analyses of historical records that suggest the existence of plague reservoirs in the continent until the th century ce . y. pestis is absent from most of europe today; specif ically, no active foci exist west of the black sea. plague is thought to have disappeared from most of europe at the end of the second pandemic ( th century ce). this finding is striking given the thousands of outbreaks that were recorded in the continent until that time , . the reasons for its disappearance are unknown, although numerous hypotheses have been put forward , includ ing a change in domestic rodent populations in europe, namely, the replacement of the black rat, rattus rattus, by the brown rat, rattus norvegicus ; an acquired plague immunity among humans and/or rodents (although this hypothesis requires an update to accommodate the recent identification of y. pestis in europe , years ago , , and the involvement of the bacterium in the first plague pandemic , ); the increased living standards such as the better nutrition and hygienic conditions at the beginning of the early modern era, which may have contributed to improved overall health conditions in europe and likely decreased the number of rats and ecto parasites in human environments , ; and the poten tial disruption of the european wild rodent ecological niche owing to habitat loss and industrialization start ing in ce . given the contribution that molecular data can offer in these discussions, future research on ancient sources of y. pestis dna will be instrumental in further revealing the history of one of humankind's most devastating pathogens. the analysis of ancient pathogen genomes has afforded promising views into past infectious disease history. for y. pestis, adna exploration of its evolutionary past has revealed how a predominantly environmental bac terium and opportunistic gastroenteric pathogen deve loped into an extremely virulent form by acquisition of only a few virulence factors. we eagerly await revelations on a similar scale for other important pathogens that are expected to arise from deep temporal sampling and genomic reconstruction, as made possible through the recent advancements discussed here. integration of ancient pathogen genomes into disease modelling and human population genetic frameworks, as well as their analysis alongside the information offered by the archaeological, historical and palaeopathological records, will help build a more interdisciplinary and com plete picture of host-pathogen interactions and human evolutionary history over time. published online april the origins of agriculture: population growth during a period of declining health emerging and re-emerging infectious diseases: the third epidemiologic transition identification of pathological conditions in human skeletal remains nd edn the global history of paleopathology: pioneers and prospects pre-columbian tuberculosis in northern chile: molecular and skeletal evidence identification of mycobacterium tuberculosis dna in a pre-columbian peruvian mummy molecular analysis of skeletal tuberculosis in an ancient egyptian population detection of -year-old yersinia pestis dna in human dental pulp: an approach to the diagnosis of ancient septicemia the use of the polymerase chain reaction (pcr) to detect mycobacterium tuberculosis in ancient skeletons ancient dna: extraction, characterization, molecular cloning, and enzymatic amplification temporal patterns of nucleotide misincorporations and dna fragmentation in ancient dna the study provides a quantitative description of adna-associated patterns of nucleotide misincorporation and fragmentation that are currently used as primary authentication criteria ancient dna: do it right or not at all absence of yersinia pestisspecific dna in human teeth from five european excavations of putative plague victims no proof that typhoid caused the plague of athens (a reply to papagrigorakis et al.) genome sequencing in microfabricated high-density picolitre reactors targeted enrichment of ancient pathogens yielding the ppcp plasmid of yersinia pestis from victims of the black death the neandertal genome and ancient dna authenticity mining metagenomic data sets for ancient dna: recommended protocols for authentication the study describes the first whole-genome sequence of an ancient bacterial pathogen through the use of high-throughput sequencing genetic analyses from ancient dna optimal ancient dna yields from the inner ear part of the human petrous bone comparing ancient dna preservation in petrous bone and tooth cementum ancient pathogen dna in human teeth and petrous bones pre-columbian mycobacterial genomes reveal seals as a source of new world human tuberculosis ancient genomes reveal a high diversity of mycobacterium leprae in medieval europe the study presents the first de novo assembled ancient pathogen genome and an analysis of m historic treponema pallidum genomes from colonial mexico retrieved from archaeological remains integrative approach using yersinia pestis genomes to revisit the historical landscape of plague during the medieval period emergence and spread of basal lineages of yersinia pestis during the neolithic decline eighteenth century yersinia pestis genomes reveal the long-term persistence of an historical plague focus early divergent strains of yersinia pestis in eurasia , years ago the study describes y. pestis genomes from bronze age human remains and provides a chronological timing of virulence determinant acquisition during the early evolution of the bacterium the stone age plague and its persistence in eurasia a high-coverage yersinia pestis genome from a sixth-century justinianic plague victim yersinia pestis and the plague of justinian - ad: a genomic analysis analysis of -year-old yersinia pestis genomes suggests bronze age origin for bubonic plague historical y. pestis genomes reveal the european black death as the source of ancient and modern plague pandemics ancient human genomes from across the eurasian steppes genomic blueprint of a relapsing fever pathogen in th century scandinavia this paper presents the metagenomic tool malt and is the first case study to demonstrate metagenomic detection of ancient pathogens in the absence of prior knowledge on the causative agent of an epidemic plasmodium falciparum malaria in st− nd century ce southern italy ancient hepatitis b viruses from the bronze age to the medieval period the studies by mühlemann (nature, ) and krause-kyora (elife, ) present a time transect of hbv genomes, spanning from the neolithic period to the medieval period, and provide an overview of the hbv population history across millennia ancient human parvovirus b in eurasia reveals its long-term association with humans this study provides an analysis of the composition of human dental calculus from ancient individuals, showing the presence of oral microbiome bacterial dna, periodontal pathogen dna and proteins associated with host immunity recovery of a medieval brucella melitensis genome using shotgun metagenomics a molecular portrait of maternal sepsis from byzantine troy the study provides insights into the genomic history of h. pylori over several millennia through a population genomic analysis of a copper age strain against a worldwide data set th century variola virus reveals the recent history of smallpox variola virus in a -year-old siberian mummy eighteenth-century genomes show that mixed infections were common at time of peak tuberculosis in europe the paradox of hbv evolution as revealed from a th century mummy tracing hepatitis b virus to the th century in a korean mummy second-pandemic strain of vibrio cholerae from the philadelphia cholera outbreak of mitochondrial dna from the eradicated european plasmodium vivax and p. falciparum from -year-old slides from the ebro delta in spain and 'patient 'hiv- genomes illuminate early hiv/aids history in north america characterization of the influenza virus polymerase genes the rise and fall of the phytophthora infestans lineage that triggered the irish potato famine reconstructing genome evolution in historic samples of the irish potato famine pathogen screening ancient tuberculosis with qpcr: challenges and opportunities genotyping yersinia pestis in historical plague: evidence for long-term persistence of y. pestis in europe from the th to the th century yersinia pestis dna from skeletal remains from the th century ad reveals insights into justinianic plague distinct clones of yersinia pestis caused the black death parallel detection of ancient pathogens via array-based dna capture ancient pathogen dna in archaeological samples detected with a microbial detection array fast and accurate long-read alignment with burrows-wheeler transform basic local alignment search tool reference sequence (refseq) database at ncbi: current status, taxonomic expansion, and functional annotation metagenomic microbial community profiling using unique clade-specific marker genes metabit, an integrative and automated metagenomic pipeline for analysing microbial profiles from high-throughput sequencing shotgun data kraken: ultrafast metagenomic sequence classification using exact alignments a robust framework for microbial archaeology complications in the study of ancient tuberculosis: presence of environmental bacteria in human archaeological remains dna sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient dna : fast approximate bayesian estimates of ancient dna damage parameters removal of deaminated cytosines and detection of in vivo methylation in ancient dna partial uracil-dna-glycosylase treatment for screening of ancient dna hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing targeted investigation of the neandertal genome by array-based sequence capture dna analysis of an early modern human from tianyuan cave application and comparison of large-scale solution-based dna capture-enrichment methods on ancient experimental conditions improving in-solution target enrichment for ancient dna genome-wide patterns of selection in ancient eurasians this study delineates large-scale population migrations into europe during the bronze age by analysis of human genome-wide data of genomic insights into the origin of farming in the ancient near east genetic origins of the minoans and mycenaeans language continuity despite population replacement in remote oceania ancient human genome sequence of an extinct palaeo-eskimo the complete genome sequence of a neanderthal from the altai mountains a high-coverage genome sequence from an archaic denisovan individual yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity the study shows a possible co-expansion of m. tuberculosis among human populations during out-of-africa migrations the bioarchaeology of tuberculosis: a global perspective on a re-emerging disease the black death transformed: disease and culture in early renaissance europe the black death advances in human palaeopathology past human infections mega : molecular evolutionary genetics analysis version . for bigger datasets a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies mrbayes : bayesian phylogenetic inference under mixed models application of phylogenetic networks in evolutionary studies ancient dna study reveals hla susceptibility locus for leprosy in medieval europeans ancient skeletal evidence for leprosy in india possible cases of leprosy from the late copper age ( - cal bc) in hungary leprosy and the adaptation of human toll-like receptor the study describes a th century salmonella enterica subsp. enterica serovar paratyphi c genome and its analysis alongside a comprehensive data set of thousands of s clonalframeml: efficient inference of recombination in whole bacterial genomes rdp : detection and analysis of recombination patterns in virus genomes inference of population structure using multilocus genotype data inference of population structure using dense haplotype data tree-puzzle: maximum likelihood phylogenetic analysis using quartets and parallel computing on the origin of leprosy dna sequencing costs: data from the nhgri genome sequencing program (gsp). genome patric, the bacterial bioinformatics database and analysis resource the paper introduces a web-based platform that performs genome assembly and multilocus sequence typing analysis and can be used for the retrieval of large data sets on enteric bacteria grapetree: visualization of core genomic relationships among , bacterial pathogens genome-scale rates of evolutionary change in bacteria beast: bayesian evolutionary analysis by sampling trees beast : a software platform for bayesian evolutionary analysis the study describes the first sequenced ancient m. tuberculosis genome and shows the presence of mixed infections in th century europe the performance of the date-randomization test in phylogenetic analyses of time-structured virus data exploring the temporal structure of heterochronous sequences using tempest (formerly path-o-gen) bayesian analysis of elapsed times in continuous-time markov chains model selection and parameter inference in phylogenetics using nested sampling improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty relaxed phylogenetics and dating with confidence the study presents a comprehensive y. pestis modern genomic data set from east asia and demonstrates extensive clock-rate variations across the y bayesian coalescent inference of past population dynamics from molecular sequences birth-death skyline plot reveals temporal changes of epidemic spread in hiv and hepatitis c virus (hcv) estimating the basic reproductive number from viral sequence data fast dating using least-squares criteria and algorithms yersinia 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yersinia pestis subsp. microtus bv. altaica strains isolated from the altai mountain natural plague focus (no. ) in russia the genus yersinia: from genomics to function complete genome sequence of yersinia pestis strains antiqua and nepal : evidence of gene reduction in an emerging pathogen genome sequence of yersinia pestis, the causative agent of plague draft genome sequences of yersinia pestis strains from the plague epidemic of surat and shimla outbreak in india comparative genomics of seasonal plague (yersinia pestis) in new mexico complete genome sequences of yersinia pestis from natural foci in china a north american yersinia pestis draft genome sequence: snps and phylogenetic analysis yersinia pestis halotolerance illuminates plague reservoirs mycobacterium leprae genomes from a british medieval leprosy hospital: towards understanding an ancient epidemic characterization of the reconstructed spanish influenza pandemic virus the authors thank c. warinner for her valuable comments to the manuscript and m. keller for his contributions in assembling comprehensive meta-information for the y. pestis modern genomic data set. in addition, the authors thank all members of the molecular paleopathology and computational pathogenomics groups at the max planck institute for the science of human history for insightful discussions during meetings. moreover, they are grateful to m. o'reilly, h. shell and r. barquera for extensive assistance with the graphics. this work was supported by the max planck society. m.a.s. researched the literature and wrote the article. all authors provided substantial contributions to discussions of the content and reviewed and/or edited the manuscript. the authors declare no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. nature reviews genetics thanks e. willerslev and other anonymous reviewer(s) for their contribution to the peer review of this work. key: cord- -xh hu k authors: dhir, bhupinder title: effective control of waterborne pathogens by aquatic plants date: - - journal: waterborne pathogens doi: . /b - - - - . - sha: doc_id: cord_uid: xh hu k the role of aquatic plants in treating wastewater contaminated with inorganic and organic pollutants is well established. recent studies have shown that aquatic plants possess potential to remove pathogens from wastewater. high removal ( %) of pathogenic microbes such as enterococci, escherichia coli, klebsiella pneumonia, pseudomonas aeruginosa, clostridium perfringens, staphylococcus aureus, and salmonella have been achieved using aquatic plant species viz. typha latifolia, cyperus papyrus, cyperus alternifolius, phragmites mauritianus, pistia stratiotes, lemna paucicostata, spirodela polyrhiza, eichhornia crassipes. pathogen removal by aquatic plants mainly occurs because of toxicity exerted by exudates produced by them or attachment of pathogens to plant roots followed by filtration. constructed wetlands have proved very efficient in treating pathogen-contaminated water. more studies are required to find out the exact mechanism of pathogen removal by these plants so that their role in phytoremediation technologies can be emphasized. water contamination has increased tremendously throughout the globe. the presence of pathogenic organisms has significantly increased in the municipal and domestic wastewater in last few decades (pandey et al., ; ramírez-castillo et al., ) . pathogens are the microscopic biological organisms capable of causing diseases. these mainly include bacteria, viruses, fungi, protozoa, helminths, and worms (olaolu et al., ) . the harmful effects from pathogens range from mild illness to chronic severe sickness. waterborne pathogens cause diseases that prove fatal and result in million of deaths annually (awuah et al., ; who, ) . according to an estimate, diseases caused by waterborne pathogens cause about . million deaths in children every year (pandey et al., ) . about species of pathogens infecting humans have been reported worldwide (ashbolt, ; jin et al., ) . these mainly include species of bacteria, types of viruses, species of parasitic protozoa, and several fungi and helminth species (table . ). some of the pathogens commonly found in wastewater include strains of shigella, salmonella, leptospira, e. coli, mycobacterium, vibrio, pseudomonas, staphylococcus, protozoa giardia, cryptosporidium, cysts of entamoeba histolytica, etc. (cabral, ; makvana and sharma, ) . intake/ingestion of contaminated water leads to waterborne infection in humans. the pathogenic microbes infect the gastrointestinal tract and cause diseases such as cholera, typhoid, paratyphoid fever, diarrhea, giardiasis, infectious hepatitis, and amebic/bacillary dysenteries. the severity of the pathogen infection depends on the dose and the susceptibility of the host (ramírez-castillo et al., ) . most of the pathogens live for short period of time outside the human body. transfer of resilient bacterial cysts, oocysts, and direct pathogen through contaminated water causes infection in humans. realizing the health risk caused by consumption of water contaminated with pathogens, various technologies for treatment of wastewater have been developed. these technologies proved useful in improving the quality of drinking water by reducing the number of pathogenic microbes via physical and chemical treatment methods (scott et al., ) . water treatment approaches aim to improve quality of water by removing pathogen contamination (zhang and farahbakhsh, ) . treatment efficacy is widely measured using the log removal value (lrv). treatment should be done in a way that removes maximum number of pathogens followed by disinfection so that high decontamination can be achieved. pathogen treatment is done mainly by two processesdremoval and inactivation (disinfection). in most of the technologies, pretreatment is done using coarse filters (gravel, sand) or removal of turbidity. this method reduces protozoa concentrations (lrv e ) effectively. pretreatment is generally followed by clarification process done by means of flocculation or coagulation. this is generally followed by sedimentation. most of the modern day water treatment technologies include primary and secondary processes. the primary treatment processes include removal or inactivation of pathogens (disinfection). the chlorination, ozonation, and exposure to uv radiation are some of the effective methods used for disinfection (johnson et al., ; gomes et al., ) . heat treatment kills pathogens by exceeding thermal tolerance. oxidation using chlorine gas and sodium hypochlorite removes pathogens by killing them. oxidation disrupts the organic structure of the pathogen. exposure to uv radiation disrupts the genetic material of the microbes and restricts their replication. the secondary processes generally followed after primary treatment includes flocculation, coagulation, and sedimentation. membrane filtration has emerged as a viable water treatment approach over the last decade. use of microfiltration (mf) and ultrafiltration (uf) membranes showed great potential for removal of pathogens (hai et al., ) . a significant removal of pathogens viz. virus, bacteria, and protozoa could be achieved by these filtration system methods (lrv of e as prescribed by the world health organization). mf removes algae, protozoa, and many bacteria effectively. lrv between and has been achieved for giardia and cryptosporidium using a . mm membrane. low virus removal is achieved by using mf. this is because of pore size considerations. the virus species are strongly associated with particles. effective virus removal is achieved using uf. the pathogen removal efficiency in all the cases depends upon the quality of the water and other parameters. apart from the physical and chemical treatment methodologies, the biological techniques such as biological reactors and filters were also evaluated for their efficacy to treat pathogen-contaminated water (chaudhry et al., ; sharma and bhattacharya, ) . both dispersed growth and attached growth biological wastewater treatment systems assist in removal of pathogens but require high hydraulic retention time (hrt) (average time water molecules stay in the system) and continuous feeding of organic matter and nutrients. moreover, the attachment of the microbes to the support medium in these systems is influenced by media composition, cellecell interaction, and polymer molecules (sehar and naz, ) . most of the treatment processes are expensive, complex to operate, and require huge setup, hence become unaffordable for developing countries. membrane filtration techniques though are inexpensive, readily available, and disposable but assist in achieving effective pathogen removal at a small scale. because of these limitations, research studies in the recent years have focused on developing alternate cost-effective sustainable technologies for wastewater treatment. phytoremediation has emerged as an eco-friendly, cost-effective technology that possesses great potential to remove various contaminants (organic, inorganic, and biological) from water at a time (from a single go). the role of terrestrial and aquatic plant species in cleaning the environment via removal of contaminants has been well established (conesa et al., ; das, ; mahajan and kaushal, ) . aquatic plants in particular have shown great capacity to treat water contaminated with inorganic and organic contaminants (zimmels et al., ; schröder et al., ; dhir et al., dhir et al., , shah et al., ; saha et al., ; el-din and abdel-aziz, ) . recent studies demonstrated that aquatic plants are also capable of treating water contaminated with pathogens (reinoso et al., ; kipasika et al., ; srivastava et al., ) . aquatic plants irrespective of algae, macrophytes showed good potential to remove pathogens from water. some studies proved that algae treat bacteriacontaminated water (abdel-raouf et al., ) . algae such as species rhizoclonium implexum (a freshwater species) when used in the quantity of and g showed maximum reduction of % of fecal coliforms from l of water. residence time of days provided % reduction of all fecal indicator bacteria. present study established that algae can prove effective for reduction of coliform bacteria from municipal wastewater. algal-based wastewater treatment system can prove as a good alternate for treatment of pathogencontaminated wastewater (ahmad et al., ) . the amount of biomass played a crucial role in removal of pathogens by algae (ansa et al., a) . the presence of algae leads to increase in ph and oxygen concentration. increase in both the parameters proves detrimental to fecal coliforms (davies-colley et al., ; awuah et al., ) . increase in the concentration of dissolved oxygen increases the rate of decay of fecal coliforms (curtis et al., ) . it is supposed that in the presence of light, toxic forms of oxygen molecules are produced (peroxides and singlet oxygen) (curtis et al., ) . the concentration of these molecules increases with increased dissolved oxygen (do) concentration. the toxic forms of oxygen are injurious to bacteria. they damage the cytoplasmic membrane of the bacteria (awuah, ) . the damage of fecal coliform increases with changes in ph. research studies have shown that algal toxins play a role in the inactivation of fecal coliforms. it has been observed that algae produce toxins when grown in waste stabilization ponds (oudra et al., ) . cyanobacteria (or blue-green algae) synechocystis sp. produced toxin microcystin in waste stabilization ponds. the toxin proved toxic for the fecal bacteria. studies have shown that green algae release some substances that prove harmful to fecal coliforms, thus contributing to their removal (ansa et al., b) . increased inactivation of fecal coliforms was also noted in darkness with increased chlorophyll-a concentration (ansa et al., b) . the studies lead to the conclusion that some substance in the algae might be contributing to the inactivation of the fecal coliforms. realizing the potential of algae in removal of pathogens from water, algal turf scrubbers (ats) were designed by engineers. they represent ecologically engineered systems that utilize the growth of filamentous algae on a submerged surface to remediate polluted water. they use energy from the sun for running the process. these scrubbers showed an ability to remove or degrade a variety of pollutants from water including nutrients, metals, and organic chemicals. ats also showed ability to treat water polluted with pathogenic bacteria. these scrubbers showed an ability to trap pathogenic bacteria through adhesion onto mucilaginous algal filaments. algal turf could remove flavobacterium columnare and e. coli from the water column with an average . log reductions in f. columnare and . log reduction with e. coli over a h period (rains, ) . some studies have also reported that high rate algal ponds reduce the level of pathogenic bacteria significantly from water. the pathogen removal in macrophyte (pistia stratiotes and l. paucicostata) and algal-based wastewater treatment systems was evaluated. the batch scale studies indicated that all treatment systems irrespective having duckweed, lettuce, and algae showed pathogen removal and biochemical oxygen demand (bod) reduction over a period of days under tropical conditions (temperature ranged between . and c, ph between . and ). high ph levels around . and do levels of about mg/l and decrease in the fecal enterococci population from . Â /ml to values below /ml in all treatment systems were observed in the algal-based system (awuah et al., ) . high removal of pathogens such as salmonella spp., e. coli, and other fecal coliforms from wastewater has been achieved using aquatic plant species, viz. t. latifolia, c. papyrus, c. alternifolius, p. mauritianus, iris pseudacorus, and s. lacustris (molleda et al., ; katsenovich et al., ; leto et al., ; abou-elela et al., ) . duckweed ponds have shown significant reduction in the number of total coliforms ( %), fecal coliforms ( %), and coliphage ( %) (karpiscak et al., ) . studies indicate that effective removal of waterborne pathogens by aquatic plants can be achieved in constructed wetlands (stefanakis and akratos, ) . the removal of pathogens by aquatic plants is supposed to occur via different mechanisms: ( ) the chemical substances, i.e., antimicrobial compounds, produced by roots of aquatic plants reduce the survival of pathogens such as bacteria (sundaravadivel and vigneswaran, ; stottmeister et al., ) . ( ) plants regulate the supply of oxygen to roots. oxygen is crucial for the activity and metabolism of microorganisms such as protozoa, nematodes, bacteria, and viruses (stottmeister et al., ; vymazal, ) . ( ) extensive roots of macrophytes or biofilms formed on the plant material/basal media provide surface area for attachment/adherence of microorganisms including bacteria such as e. coli (stevik et al., ; stott and tanner, ) . ( ) the secondary defensive compounds release on wounding of plants during pathogen or insect attack proves also affect the survival of pathogens (taiz and zeiger, ) . the submerged plants and their associated biofilms form "sticky traps" which are capable of trapping a considerable number of organisms (stott and tanner, ; weber and legge, ; kadlec and wallace, ) . the capacity of the aquatic plants in removing parasites is variable and depends upon the properties such as surface area, unique biofilms, and water quality (hogan et al., ) . temperature, ph, salinity, and pathogene sediment interaction are some other factors that also affect the removal of pathogens (microbes) (stott, ; stevik et al., ; searcy et al., ; hogan et al., ) . researchers found that seagrass meadows act as efficient systems in reducing the potentially pathogenic bacteria, viz. enterococcus by %. studies indicated that release of oxygen via photosynthesis in seagrasses proves toxic to pathogens. the chemicals isolated from leaf blades of seagrasses are supposed to kill or inhibit growth of numerous bacterial pathogens. natural biocide produced by seagrasses removed microbiological contamination (lamb et al., ) . studies have proved that eelgrass beds having zostera sp. filter and trap potentially harmful bacteria from coastal waters (webb et al., ) . recent studies have indicated that xylem tissue in plants possesses capacity to remove biological contaminants such as pathogens. xylem is a porous material and acts as membrane comprising of nanoscale pores (few nanometers to around nm). xylem conducts, uptake of fluids from their roots to shoots, can prove feasible as filter where the flow rates of sap are driven by gravitational pressure. these pores act as ideal system for filtering out pathogens and hence can prove as inexpensive water filtration devices. the xylem filter could effectively filter out bacteria such as e. coli from water with rates exceeding . % (boutilier et al., ) . xylem filters fabricated with inexpensive, biodegradable, and disposable materials could prove as ideal materials for filtering pathogens from water (boutilier et al., ) . constructed wetlands (cws) have proved as the most efficient wastewater treatment systems because of their capacity to remove various pollutants. effective treatment of wastewater released from different sources such as agriculture, food processing, tannery, stormwater runoff, landfill leachate, acid mine drainage, municipal discharges, and domestic sources has been achieved using wetlands (headley et al., ; calheiros et al., ; ahmed et al., ; vymazal, ; gruyer et al., ; zurita and carreón-Á lvarez, ; zhang et al., ; maiga et al., ; almuktar et al., ) . easy handling, operation using solar energy, self-organization, increased treatment capacity over time, and high levels of treatment with less maintenance establish them as ideal wastewater treatment systems (hench et al., ) . recent studies indicated that wetlands act as excellent biofilters and reduce significant levels of microbes such as bacteria from wastewater (gersberg et al., ; gopal and ghosh, ; meng et al., ; alexandros and akratos, ) . effective removal of pathogens (rates up to %) has been achieved using constructed wetlands and hence considered as the best wastewater treatment systems (stefanakis and akratos, ; donde and xiao, ) . significant removal of various types of pathogens, i.e., bacteria, viruses, protozoan, and helminths, has been noted in constructed wetlands (al-gheethi et al., ) . effective removal of fecal coliforms ( %e %), ms coliphages ( %e %), and protozoa ( %e %) from wastewater has been reported in wetland wastewater treatment systems (smith et al., ; Á vila et al., ; garcía et al., ; marín et al., ) . high removal capacity of eggs from parasitic organisms (nearly %) has also been noted in wetland treatment processes (Á vila et al., ) . bacteria and virus removal as high as < log has been reported in vegetated wetlands. significant reduction of waterborne bacterial pathogens namely salmonella, shigella, and vibrio (by average of %, %, and %) from domestic wastewater has been reported from a constructed wetland in different seasons of the year (rainy, winter, and summer) (makvana and sharma, ) . wetlands act as biofilters. they remove pathogens such as bacteria, virus, protozoa, and helminths via a combination of physical, chemical, and biological processes taking place in the wetland (kadlec and knight, ; decamp and warren, ; werker et al., ; kuschk et al., ; jasper et al., ) . the removal of pathogens in wetlands occurs because of filtration by plant roots, attachment to plant roots, plant microbe interaction within biofilms, adsorption to the soil/media/organic matter, sedimentation, and predation by microorganisms (jiménez, ; alufasi et al., ) . other mechanisms involved in removal of pathogens include ( ) exposure of pathogens to biocides excreted by macrophyte roots, ( ) natural die-off, ( ) predation by nematodes, protozoa, and rotifers, and ( ) oxidation and exposure to uv radiation (kadlec and wallace, ). the root zone (or rhizosphere) has been considered as active zone of constructed wetlands because of the presence of macrophytes growing there. various physical, chemical, and biological processes take place in this zone. these processes are induced by the interaction of plants, microorganisms, the soil, and pollutants. this is referred as root zone technology (preetha and senthil, ; makvana and sharma, ) . biofilms present in the plant roots are believed to supply a more effective substrate for removal of bacteria through various methods such as mechanical filtration, sedimentation, adsorption, die-off, predation, and antibiotic excretion (soto et al., ; karathanasis et al., ) removal ( %e %) of pathogen especially coliform and enteric bacteria by surface flow constructed wetlands has been reported earlier (perkins and hunter, ) . studies have indicated that beds and stand of reeds reduce pathogens such as salmonella, enterococci, and e. coli by mechanisms such as predation by protozoa and exposure to antibiotic excretions from the roots of plants (perkins and hunter, ; healy and cawley, ; nielsen, ) . the presence/absence of plants also affects pathogen removal in wetlands (arias et al., ; vymazal, ) . generally high removal of pathogens has been reported in wetlands that possess aquatic vegetation (brix, ) . a significant reduction in fecal coliform bacteria has been reported in constructed wetlands planted with aquatic species (thurston et al., ) . high removal of pathogens such as e. coli and salmonella typhimurium has been reported from wetlands having aquatic plants. about % removal of coliforms, e. coli, and streptococci has been reported from a lagoon having plantations of water hyacinth in malaysia. about %e % removal of total coliform, fecal coliform, and enterobacteriaceae has been reported in constructed wetlands located at the czech republic (ottoval et al., ) . field studies reported greater reduction of indicator bacteria, bacteriophage, and viruses from vegetated wetlands (karpiscak et al., ; mandi et al., ; green et al., ; quinonez-diaz et al., ) . the removal of pathogens in these wetlands may be due to an increased competition for nutrients or trace elements with natural microorganisms, attack by lytic phage and bacteria, or predation by nematodes, protozoa, or ciliates. about % (equivalent to log reduction) removal of fecal coliforms has been reported from a wetland planted with phragmites australis after a retention time of . days (rivera et al., ) . a significant reduction of % and % in total and fecal coliforms has been noted in a wetland system having multiple aquatic plant species such as bulrush, cattail, black willow, and cottonwood (karpiscak et al., ) . high removal of pathogens has also been reported from a vegetated constructed wetland in spain (molleda et al., ) . removal of waterborne plant pathogens such as pythium ultimum and fusarium oxysporum from the greenhouse wastewater has been reported (gruyer et al., ) . the development of microbial biofilm around the filter media and the production of cell walledegrading enzymes assist in the removal of pathogens. the removal efficiency of . % for e. coli, total coliforms, . % for fecal streptococci, % for c. perfringens, and % in the case of giardia cysts, cryptosporidium oocysts, and helminth eggs has been noted in wetlands (molleda et al., ) . plants irrespective of free-floating, planted, or emergent assist in removal of pathogens from wetlands (vymazal, ) . they help in the removal of pathogens by mechanical filtration and adsorption processes. the removal of pathogens by plants occurs by different mechanisms: ( ) influencing supply of oxygen to the microorganisms present in the root zone (stottmeister et al., ; vymazal, ) , ( ) adsorbing microbes to biofilms on the rocks, media, and plant roots (stevik et al., ; stott and tanner, ) , ( ) excreting toxic antimicrobial substances by roots that prove toxic to pathogens (sundaravadivel and vigneswaran, ; stottmeister et al., ) , and ( ) predation by nematodes and protists (gerba, ; vymazal, ; kuschk et al., ; wu et al., ) . the pathogens get attached to the root of aquatic plants, and roots filter out pathogens from wastewater. biofilms around plant rhizosphere zone support proliferation of bacterial populations. photosynthates and other compounds released by plant roots provide nutrients for these rhizosphere microorganisms. these microbes produce antibiotic compounds that lead to pathogen removal. compounds produced by algae have been shown to inhibit or restrict the growth of bacteria. the potential of the rhizobacterium can be enhanced through inoculation of antagonist bacteria. the bacteria promote rhizosphere competence and improve pathogenic bacteria removal from wastewater. in a horizontal subsurface flow constructed wetland system planted with p. australis, the inoculation of the strain saccharomyces cerevisiae pfh remarkably improved the inactivation kinetics of the pathogenic bacterium salmonella typhi in the plant rhizosphere. the single inoculation of the bacteria improved the kinetics of s. typhi inactivation by approximately log unit while multiple inoculations brought enhancement in the rate of inhibition by log units. thus this strategy proved useful in enhancing the removal of pathogenic bacteria (saad et al., ) . vegetation plays an important role in filtering contaminants from wetlands (vacca et al., ) . fibrous roots of plants construct pathogen filtration system. plants build biofilm along the roots (vankempen-fryling and camper, ) . the pathogens attach to biotic plant roots. vegetation increases the surface area of the substrate for microbial attachment. the microbial communities in the biofilms help in the transformation of the contaminant. vegetation provides biomass uptake and adsorption of nutrients (o'geen and bianchi, ) . the aeration and oxygen level around plant roots assist in effective pathogen removal. vegetated systems reported high do levels than the unplanted control (sharma and brighu, ) . more do around the root zone reduces the pathogenic activity but increases the removal efficiency. fibrous roots enable good aerated conditions required for removal of pathogens (sharma and brighu, ) . improved aeration in the root zone provides additional removal efficiency and helps to oxidize pathogens present on the root surface, thereby decreasing the pathogen load (sharma and brighu, ) . high levels of do around plant roots facilitate removal of microbes such as e. coli (avelar et al., ) . studies reported that inactivation of bacteriophage in wetlands has been caused because of secretion of microbial metabolites or the presence of proteolytic substances released by microbes or plants. certain bacteria release proteolytic enzymes that are capable of destroying the protein capsid of the viruses. the substances that enhance inactivation of viruses might also be responsible for reduction in other pathogens. thus antiviral properties of aquatic plants could be another possible mechanism causing inactivation of virus in vegetated wetlands. besides this, enhancement in the bacterial population present in rhizosphere of vegetated wetlands might also be responsible for inactivation of viruses. predation is another mechanism that plays an important role in the removal of bacteria, protozoan (oocysts), and fecal coliforms from wastewater in constructed wetlands (mandi et al., ; green et al., ) . predators involved in removal of pathogens in wetlands mainly include nematodes, copepods, rotifers, and protozoa (decamp and warren, ) . bacteriovorous activity of protozoa and ciliates such as paramecium spp., oxytrichids, halteriain, plagiopyla, and casmomorphin has been reported in wetlands (green et al., ; decamp and warren, ) . studies indicate that besides the zooplanktons, rotifers also help in the removal of pathogens such as giardia cysts and cryptosporidium via grazing because of their presence in high amounts (fayer et al., ) . the environmental variables such as sunlight, temperature, and ph regulate the removal of viral and bacterial pathogens from water. temperature plays an important role in the reduction of enteric bacteria and viruses (quinonez-diaz et al., ; winward et al., ) . studies report that high temperatures improve pathogen removal capacity by approximately log unit (ulrich et al., ) . the increase in temperature also supports the predator activity of grazing protozoa (weber and legge, ) . the constructed wetlands have a ph range between . and . . the low ph contributes to high removal of bacteria such as salmonella (pundsack et al., ) . the dissociation of carbonate and bicarbonate ions in water increases ph, thereby causing death of fecal bacteria. sunlight also contributes to the inactivation of pathogens. it mediates the process via three different mechanisms. this includes photolysis of chemical contaminants, absorption of uv-b radiation by dna, and formation of reactive oxygen species (ros). the damage to cellular dna occurs by formation of pyrimidine dimer (davies-colley et al., ) . indirect damage occurs when sensitizers absorb light and produce ros. these ros cause damage to pathogens. superoxide radical is one of the ros produced by sensitizers that cause inactivation of ms coliphage (kohn and nelson, ) . high light intensity, i.e., wavelength more than nm, causes damage to fecal bacteria because of increase in the level of dissolved oxygen and humic substances. exogenous and endogenous substances (such as humic acid) absorb light energy and transfer it to other molecules leading to formation of ros. consequently, ros react with pathogens and cause death because of cell damage (davies-colley et al., ; muela et al., ) . the process is known as photooxidation. the photolysis process is affected by change in ph of water. hydroxyl radical (oh) gets quenched by inorganic carbon under alkaline conditions and results in the formation of carbonate radical (co À ). high levels of dissolved oxygen affect the process of photolysis by removing intermediate triplet states or enhancing formation of o . the high oxygen concentration, i.e., high levels of dissolved oxygen present in wastewater, inactivates pathogens (kohn and nelson, ) . sedimentation is another factor that removes the pathogens such as protozoans, coliforms, bacteria, and helminth eggs (karim et al., (karim et al., , . pathogens such as helminth eggs possess high settling velocities in comparison to other pathogens such as protozoans and bacteria and hence get removed by sedimentation process in wetlands (sengupta et al., ) . the presence of macrophytes enhances the sedimentation process. other factors that influence pathogen removal include mechanical filtration, adsorption to organic matter, and adhesion to biofilm. the removal of pathogens (particularly subsurface constructed wetland) is also influenced by characteristics of the filter bed, i.e., nature of filter media, grain size. studies have reported that wetlands constructed with peat media removed a larger amount of pathogens such as salmonella in comparison to those having sand as media (pundsack et al., ) . high removal of log units for e. coli and log units for ms phage has been noted in a filter bed having fine soil ( e mm diameter) in a horizontal subsurface flow wetland in comparison to those having coarse soil (ushijima et al., ) . the rate and mechanism of removal varies for each pathogen. the rate of removal of pathogen depends on its structure and genetic composition. each pathogen has a different structure and genetic composition (silverman et al., ; mattle et al., ; kohn et al., ) , hence removal mechanism also varies for each species. viruses are resistant than bacteria and hence do not get easily removed by sunlight-mediated reactions (sinton et al., ; davies-colley et al., ) . viral particles can be removed by attachment or adsorption to submerged stalks of emergent plants and biofilm layer of rhizomes and roots (quinonez-diaz et al., ; nokes et al., ) , while bacteria get removed via attachment to plant roots or sedimentation (nokes et al., ; wu et al., ) . constructed wetlands irrespective of horizontal subsurface flow, horizontal free water surface flow, and vertical flow have shown efficacy in treating wastewaters (arias et al., ; mburu et al., ; jiménez et al., ) . greater pathogen removal efficiency has been noted in a subsurface wetland in comparison to free water wetlands (tables . and . ). reduction of pathogens, i.e., bacteria to be as low as < to log , viruses < to log , protozoa < to log , and helminthes < to log , has been noted in subsurface wetland silverman et al., ) . a better pathogen removal capacity has been reported in horizontal subsurface wetlands in comparison to free water surface flow wetlands (wu et al., ) . vertical flow wetlands have aerobic environment in the water and an anaerobic environment at the bottom. the removal of the cysts to the capacity log has been noted for protozoans such as cryptosporidium and giardia in horizontal and vertical flow subsurface wetlands (stott et al., ; falabi et al., ; redder et al., ) . according to literature reports, hybrid constructed wetland systems have also shown good efficiency in treating pathogencontaminated wastewater (kim et al., ; barros et al., ; reinoso et al., ) . a horizontal surface flow constructed wetland treatment system has shown great potential for treating wastewater containing domestic sewage in karachi (mustafa, ) . the pilot-scale constructed wetland planted with the phragmites karka (retz.) reduced indicator bacteria (total coliforms and fecal coliforms). the average removal of the both total and fecal coliforms ranged from % to % (mustafa, ) . furthermore, almuktar et al. ( ) assessed the possibility of recycling domestic wastewater treated by vertical flow constructed wetlands for crop irrigation. vymazal ( ) demonstrated that free water surface flow constructed wetlands that contained emergent vegetation effectively removed %e % of e. coli and %e % of fecal streptococci. this is because planted systems enhance the presence of oxygen in the root zone and plants produce excretions that exert antimicrobial properties (neori et al., ) . greater reduction of thermotolerant coliforms, enterococci, salmonella, shigella, yersinia, and coliphage populations has been reported in subsurface wetlands containing aquatic vegetation (hench et al., ) . hill and sobsey ( ) reported high removal of salmonella from a horizontal subsurface flow wetland containing aquatic plants. in contrast, some studies reported no significant difference in the removal efficiency of pathogens such as thermotolerant coliforms, e. coli, somatic coliphages, and f-specific bacteriophages from planted and unplanted vertical flow constructed subsurface wetlands (torrens et al., ; lana et al., ) . e. coli removal efficiency of . log units has been noted in planted cws in southeastern brazil as compared to . log units in unplanted cws (avelar et al., ) . this is because cws having plants show pathogen removal by several mechanisms (weerakoon et al., ) . the removal mechanism of pathogens differs for free water surface and subsurface flow wetlands. the rate of removal/inactivation varies to a great extent in different zones of the wetland. this is because efficiency of pathogen removal depends on many factors. the different zones of a wetland differ in terms of properties such as oxygen concentration, depth, and amount of planted vegetation. the hydraulic characteristics such as hrt and hydraulic loading rate influence the removal of pathogens to a great extent (garcia et al., ; stottmeister et al., ; vymazal, ) . hrt has been considered as the primary factor affecting the removal of pathogens in surface flow constructed wetlands (diaz et al., ) . the hrt for a free water surface flow wetland depends on the flow rate, presence of plants, characteristics of the removal (%) total coliforms e fecal coliforms e giardia cryptosporidium rotavirus helminth eggs porous media, and water depth. in most of the studies, a significant removal of pathogens has been noted after retention time of days. high removal of parasites has been achieved in wetlands with long retention time (sharafi et al., ) . this is because longer hrt increases the rate of pathogen inactivation because of increased exposure to sunlight, availability of more time for sedimentation, adsorption to organic matter, predation, and exposure to toxins from plants (garcia et al., ; stottmeister et al., ; ulrich et al., ; diaz et al., ) . sunlight causes inactivation of virus and bacteria (kadir and nelson, ) . studies confirmed that hydraulic overloading reduces the removal efficiency of thermotolerant coliforms because of decreased ability to adsorb to the biofilm (wu et al., ) . hence pathogen removal by wetlands occurs via physical, chemical, and biological processes. the physical processes include attachment, adsorption, sedimentation, and mechanical filtration by roots (jasper et al., ) . the chemical processes involved in pathogen removal are oxidation and exposure to uv radiation, sunlight (photooxidation), high temperature, ph, and exposure to biocides. the biological processes involved in removal of pathogens mainly include plant microbe interaction within root biofilms, root-substrate complex, and adsorption (morsy et al., ; sleytr et al., ; alufasi et al., ) . aquatic plants have shown tremendous potential to remove pathogens from wastewater. plants inactivate pathogens by mechanisms such as adsorption by plant roots, biofilm interactions, alteration in soil, or water chemistry. high removal efficiency to log ( % to %) of pathogens particularly bacteria, viruses, protozoa (cysts), and helminths (eggs) from wastewater has been noted in vegetated constructed wetlands. predation by protozoans, adsorption to plant roots, and biofilms on the rhizosphere are the major biological mechanisms responsible for reduction in the concentration of pathogens. toxins produced by plants and natural die-off because of starvation also contribute to removal of pathogens. besides this sedimentation, absorption, inactivation in presence of chemical substances or sunlight, light, temperature, and ph changes are some of the other physicochemical processes responsible for removing pathogens from wastewater treated in wetlands. all the processes contributed in getting a significant reduction ( %e %) in the concentration of pathogens from water. field studies are required to evaluate efficiency of different macrophytes in removing waterborne pathogens and to determine the exact mechanism of pathogen removal so that largescale phytoremediation technologies for 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environment key: cord- -qxkmngyk authors: kozakiewicz, christopher p.; burridge, christopher p.; funk, w. chris; vandewoude, sue; craft, meggan e.; crooks, kevin r.; ernest, holly b.; fountain‐jones, nicholas m.; carver, scott title: pathogens in space: advancing understanding of pathogen dynamics and disease ecology through landscape genetics date: - - journal: evol appl doi: . /eva. sha: doc_id: cord_uid: qxkmngyk landscape genetics has provided many insights into how heterogeneous landscape features drive processes influencing spatial genetic variation in free‐living organisms. this rapidly developing field has focused heavily on vertebrates, and expansion of this scope to the study of infectious diseases holds great potential for landscape geneticists and disease ecologists alike. the potential application of landscape genetics to infectious agents has garnered attention at formative stages in the development of landscape genetics, but systematic examination is lacking. we comprehensively review how landscape genetics is being used to better understand pathogen dynamics. we characterize the field and evaluate the types of questions addressed, approaches used and systems studied. we also review the now established landscape genetic methods and their realized and potential applications to disease ecology. lastly, we identify emerging frontiers in the landscape genetic study of infectious agents, including recent phylogeographic approaches and frameworks for studying complex multihost and host‐vector systems. our review emphasizes the expanding utility of landscape genetic methods available for elucidating key pathogen dynamics (particularly transmission and spread) and also how landscape genetic studies of pathogens can provide insight into host population dynamics. through this review, we convey how increasing awareness of the complementarity of landscape genetics and disease ecology among practitioners of each field promises to drive important cross‐disciplinary advances. its formal inception in , facilitated by technological advances that have increased the availability of molecular and landscape data in conjunction with more powerful computational and analytical approaches. landscape genetics is fuelled by a steady stream of new ideas and methodologies, which, while exciting, can contribute to a lack of consensus or consistency in some key aspects. these aspects include the formulation of research questions, sampling strategies, analytical methods (balkenhol, waits, & dezzani, ; richardson, brady, wang, & spear, ; wagner & fortin, ) and even the identity of the field itself (dyer, ; storfer et al., ) . in fact, landscape genetics has yet to develop its own comprehensive, unifying theory for linking spatial and temporal landscape heterogeneity to genetic variation . while these issues are expected to be remedied as the field matures, many suggestions have been made to facilitate this progress. these have included calls for an increase in cross-disciplinary collaboration (balkenhol, gugerli et al., ) and an expansion of the scope of landscape genetic research beyond its current emphasis on vertebrates dyer, ) and, particularly, mammals (kozakiewicz, carver, & burridge, ) . one logical avenue for cross-disciplinary expansion of landscape genetics is in disease ecology (biek & real, ) . elucidating the specific influences of landscape features on pathogen transmission can provide key insights into the processes that affect disease risk and incidence. however, accomplishing this has been a challenge for disease ecologists (ostfeld, glass, & keesing, ) . indeed, the field of spatial epidemiology has only recently begun to emphasize the use of explicit landscape approaches in studies of spatial heterogeneity in infectious disease (i.e., "landscape epidemiology"; ostfeld et al., ; meentemeyer, haas, & václavík, ) . a major challenge for the study of landscape epidemiology, a field which does not traditionally implement genetic approaches, is that it is typically dependent on the ability to identify the location and timing of transmission events such that they can be compared to landscape features of interest. transmission events are essentially impossible to observe, so disease ecologists often assume that contacts between infected and susceptible individuals are a reasonable proxy for transmission. such contacts generally must be inferred indirectly using methods such as proximity collars, mark-recapture or telemetry, often using spatial overlap as a proxy for contact (craft & caillaud, ) . these methods are logistically challenging to employ, and whether an inferred contact resulted in transmission is uncertain (craft, ) . further, much landscape epidemiological research uses infection or exposure data to indicate past transmission, but these methods provide static snapshots of pathogen prevalence and may be inappropriate for inferring how transmission or spread has occurred (or is occurring) over time (meentemeyer et al., ) . the spatial distribution and movement of hosts are major factors affecting the likelihood, timing and spatial patterns of pathogen transmission and spread (dougherty, seidel, carlson, spiegel, & getz, ) . landscape genetics can identify landscape factors that are important drivers of host population structure. these landscape factors can determine the spatial configuration of a population, its density, its connectivity with other populations, its demographic structure and its genetic health-all of which have implications for the dynamics of microorganisms infecting the host species (ellis, václavík, & meentemeyer, ; prentice, marion, white, davidson, & hutchings, ; spielman, brook, briscoe, & frankham, ) . further, pathogen dynamics can be inferred directly using pathogen genetic data (archie, luikart, & ezenwa, ; decandia, dobson, & vonholdt, ) and incorporated into landscape genetic analyses. understanding specifically how infectious agents respond to the influence of landscape factors on hosts enables us to predict how such agents might spread based on present landscape configurations, as well as under potential future landscape scenarios (real & biek, ) . this knowledge can subsequently inform management efforts at the population level (such as vaccination targeted at key regions, culling), as well as broader decisions relating to the management of the landscape itself, which is a key aim of landscape genetics generally (manel & holderegger, ; segelbacher et al., ) . landscape genetics is being applied by managers at relatively low rates compared to related ecological fields such as landscape ecology, conservation biology and telemetry research (bowman et al., ) . therefore, studies that contribute to the management of disease agents within populations could increase the practical impacts of landscape genetics significantly. however, the conceptual underpinnings of pathogen landscape genetics are not fully developed, and the methodologies employed are diverse and potentially confusing for new practitioners. here, we investigate how landscape genetic techniques are being used to better understand dynamics of microorganisms infecting host species. in conducting this review, we aim to both advocate and facilitate landscape genetic research involving disease-causing organisms. we first evaluate the use of landscape genetics in disease ecology, including the types of questions addressed, the approaches used and the infectious agents studied. we then review established landscape genetic methods and their realized and potential applications to disease ecology. at last, we identify emerging frontiers in the landscape genetic study of pathogens that hold significant potential for advancing research in this field. landscape genetics was first implemented in the study of rabies virus by real et al. ( ) , offering an approach to overcome many feasibility issues associated with understanding landscape influences on pathogen transmission. the landscape genetic approach to studying disease was later reviewed by biek and real ( ) , who were optimistic about its growth and future use. in particular, they noted that microparasites, such as viruses, are well-suited to landscape genetic study due to their rapid mutation rate and potential spatial genetic structure that can be compared to heterogeneous landscape features at fine temporal and spatial scales. analyses could be conducted using both pathogenic organisms and agents that do not cause significant diseases in their hosts (biek, drummond, & poss, ) . they also identified that methodologies such as gis, which are commonly employed both in the wider landscape genetics literature and in spatial studies of infectious disease, had not been widely implemented in molecular epidemiology (archie et al., ) . further, other popular landscape genetic tools, such as those focused on differential landscape permeability (e.g., least-cost paths), were greatly underused despite compatibility with pathogen spatial genetic data. similar to landscape genetics, landscape epidemiology is an interdisciplinary field undergoing rapid development driven by technological advancements, and arguably still working to develop clear directions for future research (meentemeyer et al., ) . it is therefore likely that the interface of these two fields (i.e., where landscape genetics is used in epidemiology) is similarly challenged, perhaps to the extent that its potential is remaining unrealized. we thus believe it is timely to revisit the body of research that combines landscape genetics and landscape epidemiology, leveraging the work done both prior and subsequent to biek and real's ( ) earlier review into clear directions for future research. we conducted a literature search in february using the isi web of science database with the following terms: ts=(("landscape genetic*" or "landscape genom*") and (disease* or pathogen* or parasit* or virus* or virol* or epidem* or infect* or transmi*)) the search returned results. we read each article and retained the empirical papers that used landscape genetic methods to address questions related to pathogens (see supporting information appendix s ). we excluded reviews (n = ), meeting abstracts (n = ), purely methods-based papers (n = ) and articles that identified as or mentioned landscape genetics but did not sufficiently incorporate landscape factors or genetic data into the study (n = ), studies that referred to any of our pathogen-related search terms without it being a primary motivation for the study (n = ), and studies that used words like "transmit" or "parasite" outside of the context of infectious agents (such as the transmission of behaviours) (n = ). one paper was excluded due to a lack of access at our institutions. studies that qualitatively discussed landscape with respect to genetic variation were kept, although one might argue that landscape genetics requires quantitative testing of landscape effects. we classified each paper according to the type of host system studied (plant, wild animal, domestic animal and human), the type of pathogen studied (bacterium, protozoan, virus, prion, fungus, macroparasite and transmissible cancer) and the source of genetic data (host, pathogen and vector), and we estimated the severity of disease that each studied pathogen causes in its sampled host or vector. we also categorized each article according to its general conceptual approach. most examples described in this study were found in our literature search, while several other examples were cited by papers from our search and subsequently also discussed here. following publication of the first study using landscape genetics to investigate disease in , there was little further research in this area until , which saw a rapid increase in the number of publications (figure a ). this increase coincided with two prominent review articles (archie et al., ; biek & real, ) that were strong proponents of a landscape genetics approach to disease ecology and expressed optimism about its future use. the rate of publication has remained relatively steady (and arguably low) since then, with none of the subsequent years recording more publications than in , when six papers were published. however, articles using landscape genetics to investigate disease were published in , potentially indicating increasing interest in this area of research. a majority of studies ( of ) used genetic data from the host for comparison with landscape features (figure b ). this is likely because dna is easier to obtain from larger, free-living hosts than for pathogens, and methods for genotyping and characterizing host spatial genetic variation are more familiar to landscape geneticists, who predominantly study free-living organisms (storfer, murphy, spear, holderegger, & waits, ) . among pathogens that are associated with a particular animal vector, the vector is often genotyped ( of studies of vector-borne diseases), as vectors such as ticks or mosquitos are also easily sampled, and vector gene flow can be used as a proxy for pathogen spread. vectors can be targeted for population control as a means of limiting pathogen spread, which makes their study of immediate relevance to wildlife and livestock managers (townson et al., ) . pathogen genetic data are used in only of pathogen landscape genetic studies, which was somewhat surprising considering that the pathogen is the primary motivation behind many of the reviewed studies. one study included both host and pathogen genetic data (talbot, vonhof, broders, fenton, & keyghobadi, ) . viruses were the most frequently studied type of infectious agent ( of studies; figure c ). in general, viruses evolve more rapidly than other microparasites, which makes them well-suited to study of genetic variation for inference of transmission history (archie et al., ; grenfell et al., ) . however, a majority of landscape genetic studies involving viruses used host genetic data, potentially reflecting the relative difficulty of obtaining viral data, which we discuss later in this section. instead, the high representation of viruses is largely due to the considerable effort devoted to studying rabies, which comprised half of all landscape genetic studies on viral systems. rabies is one of the most well-known wildlife pathogens globally, due to its negative impacts on wildlife, domestic animal and human health (gordon et al., ) . large outbreaks have occurred in north american and european wildlife in recent years, where considerable resources have been devoted to its management (holmala & kauhala, ; slate et al., ) . animals infected with rabies also often exhibit behavioural changes that may make them easier to identify (lefèvre et al., ) , potentially aiding sampling of infected individuals. f i g u r e papers using landscape genetic approaches for the study of infectious agents. (a) number of publications per year that met our search criteria. (b) number of publications using genetic data from each of the host, agent or vector species. (c) number of publications studying pathogens by type, with genetic data source indicated for each type ("unspecified" typically involves studies of a hypothetical agent or estimates of overall pathogen exposure, such as inferred by immune-linked loci). (d) number of publications adopting each of our broadly identified conceptual approaches for applying landscape genetics to the study of pathogens/ infectious agents-using host/vector genetics to predict agent spread, using host/vector genetics to explain agent spread/distribution and using pathogen genetics to directly study agent spread we broadly define three distinct conceptual approaches by which landscape genetics has been used to study infectious agents ( figure d ). these are the prediction of agent spread using genetic information from the host or vector; the use of host or vector genetic information to explain existing spatial variation in infection risk or prevalence; and the use of genetic information from the infectious agent to directly study transmission and spread. the remainder of this section will address each of these approaches in turn. because the spread of many microparasites (particularly directly was unrelated to landscape features tested, determining that current rabies oral vaccination plans should be expanded given the high potential for long-distance host movement. in another rabies study, landscape genetics was used to characterize striped skunk dispersal across riverine and highway barriers to assess their utility as barriers to pathogen spread (talbot, garant, paquette, mainguy, & pelletier, ) . using host or vector genetic data to predict pathogen spread is attractive as it avoids sampling of the agent itself, which may be substantially more difficult, especially in wildlife populations. identification of infected hosts often requires laboratory testing and may require specific, potentially invasive sampling approaches (e.g., necropsy) for accurate diagnosis. in addition, extensive sampling may be required to obtain adequate sample sizes when prevalence is low and must be conducted strategically to capture spatial heterogene- therefore, studies using host or vector data alone have limitations for inferring or predicting pathogen spread, or lack thereof, directly. however, host landscape genetic studies can provide indications of the potential risk of spread of infectious agents, and the understanding gained about host movements can inform subsequent studies of pathogen dynamics. spatial variation in pathogen prevalence or infection risk can be represented in much the same way as any landscape variable , making spatial data relating to presence of an infectious agent well-suited for incorporation into host landscape genetic models. while spatial heterogeneity in pathogen prevalence could also be considered a component of the landscape that may influence spatial genetic variation in the host, typically only adaptive loci are investigated in this context. more commonly, host neutral genetic variation is used to explain spatial patterns of infection risk or prevalence. a prominent example is a study of chronic wasting disease (cwd) in white-tailed deer. blanchong et al. ( ) found that populations with lower cwd prevalence showed higher genetic differentiation from those that had high cwd prevalence. this genetic differentiation was found to be associated with roads and rivers, which were likely barriers to both host gene flow and cwd spread. these inferences have subsequently informed and been verified by additional landscape epidemiological research (robinson, samuel, rolley, & shelton, ) . spatial heterogeneity in pathogen infection risk can also drive microevolutionary responses in the host (epstein et al., ; monello et al., ) . host species are constantly being challenged by parasitic organisms, which, if not overcome, cause disease and can have fitness consequences. this can create strong selection that acts on various genes, and geographic variation in selection at loci that are known to be associated with adaptive immune genes may reflect variation in pathogen pressure, and individual infection or disease risk (fumagalli et al., ) . this variation may be tested for association with environmental features such as temperature, humidity or urbanization (tonteri, vasemägi, lumme, & primmer, ) , enabling insights into how future changes in climate or land use might influence overall pathogen prevalence. using the sampled disease agent as the source of genetic data is the most direct way to infer pathogen spread across landscapes, but can be challenging to accomplish. genetic material may be absent from, or uninformative in some infectious agents, such as prions or clonally transmissible cancers, necessitating genetic analysis of the host (kelly et al., ; storfer et al., ) . in addition to the aforementioned difficulties with pathogen diagnosis, pathogen nucleic acid can be difficult to isolate from samples taken from the host or vector and would ideally be present in the blood, saliva or other easily collected sample. samples may also require enrichment to obtain sufficient quantities of genetic material for analysis, which can be difficult to accomplish for many pathogens, particularly viruses. however, genetic information from viruses may be particularly useful for molecular epidemiologic analyses due to their rapid mutation rate that can closely infer transmission history (archie et al., ; brunker, hampson, horton, & biek, ) . further, viruses are prominent emerging pathogens and have relatively small genomes, aiding whole genome-analysis. there are a variety of methods available for implementing landscape genetics, some designed specifically for landscape genetics, while others have been adapted from other fields. the rapid development of landscape genetics means that new methods are regularly emerging, and it is difficult to comprehensively review all of them. however, there are some well-established methodological approaches that have either seen wide use for some time or are becoming increasingly popular at the cutting edge of the field . we describe the approaches (table ) and discuss their implementation in the study of pathogen transmission and spread. in landscape genetics, simulation models are usually agent-based and spatially explicit (landguth, cushman, & balkenhol, ) . genetic data are modelled for individuals which have discrete spatial locations with respect to one another and with respect to environmental heterogeneity. individuals move, behave and reproduce according to their own attributes in response to other individuals and in response to the simulated environment, and the model simulates changes in allele frequencies in response to these parameters. landscape genetic simulation modelling has been used to test and validate methodological approaches (cushman, wasserman, landguth, & shirk, ; zeller et al., ) , address theoretical questions about how and why landscape heterogeneity influences genetics (landguth et al., ) , and evaluate and explain empirical observations (shirk, cushman, & landguth, ) . further, simulation modelling can predict how a system might respond to certain changes, such as habitat fragmentation or future management activities. simulation modelling has been widely implemented in the study of pathogenic and nonpathogenic disease, beginning with medical research in the s (elveback & varma, (rees et al., ) . the spread of particular host genes relevant to disease can also be simulated to inform management efforts. for instance, landguth, holden, mahalovich, and cushman ( ) used landscape genetic simulations to determine optimal planting regimes to maximize the spread of blister rust resistant genes among whitebark pine populations. such simulations could undoubtedly be applied to vector species in particular, such as predicting the spread of pesticide resistance genes in mosquitos (chang et al., ) and selecting appropriate sites for introduction of genetically modified vectors (lavery, harrington, & scott, ) . in addition, with the need to develop further landscape genetic frameworks for the study of pathogens, simulation modelling can prove useful in testing and validating these techniques, as it has done in the broader landscape genetics field (cushman et al., ; zeller et al., ) . for example, leo, gonzalez, millien, and cristescu ( ) used landscape genetic simulations to validate their multitaxa integrated landscape genetic framework, which appears to be a promising solution to the challenge of studying pathogens with multiple hosts and/or vectors. landscape genetic simulations may also include epidemiological parameters such as mortality or activity responses to infection, or limited infectious periods, which may otherwise confound conventional (i.e., nonsimulation) landscape genetic approaches. edge detection methods, such as monmonier's maximum difference algorithm, (monmonier, ) have also been used to detect landscape barriers to transmission in pathogen studies (carrel et al., ; joannon et al., ) . ancestry estimates from model-based clustering algorithms can assign individuals to their populations of origin, enabling inference of landscape barrier permeability through the identification of migrants and thus estimation of the risk of pathogen spread across the barrier. most of the studies implementing clustering and assignment methods did not use approaches that incorporate environmental data. instead, spatially or nonspatially explicit methods were typically used to identify genetic discontinuities and relationships with landscape barriers were inferred ad hoc, or analyses proceeded to entirely different methods that explicitly include environmental data. associations between genetic discontinuities and landscape be applied to pathogens directly without these potential constraints. resistance surfaces are commonly used in landscape genetics for modelling hypotheses concerning the influence of landscape features (from gis landscape variables) on functional connectivity using techniques such as least-cost paths (adriaensen et al., ) or circuit theory (mcrae, dickson, keitt, & shah, ) . these techniques produce measures of landscape or "effective" distance among populations or individuals for each hypothesis, which can be tested against observed genetic variation. the primary applications of resistance surface modelling in landscape genetics have been the identification of dispersal corridors and predicting the impacts of landscape and environmental change, such as habitat fragmentation or climate change, on connectivity. similar to that, landscape genetic resistance surfaces can identify transmission corridors or future patterns of spread (e.g., streicker et al., ) , and such tools have been identified previously as having great utility for pathogen landscape genetic studies (biek & real, ) . however, resistance surface modelling remains infrequently applied among pathogen studies. careful consideration is required for identifying the most relevant landscape variables to be tested and correctly parameterizing (assigning costs to) the resistance surface(s) so that these variables are represented in a biologically meaningful way. developing landscape resistance hypotheses for transmitted agents may be more difficult as their interactions with the landscape are often indirect, mediated by the ecology of hosts and vectors. pathogen ecological niche models offer an empirical approach for constructing resistance surfaces based on ecological factors influencing pathogen prevalence fountain-jones, pearse et al., ) , but these also may not adequately represent host/vector movements. our literature search returned only one study that explicitly modelled landscape resistance based on pathogen-specific biology, testing elevation (as a proxy for temperature) as a predictor of plasmodium spread, in addition to resistance surfaces that modelled human movements and mosquito vector ecology (lo et al., graph theoretical approaches, which describe connections (edges) between discrete objects (nodes) (newman, ) , are a flexible yet powerful tool for use in landscape genetics (dyer, nason, & garrick, ; garroway, bowman, carr, & wilson, ) . in landscape genetics, nodes can represent individuals, populations or habitat patches, possessing genetic parameters such as diversity measures (dyer et al., ) , or landscape parameters such as percentage habitat or habitat quality (murphy, dezzani, pilliod, & storfer, ) . similar to that, edges can represent genetic relationships between nodes such as genetic distances, gene flow or dispersal (decout, manel, miaud, & luque, ) , or spatial/landscape relationships such as geographic distance or landscape resistance (dyer et al., ) . distinct from other landscape genetic analytical approaches, graphs allow inferences based on the overall shape, or topology, of the network, which can provide unique insights into systemwide processes, such as hierarchical population structure (dyer & nason, ) . network topology may be used to identify populations or habitat patches that form important "stepping stones" for maintaining genetic connectivity across an entire system. such an approach enables experimental simulation whereby nodes may be selectively removed and the overall effect on the system's topology (e.g., overall connectivity, population structure) assessed. metrics pertaining to the importance of individual nodes to network topology can be correlated with variables such as landscape to identify important drivers of network processes. despite their unique applications, graph theory and network approaches are relatively underutilized in landscape genetics compared to methods specifically derived from population genetics and landscape ecology. however, among studies of infectious agents, network approaches in wildlife are becoming increasingly popular (craft, ; craft & caillaud, ) . epidemiological network models are typically based on host contact networks, which are usually constructed using direct observations or indirect techniques such as mark-recapture, telemetry or proximity loggers, and pathogens are simulated on these contact networks. such approaches have already incorporated landscape and other environmental features. in addition, the potential for inferring host contacts in network models using pathogen genetic markers (see below) has been acknowledged in recent reviews (craft, ; gilbertson, fountain-jones, & craft, ; white, forester, & craft, ) , and some studies have directly compared host contact network parameters to parasite genotypes (bull, godfrey, & gordon, ) . despite this, to our knowledge, no published studies have used network models to investigate pathogen movement within a landscape genetic framework. while landscape genetics initially was used to investigate spatial genetic patterns using relatively few neutral markers, the more modern advent of landscape genomics allows the study of variation across the entire genome and effectively expands the scope of landscape genetics to include the study of functional, adaptive genetic variation. next-generation sequencing (ngs) techniques such as restriction-site-associated dna sequencing (radseq) require minimal prior knowledge of the genome under study and can genotype thousands of snps randomly distributed across the genome. some of these snps will by chance be located within or near (and thus linked to) genes or regulatory regions that are under selection. genomewide association studies (gwas) can make use of this information to identify loci linked to phenotypic variation such as disease susceptibility. genotyping of candidate loci identified using quantitative trait locus mapping and gwas can be expanded across a large number of individuals using methods such as targeted sequence capture (grover, salmon, & wendel, ) , and these data can be tested in a landscape genomic framework for associations with environmental variables. loci exhibiting a signature of selection can be identified using outlier tests (excoffier, hofer, & foll, ; luu, bazin, & blum, ) , which search for loci with allelic frequencies that are outliers relative to the majority. such loci are considered potentially under selection and may then be tested a posteriori for correlations with environmental variables. newer methods have focused on explicitly incorporating environmental variables into landscape genomic analyses, known as genetic-environment association (gea) tests (lotterhos & whitlock, ; rellstab, gugerli, eckert, hancock, & holderegger, ) . gea analyses test for correlations between environmental variables and individual genotypes, which eliminates problems due to underlying population structure that must be controlled when using outlier tests. ngs approaches also generate thousands of neutral loci, which provide greater power to detect fine-scale neutral genetic structure than conventional studies based on relatively few loci (allendorf, hohenlohe, & luikart, ) . however, for studies with a particular focus on functional genetic variation, ngs approaches can also be adapted specifically for this purpose through targeted sequencing of the exome (roffler et al., ) (roffler et al., ) . the spread of functional alleles has also been incorporated into landscape genetic simulations , enhancing predictions of future pathogen spread and its effects on host populations. this small body of research is promising for expansion of landscape genomic studies designed to couple pathogen-related functional genetic variation with landscape variables. while we believe that there remains much unexplored utility in established landscape genetic methods for the study of pathogen dynamics as we have described above, we also note new frontiers with significant potential for expanding research in this area. we complete this review by discussing three particularly promising frontiers. studies relating pathogen genetic data directly to the landscape using resistance surfaces are challenged by the mediating influence of distinct host and vector traits, as well as relative differences in the contributions of multiple host and/or vector species to microparasite gene flow. this necessitates frameworks that more holistically incorporate multiple host and vector factors into studies of pathogen gene flow, which can expand the potential insights provided by landscape genetic studies of infectious agents (figure ). single or multiple host or vector species can be added as "landscape variables" (e.g., as resistance surfaces) in addition to physical landscape and environmental variables to test as factors shaping spatial pathogen genetic structure. resistance surfaces for tests of microparasite gene flow can represent host/vector distributions or abundance, ideally inferred from empirically derived ecological niche or species distribution models. optimally, host/vector movement would be represented (dougherty et al., ) , using outputs from agent-based movement models informed by telemetry or mark-recapture data, or host/vector landscape genetic data representing spatial patterns of gene flow. we note that the common issue in conventional landscape genetics of spatio-temporal mismatches between landscape processes and genetic change (anderson et al., ; landguth et al., ) would apply even more strongly here. researchers must simultaneously consider the potentially different spatial and temporal scales over which host and pathogen genetic changes (and poten- explain observed spatial patterns of prevalence least-cost path models of water bird movement estimated from ecological niche models, and road networks representing human movement, as potential predictors of avian influenza spread (young et al., the rapid mutation of microparasites relative to their hosts has potential to provide greater power to detect subtle variation in host movement patterns in response to the landscape, as well as earlier detectability of changes in host movements (such as in response to a new barrier) that are yet to be reflected in host genetic structure (landguth et al., ) . in addition, movements of nonreproducing hosts are difficult to detect using host genetic markers, but instead might be inferred using markers from directly transmitted microorganisms. such an approach has demonstrated the utility of a chronic, relatively apathogenic infection of felids (feline immunodeficiency virus) for identifying demographic structure of mountain lions and recent population history (biek et al., ) , and has identified movement of bobcats across a highway barrier that was not detectable using host markers (lee et al., ) . however, these approaches have not been broadly applied, particularly in the study of landscape effects. phylogenetic approaches can reconstruct very recent epidemic histories, providing insights into particular transmission events and pathways that may be contextualized temporally and spatially (corman et al., ; faria et al., ; carroll et al., ; magee, beard, suchard, lemey, & scotch, ; fountain-jones, packer, et al., ; fountain-jones, pearse et al., ) . the majority of such work has been conducted on rna viruses owing to their small, rapidly mutating genomes, requiring relatively little sequencing effort to detect contemporary phylogenetic signals. other pathogens that evolve more slowly, such as bacteria or fungal pathogens, require the sequencing of larger portions of their genomes to capture equivalent phylogenetic signals (biek, pybus, lloyd-smith, & didelot, ) . while this is becoming increasingly feasible (kao, haydon, lycett, & murcia, ) , more complex computational analysis is required to make meaningful conclusions. several approaches may be used for relating phylogenetic information with landscape variables. neighbour joining trees can identify clusters for quantifying population-level landscape genetic relationships (joannon et al., ) . the calculation of genetic distances based on maximum likelihood trees (carrel, emch, tung, jobe, & wan, ; real et al., ; young et al., ) results in distance matrices that can be correlated with landscape resistance matrices using conventional landscape genetic approaches. relaxed random walk phylogeographic approaches (lemey, rambaut, welch, & suchard, ) that can reconstruct pathogen dispersal have been linked to landscape predictors using a "phylogeographic glm" method (faria, suchard, rambaut, streicker, & lemey, ; jacquot, nomikou, palmarini, mertens, & biek, ) . the phylogeographic glm approach has enabled a better understanding of how landscape and hosts can constrain pathogen spread. for example, using the phylogeographic glm approach on viral genomic data, roads and rivers, coupled with dog distribution, were found to impact rabies spread in tanzania (brunker et al., ) . however, this approach is limited to discrete sampling locations and is computationally intensive (dellicour, rose, & pybus, ) . a recent framework by dellicour et al. ( ) modifies the phylogeographic glm approach to use resistance surfaces to efficiently quantify landscape resistance along transmission pathways inferred by continuous phylogeographic analyses. these landscape resistances are then correlated with temporal estimates of transmission along these routes to estimate how the landscape has shaped rates and directions of pathogen spread. such approaches are yet to be broadly applied, but appear to be important developments that should see increasing application in the future. overall, landscape genetics has been relatively underutilized in disease ecology research. we believe this is partly due to a lack of cross-disciplinary awareness between the two fields, but also a lack of a clear landscape genetic framework specifically designed for tackling pathogen systems, which are often complex and do not facilitate easy translation of existing landscape genetic tools. however, we note there has been a recent effort to develop new frameworks for such research, expanding the utility of the landscape genetic toolset. these tools will increase our capacity to study complex multihost and host-vector systems, improving the integration of multiple genetic datasets and accounting for interspecific interactions. improved understanding of host-parasite associations will facilitate the use of microparasite genetic markers to provide insights into host processes that may be difficult to detect using conventional host landscape genetics. identification of idealized systems that are designed to target specific ecological questions will also facilitate progress in this field. recent methods that enable the incorporation of quantitative landscape data into spatio-temporal phylogenetic reconstructions of recent transmission events, coupled with advances in high-throughput sequencing, hold great promise for studying how the landscape shapes transmission processes. we believe that these recent developments represent a renewed interest in advancing landscape genetic research in pathogen systems, which we expect will translate to continued growth of research in this area. we thank louis bernatchez for inviting us to contribute this paper, and nicolas bierne and two anonymous reviewers for their helpful suggestions to improve the manuscript. this work was supported by a national research foundation ecology of infectious diseases research programme grant (deb ) awarded to s.v., s.c., w.c.f., k.r.c., m.e.c. and h.b.e. c.p.k. was supported by an australian government research training program scholarship. the application of "least-cost" modelling as a functional landscape model genomics and the future of conservation genetics considering spatial and temporal scale in landscape-genetic studies of gene flow canonical analysis of principal coordinates: a useful method of constrained ordination for ecology infecting epidemiology with genetics: a new frontier in disease ecology landscape genetics: concepts, methods, applications 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population structures: implications for disease transmission in a sympatric cervid community a conceptual framework for the spatial analysis of landscape genetic data the role of parasite-driven selection in shaping landscape genomic structure in red grouse (lagopus lagopus scotica) using contact networks to explore mechanisms of parasite transmission in wildlife how's the flu getting through? landscape genetics suggests both humans and birds spread h n in egypt using simulations to evaluate mantel-based methods for assessing landscape resistance to gene flow additional supporting information may be found online in the supporting information section at the end of the article. key: cord- -j cf vzs authors: sattar, syed a. title: indoor air as a vehicle for human pathogens: introduction, objectives, and expectation of outcome date: - - journal: am j infect control doi: . /j.ajic. . . sha: doc_id: cord_uid: j cf vzs airborne spread of pathogens can be rapid, widespread, and difficult to prevent. in this international workshop, a panel of experts will expound on the following: ( ) the potential for indoor air to spread a wide range of human pathogens, plus engineering controls to reduce the risk for exposure to airborne infectious agents; ( ) the behavior of aerosolized infectious agents indoors and the use of emerging air decontamination technologies; ( ) a survey of quantitative methods to recover infectious agents and their surrogates from indoor air with regard to survival and inactivation of airborne pathogens; ( ) mathematical models to predict the movement of pathogens indoors and the use of such information to optimize the benefits of air decontamination technologies; and ( ) synergy between different infectious agents, such as legionellae and fungi, in the built environment predisposing to possible transmission-related health impacts of aerosolized biofilm-based opportunistic pathogens. after the presentations, the panel will address a set of preformulated questions on selection criteria for surrogate microbes to study the survival and inactivation of airborne human pathogens, desirable features of technologies for microbial decontamination of indoor air, knowledge gaps, and research needs. it is anticipated that the deliberations of the workshop will provide the attendees with an update on the significance of indoor air as a vehicle for transmitting human pathogens with a brief on what is currently being done to mitigate the risks from airborne infectious agents. i welcome you all to this multinational workshop! this workshop was conceived over a year ago, and the organizing committee ( table ) formally requested that astm international (www.astm.org/) hold the event under its auspices. astm's committee e , which deals with pesticides, antimicrobials, and alternative control agents, approved the proposal in april . mounting recognition of indoor air as a vehicle for infectious agents is leading government regulators, such as the u.s. environmental protection agency, to refine and update their guidelines, researchers to develop better means of studying airborne pathogens, and civil engineers and architects to find innovative means of making indoor air safer while keeping energy conservation in mind. although comprehensive guidelines and standardized means are available to study chemical pollutants in indoor air, there remains a general lack of suitable experimental facilities and standardized protocols to quantitatively assess the survival of pathogens in indoor air and to document their removal and inactivation by physical and chemical means. this workshop will address these issues, among others. the workshop's specific objectives, therefore, are as follows: the deliberations will also focus on the development of standards for assessing indoor air decontamination technologies and government regulations for registration of products to be marketed. as noted, this workshop has been organized under the auspices and with the support of astm international. the city university of new york and the university of ottawa (canada) are the academic sponsors of the workshop, and financial support has been provided by rb (montvale, nj) and microbac (sterling, va). these companies are also funding publication of the workshop proceedings. we gratefully acknowledge their generous support. the organizing committee has put together an outstanding group of speakers who will offer a comprehensive yet balanced perspective on the key issues. table lists the topics to be covered, along with the names and affiliations of the presenters. elsevier (www.elsevier.com) has agreed to publish the proceedings of the workshop after peer review. elsevier will also provide a preview of the proceedings, including the abstracts for each presentation, for release during the conference of the association for professionals in infection control and epidemiology. the workshop proceedings will also contain a summary of the concluding discussions. potential members of the audience include researchers in aerobiology, makers of air purification technologies, contract laboratories that assess air decontaminants, government regulators dealing with indoor air quality, and members of standards-setting organizations, such astm international (www.astm.org) and american society of heating, refrigerating, and air-conditioning engineers (www.ashrae.org). table is a glossary of the common terms used throughout this workshop's presentations. this is included in an attempt to create a level playing field while facilitating the understanding of the subject matter by experts in fields other than environmental microbiology. however, the emphasis here is on working definitions, recognizing that efforts are needed to develop a more comprehensive glossary for broader applications in this area. aerobiology, the study of living organisms and their components in air, became a full-fledged scientific discipline in . this was followed in by the founding of the international association of aerobiology (https://sites.google.com/site/ aerobiologyinternational/). the initial focus of this group was the study and movement of pollen, but microbes and other life forms were soon added to the mix with a corresponding broadening of the organization's scope (fig ) . the microbiologic quality of indoor air comes under the rubric of aerobiology (fig ) . this workshop will focus only on indoor air as a vehicle for human pathogens. indoor air quality exposure of humans to indoor air and its contents coincided with cave dwelling > , years ago. sharing of the human habitat with domesticated animals, such as cattle, dogs, and pigs, facilitated the rise of zoonotic infections, including airborne infections (eg, measles). exposure to pathogens of humans and animals via the agency of indoor air continues to this day. although the focus here is on indoor air, indoor air is not entirely immune to what goes on outdoors. the air from outside an edifice affects the air indoors and vice versa. in fact, the use of fossil fuels for heating the indoors contributes directly and indirectly to overall climate change. an early consequence of energy conservation was sealed buildings and houses, which eventually gave rise to sick building or tight building syndrome as a result of the trapping of airborne pollutants and higher levels of moisture inside. humans and animals are the main contributors of microbeladen particles indoors. in fact, individuals leave their own personal microbial footprint as a part of the indoor microbiome. aerosolization of microbes from biofilms and resuspension of dust are the other principal contributors to the microbial content of indoor air (fig ) . although the route by which airborne pathogens cause infections varies between microorganisms, improvements in the quality, quantity, and movement of indoor air can mitigate the airborne spread of many human pathogens by preventing pathogen this phenomenon is especially relevant in aerobiology because a host is often exposed to potentially harmful biologic, chemical, or physical agents simultaneously or sequentially. droplet nuclei airborne particles derived from larger droplets after loss of water such droplets are crucial for the spread of infectious agents by air as their relatively small size ( . - . μm) allows for their stability in air while also permitting their retention on inhalation. indoor air quality quality of the air within buildings and other enclosures, with particular reference to the health and comfort of the occupants the overall quality of indoor air is dependent on a mix of a variety of factors that may be site and time sensitive. the capacity of a microbe to infect a given host depends not only on its biology but also on the general health status of the host and the portal of entry into the host. a microbe capable of causing localized or generalized damage to the host please see "infectious agent." the totality of microorganisms and their collective genetic material present in or on the human body or in another environment a certain proportion of the microbes found in a microbiome may not be culturable but detected and identified via their genomes only. this term is now preferred over microflora. opportunistic pathogen a microbial pathogen capable of infecting hosts whose natural defenses are compromised because of advanced age, immunosuppression, or other underlying causes the number, variety, and health significance of such pathogens is on the rise in conjunction with the rising numbers of those debilitated by acquired or induced immunosuppression. pathogen (microbial) any microbe capable of causing damage to the host even an otherwise innocuous microbe can become pathogenic depending on the general resistance of the host or the microbe's entry into normally sterile areas of the body where it can become an opportunistic pathogen. perikairots environment-based opportunistic pathogens biofilm-based microbes such as legionellae and environmental mycobacteria can infect those debilitated because of age or underlying medical conditions. resident microbiota a mix of microbes normally found in or on the host many members of the resident microbiota from skin and mucous surfaces are frequently found in indoor air. particles small enough to access the alveolar space during normal breathing such particles may or may not contain viable microbes. surrogate microbe a microbe that resembles ≥ type of pathogens but is safer and easier to work with in the laboratory; also called a simulant the use of such microbes is crucial in many aspects of microbiology, in general. the body's automatic inhalation and exhalation process at rest in addition to coughing and sneezing, tidal breathing can release infectious agents into the air. transient microbiota microbes temporarily acquired by a host during normal contact with the environment inhalation and reducing the microbial load on environmental surfaces. indoor air is arguably the fastest and most highly efficient means of pathogen spread in a given setting. as depicted in figure , indoor air is a complex and dynamic mixture of numerous components in a constant state of flux influenced by many factors both indoors and outdoors. the quality of indoor air represents the outcome of the unique mix of components in a given setting that, in themselves, change temporally. one major challenge in preventing and controlling the airborne spread of infection is the presence of possibly multiple and mobile sources of pathogens at a given location and time. one or more infected or colonized persons or pets may contaminate the air in their immediate vicinity with exposure of those nearby without the air having reached any available means of pathogen decontamination. certain factors that influence indoor air quality may fall under the categories of chemical and physical. for example, smoke from burning wood for cooking fuel is, of course, chemical in nature, but respirable particles in the smoke are the primary means of lung irritation and potential damage leading to cardiopulmonary syndromes, including lung cancer (http://www.who.int/mediacentre/ factsheets/fs /en/). the study of indoor air quality received a major boost as a consequence of the severe acute respiratory syndrome outbreak in and the anthrax scares in the united states in . it also spawned much interest in the development, assessment, and application of technologies to decontaminate indoor air. as shown in figure , particles > μm in diameter entering the air may rapidly fall out of the air because of their mass under prevailing environmental conditions, particularly temperature and relative humidity, whereas smaller particles can not only remain airborne for extended periods but can also be transported readily indoors by air currents over considerable distances. respirable particles fall in the range of . - . μm in diameter, whereas smaller particles are generally exhaled because of the aerodynamics of breathing. the actual site of retention of the inhaled particles depends on their nominal size. it is noteworthy here that persons with respiratory infections breathe out pathogen-laden particles during tidal breathing. a human adult at rest breathes in an average of , l of air per day. in any given setting, one may choose not to drink the water or eat the food that is available but generally has little choice in breathing the same air as everyone else. this makes air an environmental equalizer-conferring on it the unique potential to parse out evenly whatever it may contain. further, infectious agents entering indoor air mix rapidly with no perceptible color or smell. although the potential of air to spread respiratory pathogens is well recognized, its ability to transmit enteric pathogens is not as well appreciated. airborne particles containing enteric pathogens may be retained in the tonsillar region and swallowed for relocation to the gastrointestinal tract with subsequent replication there. ijaz et al have provided a comprehensive list of human pathogens known or suspected to spread via indoor air. the following are the main topics to be covered during the workshop. despite the recognized significance of indoor air as a vehicle for human pathogens, there are major gaps in our understanding of how well these pathogens remain viable under different environmental conditions. such information is crucial to assessing the potential of a given pathogen to spread by air. construction of an aerobiology chamber (approximately m ) will be described, and data from use of the chamber to test airborne survival of staphylococcus aureus, klebsiella pneumoniae, and acinetobacter baumannii will be presented. many technologies claiming microbial decontamination of indoor air are on the market, but without proper validation of their claims. information will be presented on ways to test such technologies using standardized protocols for registration and marketing purposes. because they may not be readily available and generally are unsafe and difficult to culture in the laboratory, it is rarely possible to use actual field strains of human pathogens in testing. this necessitates the use of surrogate microbes to generate data predictive of the behavior of pathogens. however, certain surrogates that are used commonly and recommended by regulatory agencies and standardssetting organizations alike are inherently unsuitable for experimental work in aerobiology. for example, k pneumoniae, frequently used as a surrogate for airborne gram-negative bacilli, does not survive aerosolization well because it is relatively fragile and unstable in air. therefore, data generated with k pneumoniae likely will not be predictive of the behavior of actual human pathogens. this workshop will identify more suitable surrogates with supporting data. can microbial decontamination of indoor air reduce the risk for pathogen contamination of environmental surfaces? data will be presented to demonstrate that reductions in the levels of airborne microbes can indeed lead to corresponding reductions in the microbial contamination of environmental surfaces in a given setting. the use of integrated models could help analyze outbreaks, evaluate the relative importance of hygiene and infection prevention and control for policymakers, and provide guidance in environmental design for greater occupant safety and comfort. these will be illustrated using some recent examples, including severe acute respiratory syndrome, influenza, and middle east respiratory syndrome. quantitative recovery of viable microbes from air is vital in aerobiologic studies. an update will be given on available methods, including their strengths and limitations. experimentation with airborne microbes is generally quite labor intensive and costly and requires special biosafety precautions. mathematical models can assist greatly in optimizing aerobiology chamber design and in predicting the influence of furniture and other objects on the movement of microbes. data will be presented with specific reference to a chamber that fully conforms to guidance from the u.s. environmental protection agency. biofilms are not only common in the built environment, but they can be common sources of airborne pathogens. such biofilms often contain several microbial species having complex interactions between them. this will be illustrated with the example of how fungi and legionellae coexist with potential risks to human health. product performance test guidelines: ocspp . : air sanitizers-efficacy data recommendations decontamination of indoor air to reduce the risk of airborne infections: studies on survival and inactivation of airborne pathogens using an aerobiology chamber airborne spread of infectious agents in the indoor environment world health organization. who guidelines for indoor air quality: selected pollutants origins of major human infectious diseases domesticated animals and human infectious diseases of zoonotic origins: domestication time matters ventilation rates and health: multidisciplinary review of the scientific literature humans differ in their personal microbial cloud the role of super-spreaders in infectious disease secondary aerosolization of viable bacillus anthracis spores in a contaminated us senate office influenza virus in human exhaled breath: an observational study how much air do we breathe? concentration, size distribution, and infectivity of airborne particles carrying swine viruses generic aspects of airborne spread of human pathogens indoors and emerging air-decontamination technologies key: cord- -th da bb authors: gardy, jennifer l.; loman, nicholas j. title: towards a genomics-informed, real-time, global pathogen surveillance system date: - - journal: nat rev genet doi: . /nrg. . sha: doc_id: cord_uid: th da bb the recent ebola and zika epidemics demonstrate the need for the continuous surveillance, rapid diagnosis and real-time tracking of emerging infectious diseases. fast, affordable sequencing of pathogen genomes — now a staple of the public health microbiology laboratory in well-resourced settings — can affect each of these areas. coupling genomic diagnostics and epidemiology to innovative digital disease detection platforms raises the possibility of an open, global, digital pathogen surveillance system. when informed by a one health approach, in which human, animal and environmental health are considered together, such a genomics-based system has profound potential to improve public health in settings lacking robust laboratory capacity. supplementary information: the online version of this article (doi: . /nrg. . ) contains supplementary material, which is available to authorized users. in late and early , a lethal haemorrhagic fever spread throughout forested guinea (guinée forestière), undiagnosed for months. by the time it was reported to be ebola, the virus had spread to three countries and was likely past the point at which case-level control measures, such as isolation and infection control, could have contained the nascent outbreak. in , a new dengue-like illness was implicated in a dramatic increase in brazil's microcephaly cases; one year later, analyses revealed that the zika virus had been sweeping through the americas, unnoticed by existing surveillance systems, since late . although public health surveillance systems have evolved to meet the changing needs of our global popu lation, we continue to dramatically underestimate our vulnerability to pathogens, both old and new . indeed, the recent events in west africa and brazil highlight the gaps in existing infectious disease surveillance systems, particularly when dealing with novel pathogens or pathogens whose geographic range has extended into a new region. despite the lessons learned from previous outbreaks , such as the severe acute respiratory syndrome (sars) epidemic in - and the influenza pandemic -particularly the need for enhanced national surveillance and diagnostic capacity -infectious threats continue to surprise and sometimes overwhelm the global health response. the cost of these epidemics demands that we take action: with fewer than , cases, the ebola outbreak ultimately resulted in over , deaths, left nearly , children without parents and caused cumulative gross domestic product losses of more than % . as with prior crises, in the wake of ebola, multiple commissions have offered suggestions for essential reforms , . most focus on systems-level change, such as funding research and development or creating a centralized pandemic preparedness and response agency. however, they also call for enhanced molecular diagnostic and surveillance capacity coupled to data-sharing frameworks. this hints at an emerging paradigm for rapid outbreak response, one that employs new tools for pathogen genome sequencing and epidemiological analysis (fig. ) and that can be deployed anywhere. in this model, portable, in-country genomic diagnostics are targeted to key settings for routine human, animal and environ mental surveillance or rapidly deployed to a setting with a nascent outbreak. within our increasingly digital landscape, wherein a clinical sample can be transformed into a stream of data for rapid analysis and dissemination in a matter of hours, we face a tremendous opportunity to more proactively respond to disease events. however, the potential benefits of such a system are not guaranteed, and many obstacles remain. here, we review recent advances in genomicsinformed outbreak response, including the role of real-time sequencing in both diagnostics and epidemiology. we outline the opportunities for integrating sequencing with the one health and digital epidemiology fields, and we examine the ethical, legal the systematic collection, analysis and dissemination of health-related data to support planning, implementation and evaluation of public health practices and response. outbreaks and epidemics are both defined as increases in the number of cases of a particular disease beyond what is expected in a given setting. in outbreaks, the affected settings are smaller geographic regions; epidemics can span larger areas. clinical metagenomics. with its untargeted approach to sequencing, clinical metagenomics can cross disciplines in a way that clinical microbiology struggles to -identifying viral, bacterial, fungal and other eukaryotic pathogens in a single assay and coupling pathogen detection to pathogen discovery. given the current high cost of the technique -conservatively estimated at several thousand dollars -it is most often used when dealing with potentially lethal infections that fail the conventional diagnostic paradigm, such as the recent diagnosis of an unusual case of meningoencephalitis caused by the amoeboid parasite balamuthia man drillaris or the diagnosis and treatment of neuroleptospirosis in a critically unwell teenager . in the latter case, despite a high index of suspicion for infection, leptospira santa rosai was not detected by culture or pcr, as the diagnostic primer sequences were eventually found to be a poor match to the genome of the pathogen. intravenous antibiotic therapy resulted in rapid recovery. in such an example, the costs are easily justified, particularly when offset against the cost of a stay in an intensive treatment unit. however, routine diagnostic metagenomics is currently limited to a handful of clinical research laboratories worldwide; it is therefore regarded as a 'test of last resort' and kept in reserve for vexing diagnostic conundrums. substantial practical challenges hinder the adoption of metagenomics for diagnostics (fig. ) (reviewed in depth in ref. ) . chief among these is analytic sensitivity, which depends on pathogen factors (for example, genome size, ease of lysis and life cycle); analytic factors (for example, the completeness of reference databases and the potential to mistake a target for a close genetic relative); and sample factors (for example, pathogen abundance within a sample and contaminating background dna). as an example of a problematic sample, during zika surveillance, attempts to perform un targeted metagenomics sequencing on blood yielded few, or in some cases zero, reads owing to low viral titres . targetenrichment technologies (reviewed in ref. ) such as bait probes can be employed, but even these were unsuccessful at recovering whole zika genomes, necessitating pcr enrichment . in addition to sensitivity, universal pathogen detection through clinical metagenomics is complicated by specificity issues arising from misclassification or contaminated reagents, the challenge of reproducing results from a complex clinical workflow, nucleic acid stability under varying assay conditions, ever-changing bioinformatics workflows and cost. given these issues, could metagenomics replace conventional microbiological and molecular tests for infection? recent studies have used metagenomics in common presentations, including sepsis , pneumonia , urinary tract infections and eye infections . these have generally yielded promising results, albeit typically at a lower sensitivity than conventional tests and at a much greater cost. despite these problems, two factors will drive sequencing to eventually become routine clinical practice. first, the ever-decreasing cost of sequencing coupled with the potential for cost savings achieved by using a single diagnostic modality versus tens or hundreds of different diagnostic assays -each potentially requiring specific instrumentation, reagents, validation and labour -is attractive from a laboratory operations perspective. second, and perhaps most compelling, is the additional information afforded by genomics, including the ability to predict virulence or drug resistance phenotypes, the ability to detect polymicrobial infections and phylogenetic reconstruction for outbreak analysis. novel technologies: portable sequencing. given that outbreaks of emerging infectious diseases (eids) most often occur in settings with minimal laboratory capacity, where routine culture and bench-top sequencing are simply not feasible, the need for a portable diagnostic platform capable of in situ clinical metagenomics and outbreak surveillance is evident. a trend towards smaller and less expensive bench-top sequencing instruments was seen with the genome sequencer junior system (which has since been discontinued), the ion torrent personal genome machine (pgm) system and the illumina miseq system, which were released in close succession . each of these instruments costs <$ , and puts ngs capability into the hands of smaller laboratories, including clinical settings. in , the minion from oxford nanopore technologies was released to early access users , heralding the potential nature reviews | genetics outbreak response portable genome sequencing digital epidemiology one health figure | a genomics-informed surveillance and outbreak response model. portable genome sequencing technology and digital epidemiology platforms form the foundation for both real-time pathogen and disease surveillance systems and outbreak response efforts, all of which exist within the one health context, in which surveillance, outbreak detection and response span the human, animal and environmental health domains. the event through which a pathogen is transferred from one entity to another. transmission can be person-to-person, as in the case of ebola, vector-to-person, as with zika, or environment-to-person via routes including food, water and contact with a contaminated object or surface. the use of genome sequencing to understand infectious disease transmission and epidemiology. see fig. . for highly portable 'lab-in-a-suitcase' sequencing. the minion is pocket-sized and is controlled and powered through a laptop usb connection. it is provided under a model whereby the hardware is free but the consumer pays a premium for the reagent and flow cell consumables. compared with bench-top instruments, the absence of a rolling service contract or regular engineer visits makes it theoretically possible to scale this platform out to potentially unlimited numbers of labora tories. importantly, the minion has been used in field situations, including in diagnostic tent labora tories during the ebola epidemic , and in a roving busbased mobile laboratory in brazil as part of the zibra project , . others have taken the minion to more extreme environments where even the smallest traditional bench-top sequencer could not go, including the arctic and antarctic , a deep mine and zero gravity aboard the reduced-gravity aircraft (nicknamed the 'vomit comet') and the international space station . however, this technology is not yet a panacea; remaining challenges include high dna or rna input requirements (currently hundreds of nanograms), which often necessitate pcr-based amplification approaches; a flow cell cost of $ , keeping the cost per sample high despite multiplexing approaches; and high error rates, which require that genomes are sequenced to high coverage for single nucleotide polymorphism-based analysis and analysed at the signal level. moreover, although the long reads produced by the minion overcome a number of challenges in assembling eukaryotic microbial pathogen genomes, such as the presence of discrete chromosomes or long repetitive regions, the upstream nucleic acid extraction steps required to obtain genomic dna vary across microbial domains and might necessitate reagents and equipment far less portable than the minion. from transmission to epidemic dynamics. genomics is capable of informing not just pathogen diagnostics but also epidemiology. pathogen sequencing has been used for decades to understand transmission in viral outbreaks, from early studies of hantavirus in the united states of america to human immunodeficiency virus (hiv) in the united kingdom ; more recently, the approach has been successfully extended to include bacterial pathogens (reviewed in ref. ) and has come to be known as genomic epidemiology, a term encompassing everything from population dynamics to the reconstruction of individual transmission events within outbreaks . most transmission-focused investigations to date have been retrospective, with only a subset unfolding in real time, as cases are diagnosed [ ] [ ] [ ] [ ] [ ] . in transmission-focused investigations, genetic variants are used to identify person-to-person transmission figure | challenges to in-field clinical metagenomics for rapid diagnosis and outbreak response. a mobile medical unit deploying a portable clinical metagenomics platform has been established at the epicentre of an infectious disease outbreak, but the team faces challenges throughout the diagnostic process and epidemiological response. for example, in the case of zika virus, samples, such as blood, with low viral titres, a small genome of < kb and transient viraemia combine to complicate detection of viral nucleic acid by use of a strictly metagenomic approach. furthermore, obtaining a sufficient amount of viral nucleic acids for genome sequencing beyond simple diagnostics requires a tiling pcr and amplicon sequencing approach . other challenges include, for example, access to a reliable internet connection, the ability to collect sample metadata and translating genomic findings into real-time, actionable recommendations. the average number of secondary cases of an infectious disease produced by a single infectious case, given a completely susceptible population. a term describing infectious diseases that typically exist in an animal reservoir but that can be transmitted to humans. the transmission of an infectious disease, such as ebola, from a survivor of that disease who has recovered from their symptoms. a term describing infectious diseases that are transmitted to humans through contact with a non-human species, particularly those diseases spread through insect bites. an example is the zika virus, which is carried by mosquitos. geographical settings where a variety of factors converge to create the social and environmental conditions that promote disease transmission. the process by which an infectious disease changes from existing exclusively in animals to being able to infect, then transmit between, humans. see fig. . events (fig. ) , either through manual interpretation of the variants shared between outbreak cases or via modelbased approaches , with the result being a transmission network. epidemic investigations are very different -only a subset of the epidemic cases are sequenced. thus, the goal is to use the population structure of the pathogen to understand the overall dynamics of the epidemic. here, phylodynamic approaches are used to infer epidemiological parameters of interest. first conceptualized in by grenfell et al. as a union of "immunodynamics, epidemiology, and evolutionary biology" (ref. ), phylodynamics captures both epidemiological and evolutionary information from measurably evolving pathogens -those viruses and bacteria for which high mutation rates and/or a range of sampling dates contribute to a meaningful amount of genetic variation between sequences , -in other words, enough genetic diversity to be able to infer an evolutionary history for a pathogen of interest, even if that history is only over the short time frame of an outbreak or epidemic. this is possible for most pathogens, particularly single-stranded dna viruses, rna viruses and many bacterial species , , but there are certain species for which the lack of a strict molecular clock and/or frequent recombination complicate both phylodynamics studies and attempts to infer transmission events . phylodynamics relies on tools such as bayesian evolutionary analysis sampling trees (beast) , in which sequence data are used to build a time-labelled phylogenetic tree using a specific evolutionary process as a guide -often variations on a theme of coalescent theory . from the tree, one can infer epidemiological parameters, including the basic reproductive number r (ref. ). while the insights that can be gained from genomic data alone are exciting, the utility of phylodynamic approaches is greatly extended when additional data are integrated into the models (reviewed in ref. ). genomic epidemiology in action: ebola. the many genomic epidemiology studies from the ebola outbreak (reviewed in ref. ) used bench-top and portable sequencing platforms to reveal outbreak-level events and epidemic-level trends. real-time analyses published around the peak of the epidemic suggested the following: the outbreak probably arose from a single introduction into humans and not repeated zoonotic introductions , ; sexual transmission had a previously unrecognized role in maintaining transmission chains ; and survivor transmission -another un recognized phenomenon -contributed to disease flare-ups later in the outbreak . the first sequencing efforts, all of which had an effect on the epidemiological response in real time, unfolded months into the epidemic. had they been deployed earlier, we can only speculate as to their potential impact. arguably, the most compelling use of early sequencing would have been to provide a definitive ebola diagnosis in this previously unaffected region of west africa. however, even after the outbreak was underway, sequencing could have benefited the public health response. for example, ruling out bush meat as a source of repeated viral introductions could have changed public health messaging campaigns from avoiding bush meat to the importance of hygiene and safe funeral practices , potentially averting some cases. portable sequencing and phylodynamic approaches are currently being deployed in the ongoing zika epidemic; whether the real-time reporting of genomic findings is able to alter the course of a vector-borne epidemic remains to be seen. retrospective phylodynamic investigations are also useful for pandemic preparedness planning. a recent analysis of , ebola virus genomes -approximately % of all cases -reconstructs the movement of the virus across west africa and reveals drivers for its spread . the authors deduce that ebola importation was more likely to occur between regions of a country than across international borders and that both population size and distance to a nearby large urban centre were associated with local expansion of the virus. these findings may affect decision-making around border closures in future ebola outbreaks and point to the need to develop surveillance, diagnostic and treatment capacity in urban centres. the role of the environment in deploying genomics for surveillance, diagnostics and epidemiological investigation, a key question remains: where? many regions lack the diagnostic laboratory capacity to carry out basic surveillance, but continuous genomic surveillance in all of these settings would be impossible. numerous projects have attempted to describe the pool of geographic hot spots and candidate pathogens from which the next epidemic or pandemic will arise. determining these factors is key to predicting and preventing spillover events (fig. ) ). they report an increasing number of events each decade, generally located in hot spots defined by specific environmental, ecological and socio-economic characteristics. most eids are zoonotic in origin, with the highest risk of spillover in regions with high wildlife diversity that have experienced recent demographic change and/or recent increases in farming activity . a global biogeographic analysis of human infectious disease further supports the use of biodiversity as a proxy for eid hot spots , and reviews focused on systems-level, rather than ecological, factors identify the breakdown of local public health systems as drivers of outbreaks, suggesting that surveillance ought to be targeted to settings where bio diversity and changing demographics meet inadequate sanitation and hygiene, lack of a public health infra structure for deliver ing interventions and no or limited resources for control of zoonoses and vector-borne diseases . these analyses provide a shortlist of regions, including parts of eastern and southeastern asia, india and equatorial africa, on which genomic and other surveillance activities should be focused , . within these regions, sewer systems and wastewater treatment plants could be important foci for sample collection, providing a single point of entry to biological readouts from an entire community. indeed, proof-of-concept metagenomics studies have revealed the presence of antibiotic resistance genes , human-specific viruses . most were zoonotic in origin, and over one-quarter had been detected in non-human species many years before being identified as human pathogens. a later review reiterates this observation, noting that recent agents of concern -ebola, zika and chikungunya -had been identified decades before they achieved pandemic magnitude . as a result of ngs technology, the pace of novel virus discovery is accelerating, with recent large-scale studies revealing new viruses sampled from macaque faeces in a single geographic location and , new viruses discovered from rna transcriptomic analyses of multiple invertebrate species . however, understanding which of these new entities might pose a threat requires a new approach. one health. the emergence of a zoonotic pathogen proceeds in stages (fig. ) ; in an effort to better anticipate these transitions and more proactively respond to emerging threats, the one health movement was launched in . recognizing that human, domestic animal and wildlife health and disease are linked to each other and that changing land-use patterns contribute to disease spread, one health aims to develop systems-minded, forward-thinking approaches to disease surveillance, control and prevention . by investing in infrastructure for human and animal health surveillance, committing to timely information sharing and establishing collaborations across multiple sectors and disciplines, the goal of the one health community is an integrated system incorporating human, animal and environmental surveillance -a goal in which genomics can have an important role. the one health approach has been implemented through the predict project, which is part of the emerging pandemic threats (ept) programme of the us agency for international development (usaid). predict explores the spillover of selected viral zoonoses from particular wildlife taxa , and early efforts have focused on developing non-invasive sampling techniques for wildlife , estimating the breadth of mammalian viral diversity across nine viral families and at least , undiscovered species and demonstrating that viral community diversity is at least a partially deterministic process, suggesting that forecasting community changes, which potentially signal spillover, is a possibility . although the goal of using integrated surveillance information to predict an outbreak is still many years away, one health studies are already leveraging the tools and techniques of genomic epidemiology to understand current outbreaks. combining genomic data with data streams from enhanced one health surveillance platforms presents an opportunity to detect the population expansions nature reviews | genetics figure | inferring transmission events from genomic data. genomic approaches to identifying transmission events typically involve four steps. in the first step, outbreak isolates, and often non-outbreak control isolates, are sequenced and their genomes either assembled de novo or mapped against a reference genome. next, the genomic differences between the sequences are identified -depending on the pathogen and the scale of the outbreak, these may include features such as genetic variants, insertions and deletions or the presence or absence of specific genes or mobile genetic elements. in the third step, these features are examined to infer the relationships between the isolates from whence they came -a variant common to a subset of isolates, for example, suggests that those cases are epidemiologically linked. finally, the genomic evidence for epidemiological linkages is reviewed in the context of known epidemiological information, such as social contact between two cases or a common location or other exposure. recently, automated methods for inferring potential epidemiological linkages from genomic data alone have been developed, greatly facilitating large-scale genomic epidemiological investigations . and/or cross-species transmissions that may precede a human health event. for example, genome sequences from a raccoon-associated variant of rabies virus (rrv), when paired with fine-scale geographic information and data from canadian and us wildlife rabies vaccination programmes, demonstrated that multiple cross-border incursions were responsible for the expansion of rrv into canada and sustained outbreaks in several provinces ; this finding led to renewed concern about and action against rabies on the part of public health authorities . one of the first studies coupling detailed wildlife and livestock movement data with phylodynamic analysis of a bacterial pathogen revealed that crossspecies jumps from an elk reservoir were the source of increasing rates of brucella abortus infections in nearby livestock ; as the most common zoonosis of humans, brucellosis control programmes will benefit substantially from this sort of one health approach . this model, in which diagnostic testing in reference laboratories triggers genomic follow-up, represents an effective near-term solution for integrating genomics into one health surveillance efforts as the community explores solutions to the many challenges facing in situ clinical metagenomics surveillance of animal populations (reviewed in ref. ). initial forays into this area have been successful; for example, metagenomics analysis of human diarrhoeal specimens and stools from nearby pigs revealed potential zoonotic transmission of rotavirus . however, metagenomic sequencing across a range of animal species and environments yields more questions than answers. what is an early signal of patho gen emergence versus background microbial noise ? which emerging agents are capable of crossing the species barrier and causing human disease ? what degree of sampling is required to capture potential spillovers ? ultimately, a more efficient use of metagenomics in a one health surveillance strategy might be scanning for zoonotic 'jumps' in selected sentinel human populations rather than a sweeping animal surveillance strategy , with sentinels chosen according to eid hotspot maps and other factors and interesting genomic signals triggering follow-up sequencing in the relevant animal reser voirs. by combining genomic data generated through these targeted surveillance efforts with phylodynamic approaches, it will be possible to take simple presence or absence signals and derive useful epidemiological insights: signals of population expansion; evidence of transmission within and between animal reservoirs and humans; and epidemiological analysis of a pathogen's early expansion. most modern surveillance systems use human, animal, environmental and other data to carry out disease-specific surveillance, in which a single disease is monitored through one or more data streams, such as positive laboratory test results or reportable communicable disease notifications. despite marked advances over the preceding decades, testimony from multiple expert groups has repeatedly emphasized the need for improved surveillance capacity , , including the use of syndromic surveillance, a more pathogen-agnostic approach aimed at early detection of emerging disease , . syndromic surveillance systems might leverage unique data streams such as school or employee absenteeism, grocery store or pharmacy purchases of specific items or calls to a nursing hotline as signals of illness in a population. increasingly, digital streams are being used as an input to these systems, be they participatory epidemiology projects such as flu near you , the automated analysis of trending words or phrases on social media sites, such as twitter , , or internet search queries [ ] [ ] [ ] . this new approach to surveillance is known as digital epidemiology and is also referred to as digital disease detection . in digital epidemiology, information is first retrieved from a range of sources, including digital media, newswires, official reports and crowd sourcing; second, translated and processed, which includes extracting disease events and ensuring reports are not duplicated; third, analysed for trends; and fourth, disseminated to the community through media, including websites, email lists and mobile alerts in spillover, a pathogen previously restricted to animals gradually begins to move into the human population. during stage one (pre-emergence), as a result of changing demographics and/or land use, a pathogen undergoes a population expansion, extends its host range or moves into a new geographic region. during stage two (localized emergence), contact with animals or animal products results in spillover of the pathogen from its natural reservoir(s) into humans but with little to no onward person-to-person transmission. during stage three (pandemic emergence), the pathogen is able to sustain long transmission chains, that is, a series of disease transmission events, such as a sequential series of person-to-person transmissions, and its movement across borders is facilitated by human travel patterns . epidemiology platforms are currently operating , and their flexible nature and cost-effective, real-time reporting make them effective tools for gathering epidemic intelligence, particularly in settings lacking traditional disease surveillance systems. the fields of one health and digital epidemiology are increasingly overlapping. in the predict consortium, the healthmap system and local media surveillance were combined to identify health events in five countries over a -week period . predict also suggested a role for digital epidemiology in not just event detection but also the identification of changing eid drivers. eids are driven by multiple factors, many of which have digital outputs and represent novel sources of surveillance data . for example, human movement can be revealed by mobile phone data or by the patterns of lighted cities at night, hunting data collected by states can reveal interactions between humans and wildlife, and social media and digital news sources can reveal early signals of famine, war and other social unrest. a major challenge is that the number of digital data sets available for each driver varies substantially, from hundreds for surveying land use changes -many based on remote sensing data -to mere handfuls around social inequalities and human susceptibility to infection, with most data biased towards north america and europe. the digital and genomic epidemiology domains are also starting to overlap. in the ebola outbreak, digital epidemiology revealed that drivers of infection risk included settings where households lacked a radio, with high rainfall and with urban land cover , echoing the evidence from a genomic study suggesting that sites at which urban and rural populations mix contribute to disease . during the zika epidemic, majumder et al. used healthmap and google trends to estimate the basic reproductive number r to be . - . ; phylo dynamic estimates from brazilian genomic data gave similar ranges ( . - . ) , indicating that both types of data streams can be leveraged in calculating epi demiological parameters that help shape the public health response. a digital pathogen surveillance era recent reports have called for the integration of genomic data with digital epidemiology streams , . when informed by a one health approach, the epidemiological potential of this digital pathogen surveillance system is profound. imagine parallel networks of portable patho gen sequencers deployed to laboratories and communities in eid hot spots -regions that are traditionally underserved with respect to laboratory and surveillance capacity -and processing samples collected from targeted sentinel wildlife species, insect vectors and humans (fig. ) . samples would be pooled for routine surveillance -either through targeted diagnostics or, if the issue of analytical sensitivity can be overcome, through metagenomics -with a full genomic work-up of individual samples should a pathogenic signal be detected. at the same time, existing internet-based platforms such as healthmap and new local participatory epidemiology efforts would be collecting data to both identify potential hotspot regions and detect eid events, enabling both prospective and rapid-response deployment of additional sequencers. genome sequencing data coupled with rich metadata would then be released in real time to web-based platforms, such as virological for colla borative analysis and nextstrain for analysis and visualization . these sites -already used in the ebola and zika responses -would act as the nexus for a global network of interested parties contributing to real-time phylo dynamic and epidemiological analyses and looking for signals of spillover, pathogen population expansion and sustained human-to-human transmission. results would be immediately shared with the one health frontlineepidemiologists, veterinarians and community health workers -who would then implement evidence-based interventions to mitigate further spread. the pathway to such a reality is not without its roadblocks. apart from technical and implementation challenges, a series of larger concerns surrounds the rollout of genomics-based rapid outbreak response, ranging from the uptake of a new, disruptive technology to effecting systems-level change on a global scale. sequencing-based diagnostics, particularly clinical metagenomics approaches, are still straddling the boundary between research and clinical use. in this realm, uncertainty is a certainty, be it uncertainty inherent to the technology itself or informational uncertainty, such as how accurate, complete and reliable results actually are . early adopters of genomics in the academic domain are used to uncertainty, often acknowledging and appraising it, but routine clinical use requires meeting the evidentiary thresholds mandated by a range of stakeholders, from regulators to the laboratories implementing new sequencing-based tests. decision criteria that influence whether a new genomic test is adopted include the ability of the assay to differentiate pathogens from commensals, the correlation of pathogen presence with disease, the sensitivity and specificity of the test, its reproducibility and robustness across sample types and settings and a cost comparable to that of existing platforms . validation -defining the conditions needed to obtain reliable results from an assay, evaluating the performance of the assay under said conditions and specifying how the results should be interpreted, including outlining limitations -is also critical. much can be learned from the domain of microbial forensics, where sequencing is playing a large part . budowle et al. review validation considerations for ngs , noting that this technology requires validating sample preparation protocols, including extraction, enrichment and library preparation steps, sequencing protocols, and downstream bioinformatics analyses, including alignment and assembly, variant calling, the underlying reference databases and software tools and the interpretation of the data. complete validation of a sequencing assay may not always be possible, particularly for emerging patho gens. therefore, just as the west african ebola virus outbreak triggered a review of the ethical context for trialling new therapeutics and vaccines , the scale-up of ngs in emerging epidemics will engender similar conversations. rather than wait for this to happen, an anticipatory approach is best, outlining the exceptional circumstances under which unvalidated approaches might be used, selecting the appropriate approach and examining the benefits of a potentially untested approach in light of individual and societal interests. if the social landscape surrounding the introduction of a new technology is not considered, prior experience suggests that the road to implementation will be difficult, with hurdles ranging from public mistrust to moratoria on research . the enthusiasm of the scientific community for new technology must not lead to inflated claims of clinical utility and poor downstream decisions around the deployment of that technology. howard et al. outline several principles for successfully integrating genomics into the public health system, and as we pilot digital pathogen surveillance, the community would do well to keep many of them in mind: ensuring that the instruments and processes used are reliable and that reporting is standardized and readily interpretable by end users; that the technology is used to address important health problems; that the advantages of the approach outweigh the disadvantages; and that economic evaluation suggests savings to the health care system and society . it is also important to reconsider the role of the diagnostic reference laboratory in the new genomic landscape. as their mandates expand to include enhanced surveillance and closer collaboration with field epidemiologists, laboratory directors will face new challenges, from managing exploratory work alongside routine clinical care to hiring a new sort of technologist, one with basic genomics and epidemiology training. the ethical, social and legal implications of digital pathogen surveillance are an emerging area of research (reviewed in ref. ). chief among the issues that geller et al. identify is the tension that exists when a new technology has the power to identify a problem but there is limited or no capacity to address the issue. balancing the benefits and harms to both individuals and populations is challenging when the predictive insight offered by a genomic technology is variable -for example, using genomics to identify an individual as a 'super spreader' has important implications for quarantine and isolation, but that label may be predicated on a tenuous prediction. the problem is further compounded by the fact that many infectious disease diagnoses carry with them a certain amount of stigma and that an individual's right to privacy might be superseded by the need to protect the larger population . data sharing and integration. a critical need for successful digital pathogen surveillance is the capacity for rapid, barrier-free data sharing, and arguments for such sharing are frequently rehashed after outbreaks and epidemics. genomic epidemiology was born largely in the academic sphere, with early papers coming from laboratories with nature reviews | genetics in one such region, the syndromic surveillance system reports higher-than-average sales of a common medication used to relieve fever. spatial analysis of the data from the pharmacies in the region suggests that the trend is unique to a particular district; a follow-up geographic information system (gis) analysis using satellite data reveals that this area borders a forest and is increasingly being used for the commercial production of bat guano. an alert is triggered, and the field response team meets with citizens in the area. nasopharyngeal swabs are taken from humans and livestock with fever as well as from guano and bat tissue collected in the area. the samples are immediately analysed using a portable dna sequencer coupled to a smartphone. an app on the phone reports the clinical metagenomic results in real time, revealing that in many of the ill humans and animals, a novel coronavirus makes up the bulk of the microbial nucleic acid fraction. the sequencing data are immediately uploaded to a public repository as they are generated, tagged with metadata about the host, sample type and location and stored according to a pathogen surveillance ontology. the data release triggers an announcement via social media of a novel sequence, and within minutes, interested virologists have created a shared online workspace and open lab notebook to collect their analyses of the new pathogen. extensive histories in microbial genomics and bioinformatics. for this community, open access to genome sequences, software and, more recently, publications has tended to be the rule rather than the exception. indeed, a national research council report described "the culture of genomics" as "unique in its evolution into a global web of tools and information" (ref. ). the same report includes a series of recommendations on access to pathogen genome data, including the statement that "rapid, unrestricted public access to primary genome sequence data, annotations of genome data, genome databases, and internet-based tools for genome analysis should be encouraged" (ref. ). as genomics has moved into the domain of clinical and public health practice, the notion of free and im mediate access to genomic surveillance data has encountered several barriers: the siloing of critical metadata across multiple public health databases with no interoperability; balancing openness and transparency with patient privacy and safety; variable data quality, particularly in resource-limited settings; concerns over data reuse by third parties; a lack of standards and ontologies to capture metadata; and career advancement disincentives to releasing data [ ] [ ] [ ] . despite these challenges, the spirit of open access and open data remains strong in the community, with over public health leaders from around the world recently signing a joint statement on data sharing for public health surveillance . the ebola and zika responses in particular highlight the role of realtime sharing of data and samples, be it through the use of chat groups and a labkey server to disseminate zika data or github to share ebola data . in the wake of ebola, yozwiak et al. and chretien et al. outline additional issues facing data sharing, from differing cultures and academic norms to complicated consent procedures and technical limitations. they note that we as a community must agree on standards and practices promoting cooperation -a conversation that could begin by examining how the global alliance for genomics and health (ga gh) framework for responsible sharing of genomic and health-related data (box ) could be adapted for the digital pathogen surveillance community. the future: the sequencing singularity? transformative change to public and global health is profoundly difficult. complicating the existence of a rapid, open, transparent response is the fact that no matter the setting, there are often conflicting interests at work. in an outbreak scenario, conflict may result from governments wishing to keep an outbreak quiet and/or from the tension between lower-income and middle-income countries with few resources for generating and using data and the researchers or response teams from better-resourced settings . indeed, the conflicting values in outbreak responses meet the definition of a 'wicked' problem, where issues resist simple resolution and span multiple jurisdictions and where each stakeholder has a different perspective on the solution. even the international health regulations (ihr), which ostensibly provide a legal instrument for global health security, fail to effect a basic surveillance and outbreak response. as of the most recent self-reporting, only % of the member countries of the ihr are in compliance, meeting the prescribed minimum public health core capacities . in these settings, digital pathogen surveillance must be within the purview of the larger global health community and its diverse group of non-state actors rather than being solely the responsibility of nations themselves . this raises an important issue: if nations are willing to cede a certain amount of surveillance and diag nostic control box | the global alliance for genomics and health (ga gh) framework for genomic data sharing in the universal declaration of human rights, article outlines the right of every individual "to share in scientific advancement and its benefit". in this spirit, the global alliance for genomics and health (ga gh) data-sharing framework , which covers data donors, producers and users, is guided by the principles of privacy, fairness and non-discrimination and has as its goal the promotion of health and well-being and the fair distribution of benefits arising from genomic research. the core elements of the framework include the following: • transparency: knowing how the data will be handled, accessed and exchanged • accountability: tracking of data access and mechanisms for addressing misuse • engagement: involving citizens and facilitating dialogue and deliberation around the societal implications of data sharing • quality and security: mitigating unauthorized access and implementing an unbiased approach to storing and processing data • privacy, data protection and confidentiality: complying with the relevant regulations at every stage • risk-benefit analysis: weighing benefits (including new knowledge, efficiencies and informed decision making) against risks (including invasion of privacy and breaches of confidentiality), minimizing harm and maximizing benefit at the individual and societal levels • recognition and attribution: ensuring recognition is meaningful to participants, providing due credit to all who shared data and ensuring credit is given for both primary and secondary data use • sustainability: implementing systems for archiving and retrieval • education and training: advancing data sharing, improving data quality, educating people on why data sharing matters, and building capacity • accessibility and dissemination: maximizing accessibility, promoting collaboration and using publication and digital dissemination to share results to the global health community, the notion of reciprocity suggests that they should derive some corresponding local benefit. the 'trickle-down' effects of global genomic surveillance have yet to be fully articulated, but they are likely to be realized first in the zoonotic domain, where global surveillance efforts will feed back into improved animal health at a local level, in turn benefiting local farmers. outbreaks occur at the intersection of risk perception, governance, policy and economics , and outbreak response is often based on political instinct rather than data . building a resilient and responsive public health system is therefore more than just enhancing surveillance and coupling it to novel technology -it is about engagement, trust, cooperation and building local capacity , as well as a focus on pandemic prevention through development rather than pandemic response via disaster relief mechanisms . expert panels convened by harvard and the london school of hygiene and tropical medicine and by the national academy of medicine have called for a central pandemic preparedness and response agency and also underscored the need for deeper partnerships between formal and informal surveillance, epidemiology and academic and public health networks . more recently, evolutionary biologist michael worobey wrote: "systematic pathogen surveillance is within our grasp, but is still undervalued and underfunded relative to the magnitude of the threat" (ref. ). if we are to achieve the sequencing singularity -the moment at which pathogen, environmental and digital data streams are integrated into 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foundation for health research programmes. both authors contributed equally to all aspects of the article. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -vgizgq y authors: uttamchandani, mahesh; neo, jia ling; ong, brandon ngiap zhung; moochhala, shabbir title: applications of microarrays in pathogen detection and biodefence date: - - journal: trends biotechnol doi: . /j.tibtech. . . sha: doc_id: cord_uid: vgizgq y the microarray is a platform with wide-ranging potential in biodefence. owing to the high level of throughput attainable through miniaturization, microarrays have accelerated the ability to respond in an epidemic or crisis. extending beyond diagnostics, recent studies have applied microarrays as a research tool towards understanding the etiology and pathogenicity of dangerous pathogens, as well as in vaccine development. the original emphasis was on dna microarrays, but the range now includes protein, antibody and carbohydrate microarrays, and research groups have exploited this diversity to further extend microarray applications in the area of biodefence. here, we discuss the impact and contributions of the growing range of microarrays and emphasize the concepts that might shape the future of biodefence research. natural outbreaks and the wanton use of pathogenic organisms for acts of terror have had tremendous impact on human populations, especially when considering the events over the past decade. modern travel and trade have further dissipated traditional geographical boundaries that once curbed the spread of disease. infectious agents capable of spreading from human to human, such as the severe acute respiratory syndrome (sars) coronavirus, have shown us just how quickly (in a matter of weeks) a threat anywhere could become a threat everywhere [ ] . the lessons learnt from sars, avian flu and the anthrax letter attacks have emphasized the need for improved preparedness for, response to and treatment of both known and emergent biological threats [ , ] . this has also prompted heightened funding initiatives worldwide to build up national and international biodefence capabilities [ ] . among the systems and protocols to be put in place, platforms that facilitate the rapid and accurate identification of agents are particularly vital, both in confirming whether an attack has occurred and in instituting prompt measures to secure public health. the intrinsic ability of microarrays to perform multiplexed, low-volume and sensitive biological assays in a highly scalable manner is a significant advantage in biological threat analysis [ ] . developed in the early to mid s, dna microarrays stirred a technological revolution that continues to propel genomics research today [ ] . applications included the identification and comparison of mrna or dna from across tissues or organisms by hybridization against thousands of oligomeric dna probes immobilized on planar surfaces, such as glass slides (figure ). this provided researchers with an unprecedented ability to quantify genome-wide differences in gene expression or sequence changes using minimal amounts of sample. in the context of biodefence, these studies have dramatically extended our capability beyond merely detecting known pathogens; now we are able to rapidly profile and characterize pathogens, as well as identify novel strains or 'genetic islands' that manifest changes in virulence (or the evolution of resistance). such comparative phylogenomic profiling using microarrays facilitates the understanding of pathogen diversification by providing a bird's-eye view of the gene content present or absent within a given microbial genome [ ] . microarray technology has also been brought outside the laboratory to the point-of-care, a development that has widening implications for threat detection. dna-based detection systems generally rely on the ability of pcr to amplify and fluorescently tag the tiny amounts of target dna present in the specimen. advances in miniaturizing this initial pcr step, for instance the development of review glossary biodefence: defensive measures against biological threats, including natural/ emerging pathogens and bioterror agents, that have significant potential to endanger public health detection: identifying the presence of target pathogen(s) from clinical or environmental samples. diagnostics: tests used to detect a medical condition, for example to test for the causative pathogen responsible for an infection. sandwich immunoassay: a biochemical assay for detecting the presence and/ or abundance of a target substance using the antibody-antigen reaction. two antibodies are used; the first is immobilized and the other, free antibody carries a reporter group, thereby providing a positive readout when the target is recognized and 'sandwiched' between the two antibodies. sensitivity: probability of a positive result when the pathogen is indeed present; high sensitivity is related to a low type error. serovars: a group of microorganisms distinguished by the presence of specific surface antigens. specificity: probability of a negative result when the pathogen is not present; high specificity is related to a low type error. tiling arrays: tiling arrays are a type of dna microarray in which short probe segments that have been designed to cover the entire genome are used. the extent of probe overlap will translate to the mapping resolution and might range from highly overlapped probes across each individual nucleotide base (for resequencing applications) to non-overlapping probes (for genome-wide expression analysis). vaccinia virus: a poxvirus that is closely related to the virus that causes cowpox. it was the first human vaccine and has been extensively used for vaccination against smallpox. corresponding author: uttamchandani, m. (mahesh@dso.org.sg). micro-pcr (mpcr), followed by hybridization and identification against probes on dna microarrays have spun-off viable chip-based platforms that are able to successfully perform biological threat detection and analysis. such portable systems, as will be described, are already appearing on the market and competing to attract lucrative biodefence funding. alternative detection platforms have exploited quantitative pcr in 'fieldable' real-time detectors, where positive pcr amplification would indicate the presence of the corresponding target dna [ ] . these have included devices such as genexpert (cepheid), rapid (idaho technologies) or bioseeq (smiths detection), but these systems provide relatively limited throughput [ ] . microarray systems are, by contrast, more definitive and highly scalable because hundreds to tens of thousands of possible dna elements can be interrogated in a single experiment. their performance nevertheless hinges on both the adoption of robust panels of probes that can accurately identify dna from organisms of interest and the successful extraction and amplification of pathogen dna from the relevant clinical sample or isolates [ , ] . more recent developments in microarray technologies have significantly broadened the horizon for their use in the biodefence arena well beyond the confines of dnabased profiling or detection. newer microarray formats developed at the turn of the st century provide a host of other biomolecules that can be presented on chip, in-cluding proteins (whole proteomes, enzymes and antibodies) [ , ] , small molecules (drug-like molecules, peptides and carbohydrates) [ , ] and even whole cells and tissues for simultaneous, multiplexed experimentation [ ] . each format offers unique ways in which microarrays can be harnessed towards improving our understanding of pathogen biology ( figure ). the microarray paradigm has over the years contributed significantly towards these goals by not only providing a robust tool for molecular diagnostics but also by advancing biodefence capabilities in protection and therapy. this review will describe microarray-based applications for biodefence, as well as exciting new concepts and approaches that will drive future advancement and growth. probe selection and design is usually an important first step in microarray-based pathogen detection, and many issues associated with probe design for dna microarrays can impact the overall fidelity of the assay, in particular with regard to levels of specificity and sensitivity attained [ ] . these issues and considerations include cross-hybridizations, orthogonal probe binding to target dna from the specific organism(s) of interest and vice versa, uniformity of annealing temperatures (or gc content) and probe length. the occurrence of false positives and false negatives is highly problematic because they might cause an (a) as discussed in this article, dna microarrays can be applied to test for dna from pathogenic organisms or for the resequencing of pathogen genomes. (b) antibody microarrays can be used to detect pathogen proteins or antigens that might be present in environmental samples as an indication of contamination or for diagnostic purposes to determine pathogen infection in human tissues. (c) small-molecule microarrays offer novel approaches for differentiating between pathogens, for example by clustering the binding signatures obtained for each pathogen. they can also be used to identify therapeutics that could potentially disrupt the infection cycle. trends in biotechnology vol. no. unwarranted response (and potentially panic) or a missed response (and a lost opportunity for intervention), which are unacceptable when dealing with deadly biological threats. for this reason, biodefence detection platforms strive to reach near perfect accuracies, which are comparable to, if not better than, the usual gold-standard assaystypically culture-based methods. with microarrays, redundancy can, however, be easily engineered into the system to improve accuracy and analytical power simply by over-representation. the us centers for disease control (cdc) have identified a list of agents that, if spread uncontrollably, have the potential to cause a major public health crisis with heavy morbidity and mortality rates (box ). these pathogens are further subclassified into categories a, b and c according to the degree of risk imposed (table ) [ ] . in , wilson et al. fabricated a customized affymetrix microarray containing probes to detect dna amplified from different pathogenic microorganisms simultaneously, including pathogens from the us cdc's list of bioterrorism agents, such as bacillus anthracis (which causes anthrax), clostridium botulinum (which generates the botulinum toxin), yersinia pestis (which causes bubonic plague) and the ebola virus [ ] . specific multiplexed primer sets were designed to amplify unique diagnostic regions specific to each organism for hybridization against tiling arrays comprising -mer probes. the highly redundant system facilitated the accurate identification of each organism tested, with over % of the probes working as predicted. impressively, as little as fg, equivalent to the 'mass' of just two genomes (or dna from two cells), of b. anthracis could be detected after multiplexed pcr on these microarrays. even though culture methods should in theory be able to amplify and detect a single organism, the procedures are usually time-consuming and challengingdetection could take from h to several days depending on the microbe type and even longer for fastidious microbes (e.g. mycobacterium tuberculosisa very slow growing microbe) that might require specialized, or as yet undiscovered, laboratory growth conditions. molecular identification using random pcr followed by multiplexed resolution over a microarray offers a particularly attractive alternative for detection and diagnostics, generating results in - h once assays are optimized ( figure a ). wang et al. [ ] developed such a microarray approach for the screening of viral pathogens from across broad viral families. a randomized primer was used to amplify any viral rna that was present in the sample using reverse transcriptase-pcr followed by hybridization on a microarray comprising -mer probes, representing nearly virus genomes. degeneracy of probes on the microarray and cross-hybridization of certain viruses across expected patterns indicated the emergence of novel, uncharacterized strains, which could hence be identified with the aid of microarrays. similarly, sengupta and colleagues [ ] developed microarrays with probes to distinguish among various influenza viruses. primers were designed against characteristic sequences specific to influenza that targeted the viral fusion protein haemagglutinin and the glycosidase neuraminidase segments. using the affymetrix respiratory pathogen microarray, lin et al. [ ] detected the respiratory viruses influenza a and adenovirus, which were then further differentiated according to strain and species. this setup made use of a so-called 'resequencing' microarray that contained one perfectly matched and three mismatched probes per base and thus was able to identify genetic mutations at the sequence level [ ] . the array's large screening capacity meant that both the forward and reverse strands of the dna targets could be sequenced to provide added sequencing accuracy. pooled primer pairs have also been optimized for use in detecting both dna and rna targets for pathogens that cause upper respiratory tract infections, including viruses (influenza, corona viruses and others) and bacteria (bordetella pertusis, streptococcus pyogenes and others) [ ] . the small subunit ribosomal rna (ssu rrna) gene is used widely as a microbial marker for taxonomy and species classification [ ] . desantis and colleagues [ ] generated a high-density tiling microarray with oligonucleotide probes of ssu rrna with sufficient coverage to detect different orders of microbes from environmental samples, as well as novel variants that exhibited mutations in their ssu rrna. the array fluorescence intensities correlated well with the spiked pathogen concentrations, providing a means of quantifying the pathogen box . bioterror agent classification biohazardous agents that pose a significant threat require special attention, especially in the implementation of regulatory measures and controls to limit their access and distribution and to prevent them from falling into the wrong hands. in addition, such measures also aim to raise awareness within national healthcare systems and amongst healthcare providers, especially in cases where these bioterror threats are only rarely seen. the us centers of disease control (cdc) have compiled a list of biohazardous agents, which are divided into the categories a, b and c according to the level of threat they impose, and their characteristics are described here. examples of pathogens from these three categories are provided in table . the cdc defines category a agents as those that are of the highest priority for biodefence research. these organisms can be easily disseminated from person to person. they result in high mortality rates and have the potential to cause a major impact on public health. the accidental or deliberate release of these agents could cause public panic and social disruption. these agents thus require particular attention in ensuring public health preparedness because they pose a significant risk to national security. the cdc defines category b agents as those that are moderately easy to disseminate. these pathogens result in moderate morbidity and low mortality. these agents would hence require specific improvements of diagnostic capabilities as well as enhanced measures for disease surveillance. the cdc defines category c agents as those that have the potential for mass dissemination in the future because of their availability, ease of production and dissemination. this category also includes emergent threats that have the potential of causing high morbidity and mortality rates, as well as a major impact on public health. further details on the classification and prioritization of biological threat agents are available on the cdc website: http:// www.bt.cdc.gov/agent/agentlist-category.asp. levels in the original sample. a similar method was used to detect staphylococci. out of isolates taken from staphylococci species, were correctly assigned by using only s rrna sequences on microarrays, conferring a sensitivity of % [ ] . the ability to refine and expand on the existing probe set, for example by the incorporation of additional informative genetic loci, is a key advantage provided by the dna microarray platform to increase redundancy and hence improve assay performance and fidelity. miller and colleagues [ ] applied microarrays in clinical diagnostics and were able to identify pathogens in a panel of patient specimens with a % accuracy score, calculated from % sensitivity and % specificity. the microarray was designed to detect up to rna viruses, including the sars coronavirus and dengue viruses, using -mer probes. a total of such probes in replicates of seven were printed on nimblegen tiling microarrays. the authors studied ways to minimize pcr bias to ensure uniform amplification and developed a unique statistical approach to infer pathogen identity from the resulting microarray fingerprints. apart from the highdensity microarrays described here, more focused biodefence platforms have been developed for selected panels of environmental [ ] or blood-borne pathogens [ ] , using inhouse microarrays that comprise only several hundred relevant probes. such panels are designed to selectively identify targets of interest with enough redundancy to reduce the occurrence of false positives, which is infrequently controlled in other diagnostic platforms that often use either one or a handful of primer sets specific to each pathogen of interest (as is the case in the genexpert, rapid and cepheid real time pcr platforms). there are several instructive examples where dna microarrays have advanced our understanding of disease etiology and epidemiology through their use in studies into molecular evolution, host-pathogen interactions and modes of virulence. in the following section, we will review salient studies that have provided such fundamental insight, focusing on agents from the cdc categories a to c. one important application of microarrays is in the resequencing of pathogen genomes, which has been successfully applied to interrogate inter-species and/or interstrain differences (figure b ). this provides a vital alternative to traditional shotgun sequencing approaches because it is quicker and more cost-effective; however, the disadvantage is that the sequence information for the organism of interest must available beforehand. this is not a severe limitation given that many pathogenic organisms have or are being sequenced, and with the advances in de novo sequencing capabilities, new pathogens such as sars can be readily sequenced in a matter of weeks from the time of discovery [ ] . subsequent resequencing using microarrays would only take several days. early experiments on resequencing arrays showed high rates of false discovery of up to - % [ ] , but experimental improvements and the development of robust algorithms, such as the abacus (adaptive background genotype calling scheme) software package [ ] , have now greatly improved sequencing quality. adopting this approach for variation analysis, wong and colleagues [ ] tracked strains of the sars coronavirus that were rapidly resequenced using high-density microarrays. a total of probes ( -mers) were designed to cover the complete . kb genome on nimblegen microarrays [ ] . virus samples from cell cultures and patient tissues could be amplified by reverse-transcriptase pcr using optimized primers and resequenced with accuracy greater than . %. the platform was used to confirm that a lone case of a sars virus infection of a graduate student had most likely come from a laboratory source because a unique -bp deletion was detected that was not present in other sars isolates. wang et al. [ ] also developed a sars microarray that helped to characterize a novel coronavirus variant. the microarray platform is thus ideal in virus tracking should outbreaks occur and, furthermore, is readily applicable to other pathogens evolutionary perspectives lend unique insight into the genetic changes that result in differences in pathogenicity. plague-causing y. pestis is estimated to have diverged from the non-lethal yersinia pseudotuberculosis an estimated - years ago [ ] . bearing identical s rrna sequences, more comprehensive genetic analysis was required to understand the differences in disease morbidity manifested by these two bacterial species, as well as in the different modes of transmission to humans: y. pestis by flea bites and y. pseudotuberculosis by food or water. a microarray specific for y. pestis revealed three unique genomic islands and the inactivation of several genes, including those encoding the cell-adhesion proteins adhesin (yada) and invasion (inv), as well as the o-antigen biosynthetic operon. these unique characteristics potentially led to y. pestis' enhanced virulence and its adaptation from an enteric organism (y. pseudotuberculosis) to a mammalian blood-borne pathogen [ ] . equivalent findings were reported by hinchliffe et al. [ ] using a similar figure . the use of microarrays for studying emergent pathogens, exemplified by severe acute respiratory syndrome (sars)-associated coronavirus and avian influenza (h n ). various approaches have been developed for detection and diagnostics with dna and protein microarrays. (a) specific dna probes, for instance co-v (orange) for sars and h (green) for h n , can be used to detect the presence of pathogen dna or rna using pcr or reverse transcriptase (rt)-pcr, thus enabling multiple pathogens to be detected simultaneously [ ] . (b) tiling arrays can be used to resequence pathogens so that any mutations in evolving pathogens can be rapidly detected and tracked. as illustrated, four probes, one for each nucleotide base, are used to determine the genotype at a given loci. a set of probes designed for an arbitrary position xyz is shown to reveal the unknown base to be adenine (a). multiple probe sets are 'tiled' across the whole pathogen genome and, upon hybridization and analysis, can confer the complete sequence information of the pathogen with great accuracy [ , , ] . (c) antibody arrays make use of the sandwich immunoassay to screen for the presence of a pathogen. as depicted, the pathogen sample is first applied to the microarray, followed by the reporter antibody. as a result, the pathogen is sandwiched in between two antibodiesan immobilized antibody (blue) and a tagged antibody (purple) capable of reporting the presence of a pathogen on the microarray [ , ] . (d) proteome microarrays, which contain immobilized proteins from the target pathogen, can be screened against sera obtained from infected individuals. if the individual has been exposed to the pathogen, the sera will contain antibodies (blue) against specific antigens of the pathogens that will react with the immobilized protein and that can be detected using tagged secondary antibodies (purple) [ ] . this procedure also facilitates the identification of specific immunodominant antigens present in the pathogen proteome. these proteins are considered to be largely responsible for triggering the host immune response and thus have a high potential for the development of vaccines against this particular pathogen. trends in biotechnology vol. no. y. pestis gene-specific microarray. these also showed that, of the three major y. pestis subspecies, the antiqua and mediaevalis biovars showed greater divergence from the orientalis strains [ ] . similarly, variation analysis has been performed on burkholderia pseudomallei microarrays to differentiate amongst virulent and avirulent burkholderia species, specifically bioterror agents such as b. pseudomallei (which causes meliodosis), b. mallei (which causes equine glanders) and the avirulent (but closely related) b. thailandensis [ ] . in an interesting approach to reveal molecular mechanisms for pathogenesis, weiss and colleagues [ ] developed a microarray-based negative-screening approach for francisella and obtained surviving bacterial samples from infected mice to reveal genes responsible for bacterial survival and growth in vivo. in a related study, genome-wide microarrays also revealed loci that are only present in the highly virulent franscicella tularensis subspecies tularensis strain [ ] . these loci could be used to identify potential pathogenicity islands as well as to provide unique genetic markers that could be used for strain typing and identification. microarrays have also been applied to distinguish among variants of influenza a that are resistant to the antiviral drug amantidine by detecting mutations in the viral ion channel m protein [ ] . such molecular forensics experiments using microarrays have extended our capabilities to profile new or emergent threats. the differences identified might provide an understanding of the causes of increased virulence, as well as reveal vulnerabilities that can be exploited for building therapeutic defences. beyond laboratory-based applications, dna microarrays have been tested successfully in epidemic outbreak surveillance (eos). project silent guardian was a -week trial launched by the naval research laboratory during the us presidential inauguration [ ] . the challenge was to take laboratory-based microarray technology to a production-scale system capable of operationally screening up to samples per day. a custom dna microarray was designed with the capability of detecting natural (including avian flu) and biothreat agents with strain-level resolution. in total, samples were collected and screened by civilian and military laboratory personnel within the stipulated period. the trial included blinded samples that were spiked with pathogens, which were all successfully detected. this exercise showcased the feasibility of implementing microarrays as a screening tool for eos and demonstrated their use as a rapid and robust means of sample processing and pathogen identification. recommendations from the study included the development of a more user-friendly system with greater automation of the steps involved. these features are being incorporated by several recently launched commercial platforms. microarray-based platforms for biothreat screening from akonni biosystems (http://www.akonni. com) and veredus laboratories (http://www.vereduslabs. com) are now on the market for multiplexed threat detection. new advances include (i) greater integration of the sample preparation (which is considered to be one of the major bottlenecks), (ii) the use of mpcr and (iii) the development of small handheld microfluidic chips and portable readers to take microarray technology to the point-ofcare. veredus has also recently launched an influenza test chip that enables the amplification and discrimination of influenza a and b subtypes, including avian flu (h n ), within just two hours. this great improvement in detection time has been attributed to the mpcr step, which, through rapid thermocycling, significantly reduces the duration of pcr to under min. similar devices might be developed in the future for protein and other types of non-dna microarrays for applications in serodiagnostics. the national institute of allergy and infectious diseases (niaid) and the j. craig venter institute fund a programme that enables researchers to obtain, through an approval process and material transfer agreement (mta), pathogen dna microarrays for select category a-c threats at no cost to promote and accelerate research on these pathogens. the pathogen functional genomics resource centre (pfgrc) currently fabricates and distributes a range of these -mer microarrays covering agents, including b. anthracis, coronaviruses, f. tularensis, y. pestis and b. pseudomallei (http://pfgrc.tigr.org) ( table ) . such collaborative and valuable initiatives augur favourably for the future of the biodefence community worldwide and will hopefully lead to greater sharing of resources in the pursuit of knowledge on global and regional pathogens. emerging roles of protein, peptide and carbohydrate microarrays newer microarray formats have already significantly extended the scope of microarray applications. the advantages of parallelization, miniaturization and automation hold true for whichever biomolecule is presented on highdensity microarrays. it was initially difficult to believe that proteins would retain their functionality once anchored on a solid support. however, schreiber and colleagues [ ] erased this concern by developing the first small-molecule microarray in and the first protein microarray in , simultaneously demonstrating that proteins could retain their activity despite being covalently attached to glass slides. soon afterwards, other array types were developed, such as cell arrays, carbohydrate arrays and proteome arrays. some of these newer applications have brought vital capabilities to pathogen detection and biodefence, as will be described below. as before, examples are provided for key biothreat agents and are categorized according to their consequent applicationseither in detection or profiling. pathogen detection using non-dna-based microarrays antibodies have become an established denominator for disease detection, through the time-honoured use of enzyme-linked immunosorbent assays (elisas), agglutination and lateral-flow assays. as a next-generation tool, antibody microarrays are increasing in popularity, and not without reason, for they offer unparalleled throughput, minimal reagent consumption and sensitive detection of multiple targets simultaneously [ ] . the accuracy conferred is nevertheless closely linked with the quality of review trends in biotechnology vol. no. antibodies employed, and there are issues relating to crossreactivity and antibody availability, both of which are particularly crucial for finer and more accurate strain typing. notwithstanding these matters, a plethora of antibody microarrays have been developed that have growing potential for clinical, biothreat and point-of-care applications (figure c ). rucker et al. [ ] applied antibody microarrays for the detection of toxins with low-nanomolar sensitivities, such as anthrax lethal factor (lf, a metalloendopeptidase), protective antigen (pa) and tetanus toxins (endopeptidases), through the co-application of a competitive fluorescently-labelled toxin reporter. in this setting, the samples can be tested without the need for labelling by monitoring the depletion and competitive displacement of reporter signals generated by the labelled reporter. thirty-five antibodies were also arrayed to subtype the most common salmonella serovars [ ] , and a similar study was undertaken for escherichia coli [ ] . in an impressive demonstration of the detection throughput attainable, huelseweh and colleagues [ ] built an antibody microarray to detect several category a and b agents simultaneously, including f. tularensis, y. pestis, brucella melitensis (which causes brucellosis) and b. mallei, using sandwich immunoassays (figure c) . conversely, antigen microarrays have also been used to identify seropositive individuals by using the presence of defensive antibodies in the serum as a means of detecting exposure to a pathogen. pathogen proteins are typically orthologously expressed using high-throughput cloning and expression and then affinity purified to provide protein sets. in special cases, it might be necessary to consider the post-translational modifications or the way the pathogen presents itself to the host to identify and use the correct immunogenic protein variants in serodiagnostics. it is also desirable to purify the protein and check for efficient and generally uniform immobilization on the microarrays as a quality control, especially when comparisons are made across slides. in one such example, sundaresh et al. [ ] narrowed down an initial panel of f. tularensis antigens to just the top in terms of their ability to predictively discriminate seropositive sera and then used these antigens to establish a diagnostic microarray comprising whole proteins. mezzasoma et al. [ ] also developed protein microarrays for simultaneous diagnostics using parasitic and viral antigens. zhu et al. [ ] monitored the antibody profiles of sars patients using protein microarrays containing purified coronavirus proteins. antibodies present in human serum samples were detected on the protein microarrays with fluorescently labelled goat anti-human immunoglobulins. it was found that immunoreactivity against the coronavirus nucleocapsid proteins remained high for - days post infection. this provided a means to check for exposure long after infection might have occurred. over human sera samples were screened with an accuracy of % in differentiating infected and normal specimens [ ] . nevertheless, caution should be applied in protein-based diagnostics because incubation periods vary greatly amongst diseases. during the incubation period, the patient might not express antibodies at detectable levels but might still be capable of spreading the infection. in the case of sars, the incubation period (the time between exposure and onset of symptoms) has a median of - days (up to a maximum of days). antibodies might be detectable only - days after first symptoms are presented, but pcr testing might be able to pick up the virus more quickly, just - days after symptoms become apparent. it might become possible in the future to integrate dnaand protein-based microarray methods to extend the range of rapid clinical diagnostics from detection of current pathogen infections to testing for exposure long after the actual infection has occurred. as an alternative, short peptides have also been applied for serodiagnostics, and this was facilitated by the robust and routine procedures established to chemically synthesize peptide sequences up to amino acids in length. peptide microarrays have been applied for detecting immunoreactive sera against sars, amongst other pathogens, enabling the detection of low-picomolar concentrations of antibodies [ ] . lipopolysaccharide, carbohydrate-based and whole-cell microarrays have also been used for antibody-based detection of pathogens such as f. tularensis [ , ] , b. anthracis [ ] and b. pseudomallei [ ] . the surface antigens on these microbes are frequently responsible for immunoreactivity, so these methods rely on detecting the presence of such immunogenic antigens. further improvements in fabrication techniques and greater knowledge of surface chemistries might lead to the development of hybrid microarray platforms in which multiple types of antigens, such as peptides, proteins and carbohydrates, are presented simultaneously to detect host antibodies with improved efficiency and sensitivity. pathogen profiling using non-dna-based microarrays protein microarrays with a high pathogen proteome content offer a valuable platform for high-throughput serology. proteins that are immunoreactive to patient sera represent antigens that can be applied as markers in serodiagnostics or as therapeutic proteins for vaccine development. in one such example, the immunogenic epitopes of the sars coronavirus were traced to the cterminal fragments of the nucleocapsid protein by screening against human sera samples from infected individuals ( figure d) [ , ] . to address the bottleneck of protein expression, davies and colleagues [ , ] applied pcr recombinant cloning to accelerate the process of cloning and expression. this enabled more than proteins from vaccinia virus and over proteins from f. tularensis to be expressed and profiled using microarrays [ , ] . these arrays have also helped to identify antibodies against the vaccinia virus h l envelope protein, which confers protection in vivo (in a mouse model) [ ] . the vaccinia protein microarrays have also been used to derive antibody profiles in humans inoculated with the licenced dryvax smallpox vaccine [ ] , and these profiles were found to be very similar to those induced by the attenuated modified vaccinia virus ankara strain, an alternative vaccine candidate [ ] . various protein microarrays have similarly been generated for identification of the immunodominant antigens of y. pestis [ , ] . trends in biotechnology vol. no. other microarray platforms have demonstrated valuable potential in biodefence. for instance, small-molecule microarrays have been applied to functionally screen anthrax lf through activity-based binding signatures against a library of immobilized peptide-hydroxamate inhibitors. putative drug candidates were discovered that bound selectively to the anthrax lf (with a low dissociation constant, k d = . mm) and inhibited its activity in vitro [ ] . carbohydrate microarrays have also been developed to elucidate the receptor preferences of influenza viruses. stevens and colleagues [ , ] applied glycan arrays to establish the a - and a - binding selectivity of the h and h haemagglutinins, which determine influenza virulence. the selectivity was altered by specific mutations in the protein, providing a useful way of functionally monitoring viral adaptation. non-dna microarray formats have thus contributed unique ways in which we can explore pathogen biology and develop countermeasures in the event of an outbreak. it is clear that microarrays have significantly strengthened our biodefence capabilities. although the upstream cost of library creation and microarray fabrication, as well as the initial infrastructure investment of several hundreds of thousands of dollars (for microarray spotters and scanners), might represent significant hurdles for small laboratories with tight budgets, the provision of printed microarray slides through commercial or research avenues heralds a favourable trend towards the reduction of exclusivity. greater access to the technology, such as the increased availability of microarrays together with established array designs, is helping researchers worldwide to move more quickly towards the downstream application phase, for example to perform regional studies of endemic variants. the applications of microarrays described here are broad-ranging and span from dna-or protein-based detection of pathogens to epidemic outbreak surveillance and vaccine development, thus showcasing the full spectrum of microarray potential ( table ). the progress made in the past several years has brought microarrays to the forefront of rapid diagnostics and medical research. further integration of upstream sample preparation with downstream data processing is expected to transform microarrays into compact, field expedient solutions for analysis and monitoring in the near future. the large assortments of biomolecules now utilized in microarrays provide novel opportunities in molecular forensics and comparative profiling, especially for the newer and less well-understood pathogens. as we equip ourselves with better capabilities and countermeasures against potential biological threats, we hope to become more agile and responsive whenever such threats emerge in the future. microarrays have improved our confidence in this respect and will continue to play a decisive part in biodefence research and technology. molecular evolution of the sars coronavirus during the course of the sars epidemic in china chemical and biological weapons: current concepts for future defenses turning biodefense dollars into products current and developing technologies for monitoring agents of bioterrorism and biowarfare microarrays -status and prospects comparative phylogenomics of pathogenic bacteria by microarray analysis nucleic acid approaches for detection and identification of biological warfare and infectious disease agents potential applications of dna microarrays in biodefense-related diagnostics oligonucleotide microarrays in microbial diagnostics progress in protein and antibody microarray technology advances in functional protein microarray technology small molecule microarrays: recent advances and applications peptide microarrays: next generation biochips for detection, diagnostics and high-throughput screening microarrays in infection and immunity highly parallel microbial diagnostics using oligonucleotide microarrays microbiological threats to homeland security sequence-specific identification of pathogenic microorganisms using microarray technology microarray-based detection and genotyping of viral pathogens molecular detection and identification of influenza viruses by oligonucleotide microarray hybridization broad-spectrum respiratory tract pathogen identification using resequencing dna microarrays identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray potential of dna microarrays for developing parallel detection tools (pdts) for microorganisms relevant to biodefense and related research needs rapid quantification and taxonomic classification of environmental dna from both prokaryotic and eukaryotic origins using a microarray high-density dna probe arrays for identification of staphylococci to the species level optimization and clinical validation of a pathogen detection microarray multipathogen oligonucleotide microarray for environmental and biodefense applications a multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood the genome sequence of the sars-associated coronavirus microarray-based resequencing of multiple bacillus anthracus isolates high-throughput variation detection and genotyping using microarrays tracking the evolution of the sars coronavirus using high-throughput, high-density resequencing arrays viral discovery and sequence recovery using dna microarrays genechip resequencing of the smallpox virus genome can identify novel strains: a biodefense application microevolution and history of the plague bacillus, yersinia pestis dna microarray analysis of genome dynamics in yersinia pestis: insights into bacterial genome microevolution and niche adaptation application of dna microarrays to study the evolutionary genomics of yersinia pestis and yersinia pseudotuberculosis patterns of large-scale genomic variation in virulent and avirulent burkholderia species in vivo negative selection screen identifies genes required for francisella virulence genome-wide dna microarray analysis of francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent f. tularensis subsp. tularensis detection of adamantane-resistant influenza on a microarray nrl uses microarray technology to detect natural and bio-threat pathogens during silent guardian project printing proteins as microarrays for high-throughput function determination antibody microarrays for native toxin detection development of a novel protein microarray method for serotyping salmonella enterica strains use of miniaturized protein arrays for escherichia coli o serotyping a simple and rapid protein array based method for the simultaneous detection of biowarfare agents from protein microarrays to diagnostic antigen discovery: a study of the pathogen francisella tularensis antigen microarrays for serodiagnosis of infectious diseases severe acute respiratory syndrome diagnostics using a coronavirus protein microarray functional peptide microarrays for specific and sensitive antibody diagnostics lipopolysaccharide microarrays for the detection of antibodies bacterial cell microarrays for the detection and characterization of antibodies against surface antigens photogenerated glycan arrays identify immunogenic sugar moieties of bacillus anthracis exosporium polysaccharide microarray technology for the detection of burkholderia pseudomallei and burkholderia mallei antibodies antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein screening of specific antigens for sars clinical diagnosis using a protein microarray immunodominant francisella tularensis antigens identified using proteome microarray profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery vaccinia virus h l envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice proteome-wide analysis of the serological response to vaccinia and smallpox antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine modified vaccinia virus ankara is comparable to that of dryvax protein microarray for profiling antibody responses to yersinia pestis live vaccine quorum sensing affects virulence-associated proteins f , lcrv, katy and ph etc. of yersinia pestis as revealed by protein microarray-based antibody profiling quantitative inhibitor fingerprinting of metalloproteases using small molecule microarrays structure and receptor specificity of the hemagglutinin from an h n influenza virus glycan microarray technologies: tools to survey host specificity of influenza viruses the authors acknowledge funding support from dso national laboratories. key: cord- -xjnbmah authors: van goethem, n.; struelens, m. j.; de keersmaecker, s. c. j.; roosens, n. h. c.; robert, a.; quoilin, s.; van oyen, h.; devleesschauwer, b. title: perceived utility and feasibility of pathogen genomics for public health practice: a survey among public health professionals working in the field of infectious diseases, belgium, date: - - journal: bmc public health doi: . /s - - - sha: doc_id: cord_uid: xjnbmah background: pathogen genomics is increasingly being translated from the research setting into the activities of public health professionals operating at different levels. this survey aims to appraise the literacy level and gather the opinions of public health experts and allied professionals working in the field of infectious diseases in belgium concerning the implementation of next-generation sequencing (ngs) in public health practice. methods: in may , belgian public health and healthcare professionals were invited to complete an online survey containing eight main topics including background questions, general attitude towards pathogen genomics for public health practice and main concerns, genomic literacy, current and planned ngs activities, place of ngs in diagnostic microbiology pathways, data sharing obstacles, end-user requirements, and key drivers for the implementation of ngs. descriptive statistics were used to report on the frequency distribution of multiple choice responses whereas thematic analysis was used to analyze free text responses. a multivariable logistic regression model was constructed to identify important predictors for a positive attitude towards the implementation of pathogen genomics in public health practice. results: out of the invited public health professionals completed the survey. % of respondents indicated that public health agencies should be using genomics to understand and control infectious diseases. having a high level of expertise in the field of pathogen genomics was the strongest predictor of a positive attitude (or = . , % ci = . – . ). a significantly higher proportion of data providers indicated to have followed training in the field of pathogen genomics compared to data end-users (p < . ). overall, % of participants expressed interest in receiving further training. main concerns were related to the cost of sequencing technologies, data sharing, data integration, interdisciplinary working, and bioinformatics expertise. conclusions: belgian health professionals expressed favorable views about implementation of pathogen genomics in their work activities related to infectious disease surveillance and control. they expressed the need for suitable training initiatives to strengthen their competences in the field. their perception of the utility and feasibility of pathogen genomics for public health purposes will be a key driver for its further implementation. sequence information from viruses, bacteria, and other infectious organisms can be used to identify a pathogen and its specific characteristics, and compare its genetic relatedness to other pathogens [ ] . advances in sequencing technologies, especially the shift to next-generation sequencing (ngs), have made it possible to analyze pathogen genomes in much greater detail. compared to sanger sequencing, ngs technologies allow a faster and cheaper way to sequence larger lengths of nucleotides. as such, ngs makes microbial pathogen whole-genome sequencing (wgs) accessible in high throughput within a matter of days [ ] . during the last decade, ngs has expanded beyond the research settings and is being rapidly applied into routine practice for public health and food safety [ ] [ ] [ ] [ ] [ ] [ ] . in public health, integrating pathogen genomics with epidemiology provides many opportunities for improving the population-level risk assessment and management of infectious diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the main applications of wgs include ( ) retrospective (or near real-time) comparisons of pathogens' relatedness to test epidemiological transmission hypotheses of suspected outbreaks (i.e. outbreak investigations); ( ) wgs-based prospective surveillance by monitoring of cases generating alerts when clusters of pathogens with similar genomes are identified in a limited geographical area or time period or when virulent clones emerge (outbreak detection by control-oriented surveillance); and ( ) cross-sectional genomic epidemiology surveys to monitor long-term changes in epidemiology over larger geographic and population scales to inform prevention strategies (strategy-oriented surveillance) [ ] . the main added value of implementing wgs during surveillance activities or outbreak investigations is inherent in the higher resolution of the wgs output itself, leading to an increased sensitivity and specificity to identify transmission clusters compared to conventional subtyping methods [ ] . as such, there are numerous success stories of outbreak investigations applying wgs that were able to identify to the source of infection and implement targeted control measures to stop further spread, saving resources at the health protection and local authority level [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . other concrete examples of the utility of wgs for national surveillance and local infection control include the guidance of vaccination strategies [ ] [ ] [ ] [ ] and antibiotic stewardship [ , ] . besides transforming the public health approach to infectious diseases monitoring, analysis of pathogen genomics can advance the accuracy of infection diagnostics and guide the treatment of individual patients [ , [ ] [ ] [ ] [ ] [ ] [ ] . for several pathogens, ngs is able to replace current time-consuming and/or laborintensive conventional methods with a single, all-in-one diagnostic test [ ] [ ] [ ] [ ] . public health professionals play a key role in protecting the population against communicable disease threats. this requires them to give effective responses in a limited time frame, supported by adequate information resulting from applying the most appropriate tools adapted to the specific public health threat scenario. infectious disease surveillance systems build upon the cooperation between: clinicians, who are at the frontline through identification of infected patients; microbiologists, who are involved in testing specimens; molecular biologists, who study organisms at the molecular level; bioinformaticians, who develop computational approaches/algorithms to analyze genomic data; epidemiologists, who use the data to understand patterns in disease occurrence at the population level; infection control practitioners, who are responsible for local prevention and control of infectious diseases in the community; hospital hygienists, who are involved in the prevention and control of healthcare-associated infections; food safety inspectors, who monitor food products; etc. the activities of these public health experts operating at different levels in the information cycle will be impacted by the introduction of pathogen genomics as they are all connected to each other. this ranges from microbiologists adapting their laboratory workflows to epidemiologists rethinking their current data analysis approaches. typically, new laboratory technologies are adopted by data providers first, while data end-users might not be familiar enough with the new methods to effectively translate the output data into public health actions. expertise with pathogen genomics and its applications for public health practice might also differ between those in charge of national surveillance of infectious diseases and those involved in local infection control and patient management, as well as between different fields (i.e. human, animal, food, and the environment) within the one health spectrum [ , ] . differences in perceptions and needs between these different profiles should be taken into account before we can build a strategy that engages all the stakeholders in an effective collaboration. the key to success in translating pathogen genomics into public health practice is to demonstrate an added value by better addressing the needs and expectations of the whole range of public health experts. an effective exchange of expertise across disciplines (e.g., clinicians, microbiologists, epidemiologists, and bioinformaticians) is key for enabling the smooth implementation of ngs into routine public health activities. if such coordination of joint efforts cannot be accomplished, the technology shift, which is currently ongoing, might not realize its full potential [ , ] . previous surveys in the field of public health genomics focused on: human genomics [ ] [ ] [ ] ; specific aspects such as proficiency testing [ ] , the design of wgs clinical reports [ ] or data sharing [ ] ; or specific target groups such as national microbiology focal points [ ] or food safety laboratories [ ] . in this study, by organizing an online survey, we aimed to perform a wide landscape analysis of all potentially involved stakeholders in order to appraise the level of genomic literacy and to gather the opinions of public health experts and allied professionals working in the field of infectious diseases in belgium concerning the implementation of ngs in routine public health activities, in terms of its utility, feasibility, implementation, and translation into actionable results for public health decision making. an electronic questionnaire survey (see additional file ) was developed for this study using limesurvey (version . . ) [ ] for the collection of relevant information from public health professionals working in the field of infectious diseases in belgium. for the purposes of this study, a 'public health professional in the field of infectious diseases' was defined as a person with professional expertise in the field of infectious diseases and who directly or indirectly contributes to the population-level management of infectious diseases. to provide a complete picture of all involved stakeholders, the survey aimed to reach different subgroups based on professional qualification (i.e. microbiologists, molecular biologists, bioinformaticians, epidemiologists, clinicians, clinical biologists, infection control practitioners, and hospital hygienists), employing institution (i.e. governmental, private, hospital, and university), health field (i.e. human, animal, food, and environment), expertise in pathogens (i.e. bacteria, viruses, parasites, fungi, and yeasts), and level of action (i.e. national surveillance and local infection control). to identify all actors in the field of public health activities for infectious diseases, an overview was made of existing surveillance systems (i.e. data sources) in belgium (see additional file ). the set of questions was compiled based on the literature, including several review articles [ - , , , - , , ] . existing items from previous survey questionnaires [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] ) were used and adapted when relevant. most of the existing questionnaires from which some questions were adapted to be used for this survey were not validated, except for chow-white et al. as mentioned in the respective publication [ ] . the construction of the survey was discussed during several feedback rounds within a multidisciplinary team including epidemiologists, microbiologists, and molecular biologists. as a result, the survey instrument was vetted by subject matter experts. the questionnaire eventually contained eight main topics comprising background questions, general attitude towards pathogen genomics for public health practice and main concerns, genomic literacy, current and planned ngs activities, place of ngs in the diagnostic hierarchy of microbiology, data sharing obstacles, end-user requirements, and key drivers for the implementation of ngs. based on a filter question where participants indicated their level of familiarity with pathogen genomics, the respondents were redirected to different sets of questions with different levels of technicity and detail. the filter question gave access to a reduced version of the questionnaire for those participants judging themselves as not at all familiar with pathogen genomics. the responses were mainly collected as single/multiple options from a set of pre-defined answers, but also included the optional entry of free text. these qualitative open questions were included to add context to the quantitative responses. the survey tool was pre-tested by three researchers not directly involved in the development phase to ensure the acceptability and clarity of the questionnaire. participants were contacted individually by an email invitation containing a personal token to complete the survey. no monetary or other incentive was offered. the participant information statement at the beginning of the survey informed the respondents about the objective and design of the study and their rights before participation to the survey, and explained that responses are anonymized and will be kept confidential. the approval from an ethical committee was not considered necessary due to national regulations (legislation april ), as this study was not medical in nature and as participants were not subject to any actions and/or rules of conduct. the survey was available online during a two months' period during which three reminders were sent to those who had not yet responded. the first invitations were sent on the th of may and the survey remained active until the st of july . participants were invited to send any questions, feedback or comments for the survey to the organizers. only completed questionnaires were used for analysis. descriptive statistics were reported by analyzing categorical response frequencies. differences in viewpoints between the stakeholders were described using subgroup analyses and compared using a fisher's exact test. subgroups were compiled on the basis of the level of action (national vs. local), the position in the information cycle (data providers vs. data end-users), and the level of expertise in the field of pathogen genomics. the level of action was considered national when the main affiliation of the respondent concerned a national institute involved in national public health activities, whereas the local level included professionals who mainly operate at the community, hospital, or university level. subgroups based on the position in the information cycle were defined as data providers, defined here as experts in wet and dry lab procedures and (potentially) generating ngs data (including microbiologists, molecular biologists, clinical biologists, and bioinformaticians), and data endusers defined here as using ngs data to improve their activities and implementing infection control measures (including epidemiologists, local infection control practitioners, hospital hygienists, and clinicians). the level of expertise was categorized as high, middle or low, and was based on respondents' self-reported familiarity with pathogen genomics, training level, and current use of ngs. multiple logistic regression was performed to identify predictors of a positive attitude towards the implementation of pathogen genomics from a public health perspective. enthusiasm about public health agencies using genomics to understand and control infectious diseases was defined directly through a question with multiple options, each containing a clear statement (see additional file ). for the purpose of this analysis, the question asking about their enthusiasm originally consisting of multiple categories was collapsed into two levels: very enthusiastic versus all others. the following predictor variables were initially tested in the model: level of action; position in the information cycle; level of expertise; current use of ngs; institution; age group; years of professional experience; and position in their institution. model building involved a univariate analysis to select variables to be included in the multivariable model based on a χ -test (cut-off, p = . ), and variable selection from the multivariable model using backward stepwise regression based on the akaike information criterion (aic). adjusted odds ratios (ors) and % confidence intervals (cis) were calculated. quantitative analyses were performed using r software (r studio version . . ) [ ] . answers to open-ended survey questions were summarized and analyzed using nvivo qualitative data analysis software (nvivo version ) [ ] . this was done by identifying themes (codes) within the data, which were derived both deductively and inductively. following the thematic analysis framework, the text was compared and contrasted with the identified codes. the qualitative findings were summarized as a mind map linking the identified major and minor themes and a word cloud visualizing the word frequency from the qualitative responses. simultaneously, quotes were selected for the sake of illustration. out of the invited participants, did not respond at all, partially filled in the survey, and a total of participants delivered a completed survey which represents an overall survey response rate of % (fig. ). from these, participants continued after the filter question and delivered answers to all questions ( subject were redirected to a technical version of the survey and subjects to a basic version, based on the filter question). the data from the participants who preferred to quit after the filter question were only used to describe the background characteristics of the study population. the subjects who partially filled in the survey were dropped completely from the analysis. full responses to all questions as they appeared in the questionnaire are provided as an appendix to this report (see additional file ). background characteristics of the participants are presented in table . the majority of respondents had their main affiliation in the public sector ( %), followed by hospitals (including university hospitals) ( %), private sector ( %), and university ( %). the public sector was primarily represented by sciensano (belgian institute for health), comprising % of all survey participants ( / ). % of the respondents indicated that they had more than years of professional experience within the field of infectious diseases. the reported roles of respondents within their institutions included: microbiologists/molecular biologists/bioinformaticians/clinical biologists ( %); epidemiologists ( %); clinicians ( %); hospital hygienists/infection control practitioners ( %); and policy makers ( %). the survey respondents were asked to describe their level of familiarity with sequencing technologies and pathogen genomics using following classification: 'very -i am involved in the generation and/or use of ngs data' ( %), 'somewhat -i have a general sense of the applications of ngs' ( %), or 'not at all -i don't know anything about ngs and its applications' ( %). of those participants answering 'very familiar', most of them ( %; / ) indicated that they mainly used ngs in the context of wgs. of those 'not at all familiar', preferred to quit the survey and continued the survey to answer some general questions, leaving a total of participants for the remainder of the survey (fig. ) . subgroup analysis showed differences in familiarity with pathogen genomics between data providers and endusers (fig. ). data providers indicated significantly more frequently that they were 'very familiar' compared to data end-users (p < . ). the majority of respondents ( %; / ) indicated that they were very enthusiastic (i.e. 'we should be using genomics now') about public health agencies using genomics to understand and control infectious diseases, % ( / ) did not have an opinion or did not know enough of the topic to be able to give an opinion, and % ( / ) indicated that they did not see clear applications and/or an added value for public health. subgroup analysis pointed out differences in enthusiasm according to the level of expertise in the field of pathogen genomics ( fig. ). important predictors, as identified by the best fitting model, of a positive attitude related to the implementation of pathogen genomics from a public health perspective were the level of expertise, the level of action, and the position in the information cycle ( table ). participants classified as having a high level of expertise based on their self-reported familiarity with the topic, their training level, and/or the current use of ngs were significantly more likely to be enthusiastic about the implementation of pathogen genomics in a public health context compared to their peers with a low expertise (adjusted or = . , % ci = . - . ). further, public health professionals operating at the national level were more often 'very enthusiastic' about the implementation of pathogen genomics ( %) compared to those at the local level ( %). similarly, data providers were more often 'very enthusiastic' ( %) compared to data end-users ( %). a large majority of respondents considered the following public health activities as likely to be most impacted by pathogen genomics in the next five years: identifying an outbreak (clusters of related isolates) ( %; / ), nosocomial and food/waterborne outbreak investigations ( %; / ), and monitoring the spread of antimicrobial resistance ( %; / ). in contrast, only % ( / ) of respondents thought that pathogen genomics would have a major impact on making a diagnosis and selecting an appropriate treatment (individual patient management). other public health activities that will benefit from the implementation of pathogen genomics mentioned by the participants are presented in table . the most frequent concerns among participants being 'very' or 'somewhat' familiar with ngs technologies and pathogen genomics (n= ) regarding feasibility of its routine use for public health purposes, were the cost of sequencing technologies and the existing barriers to timely and open sharing of pathogen sequence data and accompanying metadata ( table ). all participants exclusively working with respiratory infections (e.g. influenza) and/or vaccine-preventable diseases (e.g. measles) (n= ) were very concerned about the cost, whereas this was only true for % of participants exclusively working with invasive bacterial diseases (e.g. neisseria meningitidis), food-and waterborne diseases (e.g. salmonella), and/or healthcareassociated infections (e.g. clostridium difficile) (n= ). further, other concerns shared by a large proportion of the participants were interdisciplinary cooperation, integration of pathogen sequence data with contextual data, access to bioinformatics expertise, and availability of typing schemes and databases. participants indicating to be 'not at all' familiar with pathogen genomics were mainly concerned about the cost of the sequencing technologies (see full responses in additional file ). other concerns provided by the participants as free text are presented in table . two-thirds of the participants ( / ) indicated that they had followed training in the fields of genomics/genetics/ molecular biology/bioinformatics. there were marked differences by position in the information cycle: % ( / ) of data end-users indicated that they had never followed any training in the field, whereas this was stated by only % ( / ) of data providers (p < . ). further breakdown of training experience by professional category is shown in fig. . the main reasons for not taking a training/course in this field (yet) were the lack of available and/or suitable trainings ( %; / ) and the lack of time ( %; / ). other reasons indicated as free text are presented in table . the vast majority of participants ( %, / ) indicated that they felt the need and/or would be interested in following (additional) courses/training/workshops covering a topic related to pathogen genomics. overall, % ( / ) participants being 'very' or 'somewhat' familiar with ngs technologies and pathogen genomics indicated that they are currently using or generating ngs data for at least one pathogen. differences between professional groups are presented in fig. . among the microbiologists, those from a national reference centre (nrc) were more likely to be currently using ngs ( / , i.e. %) compared to those from other laboratories ( / , i.e. %), however this difference was not significant (p= . ). from the public health professionals exclusively involved in human infectious disease activities, % ( / ) were currently using ngs technologies, whereas this was the case for % ( / ) of those exclusively involved in the food, animal or environmental sector (p= . ). looking forward, % ( / ) of participants indicated that they were planning to use or generate ngs data for any (additional) pathogen(s) within three years. details on the specified pathogens can be found in the appendix (see additional file ). reasons provided by participants indicating that they did not plan to implement pathogen genomics were mainly related to the cost and the lack of expertise. participants being 'very' or 'somewhat' familiar with ngs technologies and pathogen genomics (n= ) were asked to assign a score from to to the different criteria based on their increasing relative importance to decide whether or not ngs should be implemented for a particular pathogen (fig. ). clinical and/or public health significance of the pathogen were scored as the most important drivers. the different subgroups scored the different criteria similarly (see additional file ). comments provided by the participants to provide context to their scores are presented in table . centralization of sequencing and bioinformatics at nrcs organized per pathogen or per group of pathogens was most often ( %; / ) selected by respondents being 'very' or 'somewhat' familiar with pathogen genomics as the preferred wgs provision model in the belgian context. excluding participants working at nrcs slightly lowered this proportion to out of (i.e., %). there were no marked differences according to the level of action of the participants (fig. ) . illustrative quotes for the need for centralization are presented in table . public health activities, other than those provided within the survey, that will benefit from the implementation of pathogen genomics environmental monitoring "drinking water quality" "air quality, home environmental quality" metagenomics "metagenomics for patients with no identified cause of illness using conventional methods" "identification and characterization of new strains" "insights in dysbiosis" "microbiome analysis" other "discovery of a causal relation between a pathogen and a clinical disease (e.g. cancer)" "vaccine development" "phage therapy" "early diagnostics of diseases due to slow growing pathogens" "international tracking" "monitoring of antiviral resistance" concerns, other than those provided within the survey, related to the implementation of pathogen genomics for public health practice contextual data "harmonization of epidemiological datamost of the epidemiological data is very 'messy' or inconsistent, which makes systematic integration and surveillance unfeasible" "data collection is already limited so newer technologies will not automatically improve this process but be redundant if the basics are not met" "how to interpret the result at clinical level" "[…] they need to have a basic understanding (education) it order to understand and see cost/benefit of the whole picture" "appropriate training of personnel for execution and interpretation" "interpretation across sectors" "multidisciplinary knowledge" ethics "[…] healthcare workers integrity concerns" "in the hiv field, the phylogenetic analyses of virus permit to have an hindsight in paths of transmissionit is a very tricky topic in ethical and potentially legal aspects" other "does the identification prove that the pathogen poses a risk?" "the fear that some actors in the field will try to abuse their power and monopolize this new technologyto be really valuable to patient management and public health it is required to offer access to all laboratories" "high inter-laboratory variability" "[…] standardization and facilities for data sharing need to be improved" "the perceived utility and feasibility of pathogen genomics by public health practitioners is the biggest bottleneck of allall the other concerns listed above can be tackled given the drive within the field to solve them in the first place" reasons, other than those provided within the survey, for not taking a training/course in the field of pathogen genomics "lack of training adapted to public health needs" "not applicable for a clinician" "not my priority" "not relevant for my practice" "depends on the evolution in phenotypic typing" "[…] the main driver the pressure by ecdc rather than a real need for public health […] the first and main driver should be clinical significance: improve quality of care for the patient" "for bacteria, ngs will never fully replace classical methods for resistance testing, but would offer important complementary data" "cost-effectiveness (e.g. replacing multiple tests): not particularly true for viruses, but obvious for bacteria" centralization "[…] should be overall coordinated and controlled by the federal public health authority" "[…] in any scenario it will be important that sequence data are brought together in one databank for surveillance purposes" table . major themes identified within the qualitative data are utility (applications), feasibility (including capacity building, multi-disciplinary working, contextual data, costs, data sharing, ethics, timeliness, wet and dry lab), one health context, and routine implementation (including organization and translation into action). a mind map linking the identified major and minor themes is presented in fig. . a full list of identified themes and the coded text is available in the appendix (see additional file ), as well as a word cloud constructed based on the free text responses (see additional file ). this survey sought the opinion of belgian public health professionals working in the field of infectious diseases concerning the implementation of pathogen genomics in public health activities. to successfully translate pathogen genomics into public health practice, the needs and expectations of the different stakeholders should be taken into account. other questionnaire surveys related to knowledge and attitudes towards public health "the bureaucracy involved in the transmission of data" "the structure of public health in belgium will not help sharing data" "the required technical infrastructure" priority to publication "it is really a pity that priority to publication is an obstacle in the scientific world as it functions now" one health "a better collaboration between the veterinary and human side might increase the use of ngs on the veterinary side" "monitoring the emergence and spread of zoonotic pathogens has been impacted negatively, by the introduction of wgs at the human side only" genomics in specific health expert categories have been published [ , - , , , , ] . however, to the best of our knowledge this survey is the first that aimed to perform a wide landscape analysis of all potentially involved stakeholders. therefore, a strength of the current study is that it took into account a wide range of stakeholders with diverse backgrounds (epidemiologists, microbiologists, bioinformaticians, clinicians, infection control practitioners, etc.), health domains (human, food, environmental, etc.), pathogen expertise (bacteria, viruses, parasites, fungi, etc.), activity sectors (public, private, university, hospital, etc.), work positions (employee and lower/middle/high management), and degree of familiarity with genomics. besides seeking the general attitude of the participants towards the implementation of pathogen genomics in their professional activities and investigating the current and future use, this explorative study was able to touch upon multiple key topics, such as genomic literacy, data sharing obstacles, place of ngs in the diagnostic hierarchy of microbiology, and enduser requirements. familiarity with sequencing technologies and pathogen genomics varied between the different professional groups, with data providers being more familiar than data end-users. as shown before, one of the largest barriers to acceptability from the public health unit is the capacity to understand and use the data [ ] . possibly, there is a positive association between genomic literacy criteria could be assigned a score from to , or participants could indicate the 'i don't know' option. the boxplots show the median score and the interquartile range (grey boxes). the following criteria were included (top to bottom): clinical and/or public health significance, priority with respect to preventing the spread of antimicrobial resistance, local/national/international policy surveillance priorities or obligations, importance of prevention and control programs (e.g. vaccination), utility of wgs for diagnostics and/or treatment decisions (individual patient care), utility of increased resolution to infer relatedness that would not be obtained via conventional methods, availability of high-quality/complete/standardized epidemiological and/or clinical data to provide context to the wgs results, possibility to link genomic data from different sources (food-animalhuman-environment), cost-effectiveness (e.g. replacing multiple tests), time-saving compared to conventional testing methods, impact on outcomes for patients and populations (translation into actionable results), availability of wgs typing schemes and reference databases (e.g. for antimicrobial resistance), availability of validated (quality-controlled) wgs workflows (both wet and dry laboratory), availability of expertise to generate, analyze and interpret wgs data, and availability of the appropriate infrastructure (sequence technology, high-performance computing, data storage, etc.). having a high level of expertise, was the strongest predictor for a positive attitude, as was also shown in other surveys [ ] [ ] [ ] . epidemiologists and infection control practitioners should be informed about the benefits and limitations of ngs technologies in order to contribute in identifying tangible field application in public health, allowing the use of wgs output to appropriately guide public health actions [ , ] . another important challenge related to the interpretation of wgs data is the capacity to interpret signals, and thereby separating noise from public health events that require specific actions. consequently, integrating genomics into infection control and surveillance is critically linked to human resource development [ , ] . in the survey, the main reasons stated for not training in the field of genomics were lack of time or access to suitable trainings "…adapted to public health needs". however, the participants of this survey generally expressed a positive attitude towards following (additional) training courses, or workshops in pathogen genomics. educational workshops should be applied to a public health context and bring together the expertise of microbiologists, molecular experts, bioinformaticians, epidemiologists, infection control practitioners, and clinicians. the development of a new discipline called 'genomic epidemiology' integrating information on epidemiological and pathogen sequence characteristics by public health microbiologists, epidemiologists, and risk managers was recommended in the expert opinion on wgs for public health surveillance by the european centre for disease prevention and control (ecdc) in [ ] . ecdc has initiated public health genomics training workshops that bring together experts with epidemiology, microbiology and pathogen genomics backgrounds from european union (eu) member states with interest in implementing the technology in surveillance and outbreak investigations. besides, the zoonotic origin of many clinically relevant pathogens and antimicrobial resistance determinants stresses the importance of a cross-sectoral one health approach. the implementation of wgs should be synchronized and integrated between the human health and veterinary sectors [ ] allowing a better monitoring of the emergence and spread of zoonotic pathogens and antimicrobial resistance-related threats. lack of financial resources was often indicated as a principal reason for not using or planning to use wgs by the respondents of this survey, which was also reported by the european surveys conducted by ecdc [ ] and the european food safety authority (efsa) fig. mind map linking the major and minor themes identified in the qualitative responses, belgium, . codes were identified within the data deductively (i.e. themes that are expected and have been chosen in advance) and inductively (i.e. themes that are derived through analysis). during the thematic analysis the qualitative data from the survey was compared and contrasted with the identified codes. as such, the derived codes were assigned to the relevant text. next, the codes (plain boxes) were merged into categories (colored boxes). the following categories were identified: routine implementation (orange), one-health context (yellow), and feasibility (blue) [ ] . operational costs will be influenced by the processes used in current laboratory practice and differs between viruses and bacteria. whereas drug susceptibility testing and epidemiological typing are commonly performed for bacteria, this is often not the case for viruses detected in the routine laboratory [ ] . therefore, cost-effectiveness of ngs for many bacteria potentially follows from the replacement of conventional characterization methods, whereas for viruses ngs is considered as a tool providing additional complementary information without replacement of the existing methods. further, an important consideration is the added value of ngs for routine diagnostics. as long as ngs is more expensive than the conventional methods and when there is no direct benefit for the individual patient, it will not be used in routine. then the fields of application for surveillance purposes should be clearly defined to be able to justify the additional financial resources needed to perform wgs beside the diagnostic activities. to translate pathogen sequence data into truly useful and actionable information, it needs to be integrated with other types of information (i.e. clinical and epidemiological data). in belgium, most data end-users were concerned about the challenges encountered with the integration of pathogen sequence data with clinical and epidemiological data. indeed, the public health usability of any kind of lab results, including wgs data, is highly dependent on the cross-linkage with contextual epidemiological and clinical information [ , , ] . data integration is often hampered by the incomplete and/or unstandardized nature of the contextual data [ ] . the ongoing digitalization of health data such as laboratory and clinical records may represent an opportunity to review and upgrade traditional data collection processes for communicable disease surveillance. according to world health organization's (who) guidance on managing ethical issues in outbreaks [ ] , rapid data sharing is crucial during an unfolding health emergency. this suggests that pathogen sequence data should be rapidly and openly shared at the start of an outbreak, in many cases before scientific publication. however, many barriers for data sharing remain including authorship/attribution for publications, results dissemination, ethical considerations, data ownership, database access agreements, etc. [ ] . in our survey, practical barriers (lack of data standardization, poor data quality, missing metadata, etc.) seemed to be the major obstacles in belgium for sharing pathogen sequence data and associated metadata for public health purposes. participants mainly mentioned the lack of a central database and clear guidelines. this reflects a lack of information on the effective data sharing through eu-wide genomic surveillance and cross-border outbreak analysis systems managed by ecdc and efsa in support of the member states [ , , ] . finally, % of participants considered the priority to publication as a major bottleneck for sharing pathogen sequence data. publication priority is linked to the importance of guaranteeing reputational returns to research efforts [ , ] . the challenge here is to find a balanced arrangement that allows data sharing in real time and the acknowledgement of research work by giving to researchers who have been involved in data generation the possibility to use and publish their own results in priority. as the use of ngs shifts from research to routine laboratory practices, this data sharing barrier will slowly be alleviated. regarding expertise and availability of personnel, wet and dry lab experts were more concerned about the analysis of pathogen sequence data than the sequencing itself. as was mentioned in a review article of aarestrup et al. and documented in a recent european survey by revez et al., the most important limiting factor in many countries is the lack of access to bioinformatics expertise, especially when used as part of frontline diagnostics [ ] or national public health reference laboratory service [ ] . another point of discussion is the potential impact of ngs on the diagnostic microbiology pathway. traditionally, frontline clinical laboratories perform standard identification, antimicrobial susceptible testing and occasionally typing. isolates may then be referred to reference laboratories based on the need (e.g. diagnostic confirmation) or for surveillance purposes. these reference laboratories perform confirmation testing and advanced characterization. ngs was first implemented at the level of academic or reference laboratories, because of the need for investments, operational costs, and requirements for expertise [ ] while having limited added value for individual patient care. samples must be multiplexed (batching) for cost-effectiveness, which is easier to achieve in large reference laboratories with high volume of sample throughput [ , ] . however, processing delays may be present when samples are shipped to a reference center. these processing delays may result in longer turnaround times rendering this centralized approach inappropriate to support a fast response when needed. the reduced costs of sequencing facilitated the introduction of ngs technology to frontline clinical laboratories. this shift towards a decentralized use may reduce turnaround times, empower hospital-based microbiology, and strengthen local infection control efforts [ ] . this decentralized capacity will allow the inclusion of these data in the surveillance network coordinated by the epidemiologists what will compensate the reduced referral of isolates to reference centers. consequently, the implementation of ngs in routine labs is an important driver to reconsider the future role of nrcs. molecular typing for public health surveillance is undergoing a stepwise transition to ngs [ ] . current and future ngs activities represented in this national survey were mainly in the context of food-and waterborne outbreak detections and investigations, reflecting the priority for these diseases across europe and beyond [ , ] . several criteria should be considered in the process of integrating wgs in a routine laboratory setting [ ] in order to know in which situations and for which pathogens it is worthwhile to use ngs. identifying a set of key drivers that cover all aspects related to the implementation of ngs (utility and feasibility) can help to guide prioritization of pathogens and to efficiently allocate resources. clinical and/or public health significance of the pathogen was scored as the most important driver during the implementation of pathogen genomics in routine public health activities, followed by availability of expertise to generate, analyze and interpret wgs data, and priority of the pathogen with respect to preventing the spread of antimicrobial resistance. qualitative responses revealed the opinion of several participants that the assessment of the added value of new technologies for individual patient care is paramount. if pathogen genomics is routinely used to guide patient management (diagnosis and/or treatment options), the pathogen sequence data gathered for diagnostic purposes can be accumulated for public health activities [ ] . if there is no added value for routine diagnosis, the cost of wgs will have to be covered by limited public health budgets. as a limitation, the relatively low response rate induced a potential volunteer bias as those public health experts being more interested and/or experienced in the field could be more likely to participate in the survey. yet, % of the participants indicated that they were 'not at all' familiar with sequencing technologies and pathogen genomics. further, we noticed a possible underrepresentation of the food, animal and environmental field in comparison to the human field, as well as a low number of bioinformaticians in the survey. in addition, public health professionals from the belgian institute for health (sciensano) might be overrepresented. the majority of microbiologists participating in the survey are based in a nrc, emphasizing surveillance activities and hence less weight to routine diagnostics. given this potential imbalance, it is important to take into account the distribution of profiles within the study population while interpreting the results. however, it is difficult to ascertain the true underlying distribution of the different professional groups within the target population. another limitation of the study is that the specific terminology used in the questions may not have been uniformly understood or consistently interpreted by stakeholders with different professional backgrounds [ ] . public health professionals working in the field of infectious diseases in belgium were in general enthusiastic about public health agencies implementing pathogen genomics for the surveillance and control of infectious diseases. however, introducing genomic methods into public health practice is inevitably linked to the decrease in cost, the introduction in routine activities of frontline clinical labs, the identification of field applications in public health, and the necessary development of new competencies. the results of the survey confirm the need to increase genomic literacy by offering dedicated training opportunities among public health professionals, especially for the data end-users including epidemiologists, clinicians, and infection control practitioners, enabling them to critically assess the utility and feasibility of implementing pathogen genomics in their work activities. as such, those at the forefront (i.e. end-users) may act as "honest brokers" responsible for evaluating the added value of genomic application. in the end, the main driver for the advancement of pathogen genomics in public health practice depends on the added value of this information for the different clinical and public health needs. further, inter-disciplinary (between epidemiologists, microbiologists and bioinformaticians) and intersectoral (one health context) collaboration should be improved in the future to pool expertise and to ensure an integrated and cohesive system for the management of infectious diseases. in terms of feasibility, respondents in this survey were mainly concerned, like their peers in similar european surveys, about data integration, data sharing, and the cost of sequencing technologies. overall, this survey helps to better understand the perceived utility and feasibility of pathogen genomics according to public health professionals and can inform further guidance to facilitate its implementation in belgium. future challenges can be anticipated by performing a similar survey among public health experts based in a country that already progressed further in the process of implementing pathogen genomics within their public health surveillance system. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file . "questionnaire". description of data: "list of questions included in the online survey". additional file . "selection of target groups for the survey". description of data: "an overview of existing surveillance systems to identify all public health professionals who (would potentially) generate or use ngs data for the surveillance of infectious diseases based in different institutes and organizations in belgium." additional file . 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human, animal, food, feed and food/feed environmental samples in the joint ecdc-efsa molecular typing database data sharing in genomics -re-shaping scientific practice impact of food and water-borne diseases on european population health world health organization. who estimates of the global burden of foodborne diseases real-time analysis and visualization of pathogen sequence data publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank all study participants who took the time to complete the survey and provided valuable insights to reach to objectives of our study. these include the scientists from the belgian institute for health (sciensano) and the federal agency for the safety of the food chain (fasfc) working in infectious disease departments; physicians and infection control practitioners of the regional infectious disease control teams from the three regions in belgium; microbiologists from national reference centers (nrcs) and from sentinel laboratories; members from the belgian society of infection specialists and clinical microbiologists (bvikm), the belgian antibiotic policy coordination committee (bapcoc), the belgian infection control society (bics), and the belgian society for food microbiology (bsfm); clinicians, clinical biologists and hospital hygienists participating in sentinel surveillance networks; and public health experts within the ministry of health. we also would like to thank jérome ambroise, jimmy van den eynden, and boudewijn catry for pre-testing the survey, and vera cantaert, yves dupont, chloé wyndham-thomas, and karl mertens for their contributions in recruiting participants. this research was supported by the .be ready project financed by sciensano. the raw data analyzed during the current study are available from the corresponding author on reasonable request. the first page of the questionnaire explained the purpose of the research, the measures taken to protect respondents' confidentiality and the voluntary nature of participation. questionnaire respondents were asked to tick 'agree' at the start of the questionnaire to indicate that they consented to take part in the study and were willing to complete an anonymized questionnaire. ethics approval was deemed unnecessary according to national regulations (law of april ; http://www.ejustice.just.fgov.be/mopdf/ / / _ . pdf#page ), as participation was anonymous and no medical data were processed. not applicable. the authors declare that they have no competing interests. key: cord- - giy fow authors: martinez-martin, nadia title: technologies for proteome-wide discovery of extracellular host-pathogen interactions date: - - journal: j immunol res doi: . / / sha: doc_id: cord_uid: giy fow pathogens have evolved unique mechanisms to breach the cell surface barrier and manipulate the host immune response to establish a productive infection. proteins exposed to the extracellular environment, both cell surface-expressed receptors and secreted proteins, are essential targets for initial invasion and play key roles in pathogen recognition and subsequent immunoregulatory processes. the identification of the host and pathogen extracellular molecules and their interaction networks is fundamental to understanding tissue tropism and pathogenesis and to inform the development of therapeutic strategies. nevertheless, the characterization of the proteins that function in the host-pathogen interface has been challenging, largely due to the technical challenges associated with detection of extracellular protein interactions. this review discusses available technologies for the high throughput study of extracellular protein interactions between pathogens and their hosts, with a focus on mammalian viruses and bacteria. emerging work illustrates a rich landscape for extracellular host-pathogen interaction and points towards the evolution of multifunctional pathogen-encoded proteins. further development and application of technologies for genome-wide identification of extracellular protein interactions will be important in deciphering functional host-pathogen interaction networks, laying the foundation for development of novel therapeutics. the plasma membrane constitutes a critical biological interface between the cytosol and the extracellular environment of the cell, and consequently membrane-tethered proteins and secreted molecules (collectively termed extracellular proteins) are essential regulators of cellular communication. from high affinity cytokine-receptor interactions to low affinity cell-cell adhesion contacts, extracellular protein-protein interactions (eppis) are key for information processing and coordination of virtually all processes in a living organism. furthermore, given their fundamental functions and their accessibility to systemically delivered drugs, extracellular proteins are particularly suitable targets for therapeutic intervention. in fact, despite these proteins being encoded by approximately one-fourth of the human genes, at least two-thirds of the existing drugs target either secreted or membrane-bound proteins [ ] . thus, the elucidation of the eppi networks on a global scale has become crucial for the biomedical research. however, in spite of their relevance and abundance, eppis are remarkably underrepresented in available large-scale datasets. this discrepancy is due to the low sensitivity and limited compatibility of current high throughput technologies to detect extracellular interactions because of the unusual biochemical nature of the membrane proteins and the intractability of their binding partners [ ] [ ] [ ] . in particular, transmembrane domain-containing proteins are amphipathic, making it difficult to solubilize them in their native conformation, and often contain posttranslational modifications such as glycans and disulfide bonds, which are not properly added in common heterologous expression systems [ ] . in addition, interactions between cell surface proteins are often characterized by fast dissociation rates and therefore weak binding affinities, and in consequence well-established ppi methods such as yeast-two-hybrid or affinity purification-mass spectrometry (ap/ms) largely fail to detect these interactions. over the last decade, several innovative technologies have been developed to overcome the aforementioned technical challenges and allow for sensitive journal of immunology research detection of eppis [ , [ ] [ ] [ ] [ ] [ ] . nevertheless, the mapping of eppis remains a major challenge in biology. infectious diseases result in millions of deaths each year and therefore identifying new candidate targets for improved therapeutic development remains a pressing health concern. pathogens have evolved a myriad of elegant and often complex strategies to invade the host and commandeer host immune responses to allow pathogen replication, spread, and persistence in the infected organism. many cell surface molecules serve as entry receptors for initial host cell invasion, and concerted responses to the pathogenic challenge critically rely on cell functions mediated by receptors and secreted proteins. to allow host colonization, pathogens encode highly optimized protein modulators, in the form of secreted molecules or receptors expressed on the plasma membrane of the infected cells or the surface of the pathogen [ , ] . interactions between these proteins and extracellular host molecules form the foundation of communication between a host and a pathogen and play a vital role in the initiation and outcome of the infection [ , ] . characterizing host-pathogen eppi networks is therefore of utmost importance to gain a better understanding of the infection process and to inform the development of novel or improved therapeutic strategies. excellent studies on mapping hostpathogen interactions, particularly ms-based analysis of viral infection, have provided a wealth of insight into infectious diseases [ ] [ ] [ ] [ ] [ ] . nevertheless, similarly to host eppis, a significant hurdle to the elucidation of host-pathogen biology has been the shortage of datasets of extracellular interactions between host and pathogen proteins, partly due to the technical challenges that these proteins present. moreover, an additional consideration when studying pathogen-encoded molecules is that these proteins often lack any recognizable homology with any host molecules, thus precluding prediction of their functions [ , ] . robust methodologies that permit unbiased characterization of eppi in the absence of preexisting hypotheses are thus needed to elucidate the binding partners and molecular functions of most pathogenencoded molecules. excellent reviews have recently revisited the currently available technologies for proteome-wide eppi discovery [ , , [ ] [ ] [ ] . here we discuss the application of some of these technologies to the study of host-pathogen interaction and describe some of the major findings that have recently impacted research in the field of extracellular host-pathogen recognition. protein microarrays and functional genomics approaches are highlighted here as emerging techniques with unique potential for the elucidation of host-pathogen eppi networks at a genome-wide scale. classical biochemical and biophysical approaches are particularly suitable for detection of high affinity host-pathogen interactions, such as those mediated by a viral capsid protein and a host cell surface receptor, or a pathogen-encoded glycoprotein and a secreted host factor. typically, these approaches have relied on the utilization of recombinant pathogen proteins as baits to probe for host binding partners, followed by immunoprecipitation and ms, or biophysical techniques for analysis of ppi such as surface plasmon resonance (spr). spr requires prior knowledge of the possible interacting partners and is therefore unsuitable for unbiased ppi discovery, whereas immunoprecipitation and ms approaches usually fail to detect weak interactions, which often characterize eppis, particularly those that take place on the cell surface. notwithstanding, the identification of the receptors for some of the most prominent pathogens, such as the severe acute respiratory syndrome coronavirus (sars-cov) or the new world arenaviruses, was made utilizing standard immunoprecipitation techniques [ , ] . despite their inherent limitations, biochemical approaches continue to provide relevant insights into host-pathogen interactions, such as the discovery of the dipeptidyl peptidase (dpp ) as the receptor for the mers-cov just within a few months after the emergence of this virus [ ] . in addition, more recently kabanova and colleagues identified the cell surface receptor for the trimeric entry complex ghglgo encoded by human cytomegalovirus (hcmv) [ ] . several studies have shown that the ghglgo trimer is involved in the infection of fibroblasts, whereas the ghglul l pentameric complex is required for entry into endothelial, epithelial, and myeloid cells [ ] [ ] [ ] . in this work, both the trimeric and the pentameric cmv protein complexes were generated as recombinant products and used as baits to perform binding experiments on biotinylated cell surfaces, followed by immunoprecipitation and ms identification of bait-cell receptor complexes. using this approach, the authors identified the cell surface protein pdgfr as a high affinity receptor for the ghglgo trimer and demonstrated that this interaction was required for infection of fibroblasts. interestingly, in the case of the pentameric cmv complex, multiple bands were detected upon protein immunoprecipitation from epithelial cells, suggesting the existence of multiple receptors on these cells, which so far remain unknown [ ] (table ) . different biophysical techniques for detection of ppi, in particular spr, have also proven valuable in the field of host-pathogen interactions. spr offers the advantage of label-free, sensitive detection of interactions between a diversity of ligands in real time, thus allowing calculation of kinetic parameters. spr has been widely utilized to monitor antibody binding to a variety of pathogen antigens, information that has informed vaccine development [ ] [ ] [ ] . the spr technology has also been exploited for discovery of eppis. for example, viejo-borbolla and coworkers utilized spr to screen several secreted and membrane-expressed glycoproteins encoded by herpes simplex viruses (hsvs) for binding to chemoattractant cytokines (the chemokine family) and were able to identify a subset of human chemokines that bound to hsv glycoprotein g with high affinity [ ] . recently, day and colleagues utilized a combination of glycan arrays and spr and identified over host-bacterial glycan pairs characterized by a wide range of binding affinities, some of which participated in bacterial adherence to host cells in vitro, leading to the hypothesis that bacteria-host surface glycan interactions may mediate initial attachment to the target cell during infection [ ] . despite spr and related methods offering higher sensitivity for detection of transient biochemical and ms pdgfr identified as a high affinity cell surface receptor for the cmv ghglgo protein complex [ ] herpes simplex viruses (hsvs) biophysical secreted and plasma membrane-expressed glycoprotein g targets a specific set of human chemokines with high affinity [ ] human immunodeficiency virus type (hiv) monoclonal antibodies cd identified as the receptor for hiv infection of t cells [ , ] rhinovirus monoclonal antibodies icam- as the common entry receptor for most rhinovirus serotypes [ , ] plasmodium falciparum avexis basigin identified as the cell-surface receptor that mediates erythrocyte infection [ ] human adenoviruses extracellular human protein microarrays elucidation of the extracellular interactome of adenovirus-encoded immunomodulatory proteins [ ] pseudomonas aeruginosa extracellular pathogen protein microarrays (nappa) screening of patient sera against all p. aeruginosa extracellular proteins. proteins identified as potent antigens [ ] varicella zoster virus (vzv) extracellular pathogen protein microarrays (nappa) identification of extracellular viral proteins that promote humoral responses upon screening of the entire vzv proteome [ ] streptococcus extracellular pathogen protein microarrays identification of new streptococcal proteins that interact with fibronectin, fibrinogen, and c bp factors [ ] hepatitis delta virus lhdag antigen plasma membrane microarrays (mpa) candidate interactions identified between viral antigen and plasma membrane proteins [ ] simian virus (sv ) plasma membrane microarrays (mpa) candidate interactions between whole particles and plasma membrane proteins identified [ ] vaccinia virus (vacv) triceps (ms) candidate cell surface binding partners identified for vacv [ ] viral pathogens computational studies insights into global principles of virus-host ppi networks [ ] [ ] [ ] [ ] [ ] [ ] [ ] hepatitis c virus (hcv) cdna libraries cd , claudin- , and occludin as cell surface receptors and some of the players involved in hsv internalization [ ] [ ] [ ] adenovirus and coxsackievirus b monoclonal antibodies and cdna libraries car identified as a common entry receptor for adenovirus / and coxsackievirus b [ ] sindbis virus sirna screens nramp as cell surface receptor for entry into drosophila cells [ ] murine norovirus crispr/cas cd lf identified as a cell surface receptor that determines virus tropism [ ] bacterial distending toxins (e. coli) haploid cell screens sphingomyelin synthase and the putative g protein-coupled receptor tmem identified as toxin receptors [ , ] clostridium difficile and perfringens toxins haploid cell screens lipolysis-stimulated lipoprotein and the low-density lipoprotein receptor-related protein identified as the receptors for the bacterial toxins, respectively [ , ] ebola virus haploid cell screens hops proteins and the niemann-pick c (ncp ) transporter identified as endosomal receptors that mediate cytosol access [ ] lassa virus haploid cell screens lamp , lysosomal-associated membrane protein identified as an essential host factor mediating virus release to cytosol [ ] adeno-associated virus (aav) serotype haploid cell screens cell host factors identified, including heparin sulfate proteoglycan biosynthesis and intracellular transport genes. the immunoglobulin domain-containing transmembrane protein kiaa l identified as aav receptor [ ] journal of immunology research population genomics analysis p. falciparum ebl- protein binds to the erythrocyte receptor glycophorin b, a highly polymorphic gene in malaria-endemic regions [ ] streptococcus phage display bacterial proteins identified as potential fibronectin-binding proteins [ ] fusobacterium nucleatum transposon-based mutant libraries the bacterial protein fap binds to the receptor tigit and downregulates nk-mediated killing of tumor cells [ ] avexis, avidity-based extracellular interaction screen; nappa, nucleic-acid programmable protein array; mpa, microfluidic-based comprehensive protein array; crispr, clustered regularly interspaced short palindromic repeat; ms, mass spectrometry; ppi, protein-protein interaction. ppis than most biochemical approaches, these biophysical techniques have not been exploited for large-scale eppi discovery, possibly due to the low throughput of the available instrumentation and the overall difficulties for generation of the relevant protein libraries. these studies, among many others, have demonstrated the power of the classical biochemical and biophysical techniques for detection of host-pathogen interactions. nevertheless, these approaches require previous knowledge of the pathogen-encoded proteins responsible for binding and the ability to produce such proteins as recombinant reagents, which may be challenging, as exemplified by the production of hcmv entry complexes [ , ] . alternative methods have been utilized in those cases where there is no previous knowledge of the pathogen proteins required for interaction with the host cells. in this regard, the screening of large collections of monoclonal antibodies raised against membrane proteins has proven particularly useful to identify receptors that mediate viral entry. back in the early s, the discovery of cd as an entry receptor for the human immunodeficiency virus type (hiv) significantly impacted our understanding of viral pathogenesis and subsequent development of therapeutics [ , ] . in this case, the well-defined tropism of the virus determined the choice of over antibodies directed against human leukocyte differentiation antigens, of which only antibodies that recognized the surface receptor cd blocked viral infection [ ] . it is worth noting that similar monoclonal antibody screens have also been utilized for unbiased characterization of viral blockers. for example, colonno and colleagues performed a screen of more than , hybridomas from mice immunized with preparations of plasma membranes from human cells and were able to find one antibody that blocked rhinovirus binding to its cell surface receptor [ ] , identified as the intercellular cell adhesion molecule (icam- ) in subsequent studies [ ] . despite the undoubted importance of the biochemical and biophysical approaches to the study of host-pathogen interactions, the aforementioned limitations have motivated the development of alternative technologies for large-scale analysis of eppis. from the initial utilization of microarrays for detection of ppi over a decade ago, human proteome chips containing thousands of recombinant proteins have been generated, some of which are now commercially available. protein microarrays offer the unique advantage of requiring minimal consumption of protein reagents, fast readouts, and relatively more affordable instrumentation. typically, a fluorescently labeled or tagged protein of interest (the bait) is generated as a recombinant product and screened against all proteins in the array [ , ] . despite the increased availability of highcoverage protein arrays, very few are focused on extracellular proteins and therefore are not suitable for study of hostpathogen eppis. most existing microarray-based methodologies rely on multimerization of the bait protein for increased avidity and detection of weak eppis, mimicking the way these interactions occur in vivo, where proteins are arrayed in the crowded molecular environment of apposing plasma membranes. different multimerization strategies and protein microarray libraries have been developed and utilized for host-pathogen interaction discovery, some of which are described in more detail below. the wright lab developed a novel method for detection of low affinity eppis, termed avidity-based extracellular interaction screen (avexis) [ ] . in brief, avexis consists of the expression of the extracellular domain (ecd) of the bait of interest as a recombinant protein, which retains its binding properties while removing the insoluble transmembrane region of the protein. these ecds are tagged with a coiled-coil sequence from the rat cartilage oligomeric matrix protein to allow for pentamerization of the bait and therefore increased binding avidity, alongside a -lactamase tag for detection of baitprey interactions upon incubation with the colorimetric substrate nitrocefin. this multivalent strategy has been used for medium-scale screens, allowing detection of weak interactions between human receptors with low false-positive rates [ ] . notably, crosnier and colleagues applied avexis to search for the plasma membrane receptor responsible for plasmodium falciparum infection of erythrocytes [ ] . the authors compiled a library consisting of most secreted or cell surface-expressed proteins in erythrocytes and systematically assayed more than red blood cell proteins for binding to p. falciparum protein pfrh , a parasite protein essential for blood stage growth, expressed as an avexis pentameric bait. notably, the ok blood group antigen basigin was identified as a unique receptor for pfrh , and inhibition of this interaction was shown to be sufficient to block parasite invasion of the erythrocyte, findings that may importantly inform antimalarial therapies [ ] . in later studies, avexis was miniaturized making this approach compatible with the protein microarray format, thus permitting more comprehensive and lower resource-intensive screenings [ ] . although this technique should allow for high throughout and sensitive determination of eppis, this approach has not yet been applied to elucidation of pathogen-host interactions. over a decade ago, fueled by the recent completion of the human genome, genentech pioneered a significant effort to identify novel secreted or transmembrane domain-containing proteins, upon careful bioinformatics assessment and high throughput protein purification [ ] . these efforts resulted in the generation of a comprehensive human protein library, which was subsequently utilized to develop an extracellular protein microarray platform, consisting of over , secreted or single-transmembrane domain containing proteins [ ] . for the generation of this human protein library, secreted proteins or the ecd of single-transmembrane receptors were fused to different affinity tags and subsequently purified from cell culture supernatants by size-exclusion chromatography. mammalian cells or baculovirus-insect cells were preferentially used as expression systems, to maximize the likelihood of proper folding and glycosylation of the extracellular protein collection [ , ] . sds-page and multiangle laser light scattering were used to analyze noncovalent aggregation and ensure high-quality protein production. subsequently, the purified proteins were spotted on epoxysilane slides using a nanoprint arrayer, and protein immobilization on the microarray was determined by probing the slides with the relevant anti-tag antibodies [ ] . to enhance detection of low affinity interactions, a rapid method to assemble bait proteins (whose ecd was expressed as a fc tag-fusion protein) into multivalent complexes using fluorescently labeled protein a microbeads was developed. proof-of-concept assays showed high sensitivity for detection of weak eppis characterized by micromolar , a minimal off-target binding, and more than % true-positive to false-positive detection ratio [ , ] . over the years, this extracellular protein microarray has successfully identified counterreceptors for a number of human molecules, providing relevant insights into novel pathways that coordinate a multitude of cell functions [ ] [ ] [ ] . receptor discovery. recently, we applied this eppi discovery platform to the study of extracellular viral proteins ( figure ), with a focus on human adenovirus-(hadv-) encoded immunomodulatory proteins [ ] . despite the increasing relevance of hadv as both pathogens and therapeutic vectors, information on the interaction of these viruses with the host immune system remains scarce [ , ] . interestingly, the immunomodulatory proteins encoded by these viruses, termed e proteins, show substantial diversity in their ecds across and within viral species and constitute one of the most divergent regions of the hadv genome [ , ] . given this striking variability, the e proteins have been suggested to play a role in viral tropism and pathogenesis, yet the functions of virtually all e proteins have remained unknown [ ] . in our study, we took advantage of such unique variability to evaluate the effect of viral immunomodulatory protein diversity in extracellular host targeting. screening of a substantial number of e proteins encoded by different hadv species using the extracellular protein microarray platform allowed identification of over novel virus-host interactions encompassing viral species, which were fully validated by orthogonal methods [ ] . these findings revealed significant diversity in extracellular host targeting and, moreover, allowed identification of semiconserved host targets, pointing towards specific human receptors that may represent previously unrecognized hubs for viral perturbation. furthermore, most of the e immunomodulators were identified as multifunctional proteins, suggesting that viruses have evolved proteins capable of interfering with several cellular functions, a strategy consistent with the optimization of limited genomic resources. such economic targeting has been often observed in intracellular targeting [ ] [ ] [ ] , but so far few examples of widespread targeting in the extracellular environment have been reported [ , [ ] [ ] [ ] [ ] , let alone a global elucidation of eppi networks, in part due to the technical challenges associated with eppi detection. remarkably, many of the hadv e proteins preferentially interacted with host receptors that exert known or predicted inhibitory functions during the immune response (as defined by the presence of intracellular immunoreceptor tyrosinebased inhibitory motif, itim), including lilrb [ , ] , lair [ ] , and mpzl [ ] , suggesting previously unrecognized strategies of immunosuppression that may be utilized by other human pathogens [ ] . moreover, several of the receptors identified as targets for the viral proteins in this study (including the prominent cell surface molecule cd ) do not have known counterreceptors in the host, supporting the longstanding hypothesis that pathogen molecules drive the evolution of immune receptors and in many instances may represent the most relevant modulators of host receptor function [ ] [ ] [ ] . in summary, such unbiased, microarraybased study of immunomodulatory proteins represented the first large-scale analysis of the ppi landscape of a collection of extracellular immunomodulators encoded by viruses. future investigation of other pathogen-encoded molecules using similar extracellular protein microarrays will likely shape our understanding of the pathogen imprint in our immune system. microarrays. one of the main limitations of any protein microarray platform is the lower protein coverage relative to other genome-wide methods for ppi identification, due to the costs and difficulties for generation of comprehensive protein libraries to be deposited onto the microarrays. in an attempt to address this caveat, ramachandran and colleagues developed a method called nucleic-acid programmable protein array (nappa), in which dnas are directly deposited onto the array followed by protein synthesis in situ using an in vitro transcription and translation (ivtt) system, thus avoiding the need for protein purification [ ] . although this promising approach has proven superior in generating transmembrane-containing molecules as soluble proteins, it still remains to be systematically addressed if the extracellular human proteins produced in this manner present the folding and posttranslational modifications necessary for protein functionality. nevertheless, emerging data support the utility of nappa as a useful tool for the study of bacterial proteins. for example, montor and colleagues used a bioinformatics approach to predict the pseudomonas aeruginosa proteins that reside in the outer membrane of the bacteria or are secreted to the extracellular environment of the infected cell [ ] . in this work, the authors utilized the nappa approach to screen all predicted extracellular gene products for interaction with sera from cystic fibrosis patients, where p. aeruginosa establish a life-threatening lung infection. from bacterial proteins initially selected, proteins were recognized by antibodies in the sera, indicating that these bacterial proteins represent major antigens that trigger adaptive immune responses in humans. interestingly, robust antibody responses against three previously uncharacterized proteins were detected, suggesting this approach could help identify new extracellular proteins that exert unknown functions during the infection [ ] . these results confirmed the utility of the microarrays to detect immune responses against membrane proteins encoded by pathogens, and supported the use of this methodology for diagnosis applications. in this regard, several groups have developed microarrays composed of pathogen-encoded proteins [ , [ ] [ ] [ ] [ ] [ ] . such pathogen protein arrays have so far being exploited mainly for diagnosis purposes, to allow screening of antibodies present in patient sera for binding to extracellular bacterial or viral antigens on the array. nevertheless, their inherent high throughput and compatibility with multivalent bait approaches makes them a powerful tool for eppi discovery. for example, margarit et al. developed a streptococcus microarray to find novel microbial proteins capable of binding to the human proteins fibronectin, fibrinogen, and c bp and were able to identify a set of streptococcal proteins that interacted with these factors [ ] . nevertheless, despite such pathogen protein-based arrays offering great promise, this methodology remains to be systematically analyzed for eppi discovery. more recently, yu and colleagues applied nappa technology in combination with the halotag-halo ligand detection system to elucidate the interaction network of two effector proteins (sidm and lida) encoded by legionella pneumophila, a highly pathogenic bacteria that is the causative agent of legionnaire's pneumonia [ ] . similarly to many journal of immunology research pathogen proteins, these virulence factors lack significant homology to host molecules therefore complicating the assessment of their host targets and biological functions. in this work, the bacterial proteins of interest were tagged with a halotag, a modified haloalkane dehalogenase that covalently binds to synthetic halo-ligands (haloalkanes) that can be fluorescently labeled, thus allowing more robust detection of bait protein binding to interactors present on the array. in this study, more than , human proteins were expressed on the nappa array using different ivtt techniques, leading to identification of and binding partners for the lida and sidm effectors, respectively, most of them experimentally verified by pull-down [ ] . although this study focused on identification of intracellular ppi, the applicability of the nappa-halotag technology for eppi determination should be explored in the future. moreover, bait multimerization strategies should be implemented in order to make this approach more suitable for detection of transient ppis. related to the nappa technology, glick and colleagues recently built a miniaturized platform focused on human membrane proteins. by integrating the microfluidics technology, protein microarrays, and an ivtt system, this group built a new device named microfluidic-based comprehensive human membrane protein array (mpa) [ ] . a notable improvement introduced by these investigators was the addition of microsomal membranes to the ivtt system to allow for improved folding and posttranslational modifications in plasma membrane proteins, both common limitations of ivtt systems. in this work, a library of , human genes encoding for membrane proteins was built and subsequently utilized to screen the large-form delta antigen (l-hdag) encoded by the hepatitis delta virus (hda) and whole viral particles of the simian virus (sv ), a nonenveloped human pathogen. proof-of-concept assays showed encouraging results, with over % true-positive rate within a small set of proteins with known interactors and, more importantly, indicated the feasibility of this approach for expression of multitransmembrane-containing proteins, a protein type that has proven challenging given their high hydrophobicity. the mpa screens identified and over interactions for sv particles and l-hdag viral protein, respectively, and around interactions were validated by coimmunoprecipitation or protein-fragment complementation assays [ ] . to our knowledge, this is the first study to assess eppis using a comprehensive human protein library and whole viral particles (sv ) as baits, a valuable approach that may provide important insights into pathogen tropism, alongside a molecular explanation for the cell surface receptors engaged by the pathogen. further utilization of this platform followed by a more systematic analysis of the candidate hits, including nonspecific binder determination, will be needed to assess the overall performance of the mpa technology. regardless, this platform provides an extended version of the nappa approach that focuses on mammalian eppis and may therefore provide relevant insights into extracellular hostpathogen interactions. the protein microarrays have represented one of the most fruitful approaches for unbiased determination of eppis, including host-pathogen interactions. nevertheless, one of the main limitations of this technology is the need to generate comprehensive libraries, a process that is resource consuming and often not available to many researchers [ , ] . consequently, although some of the available arrays were designed to cover a significant fraction of the human proteome, any discoveries made using these platforms are limited to the proteins present in each array. the current libraries are likely to continue expanding alongside innovative approaches to facilitate sensitive detection of eppi using protein microarray formats. over the last decade, ms-based technologies have emerged as a versatile, powerful approach to decipher many aspects of the human proteome, including the characterization of protein complexes. excellent reviews on current ms-based technologies, recent improvements, and future prospects for elucidation of ppi networks are available [ ] [ ] [ ] [ ] . in this review, we briefly discuss the applications of some of these techniques to the study of extracellular host-pathogen interactions. proteins and interacting partners. the proteins expressed on the surface of pathogens mediate functions necessary for survival, replication, immunoevasion, and transmission and therefore are logical candidates for therapeutic and vaccine design. however, the study of the surface proteome in pathogens, particularly in bacteria is constrained by the fact that commonly used prediction algorithms fail to correctly predict the location of several proteins [ , [ ] [ ] [ ] . despite the characterization of the extracellular proteins and their interactions still representing the achilles heel of most proteomics methods, ms has emerged as an invaluable approach to characterize the protein composition of plasma membranes [ ] . to date, several studies have exploited ms-based techniques to gain insights into the extracellular protein composition of bacterial pathogens [ ] [ ] [ ] [ ] . for example, palmer and colleagues studied the surface proteome of the tick-borne intracellular pathogen anaplasma marginale (rickettsiales: anaplasmataceae) using liquid chromatography and tandem ms [ ] . interestingly, the authors found that the surface proteome of a. marginale isolated from tick cells, despite being less complex than that of bacteria isolated from human erythrocytes, contained a novel protein, which the authors hypothesized to play a function in human cell invasion in spite of its human counterreceptor remaining uncharacterized. this interesting observation suggests a remodeling of the bacteria surface proteome during the transition between mammalian and arthropod hosts, an aspect of the infection that could be targeted to block transmission. similarly, several studies have pursued the identification of the proteins present in viral particles utilizing ms. although these analyses suffer from several drawbacks associated with membrane protein characterization, particularly the poor solubility of these proteins and the low abundance of many plasma membrane proteins, these studies have revealed a complex composition for most of the viruses studied, alongside incorporation of many host proteins in the virions, in most cases with undetermined functions [ ] [ ] [ ] . an interesting observation from some of the studies referred above is the fact that certain bacterial proteins, predicted cytoplasmic by consensus, can be found in the extracellular environment of the cell, where they may play alternative functions. in fact, the number of proteins that are secreted through noncanonical signal sequence pathways is increasingly appreciated [ , , , ] . little is known about these bacterial proteins originally described as cytosolic proteins but capable of exerting functions on the cell surface, which some authors have named moonlight proteins, in reference to their potential to exert multiple functions [ ] . there is emerging evidence that protein moonlighting contributes to virulence of important bacterial pathogens including staphylococcus aureus or mycobacterium tuberculosis, sometimes in fascinating ways. for example, m. tuberculosis is known to encode two molecular chaperones, cpn . and cpn . , which function as modulators of myeloid cells among other regulatory functions [ ] . despite these chaperones being by definition cytosolic, cnp . has been detected in significant amounts on the bacterial surface, and either recombinant cnp . or antibodies against this protein efficiently block binding of m. tuberculosis to macrophages [ ] , through a potential interaction with the receptor cd [ ] . in addition, the protein dnak, a hsp related protein encoded by m. tuberculosis, can locate to the bacterial surface and functionally interact with cd [ ] and with the hiv coreceptor ccr [ ] . notably, dnak appears to block hiv binding to ccr in vitro, an interesting observation given the co-occurrence of m. tuberculosis and hiv infection [ ] . although a more detailed revision is out of the scope of this review, bacterial protein moonlighting, excellently revisited by henderson and martin [ ] , is a thought-provoking phenomenon that suggests a much more complex extracellular landscape than anticipated. moreover, such protein moonlighting is in line with the hypothesis that pathogens have evolved multifunctional proteins as a prominent strategy for efficient use of limited genomic resources [ , ] . another ms-based approach that holds great promise for host-pathogen eppi detection is the recently developed triceps [ ] . triceps is a chemoproteomic reagent that consists of three moieties, one that binds the ligand of interest through its amino groups, a second one that binds glycosylated receptors on the cell surface, and a biotin tag for purifying the receptor peptides for subsequent identification by ms. notably, in the initial description of the method, triceps was successfully applied to the identification of receptors for extracellular ligands of diverse nature, such as secreted glycoproteins, small peptide ligands for g proteincoupled receptors, and therapeutic antibodies. importantly, this approach has also been utilized to study cell surface molecules targeted by vaccinia virus (vacv). interestingly, the analysis of vacv binding to hela cells revealed seven candidate binding partners, including the previously identified receptors axl, chondroitin sulfate proteoglycan , and laminin binding protein dystroglycan . further, downregulation of five out of the seven candidates using short interfering rna reduced vacv infection by - %, supporting the functionality of the interactions identified, at least in vitro [ ] . although this technology is still developing and no studies on other pathogens have been published yet, future triceps-based studies promise relevant insights into pathogen interaction with distinct components of the cell surface. as an addendum to the vast amount of knowledge acquired using ms approaches and some of the additional methodologies discussed in this review, bioinformatics offers an in silico systems biology approach that reveals a global perspective on host-pathogen interactions. advances in computation have been fundamental to dissect the complex datasets generated in many genome-wide msbased studies and have enabled the reconstruction of largescale host-pathogens ppi networks, providing fundamental insights into viral disease and hence host biology [ - , - , ] . although the computational tools available for analysis of large datasets, in some cases developed in association with some of the high throughput screens mentioned in this review, certainly deserve a focused chapter, a couple of observations are specially notable. commonly observed in these studies is that the intracellular viral effectors preferentially target host proteins that act as hubs (proteins with many interacting partners) or bottlenecks (proteins central to many pathways in the network) [ , , ] . for example, dyer and colleagues built a network of host-pathogen ppi by integrating published information from pathogens [ ] . supporting previous findings, this analysis indicated that pathogen-encoded proteins preferentially interfere with host molecules that control critical cellular processes, such as cell death or nuclear transportation, possibly as a strategy to maximize control of the host machinery given limited genomic resources. interestingly, this study highlighted a small set of extracellular host proteins recurrently targeted by several of the viral and bacterial pathogens analyzed, including cell surface receptors such as vegfr /kdr and collagen, possibly indicating previously unrecognized roles in the immune response against pathogens. although informative, the analysis performed by dyer and collaborators was skewed towards viruses, with a prominent enrichment in hiv strains [ ] . more extensive analyses encompassing other human viruses and bacterial pathogens may reveal general strategies of immunomodulation and potential human targets suitable to therapeutic intervention. interestingly, increasing evidence suggests that virus-host interactions are governed by principles distinct to those that dictate within-host interactions [ , , , , ] . notably, detailed analyses carried out by the xia group highlighted significant differences between virushost and within-host (also called endogenous) interactions, such as the tendency of viral proteins to compete with host proteins for binding to a given receptor in the absence of sequence similarity with the host counterpart or the observation that viral molecules have evolved multiple short linear motifs capable of mediating a number of diverse interactions [ , ] , features that are consistent with the multifunctional capabilities of some pathogen-encoded proteins [ , , , [ ] [ ] [ ] [ ] ] . altogether, bioinformatics analysis of virushost interactions suggest that virus-mediated targeting of host proteins is characterized by signatures of pleiotropy, economy, and convergent evolution, conclusions that are supported by emerging experimental data. followed by thorough biological experimentation such computationalbased systems biology approaches will provide a unique tool to help decipher basic global principles of pathogenhost interaction and may reveal novel eppis amenable to therapeutic intervention. alongside protein microarrays-based technologies, ms, and computational analysis, the explosion of the functional genomics field in the last years has revolved the avenues to study pathogen interactions with their hosts, often in high throughput. in brief, genetic screens comprise gain-of-function and loss-of-function strategies, represented by complementary dna (cdna) libraries and rna-interference-(rnai-) based approaches, respectively. these methods were developed more than two decades ago and have been widely utilized by the scientific community, providing fundamental insights into the infection process. in particular, the cdna libraries have proven extremely successful in identifying viral receptors through a gainof-function approach, upon transduction of the cdna library from a susceptible cell line into nonpermissive cell lines. the use of cdna libraries is not reviewed in detail here in the interest of a more comprehensive revision of relative newer genomics-based approaches, such as the clustered regularly interspaced short palindromic repeat/ crispr-associated protein (crispr/cas ) or the haploid cell screens. nevertheless, these libraries have represented one of the most significant technologies to further our understanding of the pathogen-host interaction. for example, early studies made use of cdna libraries to shed light on the complex mechanism exploited by hepatitis c virus for initial invasion of the cell [ ] [ ] [ ] , identified car as a common receptor for adenovirus and coxsackievirus b [ ] , and were instrumental to identify slam and pvr as a receptors for measles and poliovirus, respectively [ , ] . in turn, the rnai technology has yielded significant insights into virus-host interactions, such as the identification of the ion transporter nramp as the receptor for the mosquito-borne sindbis virus colonization of drosophila cells [ ] . the main power of the rnai technology is that it allows high throughput genome-wide screens and therefore potential identification of essential factors that play roles in different aspects of the pathogen life cycle, including initial interaction with the host cell. although rnai screens have provided tremendous insights into host-pathogen interactions and remain widely utilized [ ] , inefficient gene depletion and off-target effects are important limitations of this methodology [ ] . technology. the increasingly popular crispr/cas technology overcomes some of the caveats often associated with genetic manipulation and holds enormous promise for genome editing and downstream applications, including host-pathogen interaction discovery [ ] . although still early days, high throughput crispr/cas screens for genome-wide studies have already displayed remarkable results, with high levels of genomic modification, hit confirmation, and strong phenotypic effects [ ] . the development of the crispr/cas technology has undoubtedly transformed the functional genetic analysis in mammals. recent studies have applied the crispr/cas technology to ablate expression of previously identified receptors for viral entry, such as the hiv coreceptors cxcr and ccr , leading to resistance to infection in primary cells [ , ] . an interesting additional application of crispr/cas is the direct editing of viral genes important for viral fitness. this approach has recently been used to target hsv- , cmv, and epstein-barr virus (ebv) essential genes, leading to a significant decrease of viral replication [ ] . these studies suggest the potential use of crispr/cas as an innovative therapeutic strategy, as aspect that will surely be further explored in the near future. another prominent example published recently is the identification of host factors that confer susceptibility to the evolutionary related type iii secretion systems, t ss and t ss , encoded by vibrio parahaemolyticus [ ] . the t sss are highly complex nanomachines utilized by gramnegative pathogens to inject a variable repertoire of virulence factors into the cytosol of the eukaryotic cells, enabling pathogen adhesion and internalization of modulation of host processes. interestingly, using genome-wide crispr/cas screens, sulfation and fucosylation of cell surface components were identified as host determinants of t ss -and t ss mediated cytotoxicity, respectively. the authors hypothesized that interactions between sulfated cell surface molecules such as host proteoglycans and bacterial adhesins act as facilitators of t ss activity, whereas fucosylated glycans on the surface may serve as receptors for t ss components necessary for insertion of the complex in the host membrane [ ] . the crispr/cas approach has just started to reveal its power as a tool for unbiased identification of novel eppis, elegantly exemplified by the identification of cd lf as the cell surface receptor for noroviruses, which, strikingly, was identified as the main determinant for the tropism of the murine norovirus [ ] . further optimization of this technology will unequivocally signify a tremendous advance for the discovery of extracellular host-pathogen ppis, the processes underlying host-pathogen interactions and its possible therapeutic applications. haploid cells, in turn, allow the study of recessive phenotypes that can be masked in diploid cells, due to the difficulties of creating true genetic knockouts in mammalian cells. despite yeast being a useful tool due to the simplicity of obtaining relevant mutants at its haploid life stage, the majority of human pathogens do not replicate in yeast therefore limiting the applicability of this approach [ ] . in recent years, human haploid cells have been increasingly utilized for genome-wide loss-of-function genetic screens using insertional mutagenesis [ , , ] . in initial studies, carette and colleagues took advantage of the kbm cell line, a derivative of the chronic myeloid leukemia cell line (cml) with a haploid karyotype except for chromosome [ ] . using gene-trap retroviruses for efficient insertional mutagenesis, the authors generated a genomewide collection of null mutants for most nonessential genes [ ] . this approach was successfully utilized to identify host factors essential for the functions of the distending toxins or cdts, potent virulence factors secreted by a number of pathogenic bacteria. in particular, mutagenized kbm cells were treated with escherichia coli-derived cdts and resistant clones were isolated, leading to identification of insertions in the sphingomyelin synthase and the putative g protein-coupled receptor tmem , suggesting that this molecule may serve as a surface receptor for the toxin [ , ] . similar haploid screens have identified novel receptors for a number of bacterial toxins, including the lipolysis-stimulated lipoprotein receptor for the clostridium difficile transferase [ ] , or the low-density lipoprotein receptor-related protein as a host receptor of the clostridium perfringens tpel toxin [ ] . in a later study, carette et al. generated a kbm -derived cell line named hap , haploid for all chromosomes [ ] . similarly to previous studies, the authors used the retroviral gene-trap approach to mutagenize hap cells followed by deep sequencing to map more than . insertions. in this study, using a replication competent vesicular stomatitis virus (vsv) carrying the ebola virus glycoprotein, a previously unknown entry receptor for ebola virus was identified. notably, these haploid cell screens identified six members of the hops complex, proteins known to play functions in endosomal/lysosomal trafficking, as well as the niemann-pick c (npc ) transporter as the most prominent hit of the assay. it is worth noting that npc is not a surface molecule but rather an endosomal receptor. these findings led the authors to propose a novel mechanism of entry by which ebola virus is internalized into the endocytic pathway, followed by endosome maturation and cleavage of the surface glycoprotein of the virus. endosome fusion, mediated by the hops complex, would allow interaction with ncp containing endosomes, triggering fusion and release of the viral genome into the cytosol. multiple cell surface receptors can lead to internalization of the ebola virus into the endocytic pathway [ ] ; such redundancy in receptor usage likely explains why these receptors were not identified in the haploid cell screen [ ] . notably, independent studies have confirmed that ncp acts an intracellular receptor for ebola, including a chemical screen approach, a study showing ncp dependence for infection of otherwise nonsusceptible cells, and more recently the elucidation of the crystal structure of this receptor bound to the ebola virus glycoprotein [ ] [ ] [ ] . interestingly, after the aforementioned haploid genetic screens identified ncp as a noncanonical entry receptor (given its intracellular localization), other filoviruses have been shown to take advantage of this receptor [ ] . the relevance of this intriguing mechanism of viral entry is further reinforced by recent work on lassa virus, an old world arenavirus that, similarly to ebola virus, causes severe to fatal hemorrhagic disease in humans [ , ] . a genome-wide haploid screen using vsv pseudotyped with lassa glycoprotein was performed in order to identify host factors essential for viral entry. although -dystroglycan (dag ) was long recognized as the cell surface receptor for lassa virus, additional factors were suspected, given the observation that certain dag -expressing cells are resistant to infection. the authors elegantly demonstrated that at a neutral ph, the lassa virus glycoprotein was bound to dag , whereas upon exposure to lower ph (resembling the lysosome environment), a receptor switch occurred leading to strong association with the lysosomal-associated membrane protein (lamp ) [ ] . thus, similarly to ebola virus, in the model suggested the virus would be incorporated into the endocytic pathway after interaction with its surface receptor dag , followed by increasingly acidic conditions that would result in interaction with lamp in the lysosomal membrane, triggering membrane fusion and release of the virus in the cytosol [ ] . more recently, pillay and colleagues applied the haploid cell screening approach to the identification of host factors essential for the adeno-associated virus (aav) serotype infection, one of the leading vectors for virus-based genes therapies [ ] . notably, the most significantly enriched gene in these screens was kiaa l, a poorly characterized type i immunoglobulin domain-containing transmembrane protein named hereafter as the aav receptor. among the host factors identified as hits, many were implicated in heparin sulfate proteoglycan biosynthesis as well as a number of proteins that participate in intracellular transport processes. aav is known to attach to the cells using heparin sulfate proteoglycans and hijacks endosomal trafficking to travel to the nucleus upon invasion of the cell; thus the authors hypothesized that these additional factors may influence virus tropism [ ] . altogether, these studies elegantly demonstrate the power of genome-wide screens in human haploid cells and the power of this approach to study virus-host interactions. future studies should further assess the applicability of this method for general detection of interactions that take place at the pathogen-plasma membrane interface. it will also be important to generate additional haploid cell lines, in order to broaden the range of pathogens and pathogen-derived molecules that can be studied using these genetic tools. in this regard, a number of haploid cell lines have been generated in mammals [ ] , unique tools to elucidate the basic aspects of human genetics. discovery. pathogens are among the most intriguing and prominent drivers of human evolution. humans have adapted to the pressure imposed by microorganisms through genomic diversification, particularly through variation of genes involved in immune system function, constantly challenged by the rapidly coevolving pathogen genomes. the advent of new technologies such as next-generation sequencing and the computational tools associated have opened new avenues for the study of human genetics, making it possible to evaluate the contribution of genetic diversity to susceptibility to infection at the genomic level. the emergence of datasets of genomic variation in multiple human populations, as well as pathogen genomes, allows detection of signatures of selection, which can be exploited to identify genes with major roles in immunity (for an excellent review see [ ] ). remarkably, the cell surface-expressed receptors are among the most polymorphic gene families in mammals, subjected to strong positive selection and rapid evolution, in many instances possibly driven by pathogen molecules that remain unknown [ , [ ] [ ] [ ] . polymorphisms in receptors and immunomodulatory genes contribute to the natural susceptibility of different individuals to infection [ ] [ ] [ ] , as illustrated by protection against hiv infection in individuals carrying homozygous polymorphisms in the viral coreceptor ccr [ ] . the identification and further study of genes under positive selection may represent a mainstream approach to dissect novel genes involved in disease and hostpathogen interaction. a notable example is the identification of glycophorin b as the erythrocyte receptor for p. falciparum protein ebl- through examination of highly polymorphic genes in populations from malaria-endemic regions [ ] . further population genetics studies promise key insights into novel immunological mechanisms and have the potential to provide molecular details that will ultimately help design effective therapies. in addition to these encouraging technologies, the generation of phagemic libraries has also represented an important tool for deciphering ppis, in this case between particular binding partners and the whole genome of specific pathogens [ ] [ ] [ ] . typically, pathogen-encoded molecules are expressed as fusions with phage envelope proteins, a method known as phage display that has been widely exploited to identify peptides with specific binding properties. for example, beckmann and colleagues built a phage display library to identify novel group b streptococci proteins capable of mediating adherence to fibronectin, a major component of the extracellular matrix often exploited for colonization of the host [ ] . from this analysis, the authors identified genes with homology to known bacterial adhesin proteins, genes involved in virulence, transport, or metabolic processes, along with genes with uncharacterized functions. interestingly, one of these genes showed significant homology with the scpb protein, a peptidase found in other streptococci that inactivates the member of the human complement system c a, suggesting that this bacterial molecule acts as a bifunctional protein, similarly to other examples of multifunctional proteins discussed above [ ] . more recently, a transposon-based insertion-inactivation mutant library was elegantly utilized to identify a bacterial protein capable of targeting the surface receptor tigit, an inhibitory molecule present in natural killer (nk) cells and t cells [ ] . fusobacterium nucleatum is a common oral bacterium that has been associated with colon adenocarcinoma and rheumatoid arthritis among other malignancies. in this study, gur and colleagues showed that different strains of f. nucleatum blocked nk-mediated killing of human tumors. using a library of f. nucleatum mutants, the authors identified fap as the bacterial protein that directly interacted with tigit, leading to inhibition of nk cytotoxicity and downregulation tumor-infiltrating t lymphocytes activation. immunoevasion is a hallmark of cancer; however whether members of the microbiome found within the tumor provide cancer cells with immunoregulatory properties has remained a major matter of debate [ ] . these interesting findings suggest that f. nucleatum present in the tumor niche may enhance tumor escape by inactivating nk-mediated killing upon interaction of the fusobacterial fap with the inhibitory receptor tigit. of note, transposon-based mutant libraries are readily available for other pathogenic bacteria and have been successfully applied to identification of bacterial genes implicated in bacterial physiology [ ] [ ] [ ] . it would be of interest to employ these libraries for unbiased identification of eppis. notably, as mentioned above, we found that hadv immunomodulators preferentially target other immunoreceptors that, similarly to tigit, also play inhibitory functions [ ] , suggesting this might represent a common immunosuppressive tactic evolved by pathogens. in fact, there is emerging evidence suggesting that may be the case, as a number of extracellular proteins from unrelated human pathogens have already been shown to target diverse immune receptors with inhibitory functions [ , , , , , ] . further exploration of inhibitory receptor targeting by other pathogens warrants exciting biological discoveries. deciphering the human genome made possible the categorization of genes that encode for the human secretome; now, the challenge of the postgenomic era is to annotate the functions of those genes and their expression patterns during health and disease. a lot has been learnt from painstaking, highly focused experiments using classical biochemistry. in recent years, the impressive technological advances in proteomics, functional genomics, and computation have revolved our understanding of cell communication and function and have collectively created a versatile platform to enable biological discoveries, from mechanistic explorations to big data and systems biology analysis. nevertheless our understanding of the molecules and mechanisms of extracellular immunomodulation and pathogen invasion remains remarkably limited. extracellular ppis between host-and pathogen-encoded molecules orchestrate an enormous diversity of cellular processes, from initial colonization of the target cell to subsequent immune responses. the elucidation of these extracellular interactomes is integral to understanding the molecular basis of infection and will guide the development of more efficient or innovative therapeutics. improvements in proteomics and genomics approaches have exponentially increased our understanding of how pathogens, particularly viruses, modulate the intracellular environment of the cell. concomitantly, we and several other groups have implemented technologies directed towards elucidation of extracellular interactomes [ , , , , , ] , which have begun to reveal fundamental principles of extracellular journal of immunology research host-pathogen interactions. notably, recent studies have revealed extensive eppi networks in model organisms such as drosophila or zebrafish [ , ] . these undertakings predict that, similarly to intracellular ppis, extracellular networks will be highly connected, with secreted and plasma membrane-expressed proteins having multiple binding partners. however, as discussed in this review, the identification of the host factors and in many cases the pathogen molecules that mediate eppis have largely defied molecular identification, in part due to the technical difficulties inherent to the study of these extracellular proteins. the elucidation of the global principles dictating extracellular pathogen-host ppis will require a coordinated effort to bring together the areas of biology and technology. there are now considerable opportunities for integrating multiple disciplines for eppi discovery, particularly proteomics and crispr/cas genome-wide screens, which should be powered by commensurable advances in bioinformatics and computation for big data analysis. the integration of orthogonal datasets coming from multiple "omics" approaches will be advantageous for elucidating the intricacies of the host-pathogen extracellular interactomes and will further enhance the rational identification of novel therapeutic targets by uncovering fundamental principles of biology. the journey from classical biochemical studies towards a systems biology approach has just begun and promises major technological breakthroughs and surprising biological findings. the development of powerful technologies for eppi discovery has already illuminated sophisticated and sometimes unexpected molecular mechanisms by which pathogens interact with their hosts and has provided unique opportunities to increase our understanding of viral and bacterial pathogenesis. further improvement of these technologies is warranted and will surely provide the scientific community with unprecedented insights into pathogen and host biology. how many drug targets are there? large-scale screening for novel low-affinity extracellular protein interactions highthroughput identification of transient extracellular protein interactions signal initiation in biological systems: the properties and detection of transient extracellular protein interactions overcoming key technological challenges in using mass spectrometry for mapping cell surfaces in tissues interactive proteomics research technologies: recent applications and advances surface plasmon resonance for high-throughput ligand screening of membrane-bound proteins strategies for membrane interaction proteomics: no mass spectrometry 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surface-exposed proteins of ehrlichia chaffeensis composition of the surface proteome of anaplasma marginale and its role in protective immunity induced by outer membrane immunization identification of proteins associated with murine cytomegalovirus virions proteins of purified epstein-barr virus identification of proteins in human cytomegalovirus (hcmv) particles: the hcmv proteome fibroblast growth factor secretion is mediated by a non-cleaved aminoterminal signal sequence secretion of fgf- requires an uncleaved bipartite signal sequence bacterial virulence in the moonlight: multitasking bacterial moonlighting proteins are virulence determinants in infectious disease mycobacterium tuberculosis cpn . and dnak are located on the bacterial surface, where cpn . facilitates efficient bacterial association with macrophages mycobacterium tuberculosis employs cpn . as an adhesin that binds cd on the macrophage surface cd is a cellular receptor mediating mycobacterial heat shock protein stimulation of cc-chemokines dendritic cell stimulation by mycobacterial hsp is mediated through ccr inhibition of human immunodeficiency virus type infection of human cd + t cells by microbial hsp and the peptide epitope - epstein-barr virus and virus human protein interaction maps structural principles within the human-virus protein-protein interaction network slam (cdw ) is a cellular receptor for measles virus cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily what have rnai screens taught us about viral-host interactions? expression profiling reveals off-target gene regulation by rnai the crispr/cas bacterial immune system cleaves bacteriophage and plasmid dna high-throughput functional genomics using crispr-cas genome editing of cxcr by crispr/cas confers cells resistant to hiv- infection a cas ribonucleoprotein platform for functional genetic studies of hiv-host interactions in primary human t cells crispr/cas -mediated genome editing of herpesviruses limits productive and latent infections crispr/cas screens reveal requirements for host cell sulfation and fucosylation in bacterial type iii secretion system-mediated cytotoxicity how yeast can be used as a genetic platform to explore virus-host interactions: from "omics" to functional studies isolation and characterization of a near-haploid human cell line ebola virus entry: a curious and complex series of events small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection ebola virus entry requires the host-programmed recognition of an intracellular receptor ebola viral glycoprotein bound to its endosomal receptor niemann-pick c cell entry by a novel european filovirus requires host endosomal cysteine proteases and niemann-pick c molecular mechanism for lamp recognition by lassa virus derivation and differentiation of haploid human embryonic stem cells population genetic tools for dissecting innate immunity in humans the natural selection of herpesviruses and virus-specific nk cell receptors pathogen-driven selection in the human genome positive selection on the human genome genome-wide scans provide evidence journal of immunology research for positive selection of genes implicated in lassa fever toll-like receptor cascade and gene polymorphism in host-pathogen interaction in lyme disease single nucleotide polymorphisms in pathogen recognition receptor genes are associated with susceptibility to meningococcal meningitis in a pediatric cohort homozygous defect in hiv- coreceptor accounts for resistance of some multiply-exposed individuals to hiv- infection cloning of ligand-binding domains of bacterial receptors by phage display m-like proteins of streptococcus dysgalactiae a fibrinogen-binding protein of staphylococcus epidermidis cancer and the microbiota the neglected intrinsic resistome of bacterial pathogens sequence-defined transposon mutant library of burkholderia thailandensis comprehensive transposon mutant library of pseudomonas aeruginosa leukocyte immunoglobulin-like receptor b is critical for antibodydependent dengue hsv- infection through inhibitory receptor, pilralpha the mammalianmembrane two-hybrid assay (mamth) for probing membrane-protein interactions in human cells the author is grateful to lino gonzalez, jennie lill, erik verschueren, and yvonne franke for critical reading of the manuscript and insightful comments and suggestions. avidity-based extracellular interaction screen aav:adeno-associated virus car:coxsackievirus receptor cmv:cytomegalovirus crispr/cas : clustered regularly interspaced short palindromic repeat/crispr-associated protein ebv:epstein-barr virus ecd: extracellular domain eppi:extracellular protein-protein interaction hadv:human adenoviruses hcv:hepatitis c virus hiv:human immunodeficiency virus hsv:herpes simplex virus itim:immunoreceptor tyrosine-based inhibitory motif ivtt:in vitro transcription/translation lair :leukocyte-associated immunoreceptor like lilrb : leukocyte immunoglobulin-like receptor mpa:microfluidic-based comprehensive human membrane protein array mpzl :myelin protein zero-like protein ms:mass spectrometry nappa:nucleic-acid programmable protein array ncp :niemann-pick c pdgfra: platelet-derived growth factor receptor alpha ppi:protein-protein interaction pvr:poliovirus receptor sars-cov: severe acute respiratory syndrome coronavirus sv :simian virus slam:signaling-lymphocytic activation molecule spr:surface plasmon resonance t ss: type iii secretion system vavc:vaccinia virus. the author declares that they have no competing interests. key: cord- - auz gi authors: aliakbar ahovan, zahra; hashemi, ali; de plano, laura maria; gholipourmalekabadi, mazaher; seifalian, alexander title: bacteriophage based biosensors: trends, outcomes and challenges date: - - journal: nanomaterials (basel) doi: . /nano sha: doc_id: cord_uid: auz gi foodborne pathogens are one of the main concerns in public health, which can have a serious impact on community health and health care systems. contamination of foods by bacterial pathogens (such as staphylococcus aureus, streptococci, legionella pneumophila, escherichia coli, campylobacter jejuni and salmonella typhimurium) results in human infection. a typical example is the current issue with coronavirus, which has the potential for foodborne transmission and ruling out such concerns is often difficult. although, the possible dissemination of such viruses via the food chain has been raised. standard bacterial detection methods require several hours or even days to obtain the results, and the delay may result in food poisoning to eventuate. conventional biochemical and microbiological tests are expensive, complex, time-consuming and not always reliable. therefore, there are urgent demands to develop simple, cheap, quick, sensitive, specific and reliable tests for the detection of these pathogens in foods. recent advances in smart materials, nanomaterials and biomolecular modeling have been a quantum leap in the development of biosensors in overcoming the limitations of a conventional standard laboratory assay. this research aimed to critically review bacteriophage-based biosensors, used for the detection of foodborne pathogens, as well as their trends, outcomes and challenges are discussed. the future perspective in the use of simple and cheap biosensors is in the development of lab-on-chips, and its availability in every household to test the quality of their food. over the last several decades, the prevalence of food poisoning has become a major world health issue. this may be due to the increase in intercontinental transportation of food. dr. oliver, at the university of tennessee, tn, usa, an expert in the foodborne pathogens, has recently reported these problems in detail [ ] . the centers for disease control and prevention (cdc), atlanta, ga, usa assesses more than million cases of the disease annually, are due to foodborne and waterborne pathogens infections [ ] . the most common causes of food poisoning are diarrhea, nausea, vomiting the biosensors are simple and rapid devices, based on organic probes, which are able to identify biological analytes, such as microorganisms, viruses, and biomolecules [ ] . the biosensor is commonly composed of a biologically active sensitive element (biological element) and an electronic part (sensor or transducer). the operating principles are as follows: "the biological element" interacts with the substrate to be analyzed and a transduction system; "the sensor" converts the biochemical response into an electrical signal. this signal digitized into a numeric value, giving the final information. biosensors can be classified according to the transduction technologies used. in the past decade, different groups of transductions have been introduced; these have led to the formation of three main classes: optical, mass-based and electrochemical transducers ( figure ) [ ] . the front part of the biosensor, the probes, plays a major role in the identification and detection of the pathogens. these give biosensors the ability to analyze a wide range of complex samples in various fields, including the diagnosis of food pathogens, clinical diagnosis and environmental monitoring. biosensors have been used and played as a useful tool for the direct detection of the pathogen in the factories during the food processed food. unlike microbiological and molecular methods, biosensors can detect the pathogen immediately and accurately, and this helps to detect the contamination level and the type of food contamination [ ] . the biosensors are simple and rapid devices, based on organic probes, which are able to identify biological analytes, such as microorganisms, viruses, and biomolecules [ ] . the biosensor is commonly composed of a biologically active sensitive element (biological element) and an electronic part (sensor or transducer). the operating principles are as follows: "the biological element" interacts with the substrate to be analyzed and a transduction system; "the sensor" converts the biochemical response into an electrical signal. this signal digitized into a numeric value, giving the final information. biosensors can be classified according to the transduction technologies used. in the past decade, different groups of transductions have been introduced; these have led to the formation of three main classes: optical, mass-based and electrochemical transducers ( figure ) [ ] . the front part of the biosensor, the probes, plays a major role in the identification and detection of the pathogens. these give biosensors the ability to analyze a wide range of complex samples in various fields, including the diagnosis of food pathogens, clinical diagnosis and environmental monitoring. biosensors have been used and played as a useful tool for the direct detection of the pathogen in the factories during the food processed food. unlike microbiological and molecular methods, biosensors can detect the pathogen immediately and accurately, and this helps to detect the contamination level and the type of food contamination [ ] . the biosensors are simple and rapid devices, based on organic probes, which are able to identify biological analytes, such as microorganisms, viruses, and biomolecules [ ] . the biosensor is commonly composed of a biologically active sensitive element (biological element) and an electronic part (sensor or transducer). the operating principles are as follows: "the biological element" interacts with the substrate to be analyzed and a transduction system; "the sensor" converts the biochemical response into an electrical signal. this signal digitized into a numeric value, giving the final information. biosensors can be classified according to the transduction technologies used. in the past decade, different groups of transductions have been introduced; these have led to the formation of three main classes: optical, mass-based and electrochemical transducers ( figure ) [ ] . the front part of the biosensor, the probes, plays a major role in the identification and detection of the pathogens. these give biosensors the ability to analyze a wide range of complex samples in various fields, including the diagnosis of food pathogens, clinical diagnosis and environmental monitoring. biosensors have been used and played as a useful tool for the direct detection of the pathogen in the factories during the food processed food. unlike microbiological and molecular methods, biosensors can detect the pathogen immediately and accurately, and this helps to detect the contamination level and the type of food contamination [ ] . bio-probes are the most important components of biosensors because responsible to bind and identify the analytes targets. the two main characteristics of bio-probes are specificity and high affinity for the analyte. figure shows the different components of a diagnostic sensor. the common bio-probe, used in biosensor devices, are antibodies, proteins, dna/rna aptamers and carbohydrates. however, many of these are usually susceptible to environmental conditions and the need for laborious immobilization methods for binding on the sensor substrate. only recent studies employed bacteriophages (or phage) or derived-phage, such as phage receptor binding proteins (rbps) and most lately phage-display peptides (pdps) like a valid alternative of standard bio-probes. nanomaterials , , x for peer review of bio-probes are the most important components of biosensors because responsible to bind and identify the analytes targets. the two main characteristics of bio-probes are specificity and high affinity for the analyte. figure shows the different components of a diagnostic sensor. the common bio-probe, used in biosensor devices, are antibodies, proteins, dna/rna aptamers and carbohydrates. however, many of these are usually susceptible to environmental conditions and the need for laborious immobilization methods for binding on the sensor substrate. only recent studies employed bacteriophages (or phage) or derived-phage, such as phage receptor binding proteins (rbps) and most lately phage-display peptides (pdps) like a valid alternative of standard bio-probes. bacteriophages (phages) are viruses ubiquitous in all environments, including soil, food ground and surface water. they specifically bind the host bacteria and inject their dna to begin the multiplication and propagation of mature virions [ ] . phages can propagate new virions in two ways: lytic or lysogenic cycle ( figure ). lytic cycle: phages attach to their host bacteria, insert their dna and take over the host machinery to dissemination new virions that lyse the bacteria and infect a new host (lytic phages). the host cells provide molecular blocks and enzymes that are needed to multiply the phage genomes bacteriophages (phages) are viruses ubiquitous in all environments, including soil, food ground and surface water. they specifically bind the host bacteria and inject their dna to begin the multiplication and propagation of mature virions [ ] . phages can propagate new virions in two ways: lytic or lysogenic cycle (figure ). the external environment can infect and destroy other adjacent bacteria. in a wide range of bacterial species, lytic phage produce infection specifically [ ] . lysogenic cycle: integrate their genome into the host dna, remain latent until they arouse for replication and dissemination (lysogenic phage). a prophage, contained in the host cell, is called lysogen. while, this dna in the host genome is called a prophage, the lysogenic cycle can continue indefinitely, except in some cases, such as adverse conditions of the environment and bacteria exposure to stress [ ] . consequently, the bacteriophage used like bio-probe in biosensor devises offer several advantages, such as ( ) specificity to host bacteria, consequently efficient bacteria screening [ ] , ( ) easy to generate mass quantities of progeny phages, due to their short replication time, ( ) ability to tolerate critical conditions, such as organic solvents and large range of ph and temperature [ ] . it is important to understand the characteristics of phages, such as the physical and chemical properties, in order to design the biosensor platform able to bind the phage without losing their ability to recognize the bacteria target. phages can bind to the gold surface trough van der waals bonding, hydrophobic bonding and covalent bonding between the gold surface and the amine and thiol groups. this strategy is also used in surface plasmon resonance (spr) sensor and quartz crystal microbalance (qcm) sensor using phage as a probe for pathogen detection [ ] . nevertheless, lytic cycle: phages attach to their host bacteria, insert their dna and take over the host machinery to dissemination new virions that lyse the bacteria and infect a new host (lytic phages). the host cells provide molecular blocks and enzymes that are needed to multiply the phage genomes and generate a progeny phage. phages produce several endolysins and holin lysis proteins inside the host cell. holins are small proteins in the cytoplasmic membrane of host bacteria that lead to peptidoglycan cell wall bacteria lysis by endolysins and release of produced phages. these phages in the external environment can infect and destroy other adjacent bacteria. in a wide range of bacterial species, lytic phage produce infection specifically [ ] . lysogenic cycle: integrate their genome into the host dna, remain latent until they arouse for replication and dissemination (lysogenic phage). a prophage, contained in the host cell, is called lysogen. while, this dna in the host genome is called a prophage, the lysogenic cycle can continue indefinitely, except in some cases, such as adverse conditions of the environment and bacteria exposure to stress [ ] . consequently, the bacteriophage used like bio-probe in biosensor devises offer several advantages, such as ( ) specificity to host bacteria, consequently efficient bacteria screening [ ] , ( ) easy to generate mass quantities of progeny phages, due to their short replication time, ( ) ability to tolerate critical conditions, such as organic solvents and large range of ph and temperature [ ] . it is important to understand the characteristics of phages, such as the physical and chemical properties, in order to design the biosensor platform able to bind the phage without losing their ability to recognize the bacteria target. phages can bind to the gold surface trough van der waals bonding, hydrophobic bonding and covalent bonding between the gold surface and the amine and thiol groups. this strategy is also used in surface plasmon resonance (spr) sensor and quartz crystal microbalance (qcm) sensor using phage as a probe for pathogen detection [ ] . nevertheless, optimal condition of physical absorption has been found for lytic phages able to detect s. aureus by spr with detection limit~ cfu/ml − , or using magnetic elastic sensors [ ] capable of detecting salmonella (detection limit~ cfu/ml − ) in milk and fat-free [ ] . however, physical absorption has several limitations, such as non-specific and weak bonding between phage and sensor surface, leading to desorption during the analytic detection and low surface coverage of deposited phages [ ] . however, the chemical bond gives greater stability to the system. one of the main factors in these methods is the creation of a strong chemical bond between the phages and biosensor surfaces, which leads to the development of a stable capture system. in this case, the purity of the suspension of phages, such as their chemical properties, are important to be aware of before performing any chemical reactions [ ] . gervais et al. developed an oriented immobilization of t phages using the commonly recognized streptavidin-biotin reaction. the tail-phage was able to detect e. coli through the expression of biotin in the t head, and using the streptavidin-coated gold surface [ ] . moreover, the t phage has been used in the functionalization of magnetic nanobeads, and is used to capture and concentrate e. coli from milk by mortari et al. the successive lysis of the bacteria-binding phage leads to the analysis of endoplasmic material through impedance, with the potential detection limit of cfu/chamber in min [ ] . however, the size of the phage particles can be a limitation in any biosensor surfaces, such as in nanoscale devises. the phages have an enzyme activity relative to their bacterial host [ ] . this kind of enzymatic activity on the biosensor surface causes contradictory signals that contribute to the efficiency of the pathogen diagnosis. moreover, results recommend that whole phage bound on sensor platforms miss their bacterial binding capacity upon drying [ ] . as the phages fall on the biosensor surface after upon drying, it likely makes their tail fibers unable to bind to their bacterial host. in order to overcome these limitations, engineered phages or derived phages have been applied in the development of biosensors devises, as discussed later in this review article. with novel advances in the field of genetic engineering, new phage probes have been designed to increase the capability or to overcome the limits of the wild-type phages. recently, n. wisuthiphaet and co-workers used engineered bacteriophage, phage t -alp, which expresses alkaline phosphatase to detect e. coli in beverage samples. after infection of the host bacteria, the overexpression of alkaline phosphatase provides, after the mission of the substrate elf- , fluorescence imaging results in high-detection sensitivity of bacteria for gram in h [ ] . in these cases, the specificity of phages, combined with engineered techniques, have permitted the easy and rapid identification of the target. reporter phages -a reporter phage is an engineered phage to produce a signal upon infection of target bacteria. this group of phages detects only live and functional cells because when the phages infect the target bacteria, they activate the cells' machinery to produce a readily detectable signal, which indicates that the target is both present and viable. this technique is an advanced method of bacteria typing [ ] . phage lambda-based cloning vectors bearing a functional bacterial bioluminescence lux operon was used to improve the reporter phage method. here, lux operon is expressed in the process of infection by phage in the host cell as part of the phage gene [ ] . "lux-" or "gfp-" expressing phages were successfully used in salmonella enterica and pathogenic e. coli o :h detection, as well as for detection of listeria and mycobacteria [ ] . in a study by zhang et al., the design of a phage that contains luciferase nanoluc (nluc), reporter phage, was able to detect e. coli :h , about cfu/ml, in a food sample by bioluminescence over h [ ] . the benefit is that the phage specificity and the strength of the analysis of the phage remove the essential for purification or lengthy sample preparation [ ] . moreover, recently has been shown a proof-of-concept drinking water diagnostic assay for low-cost, rapid and sensitive detection of e. coli using t reporter phage. t was engineered to express the luceriferase, nanoluc (nluc), after the fusion of crystalline cellulose. this novel chimeric reporter allow the detection of < cfu ml − e. coli in h from ml water sample [ ] . however, some structural disadvantages associated with this group of phages also have to be stated. to study the structure of these phages there is a need to have accurate genetic information as their structure is labor-intensive. the capacity of the phage capsid naturally creates limitations for the amount of genetic material that can be presented in the phage genome. general ways to exhibit reporter genes contain recombination, transposition, direct cloning and homologous [ ]. the phage display technology must be considered separately. since, it was first reported in in science, phage display technology has evolved as a powerful method for discovery of antigen-specific peptides. a researcher found that bacteriophages can be genetically manipulated through the incorporation of exogenous (poly) peptides into its coat proteins, making a peptides phage library. the filamentous phages, such as fd and m , are the most commonly used vector to create random peptide display libraries. filamentous phages appear as a flexible rod-shaped structure, with a total length of nm and a diameter of . nm. its circular genome, of - bases, is enclosed in a coat composed of up of copies of major coat protein (pviii), - copies each of the minor proteins (piii, pvi and pvii, pix), on the two ends of the phage particle. in particular, the foreign peptides (length from to amino acids) have been displayed fused to surface exposed n-terminus of all coat proteins. however, the most commonly-used coat proteins for phage display are piii and pviii. the phage display libraries consisted of up to different variants of phage particles with the linear random peptides [ ] . this allows ligands to be screened that have a high affinity towards the desired antigen, such as eucariotic cells, bacteria or inorganic material. the phage clones were found to bind, with high affinity, the target, which can be used as a bio-probes in biosensor devises. in this way, for all possible targets, it is possible to find a "phage-clone" able to recognize them. the advantage are robust, stable and resistant to temperature variations and hard ph conditions, such as the wild-type. moreover, the phage clone presents several copies of peptide of interest, thus increasing the avidity of the specific target binding, compared to the classic wild-type [ ] . it has introduced several opportunities in various fields, including vaccine design, targeted drug delivery and antiviral studies [ ] . these characteristics make phages an attractive choice as probes for developing biosensors in several fields [ , ] . recently bacteriophage receptor binding proteins (rbps) have been developed into tools that make use of their high specificity [ ] . rbps offer several advantages compared to other elements for example antibodies, including ligand specificity, greater stability, and affinity even against carbohydrate epitopes [ ] . the rapid detection of pathogens prevents disease progression and spread. rbps are a practical technology for bacterial diagnosis. the phage receptor proteins determine the phage-host characteristics and are also considered as suitable as biological agents. one of the advantages of the rbp is that, without lysis, the bacterial components of the bacterial cell proliferate through the agglutination and release of the dna of the pathogen. rbp often has good resistance to environmental factors, such as temperature and ph [ ] . poshtiban and coworkers anchored the phage nctc presenting rbp protein gp on magnetic beads. the modified seeds were used to extract campylobacter from milk and chicken samples. in samples infected with cfu/ml of campylobacter cells through rt-pcr, it was a prominent improvement measure of more than % in h. to confirm the specific adsorption of phage to campylobacter, s. typhimurium was used as a negative control [ ] . more effort is needed to reach commercial biosensors, given that the initial experimental results indicate that rbps are capable of being used as diagnostic agents in diagnosing pathogens. in recent years, biosensors have been developed as new diagnostic methods to minimize the limitations of common pathogen detection methods. in phage-based biosensors, bacteriophage is nanomaterials , , of attached to the sensor surface, and consequently, it can detect the pathogen in the sample [ ] . the main advantages of this method are sensitive, accuracy and reliably. bacteriophage-based biosensors have been used for direct diagnosis of pathogens in fresh foods such as milk [ , ] , and water [ ] food matrices [ ] . the table summarizes the advantages of biosensors in pathogenesis. table . ideal properties incorporated for microbial biosensor. near real-time response desired (< h desirable) no reagent addition needed operator should be automated and require minimal operator skills strain selectivity ability to distinguish an individual bacterial strain from other strains of the same species ability to detect single bacteria in a reasonably small sample volume (from to ml) ability to distinguish individual bacterial species in the presence of other microorganisms or cell. should be compatible with the transduction principle and resist nonspecific binding robustness both mechanical and chemical stability is required monitoring direct, without pre-enrichment viable cell count should discriminate between live and dead cells optical-based bioassay systems are used for rapid diagnosis of pathogens in different experimental conditions, with high sensitivity and compatibility. optical biosensors are used as more suitable diagnostic systems for the detection of pathogens. detection in optical biosensors is based on the variations induced in the light properties, such as refractive index, wavelength and polarization [ ] . currently, biacore biosensor and spreeta biosensor as commercial optical biosensors are used for the detection of foodborne pathogens. biacore biosensor is used for detecting l. monocytogenes, with sensitivity of × cells/ml in milk. salmonella enteritidis and e. coli o :h , and s. typhimurium and s. enteritidis, can be successfully detected by biacore . and spreeta biosensors, respectively [ ] . wavelengths-based biosensors enable real-time monitoring of biomolecular interactions by evaluating the kinetics and affinity of the interactions [ ] . planar optical waveguides contain an optically transparent guiding layer with a refractive index, which is higher than the substrate layers. the optical waveguide geometry provides an excellent surface for functionalization and pattering of different recognition elements, and enable the simultaneous detection of multiple analytes in a single waveguide transducer [ ] . optical techniques are separated into two major subclasses, fluorescence and label-free, based on their working platform. the most used technique for these biosensors is the measure of the changed fluorescence, in absorbance or luminescence, of the biosensor surface after analyte recognition. furthermore, one of the advantages of the optical biosensor design of label-free biosensors is the detection of a specific and susceptible bacterial pathogens [ ] . the most employed techniques for bacterial detection are fluorescence/phosphorescence spectrometry, surface plasmon resonance (spr), and bio/chemiluminescence. for the first time in , spr was used to detect a spectrum of materials [ ] . spr biosensors are optical sensors that use special surface plasmon-polaritons-electromagnetic waves, to monitor the interactions between an analyte in solution and a recognition layer, such as recognize molecules and phages, immobilized on the spr sensor surface. spr biosensing, as a spectroscopic technique, enable the quantitative and real-time detection of the binding events without labeling the interacting molecules. the optical system is comprised of an optical surface, a light emitting diode (led), a glass prism and a photodiode array. the molecular interactions at the surface cause changes in the refractive index, leading to changes in the spr angle of the reflected light. the photodiode array detects spr angle changes, and expresses the signals as a response unit (ru). ru is directly proportional to the total mass of the bound ligands. the chips in these sensors usually contains a gold surface functionalized with specific biorecognition elements by chemical bounds [ ] . advanced spr biosensors have been designed to detect pathogens using a variety of bio-probes in diagnosis of pathogens such as antibodies [ , ] , bacteriophages [ ] and lectins [ ] . bacteriophages are designed as diagnostic probes for specific detection of pathogens at the spr level. for example, singh et al. used immobilized engineered tail spike proteins derived from the p bacteriophage onto gold surfaces using thiol-chemistry, in order to selective real-time analytical detection of salmonella with the sensitivity of cfu/ml − [ ] . this technique successfully detects e. coli o :h , methicillin-resistant s. aureus (mrsa) [ ] , s. aureus, e. coli k [ ] and hepatitis b virus (hbv) [ ] . full-length recombinant det phage tail proteins (det t) are recently used in novel spr devise for detection of salmonella enterica serovar typhimurium. det t is covalently immobilized on gold-coated surfaces by amine-coupling, and can specifically bind to s. typhimurium. rapid detection (~ min) of × − cfu/ml s. typhimurium in water and % apple juice was observed by this biosensor [ ] . in table several microorganisms that have been detected using this technique are summarized. the plasmon waveguide resonance (pwr) spectroscopy, as a relatively new biophysical method, has the structures, which can couple high sensitivity of the spr sensors and the small resonance width of the dielectric wg sensors. the pwr consists a glass substrate, a thin metallic layer, and a dielectric layer on the top of the metallic layer. the metallic layer plays a very important role in exciting the dielectric waveguide modes (transverse magnetic (tm), and transverse electric (te). as an example, optical metal clad leaky waveguide (mclw) sensor can detect bacillus subtilis var. niger using index changes, scattering and fluorescence from bacterial spores bond to immobilized antibody [ ] . bioluminescence through the oxidation of organic compounds (luciferin), due to the enzyme luciferase, produces visible light in the living organisms. commonly in marine environments, some bacteria, including vibrio strains, are widely and abundantly used as luminescent organisms. the atp bioluminescence tests are a sensitive, fast and simple ways for bacterial detection. in this method, the bacterial cell is lysed and releases intracellular atp which is measured by luciferase bioluminescence reaction. the problem of this method is the low specificity in both adenylate kinase (ak) and atp diagnosis. wu et al. showed that the rate of the release of ak from the bacterial cell depends on the growth stage, phage type infection time and type of bacteria [ ] . in this way, the fluorescent combination with bacteriophage plays a role in the diagnosis of pathogens. in this method, fluorescent blended bacteriophages are involved in detecting and binding to the host bacteria. phage-bacteria is discovered using flow cytometry or epifluorescent filter technique. the average sensitivity reported so far is around cfu/ml − for flow cytometric and around - cfu/ml − for epifluorescent microscopy detection [ , ] . goodridge et al. combined this method with immunomagnetic separation. in this way they could be able to detect to cfu/ml − e. coli o : h in synthetic milk after an enrichment [ ] phase for h and cfu/ml − of e.coli o :h in broth [ ] . this method is also used to identify bacterial toxins [ ] . goldman et al. displayed a practical phage to select a -per peptide that could bind to staphylococcal enterotoxin b (seb), which cause food toxication. they could detect low . ng of seb per sample in a fluorescence-based immunoassay using a labeled seb-binding phage [ ] . amperometric biosensors are based on measuring the flow generated by oxidation or reduction in response to analyte bio-receptor reactions. in this case, a bio-receptor is usually an enzyme, such as glucose oxidase, horseradish peroxidase (hrp) and alkaline phosphatase (ap) [ ] . in general, this technology includes a thin plate of gold or platinum or carbon. the main advantage of these biosensors is that they are simple and easy to use, and at the same time, highly sensitive. the limitation of this method is low specificity due to interference with active inhibitors, and then the signal inaccuracies. in this method, a quantification of coliform e. coli k- with intermediary phages and intracellular release of bacterial enzymes, such as d-galactosidase and carbon oxides can be investigated [ ] . this sensor detected cfu/ ml of bacteria from the sample, but need to pre-incubation phase. neufeld et al. combined the phage typing technique with amperometric for the specific detection of mycobacterium smegmatis, e. coli k , and bacillus cereus [ ] . the basis of these sensors is that through phage infection, it causes bacterial leakage and the spread of intracellular bacterial content, including the enzyme. enzymatic activity is measurable in a specific substrate. the recent advanced reports, nanoparticle transducers were used to reduce the limitation of electrochemical biosensors. the gold nanoparticle is one of the most common nanoparticles used in mri, biosensors and targeting drug delivery for treating brain diseases, significantly increased electrodes sensitivity to the detection of pathogens. xu et al. designed micro-gold electrodes with phage t for the detection of e. coli. the sensitivity of this biosensor is in the range of . × - . × cfu/ml bacteria [ ] . table are summarized in several nanoparticles that have been used for detecting microorganisms. electrochemical impedance spectroscopy (eis) is a powerful technique in detecting the electrochemical system. functional sinusoidal in the system measures the changes in the electrical impedance in the medium. the analysis is carried out based on changes in capacitance, conductance, and impedance. the capacity is often reduced due to the process of microbial metabolism and impedance decreases [ ] . here, bacteriophages are used as diagnostic probes to detect pathogens at the electrode surface. in a study, the impedance was reduced as bacterial concentration enhanced, which is contrary to normal attachment of entire cells on eis, a promising electrochemical biosensor. bacteria can be detected by capturing electrons on the electrode surface. in eis electrochemical sensors with bacteriophage, as a probe, are used to detect bacteria, trough catching the bacteria target the immobilized phages on electrodes, functional groups [ , ] . the main cause of this is the activity of lytic phages on bacteria, which causes the intracellular content to drop out and decrease the conductivity. e. coli was easily detected in pure culture media or inoculated samples in a range between - cfu ml − using this method [ ] . webster et al. extend an impedimetric microelectrode array biosensor bacteriophage-based for the detection of bacteria. the results have shown that decreased the width and gap of an electrode and using the working solution with lower relative dielectric permittivity can enhance the sensitivity of impedimetric biosensors for pathogenic bacteria [ ] . graphene is wonder materials with superior properties and using graphene a screen-printed graphene electrode (spe) for the detection of s. arlettae. specific lytic phage against s. arlettae was immobilized on the sensor surface for quantitative analysis of the bacterial cells and capturing bacteria using eis biosensor. accordingly, the increase in the concentration of bacteria ( - × cfu/ml) leads to an increased quantity of charge transfer resistance (r ct ). the limit of detection was defined as around cfu/ml [ ] . in these techniques, phages are widely used as bio-probes to detect pathogens. phages are specific for host bacteria and have different characteristics, including that they are easy to amplify and cheap to produce, are resistant to temperature and ph-degradation, as well as organic solvents [ ] [ ] [ ] [ ] . moreover, cloning of engineered phage, derived from phage display libraries, make it able to develop new phages with on demand binding unites on their surfaces. this makes the phages a very promising candidate for biosensor applications [ ] . optimization of phage size, expression of binding units on phage's surface for specific binding to bacteria, are of the main troubleshooting in development of new phage-based biosensors. biosensors have already demonstrated huge potential in many fields, including detection of viruses and diseases in patients, as well as in the identification of food pathogens. many of these devices are still at laboratories' experimental bases and translation to the commercial product has been slow. the main problem, includes a signal-to-noise ratio, caused by the separation of signals from bacteria, due to the unwanted signal "noise" from the samples. therefore, sensitivity and repeatability are the major problems in the biosensors. high specificity is one of the main features of biosensors in diagnosis assisted by sensitivity, without spending time for the pre-enrichment phase. here, a summary of bacteriophage-based bio-probes and their protein receptors are discussed. at the same specificity of the antibodies and nucleic acid, the phage probes are strong, resistant and cost-effective. recent efforts have led to advances in methods in which phage, based on its chemical properties, is positioned on the surface of the sensor and provides a consistent and stable surface that results in a widespread diagnosis. although, phage-based and rbp-based systems have improved food quality control, it is still an emerging system. significant advances have been made in this area and there is a clear and promising future. finally advances in cutting edge r and d on nanomaterials and smart materials, including recent work on graphene-based biosensors [ , ] , will speed-up development and translate to commercialization. the current situation with coronavirus is a warning to all on how important of fast and reliable detection of viruses in reducing mortality. the authors declare that they have no competing interests. foodborne pathogens in milk and the dairy farm environment: food safety and public health implications estimating health care-associated infections and deaths in us hospitals chemically immobilized t -bacteriophage for specific escherichia coli detection using surface plasmon resonance climate change and food safety: a review detection and enumeration of microorganisms in ready-to-eat foods, ready-to-cook foods and fresh-cut produce in korea recent advances in bacteriophage based biosensors for food-borne pathogen detection 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salmonella typhimurium in chicken carcass specific detection of campylobacter jejuni using the bacteriophage nctc receptor binding protein as a probe development of a surface plasmon resonance biosensor for the identification of campylobacter jejuni specific assays for bacteria using phage mediated release of adenylate kinase influence of phage population on the phage-mediated bioluminescent adenylate kinase (ak) assay for detection of bacteria a high density microelectrode array biosensor for detection of e. coli o : h affinity-selected filamentous bacteriophage as a probe for acoustic wave biodetectors of salmonella typhimurium a qcm immunosensor for salmonella detection with simultaneous measurements of resonant frequency and motional resistance isolation, characterization of bacteriophages specific to microlunatus phosphovorus and their application for rapid host detection fluorescently labeled virus probes show that natural virus populations can control the structure of marine microbial communities development and characterization of a fluorescent-bacteriophage assay for detection of escherichia coli o : h phage-displayed peptides as biosensor reagents glucose biosensors: years of advances and challenges combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria use of high-affinity cell wall-binding domains of bacteriophage endolysins for immobilization and separation of bacterial cells bacteriophage-based biosorbents coupled with bioluminescent atp assay for rapid concentration and detection of escherichia coli diagnostic bioluminescent phage for detection of yersinia pestis bacteriophage-modified microarrays for the direct impedimetric detection of bacteria magnetically-assisted impedimetric detection of bacteria using phage-modified carbon microarrays impedimetric detection of pathogenic bacteria with bacteriophages using gold nanorod deposited graphite electrodes application of electrochemical biosensors for detection of food pathogenic bacteria electrochemical biosensors for rapid detection of foodborne salmonella: a critical overview nanowire labeled direct-charge transfer biosensor for detecting bacillus species computer aided modelling of an interdigitated microelectrode array impedance biosensor for the detection of bacteria bacteriophage immobilized graphene electrodes for impedimetric sensing of bacteria (staphylococcus arlettae) conductive polymers: opportunities and challenges in biomedical applications graphene-based biosensors for on-site detection of contaminants in food this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -afcgqjwq authors: ladner, jason t.; grubaugh, nathan d.; pybus, oliver g.; andersen, kristian g. title: precision epidemiology for infectious disease control date: - - journal: nat med doi: . /s - - - sha: doc_id: cord_uid: afcgqjwq advances in genomics and computing are transforming the capacity for the characterization of biological systems, and researchers are now poised for a precision-focused transformation in the way they prepare for, and respond to, infectious diseases. this includes the use of genome-based approaches to inform molecular diagnosis and individual-level treatment regimens. in addition, advances in the speed and granularity of pathogen genome generation have improved the capability to track and understand pathogen transmission, leading to potential improvements in the design and implementation of population-level public health interventions. in this perspective, we outline several trends that are driving the development of precision epidemiology of infectious disease and their implications for scientists’ ability to respond to outbreaks. the driving principle behind precision medicine is that one size does not, in fact, fit all . to date, the field has primarily focused on the use of patients' own genomic information to make personalized decisions about disease treatment . during infectious disease outbreaks, however, genomic sequence information from the pathogen is arguably more important than an individual's genomic data for designing appropriate treatment and intervention strategies . the practice of utilizing pathogen genotypic information for the diagnosis and treatment of infectious diseases is not new, but technological advances, most notably in the targeted enrichment of pathogen nucleic acids [ ] [ ] [ ] and next-generation sequencing , have greatly improved the prospect of broadly applying this approach in the clinic. in the past, practical applications of pathogen genotyping were limited by the slow pace of sequencing and its focus only on specific genes-or even portions of genes. today, in contrast, researchers can characterize entire viral and bacterial genomes from infected individuals in near real time . given enough sequence coverage, they can also characterize minor genetic variants in pathogen genomes present within an individual patient, which can be critically relevant in directing clinical care , . although not typically presented as precision medicine, pathogen genomic information has been used successfully to assess drug sensitivity and/or resistance on a patient-by-patient basis for several significant human pathogens, including hiv , influenza virus , and mycobacterium tuberculosis . this information can be used-in a manner analogous to human genotypes-to guide the design of individualized drug regimens (for example, antibiotics and antivirals) ( fig. ). applying genomic technologies during the development and usage of immunotherapeutics (for example, monoclonal antibody cocktails ) and vaccines can also provide insights into pathogen strategies for immune response evasion , and mechanisms of virulence , . by characterizing longitudinal samples from the same patients, pathogen sequencing also provides the potential for identifying genetic components involved in driving disease progression, thus providing novel drug targets . point-of-care molecular tests tailored to individual pathogens have dramatically increased the speed and specificity of infectious disease diagnosis, though there is still considerable room for improvements in sensitivity . one advantage of genomic approaches is that molecular diagnostics can be modified in light of pathogen sequence information generated during an outbreak . this, for example, was achieved during the - ebola epidemic, when rapidly generated virus genome sequences were used to update pcr-based diagnostics so that they more closely matched the makona variant of ebola virus responsible for the epidemic . in addition to the utility of genomic technologies for improving traditional diagnostic tests, metagenomic next-generation sequencing-in which all genomic information, including microbial material, is sequenced in an untargeted manner-holds great promise as a general approach for the detection and characterization of pathogens without the need for a priori knowledge of the potential causative agent , . because metagenomic approaches do not target particular pathogens, they are equally applicable to the detection of expected pathogens as they are to the detection of novel pathogens-such as the emergences of sars and mers -or to the detection of known pathogens in new places, as was illustrated by ebola virus in west africa during the - epidemic . the combination of highly multiplexed target capture and next-generation sequencing is particularly promising, as it increases both sensitivity and specificity. such an approach is feasible because it is now possible to multiplex millions of individual pathogen-specific probes, each of which can enrich for highly divergent nucleic acids (up to ~ % divergence) . pathogen genomes can also be used to inform population-level intervention strategies for infectious disease outbreaks. in contrast to the design of individual-level treatment strategies, in which the functional roles of host and/or pathogen mutations are critical, outbreak-scale genomic analyses use pathogen mutations as markers of transmission events. genomic epidemiology exploits the rapid evolution of pathogens, which often accumulate mutations on the same timescale as their epidemiological spread , to reconstruct outbreak dynamics from genomic data. with sufficient sampling, relevant metadata (such as location and date) and an appropriate statistical framework, pathogen genomes can reveal patterns of epidemic transmission at a fine-scale resolution, thus enabling the design of targeted interventions that are more precise than those based on traditional epidemiological data alone. technological advances are enabling the broad application of pathogen genome sequencing for our response to outbreaks of infectious disease. whole-genome sequencing of many pathogens can now be done directly from clinical samples and in near real time during an outbreak. by analyzing these genomes and their metadata in the context of other sequences generated from the same outbreak, as well as previously characterized variants, researchers can inform individual-and populationlevel intervention strategies to minimize the burden of infectious diseases. we term the collective approach-sequencing, analysis, and response-as precision epidemiology. one application of precision epidemiology during outbreaks is the identification of causal pathogens and their modes of transmission. large-scale virus genome sequencing efforts during the - ebola epidemic, for example, showed that it resulted from a single cross-species 'spillover' event of zaire ebolavirus, from an animal reservoir to humans, followed by sustained human-tohuman transmission . however, while human-to-human transmission typically occurs through direct contact with bodily fluids from a symptomatic individual, genomic epidemiology also demonstrated the potential for sexual transmission of ebola virus from persistently infected asymptomatic individuals . this mode of dissemination played a critical role in prolonging the ebola epidemic in west africa, and as a result of genomic studies, the world health organization (who) made an immediate change to their guidance for ebola survivors and reccomended repeated diagnostic characterization of semen samples prior to two consecutive negative results . in contrast, genomic epidemiological studies of lassa fever, which is endemic in west africa , showed that human cases of lassa fever are the result of multiple independent spillovers from a mastomys natalensis rodent reservoir, with limited human-tohuman transmission , . one of the most advanced population-level applications of precision epidemiology is food safety, where it is used for pathogen identification and source attribution. genome sequencing of foodborne bacterial pathogens now forms part of many surveillance systems, and outbreak investigations in the united states are routinely performed by the food and drug administration's genometrackr network. in recent years, this network has grown into an international collaboration among government, private, and academic research laboratories , . through near-real-time genome sequencing and public data deposition of clinical, environmental, and foodrelated bacterial isolates, this network is streamlining the process of recognizing, investigating, and reducing the impact of foodborne disease outbreaks , . the success of this approach was demonstrated recently through a broad investigation of several foodborne listeria monocytogenes outbreaks across the united states . phylogenetic analysis of pathogen genomes can also be used to elucidate the spatial and temporal scales of transmission, which are critical for the design of effective public health interventions. hiv sequences, for example, have been used to reconstruct transmission networks in detail, with the goal of focusing the use of antiretroviral drugs, along with screening and prevention education messages, in a targeted manner to interrupt community spread , . likewise, zika virus genomes have been used to determine the relative contributions to epidemic growth of local vector-borne transmission versus repeated reintroductions from travelers in sustaining zika outbreaks in the americas , . phylogenetic investigations have also been critical for disentangling the roles of community-and hospitalbased transmission of bacterial pathogens . in one example, wholegenome sequences of methicillin-resistant staphylococcus aureus (mrsa) indicated that a persistently infected healthcare worker in cambridge, uk likely played a key role in sustaining transmission within a particular hospital unit . this analysis directly led to infection control interventions, including targeted pathogen decolonization efforts. genomically informed transmission trees are also used to directly estimate key epidemic parameters (such as the basic and effective reproduction numbers of an outbreak), either independently or candida auris oxford, uk whole-genome fungal sequencing of patient and environmental isolates was used to help identify contaminated equipment as the source of many infections acquired within a hospital intensive care unit. yellow fever brazil whole-genome virus sequencing was used to show that the recent yellow fever outbreak in brazil was caused by repeated sylvatic ('jungle') spillover and not urban transmission. as sylvatic transmission involves different mosquito species than urban, this finding informs vector control strategies. zika virus florida, usa sequencing of virus genomes from cases and mosquitoes infected with zika virus in florida showed that multiple introductions of the virus from the caribbean (perhaps hundreds) were required to sustain the outbreak, suggesting that traveler education and surveillance could reduce future outbreaks. one of the earliest studies to use metagenomic sequencing of human samples to discover a novel virus responsible for a cluster of fatal hemorrhagic fever. listeria monocytogenes usa by using whole-genome sequence data, investigators were able to substantially improve their ability to identify the source and cause of listeria monocytogenes outbreaks. influenza virus worldwide this paper shows that serological changes of influenza virus can be captured by studying virus genomic sequences. such findings can be used to direct selection and design of seasonal influenza vaccines. e. coli o :h (ref. whole-genome sequencing of e. coli isolates was used to dissect a european outbreak of bloody diarrhea and hemolytic uremic syndrome caused by shiga-toxin-producing e. coli. in combination with incidence data . such analyses can provide rapid estimates of pandemic potential and are used to evaluate the effectiveness of interventions , . genomic data can even provide information on within-outbreak population structure (that is, differences in transmission dynamics between geographic locations or risk groups) and the proportion of unreported cases . finally, sequencing allows us to monitor genetic changes over time in pathogen populations, an understanding of which is critical for the design of effective diagnostics and countermeasures. vaccines, for example, are our primary line of defense against seasonal influenza. however, influenza viruses evolve quickly to evade immune responses to previously circulating variants or prior vaccinations. genetic sequencing and large-scale bioinformatic analysis provide powerful tools for tracking the evolution of influenza viruses in real time and for predicting the strains likely to be most prevalent each year. the seasonal influenza vaccine can then be regularly updated to reflect projected changes in the global population of influenza strains . advances in sequencing technologies are enabling the development and use of innovative genomic approaches for the treatment and prevention of infectious diseases. adoption of genomic epidemiology into effective outbreak responses, however, will require the establishment of improved mechanisms for coordination between academic researchers and public health agencies. this includes changes to research practice regarding the benefits for rapid and open sharing of data and results as well as a focus on building capacity for sequencing and analysis within public health agencies and the regions most severely impacted by infectious disease , . comprehensive and carefully organized sampling of pathogen genomes from patients along with rich sets of metadata (box ) are required to improve the accuracy and resolution of outbreak transmission patterns reconstructed using genomic epidemiology. sampling is typically performed or coordinated by local hospitals and departments of health, national entities such as the us centers for disease control and prevention (cdc), or international groups like the world health organization (who) and médecins sans frontières. expertise in genome sequencing, bioinformatics, and phylogenetic analysis, in contrast, is typically concentrated within academic and government research laboratories. therefore, at this point in time, for precision epidemiology to be successfully implemented, it is critical that researchers and public health agencies work together in close coordination. such collaborations were critical during responses to the recent ebola and zika epidemics; however, the approach to establishing these partnerships was largely unsystematic and, in many cases, delayed because of the need to establish relationships during the course of public health emergencies . one important approach to accelerating responses in the future is to build genome sequencing and analysis capabilities within public health agencies and hospitals as well as in developing countries disproportionately impacted by infectious disease outbreaks. several such efforts are currently underway, including the association of publich health laboratories (aphl)-cdc bioinformatics fellowship program (https://www.aphl.org/fellowships/pages/bioinformatics.aspx) and the h africa initiative, which is backed by the us national institutes of health and the uk wellcome trust . genomics programs within public health agencies and at individual hospitals would streamline the process of integrating genomic data into outbreak response efforts. genomic epidemiology, however, is a rapidly evolving field with a strong theoretical foundation, and owing to differences in priorities, academic research groups will likely continue to be at the forefront of tool development and implementation. therefore, it is imperative that researchers develop a framework of norms and rules governing research conduct during and between outbreaks , establish diverse networks of technical response teams, and produce action plans. this framework needs to be implemented in advance of an outbreak and coordinated through international organizations, like the who, and oversight committees within the united nations . it is critical that data and analyses are shared openly during infectious disease outbreaks to ensure the most comprehensive and efficient response possible while ethical constraints also receive close attention. this includes the public release of raw genome sequence data as well as analysis results, which should be provided in a format that conveys to nonspecialists the complexities and uncertainties associated with interpretation. further development of portable instruments for in-country sequencing and online analysis platforms , will continue to advance the rapid generation and open dissemination of data, analyses, and actionable insights. however, concerns regarding the perceived career benefits of slower or more limited public access to outbreak data remain a barrier to open science within the research community. despite this, there are signs of progress. during recent outbreaks, many researchers made data and analyses available and participated in open discussions via online depositories and forums, such as github and virological.org, with complete manuscripts often made available prior to publication via preprint servers such as the biorxiv . we hope that the successes of the research collaborations that followed this approach will help to increase participation in the future. these movements towards making outbreak data more openly available are also supported by several major public health agencies, including the who, which recently called for data relevant to public health emergencies to be distributed immediately and freely upon generation , . with the current capabilities, cost, and speed of sequencing technologies, the field has finally reached a point where rapid genomic surveillance and analysis can start to become a standard part of the response to infectious disease outbreaks. just as broadscale human while advances in genomics served as the initial driver of precision-based medicine, a similarly precise and comprehensive approach to analyzing pathogen phenotypes is necessary in order to fully realize the potential of genomic data for understanding and treating human disease . this realization has resulted in the development of an array of 'deep phenotyping' programs and tools focused on the collection and use of precise, standardized, and comprehensive phenotypic data obtained via wearables, wireless sensors, and other self-reporting tools , - . thus far, phenome characterization efforts have focused primarily on noncommunicable diseases, including huntington's disease , alzheimer's , sleep apnea , and copy-number-variant-based developmental abnormalities . some of these data, however, are similarly applicable for the investigation of and response to infectious diseases. even for highly pathogenic infectious agents, like ebola virus, the clinical course of the resulting disease can vary widely, and it is currently unknown what roles host and pathogen genotypes and phenotypes may play in determining outcome severity. technological advances in communication methods have also impacted our ability to respond to infectious diseases. the internet is now established as an integral part of infectious disease surveillance and as a medium for the distribution of public health information . now, with the ubiquity of smartphones and the dominance of social media, the potential exists for even more rapid and precise digital tracking of infectious disease outbreaks through a combination of traditional public health surveillance, web-based self-reporting tools , and the computational analysis of existing internet data, including search engine queries and social media-based communications , . genome sequencing revolutionized the treatment of many noncommunicable diseases, pathogen genome data are poised to drive a similar revolution in the response to infectious diseases. initial sequencing and analysis of the human genome 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guidance for managing ethical issues in infectious disease outbreaks (world health organization nextstrain: real-time tracking of pathogen evolution healthmap: the development of automated real-time internet surveillance for epidemic intelligence preprints: an underutilized mechanism to accelerate outbreak science policy statement on data sharing by who in the context of public health emergencies blueprint meeting on pathogen genetic sequence data (gsd) sharing in the context of public health emergencies possible sexual transmission of ebola virus -liberia near real-time monitoring of hiv transmission hotspots from routine hiv genotyping: an implementation case study a candida auris outbreak and its control in an intensive care setting genomic and epidemiological monitoring of yellow fever virus transmission potential genetic detection and characterization of lujo virus, a new hemorrhagic fever-associated arenavirus from southern africa prediction, dynamics, and visualization of antigenic phenotypes of seasonal influenza viruses genomic epidemiology of the escherichia coli o :h outbreaks in europe deep phenotyping for precision medicine using phenx measures to identify opportunities for cross-study analysis the human phenotype ontology: a tool for annotating and analyzing human hereditary disease mouse phenome database: an integrative database and analysis suite for curated empirical phenotype data from laboratory mice whole-animal imaging, gene function, and the zebrafish phenome project large-scale phenome analysis defines a behavioral signature for huntington's disease genotype in mice a behavioral task predicts conversion to mild cognitive impairment and alzheimer's disease an electrocardiogram-based analysis evaluating sleep quality in patients with obstructive sleep apnea large-scale objective association of mouse phenotypes with human symptoms through structural variation identified in patients with developmental disorders digital disease detection -harnessing the web for public health surveillance the value of patient self-report for disease surveillance using internet search queries for infectious disease surveillance: screening diseases for suitability using social media for actionable disease surveillance and outbreak management: a systematic literature review social media as a sentinel for disease surveillance: what does sociodemographic status have to do with it? the authors declare no competing interests. reprints and permissions information is available at www.nature.com/reprints. publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -f uvhstb authors: sintchenko, vitali title: informatics for infectious disease research and control date: - - journal: infectious disease informatics doi: . / - - - - _ sha: doc_id: cord_uid: f uvhstb the goal of infectious disease informatics is to optimize the clinical and public health management of infectious diseases through improvements in the development and use of antimicrobials, the design of more effective vaccines, the identification of biomarkers for life-threatening infections, a better understanding of host-pathogen interactions, and biosurveillance and clinical decision support. infectious disease informatics can lead to more targeted and effective approaches for the prevention, diagnosis and treatment of infections through a comprehensive review of the genetic repertoire and metabolic profiles of a pathogen. the developments in informatics have been critical in boosting the translational science and in supporting both reductionist and integrative research paradigms. "new age" infectious disease informatics rests on advances in microbial genomics, the sequencing and comparative study of the genomes of pathogens, and proteomics or the identification and characterization of their protein related properties and reconstruction of metabolic and regulatory pathways (bansal ) . the speed of microbial genome sequencing has been steadily accelerating since the introduction of modern dna sequencing methods more than thirty years ago (sanger et al. ) . the accumulation of sequenced genomes of bacteria shows a good fit to exponential functions with a doubling time of approximately months (koonin and wolf ) . despite the historical bias towards the "working horses" of bacterial genomics, such as commensals e. coli and b. subtilis (collado-vides et al. ) , the depth and breadth of the coverage of sequences belonging to different species of viral, bacterial, fungal and protozoan pathogens has been rapidly expanding. microbial genomes are thousands or millions of base pairs in length, requiring both a global view of the genome and the ability to zoom in on details for the purpose of analysis and annotation. annotation is the extraction of biological knowledge from raw nucleotide sequences (médigue and moszer ) . such decoding of the genomes allows the prediction of protein-coding genes and therefore, the proteins the organism is able to produce. desktop computer sequence editors such as chromas lite (http://chromas-lite.software.informer.com/), trace edit (http://www.ridom.de/traceedit/) or commercial products like lasergene (http://www.dnastar.com/products/lasergene.php) or sequencher (http://www. sequencher.com/) are helpful in the initial sequence assessment. the task of assembling of sequences from re-sequencing experiments, when a reference sequence is available, can be supported by tools like traceeditpro (http://www . ridom.de/traceeditpro/) or seqscape. different software pipelines have been developed to automate microbial genome annotation and assembly (table . ). the integrated microbial genome (img) system, hosted by the joint genome institute (jgi), and the rast (rapid annotation using subsystem technology) server are examples of open resources. major sequencing centers offer genome viewers and browsers through their websites (mcneil et al. ) . for example, manatee (j. craig venter institute (jcvi)) has been developed to view and to alter initial automatic annotations of prokaryotic genomes. the sanger institute's pathogen sequencing unit has been maintaining freeware for sequence analysis, viewing and annotation, such as artemis and the artemis comparison tool (act) (carver et al. ) . the alignment of genomes of three strains of staphylococcus aureus using act is shown in fig. . . alternatively, multiple genome alignments in the presence of large-scale evolutionary events, such as rearrangement and inversion, can be efficiently constructed and visualized using the mauve program (http://gel.ahabs.wisc.edu/mauve/download. php) (darling et al. ). these tools assist in the rapid identification of protein-coding informatics for infectious disease research and control genes, as well as other features like non-coding rna genes, repetitive sequences or recently acquired dna. web servers like integrated microbial genomes (joint genome institute; http:// img.jgi.doe.gov) or the bacterial annotation system (basys, http://wishart.biology. ualberta.ca/basys/cgi/submit.pl) also support comparative analysis and the automated annotation of bacterial genomic (chromosomal and plasmid) sequences (van domselaar et al. ) . they accept raw sequence data and gene identification information, and provide textual annotation and hyperlinked image output. strings of nucleotides are assembled into draft sequences that can be characterized by the following: ( ) > % of genome in contigs, ( ) average contig length > kb, ( ) > % of a set of conserved genes present, ( ) contig n length > kb, ( ) > % of bases > × read coverage, ( ) scaffold n length > kb. the information used to annotate genomes comes from three types of analysis: ( ) ab initio gene finding programs, which are run on the dna sequence to predict protein coding genes; ( ) s.aureus usa s.aureus col fig. . alignment of genomes of three strains of staphylococcus aureus. dna sequences that find a perfect match are connected with red lines or blocks. blue areas are inversions or transitions and white areas represent indels. the figure was produced using artemis software (the wellcome trust sanger institute, uk) informatics for infectious disease research and control evidence-based gene calling or translating alignments of the dna sequence to known proteins; and ( ) aligning cdnas from the same or related species. gene finding has progressed far beyond the simple identification of open reading frames. the programs aligning cdna and protein sequences to genomic dna can locate the protein coding regions by searching the publicly available databases or by applying machine learning algorithms such as hidden markov models (hmm). there is a long list of such programs including genemark, morfind, prodigal (prokaryotic dynamic programming genefinding algorithm), argon and glimmer (gene locator and interpolated markov modeller) (delcher et al. ; suzek et al. ; majoros ) . they differ in the time required for automated annotation as well as the quality of gene calling (guigo et al. ) . problems with the accuracy of current gene finders reflect not only the performance of their algorithms but also the quality of the primary resources and the abundance of non-coding dna regions in microbial genomes. genome assembly annotation methods and tools including new applications for rna genes, were reviewed in detail elsewhere (stothard and wishart ; médigue and moszer ; brent ; pop and salzberg ) . recent breakthroughs in high-throughput sequencing technologies have posed new challenges for genome assembly, annotation and analysis. these technologies make it feasible to sequence not only static genomes but also entire transcriptomes expressed under different conditions (shendure and ji ) . however, they can produce read lengths as short as - nucleotides, which cannot be analyzed with software developed for sanger data as they are often non-unique, lack neighborhood context and have a different distribution of errors. the task of linking such short-reads may be accomplished using a comparative assembly algorithm, in which new sequences are put together by mapping them onto close relatives or the "reference genomes." not surprisingly, the comparative assembly strategy works best when the two species are more than % identical. alternatively, when no "reference genome" is available, the new cohort of assembly algorithms based on de bruijn graphs -a way to transform sequence data into a network structure -has risen to the task (chaisson and pevzner ; maclean et al. ). strategies and systems that address these new challenges have recently been reviewed elsewhere (pop and salzberg ; maclean et al. ; ussery et al. the metagenomics or the sequencing of genomes of complex mixed communities has emerged at the interface of genomics, microbiology and information technology. this field examines the interplay of hundreds of microbial species present at specific sites of potential infections in space and time (hutchinson ; smarr et al. ). significantly, metagenomics has extended its focus from environmental microorganisms to microbial communities or "community whole genome sequences" of the human host (field et al. ; verberkmoes et al. ). most of the - trillion microorganisms in the human gastrointestinal tract live in the colon (turnbaigh et al. ). the genomes of these microbial symbionts have been collectively defined as the microbiome or ecosystem in which the number of microbial genes is estimated to be many folds higher than those present in the human genome. the human gut microbiome initiative, a logical conceptual extension of the human genome project, aims to discover genomes of at least new intestinal species. this approach has targeted the totality of genes involved in the gut biofilms, the mechanisms of horizontal gene transfer, and the role of the microbial pan-genome (field et al. ) . the microbiome project aims to address some of the most inspiring and fundamental scientific questions today in order to identify new ways to determine health and predisposition to diseases and define parameters in addition to conventional strings of nucleotides, large-scale sequencing can provide new types of data reflecting global genome architecture and the properties of pathogens. these data include the size of a genome and its nucleotide composition, the locations of genes and intergenic regions, gc percentage and gene density. microbial genomes are compared by the number of particular sets of genes, gene order (synteny) and the presence or absence of important genes. other metrics include gene set properties (the number of two component system regulatory genes) and nucleotide sequence-based measures (distance between paired twocomponent system genes and consensus sequence) (whitworth ; ussery et al. ). these metrics represent a global view of genomes but often have limited biological meaning. thus, "signature" sequences have been suggested as a means of identifying organisms or genes with sequence profiles correlating with the pathogen phenotype or disease outcomes. examples of genome characteristics that are more directly related to biologically important behavior are bacterial iq (a measure of the number of signal transduction proteins as a function of genome size) and extrovertedness (the proportion of signaling proteins predicted to sense external stimuli) (galperin ) . analyses of genomics data challenge the traditional taxonomy of microbial species. recent projects have focused on producing simple analytical diagnostic tools based on strong taxonomic knowledge collated in the dna reference libraries such as the dna barcode of life data system (bold; http://www.boldsystems. org). these types of data enable the acquisition, storage, analysis and publication of dna barcode results, and provide clues about the global distribution of species. their genetic diversity and structure is based on two postulates: first, that every species is represented by a unique dna barcode (indeed there are possible atgc combinations compared to an estimated million species remaining to be discovered (frézal and leblois ) ), and second, that the genetic variation between species exceeds the variation within species. dna barcoding requires a minimum sequence length of bp and more than three individual sequences per species. the initial barcode of life framework was based on the sequence of a single universal marker -the cytochrome c oxidase gene -but has evolved since then, giving rise to a flexible description of dna barcoding, a larger range of applications and the broader use of the term "barcode" (frézal and leblois ) . for example, the whole microbial genome's barcodes were defined as frequency distributions of periodic dna sequences or k-mers across the whole genome (zhou et al. ). it has been postulated that such barcode similarities are proportional to the genomes' phylogenetic closeness and could be utilized in metagenome analyses (zhou et al. ) . microbial species diversity can be also estimated by the average nucleotide identity (ani) using the list of orthologs and deriving the overall divergence of the core genome by averaging the percentages of identity at the nucleotide level (konstantinidis and tiedje ) . another approach to measure distances between genomes is based on estimating the proportion of common genes by calculating the ratio of orthologs to the total number of genes of the reference genome. more recently, similar methods such as dna content, blast distance phylogeny and the mum (maximal unique and exact matches) index have been suggested as more sensitive measures for intra-species comparisons (deloger et al. ). the true power of large-scale comparative genomic studies lies in their ability to identify and characterize biological trends and rules that explain particular phenomena (field et al. ). computational methods have become essential steps in formulating hypotheses about gene functions. the comparative approach has not only yielded fundamental insights into the function and evolution of microbial genomes, but has also led to practical results. comparative genomics has allowed the accurate estimation of the structure of genomes and the speed of gene movements, including the role of natural selection versus genetic drift, the origin of the pandemic strains, and the ecology of a pathogen in its natural reservoir yang et al. a) . computational studies identified unexpected relationships between genomic features and ecological niches, demonstrated diversity in the microbial world and helped to reconstruct evolutionary relationships among genomes (binnewies et al. ; field et al. ) . comparisons made between different genomes can also generate new hypotheses for testing, usually relating to the unexpected presence or absence of particular genes with respect to other genomes (whitworth ) . the studies of three main forces shaping genome evolution -gene loss, gain and change -have been especially fruitful in this respect (burrack et al. ; whitworth ) . discoveries of gene duplication in many bacterial pathogens, resulting in increased numbers of key gene clusters or the expansion of important protein families have led to the development of new diagnostic methods. for example, the gene clusters encode a secreted protein called the early secretory antigenic target or esat , which was identified as one of the key virulence factors in mycobacterium tuberculosis and was subsequently used in the interferon-gamma release assays for the diagnosis of tuberculosis (pallen and wren ; behr ) . comparative genomics has also revealed that pathogens undergo a process of genome decay or a reduction in the number of biosynthetic pathways, resulting in a dependence on the infected host for certain essential functions. the most surprising informatics for infectious disease research and control snapshots of genome decay have come from relatively recently emerged pathogens that have changed their lifestyles by adopting a simpler host-associated niche. for example, the genomes of yersinia pestis (parkhill et al. b) and salmonella enterica serovar typhi (parkhill et al. a ) contain hundreds of pseudogenes. these findings challenge the traditional view that bacterial genomes never contain "junk" dna and that every gene in a bacterial genome must have a function. instead, every genome should be viewed as a work in progress, burdened with some non-functional "baggage of history" (pallen and wren ) . as the smallest-scale variation in microbial genomes occurs at the level of singlenucleotide polymorphisms (snps), snp detection has been applied extensively to many pathogens (yao et al. ) . while snps are generally considered rare, at one per several thousand base pairs, two genomes of m.tuberculosis of mb each may have some , snps between two isolates (behr ) . whole-genome sequencing has been proven as an even more powerful tool to detect snps. it enabled the differentiation of escherichia coli strains that had diverged for as few as generations (shendure and ji ) and revealed genomic changes in pathogens in the process of human infection (chen et al. ; forst ; pallen and wren ). in the pre-informatics era, virulence factors were typically identified either by biochemical studies or through genetic screens. informatics has enabled innovative strategies for the recognition of virulence gene recognition through the analysis of genetic signatures (pallen and wren ) . despite the variety of microbial life styles and associated genomic and metabolic complexity, pathogen genomes share common architectural principles. as a result, computational techniques assist in exploring similarities between virulence factors and other genes with known functions. this association can then be tested using targeted genetic methods such as the inactivation of the putative virulence gene followed by the comparison of phenotypes of the original and modified microorganisms raskin et al. ) . a strategy that does not rely on sequence similarity for identifying potential genes is the detection of coding sequences, which is based the gene context "grammars" supplemented with machine learning models (garrido et al. ) . for example, functional gene recognition tools genemark and glimmer employ hidden markov models, in which the preceding nucleotide bases are used to predict the next base in a coding region, and the algorithm is trained on a trusted set of sequences. gene coding regions are then identified using probability estimates of the correct coding "grammar" in a region (dougherty et al. ) . different statistical and machine learning methods for gene prediction have been reviewed elsewhere (majoros ) . gene-gene interactions specifically associated with a phenotype or a particular disease can be explored with or without a prior biological knowledge. several techniques utilizing bayesian networks, pair-wise mutual information and graphical gaussian models have been proposed for this purpose. coupled with biological knowledge, the identification of such phenotype-specific interactions can shed light on the responsible pathways. the complexity of data handling and visualization has led to efforts to develop dedicated comparative genomics resources such as gendb (meyer et al. ) , cmr, act, (table . ) xbase and microbes online as well as data management systems such as seed (table . ) (chaudhuri et al. ). informatics has been instrumental in the change from static to a dynamic view of the microbial world. in contrast to the static view of genome annotations focused on the gene or protein prediction, the dynamic view places information obtained into a biological context to identify interactions between the genomic components and the reconstruction of regulatory networks (médigue and moszer ; sakata and winzeler ) . under the network vision of the microbial world, microbial chromosomes are not envisaged as strictly defined genotypes gradually changing in time but rather as islands of temporary, relative dynamic stability that form tightly connected (vertically and horizontally) areas of the network (koonin and wolf ) . the infection cycle should be considered as a whole and the links between growth, virulence, immune evasion and transmission should be assessed (restif ). biological interactions vary in their nature and are spatially and temporally heterogeneous. one can abstract the actions of proteins and metabolites by representing genes acting on other genes as a gene network or as genetic regulatory, transcription or expression networks. such networks can be constructed using computationally assigned functional linkages inferred by rosetta stone, operon or similar methods (rachman and kaufmann ; harrington et al. ) , and often point to highly connected and central proteins frequently referred to as "hubs" (wu et al. ) . biological interaction and communication networks share several commonalities: they are scale free (only a few nodes are highly connected) and are small world networks (highly clustered with short distances between any two nodes) (kann ) . increasingly, disease pathogenesis and the mechanisms of drug action are viewed from a biological systems perspective (wu et al. ) . from this perspective, a deeper understanding of infectious diseases may rely on an exhaustive characterization of all potential interactions occurring between proteins encoded by viruses and those expressed in infected cells. thus, the integration of all protein-protein interactions into an infected cellular network, or "infectome," offers a powerful framework for the virtual modeling and analysis of infections (navrati et al. ). the terms "interactome" and "phenomics" have been coined in this context (lussier and liu ) . numerous resources have been developed to explore host-pathogen interactions (phi) (table . ) . specifically, phi-base (winnenburg et al. ) , phidias (xiang et al. ), biohealthbase (squires et al. ) , pig (driscoll et al. ) virusmint (chatr-aryamontri et al. ) and virhostnet (navrati et al. ) have been virulence prediction lengauer et al. drug resistance prediction navrati et al. raman et al. effect of diseases on gene expression drug target identification reddy et al. squires et al. stavrinides et al. drug resistance prediction drug resistance prediction suggested to study and visualize pathogen-related pathways. for example, the virhostnet is a knowledge base for the management and analysis of proteome-wide virus-host interaction networks and a resource of manually curated interactions defined for a wide range of viral species (navrati et al. ). genomic and proteomic data is often informationally synergistic, allowing for the reconstruction of known pathways from the first principles. the combination of these forms of data have been used to identify libraries of recurring motifs, where the mixed semantics of the pattern promises to be more informative than any single data source taken in isolation in building biological networks (michael et al. ; stavrinides et al. ) . systems biology has arisen from various attempts to move away from the reductionist approach, which is hindered by the difficulty of breaking a system into separable and meaningful parts. it encompasses several high-throughput analytic technologies, including genomics, transcriptomics to measure gene expression and its regulation at the level of messenger rna and microrna production, proteomics to measure changes in protein production, and computational biology, which depends on analytic software packages for analyzing, organizing, and interpreting those data (sakata and winzeler ) . such an approach treats pathogens and their environments as a series of hierarchical levels or networks from gene products to whole organisms and integrates the time dimension in order to structure knowledge and to determine rules that would allow navigation between levels (lisacek et al. ). this approach demands new tools for data management, the integration of which offers the opportunity to correlate multiple lines of evidence and to reduce uncorrelated noise. the major difference between the pre-and post-genomics eras is that one can now potentially account for and keep track of all components at once. however, the gathering of a large collection of data does not guarantee that we can make sense of it or that new knowledge will emerge (collado-vides et al. ). the chance for enriching biomedical knowledge can be increased by mixing various streams of data and gaining robustness from the "cross-validation" of the knowledge sources (guyet et al. ). public websites like galaxy (http://galaxy.psu.edu) and interpro (http://www.ebi.ac.uk/interpro/) offer integration toolsets for genomics and proteomics analyses. as generating data remains a costly undertaking, computational models have a pivotal role to play in the integrative science. they help researchers to illuminate the underlying processes and identify the key questions that need to be addressed experimentally (restif ). compared to conventional, small-scale experimental approaches, they give a wider, often more relevant view of host responses to infections or other health insults. these computational models have the capacity to guide and direct wet lab experimental efforts complimenting traditional in vivo, in situ, and in vitro testing with the emerging in silico approach (lengauer et al. ; raman et al. ) . some impressive starts have been made on bacterial models in the form of simulation tools. for example, the reconstruction of metabolic networks gave birth to the first examples of in silico strains that can be utilized to explore alternative ways of identifying new drug targets (jamshidi and palsson ) . the end result of these simulations may be the genomic bioengineering of microorganisms based on knowledge of interacting systems and networks of genes and gene products. text mining tools are being created to query the pubmed literature database and to integrate the available genomic and proteomic information to map the genes and their interrelationship with particular networks of a disease (korbel et al. ; jelier et al. ; rzhetsky et al. ; zaremba et al. ). an unsupervised, systematic approach for associating genes and phenotypic characteristics (g p) that combines literature mining with comparative genome analysis has been successfully applied and has uncovered clusters of unsuspected g p associations (korbel et al. ). the phase of history in which biomedical science could be significantly advanced by individual researchers without data sharing has come to a close. the global, collaborative analyses of data and the exchange of the results across social, political and technological boundaries have created the demand for new cyber-infrastructures for research. there has been a major effort, in the form of e-science, to develop technologies to fulfill these demands (craddock et al. ). the chance of making a discovery or replicating the finding is greatly increased if there are effective mechanisms for different groups to share data and thereby enlarge the number of samples that are studied. this paradigm has been successful in both human genomics and infectious disease research (e.g., including the rapid discovery and identification of emerged pathogens such as the nipah virus and the novel coronavirus that caused the sars epidemic). post-genomic era solutions such as federated databases and other technologies that enhance connectivity and data retrieval have created a new knowledge environment (birkholtz et al. ; thorisson et al. ). the level of technical competence required of the users is being reduced by the provision of "off-the-shelf" solutions. for example, the gen phen project offers "database-in-a-box" installation packages, which include an open-source complete genetic association database system with the option for federation (thorisson et al. ). alternative infrastructures for e-science with significant advantages over conventional internet technologies are offered by grid and cloud computing and the semantic web (numann and prusak ; craddock et al. ) . first, grids provide unique access to high performance computing power, distributed applications and sources (see chap. for examples). second, grids increase data storage spaces, and allow data and tools to be shared by geographically dispersed users. however, developing and maintaining grid or cloud architectures remains a complex task and requires further advances in security and privacy models before they can be embraced by diagnostic laboratories (lisacek et al. ). tasks that require an e-science approach or global science that is performed in silico are typically computationally intensive and use heterogeneous resources that must be integrated across distributed networks (craddock et al. ) . increasingly, the genomic, proteomic and metabolomic data have to be integrated with traditional literature in a machine-readable way. typical sets of experimental data yield component lists with quantitative content data and a catalog of interactions and networks. this requires the establishment of a middleware to convert experimental data into a format suitable for manipulation and viewing by end-users. for example, the generic model organism database project (gmod; http://gmod.org) aims to link experimental data with corresponding contextual meta-data about experimental conditions and protocols in a multi-user, multi-center environment. it offers a collection of open source tools for creating and managing genome-scale biological databases ranging from a small database of genome annotations to a large web-accessible community database. another approach is to trade off the width of integration for more depth with regard to a particular analysis task, and to employ workflow systems such as inforsense (http://www.inforsense.com) or taverna (http://taverna.sf.net). these act as glue layers between various data sources and analysis packages and are also often referred to as pipelines, in silico protocols or e-experiments (turnbaigh et al. ) . "pipeline" is mostly used to describe executable workflows, while the other terms are dedicated to abstract workflows (lisacek et al. ) . many innovative solutions for the multi-dimensional integration of data produced by experimental laboratories have been introduced by bioinformatics resource centers for biodefense and emerging/re-emerging infectious diseases through regional biodefense centers of excellence (mcneal et al ; greene et al. ) . sets of task-and domain-specific online query and display tools are being developed to allow the end-user to view data in a number of different formats and to run informative comparisons of data with existing libraries (louie et al. ; glassner et al. ) . the most striking change in data collection and representation is expressed by the move from flat databases to atlases or collections of interconnected maps (lisacek et al. ). the uneven content and quality of data and the constant evolution of biomedical knowledge remain the main obstacles to data integration (lisacek et al. ). the quality of data is affected by a number of factors including the accuracy of the mapping algorithms and reference datasets, the standardization of data formats and the level of detail of the experiment description (stead et al. ). in addition, an increasing number of genomes are being released in "draft" form, before the finishing stage of a sequencing project, with high sequencing error rates (de keersmaesher et al. ; médigue and moszer ) . recent developments in databases and browsers for genomics have been summarized by schattner ( ) . there is an urgent need for data structures suitable for infectious disease space that can be applied to emerging "omics" data sets. the pathogen information markup language (piml) has also recently been introduced to enhance the interoperability of microbiology datasets for pathogens with epidemic potential (he et al. ) by capturing the data elements that describe determinants of pathogen profiles. however, the jury is still out on the question of which data integration architectures are best suited to assembling large scale and highly diverse genomic data. integrating high-throughput techniques with other analytic tools brings a new understanding of infectious processes and introduces an era of personalized strategies for managing infectious diseases. in this way, informatics becomes an irreplaceable platform for the constant cross-fertilization and interplay between focused and genome-wide studies. rapid and standardizable molecular identification systems have emerged during the last decade, with the development of sequence based species identification and sub-typing as the alternative to slow, labor-intensive and underpowered phenotypic techniques. molecular identification usually relies on the detection of a single gene or multiple gene targets, or requires the comparison of whole microbial genomes. for example, in the pragmatic world of diagnostic bacteriology, conserved housekeeping genes such as the s rrna gene, rpob gene and others have been accepted as reliable targets. they are found in all microorganisms and show enough sequence conservation for accurate alignment as well as enough variation for phylogenetic analyses (christen ) . furthermore, the s rrna gene based phylogeny is sufficiently congruent with those based on whole genome approaches. sequencing of six to eight genes or loci, as it typically done in multilocus sequence typing analysis, may constitute a reasonable compromise between single genebased and whole genome-based methods for species diversity studies. to streamline the process of the translation of sequencing-based identification into clinical practice, the concept of the pathogen profile has been introduced . a pathogen profile is a single, multivariate observation or set of observations, comprised of classes of specific attributes (e.g., genome, transcriptome, proteome or metabolome data), which are designed to allow the interrogation of existing or future databases, and the integration of genomics and post-genomics data with clinical observations and patient outcomes. the profile may indicate the probability that a specific marker is associated with a clinically relevant phenotype such as in vivo antimicrobial resistance or high transmissibility. this information allows the classification of strains into "risk groups" for treatment failure or a propensity to cause outbreaks of infections. it is often important to capture the quantitative information about a pathogen, in vivo, i.e. viral or bacterial loads and their units of measurement. in contrast to traditional subtyping, which is based on phenotypic characteristics such as serotype, biotype, phage type or antimicrobial susceptibility, genetic profiling describes the phenotypic potential in the nucleic acid sequence. a pathogen profile is a synthesis of various markers and clinical end-points, which can be extracted from medical charts that characterize an individual patient's clinical and public health outcomes. the profile may be heuristic, when only a single genetic marker is associated with a specific patient outcome, while more insights can be achieved when attributes from different levels of the biological hierarchy (i.e. gene detection, gene expression, metabolite profiles etc) corroborate and complement each other. machine learning algorithms, such as e-predict (urisman et al. ) , are being developed to identify viruses and bacteria present in clinical samples. these profiles are based on the microarray hybridization patterns or dna sequences of pathogens. many computerized evidence-based guidelines and decision support systems (dss) have been designed to improve the effectiveness and efficiency of antibiotic prescribing (samore et al. ; buising et al. ) . the most frequently utilized are electronic guidelines and protocols, especially for the empirical selection of antibiotics. the majority of dss result in improvement in clinical performance and, in at least half of the published trials, in improved patient outcomes (finch and low ; sintchenko et al. a) . the revival of interest in prescribing-decision-support reflects the recent change in emphasis from support for diagnostic decisions towards support for patient management, and the changing focus from systems targeting a broad range of clinical diagnoses to task-and condition-specific decision aids. despite reported successes of individual applications, the safety of electronic prescribing systems in routine practice has recently been identified as an issue of potential concern. bioinformatics assisted prescribing has become a new frontier in reducing the complexities of prescribing combinations of antimicrobials in the era of multidrug resistance. the great diversity of mutational patterns contributing to antimicrobial resistance complicates the choice of optimal therapies. a range of bioinformatics tools to predict drug resistance or response to therapy from a genotype, have been developed to support clinical decision-making (beerenwinkel et al. ; lengauer and singh ) . these tools use either a statistical approach, in which the inferred model and prediction are informatics for infectious disease research and control treated as regression problems, or machine learning algorithms, in which the model is addressed as a classification problem (sintchenko et al. a) . a statistical learning approach to the ranking of therapeutic choices often relies on a direct correlation between the baseline microbial profiles, the therapeutic decision and the patient's response to treatment (e.g., expected reduction in viral load resulting from anti-hiv combination therapy). for example, several susceptibility scores have been used for combination antiretroviral therapy. these take into account specific resistance mutations and add up the activities of individual drugs in the regimen (lengauer and singh ) . computer-assisted therapy depends on the availability of widely shared databases that can correlate quality-controlled data from genotypic resistance assays and treatment regimens with short-and long-term clinical outcomes. databases such ardb (liu and pop ) capture differences in antimicrobial sensitivities and reflect variation in the amino acid composition of resistant microbes, but simply counting mutations may not be enough to predict functional differences, which affect treatment outcomes. the molecular profiling of pathogens is based on the concept that various pathogens can be associated with different clinical outcomes. it brings together the pathogen and host factors as the pathogenesis and natural history of infection are determined by both the pathogen and human genetic susceptibility. the effectiveness of combining host and pathogen genetics in a single system or "genetics-squared" has been proven in studies of viral infections (persson and vance ) . investigations of the impact of host genetics on the susceptibility to hiv infection and the rate of disease progression have mainly used a candidate gene approach to reveal associations with a number of different genes. the genome-wide association studies look at the genetic variation across the human genome in order to uncover factors not previously suspected of influencing infection outcomes. for example, this strategy identified variants of the hiv virus associated with differences in the control of viral load at set points and in disease progression. however, unraveling the interaction between the host and microbial genetic factors requires large clinical trials, reinforcing the role of collaborative networks and data repositories. informatics methods have become critical for data mining to decipher links between genetic variation and disease pathogenesis in order to define markers of disease progression, to guide the optimum use of therapeutics and to refine the drug and vaccine development (mansmann ) . a better understanding of the function of genes and other parts of the genome has enabled the reverse engineering approach, which may lead to the characterization and discovery of potential drug targets, vaccine candidates and diagnostic or prognostic markers (davies and flower ; yang et al. b) . proteins with essential biological functions present in multiple pathogens could be the best drug targets. once the target genes essential for pathogen survival are identified, their susceptibility to specific compounds derived from large chemical libraries is examined in silico and in vitro (muzzi et al. ; biswas et al. ). increases in the use of electronic medical records and the availability of information technology tools have created opportunities for the automation of surveillance and facilitation of surveillance based on either syndromic or disease-specific signals (amadoz and gonzales-candelas ; m'ikanatha et al. ). the automation of data collection improves the time and completeness of surveillance and allows infection control professionals to focus on interventions (hota et al. ; young and stevenson ) . the comparison of chromosomal sequences allows the identification of the unique genomic signatures of pathogens for the purposes of infection control and "microbial forensics." molecular typing methodologies, in contrast to classical phenotypic methods, allow the discrimination of variations among strains within a species, the elucidation of the route of contamination, the identification of the source of infection as well as the analysis of epidemics. the identification of the natural reservoir and any possible intermediate hosts of pathogens is critical for understanding the transmission modes, designing a long-term disease control strategy, and preventing future reintroduction ). bioinformatics assisted biosurveillance addresses the inefficiencies of traditional surveillance, as well as the need for a more timely and comprehensive infectious disease monitoring and control. it leverages on recent breakthroughs in the rapid, high-throughput molecular profiling of microorganisms and text mining, as well as on the growing electronic body of knowledge about the molecular epidemiology of pathogens with epidemic potential. such a framework combines the genetic and geographic data of a pathogen to reconstruct its history and to identify the migration routes through which the strains spread regionally and internationally (cantón ; sintchenko et al. b) . computer-based geographic information systems (gis) have offered an efficient way to visualize the dynamics of the transmission of infections, especially in the setting of a community outbreak (mckee et al. ; schreiber et al. ) . another way to track infectious diseases of public health concern is to monitor health-seeking behavior in the form of queries to online search engines used by the general public or health professionals. epidemics of seasonal influenza in areas with a large population of internet users have been successfully detected using google search data and then correlated with visits to a doctor (ginsberg et al. ; brownstein et al. ). the advent of news aggregators has led to the development of new disease surveillance tools that can continuously mine, categorize, filter, and visualize multilingual online information about epidemics. the global public health intelligence network (gphin), developed almost a decade ago by health canada in collaboration with who, healthmap (http://www.healthmap.org/en) ( fig. . ) or geosentinel (http://www.istm.org/geosentinel/main.html) among many others are examples of such early warning systems. resources for infection prevention and control on the world wide web have been recently reviewed elsewhere (brownstein et al. ; johnson et al. ) the reductionist approach to biomedical research focusing on the study of cells and molecules has peaked with the sequencing of the human genome. however, it is becoming increasingly clear that "taking apart" analyses have reached their limit, and the time has perhaps come for integrative science (an and faeder ) . developments in informatics have been critical in supporting and engaging with both reductionist and integrative paradigms. on one hand, informatics has equipped comparative genomics with tools to scrutinize genes and explore genetic polymorphisms. on the other hand, informatics has enabled the generation of integrative and testable hypotheses through the discovery of knowledge in databases and through the study of gene-phenotype connections between a pathogen and its host environment. a variety of data sets can be integrated, including the patient's demographic and clinical presentation, the laboratory results, the pathogen's gene regulation and expression, and metabolic maps with different parameters reflecting the phenotypic behavior of a pathogen and host factors. in early years some skeptics saw informatics-assisted research as a distraction of effort and funding away from traditional hypothesis-driven inquiry. since then, infectious disease informatics has verified its status as a platform for hypothesis generation and testing ). new breakthroughs in infectious disease informatics (idi) are the result of cross-pollination between different disciplines that use technologies to gather and disseminate knowledge (fig. . ) . microbial genome sequence analysis and metagenomics have contributed intriguing new data types and data sources to idi. bioinformatics has brought to the idi a range of analytic tools, databases and data standards. conventional health informatics and computer science has provided high performance solutions for the data storage, sharing, analysis and visualization as well as clinical terminology libraries, data standards, decision support and technology evaluation frameworks. importantly, the infectious disease informatics community has fed the lessons learnt from the implementation of clinical and public health systems back to the broader audience. as the subsequent chapters of this volume testify, infectious disease informatics is set to lead to the more targeted and effective prevention, diagnosis and treatment of infections through a comprehensive review of the genetic repertoire and metabolic profiles of pathogens. the post-genomic era offers new opportunities for the efficient discovery of safe and efficacious subunit vaccines by shortcutting the enormous economic burden of the experimental process. our analytical capacity has already become the rate-limiting step in biomedical research. at the same time, it provides an opportunity to apply the engineering paradigm to biomedical research, thereby mandating the development of tools that can dynamically represent a body of current knowledge. however, the simplistic application of brute force computational power to massive reams of biomedical data is unlikely to result in meaningful mechanistic insight. it cannot be overstressed that informatics initiatives should compliment "wet laboratory" practices. an iterative loop of discovery and validation between the two methodologies remains the best way forward. epipath: an information system for the storage and management of molecular epidemiology data from infectious pathogens detailed qualitative dynamic knowledge representation using a bionet gen model of tlr- signaling and preconditioning bioinformatics in microbial biotechnology -a mini review geno pheno: estimating phenotypic drug resistance from hiv- genotypes mycobacterium du jour: what's on tomorrow's menu? microb infect ten years of bacterial genome sequencing: comparative-genomics-based discoveries integration and mining of malaria molecular, functional and pharamacological data: how far are we from a chemogenomic knowledge space? a bioinformatic approach to understanding antibiotic resistance in intracellular bacteria through whole genome analysis steady progress and recent breakthroughs in the accuracy of automated genome annotation digital disease detection -harnessing the web for public health surveillance improving antibiotic prescribing for adults with community acquired pneumonia: does a computerised decision support system achieve more than academic detailing alone?-a time series analysis genomic approaches to understanding bacterial virulence role of the microbiology laboratory in infectious disease surveillance, alert and response artemis and act: viewing, annotating and comparing sequences stored in a relational database short read fragment assembly of bacterial genomes virusmint: a viral protein interaction database xbase : a comprehensive resource for comparative bacterial genomics vfdb: a reference database for bacterial virulence factors identification of genes subject to positive selection in uropathogenic strains of escherichia coli: a comparative genomics approach identification of pathogens -a bioinformatic point of view bioinformatics resources for the study of gene regulation in bacteria e-science: relieving bottlenecks in largescale genome analyses mauve: multiple alignment of conserved genomic sequence with rearrangements harnessing bioinformatics to discover new vaccines integration of omics data: how well does it work for bacteria? improved microbial gene identification with glimmer a genomic distance based on mum indicates discontinuity between most bacterial species and genera microbial genomics and novel antibiotic discovery: new technology to search for new drugs pig -the pathogen interaction gateway how do we compare hundreds of bacterial genomes? a critical assessment of published guidelines and other decision-support systems for the antibiotic treatment of community-acquired respiratory tract infections host-pathogen systems biology four years of dna barcoding: current advances and prospects biosurveillance of emerging biothreats using scalable genotype clustering a census of membrane-bound and intracellular signal transduction proteins in bacteria: bacterial iq, extroverts and introverts evaluation of eight different bioinformatics tools to predict viral tropism in different human immunodeficiency virus type subtypes detecting influenza epidemics using search engine query data enteropathogen resource integration center (eric): bioinformatics support for research on biodefense-relevant enterobacteria national institute of allergy and infectious diseases bioinformatics resource centers: new assets for pathogen informatics egasp: the human encode genome annotation assessment project knowledge construction from time series data using a collaborative exploration system predicting biological networks from genomic data piml: the pathogen information markup language informatics and infectious diseases: what is the connection and efficacy of information technology tools for therapy and health care epidemiology dna sequencing: bench to bedside and beyond investigating the metabolic capabilities of mycobacterium tuberculosis h rv using the in silico strain inj and proposing alternative drug targets anni . : a multipurpose text-mining tool for the life sciences resources for infection prevention and control on the world wide web what would you do if you could sequence everything? protein interactions and disease: computational approaches to uncover the etiology of diseases analysis of mixed sequencing chromatograms and its application in direct s rrna gene sequencing of polymicrobial samples genomic insights that advance the species definition for prokaryotes genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world systematic association of genes to phenotypes by genome and literature mining mega: a biologist-centric software for evolutionary analysis of dna and protein sequences bioinformatics-assisted anti-hiv therapy bioinformatics prediction of hiv coreceptor usage proteome informatics ii: bioinformatics for comparative proteomics ardb -antibiotic resistance genes database data integration and genomic medicine computational approaches to phenotyping: high-througput phenomics infectious disease surveillance application of 'next-generation' sequencing technologies to microbial genetics methods for computational gene prediction genomic profiling: interplay between clinical epidemiology, bioinformatics and biostatistics application of a geographic information system to the tracking and control of an outbreak of shigellosis the national microbial pathogen database resource (nmpdr): a genomic platform based on subsystem annotation annotation, comparison and databases for hundreds of bacterial genomes gendb -an open source genome annotation system for prokaryote genomes building a knowledge base for system pathology the pan-genome: towards a knowledge-based iscovery of novel targets for vaccines and antibacterials virhostnet: a knowledge base for the management and the analysis of proteome-wide virus-host interaction networks knowledge networks in the age of the semantic web bacterial pathogenomics complete genome sequence of a multiple drug resistant salmonella enterica serovar typhi ct genome sequence of yersinia pestis, the causative agent of plague genetics-squared: combining host and pathogen genetics in the analysis of innate immunity and bacterial virulence bioinformatics challenges of new sequencing technology exploring functional genomics for the development of novel intervention strategies against tuberculosis targettb: a target identification pipeline for mycobacterium tuberculosis through an interactome, reactome and genome-scale structural analysis bacterial genomics and pathogen evolution tb database: an integrated platform for tuberculosis research evolutionary epidemiology years on: challenges and prospects seeking a new biology through text mining genomics, system biology and drug development for infectious diseases clinical decision support and appropriateness of antimicrobial prescribing nucleotide sequence of bacteriophage x dna genomes, browsers and databases dengueinfo: a web portal to dengue information resources next-generation dna sequencing laboratory-guided detection of disease outbreaks: three generations of surveillance systems genomic profiling of pathogens for disease management and surveillance are we measuring the right thing? variables that affect the impact of computerized decision support on patient outcomes: a systematic review decision support systems for antibiotic prescribing towards bioinformatics assisted infectious disease control building an optiplante collaboratory to support microbial metagenomics biohealthbase: informatics support in the elucidation of influenza virus host-pathogen interactions and virulence host-pathogen interplay and the evolution of bacterial effectors information quality in proteomics automated bacterial genome analysis and annotation a probabilistic method for identifying start codons in bacterial genomes genome analysis of multiple pathogenic isolates of streptococcus agalactiae: implications for the microbial "pan-genome genotype-phenotype databases: challenges and solutions for the post-genomic era the human microbiome project e-predict: a computational strategy for species identification based on observed dna microarray hybridization patterns computing for comparative microbial genomics: bioinformatics for microbiologists shortgun metaproteomics of the human distal gut flora genomes and knowledge -a questionable relationship phi-base: a new database for pathogen host interactions discovery of virulence factors of pathogenic bacteria phidias: a pathogen-host interaction data integration and analysis system genomics, molecular imaging, bioinformatics, and bio-nano-info integration are synergistic components of translational medicine and personalized healthcare research infectious disease in the genomic era bioinformatics databases and tools in virology research: an overview primersnp: a web tool for whole-genome selection of allele-specific and common primers of phylogenetically-related bacterial genomic sequences real-time surveillance and decision support: optimizing infection control and antimicrobial choices at the point of care text-mining of pubmed abstracts by natural language processing to create a public knowledge base on molecular mechanisms of bacterial enteropathogens infectious disease informatics and outbreak detection key: cord- - s akmdp authors: mubareka, samira; groulx, nicolas; savory, eric; cutts, todd; theriault, steven; scott, james a.; roy, chad j.; turgeon, nathalie; bryce, elizabeth; astrakianakis, george; kirychuk, shelley; girard, matthieu; kobinger, gary; zhang, chao; duchaine, caroline title: bioaerosols and transmission, a diverse and growing community of practice date: - - journal: front public health doi: . /fpubh. . sha: doc_id: cord_uid: s akmdp the transmission of infectious microbes via bioaerosols is of significant concern for both human and animal health. however, gaps in our understanding of respiratory pathogen transmission and methodological heterogeneity persist. new developments have enabled progress in this domain, and one of the major turning points has been the recognition that cross-disciplinary collaborations across spheres of human and animal health, microbiology, biophysics, engineering, aerobiology, infection control, public health, occupational health, and industrial hygiene are essential. collaborative initiatives support advances in topics such as bioaerosol behavior, dispersion models, risk assessment, risk/exposure effects, and mitigation strategies in clinical, experimental, agricultural, and other field settings. there is a need to enhance the knowledge translation for researchers, stakeholders, and private partners to support a growing network of individuals and agencies to achieve common goals to mitigate inter- and intra-species pathogen transmission via bioaerosols. the transmission of infectious microbes via bioaerosols is of significant concern for both human and animal health. however, gaps in our understanding of respiratory pathogen transmission and methodological heterogeneity persist. new developments have enabled progress in this domain, and one of the major turning points has been the recognition that cross-disciplinary collaborations across spheres of human and animal health, microbiology, biophysics, engineering, aerobiology, infection control, public health, occupational health, and industrial hygiene are essential. collaborative initiatives support advances in topics such as bioaerosol behavior, dispersion models, risk assessment, risk/exposure effects, and mitigation strategies in clinical, experimental, agricultural, and other field settings. there is a need to enhance the knowledge translation for researchers, stakeholders, and private partners to support a growing network of individuals and agencies to achieve common goals to mitigate inter-and intra-species pathogen transmission via bioaerosols. keywords: bioaerosols, microbes, virus, infections, viral dissemination, network, caniban, collaborations the importance of infectious bioaerosols in disease transmission has been long-acknowledged, yet poorly understood. paltry data and methodological heterogeneity limit many related studies. effective ventilation and infection prevention and control (ip & c) measures in the form of droplet and airborne isolation in healthcare institutions underscore the contribution of these modes of pathogen dispersion. moreover, recent outbreaks such as the severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) outbreaks highlight major gaps in our ability to assess and determine risk and to mitigate patient and healthcare worker (hcw) exposure alike ( , ) . the sars outbreak was eventually controlled in the absence of an effective vaccine or antiviral, strictly through public health interventions and ip & c measures aimed at controlling, among other things, bioaerosols emitted by infected patients ( ) . a decade on from this experience, mers-cov has caused community and healthcare-associated severe acute respiratory infections in the middle east and south korea, spreading by similar mechanism(s) ( ) ( ) ( ) . from an animal health perspective, pathogens such as porcine reproductive and respiratory syndrome virus may be transmitted through the air for significant distances and have significant economic impact on the agricultural sector ( ) . efforts to characterize this and other relevant organisms have been undertaken previously, specifically looking at the burden of inoculum related to transmission ( ) and the prevention of spread of aerosols through various adequate ventilation strategies ( ) ( ) ( ) . emission of african swine fever virus into the air by infected pigs raises the possibility for droplet and/or airborne transmission of this virus, which has recently produced significant alarm after documented spread in china ( ) ( ) ( ) . this hemorrhagic virus is associated with high mortality and significant loss for producers through depopulation and trade restrictions ( , ) . in recent years, new developments have enabled progress in bioaerosol research, thus establishing a community of practice in the field. although many developments have been technical, one of the major catalysts has been the recognition that crossdisciplinary collaborations across the various spheres of human and animal health, microbiology, engineering, aerobiology, infection control, public health, occupational health, and industrial hygiene are essential. a network approach has proven successful in other cross-disciplinary fields, including one health and eco-health whereby wildlife, computational and evolutionary biologists, microbiologists, virologists, epidemiologists, ecologists, environmental scientists, climatologists, and human, animal, and public health practitioners are collaborating to address challenges in zoonotic diseases research and control ( , ) . this has enhanced surveillance efforts and launched ambitious, large scale projects such as the global virome project, though gaps remain in stakeholder engagement and monitoring activities ( , ) . translation of bioaerosol research stands to benefit in a similar fashion, provided coordinated and committed efforts. potentially infectious bioaerosols are pertinent to a wide range of pathogens, some of which may be endemic and cause sporadic infections and outbreaks (e.g., mycobacterium tuberculosis and legionella pneumophila, the causative agents of tuberculosis and legionnaires' disease, respectively) and/or have potential to cause epidemics or pandemics (e.g., influenza virus a) ( ) ( ) ( ) ( ) . in addition, the importance of potentially infectious bioaerosols across different settings is underscored. these include, but are not limited to, agriculture (both crop and livestock), wastewater treatment plants, environmental reservoirs (soil and water), acute and long-term healthcare institutions, and shared public spaces (transportation hubs, recreational areas, etc.). here, we discuss the state of the science for this nascent field and identify gaps requiring urgent attention. progress in the field has been stimulated by advances in other areas which have been applied to the study of infectious bioaerosols. these include: (a) enhanced detection by molecular methods, principally real-time and quantitative pcr, nextgeneration sequencing, metagenomics, and biosensors ( ) ( ) ( ) ( ) ( ) ( ) , and (b) establishment of both conventional and novel infrastructure, such as small and large-scale wind tunnels, biocontained rotating drums for aging aerosols, and field-ready aerosol samplers ( ) ( ) ( ) . ongoing research has also facilitated the development and dissemination of procedures and protocols for experimental work, including artificial aerosols, as well as animal models of transmission including the ferret model for influenza virus transmission and macaque model for ebola virus transmission ( , ) . whilst aerosols can be used as a means of delivery of therapeutic agents directly to the respiratory tract, they may also be used for the nefarious dispersion of human or animal pathogens. scientists focused on bioaerosol generation, pathogen survival in air and aerobiological fitness must be acutely cognizant of any potential for dual use and routinely re-assess projects and proposed experiments with this perspective in mind. oversight by institutional biosafety officers and committees may also consider this aspect of bioaerosol research during risk assessments and other internal approval processes. given the role of potentially infectious bioaerosols to transmit infectious agents to human and animal populations, it stands to reason that their detection and characterization will ultimately contribute to mitigating the spread of disease. this is possible through efforts focused on the following themes: unresolved fundamental questions may be answered by studying infectious bioaerosols. areas meriting attention are numerous and a few are briefly discussed here. in the case of agents for which airborne transmission is well-recognized (i.e., m. tuberculosis), associations between particle size and generation, pathogen content and virulence, as well as the deposition within the respiratory tract may be determined. the physical and chemical aspects of aerosols including the effects of relative humidity can affect pathogen viability and understanding these aspects of bioaerosol behavior may be beneficial to understanding conditions for controlling bioaerosol dispersion ( ) ( ) ( ) . moreover, pathogens such as viruses may vary with respect to isoelectric points, possibly imparting different charges to infectious bioaerosols which may affect their behavior ( , ) . finally, although climate change and pollution have been major issues within our biosphere, little is known about the impact of pollution particulates and gases, such as ozone, on the biology and physical properties of bioaerosols ( ) . our understanding of bioaerosol production from expulsion events such as breathing, talking, sneezing, and coughing have generally been extrapolated from models, though more recent empiric evidence has become available, significantly enhancing our understanding of the transmission potential of bioaerosol emissions from naturallyinfected hosts ( , ) . awareness of factors contributing to particle velocity and penetration into space enables modeling strategies that inform engineering controls in a multitude of settings. for example, in healthcare, understanding the dispersion of potential pathogens in the environment can inform infection prevention and control practices ( ) . there is also potential benefit to determining pathogen characteristics. if enhanced infectious bioaerosol survival in air is associated with certain strains, genotypes or mutations, this provides possibilities for follow-on work to (a) determine the mechanism and (b) enhance surveillance. the latter would have implications for public and animal health. studying these factors individually or in combination can optimize air handling and other mechanical, environmental or chemical means of neutralizing bioaerosols prior to host exposure, thus alleviating dependence on personal protective equipment, which is the last means of protection prior to exposure. new understanding of the role of bioaerosols in the propagation of human and animal pathogens could lead to new practices. as it stands, it remains uncertain which bioaerosol particles are predominantly responsible for personto-person transmission. there may also be differences between pathogen populations in the infected host vs. what is emitted into the air. this poses an excellent opportunity to leverage advances in metagenomics with developments in aerobiology to extract more sophisticated data from aerosol samples and, potentially, determine genetic bottlenecks for transmission. this would feed back into more fundamental work on bacterial or viral determinants of transmission, as well as lead to novel means for surveillance and mitigation. similarly, translational studies enhance our understanding of risk and determinants of exposure. for many settings and situations, several components of risk assessments are based on limited evidence. this is of particular concern from an occupational health perspective, in healthcare (e.g., purported aerosol-generating procedures), agricultural, or specific settings such as wastewater treatment plants ( ) ( ) ( ) . developing the capacity to generate empiric data on pathogen content and biology from bioaerosols can lead to tools for outbreak investigation, surveillance, and development of risk assessment strategies and policies. as the technology for biosensors accelerates, the possibilities for rapid point-of-care testing and even remote sampling are highlighted. integration of biosensors with bioaerosol sampling ( ) has significant potential for early warning and public safety. as advances are made, capacity is built to optimize and evaluate known and novel means to control the dispersion of infectious bioaerosols. these include the use of germicidal and pulsed ultraviolet light, mechanical air filtration and respiratory protection which have both mechanical and electrostatic filtering systems ( , ) . in addition, the potential to use ozone to control indoor bioaerosols in urban and rural settings is being examined. ozone is a strong oxidizing agent having high redox potential and a short half-life, dependent on temperature. it has been used extensively in the food industry (specifically water treatment, washing of produce, and food preparation), as well as in domestic restoration to remediate odors and smoke damage. its historical use and proven efficacy demonstrate its potential to remediate contaminated indoor air ( , ) . this would present a novel application of a known treatment modality. a number of important gaps have been identified ( ) and these can be prioritized based on potential benefit: biocontainment of infectious particles is the first consideration when aerosolizing human and/or animal pathogens in an experimental setting. these systems are generally customdesigned and constructed, with few facilities capable of conducting aerosol work with risk group pathogens, and fewer still with the capacity to work with risk group or agents. as challenges in infrastructure are overcome, opportunities to optimize and improve methods and techniques in bioaerosol research must be taken. a limitation of many studies focused on viral bioaerosols is the pervasive use of nucleic acid detection, rather than infectious virus isolation. the latter is a much more accurate indicator of the infectious potential of a bioaerosol but is infrequently performed due to poor sensitivity and other technical issues ( ) . the development of sampling devices and techniques optimized to preserve pathogen viability would considerably advance the utility of studying bioaerosols for risk assessment and management. for other applications, a more rapid, field-ready point of care test would be useful and would offer remote sampling possibilities. biosensors have the potential to fill this gap and integration into aerosol sampling devices is under development ( ) . finally, the collection of nucleic acid may be leveraged to obtain more sophisticated information than is available by pcr and sanger sequencing. metagenomics on environmental samples has been well-described in other spheres and is currently being explored for air samples ( ) ( ) ( ) ( ) . to advance bioaerosol-related data interpretation, an effort must be made to standardize and share protocols and reagents to reduce the degree of data heterogeneity to enable meaningful analyses across studies. this may apply to animal models, artificial aerosolizations, collection strategies in the field and clinical settings, processing of samples, and detection methods. developing or adopting a standardized approach to aerosol sampling is challenging and requires substantial efforts to select the best sampling strategy to minimize sampling biases, optimize sample concentration and organism retrieval whilst preserving integrity ( ) . establishing standard approaches also enables training and implementation and eases knowledge translation amongst different groups (figure ) . multiple investigators have published small studies on the recovery of viral rna emitted by naturally infected humans using different approaches for recruitment, sampling, processing, and detection. unfortunately, the results are difficult to compare and, standing alone, are of limited statistical significance ( , ( ) ( ) ( ) . by standardizing approaches, a more feasible method can be used to compare separate studies allowing for larger multi-center studies that are substantially more conclusive and impactful. additional educational and operational needs, such as training of research personnel, proper use of personal protective equipment and developing decontamination protocols must be addressed. as more highly qualified personnel are properly trained, the greater the capacity for designing and utilizing experimental systems and for completing meaningful clinical and field studies. the need for cross-disciplinary experience is underscored, since robust knowledge of physics, mechanical engineering, and microbiology are required. fostering this expertise requires a collaborative effort as each discipline offers unique insight. currently, there are a limited number of workshops or accredited courses available to cultivate both interest and baseline knowledge. although the profile of bioaerosol research is rising, few trainees are exposed to the field early in their careers. courses such as bioaérosols et aérobiologie (bioaerosols and aerobiology) developed at the université laval in québec may help close this gap further given the opportunity to expand reach. the relevance of bioaerosol data to occupational health and infection prevention and control requires attention. validation of bioaerosol data for risk assessments and risk management is needed. air sampling was conducted during both sars-cov and mers-cov outbreaks, accruing important insight into the environmental distribution and persistence of these coronaviruses ( , , ) . the absence of data for more common, lower consequence pathogens represents missed opportunities to build a contextual and impactful body of knowledge during inter-outbreak and inter-pandemic periods. to develop evidence-based policy and ensure the relevance of this work, early stakeholder engagement is needed. consultation with human and animal public health, infection prevention and control, industrial hygiene, and occupational health stakeholders will ensure that the work is germane to the challenges presented by bioaerosol exposures. this pull also increases the likelihood that there will be data generated for risk assessment, policy development and implementation. part of the chaos that characterized the sars epidemic can be attributed to a lack of knowledge regarding the route(s) of transmission. this remains true for high consequence coronaviruses, as well as for a range of other respiratory pathogens, both novel and established. we propose the following to address the challenges outlined above and to further develop the field of applied bioaerosol research: . an open network approach working in isolation, microbiologists, aerobiologists, engineers, and epidemiologists have made only incremental progress. a synergistic and pluralistic approach is required for action-driven research which effects change. as capacity grows, so will opportunities for training and response. a networked approach will lead to context-specific follow-on research to ultimately inform policy for the mitigation of respiratory pathogens spread in healthcare institutions, agricultural settings and public spaces. toward this end, the present authors have established the canadian infectious bioaerosols network (caniban) that brings together members of each of these disciplines with the primary objectives of understanding transmission of pathogens. a network would form a hub around which key resources such as infrastructure, equipment, and operational procedures could be shared, thus also enabling training and underscoring best practices in biosafety and biosecurity. shared resources may encompass wind tunnels, cough chambers and mannequins, animal exposure and other biosafety enclosures, and rotating drums to examine pathogen survival in air, together with instrumentation and analysis tools for fluid flow measurement and biochemical assays. limited awareness and understanding of infectious bioaerosols among potential knowledge users, coupled with equally limited outreach by bioaerosol researchers has restricted knowledge transfer and applications. knowledgeuser engagement in research planning from the outset and ongoing involvement through to dissemination is key for effective projects. potential knowledge users include infection prevention and control practitioners, infectious disease specialists, veterinarians and animal health epidemiologists, industrial hygienists, and public health agencies. increasingly, bioaerosol sampling has been proposed and implemented in outbreak investigations and pathogen surveillance using ad hoc approaches. the development of best practices in these areas is essential to generating valid and actionable data. in summary, a collective path for researchers, stakeholders, and private partners is needed to support a network of individuals and agencies to achieve common goals. as the field of bioaerosol studies grows, applications will diversify, along with novel technologies for remote sampling and point of care testing 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particulates collected from the international space station a next generation sequencing approach with a suitable bioinformatics workflow to study fungal diversity in bioaerosols released from two different types of composting plants distribution of airborne influenza virus and respiratory syncytial virus in an urgent care medical clinic influenza virus emitted by naturally-infected hosts in a healthcare setting quantification of influenza virus rna in aerosols in patient rooms detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units airborne severe acute respiratory syndrome coronavirus concentrations in a negative-pressure isolation room we should like to thank the centre de recherche de l'institut universitaire de cardiologie et de pneumologie de québec (criucpq), assek technology, quebec frqs respiratory health network, the groupe de recherche en santé respiratoire (geser), and tsi for supporting the symposium on the transmission of respiratory pathogens in québec city, qc, canada, and symposium participants for their contributions during the course of this event. we are also grateful for support from the canadian institutes of health research, the natural sciences and engineering research council of canada and the ontario ministry of labor. key: cord- -tulne v authors: rabaa, maia a.; tue, ngo tri; phuc, tran my; carrique-mas, juan; saylors, karen; cotten, matthew; bryant, juliet e.; nghia, ho dang trung; cuong, nguyen van; pham, hong anh; berto, alessandra; phat, voong vinh; dung, tran thi ngoc; bao, long hoang; hoa, ngo thi; wertheim, heiman; nadjm, behzad; monagin, corina; van doorn, h. rogier; rahman, motiur; tra, my phan vu; campbell, james i.; boni, maciej f.; tam, pham thi thanh; van der hoek, lia; simmonds, peter; rambaut, andrew; toan, tran khanh; van vinh chau, nguyen; hien, tran tinh; wolfe, nathan; farrar, jeremy j.; thwaites, guy; kellam, paul; woolhouse, mark e. j.; baker, stephen title: the vietnam initiative on zoonotic infections (vizions): a strategic approach to studying emerging zoonotic infectious diseases date: - - journal: ecohealth doi: . /s - - - sha: doc_id: cord_uid: tulne v the effect of newly emerging or re-emerging infectious diseases of zoonotic origin in human populations can be potentially catastrophic, and large-scale investigations of such diseases are highly challenging. the monitoring of emergence events is subject to ascertainment bias, whether at the level of species discovery, emerging disease events, or disease outbreaks in human populations. disease surveillance is generally performed post hoc, driven by a response to recent events and by the availability of detection and identification technologies. additionally, the inventory of pathogens that exist in mammalian and other reservoirs is incomplete, and identifying those with the potential to cause disease in humans is rarely possible in advance. a major step in understanding the burden and diversity of zoonotic infections, the local behavioral and demographic risks of infection, and the risk of emergence of these pathogens in human populations is to establish surveillance networks in populations that maintain regular contact with diverse animal populations, and to simultaneously characterize pathogen diversity in human and animal populations. vietnam has been an epicenter of disease emergence over the last decade, and practices at the human/animal interface may facilitate the likelihood of spillover of zoonotic pathogens into humans. to tackle the scientific issues surrounding the origins and emergence of zoonotic infections in vietnam, we have established the vietnam initiative on zoonotic infections (vizions). this countrywide project, in which several international institutions collaborate with vietnamese organizations, is combining clinical data, epidemiology, high-throughput sequencing, and social sciences to address relevant one-health questions. here, we describe the primary aims of the project, the infrastructure established to address our scientific questions, and the current status of the project. our principal objective is to develop an integrated approach to the surveillance of pathogens circulating in both human and animal populations and assess how frequently they are exchanged. this infrastructure will facilitate systematic investigations of pathogen ecology and evolution, enhance understanding of viral cross-species transmission events, and identify relevant risk factors and drivers of zoonotic disease emergence. the burden of infectious diseases in human populations in low-and middle-income countries remains high (sepú lveda and murray ); in the majority of cases, disease etiologies are never determined (kotloff et al. ; mulholland ; susilawati and mcbride ) , often due to inadequate laboratory diagnostic capacity. the causative agents of diseases of unknown origin (duos) fall broadly into three groups: ( ) known pathogens that were not tested for or misdiagnosed; ( ) previously unrecognized but common pathogens (e.g., parechoviruses and human metapneumovirus); and ( ) newly emerging or re-emerging pathogens (e.g., filoviruses, henipaviruses, and coronaviruses). all three categories are relevant to policymakers when trying to allocate resources for prevention. while the third category accounts for the smallest number of infections, the impact of emerging pathogens, particularly those with potential for human-tohuman transmission, may range from disruptive to catastrophic. developing an infrastructure to tackle these issues is challenging and costly, yet interventions that are implemented without supporting data on duo frequency and population risk may prove ineffective. newly emerging pathogens in low-to middle-income countries are likely to originate from an animal source. approximately % of all human pathogen species are known to be zoonotic (woolhouse and gowtage-sequeria ) and pathogens that infect multiple species are three times as likely to emerge into human populations than host-restricted pathogens (taylor et al. ) . some pathogens undergo enzootic transmission in reservoir animal populations with occasional spillover into a human host and little to no onward transmission (jonsson et al. ; wertheim et al. ). other pathogens display human-to-human transmission after spillover and may result in large epidemics (drosten et al. ; gire et al. ; janies et al. ) . some of these emerging pathogens may eventually adapt to circulate exclusively among humans, leading to epidemic or endemic transmission cycles (holmes and twiddy ; taubenberger ) . the frequency with which such zoonotic transmission events occur and the likelihood that a pathogen will adapt to exclusively human transmission are largely determined by behavioral and immunological factors in the host, along with ecological and evolutionary factors of the pathogen (karesh et al. ) . it has been proposed that the focus of zoonosis research should move toward the early detection of zoonotic pathogens, in particular those that exhibit potential for emergence in humans (morse et al. ) . this is particularly poignant given the current epidemic of ebola virus in western africa, where sustained animal/human surveillance prior to the start of the outbreak may have had an impact on the scale of the epidemic. large-scale investigations of emerging zoonotic infections are challenging. monitoring is subject to ascertainment biases, whether at the level of species discovery, emerging disease events, or disease outbreaks in human populations. disease surveillance is often performed post hoc, driven by a response to recent events and by the availability and sustainability of detection technologies. additionally, the inventory of pathogens that exist in mammalian and other reservoirs is astonishingly incomplete (anthony et al. ) and identifying those with the potential to cause disease in humans is rarely possible in advance. finally, the nature of the species barrier and the factors that enable pathogens to cross these barriers and establish transmission among humans are largely unknown. the factors mentioned above limit our ability to study zoonotic pathogens in detail; therefore, we identified the need for active monitoring of human and animal reservoirs of infection to characterize community exposures and infections in a developing country with a prolonged history of zoonotic transmission events and outbreaks. the two major requirements for understanding the burden and diversity of zoonotic infections and the behavioral/demographic risks of infection are the establishment of surveillance networks in populations that maintain regular contact with diverse animal populations and the simultaneous characterization of pathogen diversity in human and animal populations. here, we describe a project that is currently underway in communities across vietnam in which we are collecting clinical samples and associated clinical, epidemiological, and demographic data, which will be combined with high-throughput viral genome sequences and qualitative social sciences data to address key onehealth questions with the aim of better understanding the origins, risks, and emergence of zoonotic infections. southeast asia is a global hotspot for emerging infectious diseases (morse et al. ) , and vietnam has been an epicenter of emerging disease activity over the last decade (dinh et al. ; reynolds et al. ; vinh et al. ; vu tra my et al. ; wertheim et al. ). vietnam has a large population ( , , in (statistical yearbook of vietnam, )), some of the highest livestock densities in southeast asia (gerber et al. ) , and a substantial burden of duos (ho dang trung et al. ; thompson et al. ) . furthermore, approximately % of the vietnamese population reside in rural areas and participate in small-scale animal production (statistical yearbook of vietnam, ). we hypothesize that vietnam's demography, varied animal production systems, and food consumption habits facilitate the spillover of zoonotic pathogens into humans. specifically, we predict that exotic food production systems with mixed species and limited biosecurity, abattoirs and wet markets operating with minimal basic hygiene, poor cold chains for meat distribution, limited meat inspections in the market sector, and consumption of raw/undercooked blood, meat, organ tissues, and wild animal products promote the risk of zoonotic pathogen transmission. vizions, initiated in march , is now an established platform for one-health research in vietnam. with vizions, we are aiming to integrate traditional clinical, epidemiological, and medical anthropological methods with new approaches for pathogen detection and discovery, including novel sequencing approaches combined with phylogenetic analysis to characterize pathogen populations. the principal aims of vizions are presented in box . to deliver the aims of vizions, we established two fundamental components: a hospital disease surveillance program to characterize endemic infections, novel infections and duos, and a high-risk sentinel zoonosis cohort to assess disease incidence and pathogen transfer ( figure ). the hospital surveillance component of vizions is underway in seven locations. these locations are shown in figure , and collaborating vietnamese institutions are outlined in box . over a -year period of enrollment at each site, we aim to enroll up to hospitalized cases of each of four key clinical syndromes (central nervous system (cns) infections, enteric infections, jaundice, and respiratory tract infections) that may be caused by a zoonotic pathogen. the aim to enroll cases under each clinical syndrome was based both on operational capacity and local epidemiology, specifically the ability to calculate population attributable fractions (pafs) for specific pathogens (thus providing supporting evidence of disease etiology). these samples sizes were considered to be sufficient to estimate pafs to informative levels of precision: for example, a pathogen found in % of cases and % of severe cases will give paf = % with approximate % confidence intervals of - %. from pilot data, we anticipated identifying a pathogen associated with the defined clinical syndromes in % of cns cases, % of respiratory cases, % of enteric cases, and % of hepatitis cases; roughly - % of these pathogens were predicted to be viruses. these enrollment targets should provide samples and metadata from known infections and provide > % power to detect a pathogen that is present in just / patients with a specified syndrome. upon enrollment and informed consent, data including diagnostic investigations (clinical and laboratory), age, sex, occupation, animal exposure, residential address, household size, income, and wealth indicators are recorded via a standardized case report form. the residence of each case is geo-located and will ultimately be associated with an existing suite of spatial datasets. additionally, we are collecting routine data for all hospital admissions (retrospectively and prospectively) via the international classification of diseases (icd- ) and are modeling hospital catchment populations by comparing the spatial distribution of vizions-enrolled patients to the entire population box . the principal aims of the vizions project . to establish a model international collaborative consortium with an integrated approach to human and animal health research . to estimate the burden of disease (focusing on viral and zoonotic diseases), and investigate the disease epidemiology in patients hospitalized with specified clinical syndromes and infections in a cohort of high-risk individuals occupationally exposed to animals; with targeted sampling from domestic animals and wildlife in association with these individuals . to elucidate the etiology of infectious diseases of unknown origin in the human population, and provide a repository of putative pathogens for further study . to characterize genetic diversity within virus populations on either side of the species barrier in order to understand cross-species transmission and disease emergence . to identify socio-demographic, environmental, and behavioral drivers for disease emergence . to create a platform and resource for further research on zoonotic disease agents ( ) a longitudinal cohort study of occupational risk of zoonotic infections, plus records of risk behaviors, with linked sampling of putative animal reservoirs that will generate > , specimens for molecular investigation being performed in dong thap, dak lak, khanh hoa, and ba vi (large pink circles). entering the healthcare facilities. these data are being used further to predict atypical patterns of hospital admissions to detect outbreaks ( figure sopharyngeal swab) are collected and subjected to a bacteriological and viral diagnostic algorithm for each clinical syndrome. this diagnostic process is conducted within vietnam (both at study sites and at oxford university clinical research unit (oucru) laboratories) and was designed to identify the major known causes of the four specific syndromes of interest. cases are subsequently categorized as being associated with a known causative agent or as a duo. at the time of writing (may ), we have recruited > patients across the four syndromes. working with local academic and governmental partners in three provinces (figure , box ) , we have additionally established a high-risk, sentinel cohort. we have recruited individuals that we consider to be at high-risk for zoonotic pathogen transfer as a consequence of occupational exposure to animals (figure ). cohort members were selected from ( ) farming households, especially those with mixed livestock and wildlife species, ( ) wildlife restaurants, ( ) abattoirs, ( ) wet markets, and ( ) other high-risk occupational groups such as animal health workers/veterinarians and wildlife trappers/traders. potential cohort members were identified and screened for suitability and, after providing informed consent, were asked to provide a blood sample, nose and throat swab, and a rectal swab, followed by the administration of a questionnaire to document socioeconomic factors, healthseeking behavior, occupational hazards, animal exposure, and food consumption habits. participants are then interviewed and sampled annually to document risk factors for zoonotic infections, such as animal exposure, food consumption habits, and disease episodes within their household or among their animals. throughout the three-year study, participants who develop illness are encouraged to visit the local study hospital or are visited at home by a member of the local preventative medicine centre (pmc). the medical workers determine whether the illness is likely to represent an infectious disease episode and, if so, collect clinical samples from the cohort member and administer a disease episode questionnaire related to animal exposures. additionally, the local sdah are informed and a team of animal health workers visits the site within hours of the reported disease episode for responsive sampling of representative animal species present at the study site (figure ) . this longitudinal cohort component of vizions is designed to enable detection and monitoring of cross-species virus 'chatter' between animal and human populations. further, these repositories of linked human and animal specimens will permit us to determine the incidence and seroprevalence of selected zoonoses within the cohort, define viral diversity in humans with different animal exposures and in representative non-human species, and characterize human-animal contact behavior in a variety of settings. such behavior considered in the context of this project includes the reported slaughter, butchering, and rendering of livestock and wildlife, handling or consumption of diseased animals, and consumption of raw animal products. these data and specimen repositories are expected to provide a resource for the identification of novel or unexpected agents of human disease and their potential zoonotic origins, and to deliver information that can be used to examine the barriers to and drivers of cross-species transmission. phylogenetic studies have been utilized to investigate endemic and emerging pathogen populations at various scales and offer significant insight into the evolutionary and epidemiological characteristics of pathogens involved in disease emergence (grenfell et al. ; pybus and rambaut ) . within the context of vizions, we are performing full and partial genome sequencing to characterize both ubiquitous and novel viral populations present in vietnam. using these sequences, we aim to identify principal zoonotic viral populations and investigate their dynamics in human and animal populations. initial pathogens prioritized for investigation include rotavirus, hepatitis e virus, influenza a virus, and enteroviruses. for these and other viral populations that are found to be endemic in vietnam or show extensive transmission, we will use phylogenetic methods to characterize their spatial and temporal spread through animal and/or human populations; these investigations will provide information on large-and small-scale networks of disease transmission within the country, with which we hope to gain a better understanding of the patterns and determinants of pathogen dispersal. in the case of duos, where no pathogen is detected by standard screening methods, metaviral sequencing methods (cotten et al. ) will be used to identify viral nucleic acids within the sample. to provide further context on the extent of viral exchange between human and animal populations and characterize viral diversity across species of interest, we will also use metagenomic methods to characterize the viromes of healthy human and animal specimens. viruses that show zoonotic potential in these populations will be further investigated through population-level serological studies to determine the extent and risks of these zoonotic infections across vietnam. anthropogenic factors of expanding peri-urban regions, increased natural resource use, changing scales of agriculture, and human encroachment into wildlife habitat have led to the development of intensive agricultural practices and exotic species farming in vietnam. these factors increase the frequency, duration, and intensity of wildlife, livestock, domestic animal, and human interaction (rhyan and spraker ) . vietnam now has unprecedented demands for meat from livestock, and many wildlife species are now commonly farmed (e.g., bamboo rat, wild boar, civet, porcupine) (brooks et al. ; drury ). the maintenance of large animal populations (domestic and wildlife species) generally involves supplemental feeding and the introduction of animals from other populationsall factors that facilitate pathogen transmission (wildlife conservation society ). additionally, the presence of mixed wildlife and livestock populations on farms increases the risk of introduction of novel pathogens to potentially immunologically naive populations. although human exposure to livestock is known to be an important risk factor for cross-species transmission of zoonotic pathogens in this region (dinh et al. ; ho et al. ; mackenzie ) , the risk posed by wildlife remains unknown; this is due partly to an incomplete understanding of wildlife farming and consumption practices. we aim to investigate the socio-cultural context of wildlife consumption and farming within a subset of cohort participants with exposure to wildlife. in parallel to the annual participant interviews, we are conducting qualitative research including participant observation and indepth interviews to assess contextual and behavioral risk factors in a subset of individuals with specific exposures to wildlife species, including wildlife farmers, trappers, traders, and restaurant workers. at the time of writing, the hospital component of the vi-zions project is running to schedule with respect to data collection, patient recruitment, and pathogen screening. this has been a substantial resource for collaborating hospitals, where laboratory diagnoses are seldom performed and infections are typically identified through standard clinical observations. therefore, we are developing a thorough understanding of the disease burden of many key pathogens across vietnam but still have an alarming rate of duos, ranging from . % ( of samples screened) in diarrhea cases to . % ( of samples screened) in cns cases ( / cases overall across the four syndromes have no identified pathogen). while these results are not reported back to hospitals in real time, allowing an immediate improvement of clinical assessment, they are providing vital seasonal information regarding annual fluctuations in hospital attendance and disease etiology. a key lesson from the hospital component of the study has been understanding and accounting for the gaps in diagnostic capabilities for patients who present with a disease of a presumed infectious origin in the hospital setting. a lack of diagnostic technology and funding to perform adequate diagnostics is a major issue in the healthcare systems of many developing countries and leaves treating clinicians to make diagnoses based on medical intuition and experience alone. this impacts the quality and consistency of data coming in from hospital sites, requiring additional considerations and generalizations to be made in our analyses, particularly those dependent on epidemiological data collected directly from the hospitals. furthermore, even though we are performing a considerable range of microbiological and molecular screening panels for major pathogens, the rate of duos is still high. by working in further detail on these duo specimens, we hope to uncover previously undiagnosed pathogens, thus reducing the proportion of patients hospitalized with duos. a better understanding of regional pathogens causing infections should, eventually, lead to better diagnostic approaches, which ideally should be performed in a multiplex system in a timeframe that is relevant to patient care and cost-effective for use in developing country settings. with respect to the cohort component of the vizions project, we are currently following individuals in three provinces; all of these individuals have now been in the cohort for at least year. to date, we have recorded disease episodes in these individuals, mainly manifesting as fever, diarrhea, or respiratory infections. the collections of samples (from both routine and disease sampling) from this component of the study are, at this scale, unique and many are currently in a metaviral nucleic acid sequencing pipeline. we are currently extracting and screening over fecal specimens from humans and animals that are included in the cohort study using these methods. further, we are screening for a range of endemic zoonotic pathogens including influenza, hepatitis e, rotavirus, and calciviruses. the cohort component of vizions will be complete in mid- . the formation and maintenance of vizions has been a substantial nationwide effort, but we think that the nature of the now established network will be productive and will yield a major resource for understanding zoonotic infections in vietnam. probably the greatest lesson learned has been in getting the various governmental and non-governmental organizations to work in unison. in vietnam, like in many other countries, governmental and provincial departments supporting animal and human health largely work in administrative silos, with little overlap in their political direction or interests. the relationships that have been created between the independent government departments and academic research institutions will be key for the long-term sustainability of this network. however, as a consequence of zoonotic disease outbreaks occurring relatively infrequently, continual governmental funding for such a resource is often overlooked. nevertheless, given our engagement with the various governmental departments and members of the vietnamese public, we think that maintaining this infrastructure is both practical and financially viable. through training schemes for laboratory workers, medical staff, community healthcare workers, and veterinarians, we are providing an ongoing capacitybuilding role and have provided support for several potential outbreaks and atypical clinical presentations in both the human and animal communities. conversely, we believe that assessing the amount of zoonotic transfer and identifying pathogens likely to have an impact on human health will be the evidence required to persuade continued investment. here we have described an established multidimensional platform in vietnam aimed to tackle scientific issues surrounding the origins and emergence of zoonotic infections. this countrywide project, in which several international institutions collaborate alongside vietnamese organizations, is combining clinical data, epidemiology, highthroughput sequencing, and social sciences to address key one-health questions that cannot be addressed independently. our overarching objective is to develop an integrated and sustainable approach to the surveillance of pathogens circulating in both human and animal populations. this infrastructure will facilitate systematic investigations of pathogen ecology and evolution, enhance the understanding of viral cross-species transmission events, and allow us to identify the relevant risk factors and drivers of zoonotic disease emergence. the capacity of vizions to detect and characterize unusual disease events in nearly real time will facilitate a new approach to protecting public health in vietnam. a strategy to estimate unknown viral diversity in mammals the conservation impact of commercial wildlife farming of porcupines in vietnam full genome virus detection in fecal samples using sensitive nucleic acid preparation, deep sequencing, and a novel iterative sequence classification algorithm risk factors for human infection with avian influenza a h n clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection reducing urban demand for wild animals in vietnam: examining the potential of wildlife farming as a conservation tool geographical determinants and environmental implications of livestock production intensification in asia genomic surveillance elucidates ebola virus origin and transmission during the outbreak unifying the epidemiological and evolutionary dynamics of pathogens aetiologies of central nervous system infection in viet nam: a prospective provincial hospital-based descriptive surveillance study risk factors of streptococcus suis infection in vietnam. a case-control study the origin, emergence and evolutionary genetics of dengue virus evolution of genomes, host shifts and the geographic spread of sars-cov and related coronaviruses a global perspective on hantavirus ecology, epidemiology, and disease ecology of zoonoses: natural and unnatural histories the global enteric multicenter study (gems) of diarrheal disease in infants and young children in developing countries: epidemiologic and clinical methods of the case/control study emerging zoonotic encephalitis viruses: lessons from southeast asia and oceania daszak p ( ) prediction and prevention of the next pandemic zoonosis global burden of acute respiratory infections in children: implications for interventions evolutionary analysis of the dynamics of viral infectious disease factors associated with nosocomial sars-cov transmission among healthcare workers in hanoi emergence of diseases from wildlife reservoirs the state of global health in acute undifferentiated fever in asia: a review of the literature the origin and virulence of the 'spanish' influenza virus risk factors for human disease emergence a prospective multi-center observational study of children hospitalized with diarrhea in rapid emergence of third generation cephalosporin resistant shigella spp. in southern vietnam novel porcine-like human g p[ ] rotavirus identified in hospitalized pediatric diarrhea patients in ho chi minh city streptococcus suis: an emerging human pathogen commercial wildlife farms in vietnam: a problem or solution for conservation? host range and emerging and reemerging pathogens the authors wish to acknowledge all local and international partners that form the vizions consortium: oucru hcmc, song chau, hoang nguyen van minh, corinne thompson, vu thi ty hang the authors state that they have no competing interests. a strategic award from wellcome trust of great britain funded this work (wt/ ). sb and mfb are sir henry dale fellows, jointly funded by the wellcome trust and the royal society ( /z/ /z and /z/ /z, respectively). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. this article is distributed under the terms of the creative commons attribution . international license (http:// creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord- - baxwgc authors: nan title: introduction to the immune response date: - - journal: primer to the immune response doi: . /b - - - - . - sha: doc_id: cord_uid: baxwgc in this chapter we provide an overview of the immune system and its vital role maintaining human health. immune responses require the coordinated action of leukocytes that travel the body to eliminate threats posed by trauma, infection, toxins, and cancer. leukocytes communicate via direct contact and via production and receipt of soluble proteins and intercellular messenger proteins called cytokines. complete clearance of unwanted entities may involve both innate and adaptive leukocyte responses, which influence each other. some innate mechanisms require no induction and are completely non-specific, whereas others are inducible and involve broad receptor-mediated recognition of a limited number of pathogen-associated or damage-associated molecular patterns (pamps/damps). adaptive responses involve the selective activation of lymphocytes via engagement of their antigen receptors by a specific foreign antigen. the three major subsets of lymphocytes are t helper cells (th), cytotoxic t cells (tc) and b cells, which use distinct mechanisms to recognize antigen and carry out different effector functions. immune system malfunction can contribute to many clinical illnesses including autoimmune disorders, allergies, immunodeficiencies, chronic inflammation and cancer. stated that microbes invisible to the naked eye were responsible for specific illnesses, and the first human disease-causing organisms or pathogens were identified in the late s. at about the same time, louis pasteur applied jenner's immunization technique for smallpox to the prevention of various animal diseases. pasteur demonstrated that inoculation with a pathogen that had been weakened in the laboratory could protect against a subsequent exposure to the naturally occurring pathogen. it was pasteur who coined the term vaccination (from the latin vaccinus, meaning "derived from cows") for this procedure, in honor of jenner's work. the research of pasteur and other investigators spurred the evolution of immunology as a science distinct from (but related to) the established fields of microbiology, pathology, biochemistry and histology. today, immunology can be defined as the study of the cells and tissues that mediate immunity and the investigation of the genes and proteins underlying their function. a normal healthy person's body always strives to maintain homeostasis, a natural state of balance of all its organs and the nervous and circulatory systems. when this homeostasis is disturbed by either trauma, pathogens, or the deregulation of body cells (such occurs in cancer), the immune system responds in an attempt to restore balance. at its simplest level, this response involves identifying and clearing damaged and dying cells from the body. more complex responses have evolved to counteract assault on selected landmark discoveries in immunology and immunologists who have won nobel prizes for their work are featured in appendices a and b, respectively. the body by infectious agents, including bacteria, viruses, parasites and fungi. because these foreign invaders are literally everywhere on earth and constantly seeking vulnerable hosts, the immune system is constantly occupied with containing attacks from this quarter. indeed, despite the successful eradication of smallpox and the development of antibiotics, infectious diseases remain among the biggest killers on the planet. longstanding disorders such as malaria and tuberculosis, as well as "emerging" diseases caused by new pathogens such as west nile virus, severe acute respiratory syndrome (sars) virus, and h n influenza virus, present ongoing challenges to the immune system. humans are surrounded by other organisms-in the air, in the soil, in the water, on the skin, and on the mucosae, the protective layers of epithelial cells that line the gastrointestinal, urogenital and respiratory tracts. while most of these organisms are harmless and some are even beneficial, some are pathogenic. like all species, pathogens live to reproduce. in order to reproduce, however, many must penetrate a host's body or one of its component cells. infection is defined as the attachment and entry of a pathogen into the host. once inside the body or cell, the pathogen replicates, generating progeny that spread into the body in a localized or systemic fashion. the manner of this replication determines whether the pathogen is considered extracellular or intracellular. extracellular pathogens, such as certain bacteria and parasites, do not need to enter cells to reproduce. after accessing the body, these organisms replicate first in the interstitial fluid bathing the tissues and may then disseminate via the blood ( fig. - ) . intracellular pathogens, such as viruses and other bacteria and parasites, enter a host cell, subvert its metabolic machinery, and cause it to churn out new virus particles, bacteria, or parasites. these pathogens may then also travel systemically by entering the blood. for both intracellular and extracellular pathogens, if the infectious agent overwhelms normal body systems or interferes with cellular functions, the body becomes "sick." this sickness is manifested as a set of characteristic clinical symptoms that we call an "illness" or "disease." pathogens that are not blocked by epithelial barriers can gain access to underlying cell layers. extracellular pathogens replicate outside host cells, whereas intracellular pathogens must enter host cells to replicate. in both cases, if infection is not controlled, the pathogen may spread locally in the tissues or enter the bloodstream and spread systemically. our bodies are under constant assault by harmful microbes, yet, most of the time, assaults by these organisms are successfully repelled and disease is prevented. this resistance is due to both basic and sophisticated immune responses that combat pathogens. simply put, the job of the immune response is to "clean up" infections in the interstitial fluid, tissues and blood, and to destroy infected host cells so that neighboring host cells do not share their fate. because pathogens are constantly evolving mechanisms to evade or block immune defenses, the immune system must constantly adapt to maintain its effectiveness. it is a continual horse race as to which will be the more successful mechanism: the body's immune surveillance or the pathogen's invasion and infection strategy. we note here that the immune response itself may cause limited collateral damage to tissues as part of its larger battle against pathogens, but such immunopathic effects are usually short-lived in an otherwise healthy individual. at the turn of the twentieth century, there were two schools of thought on what mechanisms underlay immune responses. one group of scientists believed that immunity depended primarily on the actions of cells that destroyed or removed unwanted material from the body. this clearance process was referred to as cell-mediated immunity. however, another group of researchers was convinced that soluble molecules in the serum of the blood could directly eliminate foreign entities without the need for cellular involvement. in this case, the clearance process was referred to as humoral immunity, a term derived from the historical description of body fluids as "humors." today, we know that both cell-mediated and humoral responses occur simultaneously during an immune response and that both are often required for complete clearance of a threat. the cells responsible for cell-mediated immunity are collectively called leukocytes ("leuko," white; "cyte," small body, i.e., a cell) or white blood cells. however, "blood cell" is a bit of a misnomer, because a majority of leukocytes reside in tissues and specialized organs, and move around the body through both the blood circulation and a system of interconnected vessels called the lymphatic system. the soluble molecules responsible for humoral immunity are proteins called antibodies, and antibodies are secreted by a particular type of leukocyte. the production of these antibodies and the mounting of cell-mediated immune responses depend on an elaborate signaling system by which leukocytes communicate with each other as well as with other cell types in the body. this signaling is mediated by small secreted proteins called cytokines, which are mainly produced by leukocytes. the mammalian immune system can mount two types of responses: the innate response and the adaptive response. recently, it has become increasingly clear that these two types of responses are less distinct and more interconnected than once thought. indeed, researchers now believe that vertebrates are capable of a continuum of immune responses that bring more and more precise weapons to bear on a threat as required. more specifically, innate immunity is involved in all levels of immune response, whereas the adaptive response is mounted only when innate mechanisms signal that there is a relatively serious infection. in all cases, the objective is to clear the body of unwanted entities and re-establish homeostasis in the most efficient way possible. innate immune responses are triggered by disruptions to homeostasis caused by either non-infectious or infectious means. with respect to the former, innate responses help to repair tissues injured by trauma (such as that caused by a cut, a blow, or surgery) and remove dead cells and cells too damaged or old (senescent) to be of further use to the body. products synthesized by leukocytes mediating innate responses (innate leukocytes) also help to prevent "bystander injury" of healthy tissues. with respect to invading pathogens, elements of the innate immune system are the first to confront the threat and work to eliminate it from the body. such elements include preexisting anatomical and physiological barriers that attempt to block pathogen entry, and other responses that are induced after the pathogen has gained entry. only if innate responses are insufficient to control the situation and restore homeostasis, as is often the case with infection by a rapidly replicating pathogen, is an adaptive response mounted. together, the innate and adaptive immune responses allow a seamless escalation of countermeasures that maintain homeostasis in the face of cellular aging, tissue trauma and/or pathogen infection. what exactly triggers the initial response by the innate system? in a word: recognition. wherever cells are damaged or dying-either in the presence or absence of infection-particular host macromolecules are released into the extracellular milieu or become accessible on the surface of damaged cells or in cellular debris. these molecules are called damage-associated molecular patterns (damps). when a pathogen attacks, it furnishes common molecular structures on its own surface, or on the surface of cells it has infected, or as part of products it synthesizes. these structures are called pathogen-associated molecular patterns (pamps). cell damage or death in the absence of a pathogen gives rise to damps only, but both damps and pamps will be present when a pathogen invades ( fig. - ) . it is the recognition of damps and pamps by innate leukocytes that initiates innate responses. damps and pamps are recognized by host proteins called pattern recognition molecules (prms), most of which are expressed by innate leukocytes. there is not a large array of different prms, meaning that there is a limited repertoire of molecular patterns that can be recognized. however, each prm recognizes a damp or pamp that is shared by many different damaged cells or pathogens. thus, prms give the innate immune response the property of broad recognition. furthermore, because innate leukocytes expressing prms are present in large numbers, these cells don't need to multiply to work effectively: the response is immediate. finally, because innate leukocytes are activated but don't multiply in response to engagement of their prms, the speed and strength of their response is exactly the same upon a second exposure to the same pathogen or type of damage. the result is a response that is said to have no memory. recognition is also the key to initiating the adaptive immune response, but there are important differences. in contrast to the broad recognition of the prms mediating the innate response, the receptors expressed by the cells participating in the adaptive response (adaptive leukocytes) are equipped with receptors that recognize unique molecular structures on pathogens. these unique structures are called antigens, and the receptors that recognize them are antigen receptors. together as a population, adaptive leukocytes express a vast array of antigen receptors capable of recognizing almost any structure, meaning that they represent a diverse repertoire of antigen specificities. however, the antigen receptor of any one adaptive leukocyte binds to an antigen exclusive to a single type of pathogen. thus, antigen receptors have the property of specific recognition. because adaptive leukocytes are very few in number, these cells must proliferate and expand their ranks after receptor engagement before they can be effective: the response is delayed. in addition, after adaptive leukocytes proliferate and act to eliminate the pathogen, some of these cells become long-lived so that the speed and strength of the adaptive response is increased upon a subsequent exposure to the same pathogen. the adaptive response is thus said to have memory. the distinguishing features of the innate and adaptive responses are summarized in table - . a damp is a common molecular structure that is produced by host cells when they are damaged or dying (with or without pathogen infection). a pamp is a common molecular structure shared by a wide variety of pathogens or their components or products. in the case of pathogen attack, both damps and pamps will be present. cell damage or death antigens got their names because they were first identified as pathogen components that could bind antibodies; i.e., they induced "antibody generation." "antigen" now refers to structures targeted by humoral or cell-mediated responses. from the point of view of the body's leukocytes, a complex pathogen represents a collection of many different pamps, which evoke an innate response, and antigens, which may evoke an adaptive response if the innate response is not sufficient to eliminate the threat ( fig. - ) . anatomically, an innate response can be triggered at any point in the body because the majority of innate leukocytes are positioned where microbes most commonly attempt to gain access: just below the skin surface or at the mucosae protecting body tracts such as the gut and respiratory system. most of the time, the innate response successfully eliminates an invader. only those entities that succeed in overwhelming the innate defenses trigger adaptive responses that are initiated by adaptive leukocytes positioned in specialized anatomical regions called the lymphoid tissues, which include the spleen, tonsils and lymph nodes (see ch. ). the general characteristics of innate and adaptive immune responses primarily as they relate to pathogens are described in more detail in the next two sections. in this section, we present a general introduction to the central elements of innate immunity, namely barrier defenses, complement activation, pattern recognition, inflammation, phagocytosis and target cell lysis. more detailed information about the innate immune response can be found in chapter on innate immunity. innate leukocytes express a small number of pattern recognition molecules (prms) of limited diversity that collectively recognize a wide range of threats adaptive leukocytes express an almost infinite number of extremely diverse antigen receptors, each of which is specific for a particular pathogen response is triggered by prm binding to widely shared damageassociated molecular patterns (damps) or pathogen-associated molecular patterns (pamps) response is triggered by antigen receptor binding to a unique antigen derived from a specific pathogen number of leukocytes responding to a given threat is large, so no proliferation is required and response is fast number of leukocytes responding to a given threat is very small, so proliferation is required and response is slower responding cells do not proliferate and are short-lived, so level of defense is similar upon repeated exposure to the same pathogen responding cells proliferate and are long-lived, so level of defense is stronger and faster with repeated exposure to the same pathogen a given pamp is expressed by a wide variety of pathogens and is recognized by prms whose engagement activates innate leukocytes. an antigen is a molecular structure that is generally unique to a specific pathogen and is recognized by a specific antigen receptor whose engagement activates a particular adaptive leukocyte. pathogen antigens pamps note: elements of an innate immune response can be found in all multicellular organisms, whereas the mechanisms of the more recently evolved adaptive immune response are present only in the higher vertebrates (fish and above). appendix c contains a table of comparative immunology that summarizes the immune system elements present in an evolutionarily diverse range of animals. the first level of innate defense is mediated by a pre-existing collection of physical, chemical and molecular barriers that exclude foreign material in a way that is totally non-specific and requires no induction. these elements include anatomical barriers and physiological barriers. an example of an anatomical barrier is intact skin, while the low ph of stomach acid and the hydrolytic enzymes in body secretions are examples of physiological barriers. should barrier defense prove insufficient, other forms of innate defense are induced. a key player in the induced innate response is complement, a complex system of enzymes that circulates in the blood in an inactive state. once activated, the complement system contributes directly and indirectly to innate and adaptive immune defenses, as described in detail in chapter . when invaders breach anatomical and physiological barriers, innate leukocytes start to take action as a result of pattern recognition mediated by the binding of prms to pamps furnished by pathogens and to damps emanating from damaged host cells. as introduced previously, and as illustrated in figure - , prms come in several different forms, some of which are membrane-bound and others of which are soluble. prms that are expressed by innate leukocytes are called pattern recognition receptors (prrs). prrs are located in the plasma membrane of an innate leukocyte, or are soluble molecules free in the leukocyte's cytoplasm, or are fixed in the membranes of intracellular vesicles called endosomes. other prms are made by non-leukocytes and are present as soluble molecules free in the extracellular milieu. when these latter prms bind to a given pamp or damp, they must then bind to another receptor fixed in a leukocyte membrane in order to trigger the appropriate clearance response. the clearance mechanisms typically induced following prm or prr engagement by a damp or pamp are inflammation, phagocytosis, and/or target cell lysis. a brief description of each of these processes follows, with more detail provided in chapter . when leukocyte prrs are engaged by their ligands, intracellular signal transduction leads to the induction of new gene transcription and the synthesis of various "proinflammatory" cytokines. these cytokines in turn drive events responsible for the influx of first innate and later (if necessary) adaptive leukocytes into the site of injury when pamps on a pathogen bind to the prrs of an innate leukocyte, the cell becomes activated and attempts to clear the pathogen through phagocytosis, target cell lysis, and/ or the induction of inflammation. the prr may be expressed on the leukocyte surface, on its endosomal membranes or in its cytoplasm. or infection. this influx is part of a process called inflammation or an inflammatory response, and the redness and swelling we commonly associate with inflammation are its outward physical signs. this inflammation is normal and helpful, and, when properly regulated, promotes a localized gathering of the cells and molecules necessary to repair tissue damage and clear pathogens. once the threat is eliminated, the inflammation resolves naturally with time. however, if the inflammation fails to resolve and becomes chronic, it may become pathological. the powerful molecules secreted by the innate leukocytes participating in the inflammatory response can eventually cause tissue damage and impair immune system function if left to operate unchecked. thus, properly controlled inflammation is part of a healthy innate response and essential for homeostasis, whereas excessive or prolonged inflammation is immunopathic and undermines homeostasis. innate leukocytes frequently use a sophisticated means of engulfing entities that is called phagocytosis ("eating of cells"). phagocytosis is carried out primarily by three types of prr-expressing cells: neutrophils, macrophages and dendritic cells (dcs); these cell types are consequently known as phagocytes. neutrophils mainly engulf and destroy pathogens, while macrophages and dcs engulf not only pathogens but also dead host cells, cellular debris and host macromolecules. the phagocytosis of foreign entities by macrophages and dcs allows these cells to "present" unique antigens from this material on their cell surfaces in such a way that the antigens can be recognized by adaptive leukocytes that have been drawn to the site of inflammation. these cells are then activated and mediate antigen-specific protection. cancer cells and cells infected with intracellular pathogens frequently express certain damps and/or pamps on their cell surfaces that mark them as "target cells" for destruction by cells of the innate system. when these molecules are recognized and bound by prrs of innate leukocytes, complex processes are initiated that result in the lysis of the cancer cell or infected cell, preventing it from causing further harm to the body. these types of innate responses are carried out primarily by neutrophils, macrophages and another type of innate leukocyte called a natural killer cell (nk cell). in this section, we introduce the main features of adaptive immunity. the adaptive leukocytes referred to in previous sections are called lymphocytes. lymphocytes are categorized as either b lymphocytes (b cells) or t lymphocytes (t cells). the fundamental functional and developmental characteristics of lymphocytes are responsible for the specificity, division of labor, memory, diversity and tolerance of the adaptive response. each of these important concepts is introduced here as a prelude to more detailed discussions of these topics in later chapters. the specificity of an adaptive immune response for a particular antigen is determined by the nature of the antigen receptors expressed on the surfaces of t and b lymphocytes. each lymphocyte expresses thousands of identical copies of a unique antigen receptor protein. interaction of these antigen receptor molecules with antigen triggers activation of the lymphocyte. in contrast to the broad recognition mediated by prms, antigen binding to a lymphocyte antigen receptor usually hinges on the presence of a unique molecular shape unlikely to appear on more than one pathogen. the antigen receptors on the surface of a b cell are called b cell receptors (bcrs), whereas those on the t cell surface are called t cell receptors (tcrs). in both cases, the antigen receptor is itself a complex of several proteins. some of these proteins interact directly and specifically with antigen, while others convey the intracellular signals triggered by antigen binding into the cytoplasm and nucleus of the lymphocyte. these signals are critical for sustaining the activation of the lymphocyte and promoting its proliferation into an army of short-lived daughter effector cells. these effector cells then undertake the effector action appropriate for eliminating the pathogen furnishing the antigen. "division of labor" in the adaptive response refers to the different but equally important contributions of b lymphocytes and two types of t lymphocytes. these two major t cell subsets are called cytotoxic t cells (tc) and helper t cells (th). the roles of b, tc and th cells in the adaptive response are defined by the distinct mechanisms by which they recognize and respond to antigen (table - ) . b cells recognize antigen in a way that is fundamentally different from that used by tc and th lymphocytes. the bcr of a b cell binds directly to an intact antigen, which may be either a soluble molecule or a molecule present on the surface of a pathogen ( fig. - a) . activation of a b cell in this way causes it to proliferate and produce identical daughter effector cells called plasma cells that secrete vast quantities of specific antibody. an antibody is a protein that is a modified, soluble form of the original b cell's membrane-bound antigen receptor. antibody molecules enter the host's circulation and tissues, bind to the specific antigen, and mark it for clearance from the body, establishing a humoral response. since antibodies are present in the extracellular milieu, such a response is effective against extracellular pathogens. however, antibodies are unable to penetrate cell membranes and so cannot attack an intracellular pathogen once it has entered a host cell. unlike bcrs, tcrs are unable to recognize whole, native antigens. rather, a tcr recognizes a bipartite structure displayed on the surface of a host cell. this bipartite structure is made up of a host surface protein called an mhc molecule bound to a short peptide derived from a protein antigen. this complex is referred to as a peptide-mhc complex (pmhc). tc and th cells are activated by the binding of their tcrs to specific pmhcs presented on the surface of a dc. dcs can acquire extracellular pathogen antigens by phagocytosis, and intracellular pathogen antigens either by infection or by phagocytosis of the debris of dead infected cells. peptides from these antigens are bound to one of two types of mhc molecules, either mhc class i or mhc class ii, and then displayed on the dc surface for inspection by t cells. tc cells recognize antigenic peptides that the dc has presented on mhc class i molecules ( fig. - b) . the activated tc cell proliferates and differentiates into identical daughter effector cells called, for historical reasons, ctls (cytotoxic t lymphocytes). ctls can lyse any host cell displaying the same peptide-mhc class i complex recognized by the original tc cell. mhc class i molecules are expressed on almost all body cells, making them all potential targets for ctl-mediated lysis. however, unlike dcs, most host cells cannot phagocytose extracellular entities. it is therefore only after infection with an intracellular pathogen that a host cell displays an antigenic pmhc complex that can be recognized by the tcr of first the tc cell and then its daughter ctls. the cell-mediated immunity directed against such infected host cells is critical to the clearance of intracellular pathogens. th cells recognize antigenic peptides that a dc has presented on mhc class ii molecules ( fig. - c) . the activated th cell proliferates and differentiates into identical th effector cells that can recognize the same pmhc structure that activated the original th cell. however, mhc class ii molecules are expressed only by certain cell types that can act as specialized antigen-presenting cells (apcs), including dcs, macrophages and activated b cells. in the course of a pathogen attack, apcs can capture the pathogen and/or its products or components, and then display peptides derived from its antigens on mhc class ii. when the tcr of a th effector cell recognizes this pmhc structure, the cell is stimulated to produce cytokines that assist b cells and tc cells to become fully activated. in this way, th cells contribute to both humoral and cell-mediated adaptive responses, and are thus crucial for protection against many extracellular and intracellular pathogens. in addition, cytokines secreted by th cells stimulate innate leukocytes, thereby reinforcing this arm of immunity. the combined efforts of tc, th and b lymphocytes and many types of innate leukocytes may be necessary to eliminate a particularly wily invader. the innate response deals with a given threat in a very similar way each time it enters the body. in contrast, the adaptive system can "remember" that it has seen a particular antigen before. this immunological memory means that an enhanced adaptive response is mounted upon a second and subsequent exposures to a given pathogen, so that signs of clinical illness are mitigated or prevented. in other words, immunity is achieved because the body has effectively "adapted" its defenses and acquired the ability to exclude a particular pathogen. immunological memory arises in the following way. a constant supply of resting b and t cells is maintained throughout the body, with each lymphocyte expressing its complement of unique antigen receptor proteins. when a pathogen antigen enters the antibody production by b cells is described in detail in chapters and . "mhc" stands for "major histocompatibility complex," a region on human chromosome that contains genes encoding several types of molecules vital for the adaptive immune response. mhc genes and proteins are described in detail in chapter . body for the first time, a process of clonal selection takes place in which only those lymphocytes bearing receptors specific for that antigen are triggered to respond. the selected cells leave the resting state and multiply to generate daughter cells all expressing the same antigen-specific bcr or tcr. the differentiation of these daughter cells gives rise to the short-lived effector cells required to eliminate the pathogen, as well as long-lived memory cells that persist in the tissues essentially in a resting state until a subsequent exposure to the same pathogen ( fig. - ) . the attack on the pathogen by this first round of effector cells is called the primary immune response. the second (or subsequent) time that the particular pathogen enters the body, it is met by an expanded army of clonally selected, antigen-specific memory cells that undergo much more rapid differentiation into effector cells than occurred during the first antigen encounter. the result is a stronger and faster secondary immune response that eliminates the pathogen before it can cause illness. new populations of memory cells are also produced during the secondary response, ensuring that the host maintains long-term or even lifelong immunity to that pathogen. the degree of diversity of antigens recognized also distinguishes the adaptive response from the innate response. whereas the innate immune system exhibits a genetically fixed and finite capacity for antigen recognition, the recognition capacity of the adaptive immune system is nearly limitless. in fact, our bodies are capable of recognizing totally synthetic antigens that do not occur in nature. this huge diversity arises from the combined actions of several genetic mechanisms that affect the genes encoding the when antigen x enters the body, only those lymphocytes with receptors that specifically recognize x will be selected to proliferate and generate daughter clonal selection is explained in detail in chapter for b cells and in chapter for t cells. note: the generation of protective memory lymphocytes is the basis of vaccination. a healthy person is immunized with a vaccine containing pathogen antigens in order to provoke the production of memory cells that will prevent disease from developing if the individual is ever naturally infected by that pathogen. vaccination is described in detail in chapter . antigen receptors. some of these mechanisms operate before a lymphocyte encounters antigen, and some after. the primary source of antigen receptor diversity is a gene rearrangement process that occurs during the development of b cells and t cells prior to encounter with antigen. the genes encoding antigen receptors are not individual continuous entities. rather, the bcr and tcr genes are assembled from a large collection of pre-existing gene segments by a mechanism called somatic recombination. a single, random combination of gene segments is thus created in each developing lymphocyte. as a result, a lymphocyte population is generated in which the antigen receptor proteins are vastly diverse in specificity because they are encoded by hundreds of thousands of different dna sequences. in the case of b cells, additional mutational mechanisms operate that result in further structural diversification of antibody proteins. the sheer numbers of b and t lymphocyte clones generated in a healthy individual guarantee that there will be at least one clone expressing a unique receptor sequence for every antigen encountered during the host's life span. as alluded to previously, these clones, with their array of antigen receptor specificities, are collectively called the individual's lymphocyte repertoire. the generation of lymphocyte clones that can theoretically recognize any antigen in the universe raises the question of how the body avoids lymphocyte attacks on molecules present in its tissues. this avoidance is called tolerance, the fifth aspect in which the adaptive response exhibits more refinement than the innate response. as stated previously, the specificity of each antigen receptor is randomly determined by somatic recombination during early lymphocyte development. by chance, some of the genetic sequences produced will encode receptors that recognize self molecules (self antigens). these lymphocytes must be identified as recognizing self and then either removed from the body entirely, or at least inactivated, to ensure that an individual has an effective lymphocyte repertoire that does not attack healthy tissue. tolerance is established in two broad stages, each of which involves multiple mechanisms. the first stage, called central tolerance, occurs during early lymphocyte development. the mechanisms of central tolerance are designed to eliminate clones that recognize self antigens, thus establishing a lymphocyte repertoire that targets "non-self." in the second stage, called peripheral tolerance, any b and t lymphocytes that recognize self but somehow escaped the screening of central tolerance and completed their development are functionally silenced by another set of inactivating mechanisms. although we described the innate and adaptive responses separately in the preceding sections, in reality, they occur in a continuum that brings stronger and stronger weaponry to bear when (and only when) it is needed. our bodies normally deal with a pathogen attack in three broad and overlapping phases, two of which involve innate responses and the last of which requires adaptive immunity ( fig. - ) . an individual pathogen, such as a single influenza virus particle, is dealt with by these phases sequentially, but because the actual assault on the host involves billions of such particles, not every particle is in the same phase at the same time. as a result, in the host as a whole, and for an infecting pathogen population as a whole, all three phases may be happening concurrently. the first phase is mediated by the innate physical barriers, which offer immediate protection. the components making up these barriers (skin, mucosae, gut enzymes) are non-inducible in that they pre-exist and do not develop or change in response to the presence of a foreign entity. should these barriers be penetrated, the second, inducible phase of innate defense becomes effective - hours after the threat enters somatic recombination, the process that shuffles the dna of the genes encoding bcrs in b cells and tcrs in t cells, is achieved by specialized gen etic mechanisms that are des cribed in chapter for b cells and chapter for t cells. the establishment of central tolerance during lymphocyte development is described in chapter for b cells and in chapter for t cells. processes regulating mature lymphocytes, including mechanisms of peripheral tolerance, are des cribed in chapter . a pathogen attempting to enter the body first encounters pre-existing barriers and enzymes that block access non-specifically. if the pathogen manages to enter the underlying cell layer, mechanisms mediated by complement and innate leukocytes are induced due to relatively broad recognition of pamps. if a more targeted, pathogen-specific response becomes necessary, elements of innate immunity then facilitate induction of highly specific adaptive responses initiated by engagement of the antigen receptors of b, th or tc lymphocytes. complement activation phagocytosis target cell lysis inflammation skin mucosal barrier ph, saliva, proteases, etc. the body. innate leukocytes activated by the binding of prrs to pamps provided by the attacking pathogen, or to damps present due to host cell injury or death, work quickly to eliminate the invader using the mechanisms of inflammation, phagocytosis and target cell lysis. complement activation also contributes to clearance at this stage. in many cases, the incursion is completely controlled by the two phases of innate defense before adaptive immunity is even triggered. however, the cells of the innate response are ultimately limited both in numbers and in recognition capacity. fortunately, even if the pathogen ultimately manages to thwart complete elimination by innate immunity, some of the invaders and/or their components or products will have been taken up by innate leukocytes and presented as foreign antigens to t lymphocytes. the third and final phase of host defense mediated by adaptive immunity is thus initiated. th, tc and b cells are clonally selected and activated, and then proliferate and differentiate into memory cells and large numbers of daughter effector cells that eliminate the pathogen via humoral and/or cell-mediated mechanisms. because it takes time for the clonally selected lymphocytes to produce effector cells, adaptive responses are not usually observed until at least hours after the start of infection. the second and third phases of immune defense are more tightly interwoven than it may first appear. innate and adaptive immunity do not operate in isolation, and each depends on or is enhanced by elements of the other ( fig. - ) . the lymphocytes of the adaptive response require the involvement of cells and cytokines of the innate system to become activated and undergo differentiation into effector and memory lymphocytes. conversely, activated lymphocytes secrete products that stimulate and improve the effectiveness of innate leukocytes. the interplay between innate and adaptive immunity is sustained by cytokine signaling and through direct intercellular contacts between innate and adaptive leukocytes. by cooperating in this way, innate and adaptive immunity combine to mount an optimal defense against pathogens. normal host homeostasis is re-established when the host wins (at least for that moment) the ongoing horse race between the host's immune system and a pathogen attempting to establish infection. pathogens replicate at widely divergent rates, with some bacteria and viruses able to double their population size within a couple of hours. fast-replicating pathogens therefore initially get ahead of the host's immune defenses, but then are caught and destroyed as the specialized cells of the immune system become activated. the mystery highlighted by the authors of this article is why slowreplicating pathogens are able to establish infections at all, and why so many of the most damaging chronic infections we experience, like those of the hepatitis b and c viruses and the bacteria causing tuberculosis, are caused by some of the slowest-replicating pathogens. the answer may lie in achieving certain threshold levels of infection that supply adequate amounts of the molecular triggers needed to spark first the innate response and then the adaptive response. if a pathogen replicates rapidly, large amounts of damps and pamps are present that immediately activate innate leukocytes, including the apcs needed to initiate t cell responses. these t cells proliferate quickly enough to overwhelm the pathogen and eliminate it. in contrast, a slow-replicating pathogen may generate only low levels of damps and pamps that do not activate large numbers of apcs, delaying the t cell response. even once the t cell response is initiated, without sufficient cytokines produced by activated apcs, t cell proliferation may be slow and insufficient to completely eliminate the pathogen. the pathogen then survives to establish a low level of infection that doesn't kill the host but makes him/her miserable. the memory t cell response is of little help, as it only controls rather than eliminates the slow-replicating pathogen. an uneasy balance is set up between the pathogen and the host immune system that results in chronic infection. the contrast in immune response kinetics between what the authors call "fast" and "slow" pathogens is illustrated in figure from their article. "the race between infection and immunity: how do pathogens set the pace?" by davenport, m.p., belz, g. t. and ribeiro, r.m., ( ) when the immune system is functioning normally, harmful entities are recognized, and the host is protected from external attack by pathogens and internal attack by cancers. some localized tissue damage may result from the normal inflammatory response that develops as innate leukocytes work to eliminate the threat, but these immunopathic effects are limited and controlled. as well, the tolerance of the healthy immune system prevents cells of the adaptive response from attacking normal self tissues. sometimes a person's immune system is incomplete, either because of primary immunodeficiency or acquired immunodeficiency. in primary immunodeficiencies, the failure of the immune system is congenital; that is, an affected individual is born with a genetic defect that impairs his/her ability to mount innate and/or adaptive immune responses. in an acquired immunodeficiency, an external factor such as a nutritional a healthy immune response is crucial for providing an individual with immunity to infectious diseases (ch. ) and may be made more robust through vaccination (ch. ). innate and adaptive immune mechanisms work together to allow a full range of responses of appropriate strength and specificity. in addition to their direct and broadly protective role, innate immune responses recruit adaptive leukocytes and contribute to their activation. once activated, lymphocytes mount humoral and cell-mediated antipathogen responses that are highly specific. cytokines are produced that further stimulate both the innate and adaptive responses. innate and adaptive immunity thus work synergistically to protect the host from all threats. cell-mediated the pace of pathogen replication may have implications for vaccination strategies. currently, vaccination often uses a harmless pathogen antigen to induce the host to respond and generate large populations of memory lymphocytes, which stand guard ready to mount a faster and stronger response should the real pathogen attack. for a fast-replicating pathogen, this tactic is successful because these types of pathogens readily generate enough pamps, damps and antigen to surpass the thresholds of apc and t cell activation. with the "head start" provided by memory lymphocytes, the pathogen may not get to replicate at all. however, a slow-replicating pathogen may not provide sufficient antigen to reach even the lower threshold required by memory t cells, meaning that the vaccine will not have offered an advantage to the host. worse, if the large numbers of memory t cells present after vaccination compete for the miserly quantities of antigen present, and so proliferate only slowly, the pathogen may in fact get a better foothold than if the host had not been vaccinated. these considerations should encourage us to experimentally test the threshold hypothesis and to refine our approach to vaccination accordingly. imbalance or a pathogen may cause the loss of an immune system component. for example, in acquired immunodeficiency syndrome (aids), the human immunodeficiency virus (hiv) destroys the t lymphocytes needed to fight infection. in both primary and acquired immunodeficiencies, patients show a heightened susceptibility to recurrent infections and sometimes develop tumors. the prognosis for most of these diseases is very poor and treatment options are limited. what happens when the immune system is complete but malfunctions, or when its normal functioning has undesirable effects? there are several instances in which inappropriate actions of the immune system can result in pathologic consequences that tip the balance from health to disease ( fig. - ) . firstly, the normal attack of a healthy immune system on a foreign tissue transplant meant to preserve life results in transplant rejection and deleterious consequences for the transplant patient. secondly, when the tolerance of an individual's adaptive immune response fails, self tissues are attacked in a way that can lead to autoimmune disease. thirdly, an immune response that is too strong or long can result in hypersensitivity to an entity. in this situation, the normal inflammation that accompanies the response gets out of control and causes significant collateral tissue damage. when an individual's normal adaptive immune system responds inappropriately to an antigen that is generally harmless, the immune response is manifested clinically as a form of hypersensitivity called allergy. lastly, if normal regulation of the inflammatory response is lost for an extended period for any reason, chronic inflammation ensues that can set up conditions allowing the development of tumors, heart disease or autoimmune disorders. the remainder of this book will take the reader on a logical tour of the immune response. chapter continues our basic immunology section, providing detailed descriptions of the various cells, tissues and cytokines involved in mediating immune responses. chapters - cover all aspects of the innate and adaptive immune responses in depth. the chapters in our clinical immunology section, chapters - , address immunological principles not only of practical value to the more medically oriented reader but also of interest to all who seek to understand the pivotal role of the immune system in health and disease. it is our hope that the reader will come away with a solid understanding of the cellular and molecular mechanisms underlying immunity and how these mechanisms can go awry to cause illness. we also provide a glimpse of how researchers seek to manipulate these mechanisms with the goal of ensuring good health for all. the pathological consequences of immune system irregularities include immunodeficiency (ch. ), cancer (ch. and ), transplant rejection (ch. ), hypersensitivity (ch. ) and autoimmune disease (ch. ). the way in which the immune system responds can maintain a healthy body or lead to disease. in a healthy individual, the immune system mounts an appropriate response that clears damaged cells and kills infectious organisms or cancerous cells. however, the immune system is a powerful and multi-faceted weapon that requires strict controls to remain in balance. when the immune system fails to respond adequately, or mounts an overzealous or inappropriate attack, or ignores normal control mechanisms, a wide variety of disease states can result. immunodeficiency autoimmune disease hypersensitivity and allergy destruction of cancer cells • the immune system is a central player in the maintenance of human health. • immune responses depend on the coordinated action of leukocytes that travel throughout the body to recognize and eliminate threats posed by trauma as well as by extracellular and intracellular pathogens, toxins and cancerous cells. • cytokines are secreted intercellular messenger proteins that mediate complex interactions among leukocytes. • complete protection from and clearance of unwanted entities often involve both innate and adaptive responses. • some innate mechanisms require no induction and are completely non-specific, whereas others are inducible and involve broad recognition of a limited number of molecular patterns (pamps and damps) by prms. • adaptive responses are mounted after innate responses have failed to remove an unwanted entity, and are dependent on cytokines produced during the innate response. • adaptive immunity involves the selective activation of lymphocytes by the engagement of their antigen receptors by antigens derived from the particular inciting entity. • the three subsets of lymphocytes are th, tc and b cells, which use distinct mechanisms to recognize antigen and carry out effector functions to eliminate specific entities. • elements of innate immunity influence adaptive immunity and vice versa. • malfunctions of the immune system are related to autoimmune disorders, allergy and other hypersensitivities, transplantation rejection, primary and acquired immunodeficiencies, chronic inflammation, and cancer development. section a ) can you define these terms? immunology, immune system, immunity, pathogen ) what was koch's theory and why was it important? ) who coined the term "vaccination" and on what basis? ) can you define these terms? homeostasis, mucosae, leukocyte, cytokine, immunopathic ) what is the difference between infection and disease? ) what is the major difference between extracellular and intracellular pathogens? ) distinguish between cell-mediated and humoral immunity. ) can you define these terms? senescent, phagocytosis, inflammation, prm, prr, mhc, lymphocyte, tcr, bcr, antibody, clonal selection, tolerance, self antigen, immunological memory ) what are three major differences between innate and adaptive immune responses? ) distinguish between damps and pamps. patient # is having surgery in the course of an elective hip replacement in a modern hospital. patient # is not so lucky and has stepped on a rusty nail in her garden. what sort of damps and/or pamps might be present in each case, and what sorts of immune responses might you expect to find in these patients? ) if a person's immune system was unable to produce memory lymphocytes why are b lymphocyte responses usually associated with defense against extracellular rather than intracellular pathogens? how might they also help protect a host against intracellular pathogens? can you extrapolate? some conceptual questions would you like to read more? overview of the immune response regulation of adaptive immunity by the innate immune system friendly and dangerous signals: is the tissue in control? key concepts in immunology key: cord- - vlxa i authors: williamson, e. d.; westlake, g. e. title: vaccines for emerging pathogens: prospects for licensure date: - - journal: clin exp immunol doi: . /cei. sha: doc_id: cord_uid: vlxa i globally, there are a number of emerging pathogens. for most, there are no licensed vaccines available for human use, although there is ongoing research and development. however, given the extensive and increasing list of emerging pathogens and the investment required to bring vaccines into clinical use, the task is huge. overlaid on this task is the risk of anti‐microbial resistance (amr) acquisition by micro‐organisms which can endow a relatively harmless organism with pathogenic potential. furthermore, climate change also introduces a challenge by causing some of the insect vectors and environmental conditions prevalent in tropical regions to begin to spread out from these traditional areas, thus increasing the risk of migration of zoonotic disease. vaccination provides a defence against these emerging pathogens. however, vaccines for pathogens which cause severe, but occasional, disease outbreaks in endemic pockets have suffered from a lack of commercial incentive for development to a clinical standard, encompassing phase iii clinical trials for efficacy. an alternative is to develop such vaccines to request us emergency use authorization (eua), or equivalent status in the united states, canada and the european union, making use of a considerable number of regulatory mechanisms that are available prior to licensing. this review covers the status of vaccine development for some of the emerging pathogens, the hurdles that need to be overcome to achieve eua or an equivalent regional or national status and how these considerations may impact vaccine development for the future, such that a more comprehensive stockpile of promising vaccines can be achieved. globally, there are a number of emerging and re-emerging pathogens. some of these cause endemic disease in regions of the globe, where they are maintained in zoonotic reservoirs and transmitted to man either by direct or indirect contact. for most of the emerging pathogens there are no licensed vaccines available for human use, although there is ongoing research and development. however, given the extensive and increasing list of emerging pathogens and the time and investment required to bring vaccines into clinical use, the task is huge. overlaid on this task is the risk of anti-microbial resistance (amr) acquisition by micro-organisms which can endow a relatively harmless organism with pathogenic potential. furthermore, climate change also introduces a challenge by causing some of the insect vectors and environmental conditions prevalent in tropical regions to begin to spread out from these traditional areas, thus increasing the risk of migration of zoonotic disease. vaccination provides a defence against these emerging pathogens. however, to date, vaccines for pathogens which cause severe, but occasional, disease outbreaks in endemic pockets have suffered from a lack of commercial incentive for development to a clinical standard. while approval of vaccines for diseases caused by such pathogens would clinical and experimental immunology review article series editor: e diane williamson make a significant impact on disease outbreaks, taking niche vaccines into clinical development, including phase iii clinical trials for efficacy, requires a large investment in time and money. an alternative is to develop such vaccines to request us emergency use authorization (eua), or an alternative status in the united states, canada and european union (eu) making use of a considerable number of alternative regulatory mechanisms that are available prior to licensing, so that the products are deployable at the first indications of a disease outbreak. this review covers the status of vaccine development for some of the emerging pathogens, the hurdles that need to be overcome to achieve eua or an equivalent regional or national status and how these considerations may impact vaccine development for the future, such that a more comprehensive stockpile of promising vaccines can be achieved. pathogens which are classed as emerging or re-emerging are identified through global surveillance programmes and organizations such as the world health organization (who). although labelled 'emerging', most of these pathogens are not new and have either been quiescent in the environment until the conditions are opportune to emerge or have evolved from a parent organism to adapt to the prevailing conditions. thus, there is an intricate relationship between the environment, the climate, wildlife and human existence and lifestyle. the coronaviruses exemplify this point: the ancestral virus possibly existed approximately years ago [ ] . coronaviruses have a wide species range infecting birds, bats, chickens, pigs, dogs, cats and rodents [ ] . however, the first human coronavirus was described only in the s [ , ] , and the coronavirus causing severe acute respiratory syndrome (sars) was discovered only in [ ] [ ] [ ] , while that causing middle east respiratory syndrome (mers) first emerged in [ ] . it is likely that warm-blooded flying birds and bats have co-evolved with the coronaviruses to aid dissemination [ ] . for example, sars is thought to have first infected old world bats, then spreading to horseshoe bats [ ] , civets and finally to man [ ] . in the outbreak of sars in china and adjacent countries, phylogenetic analysis suggested that the virus spread from bats to humans, possibly through the intermediary civet species. neither bats or civets showed any clinical signs of infection, and it is thought that bats are the main zoonotic reservoirs for the virus [ ] . organizations such as the who, national institutes for allergy and infectious diseases (niaid) and the us centers for disease control (cdc) publish lists of emerging pathogens which may be viral, bacterial or rickettsial in nature. the who priority list contains viruses which have been prioritized as the most likely to cause epidemics and for which the who will establish a blueprint programme for accelerated research and development (r&d) [ ] . the list published in february is shown in table . niaid [ ] and cdc [ ] also publish lists of pathogens of priority which comprise bacteria and viruses, but categorize these into three groups depending on pathogenicity, accessibility and the availability of vaccines and therapies. all the viruses listed by the who also occur on these lists, alongside bacterial pathogens of concern. one of these is yersinia pestis, causative of bubonic and pneumonic plague, which is recognized by all three bodies (who, cdc, niaid) as a current priority following the exceptionally large and serious outbreak between september to april in madagascar [ ] , where the disease is endemic. in addition, niaid recognizes the added threat to human health posed by the acquisition of amr by pathogens and who has also published a priority list [ ] of bacterial species for which r&d is required to develop new antibiotics (table ) . globally, there are many regions of endemic disease which are maintained by reservoirs of zoonotic pathogens in the local wild animal species. these pathogens comprise viral, bacterial and rickettsial species and some have complex lifecycles, infecting an environmental (e.g. standing water) or animal reservoir and then being transmitted either by direct contact with man or indirectly, via an insect (e.g. mosquito) or mammalian (e.g. bat, civet, camel) vector, to man, from which they may be spread by human-tohuman transmission. (fig. ). an example of a serious and widespread bacterial infection is mosquito-transmitted plasmodium falciparum, causing malaria in many tropical regions. malaria causes significant morbidity and co-morbidity in these regions in which it is endemic. due to the complex life cycle of the causative bacterium, it has been challenging to achieve a vaccine for malaria. the most advanced candidate is the rts,s/as vaccine [ ] , which is undergoing a pilot implementation in three countries in sub-saharan africa, with a view to determining its impact on disease prior to a wider implementation [ ] . despite the availability of approved vaccines [ , ] , typhoid fever and cholera remain significantly debilitating enteric diseases in regions of the world where hygienic living conditions are poor and there is little access to health care. both infections are caused by bacteria (salmonella typhi and vibrio cholera, respectively) which exist in contaminated water and food. tuberculosis (tb) is a respiratory infection which is widespread globally. caused by mycobacterium tuberculosis, and transmitted to man from zoonotic reservoirs (badgers, cattle) with a high potential for subsequent human-to-human transmission, tb is prevalent in susceptible individuals living in overcrowded conditions [ ] . although the bcg vaccine has been in routine use for many years, variable efficacy has been reported, depending on region of use [ , ] with - % reported in the united kingdom but lower levels in equatorial countries. the emergence of multi-drugresistant tb (mdr tb) in recent years raises the bar for treatment of this disease and makes a high level of vaccine efficacy even more important [ ] . examples of zoonotic reservoirs which maintain endemic viral diseases are numerous and are summarized in table . in particular, viral endemic disease is caused by the coronaviruses (mers and sars) in saudi arabia and the eastern mediterranean countries; and by the lassa arenavirus, which is endemic in west african countries and caused a serious outbreak of lassa fever in nigeria in [ ] ; other viruses which are endemic and cause viral haemorrhagic fever include dengue, which is widespread in tropical and subtropical regions worldwide [ ] , the filoviruses (ebola and marburg) which have a zoonotic reservoir in bats in sub-saharan africa [ ] ; and yellow fever virus, which is prevalent in africa, central and south america and the caribbean [ ] . interestingly, the same mosquito (aedes aegypti) which spreads yellow fever virus also transmits the dengue, chikungunya and zika viruses [ ] . chikungunya virus has a widespread distribution in africa, asia, india and south america and with occasional cases in europe, and causes a debilitating, but rarely fatal, disease [ ] . zika virus emerged in brazil in [ ] , although it was first detected in monkeys in uganda as early as [ ] , and the first documented human case occurred in nigeria in [ ] . the large brazilian outbreak of zika viral disease culminated in , with thousands of cases reported [ ] . rift valley fever virus (rvfv) is another zoonotic virus which is endemic in sub-saharan africa and primarily infects cattle, sheep and goats, but can be transmitted to man by mosquito bite to cause an acute fever [ ] . an example of a bacterium which has been classed as a re-emerging pathogen and which is endemic in global regions is y. pestis, causative of plague. bubonic [ ] [ ] [ ] . over thousands of years, y. pestis has evolved away from the enteric yersinia species to become a lethal flea-transmitted bacterium [ ] . it is transmitted to man, typically from a zoonotic reservoir in infected rats or other rodents (e.g. ground squirrels or prairie dogs), by flea-bite to cause bubonic plague [ ] (fig. ). if not detected and treated, this can develop into either septicemic plague or the most serious form of all, a secondary pneumonic plague. in turn, individuals with pneumonic plague can transmit this by aerosol droplet to others, to establish a primary pneumonic plague infection. each year, a few cases of plague are also reported in the southwestern united states, where the disease has been endemic in the rodent population since the late s [ ] . as well as infecting the rodent population, infected fleas can spread y. pestis to other wildlife species (e.g. rabbits/hares or, rarely, domesticated animals [ ] ) which, in turn, raises the potential of transmission to man by inhalation to cause a primary pneumonic plague. in endemic areas, outbreaks of plague are often associated with seasonal environmental changes, causing rodents to stray closer to human habitation. the plague outbreak in madagascar during / was particularly serious, with an estimated cases and deaths ( · % fatality rate) [ ] . this outbreak was approximately sixfold greater than usual, with an unusual predominance of pneumonic, rather than bubonic plague. although y. pestis is susceptible to antibiotics, such as the aminoglycosides (gentamycin, streptomycin), the fluoroquinolones (e.g. ciprofloxacin) or tretracyclines (e.g. doxycycline) [ ] , these need to be administered very early to a suspected plague case, and ideally before symptoms emerge. additionally, there have been reported instances of antibiotic resistance including to multiple antibiotics [ , ] . hence, there is a clear and increasingly urgent need for an efficacious vaccine. other bacterial endemic diseases include melioidosis and glanders which, although not caused by zoonotic pathogens, are endemic in southeast asia where the bacteria reside in soil and are transmitted to humans through occupational exposure, e.g. working in paddy fields [ ] . another bacterial disease which is endemic in the northern hemisphere is tularaemia, caused by the bacterium francisella tularensis, which has zoonotic reservoirs in the rabbit, hare and rodent populations in the south, central and western united states and is transmitted to man by ticks and biting flies [ ] . rickettsial species, such as coxiella burnetii, causative of q-fever, comprise bacteria which exist within another cell, and as such q-fever is contracted when humans are exposed to aerosolized droplets from the urine, milk, faeces or birth detritus from infected sheep, goats and cattle [ ] . for all these pathogens, there is a requirement for efficacious approved vaccines to curtail or prevent regular disease outbreaks. for some of these pathogens (e.g. rvfv, cchf) there are vaccines [ ] , but these are not widely available [ ] or have been used and withdrawn for safety or regulatory reasons (e.g. the live vaccine strain for tularaemia) [ ] or they are not universally suitable, requiring a screening test prior to administration due to the potential for a hypersensitivity response (as for the vaccine for q-fever) [ ] . there is no readily available licensed plague vaccine, although a series of killed whole cell vaccines (kwcv) has been produced and used in the past, mainly in the biodefence context (reviewed in [ ] ). additionally, live attenuated plague vaccines, derived from an attenuated mutant strain as the ev series, have been used in the former ussr (fussr) and are still used in asia, notably in russia and china [ , ] . ev , the most commonly cited of these, provides protective efficacy against plague, but is licensed for human use only in countries of the fussr and is documented to cause serious adverse effects in non-human primates and malaise and adverse effects in human vaccinees [ ] . whatever the context, all these diseases would be positively impacted by the availability of efficacious and approved vaccines. however, to a greater or lesser extent they are all niche diseases with no major commercial incentives to drive vaccine development programmes. this is a space that non-governmental organizations (ngos) such as the coalition of epidemic preparedness innovations (cepi) [ ] and global vaccine alliance (gavi) [ ] have entered and they are supporting vaccine efforts for some of the diseases listed above. additionally, philanthropic funders such as the gates foundation are supporting vaccine r&d efforts [ ] . in the united kingdom, and subsequent to the ebola outbreak in west africa, vaccine networks for human and veterinary vaccines have formed to prioritize vaccine efforts in these respective contexts [ ], while the department of health, together with innovate uk, has supported r&d of vaccine candidates for the prioritized pathogens [ ] . as a result of these global initiatives, a number of candidate vaccines are being developed for emerging bacterial and viral pathogens, examples of which, although by no means exhaustive, are cited here [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . who reports ongoing global vaccine r&d efforts [ ] and also tracks the progress of clinical trials for emerging pathogens [ ] . here it may be worth drawing a distinction between prophylactic vaccination, i.e. general use prophylaxis (gup), given routinely and not necessarily in the face of specific, predicted outbreaks, in contrast to post-exposure prophylactic (pep) vaccination, to be given after a suspected exposure to a pathogen, or to ring-fence an outbreak or, indeed, post-exposure therapeutic (pet) vaccination, to be given after an actual exposure. in the pep and pet contexts, the benefit of vaccination vastly outweighs the risk of disease (i.e. the risk : benefit ratio is low), while in the prophylactic context the risk : benefit ratio may be greater than, or equal to, · ). for infections with short incubation times, e.g. less than h, as for pneumonic plague, pep or pet vaccination may only be useful if administered under antibiotic cover. in the united states, once a vaccine candidate has been thoroughly tested for safety in non-clinical models, and in an escalating-dose, statistically powered, phase i design in the clinic, from then on it may be possible to pursue approval for an eua, rather than pursue the full-length pathway to biological licensing authorization (bla). this can enable the earlier availability of vaccines for use in endemic regions. however, it should be noted that eua is only available through the food and drug agency (fda) in the united states. some alternative regulatory mechanisms that may be considered prior to licensing are outlined below. from the discovery phase to the clinic, vaccine development requires the completion of a series of steps represented as a generic outline in fig. . the regulatory agencies lay down specific guidance for these steps [ , ] and this review presumes no authority in this regard, but seeks to give a generalized overview of a generic vaccine development process. exit from the discovery and preclinical phases requires substantive data demonstrating immunogenicity and efficacy in at least one suitable animal model(s). where possible, efficacy in the animal model should be demonstrated by direct exposure to the pathogen concerned. technology transfer for manufacture under good manufacturing practice (gmp) will require a demonstration of known provenance of all essential materials required in the manufacture of the vaccine candidate. this includes seed stocks of cell lines from which recombinant proteins may be expressed, e.g. escherichia coli, human embryo kidney cells, baculovirus, tobacco mosaic virus; or seed stocks of attenuated vaccine vectors, e.g. viral vectors such as adenovirus, vesicular stomatitis virus, modified vaccinia ankara; or bacterial vaccine vectors, e.g. salmonella, listeria; the genetic constructs cloned into the cell line in question; all culture media and components and all formulations and excipients. transfer of the manufacturing process to gmp may include scale-up and conversion to, for example, fermentation conditions, or to plant-based, mammalian cell line or insect cell line expression on an expanded scale. this transfer will probably require the demonstration of consistency between consecutive batches at gmp, which will also enable the development of scaled-up downstream processing methodology. vaccine components (the drug substance) from these batches may be formulated (the drug product) as required and used in safety/toxicology studies and for immunogenicity/efficacy in an appropriate second animal model, which will be as representative as possible of the human response. the second animal model is often, but not necessarily, a non-human primate, and the selection of this second model will depend entirely on the vaccine indication. there will also be a requirement to generate sufficient stability data on both the drug substance and the drug product and to demonstrate that the drug product is stable for at least the duration of the intended phase i clinical trial under prevalent conditions in that location. clearly, extended stability testing under a range of conditions, including accelerated conditions (high ambient temperature and relative humidity), will also be required to progress the vaccine through development. as well as determining its stability, both the drug substance and product will require characterization for properties such as identity, purity, isoelectric point, osmolality, endotoxin levels and potency, to demonstrate consistency between batches and to allow for release of these for clinical trials. it is essential to ensure safety of the drug product before entering a clinical trial, and the drug product may be tested in suitable small animal models for the absence of adverse effects under conditions of repeated dosing at the anticipated human dose level, as well as in the intended human schedule, but at multiples of the anticipated human dose-level. the use of rodent models (mouse or rat) for this testing allows for sufficient statistical powering of such safety/toxicological testing. if the clinical trial is to be conducted in women of child-bearing age, it may be necessary to carry out reproductive toxicology testing of the drug product; in this case it may be necessary also to use a sensitive rabbit model and to administer the vaccine to pregnant rabbits to screen for adverse effects in the mother and potential teratogenic effects in the f generation. the national regulatory authority will expect to review the existing safety data and outlines of further protocols to be used. a review of all the data pertaining to the candidate vaccine will be required by the regulators in order to proceed to a clinical trial. depending on the specific requirements of the regulatory authorities in the country of origin/manufacture of the vaccine and also the intended location of the clinical trial, this may take the form of an investigator's brochure, protocol and summaries of manufacturing, non-clinical and clinical data in a clinical trial application (for the european medicines agency, ema) or an investigational new drug (ind) application (e.g. us fda). other international regulators (e.g. in canada, japan and china) will have variations on the documentation required. in the united states, the alternative approaches to full marketing approval that are available for vaccines under development and which may be considered through the fda are to either request an eua or to request expanded access (ea), sometimes called compassionate use. the eua is issued in support of potential and actual public health, military and domestic emergencies involving chemical, biological, radiological and nuclear agents (cbrn), including emerging infectious diseases, e.g. pandemic influenza. such fda-approved medical products which may be stockpiled for use in emergencies are referred to as medical countermeasures (mcm) and include biological products, e.g. vaccines, drugs and devices. specifically, the eua authority is separate and distinct from use of an investigational medical product held under an ind and must be able to treat serious or life-threatening diseases or conditions. in contrast, ea submissions are for products used under an ind or a protocol (treatment plan), or submitted as a protocol amendment to an existing or new ind. the ea categories cover use under either an individual patient ind or protocol, individual emergency ea use, or ea for an intermediate-size population or for widespread use (large populations). other us regulatory programmes for biologicals and drugs to treat serious or life-threatening conditions include fast-track designation, breakthrough therapy designation, accelerated approval pathway and priority review designation. in canada, access to drugs through special access programmes (saps) exists for serious and life-threatening conditions, when marketed alternative products are not available or unsuitable and evidence supports the intended use. priority review of marketing submissions and 'notice of compliance with conditions' for such products may also be considered and granted by health canada. in the european union, the ema supports early patient access to new medicines and which are eligible for a marketing authorization application under the centralized procedure and either target unmet medical needs or those which have a major public health interest. the ema has launched a priority medicines (prime) scheme to facilitate early dialogue and support the development of medicines that target unmet medical needs and which promotes an accelerated regulatory assessment. in addition, the prime scheme is intended for seriously debilitating or life-threatening diseases, compassionate use of unauthorized medicines for patients with an unmet medical need (when no satisfactory treatment is available in the european union) and also provides conditional marketing authorization. the regulation and rules of access to compassionate use programmes varies between national government authorities across eu member states. in the united kingdom, the early access to medicines scheme (eams) gives patients with life-threatening or debilitating conditions access to innovative medicines without marketing authorization, but for which there is a clear medical need. a two-stage evaluation process initially considers promising innovative medicine (pim) designation before an eams scientific opinion. also in the united kingdom, the potential supply of unlicensed medicinal products (specials) may be considered by the medicines and healthcare products regulatory agency (mhra) for the importation of medicines that have marketing authorization status from countries outside the united kingdom and european union. where there is an unmet need of priority to the who for a new vaccine, the who may establish a blueprint programme [ ] . as vaccine candidates for the specific blueprint programme advance through the development process, and particularly when they have attached clinical data, the who may run a prequalification check to determine whether the candidate meets their requirements on behalf of un agencies that will ultimately purchase vaccines [ ] . to do this, who will engage with subject matter experts to draft and publish a target product profile (tpp) for a vaccine requirement. who will use its scientific advisory group of experts (sage) to review vaccine candidate performance against the tpp and to prequalify a vaccine candidate(s) which fulfils the tpp. assuming that the preclinical safety and toxicology testing has been satisfactory, a protocol for phase i testing of the vaccine candidate may be submitted to the regulatory authorities for approval. the primary objective of the phase i clinical trial is to test the vaccine candidate for safety in informed and consenting adult volunteers who will be selected according to pre-agreed inclusion/exclusion criteria. as this is the first time the candidate is being used in man, the phase i trial is typically small and cautious. if a range of dose-levels is being tested, it may be necessary to dose a sentinel group of single subjects from each arm of the study to assess safety through week of immunization and starting at the lowest level before proceeding to the next level. the data from the sentinel group would be reviewed before proceeding with the main study. then the first cohort would be dosed at the lowest level before proceeding to the next level, and so on. volunteers will be closely monitored for adverse effects in the clinic at each dosing time-point and will typically maintain a diary at home to record any symptoms arising between time-points. an independent safety monitoring panel, including medically qualified personnel, will be required to monitor the reporting of adverse effects. volunteers may also be blood-sampled to assess vaccine immunogenicity from baseline and serial samples, and depending on vaccine type may be required to supply additional samples which can be collected non-invasively, e.g. saliva/stool samples. all volunteers may be followed up for a pre-agreed period on completion of the study, to check for latent adverse events and to monitor, e.g. for maintenance of circulating antibody titres or memory response to the vaccine. the second phase of clinical trials typically allows for an enlarged study to assess vaccine safety and to monitor immunogenicity. during this phase, significantly expanded cohorts of volunteers may be dosed with vaccine at doselevel(s) selected as optimum from the phase i trial and can be monitored for safety and a more detailed immunogenicity assessment, which may include assays of both serological and cellular memory responses. once again, volunteers may be blood-sampled for baseline and serial serum/plasma samples, and depending on vaccine type may be required to supply additional samples which can be collected non-invasively, e.g. whole blood/saliva/stool. in the event that a phase iii trial for efficacy is not feasible, for ethical or practical reasons, it may be necessary to plan a blood-sampling regimen in the volunteers which will enable sufficient sample volumes to be available for animal rule studies (discussed below). if sufficient safety data are gained from a phase ii trial, the regulatory authorities may be able to consider the vaccine for eua. the third phase of clinical trials is typically designed to assess efficacy. however, where this is not feasible for either practical/logistical or ethical reasons, phase iii could be regarded as a further, significantly enlarged trial for vaccine safety with adequate follow-up of vaccinated volunteers. a phase iii field trial of vaccine efficacy may be feasible in the event of an anticipated seasonal outbreak, in which case vaccination could be given prophylactically and well in advance. as long as effective biosurveillance programmes are in place to instigate a rapid response to new infections in an unvaccinated placebo cohort, the latter group may be included in a prophylactic efficacy trial. conversely, reactive mode vaccination in the context of an actual outbreak is likely to be more complicated, as it would be unethical to omit anyone at risk of infection from the vaccination programme. hence, sufficient safety data on the candidate vaccine would be required in case of need to administer it to pregnant/nursing mothers, children and elderly people as well as to the general adult population. additionally, it may be necessary to administer an adjunct to the vaccine, such as an appropriate antibiotic, to both cohorts (vaccinated and placebo). whatever the context, careful consideration of the trial design needs to be made and approved by the regulators to achieve adequate statistical powering and to determine the need to provide supplementary antimicrobial therapy, and also the need for a placebo cohort. after the anthrax letters incident in the united states in in which anthrax was released through the postal system, resulting in five fatalities and widespread anxiety [ ] , the fda issued the animal rule (fda cfr) [ ] to expedite the development of vaccines and therapies for biothreat agents for which it is neither feasible nor ethical to carry out phase iii efficacy studies in man. in this situation, the animal rule makes provision for the substitution of animal efficacy data for human efficacy data. the animal efficacy data should demonstrate a reasonable likelihood that the candidate vaccine or therapy would be efficacious in man [ ] . the animal efficacy data should be provided ideally from more than one animal model, or from one model only if that model is well-characterized and authentically represents the human disease syndrome [ ] . in addition, the fda may require supporting data to include pharmacokinetic/pharmacodynamic (pk/pd data) and a determination of the pathophysiological mechanism of action of the biothreat agent and a 'reasonable understanding' of the protective mechanism of the proposed vaccine or therapy. thus, if the candidate is a vaccine, a reasonable understanding of the protective mechanism(s) being invoked by it requires an identification of the immune correlates of protection. immune parameters which correlate with protection in the selected animal models may include the total titre of circulating antibody induced to the vaccine and the determination of a minimum cut-off titre required to confer protection. alternatively, the titre of functional or neutralizing antibody within that total may provide the correlate, particularly where the neutralization of specific virulence factors produced by the biothreat agent is identified and quantifiable [ ] . for some vaccines, the induction of a cellular memory response instead of, or in addition to, circulating antibody will provide the correlate. this is measurable by the ex-vivo recall response of peripheral blood mononuclear cells (pbmcs) on stimulation with the vaccine antigen(s) [ ] . in the absence of direct efficacy testing in the human vaccinee, the immune correlate identified above provides a surrogate marker of efficacy. if this is a functional antibody, the titre induced in man needs to be compared with the titre required in the animal model(s) to provide protection. this can be achieved, for example, by the in-vitro neutralization of a specific virulence factor, or by a competitive enzyme-linked immunosorbent assay (elisa), where the test antibody is competed with a known neutralizing antibody for binding to the target antigen or by the passive transfer of antibody from a human vaccinee into a naive animal, followed by pathogen challenge [ ] . whatever the immune correlate may be, its effect would be expected to follow the pattern shown in fig. , where as the value increases, the likelihood of death in the vaccinee decreases [ ] . thus, vaccine efficacy = relative reduction in risk of death = − (% of vaccinated subjects at < protective level) (% of unvaccinated subjects at< protective level) . although a number of immunotherapies and pretreatments have been licensed under the animal rule, the first vaccine to be licensed in these circumstances is biothrax for therapeutic vaccination in suspected exposure to anthrax [ ] . a case for the ancient origin of coronaviruses identification of alpha and beta coronaviruses in wildlife species in france: bats, rodents, rabbits and hedgehogs cultivation of novel type of commoncold virus in organ cultures a new virus isolated from the human respiratory tract identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome the aetiology of sars: koch's postulates fulfilled the emergence of the middle east respiratory syndrome coronavirus discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats, civets and the emergence of sars review of bats and sars naiad/nih priority list of pathogens. available at cdc priority list of pathogens. available at the plague outbreak in madagascar: data descriptions and epidemic modelling who priority list of bacteria rts,s malaria vaccine efficacy and immunogenicity during plasmodium falciparum challenge is associated with hla genotype malaria vaccine implementation programme (mvip) -programme advisory group vaccines for preventing typhoid fever global economic evaluation of oral cholera vaccine: a systematic review diagnosis and treatment of tuberculosis: latest developments and future priorities efficacy of bcg vaccine in the prevention of tuberculosis variation in protection by bcg: implications of and for heterologous immunity drug-resistant tb: deadly, costly and in need of a vaccine genomic analysis offers insight into nigeria lassa fever outbreak ecological niche modeling for filoviruses: a risk map for ebola and marburg virus disease outbreaks in uganda sep yellow fever in africa and the americas: a historical and epidemiological perspective zika, chikungunya and dengue: the causes and threats of new and re-emerging arboviral diseases chikungunya: bending over the americas and the rest of the world the zika virus epidemic in brazil: from discovery to future implications zika: the origin and spread of a mosquito-borne virus zika virus outbreak of zika virus infections the pathogenesis of rift valley fever ecologic features of plague outbreak areas, democratic republic of the congo mechanism study on a plague outbreak driven by the construction of a large reservoir in southwest china (surveillance from - ) the natural history and incidence of yersinia pestis and prospects for vaccination yersinia pestis, the cause of plague, is a recently emerged clone of yersinia pseudotuberculosis plague into the st century yersinia pestis: examining wildlife plague surveillance in china and usa cat-transmitted fatal pneumonic plague in a person who travelled from colorado to arizona cdc plague treatment transferable plasmid-mediated resistance to streptomycin in a clinical isolate of yersinia pestis resistance of yersinia pestis to antimicrobial agents melioidosis in thailand: present and future tularemia transmission to humans: a multifaceted surveillance approach airborne geographical dispersal of q fever from livestock holdings to human communities: a systematic review and critical appraisal of evidence current status of rift valley fever vaccine development the use of veterinary vaccines for prevention and control of rift valley fever: memorandum from who/fao meeting protection induced by a francisella tularensis subunit vaccine delivered by glucan particles standardized guinea pig model for q fever vaccine reactogenicity plague vaccine development: current research and future trends developing live vaccines against plague russian vaccines against especially dangerous bacterial pathogens cepi new vaccines for a safer world about gavi, the vaccine alliance vaccine delivery:bill and melinda gates foundation dual route vaccination for plague with emergency use applications immunogenicity and safety of subunit plague vaccine: a randomized phase a clinical trial rift valley fever mp- vaccine phase clinical trial: safety, immunogenicity and genetic characterisation of virus isolates development of vaccines against cchf virus a monovalent chimpanzee adenovirus ebola vaccine boosted with mva effect of dengue serostatus on dengue vaccine safety and efficacy non-neutralising antibodies elicited by recombinant lassa-rabies vaccine are critical for protection against lassa fever chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice vaccine and therapeutic options to control chikungunya virus chikungunya vaccines in the pipeline a bacteriophage t nanoparticle based dual vaccine against anthrax and plague who: health products in the pipeline for infectious diseases world health organization. who vaccine pipeline tracker yjurcmg xwo kvuyedybmzdcxqbyjgdczm/pubhtml# fda guidances for vaccine development vaccines for emerging pathogens: from research to the clinic. part regulatoryinformation/guidances/vaccines/default.htm ema guideline for vaccine development who guidance for clinical evaluation of vaccines who vaccine standards: vaccine prequalification. available at the anthrax vaccine and research: reactions from postal workers and public health professionals code of federal regulations (cfr) cfr . - . evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible predictive models and correlates of protection for testing biodefence vaccines first vaccine approval under the fda animal rule the authors declare no competing interests. key: cord- -weiwksh authors: ramírez-castillo, flor yazmín; loera-muro, abraham; jacques, mario; garneau, philippe; avelar-gonzález, francisco javier; harel, josée; guerrero-barrera, alma lilián title: waterborne pathogens: detection methods and challenges date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: weiwksh waterborne pathogens and related diseases are a major public health concern worldwide, not only by the morbidity and mortality that they cause, but by the high cost that represents their prevention and treatment. these diseases are directly related to environmental deterioration and pollution. despite the continued efforts to maintain water safety, waterborne outbreaks are still reported globally. proper assessment of pathogens on water and water quality monitoring are key factors for decision-making regarding water distribution systems’ infrastructure, the choice of best water treatment and prevention waterborne outbreaks. powerful, sensitive and reproducible diagnostic tools are developed to monitor pathogen contamination in water and be able to detect not only cultivable pathogens but also to detect the occurrence of viable but non-culturable microorganisms as well as the presence of pathogens on biofilms. quantitative microbial risk assessment (qmra) is a helpful tool to evaluate the scenarios for pathogen contamination that involve surveillance, detection methods, analysis and decision-making. this review aims to present a research outlook on waterborne outbreaks that have occurred in recent years. this review also focuses in the main molecular techniques for detection of waterborne pathogens and the use of qmra approach to protect public health. tracking [ , , ] . in developing countries, the lack of financial and technological resources contributes to wbdos [ ] . currently, it is estimated that there are species of pathogens infecting humans, which includes bacteria ( species), viruses ( types), parasitic protozoa ( species), and several fungi and helminths species [ , ] . the development of a disease, when and if infection to the host is produced, depends on factors such as minimal infectious dose (mid), pathogenicity, host susceptibility and environmental characteristics. enteric bacteria have a mid in the range of to cells but it is much lower with some species, such as shigella spp., ( - ), campylobacter spp., (about ), e. coli o :h ( - ), and v. cholera ( ) [ , ] . moreover, protozoan only need a few oocysts ( - ) to produce the disease as well as the viruses which a small number of these are enough to develop a disease [ ] . the protozoa microsporidia, as the bacteria mycobacterium avium intracellulare, helicobacter pylori, tsukamurella, cystoisospora belli and viruses such as adenoviruses, parvoviruses, coronaviruses (sars), and polyomavirus are some examples of the emerging potential waterborne pathogens [ , ] . furthermore, most of these organisms appear to have certain resistance against chlorine such as the microsporidia, enterocytozoon bienusi, encephalitozoon hellem and e. intestinales. m. avium and several viruses have resistance to common disinfectants for drinking water and to inactivation by uv light and heat [ ] , which represents a higher challenge in the treatment to remove these pathogens from water sources. the major pathogens microorganisms in drinking water systems and their related diseases are listed in table . presently, there is no unified method to encompass the collection and analysis of a water sample for all pathogenic microorganisms of interest [ ] . the challenges of the detection methods are the physical differences between the major pathogen groups, low concentration of pathogens in a large volume of water which usually requires enrichment and concentration of the samples prior to detection processing, the presence of inhibitors from the sample (especially if it comes from polluted water), established general protocols for sample collection, culture-independent detection method, as well as detection of the host origin of pathogens [ ] . the most important requirements for reliable analysis include: specificity, sensitivity, reproducibility of results, speed, automation and low cost [ ] . even though culture dependent methods are extensively used for pathogens detection in water, these methods are limited by their low sensitivity and the excessive time needed to obtain reliable results. furthermore, since there is a broad environmental distribution of human pathogens that exist in a viable but non-culturable (vbnc) state such as e. coli, helicobacter pylori and v. cholerae, false negative results may arise from culture dependent methods [ , ] . in both culture and molecular methods, index pathogens for monitoring water quality have been selected in order to indicate the presence of a large amount of pathogens in water. among these, e. coli ( figure ) has been extensively used due to the fact that detection methods for these pathogens are relatively easy and inexpensive; nonetheless, they may have the disadvantage of not providing information on their host origin and, sometimes, they do not correlate with other pathogens present in the water, such as the viruses and protozoa. thus, water characterized as pathogen-free by monitoring e. coli, for example, may be contaminated with viruses or protozoa [ ] . molecular methods can be very specific for particular species and provide further phylogenetic information about pathogens [ ] . these methods allow the use of alternatives indicators which easily relate with the host source. this permits the discrimination between human and animal pathogens and tracking the source of pollution [ , ] . it has been suggested that host-origin libraries, based on a phenotypic methods, may be useful for tracking the pathogen source but the development of such libraries may incur a significant cost [ ] . therefore, molecular methods seem to better suited for health risk assessments. nowadays, there are a number of different molecular methods to detect diverse pathogens. they are used to evaluate the microbiological quality of water, the efficiency of pathogens removal in drinking and wastewater treatment plants, and as microbial source-tracking (mst) tools [ ] . several examples of detection methods and their limits of detection are listed in table . molecular techniques, such as nucleic acid amplification procedures, offer sensitive and analytical tools for detecting a variety of pathogens, including new emerging strains, present the possibility of automation, and real-time analysis to provide information for microbial risk assessment purposes [ ] . immunology-based methods e. coli o :h and s. enterica typhimurium. . × cfu/ml of e. coli and . × cfu/ml of salmonella contaminated food. [ , ] * gc logs are mean values of genome copy logs. polymerase chain reaction (pcr) is one of the most commonly used molecular-based methods for detection of waterborne pathogens [ ] . pcr operates by amplifying a specific target dna sequence in a cyclic three-step process-denaturalization, annealing and extension-in order to achieve exponential amplification of the target sequence [ ] . several variations of pcr such as multiplex pcr (mpcr) allow simultaneous detection of several target organisms by coding specific genes of diverse pathogens in the sample in a single reaction. pcr method has the advantage of quick analysis. however, it necessitates accurate primers and optimal reaction mixture to avoid the risk of false positive and negative results [ , ] . limitations of dna based methods such as pcr include the inability to discriminate between viable from non-viable cells that both contain dna, the low concentration of several pathogens in water such as cryptosporidium, giardia and viruses, and the lack of data to indicate the real infectious risk to a population. challenges of molecular techniques include: the need for water concentration methods (i.e., for virus, ultrafiltration and direct flocculation protocols are used as concentration methods), the presence of inhibitors in water samples including humid acids and metals, to which pcr is sensitive, and the reproducible purification techniques by dna or rna from heterogeneous samples. in addition, result validation is required, since as indicated by hartman and co-workers [ ] ; also, high sensitivity in molecular techniques introduces a high risk of false positive results. an example of mpcr includes the technique developed by omar and barnard [ ] to detect the pathogenic and commensal e. coli from clinical and environmental water sources. to distinguish pathogenic e. coli from commensal, the presence of genes was evaluated in environmental waters. in addition, as control to evaluate the sensitivity of the technique and the false negative due to pcr, inhibitors were controlled using mdh gene (malate dehydrogenase) and gapdh gene (glyceraldehyde- phosphate dehydrogenase). another variation of the pcr is quantitative real-time pcr (qpcr) which enables quantification of dna targets by monitoring amplified products during cycling as indicated by increasing fluorescence [ ] . this method provides high sensitivity and specificity, faster rate of detection, minimizes the risk of cross-contamination, and there is no need for a post-pcr analysis [ ] . the dual-labeled fluorescent probes such as taqman probe and the fluorescent dye sybr green are the most used techniques for detecting pathogens [ ] . qpcr approach has been used to quantify human pathogens, such as e. coli o :h [ ] , and campylobacter spp., [ ] . these qpcr systems can specifically detect and quantify pathogens at concentrations as low as one target molecule per reaction. however, most of these methods can only detect and quantify one pathogen in a single reaction [ ] . ishii and co-workers [ , ] designed a microfluidic qpcr by using taqman probes (hydrolysis probe-based qpcr) labeled with different fluorophores that can detect l. monocytogenes, v. cholerae, v. parahaemolyticus, pseudogulbenkiana sp., s. typhimurium, s. flexneri, c. perfringens and pathogenic e. coli at levels of detection of cells/l. other advances in pcr include the microfluidics and nanobiotechnology field, allowing the construction of high-density and low-volume qpcr platforms, such as the openarray system that accommodates reactions per array [ ] . qpcr protocols have also been developed for the detection and identification of cryptosporidium spp., in river water with a lower quantitation limit of . oocysts/sample [ ] . for detection of g. lamblia and g. ardeae in wastewater samples, qpcr reached a sensitivity of . cysts per reaction [ ] . moreover, because of the importance of biofilm coating pipes in drinking water systems, the detection of pathogens in microbial communities is important. l. monocytogenes has been investigated in biofilms by using qpcr techniques with a number of l. monocytogenes detected growing in biofilm of × cfu/cm [ ] . for rna virus detection, quantitative reverse-transcriptase (qrt)-pcr was developed in order to provide quantitative estimation of the concentration of pathogens in water [ ] . this technique has the advantages of detecting viable cells due to detect messenger rna (mrna), which is present only in viable organisms. however, damaged genomes may fail to be detected with this technique [ , ] . oligonucleotide microarrays are a powerful genomic technology that is widely utilized to monitor gene expression under different cell growth conditions, detecting specific mutations in dna sequences and characterizing microorganisms in environmental samples [ ] . dna microarrays are arrays containing high density immobilized nucleic acids (genomic dna, cdna or oligonucleotides) in an ordered two-dimensional matrix that enables the simultaneous detection of hundreds of genes in a single assay via nucleic acid hybridization [ ] . microarrays are made up of glass slides or chips coated with specific oligonucleotide probes which are chemically synthesized short sequences ( to bp) [ , ] . microarray technology allows the rapid detection of multiple genes of multiple organisms simultaneously in the sample due its capacity for screening large numbers of sequences [ ] , has high throughput capacity and is able to be automated. therefore, large-scale and data-intensive experiments are performed in microarrays [ , ] . microarrays have also the ability to detect antimicrobial resistance to different antibiotics, and the probes could be designed for detecting the host origin of contaminants, which represents another advantage for characterization of contamination in water. however, advanced molecular technologies such as microarrays could experience difficulties distinguishing between viable and non-viable cells, have a relatively high cost, and may have non-specific hybridization resulting in a lower specificity and low sensitivity [ ] . dna microarray coupled with pcr amplification of target genes has been developed for higher sensitivity. through these steps, signal sensitivity increased around -fold [ ] . nonetheless, the pcr-microarray technique might lack sensitivity when the amount of sample is limited, since the technique requires splitting the samples into several pcr reactions to amplify [ ] . dna microarray in a two-step pcr-dna microarray assay that first amplifies the s or s rrna gene, respectively, followed by hybridization of these products onto a low-density dna microarray was developed to detect most of the common waterborne protozoan pathogens including c. parvum, g. intestinalis, acanthamoeba spp., entamoeba histolytica, and naegleria spp. [ , ] . in , wilson and co-workers [ ] were able to identify pathogenic bacteria, eukaryotes and viruses by using species-specific primer sets to amplify multiple diagnostic regions unique to each individual pathogen in the microarray. inoue and co-workers [ ] studied the occurrence of pathogenic bacterial species in groundwater and were able to distinguish between human and animal sources. microarrays for detection of human enteric viruses in community wastewaters using cell culture coupled with multiple targets including dna and rna viruses have also been developed [ ] . the virochip, a pan-virus dna microarray containing thousands of -mer oligonucleotide probes to target all viral families to infect humans, has been described by wang and co-workers [ ] . leski et al. [ ] developed a high-density re-sequencing microarray that has the capability of detecting different types of pathogens ranging from bacteria, protozoa, and viruses, including b. anthracis, francisella tularensis and ebola virus with limits of detection of to copies per test for nearly all the pathogens with high specificity. at present, standard and custom-made dna microarrays are commercially available from companies such as affymetrix, corning and agilent technologies [ , ] . one example of these is the phylogenetic microarray "phylochip" by affymetrix which consists of , oligonucleotide probes capable of identifying strains of bacteria and archaea [ , ] . this technology is very powerful because most known bacteria can be detected in samples without culturing, and the sensitivity allows also the detection of lower-abundance organisms (detection limit of ~ . % of microbial communities) [ ] . pyrosequencing is a dna sequencing technology for short-read dna sequencing that uses enzyme-couple reaction and bioluminescence to monitor the pyrophosphate release accompanying nucleotide incorporation, in real time [ ] . pyrosequencing technology provides a large number of sequences read in a single run [ ] . in the pyrosequencing reaction, the enzymes dna polymerase, atp sulfurylase, luciferase and apyrase are needed. the nucleotides are added to form the complementary strand of the single-stranded template, to which a sequencing primer is annealed. during the nucleotide incorporation, the pyrophosphate (ppi) is released. atp sulfurylase converts the ppi into atp and the atp is then converted to visible light by luciferase and the produced light signal is detected [ , ] . the methodology includes a primary step of concentrating the water pathogens followed by a secondary concentration depending of the type of the pathogen. the third step involves dna extraction (for bacteria and protozoa) and nuclease treatment (viral dna/rna extraction). then, the dna is amplified, the pyrosequencing is taking place and finally the analysis and bioinformatics can be performed [ , ] . in recent years, a new whole genome amplification and sequencing approach called "single virus genomics", which enables the isolation and complete genome sequencing by pyrosequencing of the single virus particle, has been described [ ] . roche pyrosequencing and other commercially available high-throughput sequencing platforms such as solexa/illumina genome analyzer, applied biosystem solid sequencing as well as the most recent, the ion torrent system, have revolutionized the study of microbial diversity [ ] . pyrosequencing could identify novel pathogens associated with water and address multiple etiologies. however, the dna amount present in wastewater samples could limit the sensitivity of this tool as it requires dna templates at picomole level but a much lower amount of dna may be available in water samples. this technology is also limited by the cost, the complexity of analysis, the need for increasing availability of massive computing power and the efficiency of data generation [ ] . pyrosequencing has been used to the analysis of bacterial biofilm communities in water meters of a drinking water distribution system [ ] , mixed urban watershed [ ] , characterization of nontuberculous mycobacterium communities in unchlorinated drinking water [ ] , and the detection of bacteriophages in reclaimed and potable and lake waters [ ] . fish or fluorescence in situ hybridization is based on hybridization of the sample with rrna oligonucleotide probes labeled covalently at one end with fluorescent dye. this technique allows an enumeration of particular microbial cells by the use of confocal microscopy, fluorescence microscopy or flow cytometry in order to obtain qualitative and quantitative results [ ] . fish is commonly used for the detection and identification of different microorganism in mixed populations such as in biofilms ( figure ) and has been used to produce a quantitative description of the microbial community structure in activated sludge and wastewater [ , ] , to study mechanisms of survival, infection at cellular level and detection of emerging pathogens from water, sewage and sludge [ ] . one example is the two-color fish assay, based on species-specific probes for c. parvum (cpar probe) and c. hominis (chom probe), and has been shown to distinguish between the two species of concern to public health [ , ] . several kits of fish are now available in the market, one example of these are the commercial kit label it ® fluorescence in situ hybridization kit cy ® , fluorescein and tm-rhodamine, which optimized the preparation and hybridization of fluorescently labeled dna probes [ ] . viable but non-culturable (vbnc) cells could detected by fish couple with direct viable count (dvc) assay [ ] [ ] [ ] . in the assay, the cells are cultured in reach media with antibiotics which prevent cellular division, increases of intracellular rrna and allow the elongations of viable cells. fish is made with specific probes that target rrna labeled with fluorescent dye. the sample is stained by ,-diamino- -pheynyl indole (dapi) that is bound to double-stranded dna to count the total amount of cells in the samples, and the results are compared to culture methods to measure the proportion of vbnc cells [ ] . members of the family enterobacteriaceae and e. coli in drinking water systems, freshwater and river water have been detected by this tool [ ] . this assay is able to detect viable e. coli/ ml in more than non-e. coli/ ml [ ] . however, the technology is limited by a low sensitivity, and the necessity of pre-enrichment and concentration steps [ ] that also may increase inhibitor concentrations and lead to false negative results [ ] . vbnc cells are living cells, metabolically active, that may still retain or regain their virulence potential upon the resuscitation (return to culturable state) under favourable environmental conditions or when the stress is removed [ ] [ ] [ ] [ ] . due to the fact that vbnc cells cannot be identified by culturable methods, the number of pathogens in this state could be underestimated and, if all bacteria in the sample are in vbnc state, the sample may be regarded as pathogen-free due to non-detection [ ] . the conversion to the vbnc state is supposed to be a response to adverse environmental conditions [ ] [ ] [ ] . resuscitation of these species could be triggered by a variety of stimuli, such as rich medium, an increase in temperature, and the presence of host cells. factors as the strain used, the age of vbnc cells, and the conditions that induced the vbnc state also affect the resuscitation [ , , ] . besides fish, other tools to detect vbnc bacteria includes: immunological techniques; rt-pcr, which target the mrna that indicates the presence of viable cells that carry out transcription; dnase i protection assay, since only the viable cells have intact membranes to protect genomic dna from digestion by exogenous nucleases; enzymatic activity, as viable cells carry out metabolic reactions and respiration; bacteriophage-based assay, using a combination of fluorescence intensity and nutrient uptake analysis; as well as the commercial kit live/dead ® baclight tm assay to distinguish viable cells from dead cells based on the membrane integrity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . immunological detection with antibodies is a technology that has been employed for the detection of multiple pathogens [ , ] . these methods are based on antibody-antigen interaction, whereby a particular antibody will bind to specific antigen which comprise the use of polyclonal and monoclonal antibodies [ , ] . immunological detection includes, for example, serum neutralization tests (snt), immunofluorescence, and enzyme-linked immunosorbent assays (elisa). snt test has been used for the serotyping of viruses and involves mixing a sample, extracted from a plaque assay, with antiserum and then assessing the decrease of infectivity by plaque assay [ ] . assays based on immunofluorescence and immunomagnetic separation (ims) are used to detect protozoan parasites. the us environment protection agent (epa) has established method . for the combined detection of cryptosporidium spp. and giardia spp., or method for cryptosporidium spp., both of which use ims followed by immunostaining and fluorescent microscopy [ , ] . nevertheless, limitation such as low sensitivity, false negative results, cross-reactivity with closely related antigens and the need for a pre-enrichment in order to reduce the cell surface antigens, are present in these techniques [ ] . biosensor based methods have flourished in recent years. biosensor is an analytical device that consists of a bioreceptor that recognizes the target analyte (e.g., enzymes, antibodies, nucleic acids, cell receptors, aptameters, recombinant antibodies, imprinted polymers and synthetic catalyst) and a transducer that converts the biological interactions into a measurable electrical signal (optical, electrochemical, mass-based, thermometric, or micromechanical) [ , , ] , thereby providing selective quantitative or semiquantitative analytical information [ ] . optical biosensors rely on a change in the optical properties of the surface caused by the binding of the analytes used for detection [ ] . electrochemical biosensors are based on measuring changes in conductance, resistance or capacitance of the active surface. in these devices, one of the electrodes is immobilized with a recognition molecule. when analytes bind, a change in electrical properties occurs providing the sensor signal [ , ] . mass-sensitive biosensors include quartz crystal microbalance biosensor, which uses a quartz crystal inserted in two electrodes. as quartz is piezoelectric (used for wave propagation), the crystal can be excited by applying a voltage across the electrodes and will exhibit a resonance frequency [ , ] . biosensor methods have the advantages of automation and miniaturization of biological analytical techniques, have short analysis times, portability, real-time measurements and do not require sample pre-enrichment [ ] . nevertheless, problems such as great sensitivity to ph, change of mass, temperature, etc., represent a great challenge to the use of these methods [ ] . in , taylor and co-workers [ ] used the surface plasmon resonance (spr) detection with pretreatment of the bacteria (heat or ethanol treated or detergent-lysed cells) and the antibodies goat anti-e.coli o :h polyclonal antibody (pab) and mouse anti-e. coli o :h monoclonal antibody (mab) for signal amplification to obtain a limit of detection (lod) of cfu/ml. antibody-based indirect sensor (optical biosensor) for escherichia coli o :h with fluorescent labeling was reported by ho et al., [ ] in to reach a lod of cells/ml. detection and monitoring of pathogens in water continues to be a field with constant improvement and development of new tools that will permit relative low cost assays and rapid identification of multiple pathogens with limits of detection that meet regulatory goals. nevertheless, efforts to standardize techniques in the field have to include sample collection, sample concentration, sample purification, sample processing, analysis, and data collection are still remaining [ ] . sample collection has to be standardizing mainly if the target is on biofilms. validation of new emerging techniques has also to be completed. besides microarrays and biosensors, the integration of molecular technologies is promising. "lab-on-chip" (loc) represents an integration of sensors and microfluidic systems in a miniaturized tool that could archive real-time monitoring of samples [ ] . by this way, loc integrates several laboratory functions on a single chip and reduced sample and reagent consumption is automatized and has fast detection times and low limits of detection. however, there are still challenges related to developing practical loc components, assay procedures and validation of the on-chip detection approaches, and of course, the different scenarios that represent a water sample [ ] . since outbreaks continue to occur despite water quality monitoring, a new approach has been applied to solve the need for a conceptualization of risk and to provide guidance and legislations [ ] . quantitative microbial risk assessment (qmra) is a systematic quantitative assessment process to estimate the health risks or illness rates of human exposure to particular pathogens. the approach combines dose response information for the infectious agent with information on the distribution of exposures routes and is able to demonstrate if the health targets are met in a drinking water distribution system [ ] , which are suggested to be a health risk less than one infection per , individuals per year, or, a disease burden of − disability adjusted life year (daly) per person per year [ ] . this tool helps to predict the burden of waterborne diseases, set tolerable limits of waterborne disease, identify the means to reduce the risk to the consumers and determine measures to protect water safety [ ] . risk assessments involve five steps ( figure ) [ , [ ] [ ] [ ] [ ] . (i) hazard identification: consists in the identification and quality evaluation of the microbial hazard. this step takes into account the potential outcomes (health effects, disease outbreaks), pathogen properties (virulence, adaptation, resistance, and mutation) and hazard pathways; (ii) exposure assessment: is the estimation of the duration of human exposures to pathogens by specific routes, the determination of the size and nature of the population exposed and the barrier reduction and recontamination on water; (iii) dose-response assessment is the characterization of the relationship between dose and incidence of adverse effect in populations exposed to microbial pathogens. it comprises data of illness, secondary transmission and immunity in population. typically, the dose-response is derived from studies of exposure of human volunteers to different doses of the pathogen or is based on previous outbreaks [ ] [ ] [ ] . factors influencing the dose-response are the exposure route, exposure medium pathogen strain host endpoint and the data source. two commonly models are the beta-poisson model and the exponential model, described elsewhere [ , , ] ; (iv) risk characterization is the integration of information from hazard identification, dose-response assessment, and exposure assessment to estimate the magnitude of health effects; (v) risk management and communication is a decision-making process based in all previous steps in risk assessment [ , [ ] [ ] [ ] [ ] . in qmra, the different variants contribute to measuring the health risk but also introduce variability corresponding to dose-response sensitivity, detection methods, temporal and spatial heterogeneities in pathogen densities and uncertainty (i.e., unrepresentative population, modeling pathogen densities and knowledge of dose-response relationships) [ , [ ] [ ] [ ] . the advantage of probabilistic modeling is to distribute the uncertainties through the model [ ] . the qmra approach presents potential advantages and limitations of risk management options that could be evaluate by numerical stimulations to investigate their possible efficacy, and that risk below epidemiologically detectable levels may be estimated under specific circumstances [ ] . however, the greatest limitation on qmra is the lack of available data to carry out this assessment [ ] . qmra also depends on the method used for monitoring pathogens in water. actually, qmra approach is based exclusively on data obtained by cultivation methods, which are not allowing the complete characterization of risk, since as mentioned above, many waterborne microorganisms are not culturable or are inefficiently cultured, thus leading to underestimation of pathogens in water. therefore, selecting the optimal tool for pathogen monitoring in source water that gives the necessary information on occurrence, prevalence, virulence, and even viability of the pathogens influences the results of the qmra approach. polymerase chain reaction methods provide rapid detection of microorganisms but are complicated by the presence of inhibitory compounds in the sample and the detection of viable but non-culturable as well as non-viable pathogens. integrating the culture and pcr methods may help to overcome the weakness of each individual method [ , , ] . as mentioned before, current risk characterization approaches rely on detection of microbial indicators or "index pathogens", which does not accurately predict the occurrence of all pathogens. novel markers that better predict the presence of all pathogens of interest would improve risk assessment and management actions [ , ] . alternative indicators proposed are bacteroidales and lachnospiraceae, which are rich in host-specific bacterial organisms [ , ] , faecal bacteriophages, rotavirus, ascaris [ ] , firmicutes [ ] , bifidobacteria spp. [ ] , and methanobrevibacter smithii [ ] . commonly s rrna gene has been used as a marker to track host-specific organisms [ ] , but other authors such as villemur et al. [ ] and caldwell et al. [ ] suggest the use of dna (mtdna) to target the identification of human and animal origins. qmra has been used to estimate the health risk associated with bathers after surfing and swimming in dry weather and post-storm conditions near beaches [ ] [ ] [ ] , water distribution networks [ ] , recreational waters impacted by agricultural contamination [ , ] , and assessment of the efficiency of the applied treatment processes in drinking water [ ] . such investigations reinforce the need for preventive measures, such as those designed to prevent and take measures to reduce water contamination. a computational tool for quantitative microbial risk assessment, called qmraspot, was developed for rapid and automatic undertaking of qmra for drinking water. qmraspot uses four index pathogens: campylobacter, enterovirus, cryptosporidium, and giardia [ ] . also, a software infrastructure name frames (framework for risk analysis in multimedia environmental systems) have been created to assess the water health risk which allows describing the problem statement, integrating models and data-bases, and provides the infrastructure for performing sensitivity, variability and uncertain analyses [ ] . it is clear that qmra and management of water quality are important tools for governmental regulation and scientific analysis. nevertheless, it is also clear that these tools still are not adopted widely [ ] . efforts to encourage the use of the qmra approach have to be made to support implementation of water safety plans, improve understanding of vulnerabilities of drinking water distribution systems, assess risks associated with extreme events and establish the best management option for the given risk in order to ensure water safety [ ] . waterborne pathogens are a global concern for worldwide public health. since pathogens in water are still a major cause of severe illness and mortality, the control, monitoring and application of regulations for water quality are in urgent need and 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contamination the authors would like to thank pathogens editors and the reviewers for their helpful comments. we are gratefully acknowledge the fie grate support of the natural sciences and engineering research council of canada (to j.h, rgpin- ) and from-fonds de la recherche du québec en nature et technologies (centre de recherche en infectiologie porcine et avicole, cripa-regroupements stratégiques rs- ) and from the dfait canada's emerging leaders in the americas program. we thanks to the conacyt mixed scholarship ( / ) of f.y.r.c., and to a.l.g.b., f.j.a.g. and the universidad autónoma de aguascalientes for their funds. josée harel had the original idea for the manuscript and, with all co-authors, carried out the design. flor yazmín ramírez-castillo drafted the manuscript, which was revised by all authors. abraham loera muro and mario jacques included the fluorescence in situ hybridization detection of actinobacillus pleuropneumoniae isolated from water sources. philippe garneau critically revised the manuscript and verified the detection methods. josée harel, mario jacques, alma lilián guerrero-barrera and francisco javier avelar-gonzález revised all the manuscript and profit it. all authors read and approved the final manuscript. the authors declare no conflict of interest. key: cord- - an u authors: ijaz, m. khalid; zargar, bahram; wright, kathryn e.; rubino, joseph r.; sattar, syed a. title: generic aspects of the airborne spread of human pathogens indoors and emerging air decontamination technologies date: - - journal: am j infect control doi: . /j.ajic. . . sha: doc_id: cord_uid: an u indoor air can be an important vehicle for a variety of human pathogens. this review provides examples of airborne transmission of infectious agents from experimental and field studies and discusses how airborne pathogens can contaminate other parts of the environment to give rise to secondary vehicles leading air-surface-air nexus with possible transmission to susceptible hosts. the following groups of human pathogens are covered because of their known or potential airborne spread: vegetative bacteria (staphylococci and legionellae), fungi (aspergillus, penicillium, and cladosporium spp and stachybotrys chartarum), enteric viruses (noro- and rotaviruses), respiratory viruses (influenza and coronaviruses), mycobacteria (tuberculous and nontuberculous), and bacterial spore formers (clostridium difficile and bacillus anthracis). an overview of methods for experimentally generating and recovering airborne human pathogens is included, along with a discussion of factors that influence microbial survival in indoor air. available guidelines from the u.s. environmental protection agency and other global regulatory bodies for the study of airborne pathogens are critically reviewed with particular reference to microbial surrogates that are recommended. recent developments in experimental facilities to contaminate indoor air with microbial aerosols are presented, along with emerging technologies to decontaminate indoor air under field-relevant conditions. furthermore, the role that air decontamination may play in reducing the contamination of environmental surfaces and its combined impact on interrupting the risk of pathogen spread in both domestic and institutional settings is discussed. indoor air can be an important vehicle for a variety of human pathogens. this review provides examples of airborne transmission of infectious agents from experimental and field studies and discusses how airborne pathogens can contaminate other parts of the environment to give rise to secondary vehicles leading air-surface-air nexus with possible transmission to susceptible hosts. the following groups of human pathogens are covered because of their known or potential airborne spread: vegetative bacteria (staphylococci and legionellae), fungi (aspergillus, penicillium, and cladosporium spp and stachybotrys chartarum), enteric viruses (noro-and rotaviruses), respiratory viruses (influenza and coronaviruses), mycobacteria (tuberculous and nontuberculous), and bacterial spore formers (clostridium difficile and bacillus anthracis). an overview of methods for experimentally generating and recovering airborne human pathogens is included, along with a discussion of factors that influence microbial survival in indoor air. available guidelines from the u.s. environmental protection agency and other global regulatory bodies for the study of airborne pathogens are critically reviewed with particular reference to microbial surrogates that are recommended. recent developments in experimental facilities to contaminate indoor air with microbial aerosols are presented, along with emerging technologies to decontaminate indoor air under fieldrelevant conditions. furthermore, the role that air decontamination may play in reducing the contamination of environmental surfaces and its combined impact on interrupting the risk of pathogen spread in both domestic and institutional settings is discussed. © published by elsevier inc. on behalf of association for professionals in infection control and epidemiology, inc. air, a universal environmental equalizer, affects all living and nonliving forms on planet earth. for humans, it has profound health implications in all indoor environments where we normally spend most of our time. [ ] [ ] [ ] air quality is also forever changing because of the influence of many controllable and uncontrollable factors that are virtually everywhere. indoor air, in particular, can expose us to noxious chemicals, particulates, and a variety of infectious agents, as well as pollen and other allergens. , emerging pathogens, such as noroviruses and clostridium difficile, have also been detected in indoor air, with a strong potential for airborne dissemination. pathogens discharged into the air may settle on environmental surfaces, which could then become secondary vehicles for the spread of infectious agents indoors. the possible transmission of drug-resistant bacteria by indoor air adds another cause for concern. a combination of on-going societal changes is adding further to the potential of air as a vehicle for infectious agents. [ ] [ ] [ ] the quality of indoor air is therefore a prominent public health concern , that requires a clear understanding of the transmission processes for the development and implementation of targeted infection prevention and control measures. although direct and indirect exposure to pathogens in the air can occur by other means, infections from the inhalation and retention, including translocation and ingestion after inhalation of droplet nuclei, are generally regarded as true airborne spread. aerosols of various sizes that contain infectious agents can be emitted from a variety of sources, such as infected or colonized individuals or flushing toilets, and may expose susceptible persons either directly (droplet transmission) or by remaining suspended in the air for inhalation (airborne transmission). , contrary to the conventionally held belief, modeling work has redefined the wells evaporation-falling curve, , revealing that expelled large droplets could be carried > m away by exhaled air at a velocity of m/s (sneezing), > m away at a velocity of m/s (coughing), and < m away at a velocity of m/s (breathing), leading to potential transmission of short-range infectious agents that contain aerosols. airborne transmission requires that pathogens survive the process of aerosolization and persist in the air long enough to be transmitted to a susceptible host. aerosolized pathogens may settle onto environmental surfaces in the immediate vicinity, leading to genesis of secondary vehicles (fig ) . this review provides current information on the spread of human pathogens by indoor air, with a focus on the major classes of human pathogens from experimental and field studies, and on emerging air decontamination technologies, including test protocols developed to assess their performance under field-relevant conditions. the study of aerosolized human pathogens requires the ability to produce them experimentally at the appropriate size, store them, and sample them for residual infectious content over a predetermined time period. the equipment must also simulate naturally occurring environmental conditions and the duration of exposure to accurately assess aerosol survivability. various analytical methods and air samplers have been used to characterize airborne pathogens and overcome the challenges of collecting and analyzing them. relevant studies have been reviewed in detail elsewhere. , , aerosolized microbes must survive the prevailing environmental conditions to potentially infect a susceptible host. multiple factors affect airborne survival of microbes indoors (table ) . , the effect of these factors on different types of microbes varies, and generalizations can be difficult because of differences in the experimental methodologies used. air temperature, relative humidity (rh), and turbulence are among the more important factors affecting the fate and spread of infectious agents indoors. the analysis of air samples for microbes now includes methods that are based on the polymerase chain reaction (pcr). however, pcr-based methods typically cannot differentiate between viable and nonviable microbes. a recent study found that pcr substantially overestimated the quantity of infectious airborne influenza virus, but the differences in infectious versus noninfectious virus over time were similar to data from quantification by plaqueforming units, which determined that virus losses were evident within - minutes postaerosolization. generally, enveloped viruses survive better at lower rh, but there are many exceptions. other factors that affect aerosol activation in relation to rh include evaporative activity (ie, dehydration, rehydration), surface areas of particles, and ph. although studies with experimental animals have determined the susceptibility to airborne pathogens and the minimal infective inhalation dose of a given pathogen, there are wide variations sources of airborne pathogens indoors and potential for environmental surface contamination. these sources may include humans; pets; plants; plumbing systems, such as operational toilets and shower heads; heating, ventilation, vacuuming, mopping, and air-conditioning systems; resuspension of settled dust; and outdoor air. the yellow and red dots represent human pathogens or harmless microorganisms. adapted with permission from biomed central. in their test design. first, the number of inhaled microbes may not be known or it may be unrealistically high. second, the test protocol may not have fully excluded microbial exposure by means other than inhalation. third, there may be incomplete recording of the environmental conditions (eg, rh, air temperature) to assess their impact on microbial viability. fourth, pertinent differences may exist between laboratory-adapted strains of the tested microbe compared with strains in the field. studies using the actual pathogen aerosolized in body fluids provide the strongest evidence of pathogen survivability. in contrast, field studies face their own set of challenges, which include the noise, bulk, and expense of inefficient air collection devices. moreover, passive impingers may not adequately collect low concentrations of pathogens found in the clinical environment. slit sampling does not impose size exclusion and may be more effective at recovering viable pathogens of any size. from a methodologic perspective, field studies also must control for potential variables, such as air turbulence or human activity in areas proximate to sampling, such that sampling occurs before, during, and after an area is occupied and should include functioning ventilation systems. we have previously reviewed published studies on the airborne spread of viruses of animals and humans. , table summarizes key human pathogens with evidence of aerosol transmission. a number of these pathogens causes severe disease, and their classification as high risk by the u.s. centers for disease control and prevention and the world health organization emphasizes the need for appropriate control measures. experimental studies have used surrogates for human pathogenic enveloped and nonenveloped viruses, such as cystovirus (ϕ ) and bacteriophage ms- , respectively. enteric viruses are transmitted primarily by the fecal-oral route, but airborne transmission has been reported. airborne transmission of norovirus may be possible via aerosolization of vomitus and toilet flushing, which are regarded as potential sources of both indoor air and environmental surface contamination. enteric bacteria and viruses have been recovered from indoor air and environmental surfaces in areas sur-rounding toilets. , , we reported that aerosolized simian rotavirus sa- survived best at midrange rh. , these results contradicted a prior study by moe and harper, in which the uk strain of calf rotavirus was reported to survive best at low and high rh, but not at high temperature. subsequent studies on human rotavirus, murine rotavirus, and a uk strain of calf rotavirus, aerosolized under the same experimental setup, confirmed the behavior of all strains of rotaviruses are similar in airborne state. , furthermore, studies of different picornaviruses (poliovirus type [sabin] and human rhinovirus) and a human coronavirus (an enveloped virus) that used the same experimental conditions produced results that were consistent with the published literature, suggesting that the experimental design did not introduce bias toward the behavior of aerosolized rotaviruses. , , among the respiratory viruses, influenza virus is present in the air around infected individuals, and airborne transmission via droplet nuclei has been demonstrated in experimental models and in reports of influenza spread on-board aircrafts. low rh favors airborne survival and transmission; however, high air exchange rates facilitate dilution of virus-containing aerosols, regardless of their size. a recent study confirmed recovery of influenza virus from the air emitted by infected persons at distances of . - . m, which could reach the breathing zone of susceptible individuals, including health care workers. surprisingly, and in spite of much study, the exact mode of and the relative importance of various types of vehicles for transmission of rhinoviruses, which are the most frequent cause of the common cold, remain shrouded in mystery. , the behavior of experimentally aerosolized rhinovirus type , which represents typical picornaviruses (as previously mentioned), coupled with rhinovirus recovery from both indoor air and outdoor air, substantiate the role of air as a vehicle in spread of some of these picornaviruses. taken together, an overall assessment of the available evidence suggests a role for airborne spread and for the role of contaminated hands and environmental surfaces in rhinovirus dissemination. coronaviruses are the second leading cause of the common cold and are also responsible for the severe acute respiratory syndrome (sars) and the middle east respiratory syndrome. sars is thought to be transmitted via direct contact, but airborne transmission is also suspected because the virus has been detected in air samples that were collected from rooms where a patient was recovering from sars. , the virus is spread through droplets and can remain viable on surfaces for several days at room temperature. the use of aerosol-generating procedures, such as intubation, bronchoscopy, and oxygen delivery vents, may promote dispersal of sars via enhanced release in mists of exhaled pulmonary gases. , our earlier work on the behavior of aerosolized human coronaviruses e further substantiates the potential role of air in their aerial spread. aerosol transmission of the ebola virus is biologically plausible. the virus is present in saliva, stool, blood, and other body fluids; therefore, it could be aerosolized through symptoms associated with infection or via health care procedures. the ebola virus has been shown to survive in the air when the half-life of the virus ranged from (zaire ebola virus) to minutes (reston ebola virus), and the time for % biologic decay of the aerosolized virus held in rotating drum (at %- % rh and °c ± °c) was estimated to be between and minutes. additionally, infection of rhesus monkeys via experimentally aerosolized ebola virus has also been reported. these findings raise concerns for aerosol transmission and control of this serious pathogen; however, thus far, there is no clear evidence for the airborne spread of this virus in humans. epidemiologic evidence indicates transmission is associated with direct physical contact or contact with body fluids; however, the possibility of aerosolized spread has been postulated by ebola virologists. , bacteria approximately one-third of humans carry staphylococcus aureus, with the anterior nares as a common site of colonization, and environmental contamination plays an important role in the transmission of methicillin-resistant s aureus. shedding of the bacteria is highly variable, but transmission likely occurs via skin squames that settle out on environmental surfaces in the vicinity. smaller particles may remain airborne, particularly if there is air turbulence. , an important characteristic of the staphylococci is their ability to survive over a wide range of temperatures, rh, and exposure to sunlight. mycobacterium tuberculosis is transmitted via droplet nuclei expectorated from infected persons during coughing, sneezing, and talking. , control measures include expensive negative-pressure ventilation and less expensive, but climate-dependent, natural ventilation. upper-room ultraviolet (uv) light or negative air ionization may help reduce the airborne spread of m tuberculosis. nontuberculous mycobacteria are found in soil and water sources and can form biofilms under domestic environments, such as shower heads. transmission to humans is uncertain, but droplet aerosolization is a suspected route of pulmonary disease, with shower heads considered a common source. , contamination of hospital water supplies and medical equipment are suspected in nosocomial outbreaks of disease. similarly, legionella spp become airborne by active aerosolization of contaminated water and form biofilms in air conditioning systems. legionella-like amoebal pathogens are a subset of bacteria that grow within amoebae and often are coinfectious agents with other bacteria and fungi. clostridium difficile spores have been recovered from the air near symptomatic patients, especially those with recent-onset diarrhea. air samples were positive for c difficile in % of patients, and the highest levels of surface recovery were in areas closest to the patient. the isolates recovered from the air were indistinguishable from those recovered from fecal samples and from the environment in the same settings. additionally, c difficile has been recovered after toilet flushing, and leaving the lid open when flushing increases contamination of surrounding environmental surfaces. airborne infection of bacillus anthracis is affected by environmental factors that include room size, ventilation rate, and host factors, such as pulmonary ventilation rate. secondary aerosolization of viable b anthracis spores was reported after contamination of a u.s. senate office, with > % of particles being in the respirable size range of . - . μm. as ubiquitous microorganisms, fungi pose a health threat in indoor environments. fungal infections can be particularly serious in immunocompromised patients, especially airborne spores of aspergillus spp that are blown in from natural ventilation sources. fungal spores are aerosolized from municipal water supplies and dust and can be effectively transported over long distances by wind and air currents. , the evolution of the fungal spore has enabled them to travel long distances and be more capable of withstanding environmental insults. the most important factor of fungal growth in indoor environments is humidity ; therefore, control measures include dehumidification of the air and high-efficiency particulate arrestor filtration. recent research suggests that airborne fungal particles are heterogeneous and comprise spores and submicrometer fragments. , these fragments are of significant interest with regard to health because they remain in the air longer and are easily inhaled. there are also a variety of fungal components that have been identified in air, including mycotoxins, ergosterols, glucans, and microbial volatile organic compounds, and these require unique analysis methods. taken together, these findings provide a foundation for the definition of sick building syndrome. high humidity within sick houses and buildings allows for growth of fungi indoors, particularly species of aspergillus, penicillium, and cladosporium and stachybotrys chartarum, an indoor mold that was associated with sick building syndrome several decades ago. , , these fungi can be found in dust, furniture, carpets, and ventilation systems at concentrations ranging from - , colony-forming unit (cfu)/m . in fact, carpet has been described as a sink for fungi, but it is also a source for resuspension of fungal particles into the air. various respiratory conditions (eg, wheeze, cough, asthma) have been linked to fungi and their biologic components in the indoor environment. fungal species found outdoors include cladosporium and alternaria spp, which are responsible for triggering hypersensitivity reactions, including rhinitis, sinusitis, and asthma. , the clear recognition of indoor air as a vehicle for pathogens has incurred a corresponding upsurge in the marketing of products and technologies with claims for safe and effective decontamination of air. although many technologies are available for environmental surface decontamination, the number and variety of those for decontamination of indoor air remain limited and of questionable veracity ( table ). the air-decontaminating claims of many such technologies are not based on testing under field-relevant conditions with pathogens relevant to human health, and scientifically valid and standardized protocols to generate field-relevant data for label claims for review by regulatory and public health agencies and the public at large remain unavailable. here, we address this gap in the development of a test platform for standardized testing of commercially available devices for decontaminating indoor air of vegetative bacteria that represent airborne human pathogens. we know of only one guideline that directly relates to this topic. it specifies the size of a sealed enclosure for experimental contamination of the air with aerosols of vegetative bacteria to assess technologies for their temporary reduction. therefore, the text that follows relates directly to that guideline. the studies of microbial survival in indoor air, as well as proper assessment of methods for its decontamination, emphasize numerous challenges and highlight the need for specialized equipment and protocols. proper expertise and suitable experimental facilities for such investigations remain uncommon. several of the available sites with testing claims are neither experienced in, nor equipped to conforming with, the u.s. environmental protection agency's (epa) guidelines on testing the sanitization of indoor air. based on our considerable experience in the study of airborne human pathogens, , , , , we have built an aerobiology chamber (fig ) designed to meet the requirements of the epa guidelines and have used this to study the effects that a variety of air decontamination technologies have on the airborne survival and inactivation of vegetative bacteria, viruses (bacteriophage), and bacterial spore-formers (sattar et al, unpublished data) . additional details about the operational aspects of the aerobiology chamber, described elsewhere, are discussed briefly. any meaningful assessment of air decontamination requires that the aerosolized challenge microbe remain viable in the experimentally contaminated air long enough to allow for proper differentiation between its biologic decay or physical fallout and inactivation or removal by the technology being assessed. there- fore, initial testing is required to determine the rate of biologic decay of the test microorganism(s) under the experimental conditions to be used for testing potential air decontamination technologies. for this, the test microorganism(s) was aerosolized into the chamber, and -minute air samples were collected at different intervals using a slit-to-agar (sta) sampler over an -hour period. the culture plates were incubated at °c ± °c, the cfu on them was recorded, and the data were analyzed to determine the rate of biologic decay. the results of the tests on the airborne survival of types of vegetative bacteria are shown in figure . acinetobacter baumannii (atcc ; atcc, manassas, va) proved to be the most stable in air, followed by s aureus (atcc ; atcc) and klebsiella pneumoniae (atcc ; atcc). three types of commercially available indoor air decontamination devices that were based on uv light and high-efficiency particulate arrestor filtration were tested for their ability to reduce the levels of viable bacteria in the air of the chamber ( table ). the air within the chamber was first experimentally contaminated with aerosolized test bacterium suspended in a soil load. the test device, placed inside the chamber, was remotely operated, and samples of the chamber air were collected directly onto petri plates using sta and were incubated for cfu determinations. as shown in figures a and b , the air decontamination devices that were tested could achieve a -log reduction in viability of s aureus and k pneumoniae in - minutes (table ). so far, such testing has been conducted only once with a baumannii using device , and as the data presented in figure show, it reduced the viability of a baumannii by log in minutes (table ) . in this experiment, device was tested for its ability to manage ongoing fluctuations in the microbiologic quality of indoor air. a suspension of s aureus was nebulized into the chamber at separate time points, while the device operated continuously. as shown in figure , the device's efficacy after the challenges with aerosolized bacteria was almost the same. the times at which the device demonstrated -log reductions after each nebulization were found to be , . , and . minutes. the mean of the -log reduction times was . ± . minutes, giving an average biologic decay rate of aerosolized bacteria of . ± . cfu/m /min after the nebulizations. as previously mentioned, larger particles of aerosolized pathogens often settle onto environmental surfaces in the immediate vicinity, leading to contamination as a secondary vehicle of trans- fifteen sterile plastic plates were placed in groups of on the floor of the aerobiology chamber, with one set in each of the corners and one in the center. the lids of the plates were removed. a suspension of s aureus in a soil load was nebulized into the chamber with the muffin fan operating for minutes to evenly distribute the airborne bacterial particles. a -minute air sample was then collected from the chamber using an sta sampler to determine the initial level of airborne contamination. ten minutes were allowed to elapse for circulation of the airborne bacteria in the chamber. the muffin fan was then turned off and the airborne bacteria were allowed to settle for minutes. at the end of this period, the petri plates were retrieved and eluted for cfu to determine the titer of microbial contamination deposited on each one. such testing allowed us to determine the levels of airborne bacteria that could settle on the plates without air decontamination procedures. the experiment was repeated in exactly the same manner, but with the test device in the chamber activated and allowed to work for minutes. at the end of this period, the petri plates were retrieved and eluted for cfu to determine the titer of microbial contamination deposited on each plate. the results indicated that the nebulization of the microbial suspension for minutes produced . log cfu/m of air in the chamber. the average level of cfu on the control and test petri plates held in the chamber was ± and . ± . , respectively. the device could reduce the contamination of the plates from airborne bacteria by % as compared with the controls. recognition that human pathogens can be transmitted via indoor air emphasizes the need for the development of control procedures that limit exposure and reduce the risk of infection in susceptible individuals. this need is heightened by an increase in the aging population and numbers of the immunosuppressed. we must also be prepared for an intentional or accidental release of infectious aerosols. standardization of sampling and analytical methods is crucial to developing an understanding of airborne pathogens and technologies for their effective control. we have described the creation and application of an aerobiology test chamber that complies with the relevant guideline of the epa. the chamber was successfully used ( ) to study the airborne survival of types of vegetative bacteria under ambient conditions; ( ) to test the ability of commercial indoor air decontamination devices to abate experimentally generated aerosols of types of vegetative bacteria; ( ) to test one of the devices for its ability to deal with repeated microbial challenge with vegetative bacteria in simulation of situations in which indoor air is contaminated on an on-going basis; and ( ) to test one of the air decontamination devices for its effectiveness in reducing the level of microbial contamination of environmental surfaces as a function of reducing airborne bacteria. each of these experiments was completed successfully, thereby demonstrating the suitability of the aerobiology chamber and the protocols for aerosol generation and sampling. the use of the sta sampler proved particularly effective for providing event-related information on the levels of viable bacteria in the air of the chamber. the testing with a baumannii clearly demonstrated that it is more suitable than k pneumoniae as a surrogate for gram-negative bacteria. a baumannii is not only a relevant airborne pathogen that is more resistant to aerosolization, but it also is more stable in the airborne state. therefore, it is recommended that it be considered as an alternative for k pneumoniae by regulatory agencies, such as the epa, for testing and registration of air decontamination technologies. table regression coefficients, p values comparing decay rates of efficacy tests with stability in air, and times required to achieve log reductions device the experimental facility and test protocols described here are suitable for work with other types of airborne human pathogens, such as viruses, fungi, and bacterial spore formers. the aerobiology chamber also could be readily adapted to assess emerging technologies of indoor air decontamination. although the work reported here was performed in a sealed and empty chamber, as specified in the epa guidelines, the aerobiology chamber can be modified to represent air exchanges, and furniture can be introduced to simulate a typical room under both domestic and institutional settings. air, in general, is crucial to the establishment and maintenance of the indoor microbiome, and the continual redistribution of microbes indoors occurs at the air-surface-air nexus. although classic airborne spread of pathogens occurs via droplet nuclei, droplets can potentially contaminate environmental surfaces, depending on their size and prevailing environmental conditions, thereby creating secondary vehicles for pathogens. therefore, targeting airborne pathogens could potentially provide an additional advantage by reducing environmental surface contamination. our 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characterizing airborne fungal and bacterial concentrations and emission rates in six occupied children's classrooms evaporation and dispersion of respiratory droplets from coughing we thank dr john a. mitchell (wordsmith scientific and regulatory, llc, bozeman, mt) and elizabeth bruning (rb, montvale, nj) for their critical review and feedback. key: cord- - x zrfw authors: cherrie, mark p. c.; nichols, gordon; iacono, gianni lo; sarran, christophe; hajat, shakoor; fleming, lora e. title: pathogen seasonality and links with weather in england and wales: a big data time series analysis date: - - journal: bmc public health doi: . /s - - - sha: doc_id: cord_uid: x zrfw background: many infectious diseases of public health importance display annual seasonal patterns in their incidence. we aimed to systematically document the seasonality of several human infectious disease pathogens in england and wales, highlighting those organisms that appear weather-sensitive and therefore may be influenced by climate change in the future. methods: data on infections in england and wales from to were extracted from the public health england (phe) sgss surveillance database. we conducted a weekly, monthly and quarterly time series analysis of pathogen serotypes. each organism’s time series was forecasted using the tbats package in r, with seasonality detected using model fit statistics. meteorological data hosted on the medmi platform were extracted at a monthly resolution for – . the organisms were then clustered by k-means into two groups based on cross correlation coefficients with the weather variables. results: examination of . million infection episodes found seasonal components in / ( %) organism serotypes. salmonella showed seasonal and non-seasonal serotypes. these results were visualised in an online rshiny application. seasonal organisms were then clustered into two groups based on their correlations with weather. group had positive correlations with temperature (max, mean and min), sunshine and vapour pressure and inverse correlations with mean wind speed, relative humidity, ground frost and air frost. group had the opposite but also slight positive correlations with rainfall (mm, > mm, > mm). conclusions: the detection of seasonality in pathogen time series data and the identification of relevant weather predictors can improve forecasting and public health planning. big data analytics and online visualisation allow the relationship between pathogen incidence and weather patterns to be clarified. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. seasonality can be defined as increased or decreased observations that display a periodic pattern (e.g. week, month, quarter) of occurrence between years [ ] . microbial pathogens tend to be defined as microorganisms that can cause disease in humans and other organisms [ ] . reviews of their seasonality have been published previously [ ] . seasonal drivers are already known to produce annual peaks for a number of infectious diseases, including malaria [ ] , west nile virus [ ] , and cholera [ ] , as well as several pathogens transmissible by contact such as influenza [ ] , respiratory syncytial virus [ ] and meningococcal meningitis [ ] . seasonality may be explained by a mixture of factors including climate, social, behavioural, agricultural, environmental, stochastic changes in immune populations, and other drivers. in addition, weather can influence vector abundance, pathogen survival and host characteristics (e.g. behaviour and immune susceptibility) [ ] . the mathematical approaches to modelling have been reviewed [ ] . several studies have investigated the effects of weather and climate on pathogens in england and wales. salmonella enteritidis incidence was shown to increase by . % ( %ci; . - . ) for every °c rise over a °c threshold [ ] . similarly, campylobacter prevalence was associated with temperature in the previous weeks [ ] while other studies found little association [ ] . a systematic approach to the analysis of the potential seasonality of common pathogen serotypes and their associations with multiple weather variables is required to help narrow the focus on candidate pathogens in addition to those that have been studied in depth previously. the current analysis is well placed to address this gap given the rich data now available on a broad number of pathogens and meteorological factors. the aim of the analysis was to use several data mining techniques to identify pathogens that display a seasonal component, and describe their associations with meteorological factors as an aid to future analytical work (including forecasting) and public health planning. infectious disease data from england and wales were collected by public health england (phe) (formerly the health protection agency and before that the public health laboratory service) through a voluntary reporting system, whereby hospital laboratory records are transferred to regional epidemiology units, processed and added to the labbase national surveillance database [ ] . to avoid duplication by organism and patient, each record has a unique identifier called the organism patient illness record (opie). if a record is sent with the same patient and organism information within days ( weeks for mycobacterium spp.), then these cases are merged to ensure a single opie for the entire duration of the episode. the second generation surveillance system (sgss-formerly labbase ) voluntary national surveillance database holds records on , , reportable human infectious cases spanning from the st week in to the nd week in for root organisms and serotypes. pathogen counts were recorded at a weekly level in the database. the analysis for individual serotypes was restricted to complete years, from to , with serotypes greater than cases (above quartile one, i.e. top % in terms of total count), as a time series model could not be automatically estimated with fewer cases (n = ). we aggregated the data to a monthly level and linked with national meteorological data held on the medical and environmental data mash-up infrastructure project (medmi) platform [ ] . the analysis was performed at a national scale due to multiple factors at a local level that act as noise to obfuscate the relationship between infectious disease and weather [ ] . a range of meteorological data for the uk was downloaded from the medmi platform [ ] at a km by km resolution for - ; full details on methods used to generate data are provided elsewhere [ ] . the variables were monthly weather summaries that included: mean sunshine duration (hours per day), mean temperature (°c), mean daily maximum temperature (°c), mean daily minimum temperature (°c), mean vapour pressure (hpa), mean sea level (msl) pressure (hpa), rain ≥ mm (days), rain ≥ mm (days), total rainfall (mm), mean wind speed at a height of m (knots), mean relative humidity (%), snow lying over % of ground (days), ground frost measured as grass minimum temperature below °c (days), and air frost measured as air minimum temperature below °c (days) (additional file : figure s ). the data were imported into arcmap (esri, redwoods, ca) and aggregated (arithmetic mean) for england and wales, which enabled linkage with the infectious disease time series data. descriptive statistics were generated for the organisms including total count, crude prevalence rate per month, peak month and plots of time-series patterns (for gastro-intestinally acquired infections and those from respiratory transmission). we applied a two stage automated analysis to: a) detect seasonality and b) identify correlations with weather variables. the first stage was the seasonality detection analysis, undertaken in rstudio (ver . . ). description of the forecast package, which was used extensively in the analysis to automatically detect seasonal patterns, has been detailed elsewhere [ ] . briefly, the pathogen time series data were decomposed via box cox transformations into trend, seasonal and irregular components, which were used to forecast the time series into the future [ ] . the algorithm automatically selects model parameters such as trend (with or without a dampening parameter) and noise (arma (p,q) process) using model fit statistics (i.e. minimising akaike information criteria (aic)). a tbats model, as described above, was fitted for each organism serotype (with a non-zero count) using the weekly periodicity (i.e. the most granular temporal resolution available). the models were re-run with data aggregated at monthly and quarterly periodicities to investigate seasonality at different temporal aggregations [ ] . each time the model would provide a logical output (i.e. true/false) as to whether the model fit improved with the inclusion of the seasonal component (i.e. consistent repeating pattern over time). this is because the algorithm fits two models, seasonal and non-seasonal, and selects the seasonal model if the aic is lower than the non-seasonal model (heuristically, it selects the model that results in the best combination of good fit and lower number of parameters). to limit the seasonality definition to those whose model fit was significantly better with the addition of the seasonal component, we calculated the difference between the seasonal and non-seasonal aic (Δ i = aic nonseasonal − aic seasonal ) and excluded organisms with aic difference greater than , as suggested as a suitable cut-off by burnham and anderson [ ] . the pathogens at a monthly resolution with aic difference greater than were used in subsequent analysis with weather variables. for the second stage, we aggregated the pathogen incidence data to monthly resolution so that they were able to be merged with the weather variables previously processed to monthly values by the national climate information centre. the time series' for each of the weather variables was shown to be stationary (no significant trend from year to year) by using the augmented dickey-fuller (af) test (p < . ) and kwiatkowski-phillips-schmidt-shin (kpss) test (p > . ). we tested each pathogen time series in the same way. some were found to be non-stationary and differenced (once or twice, depending on results of af and kpss tests). cross correlation coefficients were generated between cases and weather variables for the month that they were recorded and then by the meteorological values lagged by month. the correlation coefficients were then used as input to the k-means clustering method. two clusters were generated in order to narrow the focus on those correlated with weather. the terminology for discussing the correlation coefficients was as follows: very weak (r = - . ), weak (r = . - . ), moderate (r = . - . ), strong ( . - . ) and very strong (r = . - . ). seasonality and weather correlation results were summarised and discussed in terms of differences between weather variables and within the most common genus for which serotypes were available (salmonella). supplementary to the time series analysis, an rshiny app was developed to display the results and aid future hypothesis generation. the user can filter the pathogens by seasonality, prevalence and serotype. once an individual serotype is selected, a range of descriptive information is available: wikipedia description, total number of cases, time series plot, month plot of crude rate per , (england and wales population), decomposition of time series, tbats model forecast and weather scatterplot. the weekly data on . million pathogen infections in england and wales from to were examined systematically. the minimum number for an organism to be in the database during the time period was once per week. the maximum number of cases for week was for chlamydia trachomatis. there was a non-normal distribution of total cases, from one case for organisms to , , for chlamydia trachomatis. the median number of total cases was (interquartile range quartile -quartile ; - , ). the organisms with the highest number of serotypes were salmonella (n = ) and streptococcus (n = ), although most of these had very low counts. figure shows a heat map of z-scores of crude rates by month ( fig. shows non-salmonella pathogens, and fig. shows only the salmonella genus). the months with the fewest high pathogen rates for the majority of organisms were december ( . %) and february ( . %). the months with the highest number of high pathogen rates were more evenly spread out over the summer and autumn, with july, august, september and october being the highest months for . % of the organisms. the seasonality of gastro-intestinally acquired infections (fig. ) , and pathogens acquired through respiratory transmission (fig. ) , differed substantially. the gastro-intestinal pathogens showed different distributions, with most bacteria having higher rates in summer, some viruses had higher rates in winter (e.g. norovirus, rotavirus) and others were more common in the summer (enteroviruses). some of the pathogens associated with travel overseas had a late summer increase (thought to reflect the period when people return from summer holidays). the respiratory pathogens predominated in the winter months (e.g. coronavirus, influenza, respiratory syncytial virus (rsv)). however, several of the bacterial pathogens were more frequent in warmer months (e.g. bordetella, coxiella, legionella). we detected significant seasonality in organisms using tbats models at varying periodicities ( / ; %) (additional file : table s ); with varying links with weather (additional file : figure s ). two k-means clusters (identified as the optimum number of k) were generated from the cross correlation coefficients with weather variables and represented groups of pathogens that had similar correlations with weather variables (fig. ) . the two groups were characterised by their relationship with the weather variables (additional file : table s ). group had mean positive correlations with higher temperature (min, mean, max), sunshine and vapour pressure; whilst the group had positive mean correlations with lower temperature variables (snow lying, ground frost, air frost), precipitation (rain days over mm, rain days over mm and rainfall), mean in group , pathogens had highest correlations with relative humidity (n = ) and ground frost (n = ) (additional file : figure s ). there was at least one pathogen with the highest correlation for each meteorological variable. summary information on seasonality and links with weather, by temperature cluster group are presented in table . group consisted of organisms, of which were from the salmonella genus. parvovirus b had a moderate correlation with sunshine (mean r = . ), followed by salmonella enteritidis with sunshine (r = . ) and salmonella typhimurium with vapour pressure (r = . ). group consisted of pathogens of which only two genus (influenza and trychophyton) had more than one serotype. rsv had strong correlations with air frost (r = . ), followed by moderate correlations between human metapneumovirus (hmpv) with relative humidity (r = . ) and rubella virus with lying snow (r = . ). we were interested in how the correlation coefficients varied between the weather variables that measured the same phenomenon (e.g. min, max, mean temperature). in general, there were slight differences between the different measures of temperature. the mean difference in correlation coefficients between minimum and maximum temperature was . with standard deviation of . . hmpv and rotavirus showed the largest difference between the temperature variables (comparing min temp and max temp). hmpv recorded a . higher coefficient for maximum temperature, whereas rotavirus recorded a . higher coefficient for minimum temperature. similar associations with temperature were found with vapour pressure and sunshine, although they tended to be relatively weaker when taking the mean for all of the pathogens there were also similar moderate inverse correlations with ground frost, air frost and snow lying days. for influenza a, days with lying snow had a higher correlation than the other weather variables (r = . ). notable differences in correlations between pathogens and the precipitation variables (comparing days with over mm of rain compared to days with over mm of rain), included plesiomonas shigelloides with a . higher correlation with days over mm and rsv with a . higher correlation with days over mm of rain. salmonella serotypes featured heavily with varying strength and pattern of seasonality detected. salmonella enteritidis and salmonella typhimurium had the strongest associations with meteorological variables. the remaining salmonella serotypes were split between being weakly correlated (n = ) and very weakly correlated (n = ). there is some reason to believe that the epidemiological causes of seasonality in most salmonellas is similar ( / ; % belong to group ) and the association with temperature might be linked to growth in prepared foods. in addition, the strength of association in linking the seasonality or temperature to cases will be limited to the number of isolates in each serogroup. because of this the salmonellas were grouped into four groups ( . salmonellas causing enteric fever that are usually acquired overseas (s. typhi/s. paratyphi); . seasonal salmonellas; . strains showing no evidence of any seasonality and . the remaining strains where there are insufficient numbers to determine seasonality). the remaining strains included serotypes that had so few isolates that seasonality could not be determined. when grouped thus, the seasonality of the seasonal salmonellas ( ) resembled that of the remaining strains ( ), while the overall seasonality of serotypes that individually showed little evidence of seasonality were not obviously seasonal when combined (fig. ) . the seasonality of groups and showed a high degree of correlation using data averaged over the -year period (r = . ; fig. b ). we have systematically examined a large number of human infectious disease pathogens for seasonality, and detailed potential links with weather in england and wales. this was made possible by utilising time series and clustering algorithms that can detect patterns in the data without supervision. this can lead to greater research efficiency by defining a focus for further investigations. we found that of the most prevalent organisms displayed seasonality, classified into two groups due to their association with month lagged meteorological variables. within these groups, there were well-known seasonal pathogens such as rsv, campylobacter and salmonella, as well as other less studied organisms such as aeromonas. the limitations of the big-data approach in this analysis meant that it was not possible to undertake analysis on causative weather factors on pathogen incidence. behavioural determinants that correlate with season and weather may explain the correlations found. for example, school closures for holidays can reduce transmission and therefore cases of influenza [ ] , outdoor eating, when the temperature is higher increases risk of salmonella, undercooking, raw meat contamination and recreational activities on water, are more likely to occur in summer, are associated with campylobacter [ ] . in separate work we are looking at methods to separate out the weather parameters from seasonality (and the associated behavioural determinants) using local weather data linkage, as described in 'recommendations for future research' [ ] . the study was limited by the temporal and spatial aggregation of the data, and therefore we were unable to investigate the effect of day-to-day weather in regions of england and wales. the results of the analysis were also dependent on the time-period used. for example, c. difficile have been reported to have a strong seasonal pattern previously using hospital episode statistics from england from to [ ] ; however we did not find a strong seasonal component in our study period. in our analyses, c. difficile displayed a peak in and then reduced in prevalence and seasonality. therefore, the results are presented with a caveat that the correlation coefficients with weather were sensitive to the time-period under analysis and would be expected to differ in a pathogen-dependent manner. the surveillance methods for collecting data changed over the years, with many pathogens having separate expert surveillance datasets that are independent of this data and some periods of enhanced surveillance or poor surveillance. there have also been periods where an intervention (e.g. vaccination) had been introduced, as well as those where the surveillance had improved (e.g. fungal infections; hospital infections), although we were unable to systematically account for these changes in the current analysis. furthermore, the data were lab-confirmed and therefore do not represent milder unreported or undiagnosed cases which may display a different pattern of seasonality. finally, we could not ascertain concomitant pathogens as they were not readily extractable from the database. the analysis was limited as it only considered a month lag effect and did not consider time-varying confounders. lag effects can vary for different environmental exposures. for example sunshine will induce -hydroxy-vitamin d production (the major circulating form of vitamin d) in human skin; -hydroxy-vitamin d will lag sunshine exposure by up to months due to metabolism within the body [ ] . also, the life-cycle of the pathogen or vector varies between organisms producing a lag between weather exposure and clinical manifestations of pathogen and subsequent laboratory diagnosis [ ] , but this has not been addressed in the current study. lag effects may be more pronounced for organisms that are indirectly rather than directly associated with weather [ ] , for example weather conditions that precede mosquito larvae growth do not immediately result in malaria transmission, due to development of both mosquito and pathogen being highly complex [ ] . however, given that the analysis was undertaken at a monthly resolution some short-term lagged correlations would be captured. the primary strength of the analysis is the large infectious disease dataset, which is nationally representative and has information on a wide range of pathogens. we have shown how a well-known clustering algorithm (k-means) can be applied to these data to classify pathogens by their relationship with weather variables. we have utilised a number of weather parameters from the medmi database, which allowed for subtle differences in correlation to be illustrated. the use of two methods to detail seasonal patterns was also a strength of the analysis. the advantages of using a tbats model is that it automatically selects fourier terms and other aspects of the model, whilst allowing for seasonality to change over time. wavelet analysis could be used to test for the robustness of the findings in future analysis. by sub-setting the data on the basis of seasonality detected using the difference in model fit statistics between a 'seasonal' and 'non-seasonal' model, it was less likely that the correlations with climate in the following analysis were spurious. this is akin to defining an exclusion criterion in the design of an epidemiological study to reduce the effect of bias. having detailed the strengths and limitations of the current analysis, in the following sections we aim to explain the results in relation to previously published work under headings based on the explanations for seasonality outlined by grassly and fraser [ ] . the data linkage was at the england and wales level which has certain advantages (reducing noise in the data), however public health applications often require predictions at a variety of smaller scales [ ] . analysis at a local level would complement the results presented here by showing the context in which national level predictors hold. in addition our analyses should be undertaken in different national contexts, as some pathogens shown to be non-seasonal in this context (e.g. polio, p. vivax) will be highly seasonal in non/under-vaccinated endemic regions. in particular, between salmonella serotypes, there was a clear hierarchy of strength of correlation with weather. the high prevalence of salmonella enteritidis (n = , ) and salmonella typhimurium (n = , ) contributed to high seasonality for these serotypes and strong associations with temperature and the auto-correlated sunshine and vapour pressure. the examination of salmonella data showed some of the limitations that can constrain the comparison of weather and infectious disease data. while most salmonella serotypes were seasonal, this could not be demonstrated for most of these until they were combined together with similar serotypes showing some evidence of more cases in summer months. the serotypes that showed no evidence of seasonality may be associated with contamination from reptiles kept as pets [ ] . such exposure is thought to be relatively less seasonal in its occurrence compared to foodborne salmonellosis. typhoid and paratyphoid infections in england and wales are usually associated with travel abroad, particularly to the indian subcontinent, and this is in the late spring and early autumn [ ] . temperature was most often used to explain any relationship between climate and pathogens previously [ , ] . however, there must be careful consideration of the measure of temperature used as shown in our analysis of influenza a and b. influenza a was most strongly correlated with extreme weather events (i.e. snow lying days), which may indicate specific circumstances around these events that are important for transmission of the pathogen (i.e. temperature of below °c with moisture in the air). we also found that other temperature-related variables showed consistent associations with various pathogens. vapour pressure has been used previously in a study investigating the effect of meteorological variables on the risk of legionnaires' disease in switzerland [ ] . vapour pressure may have such strong associations with several infectious diseases such as influenza [ ] , because it represents a set of meteorological parameters, i.e. warm, humid and wet conditions. similar inferences were made in a study of rsv activity in the netherlands, which found that humidity and temperature combined explained more variability than these parameters individually [ ] . this may be due to the dual impact of increased contact from lower temperature and increased immunosusceptibility associated with by higher relative humidity [ ] . the approach here was probably not optimal for linking waterborne diseases to rainfall because of the local linkage needed, as there are significant variations by geographic region. weather can influence pathogen prevalence indirectly through exerting pressure on vector abundance. we found both dengue and plasmodium falciparum had a seasonal pattern (although for dengue it was so weak that it was excluded at stage ) and for the latter weak correlation with max temperature. this can be explained by rising temperatures increasing mosquito distributions and causing seasonal peaks in dengue virus and plasmodium falciparum (i.e. the parasite responsible for cases of malaria) [ , ] , in the countries where the infection was likely acquired. other native vector-borne diseases were shown to be associated with weather in the current analysis. for example, borrelia burgdoferi, which infects ticks and causes lyme disease, had a strong correlation with sunshine. borrelia burgdoferi infected tick distribution was previously shown to correlate with season and rainfall in scotland [ ] . there is evidence to suggest that weather is a driver of faecal-oral infectious diseases, through the increased survival of pathogens in the environment [ ] . in addition to rotavirus, which have enhanced survival at low temperature, the current analysis has identified that aeromonas (a.sp, a. hydrophilia, a. sobria), bacillus (b. cereus, b. sp), coxsackie b, cryptosporidium sp., giardia lamblia, listeria monocytogenes and shigella sonnei may flourish under higher temperatures. respiratory infections transmitted by aerosols are similarly influenced by changes in weather. the high correlations between astrovirus, hmpv, mycoplasma pneumoniae, moraxella catarrhalis, neisseria meningitidis and rsv, and weather may be due to low temperatures causing increased survival and transmission or it could be lower levels of uv in the darker winter months. further work is needed to determine if specific weather thresholds control seasonality. weather may indirectly affect pathogen prevalence through host behaviour. salmonella is highest in summer months which may in part be due to changes in food handling by humans during those months [ ] . pasturella multocida, which is caused by scratches or bites from domestic animals, was shown to be highest in july in the current analysis. injuries caused by a cat or dog were shown to peak in summer in bologna, italy [ ] , which may be due to more time spent outdoors. as mentioned vector abundance will create higher incidence for certain infectious diseases such as malaria, dengue fever and cholera, which are then found to be higher in other countries due to travel behaviour. for example, uk travellers returning from countries with poor sanitation, typically india and pakistan, in summer months, have an increased risk of cholera due to the seasonal effects on the pathogen growth conditions in these other countries [ ] . several infectious diseases are more prevalent in immune-compromised individuals. previously it was found that patients (most of whom have medication, fluid or blood transferred using a central line catheter) were at increased risk of bloodstream infections caused by acinetobacter spp., escherichia coli, enterobacter cloacae, klebsiella spp., and pseudomonas aeruginosa during summer [ ] . we found associations between higher ambient temperature and enterobactor (e. sp., e. clocae, other named, e. agglomerans (pantoea agglomerans), stenotrophomonas maltophilia, acinetobacter baumannii, psuedomonas putida and pleisiomonas shigelliodes. mechanisms for seasonality in nosocomial infections need to be examined further to highlight whether meteorological factors are responsible for the primary infection, complications, or both [ ] . mystery of seasonality: getting the rhythm of nature what is a pathogen? a question that begs the point seasonal infectious disease epidemiology a weather-driven model of malaria transmission west nile virus cholera dynamics and el niño-southern oscillation dynamical resonance can account for seasonality of influenza epidemics the transmission dynamics of groups a and b human respiratory syncytial virus (hrsv seasonality and cross-protection climate drives the meningitis epidemics onset in west africa methods to assess seasonal effects in epidemiological studies of infectious diseasesexemplified by application to the occurrence of meningococcal disease the effect of temperature on food poisoning: a time-series analysis of salmonellosis in ten european countries campylobacter epidemiology: a descriptive study reviewing million cases in england and wales between climate variability and campylobacter infection: an international study evaluation of a national microbiological surveillance system to inform automated outbreak detection medmi: the medical & environmental data mash-up infrastructure project climate change and human infectious diseases: a synthesis of research findings from global and spatio-temporal perspectives the generation of monthly gridded datasets for a range of climatic variables over the uk automatic time series forecasting: the forecast package for r modifiable temporal unit problem (mtup) and its effect on space-time cluster detection model selection and multimodel inference: a practical information-theoretic approach the impact of regular school closure on seasonal influenza epidemics: a datadriven spatial transmission model for belgium association between the ambient temperature and the occurrence of human salmonella and campylobacter infections challenges in developing methods for quantifying the effects of weather and climate on water-associated diseases: a systematic review clostridium difficile infection seasonality: patterns across hemispheres and continents -a systematic review vitamin d status and its adequacy in healthy danish perimenopausal women: relationships to dietary intake, sun exposure and serum parathyroid hormone time series regression model for infectious disease and weather climate change and dengue: a critical and systematic review of quantitative modelling approaches the ecology of climate change and infectious diseases seasonal patterns of infectious diseases prevalence of salmonella spp., and serovars isolated from captive exotic reptiles in new zealand enteric fever (typhoid and paratyphoid) england, wales and northern ireland. in: travel and migrant health seasonality and the dynamics of infectious diseases meteorological factors and risk of community-acquired legionnaires' disease in switzerland: an epidemiological study absolute humidity modulates influenza survival, transmission, and seasonality variation of respiratory syncytial virus and the relation with meteorological factors in different winter seasons respiratory syncytial virus activity and climate parameters during a -year period environmental determinants of ixodes ricinus ticks and the incidence of borrelia burgdorferi sensu lato, the agent of lyme borreliosis incidence of injuries caused by dogs and cats treated in emergency departments in a major italian city seasonality in gram-negative and healthcare-associated infections not applicable. availability of data and materials data are available in the medmi database [ ] and can be visualised on the medmi rshiny app: https://thebest.shinyapps.io/seasonalpathogen/.authors' contributions gn conceived the study. mc undertook the analysis and data visualisation under the guidance of gn, with feedback throughout by cs, gl, sh and lf. the manuscript was prepared by mc and edited with important contributions from cs, gn, gl, sh and lf. all authors read and approved the final manuscript. we received permission to use the sgss pathogen count data from public health england. not applicable. european centre for environment and human health, university of exeter medical school, truro, england.received: october accepted: august in this large database of infectious diseases in england and wales, we have provided an analysis of the seasonality of common pathogens and their correlation with meteorological data. this is extremely important given the context of future climate changes. pathogens within the identified should be investigated further using the proposed meteorological variable, following recommendations proposed by imai and colleagues [ ] . in particular, future studies should be undertaken at finer spatial and temporal aggregations, using pathogen specific confounders and investigating a variety of lag effects and non-linear associations. additional file : figure s . time series plots of meteorological variables. (png kb) additional file : table s . weekly, monthly and quarterly breakdown of pathogen seasonality. (csv kb) additional file : figure s the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -gq lp authors: becker, daniel j.; washburne, alex d.; faust, christina l.; pulliam, juliet r. c.; mordecai, erin a.; lloyd-smith, james o.; plowright, raina k. title: dynamic and integrative approaches to understanding pathogen spillover date: - - journal: philosophical transactions of the royal society b: biological sciences doi: . /rstb. . sha: doc_id: cord_uid: gq lp nan pathogen spillover is the process by which a pathogen is transmitted from a reservoir host species to a recipient host species [ , ] . the term is sometimes used more broadly, particularly in public discourse, blending in elements of onward transmission in the novel host species or even pathogen adaptation to the novel host [ , ] . this theme issue focuses on pathogen spillover sensu stricto, except where explicitly noted. many of the examples considered pertain to zoonotic spillover (i.e. from wildlife or domestic animals to humans), given recent epidemics (e.g. ebola virus [ ] ) and pandemics (e.g. h n influenza virus [ ] ); however, we emphasize the general methods and mechanisms involved in understanding spillover between any two species, such as those that threaten wildlife conservation (e.g. mycoplasma ovipneumoniae from domestic sheep to bighorn sheep [ ] ) and the agricultural sector (e.g. brucella abortus from elk to cattle [ ] ). spillover requires the spatial and temporal alignment of several hierarchical factors that must occur for a pathogen to be transmitted from a reservoir or source host to a recipient host of a different species [ ] . these factors include reservoir host distribution and abundance, pathogen prevalence and shedding from reservoir hosts; pathogen survival in the environment or arthropod vector; recipient host contact with the infectious agent, reservoir host or arthropod vector; and susceptibility of the recipient host. following spillover, another suite of factors determines whether a pathogen is transmitted within the recipient host population (e.g. [ ] ). research on pathogen spillover is often focused on a single component of this process through the lens of a particular discipline. for example, the distribution of reservoir hosts is often studied through ecology, contact between reservoirs and humans is often studied via epidemiology or anthropology, and the pathogenesis of zoonoses in humans is often studied with medical microbiology and immunology. while each factor must be studied and quantified, spillover is the emergent property of these collective processes. studying each factor in isolation fails to account for the hierarchical and often nonlinear dynamics of the spillover system [ ] . pathogen surveillance, epidemic preparedness and management interventions would all benefit from integrative approaches that consider multiple components of pathogen spillover [ ] . this theme issue stemmed from a workshop on cross-species transmission of pathogens, where participants from interlinked fields including ecology, mathematical modelling, epidemiology, virology and immunology discussed how to better understand and predict pathogen spillover. here, we bring together a diverse set of perspectives-including empirical research, theory and synthetic reviews-to highlight cutting-edge research and to provide a roadmap for quantifying and integrating hostpathogen dynamics at each step in the spillover process. manuscripts are organized around three approaches. the first set of manuscripts focuses on integrating data streams to understand spillover dynamics and predict risk. the second set of manuscripts focuses on in-depth analysis of each of the factors affecting cross-species transmission: infection dynamics in reservoir hosts, pathogen survival in the environment, recipient host exposure, dose -response relationships and establishment of infection in recipient hosts. the final set of manuscripts focuses on applied perspectives, with an emphasis on surveillance and interventions. here, we summarize these contributions to highlight key insights, methodologies and future directions to improve our understanding of pathogen spillover. because spillover is the outcome of multiple ecological, epidemiological and immunological factors aligning in space and time [ , ] , predictive frameworks aim to integrate data pertinent to these factors to quantify the relative importance of these processes and to estimate risk. cross et al. [ ] review approaches for estimating spatio-temporal variation in spillover risk, focusing on the wildlife -livestock interface. the authors highlight the challenges inherent in either correlating observed spillover events with relevant covariates or integrating data on host density, distribution and pathogen prevalence using mechanistic models. they highlight that mechanistic approaches may be especially useful in systems where spillovers are infrequent, rarely observed or hard to differentiate from within-species transmission; however, linking datasets on different factors in the spillover pathway requires that such datasets be related to a common spatial and temporal resolution. the authors use case studies of brucellosis in the greater yellowstone ecosystem [ ] and of avian influenza virus in china and north america [ ] to emphasize potential solutions to these challenges for estimating spillover risk. emphasizing that statistical modelling efforts may struggle to detect nonlinear and stochastic relationships inherent in pathogen spillover, childs et al. [ ] provide a strong test of theory governing how hierarchical barriers control crossspecies transmission [ ] . the authors focus their case study on yellow fever, a mosquito-borne viral disease of historical importance in south america that persists in the region largely in sylvatic cycles that occasionally spill over to infect humans [ , ] . specifically, they use mechanistic models that incorporate spatial ecological and immunological data from brazil across years to predict yellow fever spillover in humans. the authors show that a mechanistic model of spillover risk, based on the ecology of mosquito vectors and non-human primate reservoirs, best predicts spillover events compared with models that also include human population size and immunity. this result arises because spillover occurs even in areas with low human population density and high vaccination coverage (e.g. parts of the amazon), so population density and vaccination coverage tend to inflate the predicted risk in locations with low ecological suitability. this integrated approach also highlights a key research gap-cyclical dynamics of susceptible primate populations-that could further improve prediction. this work illustrates that mechanistically modelling the interactions among the environment, viruses, vectors, nonhuman primates and humans can predict rare and seemingly stochastic spillover events with high accuracy. washburne et al. [ ] study the general statistical problems that can arise when aiming to forecast spillover risk. the authors highlight that any such statistical efforts will compile a dataset of explanatory variables expected to relate to pre-spillover processes (e.g., infection prevalence in reservoirs, human vaccination coverage) that are aligned with one of two response variables: the presence and absence of spillover or the number of spillover events at some spatial and temporal resolution (e.g., spatio-temporal counts of yellow fever spillovers [ ] ). the authors show how modelling cross-species transmission as a percolation process, in which pathogens move from infected reservoirs to recipient hosts along a graph representing various spillover pathways [ , ] , reveals first principles for how such datasets will behave and how common statistical tools can produce misleading inferences and poor predictions. for example, percolation theory reveals an inherent nonlinearity in modelling spillover counts, in which statistical inferences are driven by the dominant reservoir sources of infection and the most limiting barriers to cross-species transmission; this nonlinearity can mask the influence of alternative reservoir species or barriers, both of which could be modified through interventions but whose sensitivity as a management tool will appear reduced under linear models. percolation models provide a conceptual framework to connect statistical and mechanistic models with applications to limit risk by illuminating unexpected statistical principles governing pathogen spillover and the nonlinear impacts of management actions. the theme issue's second section uses empirical research, theory and synthetic reviews to understand the processes operating at each stage of pathogen spillover, from infection dynamics within the reservoir hosts to susceptibility and establishment of infection in the recipient host. for the former, the distribution and intensity of infection in reservoir hosts over space and time is the first determinant of spillover risk [ ] . data on these spatio-temporal dynamics help elucidate how pathogens circulate in reservoir hosts and when and where to expect pathogen excretion to be greatest [ ] . however, such field data can be expensive and difficult to collect, and researchers inevitably must tradeoff between the extent and intensity of spatial versus temporal sampling. sampling is thus often opportunistic and fails to adequately describe spillover. plowright et al. [ ] review factors that influence spatial and temporal variation in infection in reservoirs and describe sampling designs that can increase the quality and quantity of this information. although the standard prescription from sampling theory is to sample randomly in space and time [ ] , probabilistic sampling designs are rare in the study of wildlife disease, given logistical challenges and non-random distributions of hosts. the royalsocietypublishing.org/journal/rstb phil. trans. r. soc. b : authors highlight how stratified random sampling designs or adaptive sampling designs can help capture spatio-temporal pulses of infection when researchers have little a priori data on concentrations of infection or spillover events in space and time. these sampling designs can be integrated into modelling approaches and used to better quantify pathogen shedding from reservoirs. accordingly, glennon et al. [ ] present a case study for how to use mechanistic models to differentiate among transmission processes for henipaviruses in straw-coloured fruit bats (eidolon helvum). using this virulent zoonosis as a case study, the authors generalize standard frameworks common in epidemiological modelling [ ] . given that henipavirus infection dynamics in bats are poorly understood, the authors study all possible transitions among infection states in bats to produce potential models. using likelihood-based methods, they fit these models to longitudinal data from captive bats to show strong support for reinfection after virus clearance and cycles of recurrent latent infection: key areas for future empirical work. this inclusive approach to confronting epidemiological models with longitudinal data in poorly understood reservoir host systems holds promise for elucidating spatio-temporal risk of pathogen spillover. following pathogen shedding from reservoir hosts, spillover risk is influenced by the duration of pathogen survival and possible reproduction outside the host in the environment [ ] . for pathogens such as avian influenza virus, persistence in the environment (e.g. ponds) can also facilitate viral reassortment when strains co-occur, promoting co-infections during environmental exposure [ , ] . pepin et al. [ ] review and discuss how genomics, experimental ecology and epidemiological modelling can be leveraged to understand viral reassortment in environmental reservoirs. although no gold standard for capturing, isolating and identifying avian influenza virus diversity from the environment exists, environmental metagenomics and field-based viral diagnostics (e.g. field-based nucleic acid extraction, pcr and sequencing) hold promise for characterizing this context of viral reassortment [ , ] . the authors note how standardizing such field protocols and coupling these data streams with quantitative disease models and natural transmission studies should dramatically improve our understanding of viral co-occurrence and reassortment and thus, this additional process in the pathway to spillover. exposure of recipient hosts to pathogens (e.g. those persisting in the environment) can take a variety of forms; however, in a more general sense, exposure often occurs at elevated rates near boundaries between ecosystems [ ] . borremans et al. [ ] review how ecosystem boundaries can promote spillover by applying ecological theory to understand landscape permeability across ecosystems. the authors highlight that the traits of hosts and pathogens are critical for determining effects of ecosystem boundaries on crossspecies transmission. properties of ecosystem boundaries can also promote or inhibit exposure; for example, edge effects can affect species composition, diversity and population size between ecosystems, as can features of landscape configuration such as patch size and perimeter-to-area ratio [ ] . by considering the analogy between parasite flow and resource flow and by applying concepts from movement ecology, borremans et al. [ ] connect contact rates and spillover risk across ecosystem boundaries to generalize between pathogens and integrate into broader ecological theory. following the complex interactions between reservoir hosts, vectors, pathogens, the environment and recipient hosts, a crucial juncture in any potential spillover event is the point when a recipient host is challenged with a given dose of pathogen (through a particular route and sometimes over a particular duration) and a successful infection does or does not ensue [ ] . lunn et al. [ ] describe how the dose -response relationship, which quantifies the probability of successful infection in the recipient host as a function of challenge dose, can act as a filter on the aforementioned upstream dynamics to shape pathogen spillover risk. the authors integrate recent developments in the dose -response literature, as well as re-analysing data from animal challenge experiments with nipah virus and middle east respiratory syndrome coronavirus [ , ] , to highlight challenges and opportunities arising at the intersection of infectious disease ecology, microbial risk assessment and virology. lunn et al. [ ] call for closer interactions between these fields and for a new generation of pathogen transmission models that link dose -response data to epidemiological dynamics. gostic et al. [ ] next provide an example of the epidemiological insights such an approach can yield. they present a modelling analysis of dose -response experiments for leptospira interrogans, a globally important bacterial zoonosis for which environmental exposure to soil or water contaminated by urine of infected reservoir hosts is the primary transmission route [ ] . by conducting well-designed challenge experiments across a range of exposure routes, and then developing a mechanistic model to identify and quantify the key barriers to infection, gostic et al. [ ] show that intact skin is the crucial defence against leptospiral infection and that skin abrasions or wounds can increase recipient host infection risk by at least a million-fold. this close integration of experimental and modelling approaches isolates a potent and well-defined risk factor for infection with leptospira, opening the door to targeted interventions to reduce spillover risk. once a pathogen has crossed these within-host barriers to replicate and disseminate in the recipient host, the outcome of infection may range from subclinical illness to death and from dead-end spillover to sustained onward transmission [ ] . bonneaud et al. [ ] focus on the conditions favouring pathogen emergence, from the initial jump into the recipient host to adaption in the novel host environment [ ] . the authors highlight that our current understanding of host shifts stems primarily from viral infections, limiting generalizations to other pathogen taxa, given substantial differences in ecology and life history [ ] . they propose several non-mutually exclusive hypotheses to explain why novel bacterial pathogens may be less likely to specialize on their novel hosts and then test these with a mathematical model. the authors demonstrate that high levels of phenotypic plasticity, low rates of evolution and the ability to recombine should reduce propensity to specialize, suggesting that novel bacterial infections may be more likely to result in transient spillovers or increased host ranges than in host shifts. wasik et al. [ ] in turn describe the within-host barriers that pathogens, and viruses in particular, must overcome to replicate and spread in new host populations to cause onward transmission. they present three well-documented examples of viruses that have crossed these barriers to cause epidemics or pandemics in the new host species: influenza a viruses [ ] , human immunodeficiency virus [ ] and royalsocietypublishing.org/journal/rstb phil. trans. r. soc. b : canine parvovirus [ ] . the authors emphasize the role of integrated models that consider all the steps required to go from exposure to spillover to epidemic or pandemic. guth et al. [ ] expand upon these ideas through a comparative study of host and viral traits that predict virulence and the capacity for onward transmission in recipient hosts (i.e. humans). by expanding a previous global dataset of viral zoonoses [ ] , the authors show that increasing reservoir host phylogenetic distance from humans positively correlates with human mortality but negatively correlates with humanto-human transmissibility, suggesting that the virulence induced by reservoirs at high phylogenetic distance may limit viral capacity for onward transmission [ ] . in particular, distantly related reservoirs, such as bats, harbour highly virulent zoonotic viruses with a lower capacity for onward transmission in recipient human hosts, building upon prior work describing bats as special reservoirs [ ] . the theme issue's final section focuses on applied perspectives to detect early spillover events (i.e. surveillance) and the role of interventions focused upstream in the spillover pathway. in particular, early detection is critical for minimizing the spread of zoonotic pathogens following an initial spillover event [ ] . a first series of manuscripts emphasize different approaches to the surveillance of zoonoses. schmidt et al. [ ] use machine learning tools (e.g. boosted regression trees [ ] ) to predict which mammal species are more likely to play roles in ebola virus spillover events. the authors show that large-bodied, frugivorous mammals with slow life histories are likely host species, implicating some insectivorous bats, old world monkeys and forest antelopes as possible ebola virus reservoirs. predictions such as these can help prioritize future wildlife surveillance efforts (e.g. [ ] ). kuisma et al. [ ] in turn describe a community-based surveillance effort focused on wildlife mortality reporting and oriented to early detection of ebola virus disease outbreaks. spanning over a decade and covering km of challenging terrain in the congo basin, this programme has reached hundreds of villages and thousands of hunters and forest gatherers. the programme has educated community members in wildlife carcass reporting and behavioral risk reduction as well as built capacity for safe carcass sampling by trained local responders. this region was not confronted with an ebola virus outbreak during the period described here, and all reported carcasses tested negative. nevertheless, given the well-recognized fact that early intervention can avert massive human and economic costs of widespread epidemics, the low-cost and scalable surveillance programme described by the authors could provide key early detection capability more generally. two other contributions focus on zoonotic pathogen surveillance efforts in domestic animals and human populations. mwangi et al. [ ] present a real-time surveillance system that leverages the existing mobile phone network and shows immense potential to improve adaptive management of spillover. this surveillance system has been implemented in households across rural kenya, where participants are asked to report symptom syndromes in their livestock. zoonotic diseases such as rift valley fever present with severe clinical signs in domestic animal populations, but lack of active surveillance can miss these sentinels [ ] . the authors demonstrate that illnesses were more likely to be reported on mobile phones compared with standard routine household animal surveys. they also show that more severe symptoms are likely to be reported, highlighting the utility of this surveillance method for diseases such as rift valley fever. das et al. [ ] similarly describe the implementation of a surveillance system in hospitals in bangladesh that screens symptomatic patients for potential zoonoses. most patients did not have a laboratory diagnosis for their illness, indicating that unidentified pathogens are likely spilling over in human populations. broad-scale, sustainable human surveillance programmes such as outlined by the authors can play a critical role in early detection of zoonotic spillovers. following these approaches to surveillance, interventions can accordingly focus upstream or downstream in the pathway to spillover, given available data and resources, to limit cross-species transmission. at the wildlife-livestock interface, managing pathogen spillover is a main goal for animal husbandry, conservation and food security [ ] . yet, managers are often forced to make control decisions on the basis of limited evidence about intervention efficacy. manlove et al. [ ] develop a spatially explicit, stochastic model of pathogen transmission within and between wildlife reservoirs and livestock recipient hosts to improve evidence-based decisionmaking. by varying host movement patterns and epidemic growth rates, the authors show that biosecurity, containment and retroactive vaccination of the reservoir are the most effective for limiting the spatial spread and magnitude of spillover risk for fast-moving epidemics in mobile hosts. by contrast, prophylactic vaccination and depopulation of the reservoir host were more successful for fast-moving epidemics with low rates of host movement. this framework provides general intuition for how to manage different pathogens at the wildlife-livestock interface, and a flexible platform for more rigorously investigating disease control strategies. ultimately, one of the primary goals of research focused on pathogen spillover is to design interventions that can reduce or eliminate disease burden in recipient hosts. sokolow et al. [ ] explore how ecological interventions, which target the ecological context in which cross-species transmission occurs, can complement more traditional biomedical and veterinary interventions (e.g. vaccination, culling). the authors provide case studies to illustrate the potential for ecological interventions that target the reservoir host (sometimes indirectly, such as through the restoration of natural enemy populations [ ] ), pathogen survival in the environment, contact between reservoir and recipient hosts, or other aspects of risk in the recipient species. the authors also present a simple mechanistic model, parameterized for two example systems, that shows how nonlinear effects can produce counterintuitive results when comparing potential intervention strategies and highlights the importance of a detailed understanding of underlying ecological dynamics when designing and assessing interventions. lastly, the authors draw attention to the importance of social, economic and political considerations to intervention success, as these can derail even the most efficient or cost-effective intervention. in particular, aligning the benefits of an intervention with the costs incurred is crucial to motivate ecological interventions and may require working across sectors for successful implementation. royalsocietypublishing.org/journal/rstb phil. trans. r. soc. b : pathogen spillover is the result of a complex series of events that result in the successful establishment of infection in a recipient host [ ] . as highlighted in the final paper of this theme issue, developing actionable forecasts of risk is further complicated by the various phylogenetic, spatial and temporal scales over which we study and predict spillover [ ] . the authors here contextualize a diverse range of approaches to pathogen spillover within these scales to illustrate critical areas of pragmatic overlap. by focusing on an ecological perspective, the authors outline a research pipeline that connects pathogen discovery and macroecological analyses with spatio-temporal surveillance in reservoir and recipient hosts. through several case studies (e.g. lyme disease [ ] , hendra virus [ ] , plasmodium knowlesi [ ] ), the authors further demonstrate how ecologically focused research has facilitated predicting spillover of particular pathogens in space and time and facilitated design of intervention strategies. this synthesis shows how greater integration of macroecology, pathogen discovery and surveillance could ultimately generate more actionable predictions and interventions to limit spillover risks. recent epidemics, pandemics and disease emergence events all underscore the need to improve approaches to predict and prevent pathogen spillover. this theme issue highlights a range of methods and their commonalities through diverse host -pathogen systems for which researchers are assessing factors driving spillover risk across varying phylogenetic, spatial and temporal scales. contributing manuscripts further emphasize how developing a mechanistic understanding of the hierarchical factors affecting spillover can facilitate quantifying the drivers of crossspecies transmission, deriving generalizable theory and making robust predictions, even for seemingly rare and idiosyncratic spillover events. importantly, such insights can improve our ability to deploy surveillance efforts, design interventions at early stages of the pathway to spillover and manage disease cases in recipient hosts, thereby limiting or preventing further outbreaks. continued study of pathogen spillover as a repeated and hierarchical phenomenon will only improve our ability to predict, prevent and manage cross-species transmission risks. data accessibility. this manuscript has no additional data. authors' contributions. all authors contributed to the development of ideas and to the writing of this manuscript. competing interests. we declare no competing interests. funding. this work was 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pathogens in novel hosts multiple scales of selection influence the evolutionary emergence of novel pathogens cross-species virus transmission and the emergence of new epidemic diseases. microbiol onward transmission of viruses: how do viruses emerge to cause epidemics after spillover? phil host and viral determinants of influenza a virus species specificity key viral adaptations preceding the aids pandemic the emergence of parvoviruses of carnivores host phylogenetic distance drives trends in virus virulence and transmissibility across the animalhuman interface host and viral traits predict zoonotic spillover from mammals virulence evolution and the trade-off hypothesis: history, current state of affairs and the future bats as 'special' reservoirs for emerging zoonotic pathogens outbreaks in a rapidly changing central africa-lessons from ebola ecological indicators of mammal exposure to ebolavirus a working guide to boosted regression trees prioritizing surveillance of nipah virus in india long-term wildlife mortality surveillance in northern congo: a model for the detection of ebola virus disease epizootics mobile phone-based surveillance for animal disease in rural communities: implications for detection of zoonoses spillover rift valley fever: scientific pathways toward public health prevention and response hospital-based zoonotic disease surveillance in bangladesh: design, field data and royalsocietypublishing.org/journal/rstb phil taming wildlife disease: bridging the gap between science and management epidemic growth rates and host movement patterns shape management performance for pathogen spillover at the wildlife-livestock interface reduced transmission of human schistosomiasis after restoration of a native river prawn that preys on the snail intermediate host the problem of scale in the prediction and management of pathogen spillover climate, deer, rodents, and acorns as determinants of variation in lyme-disease risk hendra virus spillover risk in horses: heightened vigilance and precautions being urged this winter predictive analysis across spatial scales links zoonotic malaria to deforestation ranch (emigrant, montana) for assistance organizing the working group that led to this theme issue. we also thank helen eaton for all her assistance in preparing this theme issue for publication.disclaimer. the views, opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the department of defense or the us government. key: cord- -k pa n l authors: barros‐rodríguez, adoración; manzanera, maximino title: units for vigilance of emerging diseases based on wastewater treatment plants (wwtp‐uved) date: - - journal: microb biotechnol doi: . / - . sha: doc_id: cord_uid: k pa n l pandemics deeply affect the health and economy of the world population. a precise determination of affected communities is of great importance to establish containment measures and reduce the economic impact. here, we propose the development of units for vigilance of emerging diseases based on the screening of pathogens released to wastewater treatment plants to follow the spread of the infectious agent to determine the location of infected people. the emergence of pandemics affects people's health, the economy and many other sectors of society. the emergence of viruses (covid- , zika, h n , h n , sars, etc.), bacteria (vibrio cholerae, escherichia coli, salmonella thyphimurium, etc.) and different types of parasites (species of plasmodium, trypanosoma, etc.) frequently strike our societies (daszak et al., ) . the identification of infected individuals in one part of the country can also result in overly stringent measures affecting other distant communities due to their geographical location, even when no case of infection has ever been found in the later and the infection risk is low (gatto et al., ) . these measures may include the restriction of visitors, blocking and subjection to quarantine of local inhabitants, interruption of flights and other transport of people and goods, preventive treatments such as the use of face masks, gloves or protective glasses (feng et al., ) . curative programmes (including vaccines and drugs prescription) are also included to reduce the effect of the disease, and even the construction or adaptation of hospital facilities to respond to the demands of those affected. frequently, health and local authorities avoid performing clinical analyses such as the use of scarce detection kits, involving qpcr, or seroprevalence surveys using tests for the detection of antibodies, that determine the extent of the contagion to prevent side effects. in many cases, the results of these analyses demonstrate the lack of rigor from authorities regarding the spread of the disease in their jurisdiction, especially by those infected people who do not suffer serious effects, but who are transmitters of the disease (london and kimmelman, ) . the clear determination of which communities are affected and which are free from pathogens is of great importance to establish the best containment measures without unnecessarily affecting communities free of the infectious agent. such measures reduce damage caused by the fear of the population, and the reduction of negative economic impact, which responds with an unnecessary alarm even in unaffected areas. therefore, specific and independent protocols are required to ensure exhaustive monitoring of the pathogen's spread. we propose an analysis of local communities instead of, or in addition to, the scrutiny of individuals. patients affected by a large number of infectious pathogens including sars-cov- release them through their faeces (wu et al., ) . these faeces are finally treated in wastewater treatment plants (wwtps; randazzo et al., ) . the number of pathogens found in these wwtps is proportional to the number of affected people and their degree of affection. given that the network of households that discharge their wastewaters into each of the wwtps is perfectly defined, the determination of pathogens found in each wwtp allows us to establish a clear map of the geographical extension of the disease and to estimate the number of individuals affected in each zone in real time. bs_bs_banner today, we have enough culture-independent techniques for the extraction and massive sequencing of nucleic acids (e.g. illumina or ion torrent) and through specific amplification of pathogen genes by qpcr, to monitor the unusual presence of pathogens at any population's wwtps. we therefore suggest the creation of units for vigilance of emerging diseases (uveds) based on the continuous analysis of pathogens in wwtps in potentially affected areas and especially during epidemics and pandemics. the results potentially found by these uveds would allow collecting adequate information for the development of new therapeutic and preventive tools. for example, mass sequencing allows us to identify if certain microorganisms present in the wastewater carry antibiotic resistance genes or if a subpopulation may have changed the epitope recognized by a vaccine. this may allow to know the probability of the efficacy of a treatment in advance. we could avoid unnecessary expenses, unnecessary exposure to therapeutics such as antibiotics, preventing thus the occurrence of resistance and reduction in the efficacy of certain drugs. however, in order to provide a reliable service, some issues need to be addressed, such as developing an accurate nucleic acid (dna and rna) extraction protocol and a topological analysis of the sanitation network. for the nucleic acid extraction protocol, most of the available extraction kits are for human samples (including stools) and environmental samples such as soils, but not designed to extract rna from wastewater samples. wastewater are of heterogeneous nature and can be mixed with different concentrations of sanitation products such as bleach that could affect the stability of the nucleic acids and the efficiency of the extractive technique. therefore, buffering the wastewater sample is of paramount importance prior to the nucleic acid extraction. in addition, the extraction method should equally work for bacteria and for the different viral pathogens that can be grouped into seven different categories accordingly to the type of nucleic acid they contain by the baltimore classification (including double-stranded dna, single-stranded dna, double-stranded rna, and positive-and negative-single-stranded rna). for the topological analysis of the sanitation network, appreciation of the different discharge of wastewaters needs to be taken into consideration, since there are times when the volume of wastewater is higher and an appropriate model needs to be established to consider such variations and to be able to provide an approximate number of affected people in relation to the detected viral particles. most of the cost of an outbreak is not due to the pathogen itself, but due to the panic it causes. this is reflected in the fall in oil prices and the collapse of the stock markets. the who estimates that the expenses of this type of outbreak are approximately billion euros (e.g. approximately billion in for sars and - billion for influenza a or h n in ; goodell, ) . international agencies consider that an annual investment of between . and . billion to strengthen plant, animal and human health systems would produce a global public benefit. the fact that treated wastewater is used for agriculture might potentially disseminate plant pathogens across wide agricultural areas that are worth monitoring. current legislation in many countries requires the analysis of the presence of certain pathogens such as salmonella, faecal coliforms and e. coli. theses analyses are normally performed by wwtps managing companies whose costs are normally passed on to the consumer. therefore, we believe that the creation of such uveds for wwtps under the coordination of a central service to analyse countrywide nucleic acid samples would greatly benefit our society and should be implemented by future legislation similarly to the analysis of other pathogens in the treated wastewater. this approach would represent the beginning of a shift from personalized medicine to community medicine. a strategy to prevent future epidemics similar to the -ncov outbreak rational use of face masks in the covid- pandemic spread and dynamics of the covid- epidemic in italy: effects of emergency containment measures covid- and finance: agendas for future research against pandemic research exceptionalism sars-cov- rna titers in wastewater anticipated covid- occurrence in a low prevalence area prolonged presence of sars-cov- viral rna in faecal samples this work was funded by the spanish ministry for economy and competitiveness and the european union, within the context of the research projects ctm - -r and by the andalusian regional government and the european union under the aegis of research project p -rt- and cv - . none declared. key: cord- -dl v p authors: klein, h. g.; bryant, b. j. title: pathogen‐reduction methods: advantages and limits date: - - journal: isbt sci ser doi: . /j. - . . .x sha: doc_id: cord_uid: dl v p pathogen‐reduction (inactivation) provides a proactive approach to reducing transfusion‐transmitted infection. pathogen‐reduction technologies have been successfully implemented by plasma fractionators resulting in no transmission of human immunodeficiency, hepatitis c, or hepatitis b viruses by us‐licensed plasma derivatives since . fractionation technologies cannot be used to treat cellular blood components. although blood donor screening, deferral and disease testing have drastically reduced the incidence of transfusion‐transmitted diseases, the threat of new or re‐emerging pathogens remains. of particular concern is the silent emergence of a new agent with a prolonged latent period in which asymptomatic infected carriers would donate and spread infection. the ultimate goal of pathogen‐inactivation is to reduce transmission of potential pathogens without significantly compromising the therapeutic efficacy of the cellular and protein constituents of blood. the acceptable technology must not introduce toxicities into the blood supply nor result in neoantigen formation and subsequent antibody production. several promising pathogen‐inactivation technologies are being developed and tested, and others are currently in use, but all of them have limits. pathogen‐reduction promises an additional ‘layer of protection’ from infectious agents and has the potential to impact the safety of blood transfusions worldwide. the first decade of the st century remains an age of emerging and re-emerging pathogens that threaten the blood supply. blood collectors appreciate the dramatic reduction in risk of transfusion-transmissible infections, even as they are sobered by the failures of the th century safeguards to prevent widespread transmission of human immunodeficiency virus (hiv) and hepatitis viruses [ ] . the specter of a new, as yet undiscovered agent with an extended latent phase raises the concern that the current system of overlapping safeguards that protects patients from infectious blood components is still disturbingly vulnerable. until recently, the approach to blood safety depended upon a combination of donor education, screening, testing for selected agents and discarding components in inventory if donor exposure or illness was reported post-donation. this strategy, while effective, is reactive. pathogen reduction of blood components represents a proactive approach to blood safety [ ] . inactivation technologies promise an additional layer of protection both from infectious agents that are known as well as from those not yet recognized as threats to the blood supply (table ) . a method with broad antimicrobial activity could eliminate emerging agents before they become recognized as transfusion-transmitted pathogens. however, because blood contains numerous labile proteins and fragile cells, and because there is a wide array of potentially infectious agents, no single method of pathogen-inactivation will likely preserve all blood components, yet effectively remove all viruses, bacteria, spores, protozoa and prions. furthermore, any chemical or physical process applied to blood must be 'safe' or at least less toxic to recipients than the infectious risk of blood. many pathogens that have the potential to invade the blood supply are not yet screened by testing because of low prevalence in the general population, unknown transmission rate of infection through transfusion or the lack of a readily available test for the agent (table ) . agents with the potential to infect the blood supply are numerous and include the known viral pathogens -more than arboviruses such as the flaviviruses den- through den- and st louis encephalitis virus; the togaviruses western and eastern equine encephalitis and chikungunya; the coronavirus severe acute respiratory virus; the circovirus tt and its variant sen; and the deltavirus hepatitis d. other blood-borne viruses include the herpes viruses such as the epstein-barr virus and human herpes viruses- , - and - , as well as the human parvovirus b virus. numerous animal blood-borne agents (zoonotics) have been able to cause infection across species barriers; most do not cause disease, but have the potential to do so. of particular concern are the simian viruses such as the foamy viruses and sv that have been reported in animal handlers and in some vaccine recipients [ ] . protozoa that threaten the blood supply include the four malarial, babesia microti , found primarily in the northeastern usa, toxoplasma gondii , the causative agent of toxoplasmosis, leishmania donovani , and numerous other subspecies that result in a high disease burden in the developing world [ ] . borrelia burgdorferi , the cause of tick-borne lyme disease, has the potential for blood transmission and another tick-borne illness, human granulocytic ehrlichiosis has been reported from transfusion-transmission of the bacterial pathogen anaplasma phagocytophilum [ ] . four instances of transfusion transmission of the infectious protein or prion pr sc have been reported in the uk, at least three of which almost certainly caused the human equivalent of 'mad cow disease', variant creutzfeldt-jakob disease [ ] . there is no blood screening test for the prion diseases. most of the world does not have access to safe blood [ ] . most developing countries do not screen donor units for all viral markers, because the technology is sophisticated, the cost prohibitive and/or the incidence of infected individuals so high that little blood would be available if all markerpositive donors were excluded. approximately % of blood donations in developing nations are from family members or paid donors [ ] . each year, unsafe blood transfusions in third world countries result in an estimated - millions hepatitis b virus (hbv) infections, · - · million hepatitis c virus (hcv) infections and to hiv infections. pathogen-reduced blood could have a dramatic impact on blood safety in these circumstances. in , edwin cohn introduced what has become the most widely used commercial fractionation method for plasma proteins involving multiple steps of precipitation and physical separation by centrifugation or filtration of the precipitant and effluent using changes in ph, temperature ionic strength and ethanol concentration gradients. as pathogen-reduction occurrs after different steps of precipitation and filtration in the fractionation process, proteins isolated later in the schematic were generally safer from infectious agents [ ] . the albumin and globulin fractions proved extremely safe, particularly regarding transmission of hepatitis viruses and hiv. however, the process was not infallible and deviations from proper processing have resulted in disease transmission. some of the most important plasma protein derivatives such as factors viii and ix are separated early in the fractionation process and do not reap the benefits of added layers of fractionation and processing. pasteurization has been used effectively to inactivate viruses in albumin fractions stabilized with small chain fatty acids from as early as [ ] . other proteins, especially clotting factors, denature during the pasteurization process unless additional stabilizers are added. terminal heat inactivation of lyophilized clotting factors has brought varied results depending upon temperature and processing time. dry heat treatment of lyophilized clotting factors at - ° c does not prevent transmission of hbv, hcv and hiv. increasing the dry heat temperature to ° c for h destroys hiv, hbv and hcv, although the non-enveloped viruses, especially hav and human parvo b , may not be completely inactivated. adding humidity (vapor heating) improves viral kill. heat inactivation at ° c for as little as h has resulted in viral inactivation of both lipid enveloped and non-enveloped viruses. when this process is applied to intravenous immunoglobulin concentrates, little protein is lost. exposure to low ph inactivates many enveloped viruses, however among commercial proteins, only the immunoglobulins are stable under the necessary acid conditions (ph · ). use of organic solvents and detergents in the processing of coagulation factors inactivates the lipid-enveloped viruses, but not the non-lipid enveloped viruses [ ] . the solvent and detergent combination disrupts the lipid envelope and prevents the virus from binding to cells and replicating. incorporation of a virucidal detergent and solvent (solvent % tri-n-butyl phosphate and the detergent % triton-x- for h at ° c) into the processing of plasma from pools of approximately donors produces a product known as solvent-detergent plasma [ ] . the tri-n-butyl phosphate is removed by oil extraction, and triton-x- is removed by chromatographic adsorption. plasma protein concentrates have been made from solvent-detergent-treated plasma, and fresh-frozen plasma (ffp) equivalent has been licensed in the usa and europe. as with the heat-inactivated coagulation factors, solvent-detergent technology does not inactivate the nonenveloped viruses. solvent-detergent treatment also results in some loss of integral plasma proteins such as α - antiplasmin and protein s [ , ] . alpha- antiplasmin is crucial in maintaining haemostasis especially in patients with liver dysfunction. decreased levels of the natural anticoagulant protein s can lead to a hypercoaguable state especially when massive solvent-detergent plasma transfusions are used as in massive traumas or therapeutic plasma exchanges. these concerns, along with economic factors, resulted in the removal of solvent-detergent plasma from the us market. solvent-detergent plasma is still used widely in europe. recently, two new solvent-detergent treatment procedures have been developed for single unit or mini-pools of - units of plasma that yield > % mean recovery of coagulation factors, anticoagulants (including protein s), protease inhibitors (including α - antiplasmin), total protein, albumin and immunoglobulins. single unit and mini-pool solventdetergent treatment technologies show promise and have the potential to overcome some of the drawbacks of the original industrial solvent-detergent treatment processes [ ] . nanofiltration has proven effective in removing a wide range of viruses including the non-enveloped viruses, and may even remove viruses smaller than the filter pore size [ ] . many currently licensed plasma derived coagulation factors and immunoglobulins that are subjected to heat, pasteurization and/or solvent-detergent treatment are also nanofiltered. all classes of plasma protein fractions such as antithrombin, c- inhibitor, protein c, fibrinogen and ceruloplasmin have been nanofiltered without apparent change in the protein characteristics. methylene blue (mb) is a photoactive phenothiazine dye that has been used in europe for more than years for the pathogen-inactivation of single units of plasma. mb has an affinity for nucleic acids and the surfaces of viruses [ ] . when mb-treated plasma is exposed to ultraviolet light, most enveloped viruses are easily inactivated; however nonenveloped viruses are more resistant. intracellular viruses are not inactivated by mb/ultraviolet light, but freezing and thawing plasma often disrupts the cell membranes of leucocytes, thus liberating the viral particles and leaving them susceptible to mb pathogen-inactivation. residual intact white blood cells containing viruses are removed by a micropore filter. neither protozoa nor bacteria are inactivated by mb treatment. plasma proteins are moderately affected; fibrinogen and fviii activity is reduced by up to % [ ] . mb treatment can be used for pathogen-inactivation of single units of plasma, thus eliminating the risk of large plasma pools currently used to manufacture solventdetergent plasma. macopharma uses an in-line system consisting of a membrane filter, mb dye, illumination bag, elimination filter and storage bag. the · μ m membrane filter removes platelets, leucocytes and debris. the plasma then passes through tubing containing an mb pill that dissolves as the plasma flows through the tubing into the illumination bag. the mb-containing plasma is subjected to double-sided illumination by sodium high-intensity low-pressure lamps emitting yellow light at a wavelength of nm for - min. plasma is then passed through an mb elimination filter that removes greater than % of the residual dye and photoderivative by-products [ ] . millions of mb single unit of plasma have been transfused in europe without unexpected adverse outcomes. until recently, attempts at pathogen-reduction for cellular blood components have achieved little success. leucoreduction of blood has reportedly decreased the risks of transfusion transmitted cell-associated viruses, such as cytomegalovirus, human t-lymphotropic virus i/ii and probably epstein-barr virus and human herpesvirus- (hhv- ), as leucocytes are the principal reservoir for these infectious agents. psoralens are small, planar molecules that cross cell membranes and viral capsids and intercalate between the bases of the nucleic acids. upon illumination with ultraviolet a ( - nm), the psoralens react with the dna or rna pyrimidine bases to form covalently bonded intranucleic and internucleic acid cross-links. this cross-linking prevents replication and transcription of the rna or dna [ ] . psoralen treatment with ultraviolet a light results in the reduction of a broad array of viruses, bacteria and protozoa to a level unlikely to transmit infection. aminomethyl-trimethyl psoralen, a three-ringed synthetic psoralen known as amotosalen hydrochloride or s- , has been extensively tested in the pathogen-reduction of platelets and plasma. amotosalen and photochemical treatment have demonstrated an acceptable safety profile through extensive toxicological studies for acute toxicity, repeat dose toxicity, reproductive toxicity, phototoxicity, and mutagenic and carcinogenic potential. in order to pathogen-inactivate with psoralens, the platelet concentrate must be volume reduced and re-suspended in - % plasma and - % platelet additive solution. the amotosalen is added to the platelet and incubated for - min [ ] . the product is then exposed to ultraviolet a light after which approximately % of the psoralen have been photodegraded to by-products. the remaining psoralen and by-products are removed by a 'compound absorption disc' . intercept, an s- amotosalen system, has been evaluated in three clinical trials in europe (eurosprite) involving thrombocytopenic patients. two of these trials evaluated whole blood-derived buffy-coat platelets, and one assessed single donor apheresis platelets. these studies demonstrated that when equal platelet doses were transfused, the inter-cept and conventional platelet transfusions resulted in comparable post-transfusion platelet count increments without significant differences in adverse reactions. in the usa, the sprint trial evaluated the haemostatic efficacy and safety in thrombocytopenic oncology patients receiving intercept single donor apheresis platelets collected on the amicus separator [ ] . a total of platelet transfusions were given; intercept platelets and conventional platelets. the incidence of world health organization grade bleeding between the groups was comparable, and the incidence between the groups of world health organization grade or bleeding was equivalent. patients receiving the intercept platelets had lower post-transfusion platelet count increments, required more platelet transfusions and had a shorter interval between transfusions. explanations for the differences in post-transfusion platelet count increments in the photochemical-treated (pct) platelets were partly explained by the lower mean platelet dose and disproportionate number of transfusions containing platelet doses less that · × cells. transfusion reactions were fewer with pct platelets most likely attributed to the reduced volume of plasma in the pct units as well as increased leucocyte inactivation resulting in less cytokine production during storage [ ] . however a trend towards a higher frequency of respiratory complications has been noted and is of great concern to the us food and drug administration, but apparently less so for other national regulatory bodies. amotosalen and ultraviolet a light has been used to pathogeninactivate plasma in a system much like that used for platelets [ ] . post-thaw coagulation studies of the pct plasma demonstrate that most coagulation factors are well-preserved in the range of - % of control thawed plasma. factor viii levels, while decreased to %, are still sufficient for therapeutic use. there were no significant differences in the quantity and activity of the von willebrand factor, the pattern and distribution of the von willebrand multimers, or the activity of the adamts- . fibrinogen maintained functional activity of approximately %. protein c and s as well as antithrombin were maintained at > % pre-treatment activity levels. there was no evidence of coagulation factor activation as a result of treatment. the factor vii kinetics of post-transfusion pct plasma was compared to standard ffp in a crossover study [ ] . in a study of patients with congenital coagulation factor deficiencies, coagulation factor kinetics and therapeutic efficacy of ffp treated with amotosalen and ultraviolet a light has been shown to be consistent with that of conventional ffp [ ] . randomized controlled trials of pct-ffp supported haemostasis for the treatment of acquired co-agulopathy of liver disease and liver transplantation have revealed outcomes similar to that of conventional ffp [ ] . additional studies utilizing pct-ffp for plasma exchanges in thrombotic thrombocytopenia purpura demonstrated similar results as those with conventional plasma. cryoprecipitate can also be produced from amotosalen and ultraviolet a-treated plasma. preliminary studies indicate that pct cryoprecipitate coagulation factor levels are acceptable. riboflavin, vitamin b a naturally occurring essential nutrient, has been used as a pathogen-inactivating agent for platelets and plasma. riboflavin is a -ringed planar structure that binds to nucleic acids and intercalated between dna and rna bases. upon activation of cross-linked riboflavin with ultraviolet or visible light, guanosine bases are oxidized resulting in single strand breaks in the nucleic acids. the damaged and disrupted nucleic acids are incapable of repair and replication. toxicities of riboflavin and its photoderivative by-products do not appear to cause concern, because riboflavin and its breakdown products are present in many food and natural products. removal of the spent riboflavin and products post-illumination may not be necessary in a pathogen-inactivation system using riboflavin. the us food and drug administration has classified riboflavin as a 'generally regarded as safe' compound. the mirasol prt system contains ml of riboflavin ( μ m) in a light protective pouch and a pathogen-reduction illumination/storage bag. platelets or plasma are sterilely connected to the system, and ml of plasma or platelet product is transferred to the bag containing the riboflavin diluting it to a final concentration of μ m. the riboflavin-treated product is subjected to double-sided ultraviolet illumination [ ] . riboflavin/ultraviolet light treatment has been evaluated in preclinical studies and found to result in reduction of infectivity by many pathogens including west nile virus, intracellular hiv, bacteria and protozoa. the mirasol system demonstrated successful pathogen reduction of selected pathogen-spiked platelet units after treatment and storage for days. the viral reduction of platelets was sufficient to close the window period of transmission of hiv and chronic phase of parvo b , eliminate the viraemic period of west nile virus, prevent infection due to staphylcoccus epidermidis and escherichia coli , and result in a - log reduction of leishmania donovani infantum [ ] . additionally, leishmaniaspiked plasma units treated with riboflavin/ultraviolet light demonstrated a - log reduction in parasites. studies have demonstrated significant differences in control and treated platelets after days of storage in regards to accelerated changes in platelet morphology, increased platelet activation and induced partial platelet aggregation. riboflavin/ultraviolet light-treated plasma has been shown to retain acceptable levels of clotting factors without evidence of increased compliment activation. pathogen-inactivation of components containing red blood cells presents a particularly challenging dilemma. methods utilizing photoactivation must do so in the red wavelength region of the light spectrum above that of haemoglobin in order to avoid absorption or scattering of the light by the red blood cell. many potential methods of pathogen-inactivation easily alter or disrupt the red blood cell membrane resulting in decreased red cell survival, haemolysis or immunogenicity. s (helinx), a small molecule designed for pathogeninactivation treatment of red blood cells, is an alkylating agent derived from a quinacrine mustard that belongs to a class of 'frangible anchor linker effectors' (frale) compounds. frale compounds contain an intercalator group that inserts into the helical region of dna and rna, an effector group that permits covalent attachment of nucleic acids and a central frangible bond that orchestrates the degradation of the compound [ ] . s- is a positively charged planar structure that easily intercalates into the helical regions of the negatively charged nucleic acids. the process does not depend on light for activation. frale compounds are activated by a shift from lower ph storage environment to the higher neutral ph of red blood cells causing hydrolysis and generating s- the primary degradation product; cross-linking of the dna and rna ensues. s- is rapidly metabolized and excreted leaving no detectable parent compound. the remaining free degradation products are absorbed and removed by a compound removal step. s- binds to other proteins and cell membranes as well as nucleic acids, and up to % can potentially remain bound to the surface or contained within the red blood cells. s- has demonstrated pathogen-inactivation of a wide range of viruses, bacteria and protozoa. no unexpected toxicities have been described. assays for red blood cells storage lesions (extracellular potassium leakage, plasma-free haemoglobin, adenosine diphosphate, , -diphosphoglycerate, glucose and lactate) are comparable to control red blood cells. the red blood cell function appears to be normal, and in vivo cr-labelled survival studies exceed the standard of % at h. two randomized, controlled trials involving patients either undergoing first-time cardiovascular surgery or with haemoglobinopathies were in progress when antibodies to residual red blood cell bound s- were discovered in two subjects [ ] . the trials were suspended as a consequence of these findings. additional studies have revealed that % of patients and healthy donors that had never been exposed to s- had naturally occurring antibodies that reacted with s- treated red blood cells. modifications have been made to the s- treatment process to reduce the amount of red blood cell bound s- in attempts to eliminate immunoreactivity and immunogenicity. preliminary finding indicates that red blood cells from the modified s- treatment process were cross-match-compatible with the anti-s- antibodies formed after exposure to the original s- formulation as well as the anti-s- antibodies found in the patients and donors never exposed to s- . the antibodies do not appear to impair transfusion or to pose any clinical problem. new clinical trials have been initiated. riboflavin-based pathogen-inactivation systems for red blood cells are currently under development if found to be a successful means of pathogen-inactivation of red blood cells, riboflavin may serve as the one material to inactivate pathogens in three blood components (red cells, platelets and plasma). current leucoreduction filters are effective in removing cell-associated viruses, but remove only % of total prion infectivity in endogenoussly infected blood. a leucoreduction filter under development removes prions in exogenously and endogenously infected blood more effectively [ ] . exogenous infectivity studies were conducted using ml of red blood cells and ml of % (wt/vol) with high titre brain homogenates from hamsters infected with scrapie for a final % homogenate concentration. endogenous infectivity studies utilized red blood cells processed from ml of whole blood collected from scrapie-infected hamsters. the new prototype leucoreduction, prion reduction filter was effective in removing · logs of scrapie infectivity from exogenously infected red blood cells and all of the detectable prp sc from the endogenously infected hamsters. in the endogenous infectivity study, the pre-filtered-infected red blood cells transmitted disease to six of animals, and the post-filtration red blood cells did not transmit disease to any of animals. allogeneic blood is a critically important therapeutic, but also an inherently risky biologic source material. among the dangers is transmission of a wide range of pathogens. donor selection and blood testing have reduced this risk dramatically and will remain the cornerstone of blood safety programmes. nevertheless, infectious units still elude screening and testing; testing errors and product release errors are probably impossible to eliminate. the largest current infectious risks involve pathogens for which we do not test, for which we have no test, or for which demographic screening and testing are inadequate. the greatest fear concerns the emergence of a 'new' transmissible agent, particularly one that has not previously been associated with human disease, has a long silent period, can infect others by secondary spread and is highly lethal -as was the case with hiv. ideally, pathogeninactivation techniques would provide an additional safeguard. that has been the experience in the plasma fractionation industry. no single pathogen-reduction method will likely be effective for every class of agent and for every blood component. some combination of techniques that remove and inactivate infectious agents will probably be needed. these technologies are generally expensive and have the potential to profoundly escalate the cost of blood, but this cost may be partially offset by the elimination of some testing markers. however, if potent pathogen-inactivation techniques that preserve blood function and do not evidence some new toxic risk can be created, the developed world, which embraces the myth of zero-risk transfusion, will likely adopt them almost regardless of cost. for the developing world, in which low-risk blood donors are at a premium and elegant testing methods often not feasible, a good but not perfect pathogen reduction method, especially if relatively inexpensive and easy to implement, could save millions of lives. meeting transfusion safety expectations pathogen inactivation: making decisions about new technologies. report of a consensus conference frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates protecting the blood supply from emerging pathogens: the role of pathogen inactivation risk and prevention of transfusion transmitted babesiosis and other tick-borne diseases preclinical vcjd after blood transfusion in a prnp codon heterozygous patient who: fact sheets: blood safety and volunteer donations blood supply and demand clearance of prions during plasma protein manufacture an adventure in biotechnology: the development of haemophilia a therapeutics -from whole blood transfusion to recombinant dna to gene therapy sterilization of hepatitis and htlv-iii viruses by exposure to tri (n-butyl) phosphate and sodium cholate current status of solvent/detergenttreated frozen plasma venous thromboembolism associated with the management of acute thrombotic thrombocytopenic purpura a process for solvent/detergent treatment of plasma for transfusion at blood centers using a disposable bag system removal of small non-enveloped viruses by nanofiltration virus inactivation in blood components by photoactive phenothiazine dyes methylene blue treated fresh-frozen plasma: what is its contribution to blood safety? methylene blue and thionine in pathogen inactivation of plasma and platelet concentrates photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light therapeutic efficacy and safety of platelets treated with a photochemical process for pathogen inactivation: the sprint trial clinical safety of platelets photochemically treated with amotosalen hcl and ultraviolet a light for pathogen inactivation: the sprint trial preclinical safety profile of plasma prepared using the intercept blood system pharmacokinetic study of ffp photochemically treated with amotosalen (s- ) and uv light compared to ffp in healthy volunteers anticoagulated with warfarin fresh frozen plasma prepared with amotosalen hcl (s- ) photochemical pathogen inactivation: transfusion of patients with congenital coagulation factor deficiencies photochemically treated fresh frozen plasma for transfusion of patients with acquired coagulopathy of liver disease photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light the use of riboflavin for the inactivation of pathogens in blood products helinx technology for inactivation of infectious pathogens and leukocytes in labile blood components: from theory to clinical application therapeutic efficacy and safety of red blood cells treated with a chemical process (s- ) for pathogen inactivation: a phase iii clinical trial in cardiac surgery patients removal of exogenous (spiked) and endogenous prion infectivity from red cells with a new prototype of leukoreduction filter key: cord- -vciposnk authors: ho, zheng jie marc; zhao, xiahong; cook, alex r; loh, jin phang; ng, sock hoon; tan, boon huan; lee, vernon j title: clinical differences between respiratory viral and bacterial mono- and dual pathogen detected among singapore military servicemen with febrile respiratory illness date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: vciposnk background: although it is known that febrile respiratory illnesses (fri) may be caused by multiple respiratory pathogens, there are no population-level studies describing its impact on clinical disease. methods: between may and october , fri patients and controls in the singapore military had clinical data and nasal wash samples collected prospectively and sent for pcr testing. patients with one pathogen detected (mono-pathogen) were compared with those with two pathogens (dual pathogen) for differences in basic demographics and clinical presentation. results: in total, . % had one pathogen detected, . % had two pathogens detected, . % had no pathogens detected, and . % had more than two pathogens. multiple pathogens were associated with recruits, those with asthma and non-smokers. influenza a ( . %), influenza b ( . %) and mycoplasma ( . %) were most commonly associated with mono-infections, while adenovirus was most commonly associated with dual infections ( . %). influenza a paired with s. pneumoniae had higher proportions of chills and rigors than their respective mono-pathogens (p = . , p = . ). h. influenzae paired with either enterovirus or parainfluenzae had higher proportions of cough with phlegm than their respective mono-pathogens. although there were observed differences in mean proportions of body temperature, nasal symptoms, sore throat, body aches and joint pains between viral and bacterial mono-pathogens, there were few differences between distinct dual-pathogen pairs and their respective mono-pathogen counterparts. conclusion: a substantial number of fri patients have multiple pathogens detected. observed clinical differences between patients of dual pathogen and mono-pathogen indicate the likely presence of complex microbial interactions between the various pathogens. febrile respiratory illnesses (fri) are caused by a wide range of pathogens, most commonly by viruses and bacteria, , some of which cause more serious clinical disease and morbidity. , it may also be due to multiple pathogens co-existing in a microenvironment of complex interactions, which is not unexpected as the respiratory mucosa has abundant resident flora to begin with. for instance, one study showed that . % of ambulatory patients with influenza-like illness had two viruses detected, and another found that in . % of children with community-acquired pneumonia, the illness was due to mixed viral-bacterial infections. others also previously described respiratory viral , and bacterial co-infections , in various settings, although most focus on specific pathogen combinations, especially of the synergism between influenza and streptococcus pneumoniae (s. pneumoniae). [ ] [ ] [ ] [ ] however, there are no population-level studies describing multiple pathogens among persons with upper respiratory tract infections and their impact on clinical disease. such information is of particular importance to countries within the tropical belt where there is a predilection towards multiple pathogens due to the year-round circulation of respiratory pathogens. , a previous study documented the clinical characteristics and epidemiology of viral mono-pathogens gleaned from the respiratory disease sentinel surveillance programme of the singapore military. here, we analyse additional data from the programme, compare patients with one (mono-pathogen) and two pathogens detected (dual pathogen), and describe observed differences in clinical characteristics. all singaporean males enter national service for years after high school or equivalent. during this period, the majority spend most of their time in communal living and training quarters in military camps and return home on weekends, resulting in a semi-closed environment with community interaction. sentinel surveillance for febrile respiratory patients were performed at five major sites. the period of study was from may to oct , and servicemen who sought primary health care at these camps during regular consultation hours were recruited. the fri inclusion criterion was having a body temperature of . °c and above with cough or sore throat. after obtaining informed consent, a standardised questionnaire was administered and nasal wash sampling performed by trained personnel followed by routine clinical assessment by an attending physician. repeat consultations were excluded if the patient was deemed to not have recovered from the first episode of illness. two weeks after the initial consultation, patients were reviewed (through case records and phone calls to patients, if necessary) to determine the number of patients who eventually required referral to hospitals for further evaluation, were diagnosed with pneumonia and/or were admitted for further treatment. randomly selected unmatched controls (at a rate of - persons per week) were also obtained across the year for comparative purposes of baseline commensal rates: these are soldiers from the same camps who were reporting sick at the medical centre for reasons other than respiratory symptoms or acute infections (e.g. those with muscle sprains were selected as controls). this is to prevent mild respiratory infections from being selected and confounding the baseline rates. informed consent was also sought from controls before recruitment. nasal wash samples were obtained from trained medical staff from each side of the nose and placed in universal transport media. these were stored in fridge at °c and transported to the laboratory using carriers with ice packs within h. an iso -accredited laboratory that regularly takes part in qcmd eqa programmes was used to perform molecular diagnostic testing. detailed laboratory methods have been described in the previous publication. briefly, this was done by the extraction of nucleic acids using the dna mini kit (qiagen, inc, valencia, ca, usa) and then tested using multiplex pcr assays coupled with bead array detection technology (resplex i and ii, version . , qiagen, inc, valencia, ca, usa) which can simultaneously detect and subtype different pathogens. first, pathogens of the same genus were grouped (e.g. 'influenza a' includes its various subtypes, and 'enterovirus' also includes coxsackievirus, echovirus and rhinovirus). demographic characteristics for controls, mono-pathogens, dual pathogens and patients with more than two pathogens were analysed and compared using descriptive statistics. analyses on the prevalence of co-existing pathogens were then performed. interval/ratio variables were compared using oneway analysis of variance (one-way anova). comparison of nominal variables with expected frequencies less than or equal to was done using fisher's exact test, while comparison of nominal variables with expected frequencies more than was done using pearson's chi-square test. pearson's chi-square test was conducted to identify trend in proportions. further analysis focussed on comparing patients with one and two pathogens. in this regard, i) controls, ii) patients with more than two pathogens as well as iii) mono-and dual pathogens with sample sizes of less than observations (considered too small for analysis) were excluded. as a result, a total of mono-pathogens and dual-pathogen pairs were available for comparison. permutation tests were conducted to compare the number of symptoms observed between mono-pathogen and dualpathogen patients for each pathogen as a proxy for severity of infection. to assess differences in symptom expression, dual pathogens were compared against mono-pathogens for mean proportions of symptoms (or signs). empirical proportions of symptoms with % confidence intervals (cis) for both mono-pathogens and dual pathogens were calculated and compared using pearson's chi-square test at a significance level of . . symptoms with onsets in at least % of patients for a minimum of one pathogen or combination were described in detail. in particular, dual infections with statistically different results from their respective viral monoinfections were highlighted. r statistical software (version . . ) was used to perform all statistical analyses. ethics approval was given by the singapore military joint medical committee for research and the national university of singapore's ethics review committee. of samples of patients tested, . % had monopathogens and . % had dual pathogens detected. no pathogens were picked up in . % samples, while . % samples had more than two pathogens. among dual pathogens, virus-bacterial pairs were most common at . %, followed by bacteria-bacteria ( . %) and virusvirus pairs ( . %). demographics for patients and controls are detailed in table . gender and the prevalence of heart disease were similar across all groups. mean age was slightly higher in controls, and the number of persons with asthma was higher among patients. multiple pathogens were also more commonly detected among recruits and in those not currently smoking. table shows the differences in detection of pathogens between patients and controls. there were no significant differences in rsv, m. pneumonia, s. pneumonia and n. meningitidis between the two groups. among dual pathogens, there were virus-bacteria, bacteria-bacteria and virus-virus combinations with more than observations each. the most common virus-virus pair was that of influenza a with enterovirus; and of bacteria-bacteria pairs, it was haemophilus influenzae (h. influenzae) with s. pneumoniae. the top three virus-bacteria observations were h. influenzae, paired with adenovirus, enterovirus and coronavirus, respectively. figure depicts the incidence of dual-pathogen pairs, with further details in table s . of the samples with more than pathogens detected, h. influenzae, s. pneumoniae, adenovirus and enterovirus were most commonly involved. the most common trio was adenovirus with s. pneumoniae and h. influenzae, which accounted for . % of samples with more than pathogens. number of symptoms mono-and dual-pathogen patients had similar symptom loads (with . symptoms on average). however, among dual-pathogen patients, those involving s. pneumoniae (p = . ), neisseria meningitidis (n. meningitidis) (p = . ) and h. influenzae (p = . ) displayed a higher number of symptoms than corresponding mono-pathogen patients. nine common symptoms, not ranked by severity, are presented in figure , with further details in table s and figure s . mean body temperature of viral mono-pathogen patients was slightly higher than that of bacterial mono-pathogen patients ( mean proportion of viral mono-pathogen patients with chills and rigors was lower than that of bacterial monopathogen patients ( . vs . ; p < . ). dual pathogens with both s. pneumoniae and influenza a were associated with high proportions of chills and rigors ( . , %ci . , . ). this was significantly more than s. pneumoniae ( . , %ci . , . ; p = . ) and cough with sputum mean proportions of viral and bacterial mono-pathogen patients having cough with sputum were similar, at . and . , respectively, although mean proportion of dual pathogens with the symptom was higher, at . (p < . ). specific dual pathogens with a higher proportion of cough with sputum than both respective bacterial and viral mono-pathogen patients were h. influenzae paired with enterovirus (p = . ; p = . ), or parainfluenzae (p = . ; p = . ). mean proportion of viral mono-pathogen patients having dry cough was higher than that of bacterial mono-pathogen patients ( . vs . ; p < . ). h. influenzae with enterovirus, with higher mean proportions of cough with phlegm as described above, showed a corresponding decrease in dry cough. the proportion among dualpathogen patients was also lower than the patients infected with the virus alone or the bacteria alone (p = . , p = . , respectively). mean proportion of viral mono-pathogen patients having nasal symptoms (sneezing, blocked nose and running nose) was higher than that of bacterial mono-pathogen patients ( . vs . , p < . ). mean proportion for dual infections with nasal symptoms lay in between at . , statistically different from both viral (p = . ) and bacterial (p < . ) mono-pathogen levels. however, no specific dual-pathogen pairs had statistically different levels than their respective viral mono-pathogens. mean proportion of viral mono-pathogen patients with sore throat was only slightly higher than that of bacterial monopathogen patients ( . vs . ; p = . ). the mean proportions for dual pathogens were similar to viral monopathogen levels ( . ) and likewise statistically higher than bacterial mono-pathogen levels (p = . ). interestingly, however, dual pathogens of coronavirus with s. pneumoniae (p = . ) or h. influenzae (p = . ) were instead found to be statistically lower than patients with coronavirus alone. mean proportions of viral mono-pathogen patients with headache were similar to that of bacterial mono-pathogen patients ( . vs . ). for dual pathogens ( . ), the mean proportion were only slightly higher than viral monopathogen patients (p = . ). however, no dual-pathogen pairs had statistically different levels than their respective viral mono-pathogens. mean proportions of viral mono-pathogen patients with body aches were similar to that of bacterial mono-pathogen patients ( . vs . ). mean proportion of dual pathogens with body aches ( . ) was slightly lower than viral monopathogen patients (p = . ). however, no dual-pathogen pairs had statistically different levels than their respective viral mono-pathogens. mean proportion of viral mono-pathogen patients with joint pains were higher than that of bacterial monopathogen patients ( . vs . ; p ≤ . ). mean proportions of dual infections with joint pains ( . ) were in between these two levels, being statistically different from both viral (p = . ) and bacterial (p = . ) monopathogen patients. however, no dual-pathogen pairs had statistically different levels than their respective viral monopathogens. patients were reviewed weeks after the first consultation to ascertain whether any complications had developed in the interim. the proportion of patients referred to hospitals (for further evaluation), as well as the proportion diagnosed with pneumonia, were found to increase significantly with the number of pathogens detected (p = . and p = . , respectively) (table ) . however, there were no clear trends in the number of patients eventually requiring inpatient treatment, possibly as a result of relatively small numbers. much emphasis in respiratory illness research that is based on clinical presentations has thus far centred on monoinfections, although in reality a substantial portion of patients may actually have two or more potential pathogens. our study shows that the prevalence of patients with two or more pathogens in a tropical setting was . %, most commonly due to virus-bacteria pairs. often, it seems that the role of 'less pathogenic' co-detected microbes are casually disregardedperhaps for ease of data interpretation. yet such assumptions are questionable especially because the impact of multiple pathogens on clinical characteristics has not been well studied. this formed the impetus for our analysis of the distribution of dual pathogens in ambulatory fri patients, and comparing associated clinical presentations between mono-and dual-pathogen patients. although we cannot conclude cause-effect relationships from the study, we noted a few interesting trends. the association between new recruits and multiple pathogens is likely due to the ease of transmission within the communal environment (of increased population density) on entry into military service, as described in clinical studies among similar cohorts. , these conditions also promote shifts in predominant circulating respiratory pathogens with time, as had been previously described, sometimes culminating in outbreaks of respiratory disease. , to prevent the occurrence of such incidents, mitigating measuressuch as appropriate education on hand and respiratory hygienehave been implemented. the higher prevalence of asthma in patients and the decreased number of pathogens among current smokers may also reflect the effects of the two on the upper respiratory tract. , for example, previous studies describe the effect of cigarette smoke in causing reduced competitive commensal organisms in the respiratory tract. , among dual infections, virus-virus pairs constitute only . % of the entire data set, within the lower end of range of viral co-infection studies in ambulatory settings ( . - . %). , , this may be due to local interactions between immune and microbial mechanisms preventing the occurrence of co-existing viral respiratory pathogens. such negative correlations have been previously described, including the replacement of one virus with another when the former is removed from the general population through vaccinations. the genus enterovirus was most prevalent ( . %) among viral-viral pairs, similar to two other viral coinfection studies reporting rhinovirus rates of . % and . %. , virus-bacterial pairs were most common, with a significant proportion involving adenovirus, particularly paired with h. influenzae ( . %). such a finding had also been previously observed among hospitalised children, where % of those with adenovirus were co-infected with various bacteria. previous chinchilla models on experimental otitis media also point towards possible synergisms between adenovirus and h. influenzae, although further studies are needed to conclusively determine whether such interactions exist in the upper respiratory tract. when it came to symptoms, the increased incidences of chills and rigors and elevated body temperatures in influenza a and influenza b, respectively, when paired with s. pneumoniae correspond to previous studies showing the disposition to superinfection caused by the influenza virus on respiratory epithelium, in both laboratory and hospital studies. [ ] [ ] [ ] [ ] [ ] our results show that these apply to ambulatory patients as well. however, we also noted that these systemictype symptoms appeared to be distinct from localised upper respiratory tract symptoms (such as running nose and cough), which were not found to be significantly different from patients with influenza alone. next, a higher prevalence of cough with phlegm was correlated with a number of dual-pathogen combinations, all of which involved the bacteria h. influenzae. although there are microbiological studies on the bacteria's interactions with rhinovirus, , there is insufficient information to conclusively explain the observations noted with parainfluenzae, warranting further studies. finally, diversity in the impact of dual pathogens on clinical manifestations, as seen through the results of other symptoms, is likely indicative of complex and diverse microbial interactions between respiratory pathogens in the upper respiratory tract. bosch et al. have detailed a number of known microbiologic mechanisms, including various modalities of synergisms and competition between species. these include pathogens that are usually associated with asymptomatic colonisation in healthy individuals (e.g. s. pneumoniae and h. influenzae), which are potentially pathogenic with shifts in the respiratory tract microenvironmentfor instance, the introduction of new microbes. [ ] [ ] [ ] many of these are not yet fully understood, and it is hoped that such epidemiological data may spur greater interest in co-pathogen microbiology research. our study does not explore patients infected with more than pathogens and co-pathogen pairs with < observations. although it identifies observed correlations between pairs and symptoms, it does not determine sequence of pathogens in relation to onset of symptoms or prove causality, which require further microbiological or case-control epidemiological research. severity of symptoms other than fever was not determined, actual diagnoses by doctors were not analysed, and further differences in the actual clinical impact could not be observed. although statistically significant differences have been described, the clinical significance of these findings have to be considered alongside as small differences may not be easily translatable to clinical practice and the large number of statistical comparisons increase the chances of type i (i.e. false-positive) errors. the study predominantly involved young adult males, limiting the generalizability to other populations. it is also conducted in a tropical setting with a fairly constant climate; thus, the effect of such changes on symptomology (e.g. in a temperate country) cannot be determined. by grouping pathogens of the same genus together in analysis, it is also not possible to determine whether specific subtypes are the cause for the observations made. we are unable to detect the presence of dual-pathogen patients involving two or more viruses from the same genus, especially within enteroviruses. although we compared differences in the detection of organisms between patients and controls, we are unable to conclude on whether certain organisms (such as n. meningitidis and adenovirus) are actually commensals, and pcr is not the optimal method for diagnosis of bacterial infections. we have described the aetiology of dual pathogens causing fri in the tropical setting and compared differences with monopathogens with regard to observed clinical manifestations. the presence of higher incidences of certain symptoms with specific pathogen pairs is indicative of underlying complex microbial interactions and affirms existing microbiological co-pathogen studies. however, many of these processes are still not well explored in existing literature, opening many opportunities for further research into this area. additional supporting information may be found in the online version of this article: figure s . proportions of each clinical symptom between dual pathogens and their mono-pathogen counterparts. table s . checkerboard of dual pathogens detected among cases. the dual-pathogen pairs for further analysis of symptoms (i.e. observations or more) are in bold. table s . mean proportions and comparisons between viral and bacterial mono-pathogens and dual pathogens. acute respiratory illness in the community. frequency of illness and the agents involved 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and viral infection increased h n infection rate in children with asthma cigarette smoking and mechanisms of susceptibility to infections of the respiratory tract and other organ systems recovery of potential pathogens and interfering bacteria in the nasopharynx of smokers and nonsmokers broad spectrum respiratory pathogen analysis of throat swabs from military recruits reveals interference between rhinoviruses and adenoviruses single, dual and multiple respiratory virus infections and risk of hospitalization and mortality mixed infection is common in children with respiratory adenovirus infection synergistic effect of adenovirus type and nontypeable haemophilus influenzae in a chinchilla model of experimental otitis media immune dysfunction and bacterial coinfections following influenza insights into the interaction between influenza virus and pneumococcus influenza virus neuraminidase contributes to secondary bacterial pneumonia influenza a virus facilitates streptococcus pneumoniae transmission and disease interactions between streptococcus pneumoniae and influenza virus: a mutually beneficial relationship? influenzae potentiates airway epithelial cell responses to rhinovirus by increasing icam- and tlr expression human rhinovirus-induced isg selectively modulates epithelial antiviral immunity dynamics of nasopharyngeal colonization by potential respiratory pathogens human polymicrobial infections the ecology of nasal colonization of streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus: the role of competition and interactions with host's immune response the authors would like to acknowledge the staff from dso national laboratories for their tireless efforts in the collection of data and testing of laboratory samples, as well as staff from hq medical corps, singapore armed forces, and the centre for infectious disease epidemiology research (cider) at the saw swee hock school of public health, national university of singapore for their support. the programme was supported by a singapore ministry of defence-funded operational research programme and the centre for infectious diseases epidemiology and research in the saw swee hock school of public health of the national university of singapore and national university health system. the funders had no role in the study design, data collection, analysis, decision to publish or preparation of the manuscript. the authors declare that they have no competing interests. key: cord- -tm s wvj authors: lim, wei shen title: pneumonia—overview date: - - journal: reference module in biomedical sciences doi: . /b - - - - . - sha: doc_id: cord_uid: tm s wvj pneumonia is very common and continues to exact a high burden on health. the global burden of disease study found lower respiratory infections (lris) were the leading infectious cause of death and the fifth leading cause of death overall. pneumococcal pneumonia caused % of lri deaths in all ages ( . million deaths). novel pathogens, particularly viruses, continue to emerge as causes of pneumonia. the rise of drug-resistance among common respiratory pathogens is a further challenge. pneumonia is commonly classified according to patient location at the time of infection, leading to the categories of community-acquired, hospital-acquired and ventilator-acquired pneumonia. pneumonia may be defined as an infection of the lung characteristically involving the alveolar space. the presence of microorganisms in the alveolar space without an accompanying inflammatory response represents colonization and does not constitute pneumonia. a range of other types of infection may also affect the lung and can be classified according to their principle site of infection ( fig. ). the term lower respiratory tract infection (lrti) is often considered to include both acute bronchitis and pneumonia. however, it is sometimes used to designate non-pneumonic infections of the lower respiratory tract alone. in patients with chronic lung disease (e.g., chronic obstructive pulmonary disease (copd)), an infection of the bronchi often results in an exacerbation of the underlying lung illness. in these circumstances, the illness is usually designated as an exacerbation of disease (e.g., exacerbation of copd) rather than "acute bronchitis." in general, the more distal the infection within the respiratory tract, the greater the likelihood of bacterial infection and the greater the severity of illness. exceptions to this include acute epiglottitis, diphtheria and pertussis which may present as severe bacterial infections of the upper respiratory tract without causing pneumonia. pneumonia is further classified in various different manners. these are mainly clinical classifications that broadly describe differences in the likely range of pathogens involved (table ). the commonest grouping is according to patient location at the time of acquisition of infection. infections arising within a hospital setting may involve more drug-resistant pathogens compared to infections arising in the community. within the grouping of hospital-acquired pneumonia (hap), further distinction is usually made according to whether the patient was on an intensive care unit, or intubated (ventilator-acquired pneumonia (vap)) at the time of infection (torres et al., ; kalil et al., ) . a specific category of healthcare-associated pneumonia (hcap) was previously advocated as describing pneumonia developing outside a hospital setting that shared features of pathogenesis, causative pathogens and antibiotic resistance patterns with nosocomial (hospital-acquired) pneumonia. this category was never fully adopted internationally and the latest evidence does not support the continued use of this classification. host factors play an important role in the manifestation and management of pneumonia. pneumonia arising in immunocompromised hosts usually warrants distinct treatment. in general, the greater the degree of immune compromise, the wider the range of potential pathogens. the classic symptoms of infection, which are partly related to the host immune response, may also be absent, altering the clinical presentation. a third common classification is according to the causative pathogen(s). until recently, a microbiological diagnosis used to take days to confirm. however, with the advance of rapid, point-of-care diagnostics, microbiological confirmation within minutes/hours of clinical presentation is becoming more realistic. hence, this classification will hopefully become more clinically relevant in guiding patient management at the time of presentation. anatomically, pneumonias may be classified as bronchopneumonia or lobar pneumonia. bronchopneumonia occurs when infection leads to multiple discrete foci of consolidation within the lung, whereas lobar pneumonia is described when the area of consolidation is confined to the affected lobe which is diffusely involved. it was once thought that the pattern of consolidation (whether described radiologically or pathologically) was indicative of the causative pathogen (e.g., lobar pneumonia caused by streptococcus pneumoniae). however, it is now recognized that such discrimination is unreliable because of the large overlap in patterns caused by infecting pathogens. aspiration pneumonia refers to a specific situation when a patient who is manifestly at risk of aspiration develops pneumonia and anaerobic pathogens from the digestive tract are implicated, usually alongside multiple other microorganisms. an accompanying pleural reaction or lung abscess may develop. micro-aspiration events are common and the aspiration of microorganisms into the lower airways likely accounts for how pneumonia develops in the majority of cases (see "pathogenesis" section). hence, the presence of micro-aspiration alone in a patient with pneumonia does not invariably denote aspiration table classification of pneumonia. description/comments pneumonia. stroke-associated pneumonia has been advocated as the desired terminology for lrti occurring within days of acute stroke (smith et al., ) . the lung is not a sterile environment. the normal lung microbiome includes bacterial species that may be implicated in the development of pneumonia, such as streptococcus spp. and mycoplasma spp. (beck et al., ) . these, and other microorganisms, are normally held in check by pulmonary host defenses. disruption of these host defenses allow externally transposed pathogenic microorganisms to grow and displace the normal flora, or allow overgrowth of selected resident flora, leading to infection. there is growing interest in the role of antecedent viral respiratory tract infections as triggers for the disruption of the normal lung microbiome, providing an avenue for bacterial pathogens to take hold. the acute inflammation generated by the host immune response to infection results in an influx of inflammatory cells into the alveolar space, giving rise to the radiological pattern of consolidation (fig. ) . in most cases, the predominant inflammatory cell involved reflects the inciting pathogen; neutrophils in bacterial infections, lymphocytes in viral infections and granulomatous inflammation in mycobacterial and fungal infections. the systemic cytokine response gives rise to many of the characteristic features of infection, such as fever, myalgia and a rise in c-reactive protein levels. the introduction of microorganisms to the lung is most commonly via micro-aspiration. haematogenous spread from other sites in the body, and direct spread from a contiguous source are less common. a range of host factors that predispose to pneumonia have been identified (wunderink and waterer, ; almirall et al., ) (table ) . these factors mostly increase the susceptibility to pneumonia through reducing host defenses. some commonly used non-immunosuppressive drugs have been associated with pneumonia, but the mechanisms of action for all of these have not been fully described. immunocompromised patients are not only at higher risk of developing pneumonia but the range of possible pathogens is also wider. as the number of therapeutic interventions that modify the immune system (such as monoclonal antibodies and tyrosine kinase inhibitors) expands, patients with widely differing levels of immune integrity are being described. in addition, multiple immune insults may exist together. for instance, the severe immune defects caused by hematological malignancies are often compounded by their treatments which may include cytotoxic drugs and/or total bone marrow ablation followed by hematopoietic stem cell transplantation. immunologically, defects can be broadly grouped into (a) cell-mediated defects, (b) antibody deficiencies and (c) neutrophil dysfunction, most commonly neutropenia. understanding the type of immune defect facing a patient can aid as a guide to the likely range of pathogens involved, prior to confirmatory microbiological diagnosis. a definitive diagnosis of pneumonia comprises four aspects: (i) symptoms and signs of a respiratory tract infection, (ii) radiological changes, (iii) identification of a putative pathogen and (iv) a treatment response, or clinical course, consistent with pneumonia. it is not always easy to make this diagnosis. in settings where investigations are not readily available, such as in primary care, a clinical (or working) diagnosis of pneumonia may be made without recourse to radiological or microbiological tests. the accuracy of a clinical diagnosis of pneumonia made in primary care is reasonable; - % of patients clinically diagnosed with cap have radiologically confirmed cap. however, of patients with acute cough in whom a radiological diagnosis of cap is made, only about % are clinically diagnosed as cap (van vugt et al., ) . even in secondary care, up to % of cases of pneumonia diagnosed in the emergency department are eventually discharged from hospital with an alternative diagnosis (sikka et al., ) . the differential diagnosis of pneumonia includes other cardiac and pulmonary conditions that present acutely with features of cough and/or dyspnoea together with radiological abnormalities. (table ) in patients who are mechanically ventilated, diagnosing pneumonia amidst the wide range of differential diagnoses is challenging. patients with pneumonia usually present with a combination of (i) respiratory symptoms, specifically cough ($ %), dyspnoea ($ %), sputum production ($ %) and chest pain ($ %), and (ii) systemic symptoms including fever, rigors, myalgia and confusion. confusion is commoner in older patients and those who are severely ill. immunocompromised patients, and to a lesser extent, older patients, may not mount as rigorous an immune response and therefore may present with more subtle symptoms. about % of patients with cap present to hospital with extra-pulmonary features alone; these include falls, generalized weakness and acute abdominal pain. a high index of suspicion is required in these circumstances. until recently, identification of consolidation on a chest radiograph (cxr) has been regarded as the "gold standard" radiological investigation in the diagnosis of pneumonia. it is recognized that in patients with chronic lung abnormalities (e.g., pulmonary fibrosis, bronchiectasis, lung cancer) or in certain settings (e.g., intensive care unit), the sensitivity and specificity of a cxr for the identification of pneumonic changes can be limited. however, computed tomography (ct) scanning ( fig. ) has raised further questions regarding the reliability of the cxr for the diagnosis of pneumonia more generally. when evaluated against ct imaging, the cxr results in both over-diagnosis and under-diagnosis of pneumonia with up to a third of patients diagnosed with pneumonia on cxr having no infiltrate on ct scanning (claessens et al., ) . ultrasonography is also being evaluated for the diagnosis of pneumonia with promising initial findings when compared against cxr (orso et al., ) . it remains necessary to determine if these imaging modalities will enable discrimination of patients suspected of having pneumonia into groups that warrant different management strategies. table risk factors for pneumonia. age > years chronic co-morbid conditions copd, cancer, diabetes, chronic liver disease, renal impairment immunosuppressive disorders hiv, solid organ transplantation, immunosuppressive agents factors that increase the risk of aspiration placement of endotracheal tube, stroke, neurological disorder lifestyle factors smoking, high alcohol intake, malnutrition drugs proton-pump inhibitors, anti-psychotic medication, inhaled corticosteroids (in patients with copd) table differential diagnosis of pneumonia. identification of a causative pathogen not only aids in the diagnosis and classification of pneumonia, it also guides antimicrobial therapy and infection control measures. a wide array of microbiological tests is available (table ) . however, even with the use of modern molecular-based microbiological investigations (e.g., pcr, antigen detection tests) in patients with cap, a pathogen is identified in only - % of cases (jain et al., ) . in routine care where microbiology testing is still based mainly around cultural techniques (e.g., from blood and respiratory tract samples), a pathogen may be identified in only - % of cases. conversely, in patients with suspected vap, extensive colonization of airways creates difficulties in the interpretation of positive microbiology results. the use of highly sensitive pcr techniques can compound the difficulty. similar challenges confront the management of immunocompromised patients. in these instances, careful attention to the coherence of microbiology test results with the clinico-radiological pattern is necessary to distinguish colonization from infection. sometimes, a diagnosis of pneumonia can only be confirmed or refuted following review of the subsequent clinical course of illness, including the clinical response to empirically initiated antimicrobial agents. microbiological investigations are rarely performed in primary care settings due in part to a lack of access to laboratory facilities, low yield and delays in obtaining results in time to influence clinical management. advances in science and technology have come together to enable the development of rapid point-of-care (poc) tests that can provide microbiology test results within - min. the benefits of definitive identification of the causative pathogen (or in some cases, definitive exclusion of a specific pathogen, such as influenza) at the time of clinical presentation need to be weighed against the resource demands of these newer technologies and their costs. because lrtis can be caused by a range of pathogens, singlepathogen specific technologies are less helpful. multi-array pcr platforms may overcome some of these hurdles, but come with increased costs. the cost-effectiveness of poc-driven management strategies compared to more empirical approaches remains to be proven in many circumstances. an alternative approach to the management of patients with lrtis is to rapidly discriminate those may have a bacterial infection and who would therefore require antibiotic therapy, versus those with a viral infection or a non-infectious condition. several hostresponse biomarkers have been investigated in this regard (table ) . c-reactive protein (crp) and procalcitonin (pct) are the two most extensively studied biomarkers in respiratory tract infections. levels of these biomarkers increase more extensively in bacterial compared to viral infections. procalcitonin has a more responsive kinetic profile than crp, which means that levels of procalcitonin increase and decrease more swiftly in line with bacterial load. apart from informing the decision whether or not to initiate antibiotics, the other role of crp-and procalcitonin-guided treatment strategies in patients with pneumonia may be to guide the duration of antibiotic therapy through serial assessments of biomarker levels. a meta-analysis of individual participant data from rcts found that pct-directed treatment in the management of acute respiratory tract infections (of varying types and severity, including cap and hap) was associated with a reduction in antibiotic exposure ( . vs. . days) composed of a decrease in both (a) the proportions initiating antibiotics and (b) the duration of antibiotics ( . vs. . days), as well as a reduction in -day mortality ( . % vs. . %) (schuetz et al., ) . proadrenomedullin, neopterin, strem- and pentraxin- are other biomarkers that have been found in a small number of early studies to be potentially of value in lrtis (saleh et al., ) . measuring pentraxin- and strem- in bronchoalveolar lavage fluid table biomarkers studied in respiratory tract infections. c-reactive protein acute phase protein synthesized by hepatocytes procalcitonin prohormone of calcitonin which is induced by the activation and adherence of monocytes to the endothelial layer of blood vessels proadrenomedullin precursor for adrenomedullin which is involved in immuno-modulation strem- soluble triggering receptor expressed on myeloid cells - (strem- ) levels rise following an increase of trem- expression on neutrophils, granulocytes, monocytes and alveolar macrophages. trem- expression is increased by microbial products pentraxin- acute inflammatory marker and a component of innate immunity neopterin produced in monocytes and macrophages. a marker of cell-mediated immunity. levels rise in viral infections copeptin stable byproduct of vasopressin biosynthesis lipocalin- protein involved in innate immunity. it limits bacterial growth by sequestering iron-containing siderophores syndecan- receptor in intracellular signaling. present in alveolar macrophages midregional proatrial natriuretic peptide (mr-proanp) a byproduct of atrial natriuretic peptide (anp) biosynthesis. anp regulates macrophage activity in innate and acquired immunity samples has been found to be more discriminatory for bacterial versus viral infections than their levels in blood. other biomarkers (e.g., copeptin, lipocalin- , syndecan- ) display poorer performance characteristics. it may be that diagnostic performance can be further improved by combining different biomarkers (e.g., pct and mr-proanp) or matching certain biomarkers to selected target patient populations (e.g., hap vs. cap). most of the investigated biomarkers aim to differentiate between bacterial or viral infections according to the host's immunological response. the role of these biomarkers in patients with impairments of the immune system is therefore more limited, depending on the nature of the immune defect. data on the epidemiology of pneumonia are drawn from two broad sources-(a) datasets that rely on the coding of pneumonia following clinical episodes, and (b) cohort studies of patients with radiology-confirmed pneumonia. the accuracy of clinical coding of pneumonia reflects the difficulties with making a firm diagnosis. in the uk, up to % of coded cases of hospitalized-pneumonia may have no radiographic evidence of infection (daniel et al., ) . changes in the way pneumonia is coded can also affect the interpretation of data. in the us, there has been a shift in recent years from assigning a primary diagnosis code of "pneumonia" in patients hospitalized with severe cap, towards a primary diagnosis code of "sepsis" with "pneumonia" as the secondary diagnosis (lindenauer et al., ; ruhnke et al., ) . prospective studies of radiology-confirmed pneumonia provide more robust data but are often less comprehensive in scope. the global burden of disease study estimated that in , the incidence of lrti in children aged < years old was . episodes per child-year, and in all ages it was . episodes per person-year (collaborators, ) . most episodes of lrti/cap are treated in community settings. in the uk, cap affects approximately % of the uk adult population each year, accounting for over , hospital admissions. the average length of stay is days and estimated direct healthcare costs are £ million. in the us, the annual incidence of cap requiring hospitalization has been estimated at around per , adults (hayes et al., ) . this compares with lower estimates from other parts of the world (table ). incidence rises steeply in adults aged > year (table ) . changes in the prevalence of risk factors for pneumonia can be expected to influence the incidence of pneumonia. in the uk, a % increase in the incidence of cap requiring hospitalization was observed from to (trotter et al., ) . such trends of increasing incidence are thought to be explained by an aging population and a higher proportion of persons living with co-morbid illnesses, the latter encompassing an increase in persons with complex multi-morbidity. some have implicated alterations in the epidemiology of causative pathogens (quan et al., ) . changing patient expectations may also influence how healthcare is accessed and hospital admission policies. in contrast, in the us between and , a decrease in the annual age-adjusted rate of pneumonia-associated hospitalisations was noted despite an increase in the proportion of co-existing immunocompromising conditions from . % in to . % in (hayes et al., ) . in sub-saharan africa, the incidence of cap is dominated by the effect of hiv infection. the prevalence of hiv within cohorts of patients with cap is - %. in a community surveillance study in kenya, the annual incidence of pneumococcal acute respiratory infection was per , persons in hiv negative individuals compared to per , persons in hiv positive individuals (aston, ) . effective vaccination against respiratory pathogens has the potential to prevent infection and decrease the incidence of pneumonia. national immunization programmes involving pneumococcal vaccines have contributed towards a reduction in overall pneumococcal infections and attendant mortality. however, in some countries, replacement disease, involving pneumococcal serotypes not covered by existing vaccines, has since begun to offset the reductions in vaccine serotype disease. further vaccine development incorporating other serotypes, or effective against all pneumococcal serotypes/serogroups, will hopefully curtail this rise in replacement disease. estimates of the incidence of hospitalisations for cap (takahashi et al., ; ewig et al., ; trotter et al., ; hayes et al., ) . annual incidence (per , adults) globally, lrtis are the leading infectious cause of death and the fifth-leading cause of death overall. in , lrtis caused Á million deaths in all ages, with children < years of age bearing a disproportionate burden ( , deaths). between and , the number of deaths due to lrti decreased by . % in children younger than years, and by . % in all ages; most of these decreases occurred in countries with a low to middle socio-demographic index (sdi). in high-sdi countries, the lrti mortality rate in all ages increased by . % over the same period (from . per , to . per , ) (collaborators, ) . in the us, pneumonia remains the leading infectious cause of death, and the eighth commonest cause of death overall. since , mortality from pneumonia and influenza in the us has stayed below deaths per , population. most deaths occur in hospitalized patients. in the period - , in-hospital deaths occurred in . % of pneumonia-associated hospitalisations (hayes et al., ) . in europe, mortality rates for hospitalized patients are mostly around - % though a wide range is reported likely reflecting differences in healthcare systems, data sources and possibly microbiological patterns. in patients admitted to icu with cap, mortality rates are in the region of - %. in africa, an even wider range of mortality rates have been reported, from < % to nearly %. in most high-income countries, advancing age is associated with increasing mortality rates. however, in sub-saharan africa, this trend is not always evident; % of lrti-related deaths in adults occur in persons under years, including % in adults aged - years. rates of admission to icu vary globally according to availability of resources and admission policies. a higher proportion of hospitalized patients are admitted to icus in north american (> %) compared to europe ( - %). hospital acquired pneumonia is the second commonest nosocomial infection after urinary tract infections. estimated incidence rates are - cases per hospital admissions (torres et al., ) . these rates are influenced by the hospital ward setting and patient groups affected. the majority of hap ( %) occurs on non-icu wards, however, the incidence of hap is greater among patients in icu compared to patients on general wards. higher rates are also observed in at-risk patient groups such as the elderly, those who have had surgery and immunocompromised hosts. the incidence of vap is - episodes per ventilator-days according to data from the us. during the first days of mechanical ventilation (mv), the estimated risk of vap is % per day, decreasing to % per day from days to of mv, and to % per day from day of mv onwards. patients with brain injury and trauma are at particularly high risk of vap ( %) (kalil et al., ) . hospital acquired pneumonia is the leading cause of death from nosocomial infections in critically ill patients. the crude mortality from hap is high (up to %). however, patients who develop hap are often already severely ill and the factors that predispose to hap may also increase the risk of mortality. accordingly, mortality is higher for patients with vap than for patients with hap on general wards. in some studies of vap, - % of vap-related deaths are deemed to be a direct result of infection. the vapattributable mortality has been estimated at % with surgical patients carrying the highest associated risks (melsen et al., ) . infection with pseudomonas aeruginosa or acinetobacter spp. is associated with higher mortality as well. an extensive range of pathogens can cause pneumonia. the respiratory pathogens commonly implicated in patients with cap remain important aetiological agents in all other types of pneumonia, including hap and pneumonia in the immunocompromised host (table ). factors that modify (usually extend) the range of pathogens that might be implicated include alterations in immune status, exposure to specific environments/pathogens and prior exposure to antibiotics. s. pneumoniae is the predominant bacterial pathogen in pneumonia, especially cap. geographical differences are important in two broad regards: (a) the spectrum and frequency of likely pathogens, (b) the patterns of drug-resistance likely to be encountered. in countries where tuberculosis (tb) is endemic, acute tb pneumonia is a well-recognized cause of cap in both immunocompetent and immunocompromised persons. in thailand particularly, and to a less extent in some surrounding countries such as northern australia, burkholderia pseudomallei (a gram-negative bacillus that is present as free-living saprophytes in soil and surface waters in endemic areas) is a common cause of fulminant cap with high mortality. time from hospital admission to the development of hap influences the likely pathogens encountered. alterations in a patient's normal flora increase with duration of stay in hospital, potentially modified by illness, medical procedures and drug (antibiotic) exposure. as a general guide, the risk of infection from drug-resistant pathogens increases with duration of hospital stay. the main additional pathogens to consider are gram-negative enteric bacilli and methicillin-resistant s. aureus (mrsa) ( table ) . the additional pathogens to consider in immunocompromised patients with pneumonia depends on the degree of immune dysfunction at the time of infection. in patients with hiv, the risk of infection with s. pneumoniae (and subsequent bacteraemia) is increased over -fold even with normal cd counts (> cells/ml). similarly, the risk of infection by mycobacterium tuberculosis is increased in early hiv infection. with cd counts < cells/ml, the risk from "opportunisitc" infections increases vastly (table ) (segal et al., ) . infections with fungal pathogens are of particular concern in patients with severely impaired immune defenses. of note, some pathogens which commonly cause systemic infections in immunocompromised hosts rarely involve the lung (e.g., candida spp.). other pulmonary infections represent reactivation of disease as immune function declines rather than the acquisition of new disease (e.g., non-tuberculous mycobacteria, toxoplasma gondii). certain pathogens are associated with specific environmental exposures, or in the case of zoonosis, exposure to animal reservoirs (table ) . some pathogens display seasonal patterns of higher incidence. for instance, infections with influenza, respiratory table common microbial pathogens in pneumonia. mycobacterium tuberculosis non-tuberculous mycobacteria syncytial virus and s. pneumoniae are commoner in winter, whilst legionella infections are commoner in summer when the weather is hotter and more humid. over the years, the list of pathogens causing pneumonia has continued to increase through a combination of factors; advances in microbiological diagnostics, better recognition of clinical syndromes and the expansion of human populations into new territories. in particular, a number of viral pathogens have been recognized in the last two decades as implicated in the development of pneumonia. these include coronavirus, human metapneumovirus and enterovirus-d . infection by more than one pathogen can occur in up to a third of patients with pneumonia. these are mostly viral-bacterial pathogen combinations and may reflect recognized associations between certain infections. for instance, influenza-related pneumonia is associated strongly with the bacterial pathogens s. aureus and s. pneumoniae. particular attention is necessary with immunocompromised patients when multiple pathogens may co-exist to cause disease. in , the world health organization (who) published a global priority list of antibiotic-resistant bacteria to guide research, discovery and development of new antibiotics. the list includes a number of important respiratory pathogens, such as penicillin non-susceptible s. pneumoniae, ampicillin-resistant h. influenzae, methicillin and vancomycin-resistant s. aureus, carbapenemresistant a. baumannii, carbapenem-resistant p. aeruginosa, and carbapenem-and third-generation-cephalosporin resistant enterobacteriaceae (such as k. pneumoniae). whilst many of these pathogens are mainly implicated in nosocomial infections, some are important in cap as well. rates of drug-resistant s. pneumoniae (drsp) vary globally. northern european countries have tended to have lower numbers of drsp (< % of pneumococcal isolates) while some other countries (southern europe, japan and the united states) report figures of drsp approximating - % of isolates. the introduction of childhood pcv vaccination programmes in many countries has generally led to a reduction in rates of drsp. the epidemiology of drsp continues to change under vaccine pressure. in europe, during the era of pcv vaccination, the most frequent serotypes to display resistance to penicillin (from samples taken in to ) were serotypes , a and a. multi-drug resistance (defined as resistance to or more classes of antimicrobial agents) was commonest in serotype a, a non-vaccine serotype (yahiaoui et al., ) . an assessment of illness severity is fundamental to the management of patients with pneumonia. severity assessment guides decisions around (a) place of treatment (whether in the community, in hospital, or in intensive care), (b) depth of investigations, (c) speed of treatment and (d) type of treatment, including the choice of antimicrobial agents and route of administration. various severity assessment tools have been developed for the management of patients presenting with cap. the two most widely validated and adopted tools are the pneumonia severity index (psi) and the curb score. both of these were developed to predict shortterm, -day mortality in patients presenting to hospital with cap. prognostic tools to predict icu admission are not as widely used partly due to differences in icu admission policies influencing the predictive accuracy of tools developed in one healthcare system and applied to a different healthcare system. severity assessment tools for hap are less well validated. this reflects the much broader diversity of factors influencing prognosis in patients with hap, including type of hospital ward, reason for hospital admission, time from hospital admission, medical interventions, exposure to nosocomial pathogens and preceding antibiotic exposure. similarly, there are no pneumonia-specific tools for assessing severity in immunocompromised patients. in these patients, the degree of immune compromise is often the dominating factor in severity assessment. patients who are severely immunocompromised may lack many of the symptoms or signs associated with severe illness. a high degree of vigilance is therefore important. early treatment with appropriate antimicrobial agents is the goal. in patients who are severely ill, earlier treatment (measured in hours) is associated with improved clinical outcomes, such as mortality. in patients presenting with signs indicative of severe sepsis, antibiotic administration within an hour is advocated. the impetus for early antimicrobial treatment limits the window in which to complete investigations to confirm the diagnosis of pneumonia and associated microbiology, prior to antimicrobial administration. in many instances, empirical broad-spectrum treatment must be started whilst awaiting results. in the case of influenza infection, time from onset of illness to antiviral treatment is the critical factor. viral load peaks rapidly within the first days of illness. in line with this, evidence of clinical benefit from neuraminidase inhibitors is greatest when treatment is started within h of symptom onset. concepts such as "start smart, then focus" acknowledge the inevitable uncertainty that often exists at the time of commencement of antimicrobials, and emphasis the equally important role of reviewing the diagnosis and treatment plan in the light of emerging results and response to empirical treatment. this pertains not only to antibiotics, but also to antiviral and antifungal agents. the optimal duration of antimicrobial therapy in the treatment of pneumonia has not been adequately studied. traditionally, a -day course of antibiotics has been standard. increasing awareness of the importance of good antimicrobial stewardship has led to efforts to refine the required duration of antimicrobial therapy. shorter -day courses of treatment are gaining acceptance, as are biomarker-driven antibiotic prescribing strategies (see section on "biomarkers"). some infections, such as legionella pneumonia, are still treated with longer courses of antibiotics ( - days) based on clinical experience rather than evidence derived from clinical trials. in the treatment of pneumonia, adjuvant therapy refers broadly to interventions that are aimed at modulating the immune response to infection. the use of systemic corticosteroids has been extensively tested in patients with severe sepsis, a large proportion of whom have pneumonia. a meta-analysis of trials involving > , patients found, with low certainty, a small mortality benefit in favor of low-dose corticosteroids (rochwerg et al., ) . fewer trials have been conducted in patients with cap and the existing debate around the value of corticosteroids for this specific indication reflects the weaker evidence base; evidence from trials involving > patients indicate a mortality benefit in patients with severe pneumonia, but not in those with non-severe pneumonia (stern et al., ) . macrolide antibiotics have immunomodulatory properties apart from their antibiotic effects. large observational cohort studies of patients with both all-cause pneumonia (i.e., involving a range of respiratory pathogens) and pneumococcal pneumonia suggest the combination of macrolide with beta-lactam antibiotics is associated with improved prognosis. in contrast, data from available randomized controlled trials are conflicting. hmg-coa reductase inhibitors (statins) have a range of immunomodulatory effects and in murine models of sepsis, pre-dosing with statins has been associated with improved outcomes. studies in adults with pneumonia support the notion that patients already taking a statin at the time of infection have a better prognosis, but the value of statins started as adjuvant therapy in patients presenting with pneumonia has not been established. immunization programmes against common respiratory pathogens are cost-effective public health interventions for the prevention of cap in many countries. in relation to s. pneumoniae, two types of pneumococcal vaccines are commercially available. the current multivalent pneumococcal polysaccharide vaccine (ppv) was first introduced in and covers of over pneumococcal serotypes/serogroups ( , , , , , b, f, , n, v, a, aa!, f, , b, f, c, a, f, , f, f, f.) . the -valent ppv offers good protection against invasive pneumococcal disease but relatively weak protection against pneumococcal pneumonia (falkenhorst et al., ) . in older persons who are most at risk of pneumonia, immunosenescence adversely influences the protective effect of these vaccines. pneumococcal conjugate vaccines (pcvs) promote a more robust immune response and are able to reduce nasopharyngeal carriage of vaccine-type s. pneumoniae strains, but cover a smaller range of pneumococcal serotypes. as children are the main carriers of s. pneumoniae, vaccination of children with pcvs has been associated not only with a reduction in the incidence of childhood pneumococcal infections, but also in the incidence of adult pneumococcal pneumonia (herd protection) (tsaban and ben-shimol, ) . countries vary in the target groups for pneumococcal vaccination and the vaccines offered. trivalent influenza vaccines against of the main circulating influenza strains ( influenza a strains and influenza b strain) have been available for many years. due to antigenic shifts within influenza viruses, the components of these vaccines are reviewed (and renewed) each year to maximize vaccine-pathogen "match." vaccines against all four of the common seasonal influenza viruses (quadrivalent vaccines covering influenza a strains and influenza b strains) are now available. in addition, and perhaps more importantly, is the development of newer conjugated vaccines and high-dose vaccines that promote stronger immune responses in older at-risk persons compared to previous "standard" influenza vaccines, hence offering greater protection. influenza vaccines that are not strain-specific (so-called "universal" vaccines) and therefore less subject to mismatch are also being developed as are vaccines against respiratory syncytial virus. the h. influenzae type b (hib) vaccine is available for the prevention of childhood pneumonia. however, in adults, non-typeable h. influenzae (nthi) is much more important and there are some data to suggest that childhood carriage of nthi may be increasing. an effective vaccine for nthi is not currently available. smoking cessation and a reduction in alcohol use are important modifiable lifestyle factors for the prevention of pneumonia. current tobacco smokers are over two times more likely (pooled odds ratio . , from meta-analysis) to develop cap compared to adults who have never smoked, while people who consume alcohol (or in higher amounts) have a % increased risk of cap compared to those who consume no (or less) alcohol (simou et al., ) . for every - g higher alcohol intake per day, there is an % increase in the risk of cap. a large number of specific interventions have been advocated in the prevention of vap. these may be grouped as functional (e.g., semi-recumbent position), mechanical (e.g., silver-coated endotracheal tube) and pharmacological (e.g., selective decontamination of the digestive tract) interventions. the institute of health improvement (ihi) developed the concept of "bundles" of care in the icu to improve clinical outcomes. implementation of a vap prevention bundle may enable the ideal goal of "zero vap" to be achieved (Álvarez-lerma et al., a,b) . recovery from pneumonia usually takes several weeks. at - weeks following discharge from hospital for an episode of cap, one or more symptoms continue to be reported by % of patients, including cough, dyspnoea and fatigue in roughly equal proportions. functional impairment is reported by - % of patients at weeks (pick et al., ) . cardiac complications (including heart failure, acute coronary syndrome and arrhythmias) have been observed in % of patients within days following occurrence of cap, with higher rates among patients who are hospitalized compared to those treated in the community (corrales-medina et al., ) . the risk of cardiac complications remains high for at least the first - years following hospitalization (corrales-medina et al., ) . higher rates of long-term mortality ( % at year, % at years) have been observed in patients following hospitalization for cap compared to patients hospitalized for other reasons. the predominant causes of long-term mortality are vascular and respiratory in nature. although studies have attempted to adjust for co-existing medical illnesses, it remains uncertain whether the association of pneumonia with long-term mortality is causal or whether pneumonic events are simply markers of poorer overall health (wagenvoort et al., ) . many of the current treatment strategies for pneumonia have remained broadly unchanged over the last decade. efforts at reducing the burden of disease through vaccination have been rewarded with some success but continued innovation is required to maintain gains made so far. the rise of antimicrobial resistance threatens our ability to treat infections that occur, urging a more judicious approach than the empirical antimicrobial strategies that characterize much of current clinical practice. the future expectation is a shift towards targeted treatments supported by rapid diagnostics, thus enabling the use of non-antibiotic pathogen-specific therapies (such as anti-toxins) and promising the eradiation of inappropriate antimicrobial use. immune-modulating agents may offer further benefits in relation to short-term recovery, and improved post-pneumonia care could translate into better longer-term outcomes. risk factors for community-acquired pneumonia in adults: a systematic review of observational studies prevention of ventilator-associated pneumonia: the multimodal approach of the spanish icu "pneumonia zero" program the multimodal approach for ventilator-associated pneumonia prevention"-requirements for nationwide 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hospitalisation corticosteroids in sepsis: an updated systematic review and meta-analysis mortality reduction among pneumonia patients still substantial despite the impact of coding changes host-response biomarkers for the diagnosis of bacterial respiratory tract infections procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections hiv- and bacterial pneumonia in the era of antiretroviral therapy diagnosis of pneumonia in the ed has poor accuracy despite diagnostic uncertainty alcohol and the risk of pneumonia: a systematic review and meta-analysis diagnosis of stroke-associated pneumonia: recommendations from the pneumonia in stroke consensus group corticosteroids for pneumonia the incidence and aetiology of hospitalised community-acquired pneumonia among vietnamese adults: a prospective surveillance in central vietnam international ers/esicm/escmid/alat guidelines for the management of hospital-acquired pneumonia and ventilator-associated pneumonia: guidelines for the management of hospital-acquired pneumonia (hap)/ventilator-associated pneumonia (vap) of the increasing hospital admission for pneumonia indirect (herd) protection, following pneumococcal conjugated vaccines introduction: a systematic review of the literature diagnosing pneumonia in patients with acute cough: clinical judgment compared to chest radiography long-term mortality after ipd and bacteremic versus non-bacteremic pneumococcal pneumonia advances in the causes and management of community acquired pneumonia in adults distribution of serotypes and patterns of antimicrobial resistance among commensal streptococcus pneumoniae in nine european countries key: cord- -mk mzc z authors: morris, cindy e.; bardin, marc; kinkel, linda l.; moury, benoit; nicot, philippe c.; sands, david c. title: expanding the paradigms of plant pathogen life history and evolution of parasitic fitness beyond agricultural boundaries date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: mk mzc z nan how do pathogens, whether they parasitize plants or animals, acquire virulence to new hosts and resistance to the arms we deploy to control disease? the significance of these questions for microbiology and for society at large can be illustrated by the recent worldwide efforts to track and limit the emergence of human transmissible strains of swine and avian influenza virus and of multidrug-resistant lines of human pathogenic bacteria, and to restrain the spread of ug , a strain of stem rust of wheat. recent research in medical epidemiology has elucidated the impact of pathogen ecology in environmental reservoirs on the evolution of novel or enhanced pathogen virulence. in contrast, the evolution of virulence in plant pathogens has been investigated from a predominantly agro-centric perspective, and has focused overwhelmingly on evolutionary forces related to interactions with the primary plant host. here, we argue that current concepts from the field of medical epidemiology regarding mechanisms that lead to acquisition of novel virulence, biocide resistance, and enhanced pathogenic fitness can serve as an important foundation for novel hypotheses about the evolution of plant pathogens. we present numerous examples of virulence traits in plant pathogenic microorganisms that also have a function in their survival and growth in nonagricultural and nonplant habitats. based on this evidence, we make an appeal to expand concepts of the life history of plant pathogens and the drivers of pathogen evolution beyond the current agro-centric perspective. the classification of diseases in terms of their epidemiology is a useful starting point for a comparison of plant and human pathogens [ ] . in medical epidemiology, anthroponoses are diseases trans-mitted among humans that have no other known reservoirs for multiplication. typhoid fever, smallpox, and certain venereal diseases are examples. zoonoses, such as rabies, lyme disease, severe acute respiratory syndrome (sars), and avian and swine influenzas, are transmitted to humans from living animals. sapronoses are diseases transmitted to humans from environmental reservoirs where the pathogen thrives saprophytically. these habitats include soil, water, and decaying plant and animal matter. examples include legionnaire's disease, cholera, aspergillosis, and the emerging epidemics of melioidosis (burkholderia pseudomallei). human pathogens with saprophytic phases or residing in environmental reservoirs are also referred to as ''environmental pathogens'' [ ] [ ] [ ] [ ] [ ] . studies of virulence factors of human pathogens in environmental reservoirs have begun to reveal the importance of alternate hosts, of dual-use virulence factors, and in general of how environmental habitats can select for traits that confer enhanced fitness as human pathogens. for example, interactions with microbial eukaryotes seem to have led to the acquisition of traits useful for pathogenicity to mammalian cells. numerous environmental pathogens, including cryptococcus neoformans, legionella spp., chlamydophila pneumoniae, mycobacterium avium, listeria monocytogenes, pseudomonas aeruginosa, and francisella tularensis, might have acquired virulence traits via their resistance to predation by amoebae. this resistance, associated with the ability to grow inside the amoebae-which are essentially alternate hosts-has likely led to the selection of traits conferring survival in macrophages [ ] . resistance to macrophages involves the capacity of the bacteria to resist or debilitate the macrophage's phagosomes and to multiply in the cytoplasm. many of the traits essential for virulence to humans likewise seem to play roles in adaption to the environments where the organisms are saprophytes (table ). these traits have dual roles in environmental and parasitic fitness and are thus referred to as ''dualuse traits''. melanins, siderophores, and the capacity to form biofilms are among the frequently cited examples. c. neoformans provides one of the richest examples of dual-use traits. this fungus, frequently found in soils that contain high levels of bird guano and in association with certain plants, causes meningoencephalitis. a nonexhaustive list of its dual-use traits includes capsule formation and production of melanin, laccase, phospholipase, proteases, and ureases [ ] . in the environment these traits contribute to survival and in human hosts they contribute to the capacity of c. neoformans to avoid host resistance mechanisms and to attack host tissue. microbial efflux pumps have also evolved dual uses. these transport systems are used for managing toxic compounds in the environment of the microorganism and can have a broad spectra of activity leading to multidrug resistance among environmental microorganisms [ ] . human activities resulting in the disposal of a wide range of chemical products into the environment, including household cleaners that contain the broad spectrum antimicrobial triclosan, may be inadvertently exacerbating the abundance of multidrug-resistant bacteria [ ] . virulence of environmental pathogens has been described as a set of cards, or a diverse set of attributes acquired as a function of the life history of a pathogen and its adaptation to different environments [ , ] . it is becoming increasingly clear that evolutionary forces outside the context of human-pathogen interactions are responsible for the acquisition and maintenance of some virulence factors [ ] . genomics and phylogenetics are revealing the evolutionary link between, for example, commensal strains of escherichia coli and modern pathogens such as enterohaemorrhagic strains of this species (such as o ). the mechanisms proposed to explain how these commensals have become pathogens are grounded in their ecology and life histories, culminating in the notion of ecological evolution (''eco-evo'') [ ] . the eco-evo approach to understanding the emergence of pathogens gives credence, from the perspective of genomics, to evolutionary and adaptive scenarios that are surmised from a thorough understanding of the ecology and life history of pathogens. at present, epidemiological classifications of plant diseases are based on the interaction of the pathogen and the host (biotrophic or necrotrophic, obligate or facultative), on the number of cycles of propagule production (mono-and polycyclic diseases), on the importance of latency in symptom expression, and on the role of vectors, but there is no formalized equivalent of ''sapronoses''. nevertheless, numerous plant pathogens are present in diverse nonagricultural habitats or survive saprophytically in agricultural contexts. these include a range of bacteria, fungi, and stable viruses (a nonexhaustive list of examples is presented in table ). a striking characteristic of many of the virulence factors of these plant pathogens is that they are linked to-or are in themselves-traits critical to adaptation to the nonplant environment, as will be illustrated below. this provides a compelling reason to adopt a holistic view of the life history and evolution of plant pathogens, to move beyond the traditional borders of agriculture and the presumed ''primary'' plant host. adaptation to biotic and abiotic stresses, within or outside of agricultural habitats, likely plays as important a role in the evolution of parasitic fitness of plant pathogens as it does for human pathogens. as illustrated above, traits that confer fitness in response to biotic and abiotic environmental stress can have dual-use as virulence factors in human pathogens. toxins and toxin transport systems (including efflux pumps, in particular) are among the common adaptations for antagonizing and defending against the co-inhabitants of a habitat. in plant pathogens, the transport systems for toxins and antimicrobials can have broad spectrum activity, leading to resistance to agricultural fungicides and also contributing to virulence [ ] . genes coding for wide spectrum efflux pumps are present in the chromosomes of all living organisms [ ] . the efflux pump bcatrb of botrytis cinerea confers resistance to antimicrobials produced by soil and plant microflora ( , diacetylphloroglucinol and phenazine antibiotics) [ , ] and also to the fungicide fenpiclonil and the plant defensive phytoalexin resveratrol [ ] . the transporter abc from magnaporthe grisea protects the fungus against azole fungicides and the rice phytoalexin sakuranetin [ ] . numerous plant pathogenic bacteria, including erwinia amylovora, dickeya spp. (formerly the multiple biovars of e. chrysanthemi), and agrobacterium tumefaciens, also produce efflux pumps that are involved in their resistance to plant antimicrobials (reviewed by martinez et al. [ ] ). toxins themselves can have a broad spectrum of action. for example, mycotoxins, well known for their human and animal toxicity, have broad spectrum activity and are thought to have evolved as a defense against predators (nematodes) and antagonists (other microorganisms) [ ] . one family of these, the trichothecenes, contributes significantly to the virulence of many gibberella (fusarium) species [ ] . adaptation to biotic stress also implicates systems for the detection or inhibition of arms of aggression used by co-inhabitants. recent work on fungi suggests that systems to detect enzymes that degrade fungal cell walls are also deployed as virulence factors. lysin motifs (lysms) are carbohydratebinding protein modules that have been found in mammalian and plant pathogenic fungi as well as in saprophytes [ ] . bolton et al. [ ] demonstrated that the lysm protein ecp acts as a virulence factor in the plant pathogenic fungus cladosporium fulvum. as virulence factors they may suppress host defenses by sequestrating chitin oligosaccharides that are known to act as elicitors of plant defense responses [ ] and also as activators of host immune responses in mammals [ ] . de jonge and thomma [ ] suggest that these proteins may also have a role in the protection of saprophytic fungi against chitinase-secreting competitor microbes or mycoparasites. protection against abiotic stress can involve molecules that have also become virulence factors. siderophores [ ] [ ] [ ] and various pigments including melanins [ ] are virulence factors in some human pathogens. siderophores contribute to resistance to oxidative stress and sequestering iron when it is rare in the environment. in the plant pathogens alternaria brassicicola, cochliobolus spp., fusarium graminearum [ ] , and m. grisea [ ] , siderophores or their precursors are virulence factors. melanins offer protection from extreme temperatures, uv radiation, and antimicrobials. in the plant pathogens m. grisea and colletotrichum spp., melanins are also virulence factors via their essential role in the formation of tissue-penetration structures such as appressoria [ ] . in many cases, toxins and siderophores are produced by nonribosomal peptide synthase or polyketide synthase pathways. these pathways, widely distributed in the microbial world, are highly adaptable and have given rise to a wide range of compounds with a plethora of activities, including many of pharmaceutical importance [ ] . hc-toxin of cochliobilus carbonum, victorin in c. victoriae, and t-toxin in c. heterostrophus are products of these pathways [ ] . the key virulence factor of streptomyces spp., thaxtomin [ ] , and the multitude of host-specific and nonspecific toxins in pseudomonas syringae pathovars [ ] are also produced by these pathways. the capacity to detect changes in conditions of the abiotic environment has also become part of the virulence factors of some plant pathogens. for example, to detect changes in environmental conditions, organisms exploit two-component histidine kinase complexes. these are key elements of the machinery for signal sensing, allowing bacteria, yeasts, fungi, and plants to adapt to changing environments. in the plant pathogen b. cinerea, one of its multiple histidine kinases, bos , not only mediates osmosensitivity and resistance to fungicides, but is also essential for formation of macroconidia and expression of virulence [ ] . recognition and understanding of the full complexity of the life history of plant pathogens will enhance our capacity to evaluate the diversity and intensity of environmental stresses that microorganisms face and will contribute novel hypotheses concerning the role of environmental stresses in the evolution of pathogenicity. stress is considered to play an important role in adaptive evolution in general, in particular via its effect on mutation rates [ ] . for certain fungi and bacteria, including plant pathogens, stress increases the activity of transposable elements [ ] [ ] [ ] and induces the sos response and other systems involved in the modification or repair of dna [ ] . mutations can target the ensemble of the microbial genome. however, it has been suggested that adaptation of bacteria to multiple stresses can lead, in particular, to the acquisition of virulence factors and to the emergence of pathogenic variants [ ] . adaptation to specific habitats-which involves adapting to a particular ensemble of biotic and abiotic parameters-could also influence the evolution of parasitic fitness. available examples focus on soilborne and rhizosphere microorganisms. the rhizosphere is a dynamic soup whose chemistry changes as plants grow, die, and degrade. chemicals in the rhizosphere are food substrates and means of communication, antagonism, and collaboration among microorganisms, among plants, and between plants and microorganisms. to decompose dead plant material and recycle carbon, microorganisms have developed a range of cell wall-degrading enzymes, without which our planet would be quite encumbered by the accumulation of tissue from dead plants. pectolytic, cellulolytic, and lignolytic enzymes are also well-known pathogenicity factors [ ] [ ] [ ] . to hone the efficiency of these enzymes in planta, pectinolytic fungi are adept at modulating the surrounding ph. alternaria, penicillium, fusarium spp., and sclerotinia sclerotiorum also exploit these ph changes to enhance the action of these enzymes as virulence factors [ ] . streptomyces spp. are considered quintessential soil inhabitants. their ability to degrade biopolymers, including cellulose and chitin, contributes greatly to nutrient cycling, and their vast array of antimicrobials contributes to survival and microbial communication in soil [ ] . some streptomyces species are pathogenic to root crops and to potatoes in particular. a recently discovered virulence factor in streptomyces, a saponinase homologue [ ] , may be the result of adaptation to the rhizosphere. saponins are plant glycosides that contribute to resistance against fungi and insect herbivores. bacteria, and especially gram-positive bacteria, can also be sensitive. saponins are also exuded from the roots of some plant species where they have allelopathic as well as antimicrobial activity [ , ] . key vital functions, housekeeping functions, and basic life cycle processes should also be considered for their potential to give rise to pathogenicity factors. traits fundamental to fitness and survival in general can confer or enhance pathogenic fitness. in plant pathogenic bacteria these include flagella, motility, lipo-and exopolysaccharides, o-antigens, fimbriae, mechanisms for iron acquisition and for quorum sensing, toxin production, cell wall-degrading enzymes, and resistance to oxidative stress [ ] . motility, for example, is essential to dispersal and for attaining new resources. in ralstonia solanacearum it is also essential for early stages of plant invasion and colonization during pathogenesis [ ] . in the fungus aschochyta rabiei, kinesins that are essential for polarized growth and transport of organelles are suspected to be a virulence factor [ ] . an f-box protein of giberrella zeae has been reported to be involved in sexual reproduction and in pathogenicity [ ] . the enzymes that allow fungi to detoxify compounds resulting from plant defense mechanisms are probably also simply means of acquiring nutrients [ ] . for example, detoxification of tomatine in tomatoes by septoria lycopercici and by fusarium oxysporum f. sp. lycopersici is achieved by the deployment of glycosyl hydrolases by these fungi; gaeumannomyces graminis detoxifies avenacins in oats via a beta-glucosidase [ ] . another example of adaptation of basic cellular functions into pathogenicity factors concerns elicitins. elicitins are part of one of the most highly conserved protein families in the phytophthora genus and are widespread throughout phytophthora species. elicitins of p. infestans induce hypersensitivity in plants. recent work from jiang and colleagues [ ] suggests that a primary function of elicitins is the acquisition of sterols from the environment. how can we make sense of the processes that have led to the wide variety of pathogenicity factors in plant pathogens and that continue to drive the evolution of pathogens? bacterial plant pathogens are particularly illustrative of the differences in suites of secretion systems [ , , , ] and of effectors [ , , , , , ] among members of different genera, species, or strains of the same species that attack plants. effectors are proteins secreted by plant pathogens that modulate plant defense reactions, thereby enabling the pathogen to colonize the plant tissues. it is tempting to wonder if the effectors and secretion systems have critical roles in fitness elsewhere other than in association with the host plant. the examples listed above that describe traits that play roles in both environmental fitness and virulence to plants provide a compelling incentive to expand our paradigms concerning the forces that drive evolution of plant pathogenicity. the evolutionary forces that have been described to date for plant pathogens [ ] need to be extended beyond the current agro-centric paradigm. to expand this paradigm we propose that the life cycles and life histories of plant pathogens be reconsidered. studies of pathogen ecology, evolution, and life history should include the full range of habitats and reservoirs these organisms can inhabit. this in turn will permit testing a range of novel hypotheses about the role of ecological contexts-other than direct interaction with host plants-as forces of evolution. in table we propose some such hypotheses. for example, rates of mutation and of transposition of insertion sequences or of transposable elements including phages might be different when a microorganism inhabits nonagricultural habitats (biofilms, lake water, or inert surfaces exposed to uv, for example) than when it colonizes plants. the consequences of these mutations for pathogenicity might in turn be markedly different than for fitness in nonagricultural habitats. likewise, the formation of spores or aggregates that can be released into the air and their survival over long distances might be highly influenced by the nature of the reservoir that the pathogen colonizes, resulting in direct effects of habitat on gene flow. furthermore, the biotic and abiotic stresses endured in nonagricultural habitats might exert positive selection for adaptive survival traits that have dual-use as virulence factors as illustrated in the examples above. these questions are clearly pertinent for pathogens that are not obligate biotrophs. however, the complexity of the biotic and abiotic environment perceived by obligate biotrophs during colonization of plants (powdery mildews on leaf surfaces inhabited by other microorganisms, for example) or during their dissemination (survival in air or in association with vectors) are also likely to exert selection independent of that due to the host plant genotype per se. these are only some of the ways in which environmental parameters other than the host plant are expected to have a marked influence on the diversification of plant pathogens. if nonagricultural environments can foster the evolution of traits that contribute to pathogen virulence, other scenarios are also probable where i) crop plants foster the emergence of traits antagonistic to survival outside of agricultural contexts ii) or nonagricultural environments foster the emergence of traits that are detrimental to pathogen virulence in crops. understanding the prevalence and significance of alternative habitats to pathogen life history is crucial to determining the broad costs of virulence for pathogen fitness. the cost of virulence in terms of fitness in association with plants has been explored extensively for several obligate parasites such as rusts and powdery mildews. work by thrall and burdon [ ] has shown clear fitness tradeoffs between pathogen aggressiveness (capacity to induce intense disease symptoms) and dissemination (via intense spore production). for nonobligate pathogens we do not know the cost of fitness outside of agricultural habitats. the interplay between evolutionary forces and habitat has not been explored for plant pathogens and might be a key feature in the emergence of certain diseases. by expanding our paradigms concerning pathogen life history and the selective forces that drive plant pathogen evolution, we will enhance our understanding of how table . novel hypotheses to be tested concerning the impact of substrates other than host plants on the evolutionary potential of plant pathogens. evolutionary force a novel hypothesis arising from expanded paradigms about the evolution of plant pathogenicity concerning: modifications of the genome. relative to its association with cultivated plant hosts, association of the pathogen with a given nonagricultural substrate leads to: n a significantly greater overall mutation rate. n a greater rate of transposition of insertion sequences or of transposable elements. n more frequent mutations or transpositions that target genes involved in pathogenicity. n a higher probability of acquisition of alien nucleic acids. n genetic exchange with more phylogenetically diverse microbes. effective population size. the effective sub-population size of a pathogen associated with a given nonagricultural (or nonplant) substrate is significantly different from that for sub-populations from cultivated host plants. this could lead to genetic and/or phenotypic differentiation of sub-populations based on substrate of origin. dissemination. the habitats occupied by the plant pathogen influence the mode(s) of dissemination, thereby influencing the distance of dissemination and the spatial and temporal scales of gene flow. mode of reproduction (recombination) genetic recombination. the frequency of recombination (via sexual cycle or other means) varies among strains of plant pathogens as a function of the habitat or substrate. selective pressures and impact on fitness. strains of pathogens adapted to a broad range of habitats have the greatest parasitic fitness. a the evolutionary forces listed here are those that have been considered for plant pathogens in agricultural contexts [ ] . these hypotheses concern pathogens with a marked saprophytic phase or for which nonagricultural or nonplant substrates can be a notable reservoir for survival. reservoirs can include irrigation water, natural waterways and bodies of water, biological vectors (animals, fungi, etc.), abiotic vectors (aerosols, clouds, precipitation), wild plants and weeds, soil, and physical structures in agricultural systems (greenhouse materials, tubing, plastics). doi: . /journal.ppat. .t pathogens survive in the absence of hosts, how and where new pathotypes are likely to emerge, and the significance of natural habitats to agricultural epidemics. insights will come from fundamental research to identify the mechanisms that drive the evolution of pathogenic traits and to explore the ecological significance of pathogenic traits to microbial fitness apart from the plant host. distinguishing the role of adaptation sensu stricto in the emergence of plant pathogenicity relative to that of exaptation [ ] , the useful cooptation of phenotypes that have arisen under natural selection due to forces unrelated to interaction with the primary host plant, will yield critical insight into how plant pathogens evolve independently of agricultural practices. a more complete understanding of the forces that drive plant pathogen evolution will be critical to enhancing and diversifying sustainable 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soil microfungal assemblages in ontario lack of host specialization in aspergillus flavus ecology of the fungus, fusarium: competition general ecology of the fusaria biodiversity of the genus fusarium in saline soil habitats effects of water potential on spore germination and viability of fusarium species metabolites of fusarium the stem canker (blackleg) fungus, leptosphaeria maculans, enters the genomic era insectassociated fungi in soils of field crops and orchards diversity, host, and habitat specificity of oomycete communities in declining reed stands (phragmites australis) of a large freshwater lake penicillium mycobiota in arctic subglacial ice detection of infectious tomato mosaic tobamovirus in fog and clouds detection of tomato mosaic tobamovirus rna in ancient glacial ice detection of infectious tobamovirus in forest soils we thank the three anonymous reviewers for their constructive comments and for the suggestion of additional materials to incorporate into the text. we also thank dr. melodie putnam (oregon state university, united states of america) for useful discussions about the ecology of bacterial plant pathogens. key: cord- - rit h authors: chinchio, eleonora; crotta, matteo; romeo, claudia; drewe, julian a.; guitian, javier; ferrari, nicola title: invasive alien species and disease risk: an open challenge in public and animal health date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: rit h nan protecting public and animal health. for example, empirical research on ias pathogens, which would be needed to assess the risk of infectious disease emergence, is skewed toward a few species (e.g., vector species like the tiger mosquito aedes albopictus) or toward selected pathogens known to harm biodiversity conservation, while a global vision of ias-associated health threats is still not available [ , [ ] [ ] [ ] . in this context, it is urgent to raise awareness in people working in the fields of animal and public health of the need to consider ias as a health threat. to this aim, we provide here an overview of how animal ias may affect local disease dynamics both directly and indirectly, i.e., acting as pathogen hosts or disrupting the recipient ecosystem structure, through real-case examples from the ecological literature, and, in the last paragraph, we propose future initiatives aimed at improving our capacity for targeted actions toward the ias most likely to threaten human and animal health, calling for an increased involvement of people working in the fields of animal and public health in a new invasion epidemiology field. ias may host pathogens that are absent in the area of release and cause their establishment and subsequent spillover to local species, possibly resulting in an increase of disease risk for humans, domestic animals, and native wildlife. the north-american raccoon procyon lotor, for example, introduced to central europe baylisascaris procyonis [ ] , a nematode causing larva migrans syndromes potentially inducing severe central nervous system disease in humans ( fig a) . introduction to europe of north-american crayfishes procambarus clarkii infected with the fungus aphanomyces astaci caused huge economic losses to fisheries, being the pathogen lethal to native crayfishes [ ] . similarly, squirrelpox virus, introduced to the united kingdom along with the american eastern gray squirrel sciurus carolinensis, is significantly contributing to the increased mortality of native red squirrels sciurus vulgaris [ ] . however, while pathogen co-introductions occur over a wide range of parasite and host taxa [ ] , some pathogens are lost during the invasion process [ ] : for example, there is no evidence for poxvirus in italian gray squirrel populations [ ] . pathogen loss may be due to the absence of the pathogen in the individuals of the founding populations or to its inability to survive to translocation or establish in the area of release. the outcome depends on several factors related to the ias (e.g., founding population origin), the pathogens (e.g., host specificity), and the area where the species is released (e.g., environmental conditions, presence, and density of local hosts) [ ] . as shown by a study on ectoparasites of introduced birds, factors related to transmission efficiency, such as the number of host introduced and host longevity, are likely to play a major role [ ] . an increase of local disease risk may also occur if the introduced ias is susceptible to, and able to transmit, local pathogens. pathogens acquired by ias may be amplified and possibly spill back to humans and local species [ ] . a case in point is the australian brushtail possum, trichosurus vulpecula, in new zealand ( fig b) . invasive possums probably became infected with mycobacterium bovis, the causal agent of tuberculosis in cattle, from wild deer, after the beginning of commercial deer hunting in . currently, they are the most important maintenance host for bovine tuberculosis, supporting higher transmission rates compared to local species and, being sympatric with cattle, providing interface for transmission between livestock and forest residents [ ] . another case is represented by invasive raccoon dogs nyctereutes procyonoides, which may amplify rabies circulation in eastern europe or cause its reemergence in currently rabies-free countries [ ] . ias competence for pathogen transmission plays a major role in defining the outcome of pathogen acquisition, and, as the possum-tuberculosis case exemplifies, it is the result of both ias-pathogen interaction (e.g., ias susceptibility, period of communicability, and pathogen excretion rate) and ias behavioral patterns (e.g., habitat, home range extension, and intraand interspecific contact rates). based on ias competence, the acquisition of a local pathogen may even lead to the reduction of disease risk (the so-called dilution effect [ ] ) or to no consequences at all. for example, in ireland, the invasive bank vole myodes glareolus has been found to divert fleas from the native wood mice apodemus sylvaticus, which is a more competent host for bartonella spp. [ ] . however, the identification of the contexts in which a dilution effect may occur is still highly debated in ecology, as it strongly depends on local host species diversity and on the interactions occurring between the species involved in the transmission cycle [ ] . introduced species may disrupt local infection dynamics also indirectly, i.e., nonacting as pathogen hosts but through competitive and trophic interactions with native species or modification of local habitats, thus altering the abundance and/or contact rates among local host species, parasite infective stages, or vectors. in southern florida, the invasive python python bivittatus caused the decrease of several mammal species, inducing the local mosquito vector of zoonotic everglades virus to feed almost exclusively on the virus' main reservoir host, the hispid cotton rat sigmodon hispidus, potentially leading to an increase in pathogen circulation (fig c) [ ] . an example of habitat alteration is given by the activity of invasive feral pigs sus scrofa on the island of hawaii: they create wallows and cavities in tree fern trunks improving habitat suitability for mosquito vectors for avian malaria plasmodium relictum [ ] , one of the main threats to native hawaiian forest birds' conservation. again, ias indirect effects on local infection dynamics are highly context dependent, and mechanisms presented so far may act in concert, producing unpredictable outcomes. in scotland and northern england, for example, the invasive gray squirrel has been found to harbor several local strains of borrelia burgdorferi [ ] . however, in those areas, gray squirrels are also causing the decline of another competent host for b. burgdorferi, the red squirrel, and the effect of these concurring mechanisms on human lyme disease risk remains unknown [ ] . during the last centuries, more than , ias introduction events have been recorded worldwide, and this number still presents an increasing trend [ ] . in such context, the identification of those species deserving priority attention, based on their actual and potential impacts, is essential to support decision-making [ ] . several tools to inform preventive and management actions on animal ias, including horizon scanning protocols, risk assessments, and impact assessments, have been developed in the last years (see [ ] for a recent review), but the majority of them focus on environmental impacts, not specifically considering disease emergence risks in humans and local animal populations [ , ] . some authors have called for a greater attention on the potential health risks posed by biological invasions [ ] [ ] [ ] ] , highlighting the need for a better integration between biological and health sciences, surveillance actions, and coordinated policies. we support their appeal, arguing that an increased awareness of people working in the fields of animal and public health on the risks concerning biological invasions and their consequent involvement in the invasion biology field is the first step toward a complementary invasion epidemiology field. such field would be integrated with invasion ecology, but more specifically aimed at the prevention of the emergence of diseases in human and animal populations consequent to ias introduction and establishment. to this aim, we propose some initiatives that should be addressed by future research work. a first major constraint in addressing the issue of disease emergence connected to ias is given by the lack of comprehensive data on pathogens affecting ias. in this sense, we recommend the gathering in ad hoc databases of all the available information on ias pathogens affecting human and animal health, including their geographical distribution and prevalence in ias populations, in both native and introduced ranges. it would also be advisable to improve our understanding of the key epidemiological events and factors driving the emergence of infectious diseases following ias establishment, for example, through ex-post analyses on the already established ias. in particular, as the emergence process of a disease is composed of several stages (introduction in a new area/host population, establishment, and spread) [ , , ] , the key factors involved in the process and related to ias biology, pathogenic features, and the biotic and abiotic components of the area of release should be identified for each of these stages. we also suggest urgently directing research efforts at developing transparent and flexible tools able to prioritize ias based on the risk of transmitting pathogens with the potential to impact the health of humans, production animals, and native wildlife. such tools could be based on the framework of the world organisation for animal health (oie)/international union for conservation of nature (iucn) for wildlife disease risk analysis and readapted to account for the main mechanisms through which alien species may affect local health, in particular the introduction of new pathogens and the acquisition and spread of local ones. the lack of data on ias pathogens is certainly an obstacle in underpinning in-depth risk assessments [ ] , in particular, quantitative ones. however, a simple and transparent qualitative disease risk assessment procedure would enable the prioritization of empirical research needed to cover these knowledge gaps, while at the same time guiding local health administrators in the allocation of resources for management and preventive actions toward ias. the issue related to irregular data availability could be partially overcome, as a first step, by eliciting opinions from experts. finally, awareness and action will be influenced by, and need to consider, the wider public perspective, not just researchers and institutions. initiatives aimed at sensitizing citizens about the health threats of ias will be needed to promote responsible behaviors when crossing borders and to improve the general public attitude toward ias control and eradication programs. all the suggested initiatives, to be successful, necessitate a stronger connection between ecologists, biologists, and other people working in the fields of animal and public health and beyond. only through wider collaboration and dialogue will the potential health impacts of biological invasions be fully appreciated and, perhaps, ameliorated. emerging infectious diseases of wildlife-threats to biodiversity and human health global trends in emerging infectious diseases alien species as a driver of recent extinctions invasive species database impact of the invasive brown marmorated stink bug in north america and europe: history, biology, ecology, and management aquatic invasive species and emerging infectious disease threats: a one health perspective the spread of invasive species and infectious disease as drivers of ecosystem change invasive species challenge the global response to emerging diseases invading with biological weapons: the importance of disease-mediated invasions is invasion success explained by the enemy release hypothesis? indirect effects of parasites in invasions parasites as drivers and passengers of human-mediated biological invasions co-invaders: the effects of alien parasites on native hosts parasites lost-do invaders miss the boat or drown on arrival? natural history of host-parasite interactions the effects of invasion on parasite dynamics and communities wildlife diseases: from individuals to ecosystems horizon scanning for invasive alien species with the potential to threaten biodiversity and human health on a mediterranean island alien species and public health impacts in europe: a literature review alien pathogens on the horizon: opportunities for predicting their threat to wildlife first detection of baylisascaris procyonis in wild raccoons (procyon lotor) from leipzig biological invaders in inland waters: profiles, distribution, and threats ecological replacement of native red squirrels by invasive greys driven by disease introduced species and their missing parasites invasions and conservation: no evidence of squirrelpox virus in grey squirrels introduced to italy parasite spillback: a neglected concept in invasion ecology? epidemiology and control of mycobacterium bovis infection in brushtail possums (trichosurus vulpecula), the primary wildlife host of bovine tuberculosis in new zealand rabies in northeastern europe-the threat from invasive raccoon dogs effects of species diversity on disease risk disruption of a host-parasite system following the introduction of an exotic host species mammal decline, linked to invasive burmese python, shifts host use of vector mosquito towards reservoir hosts of a zoonotic disease managing disease an invasive mammal (the gray squirrel, sciurus carolinensis) commonly hosts diverse and atypical genotypes of the zoonotic pathogen borrelia burgdorferi sensu lato no saturation in the accumulation of alien species worldwide prioritizing species, pathways, and sites to achieve conservation targets for biological invasion developing a framework of minimum standards for the risk assessment of alien species a comparison of impact and risk assessment methods based on the imo guidelines and eu invasive alien species risk assessment frameworks review of risk assessment systems of ias in europe and introducing the german-austrian black list information system (gablis) novel organisms: comparing invasive species, gmos, and emerging pathogens parasites and biological invasions: parallels, interactions, and control key: cord- -xc ythgp authors: van den wijngaard, cees; van asten, liselotte; van pelt, wilfrid; nagelkerke, nico j.d.; verheij, robert; de neeling, albert j.; dekkers, arnold; van der sande, marianne a.b.; van vliet, hans; koopmans, marion p.g. title: validation of syndromic surveillance for respiratory pathogen activity date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: xc ythgp syndromic surveillance is increasingly used to signal unusual illness events. to validate data-source selection, we retrospectively investigated the extent to which respiratory syndromes (based on different medical registries) reflected respiratory pathogen activity. these syndromes showed higher levels in winter, which corresponded with higher laboratory counts of streptococcus pneumoniae, respiratory syncytial virus, and influenza virus. multiple linear regression models indicated that most syndrome variations (up to %) can be explained by counts of respiratory pathogens. absenteeism and pharmacy syndromes might reflect nonrespiratory conditions as well. we also observed systematic syndrome elevations in the fall, which were unexplained by pathogen counts but likely reflected rhinovirus activity. earliest syndrome elevations were observed in absenteeism data, followed by hospital data (+ week), pharmacy/general practitioner consultations (+ weeks), and deaths/laboratory submissions (test requests) (+ weeks). we conclude that these syndromes can be used for respiratory syndromic surveillance, since they reflect patterns in respiratory pathogen activity. syndromic surveillance is increasingly used to signal unusual illness events. to validate data-source selection, we retrospectively investigated the extent to which respiratory syndromes (based on different medical registries) refl ected respiratory pathogen activity. these syndromes showed higher levels in winter, which corresponded with higher laboratory counts of streptococcus pneumoniae, respiratory syncytial virus, and infl uenza virus. multiple linear regression models indicated that most syndrome variations (up to %) can be explained by counts of respiratory pathogens. absenteeism and pharmacy syndromes might refl ect nonrespiratory conditions as well. we also observed systematic syndrome elevations in the fall, which were unexplained by pathogen counts but likely refl ected rhinovirus activity. earliest syndrome elevations were observed in absenteeism data, followed by hospital data (+ week), pharmacy/general practitioner consultations (+ weeks), and deaths/laboratory submissions (test requests) (+ weeks). we conclude that these syndromes can be used for respiratory syndromic surveillance, since they refl ect patterns in respiratory pathogen activity. e arly warning surveillance for emerging infectious disease has become a priority in public health policy since the anthrax attacks in , the epidemic of severe acute respiratory syndrome in , and the renewed attention on possible infl uenza pandemics. as a result, new surveillance systems for earlier detection of emerging infectious diseases have been implemented. these systems, often labeled "syndromic surveillance," benefi t from the increasing timeliness, scope, and diversity of health-related registries ( ) ( ) ( ) ( ) ( ) ( ) . such alternative surveillance uses symptoms or clinical diagnoses such as "shortness of breath" or "pneumonia" as early indicators for infectious disease. this approach not only allows clinical syndromes to be monitored before laboratory diagnoses, but also allows disease to be detected for which no additional diagnostics were requested or available (including activity of emerging pathogens). our study assessed the suitability of different types of healthcare data for syndromic surveillance of respiratory disease. we assumed that syndrome data-to be suitable for early detection of an emerging respiratory disease-should refl ect patterns in common respiratory infectious diseases ( ) ( ) ( ) ( ) . therefore, we investigated the extent to which time-series of respiratory pathogens (counts per week in existing laboratory registries) were refl ected in respiratory syndrome time-series as recorded in medical registries in the netherlands. we also investigated syndrome variations that could not be explained by pathogen counts. as an indication for syndrome timeliness, we investigated the delays between the syndrome and pathogen time-series. we defi ned syndrome data as data in health-related registries that refl ect infectious disease activity without identifying causative pathogen(s) or focusing on pathogenspecifi c symptoms (such as routine surveillance data for infl uenza-like illness [ ] or surveillance of acute fl accid paralysis for polio [ ] ). registries for syndrome data were included if they met the following criteria: ) registration on a daily basis; ) availability of postal code, age, and sex; ) availability of retrospective data (> years); and ) (potential) real-time data availability. six registries were selected ( table ) that collected data on work absenteeism, general practice (gp) consultations, prescription medications dispensed by pharmacies, diagnostic test requests (laboratory submissions) ( ), hospital diagnoses, and deaths. in all registries, data were available for all or a substantial part of - . for the gp, hospital, and mortality registry, defi nition of a general respiratory syndrome was guided by the case defi nitions and codes found in the international classifi cation of diseases, th revision, clinical modifi cation (icd- -cm), as selected by the centers for disease control and prevention (atlanta, ga, usa) (www.bt.cdc.gov/surveillance/syndromedef). for the laboratory submissions and the pharmacy syn-drome, we selected all data that experts considered indicative of respiratory infectious disease (for detailed syndrome defi nitions, see online technical appendix, available from www.cdc.gov/eid/content/ / / -techapp.pdf). as a reference for the syndrome data, we included specifi c pathogen counts for - from the following sources: ) weekly sentinel surveillance system of the dutch working group on clinical virology (which covers %- % of the population of the netherlands [ ] respiratory disease-related counts of streptococcus pneumoniae (data in - were interpolated for laboratories during short periods of missing data; total coverage %); and ) national mandatory notifi cations of pertussis. the networks for respiratory pathogen counts are other networks than the earlier described laboratory submissions network for syndrome data. data were aggregated by week and analyzed by using sas version . (sas institute inc., cary, nc, usa). for the gp, pharmacy, and laboratory submissions registries, we expressed the respiratory counts as a percentage of total weekly counts to adjust for the infl uence of holidays and, for laboratory submissions, changes in the number of included laboratories over time. by looking at the graphs, we explored the relationship between the time-series of respiratory pathogens and syndromes and calculated pearson correlation coeffi cients. to investigate whether the respiratory syndromes refl ect patterns in respiratory pathogen counts, we constructed multiple linear regression models. these models estimated respiratory syndrome levels at a certain time with, as explanatory variables, the lagged (range of - to + weeks) pathogen counts as explanatory variables. we used linear regression of the untransformed syndrome to estimate the additive contributions of individual pathogens to the total estimated syndrome. we assumed a constant syndrome level attributable to factors other than the respiratory pathogens and constant scaling factors for each of the lagged pathogens. a forward stepwise regression approach was used, each step selecting the lagged pathogen that contributed most to akaike's information criterion of model fi t ( ) . each pathogen entered the model only once and only if it contributed signifi cantly (p< . ). negative associations (e.g., between enteroviruses, which peak in summer, vs. respiratory syndromes, which peak in winter) were excluded to avoid noncausal effects. to discriminate between primary and secondary infections by s. pneumoniae (as a complication of respiratory virus infection) ( ) ( ) ( ) ( ) , we used the residuals from regressing s. pneumoniae counts on other pathogens as the variable for s. pneumoniae (instead of its counts) for all the earlier described models for respiratory syndromes. we checked for autocorrelation in the residuals of the models with hierarchical time-series models (using splus . ) ( , ). we calculated r values to estimate to what extent respiratory pathogen counts explain variations in syndromes. to explore to what extent seasonal variation could be a confounder, we also calculated r values of the models after adding seasonal variables (sine and cosine terms) and r values for seasonal terms alone. we also investigated the pathogen-specifi c effects in the models, by calculating the standardized parameter estimates before and after adding seasonal terms. the models were used to estimate the expected syndrome level with % upper confi dence limits (ucls). we considered distinct syndrome elevations that exceeded the ucls, as unexplained by the models (for model details, see online technical appendix). we investigated the timeliness of the registry syndromes in ways: ) as a measure of differences in timeliness between registries, we evaluated the time delays of the syndromes relative to each other by calculating for each of the syndromes the time lag that maximized pearson correlation coeffi cient with the hospital registry (as a reference); ) by estimating the time delays between each of the syndromes and the lagged pathogens included in its regression model. respiratory syndrome time series were plotted for all registries (figure ). the christmas and new year holidays coincided with peaks and dips in the pharmacy and absenteeism syndromes (not shown). because these results were probably artifacts, we smoothed these yearly peaks and dips and censored them in the analyses performed on the absenteeism registry, in which they had a strong infl uence on outcomes. for all registries, the respiratory syndromes demonstrated higher levels of activity in winter, which overlapped or coincided roughly with the seasonal peaks of infl uenza a, infl uenza b, rsv, and (albeit less pronounced) s. pneumoniae laboratory counts ( figure ). infections with parainfl uenza virus, m. pneumoniae, adenovirus, and rhinovirus were detected slightly more frequently during winter (data not shown). bordetella pertussis and enterovirus showed seasonal peaks only in summer (data not shown). the seasonal peaks in laboratory counts of infl uenza a, infl uenza b, and rsv corresponded with peaks in the gp, pharmacy, and hospital syndromes. other syndromes did have less obvious correspondence. each year, around october, the respiratory syndrome showed a peak in the gp we calculated pearson correlation coeffi cients between the different unlagged time series of respiratory pathogens and syndromes (table ) . syndrome time series in all reg-istries correlated strongly with s. pneumoniae (unadjusted total counts). the hospital, gp, pharmacy, and laboratory submissions data strongly correlated with rsv and infl uenza a counts (table ) . mortality data correlated strongly with infl uenza a (r = . ) and infl uenza b (r = . ) infections. the highest correlations between pathogen time series were between s. pneumoniae and the other pathogens (up to . with infl uenza a, table ). table presents, for each registry, the time lag (in weeks) that maximized the model fi t of regressing syndrome on pathogens. for the gp, hospital, mortality, and pharmacy data, the respiratory pathogens explained the syndrome variation very well ( %- %). variations in the absenteeism syndrome could be explained for % by variations in the pathogen counts. although the laboratory submissions syndrome had the lowest explained variance, still % of the variations in this syndrome were explained by variations in pathogen counts. hierarchical time-series models did not show signifi cant autocorrelation in the residuals of the models with pathogen counts as explanatory variables ( , ) . when seasonal terms were added to the model, the variations in the mortality syndrome were just as well ex- emerging infectious diseases • www.cdc.gov/eid • vol. , no. , june plained as by the model with only pathogen counts (table ; r remains %), while by the model with only seasonal terms, the explained variance was much lower (only %, table ). for the hospitalizations, laboratory submissions, and gp data, only slightly more syndrome variation was explained by adding seasonal terms. with only seasonal terms, the explained variance for these syndromes was clearly lower than with only pathogens in the models ( %- % lower, table ). however, for the absenteeism and, to a lesser extent, the pharmacy data, the model with both pathogen and seasonal terms clearly explained more syndrome variations (table , absenteeism % vs. %; pharmacy % vs. %). furthermore, for the absenteeism data, the model with only seasonal terms had an even higher r than the model with only pathogens, whereas for the pharmacy data, the r with only seasonal terms was only slightly lower ( %, table ). table shows that for mortality, hospitalizations, laboratory submissions, and gp data, the pathogens with the highest effect clearly were rsv, infl uenza a, and infl uenza b, with no or only modest decline in standardized parameter estimates after adding seasonal terms. for the gp and hospital data, some pathogens became insignifi -cant after seasonal terms were added (gp: rhinovirus and adenovirus; hospital: parainfl uenza virus). for the pharmacy data, half of all pathogen variables became insignifi cant after seasonal terms were added, whereas for the absenteeism data, almost all pathogens became insignificant (table ) . several syndrome observations exceeded the % ucls of the models ( - /registry/year), which indicates that those syndrome observations deviated strongly from model predictions. the recurrent elevation in october of the absenteeism, gp, and pharmacy syndrome several times exceeded the ucls (october : pharmacy and gp; : absenteeism; : gp, absenteeism; not shown), which indicated that the model could not explain these elevations. in figure , for each registry, the difference in timeliness with the hospital registry is indicated by the lag that maximizes r . the absenteeism syndrome (green line) preceded the hospital syndrome by week, followed by the gp-based and prescription-based syndromes at + week and the syndrome based on mortality and laboratory sub- mission data at + weeks after the hospital syndrome (projected on x-axis, figure ). the differences in timeliness between the syndromes and the pathogen surveillance data were refl ected by the regression models relating the syndromes to the (positive or negative) lagged pathogens (table ). infl uenza a and infl uenza b had lags of - weeks, which suggests that the registry-syndromes were - weeks ahead of laboratory counts for these infections. fluctuations in the time series of respiratory hospitalizations and the laboratory rsv counts seemed to appear in the same week (lag = ). all other syndromes appeared to be - weeks later than the rsv counts, except absenteeism, which is weeks earlier. again, absenteeism seemed to be the earliest syndrome ( - weeks earlier than rsv, infl uenza a, and infl uenza b), followed by the hospital syndrome ( - weeks earlier), the gp-based and prescription-based syndromes ( weeks earlier until week later), the laboratory submission syndrome ( week earlier until weeks later), and the mortality syndrome ( - weeks later than rsv, infl uenza a, and infl uenza b). we explored the potential of dutch medical registries for respiratory syndromic surveillance. although several other studies also evaluated routine (medical) data for syndromic surveillance purposes ( ) ( ) ( ) ( ) ( ) ( ) , most evaluated only syndrome and correlated this only to infl uenza data. an exception is bourgeois et al. ( ) , who validated a respiratory syndrome in relation to diagnoses of several respiratory pathogens in a pediatric population, and cooper et al. ( ) , who estimated the contribution of specifi c respiratory pathogens to variations in respiratory syndromes. both studies concluded that rsv and infl uenza explain most of the variations in these syndromes, consistent with our fi ndings. our study shows that all syndrome data described in this study showed higher levels in winter, which corresponded to the seasonal patterns of rsv, s. pneumoniae, and infl uenza a and b viruses. linear regression showed that the syndromes can be explained by lagged laboratory counts for respiratory pathogens (up to %, highest effect of infl uenza a, infl uenza b, and rsv), which indicates their potential usefulness for syndromic surveillance. timeliness differed, with up to weeks potential gain in early warning by syndromic data, compared with routine laboratory surveillance data. a limitation of our study is the short duration of our time series, especially for absenteeism and pharmacy data. therefore, whether our observed associations between syndromes and pathogen counts can be generalized remains unclear. we relied on laboratory pathogen counts as a proxy for their prevalence and the illness they cause. changes in test volume over time would result in misclassifi cation bias (as noncausative pathogens will be detected as well). however, such changes are presumably dwarfed by changes during "truly" epidemic elevations of common respiratory pathogens. additionally, laboratory diagnostics are mostly performed on hospitalized patients, and thus results inadequately refl ect activity of pathogens that predominantly cause mild illness. by adding seasonal terms, we observed that for the absenteeism and, to a lesser extent, the pharmacy registry, the associations between the respiratory syndromes and the pathogen counts might be biased to some extent. for the gp, hospital, laboratory submission, and mortality data, emerging infectious diseases • www.cdc.gov/eid • vol. , no. , june absenteeism ---gp - - -- pharmacy - - hospitalization -- ---laboratory submissions - - - - mortality ---- --*s. pneumoniae, streptococcus pneumoniae; rsv, respiratory syncytial virus; rv, rhinovirus; piv, parainfluenza virus; gp, general practice; -, pathogen not included in model. †the lag time (in weeks) is indicated, that showed optimal fit between syndrome time-series and lagged pathogen counts included in the linear regression model; e.g., according to the model, the trend in hospitalizations precedes the influenza a laboratory counts by weeks. season is probably not an important confounder for the association between the syndromes and pathogens, because including seasonal terms in the models resulted in the same or only slightly higher explained syndrome variance (measured by r ). models with seasonal terms alone mostly had lower explained variance than the pathogen models. for the gp and hospital data, some pathogens became insignifi cant after seasonal terms were added (table ) but not those pathogens with the largest effect estimates (rsv, infl uenza a and b). therefore, we are confi dent in concluding that the gp, hospital, laboratory submission, and mortality syndromes do refl ect pathogen activity suffi ciently for use in syndromic surveillance. the higher r value of the absenteeism model with seasonal terms alone suggests seasonality of absenteeism caused by several nonrespiratory conditions ( , ) . to some extent, this also applies to the pharmacy syndrome, which includes medications that are not specifi c for respiratory infections (e.g., antimicrobial drugs). this could be validated in future studies by linking medications to illness. however, for both the absenteeism and pharmacy syndromes, the variation explained by seasonal terms is probably overestimated to some extent because data for only and years were used. consequently, these time series contained less information on variation between different years than for the other registries, which benefi ts fi tting of a model with several sine and cosine terms. to our knowledge, laboratory submission data (test requests) have not been evaluated before as a data source for syndromic surveillance. the modest explained variance for the laboratory submissions syndrome could possibly refl ect the limited use in our country of laboratory testing algorithms, which leads to substantial differences in diagnostic regimes for patients with similar clinical symptoms. in addition, occasional extra alertness by clinicians can make these data unreliable for surveillance. for instance, an unusual peak was observed in the laboratory submissions syndrome in , after the offi cial announcement of an outbreak of legionnaires' disease ( ) . an unexpected increase was also observed in the absenteeism, gp, and pharmacy syndromes, which occurred consistently each year around october ( ) ( ) ( ) ( ) . these peaks preceded the syndrome peaks concurring with peaks in infl uenza a, infl uenza b, and rsv counts and may be caused by rhinovirus activity-and asthma exacerbations caused by rhinovirus-which usually rises in the fall ( ) ( ) ( ) . rhinovirus might go undetected because gp physicians rarely ask for diagnostics if they suspect a nonbacterial cause for relatively mild respiratory disease. although (table ) . measured by the syndrome lag with the maximized r , the timeliness differed between the registries in the following order: absenteeism, hospital, pharmacy/general practice (gp), mortality/ laboratory submissions (as projected on the x-axis). specifi c asthma diagnoses were excluded from the respiratory syndrome defi nitions, exacerbations of asthma might affect other respiratory categories in the gp or pharmacy syndrome. this observation illustrates that additional diagnostics are needed for identifying the causes of unexplained respiratory disease elevations. several novel respiratory pathogens for which diagnostics are not yet widely available have been discovered in recent years, underlining that it is quite possible that "hidden" epidemics occur ( ) ( ) ( ) . the extra october peak and several other syndrome elevations above the % ucls in our study may well refl ect such hidden epidemics. the fact that these occur is supported by studies showing that many individual syndrome cases cannot be linked to known pathogens. for example, cooper et al. ( ) , who investigated syndromic signals by using patient self-sampling (at home), could only obtain diagnostic results for % of these cases. for early warning surveillance, timeliness is crucial. absenteeism data seem to have the best timeliness, but their lack of medical detail complicates interpretation. unexpectedly, the hospital data refl ect respiratory pathogen activity earlier than the gp data. although in the netherlands patients are encouraged to consult their gp before going to the hospital, elderly persons, for whom respiratory infections are more likely to cause severe illness, may often go to a hospital directly. therefore, hospital data may prove to be an earlier marker for respiratory disease than gp data, but this possibility needs further exploration. an important concern when using syndromic surveillance is that it may generate nonspecifi c alerts, which, if they happen regularly, would lead to lack of confi dence in a syndrome-based surveillance system. here, we see a clear advantage of using data from multiple registries in parallel so that signal detection can be made more specifi c by focusing on signals that occur concurrently in > data source. to illustrate this we defi ned every exceeding of the ucls of the regression models as a "signal," i.e., a syndrome elevation unexplained by known pathogen activity and therefore possibly refl ecting activity of underdiagnosed or emerging infectious disease. over - (the period that all registries were in the study), only "concurrent" signals occurred versus "single" signals over all registries. we did not evaluate whether the syndromes indeed detect outbreaks of infectious diseases earlier than clinical or laboratory pathogen surveillance. such an evaluation is often performed by testing the ability to detect historical natural outbreaks or simulated outbreaks ( , ) . however, historical natural outbreaks are rare and simulated outbreaks may be unrealistic. nevertheless, further research into the outbreak detection performance of these syndromes would be worthwhile. the results of this study suggest that it might be best to combine syndromic data and pathogen counts in a prospective surveillance system. such surveillance can identify distinct syndrome elevations that cannot be explained by respiratory pathogen activity as indicated by routine laboratory pathogen surveillance. overall, the gp, hospital, mortality and, to a lesser extent, laboratory submission syndromes refl ect week-toweek fl uctuations in the time-series of respiratory pathogens as detected in the laboratory. registries monitoring trends of these syndromes will therefore most likely refl ect illness caused by emerging or underdiagnosed respiratory pathogens as well and therefore are suited for syndromic surveillance. further research would be required to assess to what extent absenteeism and pharmacy data refl ect respiratory illness. investigating the actual outbreak detection performance of the syndromes in this study would also be worthwhile. data from the registries in this study are not yet realtime available, although given modern information technology, this availability is clearly feasible. our study can help prioritize which type of healthcare data to include in future syndromic real-time surveillance systems. syndromic surveillance and bioterrorism-related epidemics using automated medical records for rapid identifi cation of illness syndromes (syndromic surveillance): the example of lower respiratory infection surveillance of the bioterrorist threat: a primary care response evaluation of australia's national notifi able disease surveillance system experimental surveillance using data on sales of over-the-counter medications-japan syndromic surveillance in public health practice use of ambulance dispatch data as an early warning system for communitywide infl uenzalike illness public health assessment of potential biological terrorism agents use of automated ambulatory-care encounter records for detection of acute illness clusters, including potential bioterrorism events outbreak detection through automated surveillance: a review of the determinants of detection surveillance of respiratory pathogens and infl uenza-like illnesses in general practices-the netherlands, winter - the global eradication of polio by the year automated, laboratory-based system using the internet for disease outbreak detection, the netherlands reporting virus diagnostics in the netherlands: representativeness of the virological weekly reports a new look at statistical model identifi cation respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species-and cell type-dependent manner enhanced adherence of streptococcus pneumoniae to human epithelial cells infected with respiratory syncytial virus association of invasive pneumococcal disease with season, atmospheric conditions, air pollution, and the isolation of respiratory viruses respiratory viral infection predisposing for bacterial disease: a concise review automated detection of infectious disease outbreaks: hierarchical time series models bayesian statistical instrument for trend detection and time-series modelling syndromic surveillance for infl uenzalike illness in ambulatory care network modeling emergency department visit patterns for infectious disease complaints: results and application to disease surveillance validation of syndromic surveillance for respiratory infections developing a national primary care-based early warning system for health protection-a surveillance tool for the future? analysis of routinely collected data medication sales and syndromic surveillance the contribution of respiratory pathogens to the seasonality of nhs direct calls seasonality of infectious diseases seasonal variation in cause-specifi c mortality: are there high-risk groups? -year follow-up of civil servants from the fi rst whitehall study a large outbreak of legionnaires' disease at a fl ower show, the netherlands rhinovirus infections in an industrial population. i. the occurrence of illness respiratory infections and the autumn increase in asthma morbidity a case-control study of acute respiratory tract infection in general practice patients in the netherlands cloning of a human parvovirus by molecular screening of respiratory tract samples identifi cation of a novel coronavirus in patients with severe acute respiratory syndrome identifi cation of a new human coronavirus linking syndromic surveillance with virological self-sampling systematic review: surveillance systems for early detection of bioterrorism-related diseases we thank daan notermans for his expert opinion on providing syndrome defi nitions; mariken van der lubben for reading and commenting on the manuscript; statistics netherlands (cbs, ingeborg deerenberg and john kartopawiro), the foundation for pharmaceutical statistics (sfk, jan-dirk kroon and fabiënne griens), the dutch national medical register (lmr, willem hoogen stoevenbeld), and the national information network of gps (linh, robert verheij) for providing data; and the members of the dutch working group on clinical virology for collecting and providing weekly positive diagnostic results.mr van den wijngaard is an epidemiologist at the center for infectious disease control at the national institute of public health and the environment (rivm), bilthoven. his main research interests include the use of healthcare data for infectious disease surveillance and monitoring. all material published in emerging infectious diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required. key: cord- -z bxsjug authors: martin, r. r.; tzanetakis, i. e. title: pathogen-tested planting material date: - - journal: encyclopedia of agriculture and food systems doi: . /b - - - - . -x sha: doc_id: cord_uid: z bxsjug abstract certification programs have been developed to provide plant material that meets a predetermined level of plant health. the primary objectives of these programs are to limit pathogen incidence in plant material in order to minimize losses by growers and prevent movement of harmful pests and pathogens that may harm the environment. for many fruit and nut crops, orchards are expected to remain productive for years or decades; thus, starting with plants of high health status is essential. the components of certification programs in terms of plant health will be outlined, along with the benefits of harmonizing these programs where possible to facilitate plant movement without increasing trade in plant pathogens. buffer zone an area surrounding or adjacent to an area for production of plants in a certification scheme designed to minimize the probability of spread of the target pathogens, pollen, or seed into or out of the block, to meet phytosanitary or other control measures as defined in a certification standard. certification standard comprehensive process established and authorized by a regulatory entity for the production of plants free of regulated pathogens, with predefined trueness-to-type or genetic purity. these regulations also define plant production, plant identification and labeling, and quality assurance requirements. generation or g-level it indicates the relationship of plants in a certification scheme to the original pathogentested plant material at the top of the scheme. regulations developed by certifying agencies specify the conditions under which each generation (g) level must be maintained in order to meet the standard. heat therapy a method used to eliminate viruses in which plants are grown at - c for - weeks. after this meristem tips ( . - . mm) are removed and used to regenerate plants. this results in the production of plants that are often free of targeted pathogens. once grown in tissue culture, rooted and planted in soil, the plants are retested for the targeted pathogens to determine their health status. index procedure to determine whether a given plant is infected by a targeted pathogen. it involves the transfer of a bud, scion, sap, etc. from one plant to one or more indicator plants sensitive to the virus. international standard for phytosanitary measures it is an international standard adopted by the conference of the meristem-tip (tissue) culture a pathogen-elimination technique whereby tissue pieces (cells) are separated from an organism and cultured in a sterile growth media apart from that organism. one of the preferred pathogenelimination methods for plants is 'microshoot tip culture,' which is effective for eliminating most viral, bacterial, and fungal contaminants. in this technique a meristem tip of less than . - . mm is extracted from the shoot and placed in sterile tissue culture growth media; the meristem then grows to a complete plant. phytosanitary measure any legislation, regulation, or official procedure with the purpose to prevent the introduction or spread of pests or diseases. large-scale production and globalization of agriculture has resulted in a narrow germplasm base for all major food crops being grown over vast areas, which increases the risk of epidemics that could impact food security on a broad scale (strange and scott, ) . in addition, the majority of the production of these crops is in areas far from their centers of origin and subjected to many plant pathogens that they did not coevolve with. cassava is an excellent example of this, as its center of origin is in south america, but african cassava mosaic virus, which causes a devastating disease in africa, is not present in south america (fauquet and fargette, ; thresh et al., ) . most of the production of these crops is isolated from new strains of pathogens as they evolve at centers of origin and are at risk of severe disease outbreaks should new strains of a pathogen be distributed with planting material. this is also true for most of the fruit, vegetable, and ornamental species that are grown commercially. concomitant with the globalization of crop plants is an increased risk of globalization of plant pathogens that can lead to serious epidemics. the classic example of such movement of a pathogen is phytophthora infestans, the causal agent of potato late blight in the mid-nineteenth century, where its impact on human suffering and migration is well documented (schumann, ) . yet, years later, new strains of this pathogen have emerged and continue to cause serious plant disease problems (vleeshouwers et al., ) . there are examples of outbreaks of all types of pathogens (fungi, bacteria, nematodes, and viruses) that have resulted from movement of plant pathogens with the movement of plant propagation material. to protect agricultural production from introduced pathogens, two types of phytosanitary systems have been employed. plant quarantine is generally used at the national level to restrict introduction of plant pathogens that are not present in a country or have limited distribution and active programs to eradicate or delimit the pathogen. there are also domestic quarantines that restrict the movement of plant material within a country to address pests and pathogens that have limited distribution or may have been deregulated as a federal quarantine pest. plum pox virus (ppv), golden nematode, light brown apple moth, ralstonia solanacearum race biovar , and phytophthora ramorum are examples of quarantine pathogens and pests in the united states. certification programs are national, state, or provincial programs used to produce planting materials, vegetative or seed, that are free of or have less than some predetermined threshold of various pathogens (waterworth, ) . these programs primarily target domestic pathogens for plants at the g -g ( figure ) level of certification programs. these programs aim to ensure that the planted crop has a pathogen(s) level low enough to minimize the risk of severe crop loss, increasing the chance of harvesting an acceptable crop. the pathogen tolerances allowed in certified plants depends on the pathogen, crop, and agroecosystem where the crop is grown. the primary goal of quarantine and certification programs is to facilitate movement of plants without increasing trade of plant pathogens. the objective of all plant certification programs is to provide a supply of healthy plants, which is accomplished through adherence to best management practices (bmps) that are science based together with a defined level of testing to monitor the effectiveness of the program. the application of bmps to the production system combined with audit testing has proven to be a very useful means of producing plants of high health status. certification programs should be reevaluated periodically to take into account new pathogens or vectors that have been reported in the region, or other changes that affect the pathogen, vector, host, or environment that may impact the integrity of a certification scheme. bmps require that a hazard analysis and critical control points evaluation of the entire certification system be completed. this process is used to: ( ) identify weak points and greatest risk factors to the system in terms of introduction of pathogens or pests, and ( ) establish inspection, testing and mitigation procedures, and record-keeping requirements (parke and grünwald, ) . once the weak points are identified, management practices are designed to prevent intrusion of pests and pathogens. mesh size on a screenhouse might be adjusted to account for the primary virus vectors in the region to protect a g block. for example, if pathogens in a region are vectored by aphids the screen size might be larger than in another region with pathogens vectored by thrips. vector management in field blocks of certified plants will be adjusted based on the vectors and pathogens present in the region. knowledge of the phenology of vectors is also important, because times of peak vector movement present the greatest risk of pathogen intrusion into the system; thus, vector control measures may well change during the year to take into account the risk of vector migration into a nursery. knowing which of the targeted pathogens are most prevalent in a region and how they are vectored are important considerations when developing bmps for a nursery (martin and tzanetakis, in addition to certification programs that are designed for mass production of planting materials of high health status for plant, food, and fiber production, there are extensive quarantine programs that have been developed in many countries to reduce the risk of introduction of exotic pests. though not the focus of this article, quarantine goes hand-in-hand with certification to provide an integrated system to protect agriculture and environment from plant pests and pathogens, thus its relation to certification will be touched on briefly. the rationale for a brief overview of quarantine is that many certification programs focus on domestic or endemic pathogens, with the assumption that quarantine pathogens are detected and eliminated at the stage where plants enter the country and need not be included in the domestic certification program. quarantine programs are operated primarily at the national level and deal with pathogens that are not known to occur in the country or are present at a very restricted level and have active programs targeted to eradicate or prevent further spread of the pest or pathogen (foster and hadidi, ; reed and foster, ; anonymous, ) . as examples, plant material of berry crops, citrus, grape, potato, sweet potato, pome, and stone fruit, etc. coming into the united states under quarantine are tested for exotic pathogens before they are released for propagation, but they could be released if found to be infected only with viruses already endemic in the country. in some cases plants may be held in quarantine for - years while graft indexing on indicators is carried out. to be effective, quarantine programs are dependent on the public understanding the risks associated with introducing exotic pathogens into natural and agricultural ecosystems. public in this sense includes anyone who may wish to transport plant material from a foreign country to their homeland, including hobbyist interested in a new ornamental or food crop to add to their garden or plant collection, plant breeders interested in new potential germplasm, plant pathologists interested in adding to their pathogen collection for scientific study, germplasm curators attempting to broaden the diversity of their collections, or growers interested in getting a head start on a new cultivar developed in a foreign country. also, an effective quarantine program needs to have an effective and efficient mechanism to introduce plant material into the country. this combination of an educated public that understands the risks of introducing foreign pathogens and pests, together with an efficient system to bring plant material through approved quarantine and testing facilities will reduce the temptation by individuals to do 'suitcase' imports that could threaten local environments and agriculture. the introduction of ppv into pennsylvania highlights the benefits of quarantine systems to exclude exotic pathogens. plum pox is a devastating disease first identified in the early s in bulgaria that has since spread through much of europe and the mediterranean countries, where it is the most serious disease of stone fruits. it was detected for the first time in the western hemisphere in chile in (herrera et al., ) and is now considered to be widespread there and has since been detected in argentina. ppv was detected in a localized area of pennsylvania in the united states in (levy et al., ) and an eradication program was implemented immediately. after years, the eradication effort was deemed successful, but the cost was in excess of $ million us dollars to eliminate the pathogen from a relatively small geographic area (welliver, ) . thus, quarantine programs can be an effective and economical way to reduce international movement of plant pathogens and pests to protect local agriculture and native flora. the international plant protection convention (ippc) is an international agreement on plant health to which countries are signatories. the secretariat of the ippc is provided by the food and agricultural organization of the united nations. the ippc has the mission to protect cultivated and wild plants by preventing the introduction and spread of pests and pathogens. they develop standards (international standards for phytosanitary measures (ispms)) that are recognized by participating countries and provide for science-based standards for the safe movement of plants and plant products. as an example, ismp no. (anonymous, b) provides recognized standard treatments to eliminate plant pathogens and pests, including fumigation, cold treatment, heat treatment, and irradiation; and ismp no. (anonymous, ) provides agreed on sampling levels that participating countries use when shipping plants or plant products internationally to protect against movement of quarantine pests and pathogens. the ippc develops standards for range of issues related to plant protection. once the standards are approved by the ippc member countries, they become an ismp. certification programs have been developed for many vegetatively propagated food crops over the past years hadidi et al., ) . the first certification programs were developed in the netherlands and germany in the early s for potato production when they realized that leaf crinkling, rolling, and mosaics were transmitted through the tubers to the next generation and that by selecting the most vigorous and healthy-looking plants for propagation, tuber production was improved greatly. they developed a program for plant inspection and production, which was adopted in the united states and other countries long before it was known that many of the diseases were caused by viruses. in the united states and canada the first seed potato production programs were established in (slack and singh, ) . visual inspections still play a major part in seed potato certification programs with inspections carried out early and midlate in the growing season to observe foliar symptoms, at harvest for observing tubers symptoms, and a postharvest inspection of plants grown from tubers to look for late-season infections (frost et al., ) . visual inspections are part of all plant certification programs. trained inspectors also watch for other problems, such as herbicide damage, cultivar mixtures or trueness-to-type issues, physiological disorders, etc. during visual inspections, thousands of plants can be observed in a relatively short time. in most programs there is also laboratory testing that is carried out on each tuber or 'seed' lot to identify viruses that may be present and to monitor for symptomless pathogens. as laboratory-based diagnostics are improved in terms of sensitivity, specificity, and costs, more programs are incorporating their use to improve the quality of the program. certification programs have been developed for a wide range of crops . testing methods including mechanical transmission to herbaceous hosts, immunological and molecular techniques (enzyme-linked immunocapture assay (elisa), polymerase chain reaction (pcr), etc.), isolation of fungi or bacteria, etc. that are used for detection of various pathogens are similar in all programs. with woody crops there is often the need to do graft transmission onto indicator plants to test for uncharacterized graft-transmissible agents (converse, ; hadidi et al., ) . there are many definitions for certification programs. north american plant protection organization (nappo) defines a certification program as: "a domestic program consisting of maintenance, multiplication, distribution, and production of plant materials intended for release either domestically or for export, under an officially sponsored certificate attesting to the status of the material" (lanterman et al., ) . many certification programs are based on a published standard that defines site selection and preparation, isolation distances from plants of the same species and other vegetation, number of inspections, record keeping on plant traceability so that tracebacks or traceforwards can be done if a problem should arise, a pest and disease management plan, records of all pest management activities, the conditions and protocols to be followed during plant or seed production, and types and amount of testing that needs to be done at each level in the propagation cycle. the selection of plant material that is trueto-type is an essential first step. the selected plant(s) is then subjected to pathogen testing using a range of laboratory and biological indexing. if infected with any of the targeted pathogens listed in the standard, the plants are subjected to 'cleanup.' after 'cleanup' the plants are retested to ensure the pathogen(s) have been eliminated. once determined to be free of targeted pathogens and true-to-type, this plant can be designated as a g (figure ) plant that enters into the certification program. in many cases these plants are maintained in protected culture (screenhouse) and become the source plants that are propagated in certification programs. there is an effort in the united states to develop auditbased certification programs that rely on bmps outlined in the certification standard (thompson, ) . an audit-based program relies on compliance monitoring and requires a reasonable level of trust between the regulatory agency (inspectors) and nursery managers for the certification systems to function. the audit or inspection assesses the degree of compliance of the nursery to the certification standard, so that plants can be certified to meet that standard. an auditor or inspector designated by the certifying agency is responsible for the audit process. all records of the nursery can be reviewed by the auditor (thompson, ) . with such a system in place, record keeping by the nursery becomes even more critical as the inspector's decision will be based on the records reviewed, visual inspection of the nursery, and some limited testing. the goal is to have a systems approach that uses science bmps for nursery production of plant materials (parke and grünwald, ) . the use of pathogen-tested planting material is the first and arguably the most important step for the control of many systemic plant pathogens. most effort in this area has focused on viruses, viroids, systemic bacteria, and phytoplasmas because there are no postinfection treatments that can be used in plant or food production systems to rid plants of these pathogens. many important systemic pathogens are not transmitted through the seed or transmitted to less than % of the seedlings, or transmitted as seed coat contaminants that can be controlled with various seed treatment methods (ling, ; liu et al., ) . in this case, seedlings free of the targeted pathogen(s) can be identified and then grown in isolation to produce seed free of these pathogens or with an incidence below some predetermined threshold. with seed coat contaminants the seed can be treated to inactivate pathogens on the seed surface (ling, ) . in the case of vegetatively propagated crops it is often necessary to eliminate a specific pathogen, usually through thermal-, cryo-, or chemotherapy combined with meristem-tip culture to produce plants free of the targeted pathogens (mink et al., ; laimer and barba, ) . the plants regenerated from such meristems are then thoroughly tested, and a single plant free of pathogens of interest is the starting point for massive vegetative propagation. in the case of seed or vegetatively propagated plants, the plants are propagated under a defined set of conditions described in a certification standard. certification standards often have tolerance levels of some small percentage of infection, defined requirements for the site where the crop (seed or vegetative) is grown, required field inspection(s) during the production cycle that include cleanliness in terms of weeds, other crops that may serve as contaminants in seed lots or as hosts for targeted pathogens and freedom from pathogen vectors. tolerances for pathogens depend on the rate of pathogen spread for annual crops, but tolerances are much lower for perennial crops. crops that are only grown for a single year or a few years in production fields, such as most seed crops and potatoes, strawberries, sweet potatoes etc., may have higher tolerances than most of the fruit and nut crops, such as citrus, tree fruits, grapevines, or berries, which are expected to be productive for many years or decades in the same field. to set tolerances for various pathogens requires sampling and testing, and a major concern is how many samples to test. the number of samples that need to be tested per lot to achieve a confidence level (i.e., % or %) that a pathogen is not present is outlined in ispm no. (anonymous, ) . in some countries, certification schemes are managed at the national level and in others at the state or provincial level. plant pathogens are recognized as major constraints to agriculture production worldwide. most fruit and nut crops; major food crops such as potato, cassava, sweet potatoes; and many ornamentals are vegetatively propagated, using cuttings, tubers, or rhizomes. more recently, tissue culture propagation has become a major component of mass propagation for many of these crops. elaborate protocols are used to eliminate targeted pathogens from one or a few infected plants to provide a source of plant material free of these pathogens (mink et al., ; laimer and barba, ) . plants entering a certification program are often advanced selections, cultivars, or varieties developed in breeding programs. in some cases breeders work closely with the cleanup programs and get plants into the testing and cleanup phase before cultivar release, in an effort to have certified plants available at the time of release. another source of material entering certification programs are 'heritage' cultivarscultivars that have not been used for many years but are maintained at germplasm repositories. as requests for these cultivars have increased, there is a need to confirm that they meet current certification standards before commercial nurseries are willing to add them to their production systems. in the united states, there is an effort to have new cultivars coming into the country, as well as those developed in-country go through one of the 'clean plant centers' that have been developed over the past years. increased funding for these programs at the federal level since has provided for capacity building and the ability to process materials in a more timely manner to meet the needs for food production. these plants are tested for target pathogens before they enter into a g block, then a certification program and made available for production. in many cases, the g block is maintained by federal, provincial, or state government agencies, or some type of government/private entity, though recently private companies are maintaining their own proprietary g germplasm. these plants are tested on a predetermined schedule to monitor for reinfection, in addition, these blocks are retested if a new virus is discovered in the crop that may have been missed with the testing procedures used previously. producing g plants . candidate plant (advanced selections, heritage, new, or imported cultivars) arrive at clean plant center, (time ¼ , t¼ ). . testing program to determine the health status of the plants with respect to targeted pathogens listed in the certification standard, grafting for many fruit and nut crops. this process can take up to three years for crops, such as grapevines or fruit trees, but less time for other crops (t ¼ . - years). if no grafting is required this can be completed in several weeks; this is the case for some crops, such as potatoes. . if negative for all targeted pathogens, the plant enters the g block. if infected, the plants are put through therapy treatment ( - months), meristem-tip cultured and whole plants regenerated ( - months). . these plants are then retested and, if 'clean,' enter the g block; but if still positive they go through the therapy again (t¼ . - years). this is repeated until 'clean' plants are obtained. each cycle of testing can take up to - years for crops like grapevines or fruit trees, - years for strawberry or rubus. in most cases protocols have been developed such that there is a reasonable likelihood of having 'clean' plants after the initial cycle of therapy. however, if three cycles of cleanup were needed to get plants free of targeted pathogens this can easily require - years, depending on the crop. there is a misconception among many growers and researchers that 'tissue culture' propagated is synonymous with virus free. this is not the case. to eliminate viruses it is necessary to regenerate plants from meristematic tip, generally less than . mm and often in combination with some type of therapy (thermal, cryo, or chemo) before removing meristems (mink et al., ) . some viruses move into the meristematic tissue quite effectively, and a combination of thermal therapy or chemotherapy are required to get meristematic tissue free of the virus (chen and sherwood, ) . in the past these were referred to as 'heat stable' viruses and were 'difficult' to eliminate using thermal therapy and meristem-tip culture, whereas 'heat labile' viruses were much easier to eliminate. one now know that many of the 'heat labile' viruses are phloemassociated viruses, such as luteoviruses and closteroviruses, and the reason they are 'heat labile' is that the phloem tissue is not differentiated in the meristematic dome. thus, the virus does not move readily into the meristem. this is the case for the grapevine leafroll-associated viruses, which are in the closteroviridae family. in raspberry, raspberry leaf mottle and raspberry leaf spot were considered 'heat labile' as they were relatively easy to eliminate. these two diseases are caused by strains of raspberry leaf mottle virus, which is a closterovirus and phloem limited. raspberry bushy dwarf virus (rbdv) was considered a 'heat stable' virus because it is difficult to eliminate using thermal therapy and meristem-tip culture. rbdv infects most cell types and is not restricted to phloem tissue, thus, it likely moves into the meristematic dome much sooner than phloem-restricted viruses. the ease of obtaining meristems free of a virus in combination with heat therapy is not related to the heat stability of the virus, but rather how rapidly or effectively it can invade the meristematic tissues. the 'cleanedup' g plants are maintained under clearly defined conditions to minimize the risk of reinfection and should be free of all targeted pathogens outlined in the certification standard. in many cases the g plants are maintained in protected culture, such as in a screenhouse. the g plants are then sold to private nurseries for mass propagation under a set of conditions outlined in a certification program that is managed by a regulatory agency. the goal is to have a systemsbased approach that addresses risks of reinfection and pathogen spread during plant propagation. there are multiple cycles of plant increase for vegetatively propagated crops and unfortunately a wide range of terms have been used to identify each cycle (figure ). in , the nappo (rsmp no. ; anonymous, ) suggested the use of a simple terminology for the cycles of vegetative propagation. g plants are the plants that have tested negative for all targeted pathogens outlined in the certification scheme. g plants propagated by tissue culture or by traditional vegetative propagation methods become g plants, and multiple cycles of tissue culture can be carried out and still retain g status. the use of tissue-culture propagation and the number of propagation cycles allowed in tissue culture should be defined in the certification standard and will vary depending on the crop and certifying agency. g plants are derived from g or g plants. g plants are grown as potted plants that are propagated from g , g , or g plants and grown at another location. most existing programs use the various terminologies shown in figure , but follow this basic scheme of scale up in stages - to produce plants that are sold to growers. with the application of tissue culture in some propagation schemes, nurseries are able to sell g or g plants to growers, which should translate into higher health-status plants being used by the industry. however, for many crops conventional vegetative propagation is still the primary means of plant multiplication that is used. there are several reasons for this: ( ) costs for conventional propagation of crops, such as strawberry, where a - fold increase can be obtained in field production each year, is relatively inexpensive to produce millions of plants; ( ) the agency that regulates the certification program may prohibit or limit the number of cycles of tissue culture as a means of propagation due to concerns about somatic mutations; ( ) grafted plants may use a rootstock that is generated from seed, which is inexpensive. in such cases, the rootstock may be seed-propagated and the scions by tissue culture or by conventional vegetative propagation methods. during propagation it is important that care is taken to prevent contamination of tools with viruses that are readily mechanically transmitted. tools are sterilized during tissue-culture propagation to maintain sterile conditions during transfers of tissue-culture plants. it is also necessary in conventional propagation to prevent spread of viruses during the cutting of tubers, rhizomes, taking cuttings, etc. (lewandowski et al., ) . contamination using cutting tools is much more likely in herbaceous crops, such as potatoes, than in woody crops like grapevines, tree fruits, nut crops, etc. there are efforts among regional plant protection organizations (rppos) to harmonize quarantine standards between the member countries. there are nine rppos (asian and pacific ppo; caribbean plant protection commission; comite de sanidad vegetal del cono sur; comunidad andina; european and mediterranean ppo (eppo); interafrican phytosanitary council; near east ppo; north american ppo; organismo internacional regional de sanidad agropecuaria and pacific ppo (roy, ) ). there are also efforts at harmonizing certification standards across some of the rppos, such as for fruit trees and grapevines in nappo countries, and the eppo countries have adopted certification schemes for crops (roy, ; eppo website) . these rppo-developed standards are often a minimum standard that is required, but member countries can require a more stringent standard internally. although this is happening at the international level, there are many cases where the harmonization of certification programs within countries has not happened. in countries where these programs are regulated at the province or state level, there are often significant differences between the certification standards. for example, in the united states some states require that the g plants for some crops be maintained within protected culture (screenhouse) to minimize vector transmission of pathogens, whereas other states do not require that same level of protection. also, the type and level of testing required at each level in the certification scheme can vary between states. there is an effort underway to harmonize certification standards for some of the fruit crops across the united states. as these programs are developed, they are looking at certification schemes in other countries and rppos with the goal of harmonizing standards on a broader scale if possible. for seed propagated crops, seed is often increased in a country other than where the seed will be planted for food or fiber production. to speed up increase of seed for commercial production many companies grow seed in the northern and southern hemisphere to get two cycles of increase in a single year. as a result, seed is moved between countries on a frequent basis and are potentially exposed to quarantine or certification pests of concern in the country where the final crop is grown. also, because seed has a long shelf life, it may be moved to several countries before it is planted, or a seed lot may be subdivided and shipped to multiple countries. many countries have phytosanitary requirements for the movement of seed though requirements for field inspections, sampling, and testing can vary and cause problems if trying to move the seed internationally. the ippc's ispm no. provides information on agreed on treatments for a range of regulated plants pathogens and pests (anonymous, a) . the preparation of a phytosanitary certificate is done by the exporting country but must meet the requirements of the importing country.thus, if a seed lot is being shipped to multiple countries the documentation can become very complicated. ippc has also developed ispm no. , which covers the reexport of the seed to a third country, where the phytosanitary requirements would have to be met by the original exporting country (anonymous, ) . for these reasons, harmonized standards could greatly facilitate seed trade to meet the needs of the increasing globalization of agriculture. there are a number of agencies for seed testing and accreditation of certification schemes. the testing and accreditation by these agencies are recognized by various countries or groups of countries and provide a mechanism to harmonize seed certification standards for international movement of seed among a group of countries. the association of official seed analysts, association of official seed certifying agencies, international seed trade association (ista), national seed health system, oecd seed schemes (us), society of commercial seed technologists are all involved in seed testing and are recognized by multiple countries. ista testing and accreditation is recognized by more than countries. nappo regional standards for phytosanitary measures no. provides information on the movement of seed between countries in north america (anonymous, ) . for seed production, the emphasis is on eliminating pathogens from the elite germplasm that can be transmitted through seed or contaminate the seed surface, including viruses, bacteria, and fungi. in many cases, elite germplasm of seed crops is produced and maintained by private companies. with seed certification, genetic purity is often as great a concern as pathogen contamination. there are multiple levels in certification programs for seed crops, similar to those used for vegetatively propagated crops. the seed crops would clearly fit under the g terminology shown in figure , and in this case g would stand for generation in the true sense of the word. the four common levels in seed certification (aosca) include: breeder seed (seed controlled by the plant breeder, or the institution or company where the breeder works), foundation seed (propagated from breeder seed under conditions that retain genetic purity, identity, and health status), registered seed (propagated from breeder or foundation seed under conditions that maintain genetic purity, identity, and health status), and certified seed (progeny of breeder, foundation or registered seed and grown under conditions that maintain genetic purity, identity, and health status). in addition to genetic purity, identity, and health status, seed certification also requires information on percentage germination, date of germination test, and percentage contamination with other seed. for pathogens that are transmitted internally in the seed, it is often possible to grow out seed and identify plants free of the pathogen because this type of transmission is rarely % efficient (liu et al., ; mink, ) . the exceptions to this are the cryptic viruses, which are seed transmitted at %. these viruses are not known to cause disease and are not considered in quarantine or certification programs. heat treatments for eradication of embryo infection by virus have not been successful without a loss in seed viability (maury et al., ) . for seed contaminants, various types of seed treatment have been used to eliminate or reduce pathogen level below set tolerances. dry heat at c for h, followed by c for h, and finally c for h was very effective at controlling very stable viruses, such as tobamoviruses (tobacco mosaic virus, tomato mosaic virus, and cucumber green mottle mosaic virus, lee, ) and potexviruses (pepino mosaic virus, ling, ) as well as for a wide range of fungal and bacterial pathogens with little or no effect on seed germination (lee, ) . soaking tomato seed for min in a % sodium hypochlorite solution (dilution of commercial bleach, depends on the percentage active ingredient in the bleach product) containing . % triton-x- as a wetting agent completely inactivates potexviruses (pepino mosaic virus; ling, ) on the seed surface with little or no impact on seed germination. hydrochloric acid or trisodium phosphate were not as effective as bleach in eliminating virus from seed coats (ling, ) . with woody plants that involve graft transmission assays for detection of viruses, grafted plants may need to be observed for two or more years for symptoms before the plant is determined to be free of the pathogen. these potentially long intervals for detection using biological indexing has prompted most quarantine facilities to adopt laboratory-based testing where possible (rowhani et al., ; martin et al., ; , including: electron microscopy, elisa, immunospecific electron microscopy, nucleic-acid-based techniques such as pcr or reverse transcription -pcr (rt-pcr) combined with gel electrophoresis and sequencing of the any amplicons obtained, double-stranded ribonucleic acid (dsrna) detection; and mechanical transmission to herbaceous hosts (reed and foster, ; miller et al., ). however, with widely used tests currently available, there is still a need for biological indexing onto woody indicators for some exotic pathogens. for many of the crops there are various laboratory tests available for the major pathogens of concern. thus, in some cases, once all laboratory tests have been completed and found negative, plants are released to the importer on a provisional basis while the biological indexing is completed. this is done with an agreement that in case the tests are positive, the plant will be destroyed. this process allows the importer to begin multiplication of a new cultivar, which in some cases can reduce the time to get the materials to production fields by - years. the plants must be maintained and propagated under quarantine-approved conditions until all testing is completed. the currently used assays for virus detection (elisa, pcr, and q-pcr) are great for detecting a virus in a large number of samples, such as in surveys or ecological or epidemiology studies. however, for quarantine and certification programs a method that was capable of detecting all viruses in a single test would be ideal, rather than doing individual tests for each virus known to infect the host. in some crops, this means - tests per plant (berries or grapevines). recent work with macroarrays has shown great promise for detecting the most common viruses in grapevine, with viruses detected in a single test (thompson et al., ) . potentially, within the next five years as 'deep' or 'nexgen' sequencing becomes more widely adapted (studholme et al., ; kreuze et al., ) , and universal plant microarrays are perfected (hammond, ) they could replace indexing. this would require extensive validation of these technologies to ensure they are as good or better than current methods. the advantage of these technologies is that they have the potential to provide information on any virus in a plant without any a priori knowledge of the virus, in contrast to other laboratory techniques that detect known viruses (kreuze et al., ; al rwahnih et al., ; kashif et al., ; thekke-veetil et al., ; seguin et al., ; hammond, ; esteban et al., ) . wang et al. ( ) demonstrated the feasibility and utility of the microarray technology to identify and characterize new viruses. they developed microarrays containing oligonucleotides that represented conserved sequences of all fully sequenced human respiratory viruses, which at the time represented a few hundred viruses. using this array they identified a novel coronavirus and showed that it caused severe acute respiratory syndrome, a newly emerging disease at the time (wang et al., ) . nexgen sequencing has a huge impact on many aspects of biology and is used in virus discovery and is being investigated for virus diagnostics. sequencing of total nucleic acids in plants has led to the identification of multiple viruses and viroids in single plants (adams et al., ; al rwahnih et al., kreuze et al., ; sequin et al., ) and offers the potential to certify plants as virus-free rather than virus-tested. obtaining the 'virome' of a plant provides much information on the range of viruses and their diversity within a single plant. however, work with grapevines has shown that many of the viruses were related to mycoviruses rather than plant viruses (al rwahnih et al., ; coetzee et al., ) . this leads to questions on the significance of these viruses in plant health. the new technology will allow for rapid discovery of many new viruses; unfortunately, characterizing the biological significance of these viruses will take much longer. mycoviruses in grapevines may actually be modifying endophytes and indirectly impacting the health status of grapevines. thus, eventually how viruses and other microorganisms impact the whole plant (understanding the microbiome) may be important in certification and quarantine, but we are not there yet. although it is good to know what is in the plant, this technology is resulting in the identification of many new viruses for which there is no biological information. for the next decade it is likely that the biological significance will lag behind the discovery of microorganisms in plants, and it is important that for certification and quarantine programs, any changes are made in response to the biology of these organisms rather than their presence. it seems reasonable that new plant viruses related to known plant pathogenic viruses should be considered as the highest priority for evaluating their biological significance and determining if they should be part of quarantine and certification regulations. certification standards vary widely among crops and regulatory authorities. the viruses included in a certification program can vary from country to country or state to state. an excellent example is grapevines where certification in italy includes more viruses than programs in germany or france; or grapevine certification in washington state is different from california. some of these differences are due to environmental considerations, where infection by a pathogen may cause severe disease in one setting, such as crown gall in grapevine in new york state, but be latent or symptomless under different environmental conditions, such as crown gall in grapevine in california. attempts to harmonize certification schemes across boundaries to facilitate trade of plants without increasing trade in plant pathogens will require certification programs to account for disease expression by pathogens in areas where the certified plants may be sold, rather than only where the nursery stock is produced. in the united states, where certification programs are regulated by individual states, there are efforts underway in multiple crops (blueberry, grapevines, hops, rubus, strawberry, and fruit trees) to develop a single certification standard that all states with programs for that crop would adopt. if successful, this in essence would provide a national program for certification of these crops. as this process is developing, there is an ongoing communication with trading partners to harmonize these standards as much as possible with their certification programs to facilitate international trade. next generation sequencing and metagenomic analysis: a universal diagnostic tool in plant virology deep sequencing analysis of rnas from a grapevine showing syrah decline symptoms reveals a multiple virus infection that includes a novel virus deep sequencing evidence from single grapevine plants reveals a virome dominated by mycoviruses rspm no. , guidelines for international movement of pome and fruit trees in a nappo member country phytosanitary principles for the protection of plants and the application of phytosanitary measures in international trade ispm no. , phytosanitary treatments for regulated pests glossary of phytosanitary terms ispm no. , international standards for phytosanitary measures ispm no. . phytosanitary certificates rspm no. , phytosanitary guidelines for the movement of seed evaluation of tip culture, thermotherapy and chemotherapy for elimination of peanut mottle virus from arachis hypogeae deep sequencing analysis of viruses infecting grapevines: virome of a vineyard virus diseases of small fruits. u.s. department of agriculture agriculture handbook no. a diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses african cassava mosaic virus − etiology, epidemiology, and control plant virus disease control integrated control of potato pathogens through seed potato certification and provision of clean seed potatoes virus and virus-like diseases of pome and stone fruits plant virus disease control universal plant virus microarrays, broad spectrum pcr assays, and other tools for virus detection and identification survey of sharka disease (plum pox virus) on stone fruit trees in chile detection of viruses in sweetpotatoes from honduras and guatemala augmented by deep-sequencing of small-rnas complete viral genome sequence and discovery of novel viruses by deep sequencing of small rnas: a generic method for diagnosis, discovery and sequencing of viruses elimination of systemic pathogens by thermotherapy, tissue culture, or in vitro micrografting disease control through crop certification − woody plants advances in seed treatments for horticultural crops first report of plum pox virus (sharka disease) in prunus persica in the united states surprising results from a search for effective disinfectants for tobacco mosaic virus − contaminated tools effectiveness of chemo-and thermotherapeutic treatments on pepino mosaic virus in tomato seed pollen and seed transmission of cucumber green mottle mosaic virus in cucumber viruses and virus diseases of rubus new and emerging viruses of blueberry and cranberry high risk strawberry viruses by region in the united states and canada: implications for certification, nurseries and fruit production seed certification for viruses plant disease diagnostic capabilities and networks pollen-and seed-transmitted virses and viroids heat treatment of perennial plants to eliminate phytoplasmas, viruses, and viroids while maintaining plant survival a systems approach for the management of pests and pathogens of nursery crops exclusion of pome and stone fruit viruses, viroids, and phytoplasmas by certification and quarantine pathogen testing and certification of vitis and prunus species control measures of pome and stone fruit viruses, viroids, and phytoplasmas: role of international organizations plant diseases: their biology and social impact de novo reconstruction of consensus master genomes of plant rna and dna viruses from sirnas control of viruses affecting potatoes through seed potato certification programs plant disease: a threat to global food security application of high-throughput dna sequencing in phytopathology molecular characterization and population structure of blackberry vein banding associated virus, a new ampelovirus associated with blackberry yellow vein disease certified − feasibility of audit-based certification to prevent invasive plant pests in the horticultural industry profiling viral infections in grapevine using a randomly primed reverse transcription-polymerase chain reaction/macroarray multiplex platform the control of african cassava mosaic virus disease: phytosanitation and/or resistance? understanding and exploiting late blight resistance in the age of effectors microarray-based detection and genotyping of viral pathogens viral discovery and sequence recovery using dna microarrays certification for plant viruses − an overview plum pox virus case study: the eradication road is paved with gold key: cord- -i j tctr authors: koon, kassi; sanders, catherine m.; green, jessica; malone, leslie; white, holly; zayas, delineliz; miller, rebecca; lu, stanley; han, jian title: co-detection of pandemic (h n ) virus and other respiratory pathogens date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: i j tctr from may through october , a total of , clinical samples from us states were screened for multiple respiratory pathogen gene targets. of , ( . %) samples positive for pandemic (h n ) virus, % contained > other pathogen, most commonly staphylococcus aureus ( . %), streptococcus pneumoniae ( . %), and haemophilus influenzae ( . %). of the respiratory specimens shipped by overnight mail from the states, > % were nasopharyngeal swabs in transfer buffer. high-throughput nucleic acid extraction was performed automatically by using kingfisher instrumentation (thermo scientifi c, hudson, nh, usa) and magnetx chemistry (scigenix, marietta, ga, usa) according to manufacturers' specifi cations. multiplex pcr amplifi cation and luminex (austin, tx, usa) liquid suspension detection methods were based on internally validated protocols. reactions were amplifi ed by using abi thermocyclers (applied biosystems, singapore), and the resulting pcr products were detected by using the liquichip workstation (luminex) according to previously described protocols ( , the main fi nding of this large-scale clinical study was the co-detection of multiple pathogens with the pandemic infl uenza virus strain. in % of samples, no pathogens were detected, which may represent infection with common pathogens not detected by the assay. for example, bocavirus and all coronavirus groups not detected by the assay account for ≈ % and %- %, respectively ( , ) , of respiratory infections. an expanded test menu may improve the detection rate for such pathogens. this study raises questions. first, does co-detection equal co-infection? second, and more practical, does codetection change the clinical outcome? we chose the word co-detection rather than co-infection or co-colonization because co-infection means all identifi ed microorganisms contributed to the pathogenic effect, and co-colonization may not indicate the causative agent. co-detection indicates that > other pathogen was detected in a sample. the differences among the defi nitions have etiologic meaning, but the data presented here cannot be used directly to address etiology. most samples in this study were nasal swabs rather than upper or lower respiratory tract samples. nasal swab samples have greater potential for contamination with normal fl ora, particularly s. aureus. no data on asymptomatic carriers were available because these persons rarely seek healthcare. however, these fi ndings raise questions about the effectiveness of the single-agent etiology approach toward infectious diseases. pandemic (h n ) virus and multiple other pathogens are often detected during autopsy ( , ), indicating that co-infection may play a major role in the disease process. in addition, detection of multiple pathogens is associated with increased critical illness in children ( ) . the centers for disease control and prevention identifi ed "the need for early recognition of bacterial pneumonia in persons with infl uenza" ( ) . however, no suggestions were provided for meeting this need. furthermore, the centers "underscore the importance of managing patients with infl uenza who also might have bacterial pneumonia with both empiric antibacterial therapy and antiviral medications" ( ) without identifying measures that would make this task tangible. current practices of clinical diagnosis based on signs and symptoms inherently lack this type of information. the true value of a multiplex molecular method of screening for infectious respiratory agents depends on the clinical relevance. among the samples with > positive results, % had positive results for viral pathogens without co-detection of bacterial pathogens. for these patients, prescription of antimicrobial drugs on the basis of clinical fi ndings alone could serve to spread drug resistance through selective pressure on normal fl ora. furthermore, limited secondary treatment resources, such as oseltamivir administration during a pandemic, could be prioritized on the basis of screening results. of the , samples studied, . % were negative for the pandemic (h n ) virus strain. our fi ndings suggest that multiplex screening for respiratory pathogens is useful for providing rapid surveillance information to inform physicians who would otherwise base decisions on clinical signs and symptoms alone. electronic reporting of empirical laboratory respiratory pathogen detection provided by a clinical laboratory improvement amendments-approved laboratory can greatly enhance surveillance data collection ( ) . because most states have the authority to collect data of public health relevance ( ), the screening service provided by the diatherix laboratories could facilitate reporting of notifi able diseases. predominant role of bacterial pneumonia as a cause of death in pandemic infl uenza: implications for pandemic infl uenza preparedness centers for diseases control and prevention. bacterial coinfections in lung tissue specimens from fatal cases of pandemic infl uenza a (h n )-united states direct screening of clinical specimens for multiple respiratory pathogens using the genaco respiratory panels and simultaneous detection and high-throughput identifi cation of a panel of rna viruses causing respiratory tract infections human infl uenza a virus (h n ) detection by a novel multiplex pcr typing method development and evaluation of a novel multiplex pcr technology for molecular differential detection of bacterial respiratory disease pathogens evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections high prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol clinical disease in children associated with newly described coronavirus subtypes a review of strategies for enhancing the completeness of notifi able disease reporting mrs koon is pursuing a doctorate degree in public health at walden university, minneapolis, mn, usa. her research interests include developing multiplex amplifi cation assays for respiratory pathogens and infectious disease surveillance. key: cord- - jv em authors: alegbeleye, oluwadara oluwaseun; singleton, ian; sant’ana, anderson s. title: sources and contamination routes of microbial pathogens to fresh produce during field cultivation: a review date: - - journal: food microbiol doi: . /j.fm. . . sha: doc_id: cord_uid: jv em foodborne illness resulting from the consumption of contaminated fresh produce is a common phenomenon and has severe effects on human health together with severe economic and social impacts. the implications of foodborne diseases associated with fresh produce have urged research into the numerous ways and mechanisms through which pathogens may gain access to produce, thereby compromising microbiological safety. this review provides a background on the various sources and pathways through which pathogenic bacteria contaminate fresh produce; the survival and proliferation of pathogens on fresh produce while growing and potential methods to reduce microbial contamination before harvest. some of the established bacterial contamination sources include contaminated manure, irrigation water, soil, livestock/ wildlife, and numerous factors influence the incidence, fate, transport, survival and proliferation of pathogens in the wide variety of sources where they are found. once pathogenic bacteria have been introduced into the growing environment, they can colonize and persist on fresh produce using a variety of mechanisms. overall, microbiological hazards are significant; therefore, ways to reduce sources of contamination and a deeper understanding of pathogen survival and growth on fresh produce in the field are required to reduce risk to human health and the associated economic consequences. foodborne diseases are rife in many regions of the world, with at least in people falling ill yearly from consumption of contaminated food and , deaths occurring as a result, according to the world health organisation (who) ( ) . foodborne diseases have exerted pressure on medical services, contributed to economic and political distress, exacerbated malnutrition and led to human suffering. there are several agents such as chemicals, pathogens, and parasites, which may adulterate food at different points in the food production and preparation process (allos et al., ) . many of these agents have been extensively characterized and investigated by numerous studies (farber and peterkin, ; zhao et al., ; le loir et al., ; ehling-schulz et al., ; adzitey et al., ; botana, ) . strategies and protocols to prevent occurrence (and outbreak) of foodborne diseases have been devised and implemented by many researchers, regulatory bodies, and governments. however, despite the considerable progress achieved scientifically, foodborne diseases continue to occur, representing a significant cause of morbidity and mortality globally (mead et al., ; murray et al., ) . although foodborne diseases are more common in developing countries particularly in africa and south east asia with specific groups of people such as children, the immunocompromised, pregnant and aged being particularly at risk, foodborne diseases are not limited to these regions or groups of people (who, ) . for instance, according to the centres for disease control and prevention (cdc), between and , there were . million episodes of domestically acquired foodborne gastroenteritis caused by unspecified agents in the united states alone (cdc, ) . approximately . million acute gastroenteritis occurred, and there were at least , hospitalizations in the us each year and hospitalizations caused by the known gastroenteritis pathogens. an estimated persons died of acute gastroenteritis each year, of which deaths were caused by the known foodborne pathogens (scallan et al., ) . health canada ( ) estimates that e million cases of foodborne illnesses occur in canada every year. although the conventional notion is that foodborne diseases typically originate from meat and poultry products, vegetables and fruits have been implicated in various foodborne outbreaks (westrell et al., ; lynch et al., ; [european food safety authority (efsa), ] . a significant increase in foodborne disease outbreaks or cases associated with consumption of fresh produce has been reported. this increase has been largely due to a general increase in produce consumption, globalization of the produce industry and more effective surveillance (tauxe et al., ; lederberg et al., ; havelaar et al., ) . increased consumption of fresh produce is likely due to global government efforts to promote healthy eating, the associated health-promoting benefits of consuming fresh produce and ease of access to fresh local produce (pollack, ; regmi, ; berger et al., ; painter, ) . since fresh produce is mostly eaten raw or after minimal processing, pathogen contamination constitutes a potential health risk (callej on et al., ; li et al., ) . there are numerous factors capable of compromising the microbiological integrity of produce along the farm to fork continuum, all of which have potentially fatal outcomes. however, pre-harvest hazards to produce have been recognized as important because usually, once pathogen contamination is established in the field, it can be challenging to decontaminate produce. there are numerous circumstances that can undermine the safety of produce on farms. many of these arise because agriculture has grown more intensive over the years, and produce fields are often located near animal production zones thus entwining the ecological connections between wild animals, livestock and produce (strawn et al., a,b) . this, in many cases, predisposes fruits and vegetables to pre-harvest hazards. some important pre-harvest hazard sources to produce include the use of contaminated soil, irrigation water and manure for produce cultivation. wild animals and insects have also been implicated as vehicles of pathogens to produce. to ensure produce safety on a sustainable scale, it is imperative to correctly understand the routes of entry, fate, transport, establishment, and survival of pathogens in the agricultural environment such as soil, irrigation water and manure. the knowledge gap in this regard is being filled rapidly, as many studies have attempted to explain the behavior of foodborne pathogens in agricultural media and describe the associations among pathogens, produce and the agrarian environment. in this review, the extent of the produce contamination problem is discussed as well as the sources and routes of contamination of soil, irrigation water, fruits, and vegetables. also, the various mechanisms and strategies through which bacterial pathogens become established on fruits and vegetables are briefly examined. the nutritional and health benefits of consuming fruits and vegetables have been recognized and widely publicized. this has elicited changes in human dietary habits, with many consumers incorporating more fruits and vegetables into their meals. consequently, the global production of fruits and vegetables has surged exponentially in recent decades. the increased demand for produce has led to modifications such as increased use of soil amendments, utilization of alternative water sources and increased imports and exports in agriculture-spanning across agronomic practices, processing, preservation, packaging, distribution, and marketing . some of these modifications, however, have great potential to compromise the safety of fruits and vegetables. the biological hazards that are most relevant to fresh produce safety are either zoonotic or human in origin and can be classified into sporeforming bacteria, non-spore forming bacteria, viruses, parasites and prions (james, ) . most studies/surveillance efforts have identified bacterial contaminants in produce-borne illness outbreaks. there is, therefore, a disproportionately higher abundance of information regarding bacterial contamination in the literature. this may be because bacterial species have in fact caused many more outbreaks, but other microbial groups-viruses and parasites have been understudied. the most commonly implicated etiologic agents are presented in table . although data and information available on outbreaks associated with fresh produce are diverse and patchy, the available research evidence indicates that the foodborne illness burden due to contaminated produce has increased, in recent decades. in the united states, between and , approximately % of total foodborne illness outbreaks were produce related (jung et al., ) . in europe, from to , produce was linked with % of the outbreaks, % of the hospitalizations and % of the deaths (efsa, ) . in australia, fresh produce was linked to % of all foodborne disease outbreaks informed from to (lynch et al., . specific produce items are more commonly linked to foodborne illness incidents; for example, leafy greens such as lettuce and spinach, as well as fresh herbs such as parsley and basil are conventional sources of bacterial infections (who, ; berger et al., ; denis et al., ) . berries, green onions, melons, sprouted seeds, and tomatoes are similarly high-level priority produce items (olaimat and holley, ; denis et al., ) . in the us, between and , of multistate foodborne outbreaks were associated with vegetables (cdc, ) . a list of recent produce-related outbreaks is presented in table . most industrialized nations especially the united states have extensive and exhaustive datasets indicating the magnitude of outbreaks, the extent of severity and casualties incurred, the implicated pathogen and produce item as well as documented preventive protocols to avoid future outbreaks. unfortunately, however, the same is not true of many other countries especially african countries, the majority of which are still grappling with other challenges and hence, lack the resources to efficiently track and trace foodborne illness incidents (who, ) . many conventional foodborne detection methods are time consuming and laborious, and advanced techniques have therefore been developed and optimized as alternatives to or for use in combination with these traditional techniques. many of these are rapid, sensitive, reliable and standardized. they can be categorized into nucleic acid based, biosensor-based and immunological based methods (croci et al., ; adzitey et al., ; law et al., ) . typical examples include simple polymerase chain reaction (pcr), multiplex pcr, real-time pcr, nucleic acid sequence-based amplification (nasba), loop-mediated isothermal amplification (lamp) and oligonucleotide dna microarray. other examples are optical, electrochemical and mass-based biosensors, and enzyme-linked immunosorbent assay (elisa) and lateral flow immunoassay (law et al., ; gilchrist et al., ) . these advances in epidemiological investigation approaches and techniques have made it possible to explore the crucial associations between produce and pathogens. in spite of this, however, prompt identification of implicated produce vehicles, location or point of contamination in fresh produce associated outbreaks is still a significant challenge. one prime constraint is the relatively short shelf life of fresh produce, which is often discarded by the time an outbreak is identified (strausbaugh and herwaldt, ; lynch et al., ) . therefore, most of the time, the real source of contamination is not ascertained causing investigators to speculate or assume a source. this is, however, dangerous because, in addition to the possibility of being wrong, there is empirical evidence that once a particular transmission pathway is identified, repeated investigations are bound to be biased in causation (lynch et al., ) . another important consideration is that usually, outbreaks receive widespread attention if the event (i) has severe public health impacts (ii) is unusual or sudden (in that the etiological agent and/produce type are unanticipated; making the circumstances of the outbreak unique and (iii) poses a significant risk of international spread with consequences for international travel or trade. invariably, the smaller, 'less significant' outbreaks are never investigated. more importantly, foodborne illness incidents occur sporadically in populations, and these cannot be captured in routine epidemiological surveillance or outbreak investigations (scallan et al., ) . this means that the data available may not be a valid representation of the problem. it is likely that the foodborne illness burden related to consumption of contaminated produce is still largely underestimated. the possible routes and sources of produce contamination are numerous, and intensive efforts have been made to accurately understand the exact mechanisms through which pathogens are introduced into fresh produce (kotzekidou, ) . sources and routes of produce contamination vary for different production zones. this is because each farm has a distinct combination of environmental risk factors such as topography, land-use interactions, and climate. combinations of these peculiar environmental risk factors influence the frequency and transmission of foodborne pathogens and subsequently impact the risk of produce contamination (strawn et al., b) . primarily, pathogens may contaminate produce 'on-field' via various routes including; atmospheric deposition, uptake from contaminated soils and groundwater (harris et al., ; lynch et al., ; mei soon et al., ) , use of raw (or poorly treated) manure and compost, exposure to contaminated water (irrigation or flooding), transfer by insects, or by fecal contamination generated by livestock or wild table the most commonly implicated etiological agents in fresh produce borne illnesses (brackett, ; buck et al., ; heaton and jones, ; jung et al., ; callej on et al., ) . animals (cooley et al., ; uyttendaele et al., ) . a schematic representation of the main entry points for pathogens to humans via produce is provided in fig. . the use of organic materials such as livestock excreta, slurries, abattoir wastes, sewage sludge as well as municipal and industrial waste treatment residuals as soil amendments is widespread goss et al., ) . although these serve as a costeffective source of nutrients for agricultural purposes, research demonstrates that raw manure as well as contaminated (or improperly treated) manure constitute a significant risk of pathogenic contamination for produce (james, ; manyi-loh et al., ) . public health relevant bacteria, viruses and parasites such as e. coli o :h , salmonella spp., l. monocytogenes, campylobacter spp., porcine enteroviruses, bovine coronavirus, bovine virus diarrhoea cryptosporidium parvum and giardia have been isolated from raw/poorly treated manure (derbyshire, ; derbyshire and brown, ; sellers, ; strauch, ; pell, ; grewal et al., ) . pathogens may be spread through direct interaction of vegetable surfaces with manure, or by splashing of (contaminated) soil/manure particles from the soil on vegetables via rainfall and/ overhead irrigation or by vectors. additionally, manure piles stored next to growing areas may constitute contamination risk due to run-off (james, ; warriner et al., ) . manure application could be by broadcasting as a solid, semisolid or liquid throughout the field or by the introduction of livestock or wildlife feces at distinct locations (jung et al., ) . in many parts of the world, organic cultivation systems use more manure than conventional growers, and chemical treatment against pathogens is prohibited in organic farming. there have thus been some assertions that organic produce represents a more significant safety risk than its non-organic counterpart, although, there is no unequivocal research evidence supporting this claim loncarevic et al., ; warriner et al., ; ivey, ; maffei et al., ) . the survival of pathogens in manure and biosolids depends on factors such as the manure source, production process, and characteristics, treatment technique applied, physicochemical factors like ph and relative humidity, incidence of antagonists or predators, weather conditions, desiccation, aeration, soil type, degree of manure incorporation, amongst others (ingham et al., ; wood, ) (table ) . the manure composition, which is determined in large part, by the feed formulation, dictates the profile of pathogens occurring in manure as well as their ability to persist even posttreatment (franz et al., ) . certain workers have proposed that cattle diet may influence the incidence of representative bacterial species; e. coli o :h and salmonella in manure. these pathogens have been reported to persist longer in manure obtained from cattle fed diets rich in energy but low in fiber content such as high digestible grass silage and maize silage compared to animals that received diets with low energy and higher fiber content such as straw . it has also been suggested that feeding cattle with hay may significantly reduce shedding of acid-resistant e. coli (diez-gonzalez et al., ; . how effective these strategies are in reducing pathogen load in (animal-derived) manure, is however not clear. manure treatment techniques such as composting, aerobic and anaerobic digestion, pelleting, alkaline stabilization, conditioning, dewatering and heat drying have been used to treat manure before application as fertilizer for a long time. while many of them are reasonably efficient, concerns have been raised about their ability to satisfactorily eliminate pathogenic bacteria (day and funk, ; lu et al., ; lorin et al., ) . tailing of pathogen inactivation curves, as well as apparent regrowth or recontamination of bacteria after treatment, have been reported. many pathogens have been shown to be capable of withstanding manure treatment processes, thereby, constituting a major risk of contamination (brackett, ) . composting is a popular manure treatment and composting temperatures that exceed c for three days are considered sufficient to kill most pathogens (grewal et al., ) . however, few studies have demonstrated that the heat-induced death of bacteria in composted materials is a complex phenomenon (ingham et al., ; gupta, ) . bacterial regrowth and recontamination in cooled compost have been reported (hassen et al., ; ingham et al., ) . pelletizing is another common treatment available and is commonly applied to chicken manure (chicken manure pellets). pelletizing the manure reduces the off-odor and facilitates transport and storage. although the process usually involves a thermal procedure, more studies are required to validate whether the process efficiently inactivates clinically relevant pathogens (chen and jiang, ; jung et al., ) . the use of a fish emulsion as fertilizer has raised similar concerns; although most preparation methods available include a thermal process, the ability of this to inactivate enteric bacteria and viruses needs to be rigorously validated (jung et al., ) . due to the diverse range of variables associated with manure composition, treatment, pre-application storage, application and incorporation, regulatory bodies have stipulated minimum manureto-harvest time intervals necessary to ensure microbiological safety. the united states department of agriculture (usda) 'organic production and handling' specifies that unless composted, raw animal manure must be incorporated into the soil not less than days prior to harvest of a product whose edible portion has direct contact with the soil surface or soil particles, or days if there is no direct contact (usda, ) . canadian authorities specify , and months for tree fruits and grapes, small fruits and vegetables respectively as the minimum time delay between manure application and harvest for these crops (olaimat and holley, ) . irrigation water has been identified as a potential source of produce contamination (benjamin et al., ; uyttendaele et al., ; faour-klingbeil et al., ) . being a common and essential requirement for crop production, water must be supplied to plants when necessary, and irrigation water sources are used to supplement limited rainfall in many areas (kirby et al., ) . epidemiological investigations of food poisoning outbreaks, experimental studies examining pathogen contamination of fruits and vegetables as well as observations of increased incidence of disease in areas practicing wastewater irrigation with little or no wastewater treatment indicate that contaminated irrigation water might indeed be a source of foodborne pathogens on fresh produce (norman and kabler, ; hern andez et al., ; steele and odumeru, ) . for example, hepatitis a outbreaks associated with lettuce (seymour and appleton, ) and spring onions (josefson, ) were linked to sewage-contaminated irrigation water (heaton and jones, ) . various factors including irrigation regime (method and timing of irrigation), irrigation water sources, type of crop and land use practices in the farm influence the extent and frequency of pathogenic contamination of produce ( such as pathogen concentration, pathogen strain, weather patterns, plant state, and physiology also have significant implications for produce safety (marvasi et al., ; uyttendaele et al., ; decol et al., ) (table ) . there are several types of irrigation systems available, each of which is typically complex and has its own drawbacks. most irrigation systems create complicated ecological environments with multiple potential sources and routes of pathogenic contamination (pachepsky et al., ) . each irrigation subsystem: collection, replenishment, storage, conveyance, distribution off and on-farm, as well as on-farm application involve processes that have great potential to compromise the microbiological integrity of the irrigation water in unique ways. during transportation from the source to the field, water is susceptible to significant microbiological depreciation (pachepsky et al., ) . the prevailing deterioration dynamic will depend on the transportation mode. for instance, irrigation water transport via irrigation ditches and canals involves interaction with microbial reservoirs of bottom sediments, bank soils, algae and periphyton, whereas water transport via pipes involves interactions with biofilms in the transport pipes pachepsky et al., ) . this sort of contamination is particularly prominent in reclaimed water distribution systems . the method of storage for irrigation water can have a profound effect on pathogen transmission. for example, certain studies have demonstrated that water quality is rapidly degraded in storage ponds and tanks due to inputs from avian species or other wildlife (field and samadpour, ; mclain and williams, ; higgins et al., ) . other storage systems such as check dams, impoundments, inter-basin transfer schemes, abstraction schemes and reservoirs have been identified as places where indicator and pathogenic microorganisms can survive and proliferate (abbasi, ; kirubel, ) . the mode of application also has significant impacts on the risk of microbiological contamination (berger et al., ) . compared with furrow and subsurface drip irrigation systems, sprinkler irrigation poses a higher risk of microbiological contamination (kisluk and yaron, ; pachepsky et al., ) . surface furrow and drip irrigation systems minimize contact between edible portions of certain plants (leafy vegetables provide larger surface area for contact and possible microbial attachment) and irrigation water (directorate, ; fonseca et al., ; mei soon et al., ; uyttendaele et al., ) . hydroponic growing systems also offer this advantage (jung et al., ; allende and monaghan, ) . the irrigation application method has been determined to influence the internalization of some pathogens in produce such as spinach plants. according to some studies, the likelihood of internalizing pathogens increases when the organisms are introduced by water sprinkling systems as opposed to when the water is directly applied to the soil (solomon et al., ; stine et al., ; mitra et al., ; warriner et al., ; erickson et al., a; kisluk and yaron, ; zheng et al., ) . more details on pathogen internalization are provided in section (below). depending on the geographical location, the irrigation regime with respect to time of day, season and harvest time may influence the likelihood of pathogenic contamination. for example, kisluk and yaron ( ) in a study conducted in haifa, israel demonstrated that night-time irrigation and irrigation during the winter season is more likely to contaminate plants with enteric bacteria. contaminated irrigation water poses the most significant risk when crops are irrigated close to harvest time, because harvesting of produce containing viable pathogens is more likely. therefore, an adequate time interval between irrigation and harvest should be conscientiously followed. the microbial quality of irrigation water depends mostly on the source of the water. in order of increasing risk of microbial contamination hazard, irrigation water sources can be ranked as follows: potable or rainwater, deep groundwater, shallow groundwater, wells, surface water and raw or inadequately treated wastewater (james, ; pachepsky et al., ) . the microbial quality of rainwater or rain-harvested water is relatively good. the quality and safety of use, however, depends largely on the collection, transportation and storage means. this can be illustrated with roof-harvested rainwater, which may become contaminated with pathogenic bacteria and protozoan parasites because of the occurrence of animal droppings on roofs, particularly immediately after relatively long periods of drought . groundwater (or borehole water) is usually microbiologically safe, except if it has been contaminated with surface runoff or other sources of contamination close to the aquifer. certain farm operations such as intensive dairying and border-strip irrigation (a type of surface irrigation, which is a hybrid of level basin and furrow irrigation) (valipour et al., ) lead to leaching of pathogens such as e. coli and campylobacter to shallow groundwater, thereby contaminating it (close et al., ) . water from wells that are free from leaks and have sound casing are expected to be microbiologically safe. factors such as the design of wells, nature of the substrata, depth to groundwater and rainfall may affect the microbial quality of good water (james, ; gerba, ) . surface waters; which are the predominant source of irrigation waters in many countries, including open canals, ponds, lakes, rivers and streams are much more susceptible to pathogenic contamination compared to groundwater (allende and monaghan, ; uyttendaele et al., ) . sewage discharges, septic tank contamination, storm drains, wild and livestock defecation, run-off from contaminated fields, industrial and municipal effluents can all potentially contaminate surface waters (steele and odumeru, ; james, ) . wastewater is usually of poor chemical and microbiological quality. therefore, it requires extensive treatment before it can be safely used to irrigate crops. water sources (other than rain) used to irrigate produce is usually only minimally treated or untreated in many cases (steele and odumeru, ; jung et al., ) . it is expensive and time-consuming to treat irrigation water up to drinking water standards, which is the ideal recommendation (crook and surampalli, ; forslund et al., ) . although awareness of the potential dangers of using microbiologically compromised water for irrigation has increased in recent times, scarcity of water resources in certain regions has contributed enormously to the use of sub-optimal supplementary irrigation water sources. in such cases, irrigation water represents a greater microbiological risk to produce (sundstr€ om et al., ) . one of the most frequent pathogens implicated in water-related outbreaks is e. coli o :h (cdc, ; hilborn et al., ) . the organism can survive for a protracted period in water (even in deionized water) depending on temperature conditions (chalmers et al., ; islam et al., a) . it also exhibits a remarkable ability to withstand extreme environmental conditions such as high acidity and extremely low-temperature conditions. the ability of a pathogen to survive (or persist) in the environment (and on produce) is an essential determinant in the risk of human infection. the actual risks associated with pathogens occurring in irrigation water depend on numerous variables including environmental conditions such as temperature, ph and uv light (sant'ana et al., ) . other factors such as the excreted load of the pathogen, its latency period before it becomes infectious, its ability to efficiently multiply outside a mammalian host, its infectious dose for humans, inhibitory competition from the indigenous microflora as well as host response also play a relevant role (steele and odumeru, ) . bacteria and viruses survive for lengthier periods in groundwater compared to surface water because groundwater tends to be cooler, offers protection from sunlight, and has less biological activity (steele and odumeru, ) . these groups of microbes only typically last no longer than and days in surface water and sewage, respectively. conversely, parasites (eggs/cysts) may survive for as long as days or even several months in surface water and wastewater (lefler and kott, ; sagik et al., ; bihn, ) . this suggests that pathogenic microorganisms are capable of surviving for extended periods, which constitutes a profound threat to produce safety. regardless of the source or route of exposure, one potentially fatal consequence of pathogen contamination of irrigation water is the repeated inoculation of plants with the pathogens. the fate and transport of these pathogens once introduced into the produce vary widely (table ). some pathogens are capable of adhesion to surfaces of produce while some others can rapidly internalize into plant tissues under certain conditions, translocate and persist until consumed (wariner et al., ; bernstein et al., a; doyle and erickson, ) . this has rendered many conventional processing and chemical sanitizing methods ineffectual (hong and moorman, ) and is a growing public health concern. although the potential for produce contamination via irrigation water has been identified, it is difficult to estimate the magnitude of the problem (groves et al., ; antwi-agyei et al., ) . despite the fact that numerous studies have linked poor microbiological quality of irrigation water with the incidence of human pathogens on fruits and vegetables, direct evidence of irrigation water causing foodborne disease is relatively rare (harris et al., ) . this is because a substantive "cause-effect" relationship is yet to be established as it is required that the same pathogenic strain is isolated from the patient, produce, and irrigation sources (pachepsky et al., ) . furthermore, there must be a clear sequence of events connecting patient, produce, and irrigation source (steele and odumeru, ) . this is difficult to achieve due to certain limitations such as an inability to promptly identify the locations associated with produce contamination and delays inherent in foodborne outbreak investigations (pachepsky et al., ) . in the absence of direct confirmation, the "cause-effect" relationship can only be deduced based on circumstantial or subjective evidence (pachepsky et al., ) . also, it is apparent that there is no valid link between detected pathogen levels in irrigation waters and disease risk. some studies have demonstrated a lack of correlation between pathogen prevalence in waters used for irrigation and disease incidence due to consumption of irrigated produce (cooley et al., ; mcegan et al., mcegan et al., , . there is an abundance of laboratory studies elucidating potential mechanisms of produce contamination from waterborne pathogens. however, field studies showing the exact process of produce contamination via this medium are relatively scarce. it is thus expedient to generate more field data in this regard. soils typically harbor an abundant consortium of microorganisms, some of which are human pathogens such as b. cereus, clostridium botulinum, c. perfringens, listeria monocytogenes and aeromonas (nicholson et al., ; warriner et al., ; jay, ) . they may, therefore, serve as a medium of plant contamination through seeds, roots or surfaces. many soil resident pathogens have adapted to survival in soil with spores persisting indefinitely. however, since many agricultural soils are predisposed to point and nonpoint sources of pathogenic contamination, allochthonous pathogens may continuously be introduced into soil environments (santamaria and toranzos, ) . some of the primary sources of pathogens into soil include the use of contaminated irrigation water and manure, animal grazing, municipal solid wastes and other effluents (santamaria and toranzos, ; sant'ana et al., ) . the fate, survival and recalcitrance of pathogens in soil depend on factors such as soil type, soil moisture, ph, temperature, nutrient availability, agronomic practices, as well as soil biological interactions (table ) . soil matric potential (moisture levels) is determined by soil properties and water inputs through precipitation and/irrigation and has been demonstrated to be one of the most critical factors influencing microbial transport and survival in soil . cool, moist environments are favorable for the survival of bacteria and viruses. under dry soil conditions, a reduction in bacterial and viral population densities are usually observed (santamaria and toranzos, ; ghorbani et al., ) . escherichia coli survival has been reported to be highest in organic soils under flooded conditions, and peak populations recorded after a rise in the water-table accompanying significant rainfall events (tate, ; rochelle-newall et al., ) . some pathogens such as streptococcus faecalis have been proven to thrive poorly under low soil moisture conditions (kibbey et al., ; jamieson et al., ; cabral, ) . increased rates of virus inactivation at low soil moisture levels have been demonstrated (yeager & o'brien, ) . also, decreased recovery of viral (poliovirus type and coxsackievirus b ) infectivity in dried soils was attributed to evaporation of soil water in the same study by yeager & o'brien ( ) . in addition, experimentation by hurst et al. ( ) correlated inactivation of enteroviruses [echovirus type (strain wallace), coxsackievirus b (strain nancy) and poliovirus type (strain lsc)] in sludge-amended soils with moisture loss in the sludge piles. soil ph influences microbial diversity and the biogeochemical processes, which they mediate (fierer and jackson, ; nicol et al., ) . optimum ph for bacterial survival seems to be neutral, but fungi are known to be more tolerant of acidic conditions, compared to bacteria (leahy and colwell, ) . amino acids (most viruses behave as proteins) have different pk values and so the ratio of positive to negative charges on proteins vary with ph (yates et al., ) . in an experiment that lasted days using poliovirus type , echovirus , echovirus and coxsackie b , viruses were detected up till the th e th day at ph . while at ph . , the viruses died off between the th and th day depending on virus type (bagdasaryan, ) . soil types vary depending on organic matter content, water release characteristics, particle size distribution and moisture retention capacity. these variations significantly influence the survival of enteric pathogens in soil atkinson et al., ) . clay soils support the adsorption of microorganisms onto soil particles, and this reduces microbial die-off rates (reddy et al., ) . clays protect bacterial cells, and possibly viral particles, by creating a barrier against microbial predators and parasites (santamaria and toranzos, ) . viruses, which are mostly large proteins possessing various charges, are capable of forming numerous bonds with clay minerals (stotzky, ) . for example, the survival of e. coli is prolonged in clay soils where adsorption of cells to the soil particles protects it against protozoa (mosaddeghi et al., ) . escherichia coli can persist for up to weeks in clay and loam soils, but for much less ( weeks) in sandy soils (lang and smith, ) . results of a study that compared rotavirus survival in three soil fractions (whole soil, sand and clay) at temperatures , and c for days showed least survival in sand fractions (davidson et al., ) . in the absence of soil particles, rotavirus survived best at c with survival decreasing, with an increase in temperature, except in whole soil, where it survived better over the entire temperature range and for more than a week at c, indicating that whole soil offered some protective effect (davidson et al., ) . conversely, though, there is a report of shorter survival duration of enteroviruses (poliovirus type , echovirus , echovirus and coxsackie b ) in loamy soil than in sandy soil (bagdasaryan, ) . a link between higher organic matter content and enteric pathogen persistence has been established (jamieson et al., ; williamson et al., ; . there is overwhelming research evidence in this regard, seeing that many of the studies that compared the persistence of enteric pathogens in top and sub-soils recorded higher survival rates in topsoil (zhai et al., ; nyberg et al., ) . research has also shown higher pathogen levels in organic soils after manure application compared to sandy soils (tate, ; jamieson et al., ) . therefore, the rates of pathogen survival are lower in sandy soils, which have a low water-holding capacity (mubiru et al., ; erickson et al., a) . lower temperatures are more suitable for bacterial and viral survival. the ultraviolet radiation from the sun inactivates viruses on the surface of the soil, but viruses in deeper soil strata are protected from this (rodríguez-l azaro et al., ; zablocki et al., ) . in loamy soil samples, at ph . , poliovirus and echovirus were recovered after e days at e c compared to recovery e days at e c (bagdasaryan, ) . similarly, cfu/g amended with dairy manure persisted for e d in fallow soils, manure did not seem to affect persistence, clay seemed to improve pathogen persistence and activity gagliardi and karns, (continued on next page) poliovirus type and coxsackievirus b pfu were recovered for up to days at c whereas pfu were recovered from soil for up to days at c (temperature profiles tested were , and c) (yeager & o'brien, ) . the persistence of poliovirus in sludgeamended soil was assessed in a field study where appropriately cultivated and irrigated plots were treated with virus-spiked effluents by flooding. this was done for days spanning through spring, summer and winter seasons. poliovirus survived best during winter (when it was detected after days), but during summer, the longest survival period was days (tierney et al., ) . parasites seem to prefer warm temperature conditions. prevalence of hookworms have been correlated to warm temperatures, relatively high rainfall and low clay content (sandy soils with clay content of less than %) (mabaso et al., ) . nutrient availability is essential for the survival of microbes in the soil. the presence of organic matter promotes the survival, and in many cases, the regrowth of enteric bacteria looney et al., ) . organic matter improves nutrient retention, serves as carbon sources for bacterial species and enhances moisture retention (gerba et al., ; schoonover and crim, ) . apart from environmental stress responses, foreign enteric bacteria must compete with the endogenous microflora to become established in the soil environment (jiang et al., ) . some autochthonous soil organisms have been shown to be resistant to newly introduced microorganisms in their environment (ellis and mccalla, ) . also, certain bacteriophage, some protozoa, nematodes and free-living soil organisms such as bdellovibrio can parasitize non-indigenous pathogens, thereby limiting their survival (klein and cassida, ; goss and richards, ) . additionally, increased pathogen survival, and regrowth in some instances, in sterile soils and soils with relatively low biological activity has been reported (gerba et al., ; tate, ) . there is some research evidence that alien enteric pathogens compete poorly for nutrients and are thereby susceptible to inhibition by soil-borne bacteria (jiang et al., ) . the effects that this has on the persistence of pathogens (especially pathogens introduced via contamination) in soil is however not yet fully understood. the impacts that soil edaphic and biotic conditions have on the occurrence, fate and persistence of microorganisms in soils should not be underestimated. these factors can collectively or independently stifle or encourage foreign pathogens. for instance, members of listeria possess advantageous intrinsic factors such as an extensive repertoire of transport systems (like phosphotransferase system and transcriptional regulators) which makes them capable of successfully persisting in the soil ecosystem (newell et al., ) . however, these species are highly sensitive to extrinsic factors and this affects their ability to survive in soil environments (newell et al., ; locatelli et al., ) . although studies have been conducted on the occurrence of l. monocytogenes in various ecological niches, including soil, more emphasis has been placed on the occurrence of listeria spp. in fresh vegetables under storage conditions, food processing and packaging environments. the expression of genes and induction of proteins such as cold shock and cold acclimation rapid decline in cell numbers with no difference identified for soil type. increase in retention at both rainfall rates. campylobacter jejuni clay loam silt cfu/g cfu/g cfu/g proteins, as well as tolerance for low ph and high salt concentration in these environments have received much research attention. there is however, need for more research to understand the dynamics of listeria survival in soils. agronomic practices such as soil improvement and manure application method influence the survival of pathogens in the soil (table ) . soil improvement strategies (inorganic and organic fertilizer, compost, biosolids and other residuals application), significantly enhance the nutrient loads of soils (diacono and montemurro, ) . in varying degrees, these are important sources of primary nutrients such as n and p as well as secondary nutrients such as ca, mg and s to the soil. a ready supply of essential nutrients encourages the growth of pathogens. compost application modifies the long-term soil conditions by increasing the ph steadily, this, therefore, affects pathogen survival in soil (weller, ; sharma and reynnells, ) . bacteria tend to decline more rapidly when manure is applied superficially as opposed to when incorporated into the soil immediately after application (solomon et al., ; islam et al., a) . this is probably due to the elimination of drying conditions and exposure to uv at the soil surface (schulze-makuch and irwin, ) or because incorporation of manure disrupts macropores and boosts soil-bacteria contact . after manure application on land, if applied manure is contaminated, it is probable that the pathogens will move through the soil matrix, either vertically or horizontally. vertical movement of pathogens through the soil is influenced by the amount and intensity of rainfall, climatic conditions as well as the season of application. horizontal movement is known to be influenced by soil type, moisture levels, temperature, microbial activity, transport through plant roots, rainfall patterns, soil ph amongst other biophysical factors. it is, however, apparent that water flow is the most important dispersal factor for percolation of manure-derived pathogens in soils, regardless of type and structure although more quantitative information regarding this is desirable (mawdsley et al., ; jiang et al., ; jamieson et al., ; islam et al., b; you et al., ; semenov et al., ) . the extent of movement will affect the distribution and eventual fate of the pathogens. some will spread in soil and attach to roots. others may be washed off to surface waters or percolate to aquifers, potentially contaminating irrigation water sources (fig. ) vinten et al., ; avery et al., a, b; islam et al., b) . pathogens occurring in contaminated manure, therefore, can be rapidly transported within soil systems (gagliardi and karns, ; kisluk and yaron, ) . the success of conveyance and distribution, however, further depends on inherent survival capabilities of the pathogen as well as the presence and structure of plant root systems (fig. ) (kemp et al., ; mubiru et al., ; avery et al., a; . there is some evidence that pathogens may indeed survive longer in manure-amended soils than actual manure samples, and this has been illustrated for enteric species such as s. typhimurium and e. coli o :h . salmonella typhimurium, has, however, exhibited superior persistence capabilities compared to e. coli o :h in manure-amended soils (islam et al., b; you et al., ; fremaux et al., ; pornsukarom and thakur, ) . there is a paucity of data on the persistence of pathogens in manure amended-soils in the tropics (ongeng et al., ) . one interesting study provides an insight into the survival of e. coli o :h and salmonella typhimurium under tropical climatic conditions . the study showed that survival periods were mostly shorter than the observed record in temperate regions indicating that biophysical conditions in the tropics may be more injurious to these pathogens. it is, therefore, not prudent to predict the survival of e. coli and s. typhimurium in tropical soils from data obtained in temperate locations. the soil is the most important cultivation medium and represents a relevant risk for produce contamination. a myriad of studies regarding the behavior of pathogens in various kinds of soil ecosystems is available. however, validated consensus protocols for conducting and interpreting experimental studies as well as for evaluating the effects of environmental and soil characteristics on fate of pathogens in soils are not yet available. it is important to further understand the effects of soil types, environmental factors, biological processes and interactions, cultivation and management practices on the behavior of (indigenous and foreign) enteric pathogens in agricultural soils. apart from farm animals, whose roles as reservoirs of enteric pathogens has been established, wild animals such as birds, reptiles, rodents, amphibians, some helminths, and insects like flies and beetles can also serve as vehicles of pathogens to contaminate cultivation media and produce (beuchat, ; lim et al., ) . livestock and wild animals may gain access to cultivation areas either because of adjacent land use (livestock rearing) or by intrusion (jay-russel, ) . birds such as gulls, pigeons, chickens, starlings, canada geese, migratory ducks and sandhill cranes (pacha et al., ; hald et al., ; ekdahl et al., ; humphrey et al., ) have been determined to be carriers of pathogens such as e. coli, salmonella and campylobacter (wallace et al., ; schmidt et al., ; wani et al., ) . insects are typically ubiquitous in cultivation fields, and hence, have unrestricted access to produce. they are usually found in manure piles, feedlots and other habitats near cultivation fields, and so farms practicing mixed farming represent a more significant risk (martínez-vaz et al., ) . many bacterial species have evolved to exploit insects as hosts or vectors. filth flies, fruit flies, cockroaches and other insects act as mechanical and biological vectors to contaminate fruits and vegetables on the field (sasaki et al., ; mpuchane et al., ; alam and zurek, ; humphrey et al., ) . many pathogens use flies as vectors for cross-transmission. for example, the transient survival of pectobacterium carotovorum subsp. carotovorum in the gut of the fruit fly drosophila and subsequent transmission to other plants has been observed (nadarasah and stavrinides, ; lim et al., ) . under laboratory environment, direct bacterial transfer from contaminated flies to fruits or plant leaves was shown to occur (sela et al., ; talley et al., ; lim et al., ) . members of muscidae and calliphoride which are usually abundant in production fields adjacent to cattle rearing lots have been associated with the transmission of e. coli o :h . insects that feed on plants also play significant roles in produce contamination by providing direct routes for internalizing pathogens from manure to plants in the field . insect deterioration creates openings that aid the ingress of pathogens into inner plant tissues, thereby enhancing colonization of spoilage and pathogenic bacteria on produce (warriner and namvar, ; lim et al., ) . a seasonal trend to contamination by insects has been identified. there is increased insect and animal activity during the warmer months of the year. moreover, peak incidences of pathogens have been reported during the warmer months (liang et al., ) . reptiles including snakes, lizards, chameleons, turtles, as well as other ophidians, saurians and chelonians have been found harboring enteric bacteria like salmonella (corrente et al., ; beuchat, ) . many wild rodents are asymptomatic carriers of pathogens like salmonella and campylobacter. the occurrence of rodents on farms are often associated with infrastructural impairment, and although their destructive tendencies have been widely recognized, their zoonotic risks are often primarily underestimated. they are capable of amplifying the number of pathogens in the environment and transferring them to other farm animals and produce (meerburg and kijlstra, ) . commensal rodents (house mice and rats) pose a particular threat because of their ecology (they live close to livestock) and high fecundity (brooks and jackson, ; witmer et al., ) . foodborne illness resulting from the consumption of contaminated produce is dependent on specific factors. first, the produce must be contaminated with a pathogen, which must survive until the time of consumption at levels sufficient to induce illness (harris et al., ) . the dose required to cause illness in many cases, is very low, which indicates that the microorganism needs only to contaminate the food to survive without necessarily reproducing. for instance, pathogenic parasites and viruses are not capable of multiplying outside a human or animal host and only need to survive in sufficient numbers to cause illness (harris et al., ) . the survival and or growth of pathogens is influenced by the kind of organism, produce type, on-field environmental conditions, as well as the physiological state of the plant and pathogen. the possible routes of entry into plant tissues include: natural apertures (such as stomata, lenticels, sites of lateral root emergence), wounds caused by biotic or abiotic circumstances and following the flow of water from roots to leaves, where pathogens can efficiently survive and multiply (steele and odumeru, ; deering et al., ; hirneisen et al., ) . the popular opinion is that pathogens will survive but not thrive on intact (uninjured) outer surfaces of produce, primarily due to the protective effects of natural plant barriers (such as cell walls and wax layers) (mathews, ; heaton and jones, ) . survival and proliferation of enteric pathogens on produce is significantly enhanced if the protective barrier becomes compromised either by physical or biological damage (such as punctures or bruising), insect ruination or through degradation by plant pathogens. it is vital to understand the microbe-microbe and plant-microbe interactions that occur in the phyllosphere and rhizosphere which influence the adaptation, colonization, attachment is pre-requisite for the colonization and subsequent transmission of enteric pathogens throughout plants including the edible portions (berger et al., ) . it is important to note that attachment onto the surface of intact produce is limited in contrast to the attachment on other food commodities such as processed meat tissues . however, the attachment does indeed occur and is facilitated by stomata, lenticels, broken trichomes, as well as bruises and cracks occurring on produce surfaces. the incidence of scars and cracks (which may set in late in the growing season while the fleshy portion is enlarging rapidly) in certain fruits also aids pathogen attachment (bhagat et al., ) . cracks tend to occur in or on the weak areas on plant surfaces such as around lenticels and trichomes, and hence, these areas are more susceptible to invasion by pathogens. cavities within the epidermis may also develop from cuticular cracks as the fruit develops, thereby entrapping pathogens and shielding them from desiccation and disinfection. the initial phase of bacterial attachment is a rapid process initiated once the bacteria establishes contact with the plant surface (phyllosphere) (sant'ana et al., ) . the phyllosphere, also known as the aerial parts of plants pose challenges for microbial survival. exposure to high uv doses, temperature and relative humidity fluctuations sabotage viability (brandl et al., ; heaton and jones, ) . epiphytes that exist within the phyllosphere have, however, evolved specialized mechanisms to improve stress tolerance and nutrient acquisition. for instance, pseudomonas spp. produce pigments to insulate against uv and pectolytic enzymes to gain nutrients (heaton and jones, ) . the ability of the pathogen to persist on the phyllosphere improves the chances of a viable or infectious dose remaining post cultivation (heaton and jones, ) . the successful attachment on the phyllosphere also depends on the crop and pathogen type. a classic illustration is salmonella invasion of lettuce and tomatoes. salmonella contamination of lettuce and tomatoes via soil is usually quite low, implying that salmonella does not readily attach to or grow in the phyllosphere of these crops (critzer and doyle, ) . also, attachment of salmonella and e. coli o :h is observed more frequently with brassicaceae compared to lettuce, carrots, and tomatoes, which has generated the theory of selective attachment, suggesting that certain produce types are more prone to contamination than others (warriner and namvar, ). specific pathogens such as salmonella have surface epitopes that can bind to plant structures such as stomata to aid attachment (warriner and namvar, ). some also have higher capabilities to metabolize nutrients contained within the apoplastic fluid of plants (warriner and namvar, ) . these traits significantly enhance their attachment abilities. finally, hydrophobic interactions between a plants' epidermal layer and microbial cells are believed to play a major role in facilitating this initial phase of attachment (burnett and beuchat, ) . surface colonization is the final phase of attachment during which biofilms may be formed. biofilms are microbial colonies, which form when single microorganisms attach and aggregate on a hydrated surface and undergo a "lifestyle switch," giving up life as a single cell to live on a surface in an adhesive cell matrix with other microorganisms (lemon et al., ) . cells in a biofilm have a better chance of adaptation and survival (especially during periods of stress) as they are protected within the matrix (decho, ) and are usually resistant to antimicrobial agents (lemon et al., ) . naturally occurring biofilms are present in many fruits and vegetables, but the ability of foodborne pathogens to associate with them and persist is not yet fully understood (brackett, ; ferreira et al., ; larsen et al., ) . pathogen serovars that are strong biofilm producers have been shown to attach better to both intact and injured produce surface compared to strains that are weak biofilm producers (lindow and brandl, ; kroupitski et al., ) . the occurrence of biofilms improves the chances of transient occupants of leaf surfaces such as enteropathogens of becoming effectively incorporated into phyllosphere biofilms (heaton and jones, ) . bacterial appendages such as curli, pili, fimbriae, and flagella, as well as proteins in outer membranes and genes, may also facilitate the surface colonization by pathogens. increases in the expression of flic, flagellin-encoding gene have been observed in certain produce contamination studies. after attachment, it becomes very difficult to remove the pathogens from produce by surface washing (beuchat and scoutten, ) . overall, enteric soil pathogens may reach the edible portions of fruits and vegetables via numerous mechanisms and routes and these have been elucidated by several studies (natvig et al., ; johannessen et al., ; barak and liang, ; tyler and triplett, ) . some of these routes include germination of seeds in contaminated soils, which leads to bacterial colonization of roots and edible parts, direct transfer of pathogens within the soil to crops when heavy rain or water gun irrigation causes leaf splash, bacterial infiltration through roots, amongst others. attached pathogens can reach the interior of fruits and vegetables via a variety of pathways. the extent of internalization depends on factors such as the route and mechanism of entry, the type and age of the plant, the aerial and/ or root morphology and exudates, the soil type and biology and the strain and/serovar of bacteria (hirneisen, ; brandl et al., ; lim et al., ) . the mechanism could be either passive or active (sant'ana et al., ) . passive internalization involves the uptake of bacteria mainly through roots and seeds. mechanistically though, enteric pathogens may be internalized via the root system and transported to edible tissues, but the risk of contamination by this route is likely low . this is because in the environment, particularly areas that are not prone to contamination events, the levels of enteric pathogens are likely to be extremely low (cooley et al., ; matthews et al., ) . in contaminated zones, however, human pathogens may indeed invade root tissues and subsequently translocate to edible portions (solomon et al., ; solomon and matthews, ) . depending on the age of the plant, pathogens may invade external root surfaces (main and side roots, as well as root hairs) and subsequently internalize. the developmental stage of plant root systems when contamination occurs influences the capability of pathogens to interact with, penetrate plant roots and migrate to other tissues (mootian et al., ) . the physiological characteristics of the roots may also determine the success of internalization; for example, some root vegetables possess antimicrobial properties, which limits the growth and internalization of enteric bacteria . pathogens like e. coli o :h have been demonstrated to survive longer in the soil in the presence of rye and alfalfa roots (gagliardi and karns, ) . other work has demonstrated that pathogens enter root tissues at sites of lateral root emergence or through damaged roots (mendes et al., ) . salmonella and e. coli o :h have penetrated arabidopisis and lettuce plants' roots, while klebsiella pneumoniae have been detected on numerous plants' roots (tyler and triplett, ) . other examples include the invasion as well as (endophytic and systemic) colonization of barley roots by s. typhimurium, the shoots of black pepper stem cuttings by pseudomonas aeruginosa, as well as roots and shoots of tomato seedlings by p. aeruginosa (kutter et al., ; kumar et al., ) . it is, however, important to note that successful invasion of the root and shoot system may not guarantee translocation to the edible or foliar portions of produce. in some surveys, bacterial pathogens were detected in roots but not leaves of crops examined (watchel et al., ; warriner et al., ; bernstein et al., a; mitra et al., ; sharma et al., ) . a growing body of evidence suggests that seeds may serve as primary inoculum source in produce contamination. in the case of vegetables, seed sprouts have been implicated as the initial inoculum source, severally (warriner et al., ; deering et al., ; kumar et al., ) . in recent time, seeds have been recognized as a significant source of inoculum for foodborne illnesses associated with sprout consumption (mahon et al., ; national advisory committee on microbiological criteria for foods, ; buck et al., ; yang et al., ) . it is possible that enteric bacteria may be transmitted from contaminated seeds to sprout to mature plants, throughout entire plant life cycle up to consumption. the contamination may be transferred from seed again, thus persisting in produce cultivation cycles, for a long time. there is a record of e. coli :h adherence to outer surfaces and subsequent successful internalization of radish sprouts produced from contaminated seed during sprout growth (itoh et al., ) . rate and efficiency of uptake also depends on the type of produce, and the level of internalization varies widely among plants and even among different species of the same crop due to variations in intrinsic factors, which affect pathogen survival and proliferation (golberg et al., ) . for instance, certain produce items, like fully ripe tomatoes are typically in the ph range ( . e . ) which conventionally impedes growth of most enteric bacteria, whereas, the ph of numerous vegetables, melons, and soft fruits is usually . or higher, which is conducive for bacterial growth gagliardi et al., ) . therefore, gram-negative bacteria are more commonly associated with vegetables while molds and certain yeasts mostly occur on fruits, due to the differences in ph requirements of the respective groups of microbes (jay, ) . members of the brassicaceae family (radish, turnip and broccoli) were demonstrated to have a higher prevalence of salmonella contamination than lettuce, tomatoes and carrots when grown in contaminated soil (barak et al., ) . among leafy greens, radicchio and endive may be more likely to be contaminated than lettuce, spinach, parsley or cilantro (barak et al., ) . salmonella typhimurium has been demonstrated to internalize more efficiently in iceberg lettuce and arugula leaves compared to romaine, red lettuce, fresh basil, parsley and tomato leaves, which displayed only marginal internalization. listeria monocytogenes seems to exhibit a selective preference for certain vegetables like radishes and potatoes, as certain studies reported that although l. monocytogenes successfully invaded tissues of a wide variety of vegetables, radishes and potatoes appeared to be more often and severely contaminated (beuchat, ) . it is also apparent that l. monocytogenes does not survive and internalize satisfactorily on fresh carrot, in fact, low doses of raw carrot juice have been demonstrated to inhibit the growth of the pathogen (beuchat et al., ; farber and peterkin, ; oh, ; benkerroum, ) . internalization is believed to be a plant-pathogen specific interaction, and therefore, internalization success varies from pathogen to pathogen. a comparison of the internalization of l. monocytogenes to s. typhimurium on inoculated seeds of cress, radish, spinach, lettuce, mustard, carrots, and tomatoes showed significant variations in the rate and efficiency of internalization by the pathogens. under identical experimental conditions, s. typhimurium internalized into the roots of the vegetables, whereas, l. monocytogenes did not (jablasone et al., ) . similarly, while s. typhimurium was found to be associated with the internal portions of barley sprouts, l. monocytogenes, l. ivanovii and l. innocua were not (kutter et al., ) . furthermore, the degree of internalization is contingent on the serovar/strain (larsen et al., ) . gene expression, metabolic and antimicrobial capacities vary among strains. certain strains manifest up-regulation of peculiar genes like the pdu, cob-cbi, and out which improve carbon source utilization and may confer a competitive edge, thereby enhancing the survival and persistence of these strains (fox et al., ) . some e. coli strains possess metabolic capacities, which foster their survival in certain agroecosystems such as soils (franz et al., ) . in a bid to explain the strain-specific internalization dynamics, a five serovar salmonella cocktail (montevideo, michigan, poona, hartford and enteritidis) was inoculated into hydroponic growth substrates. serotypes montevideo and michigan were most prevalent, while enteritidis, hartford and poona were not detected in any of the tomato tissue samples (guo et al., ) . this is a quintessential illustration of internalization variation among serovars. likewise, salmonella serovars; cubana, infantis and typhimurium exhibited varying capabilities to internalize and colonize alfalfa sprouts when seeds were inoculated under identical environmental conditions (dong et al., ) . some scholars have endeavored to compare the survival of two arguably most prominent foodborne pathogens: e. coli and salmonella. serovars of both can proficiently adapt to environmental stress; -numerous strains are known to become habituated to low ph conditions and subsequently manifest remarkable tolerance to stress conditions. escherichia coli can perpetually evolve new varieties that have neither been previously reported nor characterized and which are capable of exploring and inhabiting previously unrecognized niches (newell et al., ) . both seem to be capable of long-term survival in the agricultural environment and on produce, but it is quite apparent that salmonella survives better than e. coli (brandl, ; mandrell, ; newell et al., ; schikora et al., ; ongeng et al., ) . many salmonella serovars bind to plants significantly better than e. coli strains. escherichia coli's inability to lower its metabolic rate to suit the low availability of accessible organic carbon and to competently cope with low nutrient conditions contributes significantly to its die-out in soils and on produce, and therefore, lowers its competitiveness (survival) compared to salmonella franz et al., , . internalization has been correlated with motility and chemotaxis. flagella mutants (flighi:tn , chey) deficient in motility and chemotaxis respectively have exhibited reduced attachment and penetration of lettuce leaves (kroupitski et al., ; lim et al., ) . it has also been hypothesized that products of photosynthesis serve as nutrients to aid internalization of pathogens (lim et al., ) . active internalization typically involves the penetration of bacteria through natural openings. the ability of foodborne pathogens to internalize in produce represents a significant public health risk because internalized pathogens are protected against optimized disinfection modes (meireles et al., ) except irradiation which seems capable of reasonably eradicating internalized pathogens in produce. the technique penetrates produce tissues to eliminate internalized pathogens, and gram-negative bacteria are very susceptible to even low doses (saroj et al., ; o'bryan et al., ) . however, treatment with irradiation may produce off flavors, colors and odors and may inactivate some of the nutrients (fan and sokorai, ) . it is, therefore, not accepted and endorsed for produce treatment. there are other relatively new technologies such as modified atmosphere packaging, ozone, ultrasound and ultraviolet treatments, which seem promising in ensuring the microbiological safety of fresh fruits and vegetable products (shayanfar and pillai, ) . however, limited commercial applications have been described for most of these new technologies. electron beam technology is another up-and-coming treatment option, which according to experts, can play a pivotal role in mitigating some of the contemporary microbiological risks facing the produce industry (shayanfar and pillai, ; lung et al., ) . it is an environment friendly, cost and time effective decontamination strategy that uses low-dose ionizing radiation to treat crops (-as well as other food items), to eliminate microbial contamination. it is capable of inhibiting the germination of crops and controls the rate of ripening of fruits and vegetables, thereby extending their shelf life (lung et al., ) . it inhibits a variety of enteric pathogens without compromising food sensory and nutritional qualities and can be used in combination with other traditional or non-traditional food processing technologies (lung et al., ) . regulatory authorities such as the us food and drug administration have approved it, but the full import of the safety of use is not yet conclusive. given the amount of evidence indicating that enteric pathogens (that are not plant pathogens) can invade and be internalized into plants, it is important to understand how such microbes establish access to plant tissues, as this may facilitate the development of strategies to reduce internalization. for successful colonization, major interactions take place between pathogens and their plant hosts that determine the success of the pathogenic attack (warriner and namvar, ). many enteric pathogens have devised mechanisms to overcome plants' basal defense mechanisms and innate immune responses (lim et al., ) . plants first line of response to foreign invasion is by the innate immune system. this consists of two main branches: pamp-triggered immunity (pti) and effector-triggered immunity (eti). in the first stage, microorganism associated molecular patterns (pamps or mamps such as flagellin, peptidoglycan, lipopolysaccharide) are identified by plant host receptors popularly known as pattern recognition receptors (prrs) (deering et al., ) . these batteries of receptors deployed by the host are designed to curb the growth and spread of the pathogen (ausubel, ) . pti response is broad-spectrum; sensitive to molecules familiar to many classes of microorganisms including non-pathogens. upon recognition, plant defense signal pathways are activated among which, jasmonate, salicylic acid and ethylene play essential roles. virulent plant pathogens may through diverse strategies, such as the production and secretion of effectors, efficiently override pti, for example, there are some 'effectors' that can overcome pti by interfering with mamp detection and subsequent defense signaling (kazan and lyons, ) . this results in effector-triggered susceptibility (ets). for susceptible interactions, effectors produced and released by the microorganism are transferred into the plant cell through the ttss (type iii secretion system). specific nucleotidebinding leucine-rich-repeat (nb-lrr) proteins encoded by resistance genes, resulting in eti and limitation of pathogen transmission to other tissues, recognize these effectors. while pti is considered the first line of defense against pathogenic infection, eti is an accelerated and amplified response, the outcome of which is often a hyper-sensitive response (hr) (spoel and dong, ) . the ability of pathogenic bacteria to colonize a plant may also be influenced by their interactions with other microorganisms either positively or negatively (deering et al., ) . if other microorganisms supply carbon sources (via degradation of cell wall polymers or induced secretion of sugars), or sequester antimicrobials, this can enhance pathogen colonization (bais et al., ; warriner et al., ; augimeri et al., ) . alternatively, plant pathogens that wound or destroy living tissue may create a microenvironment that is suitable for the survival and/replication of human pathogens (rashid et al., ) . pathogens are often associated more with plants whose tissues have been damaged by soft-rot pathogens compared to those with healthy tissues (brandl, ) . before pathogenic bacteria can colonize the surface or interior of a plant host, they have to contend with the naturally occurring microflora that is already established (deering et al., ) . the ability of the indigenous bacterial community to inhibit the growth of introduced enteric pathogens has been demonstrated by numerous studies (liao and fett, ; matos and garland, ; schuenzel and harrison, ; cooley et al., ; johnston et al., ) . there is direct evidence that the stomata play essential roles in internalization, host immunity and pathogen virulence of pathogens (kroupitski et al., ; zeng et al., ) . some researchers have reported that plant stomata close in response to plant pathogens and some human pathogens (melotto et al., ; roy et al., ) . escherichia coli o :h has been reported to trigger stomatal closure even under high relative humidity, a stressful environmental condition that generally weakens plant defenses against bacteria in field and laboratory conditions (roy et al., ) . stomata closure could be triggered by certain peptides such as flg produced by bacterial flagellin and lipopolysaccharides which are recognized by pamps or mamps in a salicylic acid-dependent manner. in the case of some plant pathogens such as xanthomonas spp. and pseudomonas syringae, virulence factors produced are capable of overcoming this innate immunity and counter stomata defense. for example, pst dc and several other pathovars of pseudomonas syringae, produce coronatine (cor), a phytotoxin which can reverse stomatal closure induced by bacteria or mamps (zeng et al., ) . stomatal immunity can diminish the penetration of human pathogens through the leaf epidermis, resulting in low bacterial titers in the plant apoplast (roy et al., ) . however, plant defense responses induced by pathogens vary and plants may recognize and respond to some human pathogens more effectively than others (roy et al., ) . for example, comparison of plant defense responses induced by e. coli o :h and s. typhimurium sl in arabidopsis thaliana and lettuce (lactuca sativa) revealed some variations. while e. coli o :h triggered stomatal closure, sl only induced a transient stomatal immunity. also, pr gene expression was significantly higher in arabidopsis leaves infected with e. coli o :h compared with sl (roy et al., ) . although, numerous studies have examined the intricacies of internalization in fresh produce, many of these are laboratory based. the few available field studies, which have mostly studied e. coli, indicate that internalization of pathogens may be not be very common in field settings erickson et al., b erickson et al., , erickson et al., , b . more field studies are therefore, required to properly understand the potential/likelihood of enteric pathogens to internalize in fresh produce as well as the actual factors that influence the success of internalization. to successfully achieve an acceptable level of microbiological safety for fresh produce, it is essential to control environmental contamination in the field by taking appropriate pre-harvest precautions. one fundamental factor to consider is the state or quality of the growing fields. fields on which wild or domestic animals have been recently grazed that have been subjected to flooding or may have been previously contaminated with manure constitute an unacceptable microbiological risk (turb e et al., ) . therefore, growers need to scrupulously investigate land history when selecting a location for produce cultivation (islam et al., a, b) . cultivation areas should be safeguarded from flooding, and fecal contamination and manure should be adequately treated (using popular methods like composting and aging) before application as fertilizer. also, suitable buffer zones (physical barriers) such as mounds, diversion berms, vegetative buffers as well as ditches should be erected between animal grazing regions and produce cultivation areas (james, ; olaimat and holley, ) . appropriate livestock waste disposal and farm general waste management should be adopted to ensure safety. numerous experts have highlighted the need for monitoring, regulation and control of the microbiological quality of irrigation water. several regional and international standards exist for irrigation water use and practices to prevent incidence of bacterial contamination. the use of potable water for irrigation (and other cultivation operations) is highly recommended. certainly, this is not economical in many instances and may increase production costs, which will raise prices; it is however, pertinent to public health safety. in developing countries, a myriad of safety regulations exists such as cessation of irrigation prior to harvesting, lowering of watering cans to reduce splashes from (contaminated) soil, adoption of furrow irrigation system over the use of sprayers which expose edible portions of leafy vegetables directly to irrigation water, and so on (keraita et al., ; amoah et al., ; uyttendaele et al., ) . in cases where surface water is the irrigation water source, drainage of contaminated water into the surface water reservoir may be prevented by constructing ditches, buffer strips, as well as retention and drainage systems. potential overflow points should be identified and eliminated. it is also important to determine (potential) points of contamination because control measures are bound to be more effective if focused on eliminating contamination at the source (madramootoo et al., ; pachepsky et al., ) . irrigation wells, functional septic, water and sewage systems should be installed and properly maintained especially during periods of excessive rainfall to prevent pathogen contamination (buck et al., ; olaimat and holley, ) . surface and groundwater resources should be protected from any potential sources of contamination including wildlife, animal waste, agricultural run-off, human activity, sewage, or industrial effluents. other management practices like; removal of riparian areas, erection of fences, and treatment of irrigation water (for example, using uv treatment) can be considered to enhance safety assurance of irrigation water. these precautions will minimize contamination risks on produce farms and should be applicable not just to supposed high-risk crops (such as leafy greens) but all produce (squash, and others) (strawn et al., b) . implementing some of these may, however, be costly and have negative impacts on landscape health. irrigation water sources should be routinely monitored to ensure microbiological safety (brackett, ; islam et al., b) . ideally, there should be more regular reporting on the microbiological quality of irrigation waters in different world regions. such surveys should reflect the true levels of actual pathogens rather than indicators, and bias should be avoided towards contaminated samples by intensively monitoring every irrigation source possible, and not just sites where extensive contamination has been known to occur (stoeckel, ) . as part of a total package of hygiene measures to prevent the transfer of foodborne pathogens, wild animals, birds, flies and rodents should be controlled in cultivation areas. interventions to mitigate wildlife intrusion of a farm may be costly and not entirely effective, especially if not done properly, thereby allowing certain animals direct access to crops. in many cases, it is not economical to fence large farms, but small farms can be fenced to restrict wild animals (jung et al., ) . other mechanical/biological control methods include the use of scarecrows, reflective strips, monitoring of animal tracks and field intrusion as well as gunshots to ward off pests and animals. mechanical traps and baits can be used to control mice and rodents. overall, practical, cost-effective methods should be adopted to mitigate wild sources and routes of produce contamination. considering that, in many important outbreaks, vegetable seed sprouts have been implicated as the initial inoculum source, the elimination of bacteria from seeds before planting has become crucial (buck et al., ) . chemical or physical treatment methods are usually used to decontaminate seeds, in a bid to reduce the risks of sprout borne disease outbreaks. however, this poses some challenges for growers, as the chosen decontamination method has to fulfill certain conditions. one important consideration is the preservation of seed viability. selected treatment dosage should be able to inactivate pathogens without adversely affecting seed viability (buck et al., ) . also, the treatment must be able to penetrate and access bacteria that may be residing in protected seed tissues, and finally, certain treatments may be inactivated by seeds, rendering them less effective (buck et al., ) . nevertheless, the efficacy of chemical seed treatments for sprout seed including chlorine compounds (commonly calcium and sodium hypochlorite), ethanol, hydrogen peroxide, calcium edta, -hydroxybenzoic acid, ozonated water and other commercial disinfectants have been extensively documented. it is also possible to use gaseous chemicals and thermotherapy (e.g., hot water treatment), although excessively high temperatures may affect sprout vigor. another potential issue with hot water treatment is that when treating large batches of seed, it is practically impossible to achieve temperature uniformity throughout the water bath. therefore, while a portion of the seeds receives the appropriate temperature-time exposure, some will still contain viable bacteria after 'treatment.' also, there is a potent risk of cross-contamination with this technique. other viable options include seed treatment with bacteriophage, combinations of thermotherapy with chlorine and the use of ionizing radiation. radiation is particularly appealing because it can penetrate seed tissues and possibly eliminate bacteria localized within protected tissues (buck et al., ) . however, it has been postulated that high levels of irradiation may distort the physiology and organoleptic properties of seedlings, more research is therefore, needed to evaluate the prospects and risks of this approach. other precautionary measures include testing seed lots for purity and germination rate prior to marketing, proper warehouse storage (in metal bins) until bagged, as well as ensuring general facility sanitation and employee hygiene (national advisory committee on microbiological criteria for foods, ). safety criteria and regulations are mostly region specific, it is however, critical to enforce these regulations, ensure that growers adhere to such and there is a need to constantly improve standards; if new information becomes available, regulations should immediately be updated (k€ opke et al., ) . most of the available data is from the developed world mainly from the us and certain parts of europe. it is necessary to develop surveillance and tracking systems and generate robust databases for other regions as well. more studies should be conducted under field conditions, rather than laboratory or greenhouse simulations, as this will provide a better understanding of how enteric pathogens behave in agricultural production environments. finally, and more importantly, it is necessary to ensure producers are mindful of their roles in assuring food safety. growers should be encouraged to adopt the best possible agricultural practices to ensure produce safety. it is also important to enlighten consumers about possible risks and appropriate mitigation strategies. there are wrong notions and misconceptions, which have to be corrected promptly, for example, many people believe it is not necessary to wash organically grown fruits and vegetables (leifert et al., ). it is evident that epidemiologic investigations are worthwhile as public health directives and policies based on investigation output have averted impending foodborne disease crises in many cases. the relevance of epidemiological surveys globally and regionally, therefore, cannot be overemphasized. this means that epidemiological investigation tools and systems need to be objective, updated, precise, flexible and timely. while significant progress has been achieved in the area of epidemiology, there are still certain cracks that need to be addressed. the use of routine, optimized clinical pathogen identification techniques may mean that new pathogens may likely be missed. this is a potentially grave issue, because periodically, since the development of foodborne disease surveillance, the list of foodborne pathogens has continued to expand. care should, therefore, be taken to avoid research bias since it is likely that produce items that have been previously associated with foodborne illness outbreaks and product recalls may receive particular scrutiny. new pathogens emerge due in part, to evolving ecology and technology while already recognized strains continue to evolve, potentially becoming smarter, evading and subverting detection, sanitization and plant host defenses. it is important to further understand the evolution dynamics and emergence of new pathogens, as well as develop and optimize methods to meet the emerging challenges. awareness and surveillance of viral and parasitic enteric pathogens need to be more robustly developed. although noroviruses, hepatitis a, rotaviruses as well as certain emerging viruses such as sars are well known, they are rarely routinely screened for in fresh produce in most countries. also, their ecology in fresh produce is poorly understood, for instance, the knowledge of the stability and persistence of human norovirus in foods has been garnered mostly from the study of surrogate viruses. more importantly, their significance in foodborne disease incidence remains undetermined. parasitic pathogens like ascaris, giardia, entamoeba, cyclospora, cryptosporidia and trichinella are recognized (newell et al., ; robertson et al., ) , but not all are routinely monitored in produce. the roles that livestock and wildlife play in pathogenic contamination of fruits and vegetables as well as their epidemiology through the food chain is poorly understood. it is difficult to compare the available studies because some have used naturally contaminated animals, while others used experimentally inoculated animals. the exact transport/transfer mechanisms of pathogens from animal fecal material or contaminated manure/soil to fruits and vegetables via splash are not yet properly understood. for example, it will be helpful to understand the specific spatial factors that influence the transfer of pathogens from fecal pellets to fruits and vegetables. the survival times for pathogens in fecal contaminants, manure, and manure-amended soils are inconsistent, reflecting the varying conditions under which many of the available studies have been conducted (these variations are demonstrated in tables e ). the fate of pathogens on the soil surface, the relationship between manure-derived pathogens and soil particles, as well as the states in which pathogens occur in soil slurry or manure mixtures, should be further explored. the exact mechanisms of uptake or (transmission) of pathogens from contaminated manure or manure amended soils to plants, particularly in field settings should be studied. this will facilitate the design of scientifically sound produce safety standards. the majority of studies available on pathogen transport in soils have been conducted using homogenized natural soils in laboratory designed soil columns. these may not be a true representation of field conditions and diversifying the experimental conditions will aid the development of efficient, grower-level interventions that will effectively reduce the likelihood of on-field contamination of produce. there are dissenting opinions among experts on a variety of issues pertinent to produce safety. with regards to the factors, mechanisms as well as principles that aid competent internalization and persistence of pathogens on produce, there are many variations. the available studies are difficult to compare largely because they have been conducted under varying physicochemical circumstances, types of microcosms, experimental conditions and used distinct strains (shown in tables e ). most studies were conducted under disparate environmental conditions, and accurate weather data necessary to interpret results from the varying sources is lacking. study results for one crop variety may indeed not hold true for other varieties, for instance, data for apples may not necessarily apply to all pome fruit and data for romaine lettuce may not apply to all leafy greens. when possible, varieties exhibiting greater potential for pathogen survival should be selected for experimental investigations. another relevant consideration for crop selection is preference for varieties that are indigenous to the region in question. some other seemingly trivial controversial issues include whether outer leaves are significantly more likely than inner leaves to become contaminated via splash and whether or not the potential for survival on the abaxial side of leaves is higher than on the adaxial side. the implications of dormant, non-dividing 'persister' cells occurring in certain plant pathogens on the ability to withstand environmental stresses and extensive survival as well as the issues surrounding linked resistance is still an important research debate. also, even though atmospheric deposition seems to be an uncommon route of pathogenic contamination for produce, it has been documented as a potentially important route (beuchat and ryu, ; harris et al., ; mei soon et al., ) . it will be worthwhile exploring how relevant this is for produce safety. while many of the available studies have made stringent efforts to simulate produce cultivation circumstances, it is extremely challenging to create precise/accurate environmental conditions in a laboratory setting. most studies are conducted under controlled laboratory conditions. factors like the biological activity of the soil, manure, water and crops, soil and water chemistries as well as meteorological elements such as wind, uv intensity, temperature, rainfall are simply impossible to replicate under laboratory conditions. laboratory scale model systems may provide important details about the roles of environmental variables on pathogen growth and survival in agricultural environments, but the slightest tweaks in experimental protocols can affect pathogen survival in agroecosystems. unfortunately, actual field-based studies are subject to disruption from unforeseen environmental events such as weather extremes and damage triggered by biological agents including insects or onset of plant diseases. more field studies (where typical agricultural practices and conditions are closely simulated) are therefore, highly desirable to further understand the persistence phenomenon. safety and ethical issues however restrict the use of pathogens in the greenhouse and field-based research. strategies to improve existing biocontainment and decontamination processes should be developed and optimized as soon as possible. another possible solution is to develop and optimize strategies that will cater for the experimental variations in model system development. an assessment and identification of environmental variables that influence the fate of test organisms should be included in experimental designs. despite meticulous planning however, a field trial may fail to yield serviceable results due to factors that are out of the researcher's control. consequently, more replicate trials may need to be conducted. furthermore, agronomic and farm management practices are not uniform in all regions, and production practices significantly differ from region to region depending on seasons and weather patterns within the same region. these often depend on operation scale, type of farming practices et cetera. the risks associated with conventional cropping systems are bound to differ from those of systems that combine intensive livestock farming with arable farming. in addition to general studies, a case-by-case approach should be considered where possible (if financial and technical resources, as well as other circumstances, permit) because farming operations vary widely from farm to farm and this influences the potential for pathogen occurrence, survival, proliferation and dissemination. the potential of fresh produce to harbor pathogens is now well recognized, and fresh produce has been established as a vehicle of foodborne disease. the diverse and complex sources and routes of enteric pathogens to fruits and vegetables have been widely researched. the interplay of land use, water management, weather patterns and specific pathogen properties and sources have been illustrated to have significant consequences for the microbiological safety of fresh fruits and vegetables. attempts have been made to understand the general microbial profile of fresh produce, the behavior, fate and transport of pathogens, as well as their location in and on plant parts. the facts gleaned from these studies have been the subject of many extensive reviews. there is abundant information about the factors that affect the contamination and persistence of pathogens on fresh produce. in light of the available evidence, significant effort must be made to efficiently monitor and illustrate recent trends in the occurrence of foodborne diseases associated with the consumption of fruits and vegetables. partnerships and collaboration among all relevant stakeholders; commercial growers, public health practitioners, veterinary and food safety experts and field biologists is necessary in order to ensure the safety of fruits and vegetables delivered to consumers. on a final note, the need to control all potential pathogen entry pathways has been established and is being continuously stretched by regulators and other specialists. there are numerous other factors along the food production chain that may predispose produce to microbial contamination. however, it is of utmost importance to avoid and control microbial contamination of produce at the preharvest stage. this is because contaminated manure, water and soil have been shown to indeed contaminate produce, and decontamination of produce, polluted arable soil and groundwater has proven to be a very challenging and expensive endeavour. the authors declare no conflicts of interest. . . effect of soil properties and environmental variables on the incidence of pathogens in soils water resources projects and their environmental impacts molecular techniques for detecting and typing of bacteria, advantages and application to foodborne pathogens isolated from ducks kinetics of pathogen destruction during storage of dewatered biosolids association of escherichia coli o : h with houseflies on a cattle farm irrigation water quality for leafy crops: a perspective of risks and potential solutions surveillance for sporadic foodborne disease in the st century: the foodnet perspective inactivation of ms- phage and poliovirus in groundwater lowcost options for reducing consumer health risks from farm to fork where crops are irrigated with a farm to fork risk assessment for the use of wastewater in agriculture in influence of temperature on salmonella survival in hog manure slurry and seasonal temperature profiles in farm manure storage reservoirs potential mechanisms for achieving agricultural benefits from biochar application to temperate soils: a review establishing a role for bacterial cellulose in environmental interactions: lessons learned from diverse biofilm-producing proteobacteria are innate immune signaling pathways in plants and animals conserved? escherichia coli o survival following the surface and sub-surface application of human pathogen contaminated organic waste to soil fate of escherichia coli originating from livestock faeces deposited directly onto pasture survival of e. coli o : h in organic wastes destined for land application persistence of a salmonella enterica serovar typhimurium dt clone in a piggery and in agricultural soil amended with salmonella-contaminated slurry effect of irrigation regimes on persistence of salmonella enterica serovar newport in small experimental pots designed for plant cultivation role of soil, crop debris, and a plant pathogen in salmonella enterica contamination of tomato plants occurrence of generic escherichia coli, e. coli o and salmonella spp. in water and sediment from leafy green produce farms and streams on the central california coast large multistate outbreak of norovirus gastroenteritis associated with frozen strawberries presence and public health implications of listeria monocytogenes on vegetables listeria monocytogenes: incidence on vegetables. food contr. 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the authors are grateful to the national council for scientific and technological development (cnpq) (grant # / - ) and the coordination for the improvement of higher education personnel (capes) (grant ## p ) for the financial support. oluwadara alegbeleye wishes to acknowledge the financial support of conselho nacional de desenvolvimento cientifico e tecnol ogico (cnpq) through award grant number / - . supplementary data related to this article can be found at https://doi.org/ . /j.fm. . . . key: cord- -d v b j authors: cheong, chang heon; lee, seonhye title: case study of airborne pathogen dispersion patterns in emergency departments with different ventilation and partition conditions date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: d v b j the prevention of airborne infections in emergency departments is a very important issue. this study investigated the effects of architectural features on airborne pathogen dispersion in emergency departments by using a cfd (computational fluid dynamics) simulation tool. the study included three architectural features as the major variables: increased ventilation rate, inlet and outlet diffuser positions, and partitions between beds. the most effective method for preventing pathogen dispersion and reducing the pathogen concentration was found to be increasing the ventilation rate. installing partitions between the beds and changing the ventilation system’s inlet and outlet diffuser positions contributed only minimally to reducing the concentration of airborne pathogens. emergency departments (ed) are visited by patients with various diseases, and thus, are vulnerable to the dispersion of airborne pathogens [ ] [ ] [ ] . in particular, if the hospital does not have isolation rooms [ ] [ ] [ ] for the treatment of patients carrying airborne pathogens or has more respiratory patients visit the hospital than the number of beds available in the isolation rooms, it is possible that patients with respiratory problems will be assigned to normal beds in the ed. in this situation, it is anticipated that the possibility of airborne pathogen dispersion in the ed increases. airborne pathogens are particles that are µm or smaller (aerosol) [ ] . as they travel in the air, their dispersion to nearby areas is possible, depending on the indoor air conditions. controlling the indoor air patterns, installing partitions between units to prevent airborne pathogen dispersion, and installing uv lamps to sterilize the pathogens can be considered as alternatives to prevent the dispersion of airborne pathogens inside eds. partitions are used in target ed in hospitals as a means to control the dispersion of airborne pathogens [ ] . among these options, we focused on the effects of arrangement of diffusers, the installation of partitions between beds, and increased ventilation rate affect the airflow patterns in an ed. each of these factors was applied to the bed areas of an ed, and the evolution of airborne pathogen dispersion was analyzed through a cfd (computational fluid dynamics) simulation. as mentioned previously, the objectives of the research were to investigate the effects of the locations of ventilation system diffusers, the presence of partitions between beds, and increased ventilation rate on the dispersion of airborne pathogens. therefore, the following scenarios were analyzed. first, the ventilation around the beds in the ed was increased from the regulated ventilation level of three air change per hour (ach) to the ventilation level of six ach, which is required for a negative-pressure isolation chamber. isolation rooms for patients with airborne pathogens require a minimum ventilation rate of - ach [ , ] . then, the effect on the pathogen dispersion control was analyzed. second, the location of diffusers was changed around the beds in the ed, and the consequential changes observed in the pathogen dispersion patterns were analyzed. in this case, the conditions were largely in one of two categories: supplying and exhausting the air over the respiratory system of a patient. an alternative was designed by considering the general design of inlets and outlets for negative pressure chambers, in which the air is exhausted at the restroom side or around the respiratory system of the patient. last, an additional partition was installed between the ed beds, and the degree of change observed in the pathogen dispersion patterns was analyzed. this approach is thought to be an alternative that can prevent the dispersion of airborne pathogens to adjacent beds. in south korea, generally, ed is designed with an open plan and the airborne pathogen can freely migrate from infected patient to any place in ed. the conventional fabric partition or curtains between beds easily pass the airborne pathogens smaller than µm. the impermeable partitions that have benefits such as easy installation and cheap price are thought to be useful to prevent the air pathogen transfer from a bed to another. however, airflow pattern and ventilation rate can influence the effects of the partition regarding airborne pathogen dispersion. this study investigates the combined effects of ventilation rate, inlet/outlet location and existence of partition in ed on airborne pathogen dispersion and consequent infection risk. the results of this study are expected to provide an effective architectural design guide for ed to prevent airborne infections. the subject for analysis was an ed based in seoul. the ed was consisted of faculty area, nurse station, laboratory, family waiting area, acute treatment area and general treatment area. the general treatment area where the emergency beds were located in the ed was selected as the analysis model. figure illustrates target area with ten beds without partitions. in the actual conditions, the space was open to some other areas in the department. the conditions concerning the surrounding areas, which cannot be specified, were excluded and the subject space was independently modeled in the analysis. a cfd analysis program, star-ccm+ v , was used to achieve the research objectives. star-ccm+ is a cfd simulation tool to fluid dynamics, particle/gas flow and heat transfer in a controlled volume. this software provides lots of physics models and analysis tools for the investigation. the airflow dispersion patterns and the airborne pathogen density distribution were analyzed in the study to estimate airborne infection risk. the subjects of the cfd analysis were the airflow and ventilation conditions of the bed area in the ed, and variation in the pathogen dispersion trend with the installation of partitions between the beds. for the star-ccm modeling, only the indoor air area was modeled as a solid object in autocad (computer-aided design, autodesk, san rafael, ca, usa), and imported to star-ccm+. a polyhedral mesh was selected for the analysis model, and a prism layer mesh was applied. for the equations governing the flow field of the three-dimensional model, the continuity equation, momentum equation, and equations related to the degree of turbulence were applied. for the turbulence analysis, the realizable k-ε turbulence model was used. the segregated flow model was selected for the fluid model. the three-dimensional model, the continuity equation, momentum equation, and equations related to the degree of turbulence were applied. for the turbulence analysis, the realizable k-ε turbulence model was used. the segregated flow model was selected for the fluid model. in this research, two analysis models were established based on the installation of partitions. figure shows the image of each simulation model with and without partition. the images of meshing conditions are also depicted in figure . the mesh and number of cells for each condition, and the boundary conditions are listed in tables and , respectively. the air supply and exhaust were in the upper area shown in figure . all patients were lying in the beds with their heads near the wall. according to the literature by sung ( ) , the minimum ventilation requirements for hospitals in south korea, as defined by current regulations, is approximately . ach. based on this, the baseline ventilation condition without airborne pathogen control was set at ach in this study. the entire air supply was assumed to come from the outside [ ] . the ventilation condition for pathogen control was set at ach. this is the minimum value in the range of ach- ach, which is the required ventilation rate inside negative pressure units suggested by the "plan for extended installation of negative pressure units at nationally designated isolation hospitals" published by the korea centers for disease control & prevention in [ ] . in this research, two analysis models were established based on the installation of partitions. figure shows the image of each simulation model with and without partition. the images of meshing conditions are also depicted in figure . the mesh and number of cells for each condition, and the boundary conditions are listed in tables and , respectively. the air supply and exhaust were in the upper area shown in figure . all patients were lying in the beds with their heads near the wall. according to the literature by sung ( ) , the minimum ventilation requirements for hospitals in south korea, as defined by current regulations, is approximately . ach. based on this, the baseline ventilation condition without airborne pathogen control was set at ach in this study. the entire air supply was assumed to come from the outside [ ] . the ventilation condition for pathogen control was set at ach. this is the minimum value in the range of ach- ach, which is the required ventilation rate inside negative pressure units suggested by the "plan for extended installation of negative pressure units at nationally designated isolation hospitals" published by the korea centers for disease control & prevention in [ ] . in this research, two analysis models were established based on the installation of partitions. figure shows the image of each simulation model with and without partition. the images of meshing conditions are also depicted in figure . the mesh and number of cells for each condition, and the boundary conditions are listed in tables and , respectively. the air supply and exhaust were in the upper area shown in figure . all patients were lying in the beds with their heads near the wall. according to the literature by sung ( ) , the minimum ventilation requirements for hospitals in south korea, as defined by current regulations, is approximately . ach. based on this, the baseline ventilation condition without airborne pathogen control was set at ach in this study. the entire air supply was assumed to come from the outside [ ] . the ventilation condition for pathogen control was set at ach. this is the minimum value in the range of ach- ach, which is the required ventilation rate inside negative pressure units suggested by the "plan for extended installation of negative pressure units at nationally designated isolation hospitals" the numbers of inlets and outlets, as shown in figures and , were slightly different in the base model and the type model. this was because the diffusers were placed in the spaces between the beds in the base model so that the ventilation would not occur directly in the face of the patient. the type model applied walls between each bed, and the ventilator supply was placed at every bed. the inlet condition for the star-ccm+ simulation model was set to the velocity inlet, and the outlet condition was set as a split ratio outlet. the buoyancy effect was considered, and the detailed thermal conditions of the simulation models are shown in table . assuming that the patient breathed times/min, had a breathing capacity of ml (about l/min), and their mouth measured . m × . m, the average air velocity of exhalation was calculated to be approximately . m/s. based on this, therefore, the average exhalation velocity was set in the model to be . m/s (red arrow in figure .). patients were assumed to be lying in the center of their beds. the exhalation was emitted from one patient, and the analysis was conducted accordingly. a passive scalar was selected to represent the pathogen included in the exhalation, and the concentration was . . according to previous study [ ] , the passive scalar method is also a good approach of small particles bioaerosols. the airborne pathogen dispersion pattern resulting from the exhalation, mixing of airflows, and turbulence patterns was visualized for analysis. figure shows the data measuring points for the analysis of pathogen concentration. the points were set at a height of . m, in consideration of the location of the respiratory system of a patient lying in a bed. the meshing conditions of the base ( , , cells) and type ( , , cells) models are provided in table . the black boxes above the beds are inlets and outlets of the ventilator. the bulge of the bed is . m × . m × . m. human body is expressed in figure and mouth is modeled top of the human body( . m × . m, . m height). simulation cases for analysis were established, as shown in table , to conduct a case study that analyzed the range of airborne pathogen dispersion according to the location of diffusers in the bed area of the ed, the ventilation rate, and the installation of partitions between beds. case is the condition that best represents the usual bed conditions of existing eds. the ventilation rate was ach, per regulation, with no partitions between the beds. fabric curtains are usually installed between the beds, but it was assumed that the curtains were left open, as in situations where undressing or exposing the body was not necessary. the inlets were around the periphery of the ed (region a), and the outlets were in the central area (region b). the inlet was located near the side of the patient's respiratory system. the conditions of case were the same as in case , only with the locations of the inlet and outlet switched, so that the outlet was located over the patient's respiratory system. cases and were the same as cases and , respectively, with the ventilation rate increased to ach. cases - had the same conditions as cases - , except for having partitions in place between the beds. as in the previous explanation of the analysis model, the total number of inlets and outlets was increased, and the air velocity at the inlets decreased accordingly with the installation of a diffuser for every bed. simulation cases for analysis were established, as shown in table , to conduct a case study that analyzed the range of airborne pathogen dispersion according to the location of diffusers in the bed area of the ed, the ventilation rate, and the installation of partitions between beds. case is the condition that best represents the usual bed conditions of existing eds. the ventilation rate was ach, per regulation, with no partitions between the beds. fabric curtains are usually installed between the beds, but it was assumed that the curtains were left open, as in situations where undressing or exposing the body was not necessary. the inlets were around the periphery of the ed (region a), and the outlets were in the central area (region b). the inlet was located near the side of the patient's respiratory system. the conditions of case were the same as in case , only with the locations of the inlet and outlet switched, so that the outlet was located over the patient's respiratory system. cases and were the same as cases and , respectively, with the ventilation rate increased to ach. cases - had the same conditions as cases - , except for having partitions in place between the beds. as in the previous explanation of the analysis model, the total number of inlets and outlets was increased, and the air velocity at the inlets decreased accordingly with the installation of a diffuser for every bed. simulation cases for analysis were established, as shown in table , to conduct a case study that analyzed the range of airborne pathogen dispersion according to the location of diffusers in the bed area of the ed, the ventilation rate, and the installation of partitions between beds. case is the condition that best represents the usual bed conditions of existing eds. the ventilation rate was ach, per regulation, with no partitions between the beds. fabric curtains are usually installed between the beds, but it was assumed that the curtains were left open, as in situations where undressing or exposing the body was not necessary. the inlets were around the periphery of the ed (region a), and the outlets were in the central area (region b). the inlet was located near the side of the patient's respiratory system. the conditions of case were the same as in case , only with the locations of the inlet and outlet switched, so that the outlet was located over the patient's respiratory system. cases and were the same as cases and , respectively, with the ventilation rate increased to ach. cases - had the same conditions as cases - , except for having partitions in place between the beds. as in the previous explanation of the analysis model, the total number of inlets and outlets was increased, and the air velocity at the inlets decreased accordingly with the installation of a diffuser for every bed. the wells-riley model is a method of expressing the probability of air-borne infection using the concentration of a steady-state infection source and thread ventilation [ ] [ ] [ ] [ ] [ ] . equation ( ) shows the expression of the wells-riley model. equation ( ) shows the modified wells-riley model for infection risk by spatial area. where: p i : airborne infection probability of a susceptible person; c: number of infection cases; i: number of infectors; p: breathing rate of a susceptible person; and q: quantum generation rate by an infector. where n: expected number of quanta. the quanta generation rate of influenza was set to particles/h [ , ] . the existing literature suggests that the quanta generation rate of influenza is - particles/h [ ] . in south korea, after the middle east respiratory syndrome (mers) outbreak in , only one patient companion can stay in the ed and take care of the patient near their bedside. other patient companions must wait in the waiting room. the duration of a patient's stay in the emergency room depends on the patient's condition, but in the worst case, a patient may stay for two or three days. however, in this study, we assumed that the condition is treated for h: h for blood tests and h of other medical treatments by hospital members' interview. the quanta generation scenario is depicted in figure . airborne pathogen concentration of each monitoring point is used to calculate airborne infection risk of neighboring non-infected patient. airborne pathogen concentration at x -y is used to evaluate companions' airborne infection risk. figure . figure shows the average concentration at all measuring points together with the beds of the patient with a respiratory problem who generated the pathogen (x -y and x -y ), the beds opposite to the pathogen source patient (x -y and x -y ), and the average pathogen concentration at the rest of the beds. according to figure , there was a large difference between the pathogen concentrations at the pathogen source bed and that at the rest of beds. first, the reduction of pathogen concentration with increased ventilation rate is well demonstrated in all cases analyzed (cases and vs. cases and and cases and vs. cases and ). the effect of switching the ventilation outlet from region b to region a varied depending on the existence of partitions. in the condition without partitions, there was minimal effect on the average concentration at the periphery, and the pathogen concentration at the pathogen source bed showed a slight increase (x ). however, the change of outlet location (from region b to region a) reduced the dispersion of the pathogen to the adjacent beds (x , x , x , and x ). placing a partition between the beds when the outlet location was at region b did slightly decrease the concentration at the pathogen source beds (x -y ) as compared to the condition without partitions. in the case of the condition with the partitions, switching the outlet location from region b to region a increased the concentration at the pathogen source bed. in addition, comparing cases and , changing the outlet location from region b to region a when partitions were between the beds contributed to a significant increase in the pathogen concentration at the adjacent bed (x -x ). the pathogen concentration at x -y and x -y in case exceeded the corresponding values in case and . however, installing partitions between the beds (case vs. cases and ) was confirmed to slightly reduce the average concentration levels at the rest of the beds on the periphery. figure shows the expected infection risk at each measuring point. the infection risk reflects the tendencies of the pathogen concentrations in each case; a higher pathogen concentration means a higher infection risk. increasing the ventilation rate is always desirable for the reduction of infection risk. figure illustrates that installing partitions contributed to a reduced infection risk in the periphery (a) and a clear reduction in the average infection risk at the increased ventilation rate of ach. however, the increased infection risk at the bed adjacent to the airborne pathogen generation source should not be neglected in the cases with partitions between the beds. estimated number of airborne pathogens and airborne infection risk at each monitoring point is demonstrated in appendix a. figure . figure shows the average concentration at all measuring points together with the beds of the patient with a respiratory problem who generated the pathogen (x -y and x -y ), the beds opposite to the pathogen source patient (x -y and x -y ), and the average pathogen concentration at the rest of the beds. according to figure , there was a large difference between the pathogen concentrations at the pathogen source bed and that at the rest of beds. first, the reduction of pathogen concentration with increased ventilation rate is well demonstrated in all cases analyzed (cases and vs. cases and and cases and vs. cases and ). the effect of switching the ventilation outlet from region b to region a varied depending on the existence of partitions. in the condition without partitions, there was minimal effect on the average concentration at the periphery, and the pathogen concentration at the pathogen source bed showed a slight increase (x ). however, the change of outlet location (from region b to region a) reduced the dispersion of the pathogen to the adjacent beds (x , x , x , and x ). placing a partition between the beds when the outlet location was at region b did slightly decrease the concentration at the pathogen source beds (x -y ) as compared to the condition without partitions. in the case of the condition with the partitions, switching the outlet location from region b to region a increased the concentration at the pathogen source bed. in addition, comparing cases and , changing the outlet location from region b to region a when partitions were between the beds contributed to a significant increase in the pathogen concentration at the adjacent bed (x -x ). the pathogen concentration at x -y and x -y in case exceeded the corresponding values in case and . however, installing partitions between the beds (case vs. cases and ) was confirmed to slightly reduce the average concentration levels at the rest of the beds on the periphery. figure shows the expected infection risk at each measuring point. the infection risk reflects the tendencies of the pathogen concentrations in each case; a higher pathogen concentration means a higher infection risk. increasing the ventilation rate is always desirable for the reduction of infection risk. figure illustrates that installing partitions contributed to a reduced infection risk in the periphery (a) and a clear reduction in the average infection risk at the increased ventilation rate of ach. however, the increased infection risk at the bed adjacent to the airborne pathogen generation source should not be neglected in the cases with partitions between the beds. estimated number of airborne pathogens and airborne infection risk at each monitoring point is demonstrated in appendix a. case and had the same ventilation rate of ach, but the ventilation system inlet and outlet locations were switched to compare the consequent pathogen dispersion patterns. figure shows the analysis results for cases and in plan (at . m height) and sectional (x ) views. figure a ,b are the analysis results, in plan and sectional views, respectively, for case , in which the air was supplied from the periphery and exhausted at the center of the room. figure c ,d show the results of a central air supply and peripheral exhaust, in other words, exhaust over the respiratory systems of patients, as in the conditions of case . white parts of figure b ,d are the bed and human body section. in terms of the pathogen concentration distribution, the pathogen normally dispersed towards the adjacent beds, in both cases and . the concentration in the area near the bed of the patient who released the airborne pathogen was slightly higher in case , where the air was supplied from the periphery (region a) that was above the patients' respiratory systems. the same pattern can be observed in figure a ,c, which show the sectional views. these figures show that, in both cases and , the pathogen dispersed towards the beds on the opposite sides. the pathogen concentration at the hallway side was higher in case than in case . it is therefore anticipated that the probability of infection for the people in the hallway and the patient on the other side would be greater in case than in case . case and had the same ventilation rate of ach, but the ventilation system inlet and outlet locations were switched to compare the consequent pathogen dispersion patterns. figure shows the analysis results for cases and in plan (at . m height) and sectional (x ) views. figure a ,b are the analysis results, in plan and sectional views, respectively, for case , in which the air was supplied from the periphery and exhausted at the center of the room. figure c ,d show the results of a central air supply and peripheral exhaust, in other words, exhaust over the respiratory systems of patients, as in the conditions of case . white parts of figure b ,d are the bed and human body section. in terms of the pathogen concentration distribution, the pathogen normally dispersed towards the adjacent beds, in both cases and . the concentration in the area near the bed of the patient who released the airborne pathogen was slightly higher in case , where the air was supplied from the periphery (region a) that was above the patients' respiratory systems. the same pattern can be observed in figure a ,c, which show the sectional views. these figures show that, in both cases and , the pathogen dispersed towards the beds on the opposite sides. the pathogen concentration at the hallway side was higher in case than in case . it is therefore anticipated that the probability of infection for the people in the hallway and the patient on the other side would be greater in case than in case . cases and are the same as cases and , respectively, except for the ventilation rate. the ventilation rates for cases and were increased to ach. the analysis results for cases and are depicted in figure . the concentrations of airborne pathogens in cases and were clearly lowered when the ventilation rate was ach, as compared to ach. in other words, pathogen control by increasing the ventilation rate would have a greater effect on the pathogen concentration than changing the ventilation inlet and outlet locations would. cases and are the same as cases and , respectively, except for the ventilation rate. the ventilation rates for cases and were increased to ach. the analysis results for cases and are depicted in figure . the concentrations of airborne pathogens in cases and were clearly lowered when the ventilation rate was ach, as compared to ach. in other words, pathogen control by increasing the ventilation rate would have a greater effect on the pathogen concentration than changing the ventilation inlet and outlet locations would. cases and have the same ventilation conditions as cases and , respectively, with partitions added between the beds. the upper and lower parts of these partitioning walls were fixed to the ceiling and the floor, thus spatially separating the pathogen source bed from the adjacent beds. the front side remained open. this partitioning format not only controlled the dispersion of airborne pathogens from the source bed to the neighboring beds but also limited the visual field of the medical personnel. figure a ,c show, in plan view, the pathogen dispersion for the conditions of changing the inlet locations at the ventilation rate of ach. in case , the air was supplied over the patient's respiratory system (region a), and the air supply pattern in sectional view was as shown in figure b . the pathogen concentration for case in plan view is shown in figure a . in case , the air was supplied from the hallway side (region b), resulting in concentrations in sectional view as shown in figure d , and plan view as shown in figure c . considering figure , it is evident that the partition delayed the airborne pathogen dispersion to the adjacent beds. when the ventilation system inlets were installed over the patients' respiratory systems (region a), the airflow into the inlet acted like an air-curtain and prevented the airborne pathogen dispersion from the emitting source to other places, and the outlet at the hallway provided a secondary barrier (case ). interestingly, in case , with a ach ventilation rate, the airborne pathogens were not sufficiently exhausted at the outlet above the patients' respiratory organs. the rest of the airborne pathogen moved to the hallway, and the airflow from the inlet installed in the hallway accelerated the dispersion of the airborne pathogen to the adjacent beds. as a result of these dispersion patterns, installing partitions caused airborne pathogen concentration to increase at x and x . however, installing partitions contributed to a decrease in the airborne pathogen concentrations at x and x . in case , the pathogen concentration in the pathogen source bed greatly increased compared to the other cases, which could increase the infection risk for medical staff working in the source bed area. cases and have the same ventilation conditions as cases and , respectively, with partitions added between the beds. the upper and lower parts of these partitioning walls were fixed to the ceiling and the floor, thus spatially separating the pathogen source bed from the adjacent beds. the front side remained open. this partitioning format not only controlled the dispersion of airborne pathogens from the source bed to the neighboring beds but also limited the visual field of the medical personnel. figure a ,c show, in plan view, the pathogen dispersion for the conditions of changing the inlet locations at the ventilation rate of ach. in case , the air was supplied over the patient's respiratory system (region a), and the air supply pattern in sectional view was as shown in figure b . the pathogen concentration for case in plan view is shown in figure a . in case , the air was supplied from the hallway side (region b), resulting in concentrations in sectional view as shown in figure d , and plan view as shown in figure c . considering figure , it is evident that the partition delayed the airborne pathogen dispersion to the adjacent beds. when the ventilation system inlets were installed over the patients' respiratory systems (region a), the airflow into the inlet acted like an air-curtain and prevented the airborne pathogen dispersion from the emitting source to other places, and the outlet at the hallway provided a secondary barrier (case ). interestingly, in case , with a ach ventilation rate, the airborne pathogens were not sufficiently exhausted at the outlet above the patients' respiratory organs. the rest of the airborne pathogen moved to the hallway, and the airflow from the inlet installed in the hallway accelerated the dispersion of the airborne pathogen to the adjacent beds. as a result of these dispersion patterns, installing partitions caused airborne pathogen concentration to increase at x and x . however, installing partitions contributed to a decrease in the airborne pathogen concentrations at x and x . in case , the pathogen concentration in the pathogen source bed greatly increased compared to the other cases, which could increase the infection risk for medical staff working in the source bed area. figure shows the analysis result of the cases considering the conditions of installing partitions between the beds, changing the ventilation system inlet/outlet locations, and increasing the ventilation rate to ach. the simulated results of cases and demonstrate that the increase in airborne pathogen concentration occurred mainly at x . the increased ventilation ensures a solution for airborne infection prevention. the effects of installing partitions at a ventilation rate of ach varied depending on the ventilation system's inlet and outlet locations. locating the inlets above the patients' respiratory organs still generated turbulence and increased the airborne pathogen concentration at the adjacent beds. the simulation results of case clearly show that the increased ventilation rate of ach and partition installation effectively reduced the airborne pathogen figure shows the analysis result of the cases considering the conditions of installing partitions between the beds, changing the ventilation system inlet/outlet locations, and increasing the ventilation rate to ach. figure shows the analysis result of the cases considering the conditions of installing partitions between the beds, changing the ventilation system inlet/outlet locations, and increasing the ventilation rate to ach. the simulated results of cases and demonstrate that the increase in airborne pathogen concentration occurred mainly at x . the increased ventilation ensures a solution for airborne infection prevention. the effects of installing partitions at a ventilation rate of ach varied depending on the ventilation system's inlet and outlet locations. locating the inlets above the patients' respiratory organs still generated turbulence and increased the airborne pathogen concentration at the adjacent beds. the simulation results of case clearly show that the increased ventilation rate of ach and partition installation effectively reduced the airborne pathogen the simulated results of cases and demonstrate that the increase in airborne pathogen concentration occurred mainly at x . the increased ventilation ensures a solution for airborne infection prevention. the effects of installing partitions at a ventilation rate of ach varied depending on the ventilation system's inlet and outlet locations. locating the inlets above the patients' respiratory organs still generated turbulence and increased the airborne pathogen concentration at the adjacent beds. the simulation results of case clearly show that the increased ventilation rate of ach and partition installation effectively reduced the airborne pathogen dispersion. however, the large increase in the pathogen concentration at the pathogen generation bed must be mitigated. the study focused on the effects of some potential solutions for preventing the spread of airborne pathogens in eds: changing the ventilation inlet/outlet locations, installing partitions, and increasing the ventilation rate. in terms of ventilating the ed, supplying the air from the center (region b) and exhausting over the patients' respiratory systems (region a) helped prevent dispersion of the airborne pathogen when partitions were not present. changing the ventilation inlet and outlet locations in conditions of low ventilation rate and partition installation could cause the undesirable dispersion of the airborne pathogen (case ). installing the outlet of the ventilation system over the patients' respiratory systems was found to greatly increase the pathogen concentrations around the airborne pathogen source. yang et al. ( ) analyzed by cfd, it was said that setting the parallel bed layout and the exhaust port near the patient's head will lower the probability of air infection in the isolating room [ ] . hospital managers should be considered the placement of beds and the direction of airflow to reduce airborne infections. ordinary breathing generates lots of bio-aerosol ( µm or smaller) that can be transferred via air [ ] [ ] [ ] . thus, it is concluded that switching the ventilation inlet/outlet locations presents a high risk of increasing the probability of infection for medical staff providing medical treatments, such as endotracheal intubation [ , ] and nebulizing [ ] , to patients. installing partitions between the beds generally offered the advantage of reducing the average concentrations of pathogens in the ed (cases , , and ). however, it also had the shortcoming of increasing the pathogen concentration at the beds opposite and adjacent to the pathogen-source bed. these results suggest that patients who are at high risk of airborne infections may be able to provide appropriate patient placement in the ed. physical blocks primarily prevent the spread of airborne pathogen between the emergency beds [ ] . according to the cfd analysis, hospital curtains were presented in a simple and effective way for the spread of infection [ ] . thus, the airborne pathogens tend to accumulate around the source. increased ventilation should, therefore, accompany partition installation in the ed. it should be ach recommended by who rather than korean ventilation's criteria. increasing ventilation rate requires additional hvac system installation and surplus energy consumption. natural ventilation or hybrid ventilation method may be implemented to reduce energy consumption. however, natural ventilation has limitation or disadvantage regarding airflow control, heat recovery, outdoor contaminants incoming [ ] , difficulty in securing sufficient and consistent ventilation rate by season changing [ , ] . to reduce energy consumption, partial fan assisted natural ventilation with appropriate filtering system, dedicated outdoor air system (intermediate season) and heat recovery system (heating season) in hvac system can be good solutions. most hospitals use partitions, but each hospital has a different material. future research of the effect of airborne infection of the partition is also necessary. future research needs to investigate the influence of airborne infection depending on the material, installation structure and usage method of the partition. the south korean government tries to enhance the quality of ed facilities in major hospitals to prevent airborne pathogen infections. however, the korean building code still does not require the performance enhancement of small hospital and ed building equipment with respect to airborne infection prevention. this study provides the basis for design guidelines for airborne-infection prevention for small hospitals and eds. the limitation of this study did not take into account the various factors affecting the airflow, such as natural ventilations. future research needs to consider the airflow around the ceiling diffuser and approach the multidisciplinary research on hospital facilities and built environment will be conducted. our research investigated the effect of various architectural features in eds on indoor pathogen concentration distribution. the major variables considered were ventilation rate, installation of partition walls between beds, and changing ventilation inlet/outlet locations. the overall analysis is summarized as follows. ( ) the most effective method for controlling airborne pathogen dispersion is increasing the ventilation rate. ( ) changing the ventilation inlet/outlet locations generally results in good prevention of airborne pathogen dispersion. however, it can also cause undesirable airborne pathogen dispersion in conditions with low ventilation rates and partitions (case ). ( ) installing partitions could contribute to decreasing the average airborne pathogen concentration (cases , and ). however, it was also observed that the partitions could increase the pathogen concentrations in the beds opposite and adjacent to the pathogen source. increasing the ventilation rate can enhance the effect of installing partitions. ( ) in the analysis, the most effective method for pathogen control was to use all the methods studied: increasing the ventilation rate, installing partitions, and positioning the ventilation system outlets over the patients' respiratory organs (region a). further research will be conducted to improve architectural techniques for preventing the dispersion of airborne pathogens. our research only analyzed the concentrations of the airborne pathogen; therefore, it has the limitation of not indicating the real probability of infection. in our future research, an analysis of the probability of infection by the airborne pathogen will be included. author contributions: chang heon cheong designed the research plan, performed the cfd simulation and drafted the manuscript. seonhye lee reviewed the results of the study relating to the hospital environment, airborne infection, and public health. all authors read and approved the final version of the manuscript. the authors declare no conflict of interest. emergency nurses' perception and performance of tuberculosis infection control measures influencing factors on the compliance about standard precautions among icu and er nurses press release for th regular session of the national assembly of korea guidelines for preventing the transmission of mycobacterium tuberculosis in health-care settings american institute of architects (aia). - guidelines for design and construction of hospitals and health care facilities isolation rooms: design, assessment, and upgrade hvac system and contamination control in hospital relating mers natural ventilation for infection control in health-care settings improvement of airborne infection prevention methods in emergency room by design case study korea centers for disease control & prevention. plan for extended installation of negative pressure units at nationally designated isolation hospitals cfd simulation of airborne pathogen transport due to human activities an advanced numerical model for the assessment of airborne transmission of influenza in bus microenvironments modelling the risk of airborne infectious disease using exhaled air risk of indoor airborne infection transmission estimated from carbon dioxide concentration airborne contagion and air hygiene spatial distribution of infection risk of sars transmission in a hospital ward influenza outbreak related to air travel review and comparison between the wells-riley and dose-response approaches to risk assessment of infectious respiratory diseases hvac filtration for controlling infectious airborne disease transmission in indoor environments: predicting risk reductions and operational costs simulation of the aii-room for preventing spread of the air-borne infection in hospital inhaling to mitigate exhaled bioaerosols the size distribution of droplets in the exhaled breath of healthy human subjects airborne infectious disease and the suppression of pulmonary bioaerosols compliance with nosocomial infection control and related factors among emergency room nurses the relationship between empowerment and performance of infection control by emergency department nurses evaluation of droplet dispersion during non-invasive ventilation, oxygen therapy, nebulizer treatment and chest physiotherapy in clinical practice: implications for management of pandemic influenza and other airborne infections reducing risk of airborne transmitted infection in hospitals by use of hospital curtains quantifying the impact of traffic-related air pollution on the indoor air quality of a naturally ventilated building assessment of natural ventilation potential for residential buildings across different climate zones in australia the effect of the london urban heat island on building summer cooling demand and night ventilation strategies key: cord- - vjfkfd authors: peng, shanbi; chen, qikun; liu, enbin title: the role of computational fluid dynamics tools on investigation of pathogen transmission: prevention and control date: - - journal: sci total environ doi: . /j.scitotenv. . sha: doc_id: cord_uid: vjfkfd transmission mechanics of infectious pathogen in various environments are of great complexity and has always been attracting many researchers' attention. as a cost-effective and powerful method, computational fluid dynamics (cfd) plays an important role in numerically solving environmental fluid mechanics. besides, with the development of computer science, an increasing number of researchers start to analyze pathogen transmission by using cfd methods. inspired by the impact of covid- , this review summarizes research works of pathogen transmission based on cfd methods with different models and algorithms. defining the pathogen as the particle or gaseous in cfd simulation is a common method and epidemic models are used in some investigations to rise the authenticity of calculation. although it is not so difficult to describe the physical characteristics of pathogens, how to describe the biological characteristics of it is still a big challenge in the cfd simulation. a series of investigations which analyzed pathogen transmission in different environments (hospital, teaching building, etc) demonstrated the effect of airflow on pathogen transmission and emphasized the importance of reasonable ventilation. finally, this review presented three advanced methods: lbm method, porous media method, and web-based forecasting method. although cfd methods mentioned in this review may not alleviate the current pandemic situation, it helps researchers realize the transmission mechanisms of pathogens like viruses and bacteria and provides guidelines for reducing infection risk in epidemic or pandemic situations. volume, contact angle and environmental temperature were analyzed and the lifetime of droplets under those conditions was investigated. the evaporation of droplets will also be affected by the dust in the air, and this factor should be also considered in the future. different from other particles, pathogens are much smaller and their diameters are generally no more than nm and their motion is largely affected by flowing air, hence it is difficult to analyze their trajectories directly in the atmospheric environment. with the development of computer science, a new method based on computation fluid dynamics (cfd) can be used to solve this problem and it has been already well developed over the years. [ ] and in the next decades, an increasing number of investigations about air pollution, atmosphere environment and pathogen transmission can be found. as mentioned above, the droplet can bring the pathogen into the airflow and hence cause infectious diseases due to the spread of it. normally the droplet with pathogens is generated by coughing or sneezing from the infected and the procedure of droplets generated by sneezing is shown in fig. : the impact of covid- is global and the pandemic situation is closely related to the health of every individual. it does not mean that there is no way to prevent or control it effective vaccines are unavailable though. understanding the transmission of infections such as covid- in various media is of great importance. in this review, the principles of different cfd algorithms are described concisely and intuitively; theories and applications of cfd in investigations of pathogen transmission are summarized. the objective of this research work is to indicate the important role of cfd method in analyzing pathogen transmission. through summarizing various applications of cfd method, the transmission mechanism of pathogens and prevention methods are also concluded in this work. three steps are necessary for numerical analysis by using cfd tools: ( ) generating the mesh model with high quality is the key to ensure the accuracy of calculation; ( ) boundary conditions are required to define variables at the boundary. ( ) different algorithms that can be selected in cfd determine the way of iteration. this work carried out in this section is to summarize the feature of cfd from three aspects: simplification, algorithms diversity and maneuverability. different from experimental methods, the cfd method based on mathematical models that can be operated on computers is effective and cost-saving. in numerical simulations, the pathogens are carried by small particles like solid particles or droplets can be defined by calculation models. although the biological properties of the pathogen are complicated and various, the shape feature of the pathogen carrier is relatively simple to describe. in cfd, those particles can be defined as spheres, tetrahedrons, hexahedrons and even by the shape factor, then the pathogen transmission in different environmental fluids can be solved by utilizing multiphase models. moreover, the transport species model can be also applied to simulations of it. in this model, the infectious pathogen in the air is defined as the pollutant source with a constant concentration (generally measured by the field experiment). characteristics of fourier heat transfer law used to describe the heat exchange: fick law used to describe the mass exchange: (j j is the source diffusion relative to coordinates, d is the diffusion coefficient between two sources) () reynolds transportation law calculates the source quantity in control volume at time "t", which can be described as: introducing the continuous equation: and guass law (divergence law) then, the improved transportation can be written as: combining the balance equation of forces: then, the equation can be written as the form shown below: considering the acceleration caused by forces except the drag force and solving the particle motion in each step by iterative calculation: besides, the volume of fluent (vof) model performs well in simulating pathogen transmission, especially in the gas-liquid interface. in a control volume, the total value of each phase equals % and there are three situations in vof: if the volume fraction of "q" phase is  ,then: ( ) α  , no "q" phase in this cell; ( ) α  , the cell is full of "q" phase; ( ) < α < , interface between "q" phase and other phases can be found in the cell. the momentum equation and the energy equation of vof are determined and shared by each phase. the momentum equation mainly depends on characteristics and volume fraction of each phase and it can be written as below: energy equation of vof can be written as: j o u r n a l p r e -p r o o f k eff is effective thermal conductivity; j j,q is diffusion flux of "j" phase in "q" phase while the h j,q represents the enthalpy of it; s h is the volume of the heating source defined by users. the energy was defined as a variable relating to the average quality in vof: ( ) resistance characteristics in all directions are assumed the same, and the equation can be written as: generally, the inertia resistance coefficient c and viscous coefficient   are needed as the parameter of the boundary condition. taking the medical mask as an example and the way of obtaining these parameters are shown in fig. : the pressure difference (p -p ) between both ends of material from the mask as shown in fig. j o u r n a l p r e -p r o o f can be measured under a velocity input (v i ). the relationship between pressure difference and the input velocity can be described by the equation as: then the coefficient c and   can be calculated in the case of density  and thickness n  are known. some of investigations about transport species model and multiphase model are listed in tab several algorithms are mainly used in recent as summarized in table. . although the mechanism of pathogen transmission in the fluid is complex, the motion of pathogens still follows the hydrodynamics law and can be solved by mathematics models of cfd algorithms. for example, the lbm method can be used to solve pathogen transmission in small-scale while fdm method can be applied on the large-scale transmission of the pathogen. cfd tools are in high compatibility and their computing files can be transferred in a variety of software. in general, the structure of cfd software consists of three parts: pre-processing, solver and post-processing and fig. below shows some options of each part. pathogen transmission in the environment is a complex process. for the sake of the accurate simulation result, the calculation model and parameters of the simulation are necessary. furthermore, epidemic models should be taken into account in numerical simulations. by using experimental methods to get the data that is required in the boundary condition is important, besides, experimental data are also needed to validate the simulation result. this section summarizes various epidemic models that should be used in the simulation. moreover, experimental methods which can be applied to analyzing pathogen transmission are presented in this section as well. although some of the experiments summarized were not used in combination with cfd methods, they can provide valuable references for similar studies by using numerical simulations. poussou, et.al [ ] investigated the effects of moving people on pollutant diffusion and airflow. by combining the piv experiment technology, they used cfd method with a second-order upwind scheme to simulate the airflow. the re-normalization group (rng) k-ε was used in simulation in order to solve the turbulence with the good performance of accuracy, efficiency and robustness; in gao and niu [ ] study, rng k-ε model including the effect of low-reynolds-number is used to solve the airflow and the diffusion of tracer gas which can represent the contaminant transmission are calculated by the equation below: where t,  and  are time, air density and tracer gas concentration respectively. unsteady flow is a big challenge in accurate simulation as zhang, et.al [ ] indicated. flow in the environmental channel is always unsteady, and it hence increases the complexity of simulation on pathogen transmissions. how to treat an unsteady flow as the steady flow in practice is still a difficulty. by defining the wave in hydraulic calculation can effectively simplify the disturbance in unsteady flow. capillary wave [ ] can reflect the disturbance brought by some factors to surface of fluid which can be written as: ( ) for shallow waves: ( ) for deep waves: ku [ ] presented a "waveform" method to model unsteady flow in blood which reflects the relationship between time and volumetric flow rate: fig. volumetric flow rate mantha, et.al [ ] used this method in the simulation of biological flows and found the relationship between wall shear stress and the location of the aneurysm; nanduri, et.al [ ] also used "waveform" to solve the unsteady laminar flow and the objective of this study is to build a human body surface model to simulate the airflow around the body. although this method is useful for analyzing particle transportation in biological flows, it is not suitable for simulating unsteady flow in the atmosphere due to greater disturbance. more, in order to simplify the model for analyzing the airflow in building, axley [ ] presented a multi-zone model which allows users to calculate the hourly rate of airflow between various rooms and dols and walton et.al [ ] improved this model by providing the equation of mass conversion as: based on the dispersal theory which is not limited to the wall-mixed region, multi-zone model parameters should also be considered as well. airflow caused by temperature difference will affect pathogen transmission. chen, et.al [ ] simulated the three cases by combing the multi-zone model and two-way air flow effect in order to demonstrate the effect of temperature difference on air quality of indoor. it can be found from one of the cases they studied, the airflow generated by the temperature difference between bathroom and corridor can transport infectious pathogens, and hence the door of infected zoom should be closed as they suggested. closing doors and windows in a room is not equivalent to obtaining a closed space. the crack of the door and windows is always ignored by researchers when simulating the airflow or pathogen spreading in the building or a single room. by using multi-zone method in cfd simulation, yang, et.al [ ] analyzed the effect of stack and wind effect on contaminating dispersion and found that these factors will cause the contamination horizontally or vertically spread. their research also indicated that the pollutant gas can be transported through cracks of doors and windows and may cause infectious disease. because of the great effectiveness, multi-zone method was widely used in many cases which can be found in wu ( ) where s is the susceptible people in an area, i is the number of infectious people and p represents the pulmonary ventilation rate of susceptible people. zhu, et.al [ ] investigated the potential risk of infection in public transportation by using wells-riley model in cfd simulations. it was proved in their study that the closer to the operating exhaust in the bus the infected person is, the smaller the infection risk bringing to others is. besides, this study indicated that the ventilation system of most of buses is not effective because there is only one single exhaust was located in the middle of the cabin or the back wall. yan, et.al [ ] studied the transmission of coughing particles in the breathing zone of people. in their investigation, the method that combines the wells-riley model and the lagrange model in cfd was used. it was illustrated from this study that the location of releasing particles will affect the particle travel distance. based on the well-riley model, this research work has also presented a quantifiable approach to assess the infection risk of passengers. these studies are helpful for improving the design of the vehicle ventilation system and hence reduce the infection risk though, they did not consider the effect of altitude on airflow patterns in vehicles. the wells-riley model can also be applied to building simulation. niu based on physical characteristics like aerodynamics of respiration droplets, chaudhuri, et.al [ ] proposed a numerical model for the early state of covid- pandemic by integrated the chemical mechanism and pandemic evolution equations. the " " this work derived by using the theory of collision rate represents the lifetime of the droplet. it can be written as: some investigations based on these models are listed below in tab. : more, hathway [ ] combined the cfd method and sir model in order to analyze pathogen transmission in hospital space and asanuma and kazuhide ito [ ] predicted the exposure risk of the population in the hospital by using cfd with considering the sir model. from these investigations, it can be found that this epidemic model is well performed in simulating the spread of infectious diseases. however, the number of researches that applied these models to cfd simulation is still a small amount due to the complexity of modelling and calculation in simulating the airflow or particle transport among a crowd of people. in cfd simulation, not only the mesh model is crucial but also the parameter of simulation is of great importance to let users obtain the results they require. generally, the boundary condition such as velocity, pressure, turbulence intensity can be measured from experiments. in recent, micro-particles experiments and tracer gas experiments are most used in investigations of airborne transmission. romano, et.al [ ] simulated the airflow pattern and concentration of airborne particles in an operating theater (ot) by using cfd method. they also conducted an experiment in order to verify the accuracy of simulation results and in their experiment, a six-way aerosol distributor was used to convey the generated aerosol particles; opc (optical particles counter) equipped with a dilution system was used to measure the particle concentration; a rotating vane anemometer and a thermo-anemometer were used to measure the velocity and temperature respectively. they also validated the simulation result by comparing the data measured from the experiment and found that the experimental and numerical data were well coincided (error is less than % for temperature and % for velocity). the value of mean absolute percentage error for particle concentration is % though, the experimental curve and the numerical curve are similar in changing trends. therefore, experiments involving particle-fluid flow are more suitable for qualitative analysis, because it is hard to accurately control conditions such as temperature, pressure, stable velocity of flow. zhou, et.al [ ] established a model which can be used to predict the distribution of negative ions produced by the air ionizer and the efficiency of this device. in their experiment, an emission system consisting of a compressor and nebulizer was used to compress the filtered air and aerosolize the j o u r n a l p r e -p r o o f bacteria; an ion counter was used to test the emission concentration. in order to present their experiment clearly, the installed experimental system is shown below in fig. : fig. the detailed experimental setup [ ] the objective of the experiment carried out in this work was to measure the susceptibility besides, it was proved that the bacterial load in the shower air will increase while turning on the shower spray. the effect of droplet velocity and distribution on aerosolized bacterial groups was not given this study, more, the parameter of shower such as water temperature, nuzzle structures should be also considered in the experiment as well. choi, et.al [ ] classified the airborne particle according to their optical properties by using experimental methods. ink-jet aerosol generator (ijag) was used to generate, dry the airborne particle, the light-scattering signal was used to estimate the correlation value in the classification analysis of airborne particles. the correlation value proposed in this work is helpful for particle detection and classification though, how to apply this method to detect other airborne pathogens with more complicated biological characteristics is required to be furthered. mei ( ) conveyed air from experiment needs to be filtered; ( ) particles should be uniformly delivered. j o u r n a l p r e -p r o o f experiments to analyzed the particle are useful for understanding the motion law of it. however, it is difficult to massively measure the characteristic of nano-scale particles. the tracer gas method is also a common method in analyzing the pollution diffusion and airflow patterns. tracer gas can be mixed with air without any changes and it can be easily detected because of special physical characteristics. helium, nitrogen, argon and carbon dioxide are always chosen to carry out the experiment as a tracer gas. gao, et.al [ ] combined the use of experiment and cfd method to study airborne transmission in different flats of a high-rise building and to verify their simulation, the data of tracer gas experiment from denmark aalborg university [ ] is used. the analysis of this work is comprehensive by illustrating the transmission mechanism of the airborne virus and how to control virus transmission in a high building based on this investigation is needed to be furthered. to investigate airborne transmission between horizontal adjacent units, wu, et.al [ ] analyzed influence factors of transmission route especially the contribution of wind force and thermal buoyancy force and found from the result that the wind force is the main driving force to affect the inter-unit dispersion. the experiment conducted in this work is conducted in a slab-type building in hongkong, sf was used as the tracer gas and injected by the air samples; co was used to calculate the ventilation rate and monitored by tsi q-trak and co sensor. although the spread risk may be overestimated in the analysis because the crack of the door and windows can cause the pathogens aerosol deposit, this work still provides a valuable study in identifying the possible transmission route of the airborne. ai, et.al [ ] used a tracer gas (no ) experiment to examine the characteristics of airborne transmission of the exhaled droplet between two people in an experimental room. two manikins were used to represent an exposed people and an infected people; air velocity was measured by the swema omnidirectional anemometer; pt sensor was used to monitor the air temperature; to test the tracer gas concentration, a faster concentration meter (fmc) and innova multi-gas sampler and monitor are used. this work has indicated an interaction between exhaled gas and supply flow and analyzed the impact of these factors on infection risk for an exposed person facing an infectious person. although the experiment carried out in this work was based on a steady-state condition without taking the impact factor of time into consideration, it provided an effective method for researches afterward. ( ) culturing and filtering the suitable bacteria; ( ) aerosolizing the bacteria particles and conveying into measuring environment; ( ) analyzing the airborne transmission of e. coli by using pcr. and the flow chart given by them is shown below in fig. : fig. experiment process of tracing bacteria [ ] it will be more persuasive if this process can be carried out in an experiment of researches by using the tracer gas method or particle experiment. however, it will also increase the risk in conducting experiments if the bacteria or virus are highly infectious. overall, both the particle experiment and tracer gas experiment can help people understand the process of pathogen transmission, moreover, it provides crucial information for cfd users. on the one hand, the information including experimental data can be used as boundary conditions in cfd simulation; on the other hand, the results of the experiment can be quantitively or qualitatively verified to ensure the accuracy of cfd simulation. therefore, designing an effective experiment in analyzing pathogen transmission is necessary, it makes the simulation result more convincing. j o u r n a l p r e -p r o o f transmission of pathogens can be different in various spaces and when the epidemic outbreaks caused by infectious pathogens, hospitals will become a high-risk place and may lead to a second infection. how to control the pathogen in hospitals by using an effective ventilation system becomes a great concern. kao et.al [ ] [ ] they used the tracer gas no to replace the viral gas emitted from the patient and simulated three cases under different volumes of supplied air and exhausted air, the simulation results presented the diffusion process of tracer gas as in fig. : fig. the simulation of tracer gas diffusion [ ] in the same year, this research group studied a similar topic by using the tracer gas and cfd method. in this analysis, the stack effect of high rise building on airflow is considered and the simulation model is based on the general hospital k in korea as shown in fig. . fig. the prince of wales hospital and the simulation model of it [ ] they have simulated the spread of tracer gas in the wards of both on the lower floor ( f) and higher floor ( f) to demonstrate the stack effect. some of the simulation results are shown as shown in fig. : figure. simulation results of the tracer gas transmission in wards of different floors [ ] j o u r n a l p r e -p r o o f these researches above mainly investigated pathogen transmission inside the hospital and they are meaningful in protecting patients and hospital staff. however, not only pathogen transmission inside the hospital is dangerous, but the pollutant emission from the hospital is also a great concern for public health. chang et.al [ ] by using cfd modeled the atmospheric environment out the hospital and simulated the spread of the viral (sars) gas emitted from the hospital. the mesh model of simulation is shown in fig. : fig. mesh model of simulation [ ] this model was generated by tetrahedral grids; the wind velocity as a boundary parameter was measured by the hot-film probe and anemometry equipment; wind directions were considered in the calculation. moreover, in order to verify this model, tracer gas was used in the experiment model with a : scale. the simulation result below in fig. respectively shows the concentration contour of pollutant gas at both the height of the roof chimney (right) and . m (left) above the ground. fig. diffusion of pollutant gas emitted from hospital [ ] by the simulation results, they indicated that both the maximum concentration and mean concentration of pollutant gas in small and would not affect residents' health. however, when a large number of sars patients were arranged in the hospital, it is still a bit risky for people who actives in the high-concentration area on the ground level. research works above were mainly focused on the airflow pattern or impact of ventilation on pathogen transmission. however, cross-infection frequently happened in hospitals and should be paid attention to in case studies of pathogen transmission. based on eulerian-lagrangian method, a case study proposed by wang, et.al [ ] has illustrated that the sneezing process from a virus carrier is ventilation is easier to be controlled, hence, it is necessary to ensure safety when emitted the viral gas from the exhausted system from the hospital. without professional medical equipment, the buildings with high population density such as residential buildings, commercial buildings and campus buildings are in higher infection risk. yang, et.al [ ] studied natural ventilation in teaching buildings by using cfd method. in their investigation, phoenics with rans model was used to simulate the ventilation; the simple algorithm was used to calculate and presto scheme was used to staggered the pressure interpolation; the wind profile at inlet boundary of the simulation was determined by the equation of ashrae [ ] as: . () ref u y y uh     ( ) through the simulation, they indicated that the ventilation of the teaching building with a "line-type" corridor is better than that of the inside corridor; they have also presented an optimization design for better ventilation in teaching buildings by determining the best wind angle. moreover, cuce, et.al [ ] studied the natural ventilation in school buildings based on its working principles and limitation of passive ventilation; in a crowded room, the concentration of volatile organic substances generated by human skin oil is high, xiong, et.al it can be observed from simulations that particles will spread in flushing because of the turbulence generated by the high speed of flowing water. more, it was obtained that %~ % of particles can reach above the toilet seat. the research of this work is meaningful and it was indicated that before flushing, laying down the lid is useful for preventing the virus transmission. more, washing the seat of the toilet is necessary because the floating virus may deposit on the surface of it. this research group has also analyzed the movement of a virus-laden particle in the process of urinal flushing [ ] . without the prevention, over % of particles can escape from the urinal and the particle can reach the highest position of . m at only . s . so it is mandatory to wear a mask in public to reduce infection risk. furthering this study about virus transmission in the squat toilet by applying the method j o u r n a l p r e -p r o o f proposed in this work is important because, in many places such as china, the use of the squat toilet is higher than that of the sitting toilet in public. some investigations of cfd simulations of ventilation or pathogen transmission in the building environment are summarized below in tab. : it can be obtained from these investigations: ( ) ventilation is one of the most effective methods to control the pathogens transmission and the reasonable arrangement of the ventilation system is necessary. ( ) the effect of the stack effect should be considered when analyzing the ventilation in high-rise buildings. ( ) rooms with infected patients need to be diluted with plenty of fresh air. traffic vehicles are also dangerous when there are infectious patients. under the high personnel density and weak ventilation system, it is difficult to control the pathogen such as the airborne virus. according to this problem, more and more researchers investigated airflow in various kinds of vehicles by using cfd methods. ( ) two particles collisions are mainly considered; ( ) the velocity distribution of each particle exists independently; ( ) the external force does not affect the dynamic behavior of the local collision. there are various models that can be used in the simulation of lbm and these models can be defined by the layout of lattice, some models which are in common use are shown in fig. ( d) and face masks have been used to prevent virus transmission and it is necessary for the epidemic situation. li [ ] simulated the aerodynamic behavior of a gas mask which consists of two filter layers. fig. : fig. grid model of the gas mask (left) and the flow field of simulation (right) [ ] this research indicated that the design of the mask such as the hole properties is important: larger hole area and greater hole distribution lead to a lower pressure drop, a smaller dead zone, and so on. theoretical analysis was mainly studied in this work and it has also provided a reference in designing a sufficient mask. dbouk, et.al [ ] analyzed the role of the mask in preventing the droplet transmission by utilizing openfoam with a combination of the use of turbulence model and porous model. in the simulation model, the mask fitting to the face was considered which is shown as in fig. : turbulent flow in the atmosphere is unsteady due to the changing weather and it is difficult to measure the airborne transmission in the atmospheric environment directly. although aerodynamics models of airborne transmission based on cfd method have been greatly developed, it is still a big challenge for applying them to the large-scale environment. seo, et.al [ ] presented a method based on meteorological information from web-system that can help for solving this problem. this research group analyzed the relationship between foot-and-mouth disease (fmd) spread and hourly wind in anseong. moreover, they collected the infection data and built a model by using the gis method. then, they used a code division multiple access (cdma) to send the weather data to a weather data acquisition server (wdas) in every minutes and interlock the data with geographical information. the openfoam code was used to simulate the spread of the airborne virus the simulation result can well describe the virus transmission. the process of the cfd simulation based on web-based forecasting system can be described as below in fig. : fig. detailed process of cfd simulation based on web-based forecasting system the web-based forecasting system has been widely used in various cases such as flooding (li, j o u r n a l p r e -p r o o f et.al [ ] ), tourism demand (song, et.al [ ] ), monitoring of marine pollution (kulawiak, et.al [ ] ), etc. however, there are few studies about pathogen transmission based on combing the web-based forecasting system and cfd method. hence, more databases of pathogen transmission and meteorological information are needed to develop the web-based forecasting system in the analysis of pathogen transmission. from investigations summarized in this review, it can be found that ventilation is one of the most effective methods to control pathogen transmission in the air. different environments require different ventilation systems, the building environment such as teaching building and residential building and the natural ventilation method is the main way to dilute the concentration of the pathogen. however, in high-risk zones such as hospitals, not only the reasonable ventilation of indoor is required, but also the infectious risk due to emission needs to be considered. besides, pathogen transmission in j o u r n a l p r e -p r o o f journal pre-proof different vehicles is distinct, a proper strategy of ventilation is necessary for transportation especially the airplane and high-speed train with an enclosed environment. this review also presented some advanced methods for cfd application on pathogen transmission according to recent investigations as: ( ) lbm simulation allows researchers to investigate pathogen transmission from the mesoscale level; ( ) based on the porous media model, researchers can better analyze the transport of pathogens in complex media, such as medical masks, human organs, etc. ( ) web-based forecasting system can be combined with the cfd method to analyze the transmission of infectious pathogens in the atmospheric 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transport and deposition multi-block lattice boltzmann simulations of solute transport in shallow water flows large-scale oil spill simulation using the lattice boltzmann method, validation on the lebanon oil spill case lattice boltzmann simulations of anisotropic permeabilities in carbon paper gas diffusion layers drag correlation for dilute and moderately dense fluid-particle systems using the lattice boltzmann method lattice boltzmann simulation of liquid water transport in microporous and gas diffusion layers of polymer electrolyte membrane fuel cells an immersed boundary-lattice boltzmann model for simulation of malaria-infected red blood cell in micro-channel aerodynamic behavior of a gas mask canister containing two porous media on respiratory droplets and face masks -d numerical simulation of main sieve diaphragm with three types passageway design in a gas mask canister the role of porous media in modeling fluid flow within hollow fiber membranes of the total artificial lung web-based forecasting system for the airborne spread of livestock infectious disease using computational fluid dynamics a web-based flood forecasting system for shuangpai region developing a web-based tourism demand forecasting system interactive visualization of marine pollution monitoring and forecasting data via a web-based gis the authors are grateful for the research support received from applied basic the authors declared that they have no conflicts of interest to this work as: we declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.j o u r n a l p r e -p r o o f key: cord- -i kx n m authors: raison, charles l; miller, andrew h title: pathogen–host defense in the evolution of depression: insights into epidemiology, genetics, bioregional differences and female preponderance date: - - journal: neuropsychopharmacology doi: . /npp. . sha: doc_id: cord_uid: i kx n m significant attention has been paid to the potential adaptive value of depression as it relates to interactions with people in the social world. however, in this review, we outline the rationale of why certain features of depression including its environmental and genetic risk factors, its association with the acute phase response and its age of onset and female preponderance appear to have evolved from human interactions with pathogens in the microbial world. approaching the relationship between inflammation and depression from this evolutionary perspective yields a number of insights that may reveal important clues regarding the origin and epidemiology of the disorder as well as the persistence of its risk alleles in the modern human genome. major depressive disorder (mdd) is about relationships. or so one would think based on an extensive literature that seeks to explain how a condition with genetic underpinnings could be so widespread when it is so apparently inimical to the darwinian mandates of survival and reproduction. this literature on adaptive theory bifurcates over whether depression is adaptive in and of itself or whether the genes that increase the risk of depression also promote other types of social behavior (ie, avoiding conflict with more powerful individuals) that promoted survival and reproduction sufficiently to have been retained in the human genome (supplementary table s ). despite differences, however, these theories all share in common the notion that the adaptive value of mdd is to be found in its effects on our relationships with other people. we agree that the adaptive value of mdd is to be found in our relationships, but not primarily in our relationships with other humans. rather, in this review we argue that mdd, and the genes that subserve it, evolved to help us manage relationships with the wide range of pathogens with which humans co-evolved. specifically, at least some of the genes that promote mdd evolved and have been retained in the human genome because-on average-they helped hominids avoid death from infection. importantly, we are not suggesting that these genes promoted host defense against pathogens at the cost of inducing depression. rather, the tendency of these genes to induce depression and other related behavioral changes including arousal, anxiety and alarm is integral to their anti-pathogen benefits, becauselike sickness from which it evolved-depression conferred protection from pathogens in the environments in which humans evolved. said differently, depression may itself be an anti-pathogen defense strategy, one that is both activated by inflammation and, somewhat paradoxically, one that across evolutionary time may have made inflammatory activation less necessary, as we shall see in our discussion of sex differences in the prevalence of mdd. from this perspective, even environmental risk factors for depression, such as psychosocial stress, promote mdd primarily because across evolutionary time these adversities reliably served as warnings that an individual's risk for infectious morbidity/ mortality were heightened. although this perspective may be most relevant to sub-types of depression associated with increased inflammation, we highlight in this review the possibility that other immune and/or behavioral responses relevant to pathogen-host defense may also be associated with mdd in ways that extend the role of microbial-human interactions in the evolution of depression beyond its now well-recognized associations with inflammation. consistent with earlier publications from our group miller, , ; miller and raison, ) , we refer to this constellation of ideas as the pathogen-host defense theory of depression, or pathos-d. this position overlaps, but has significant differences from a pathogen defense theory of depression first articulated by kinney and tanaka, ( ) (anders et al, ) . table situates pathos-d within other possible explanatory frameworks for understanding why mdd and alterations in immune function should be related. supplementary table s provides a comparison of different adaptive explanations for the persistence of mdd and its risk alleles in human populations. as with all theories, the value of pathos-d will eventually be determined by its ability to make novel predictions and to provide coherent and parsimonious explanations for a wider range of variables than are accounted for by competing hypotheses. our aim in this review is to evaluate pathos-d by these criteria. to do this, we list a series of concepts consistent with pathos-d theory, in each case evaluating the evidence for and against the theory's explanatory and predictive potential. in addition, we explore the theory's possible explanations for sex differences in the prevalence of mdd. in all these areas, we are careful to note the degree of data in support of each idea, and which ideas are frankly speculative. figure provides a graphic representation of epidemiological, biological, and genetic domains relevant to mdd that are addressed by pathos-d. of all the predictions of pathos-d, this one is the best established. indeed, it was the effort to provide an evolutionary perspective on the association between inflammation and depression that provided the initial impetus for the theory. taken as a whole, studies suggest that mdd is associated with all the cardinal features of an inflammatory response including increased proinflammatory cytokines and their receptors and increased acute phase reactants, chemokines, and soluble adhesion molecules in peripheral blood (lieb et al, ; calabrese et al, ; sluzewska et al, ; song et al, ; howren et al, ; dowlati et al, ; liu et al, b; valkanova et al, ; black and miller, ; haapakoski et al, ; eyre et al, ; goldsmith et al, b; van dooren et al, ) . several studies provide evidence of increased inflammatory activity in the central nervous system of depressed individuals as well, as indexed by increased cerebrospinal fluid (csf) concentrations of interleukin (il)- -β and neural adhesion molecules, as well as by increased prefrontal cortex gene expression of il- -α, il- , il- , il- , il- , il- , il- , il- a, il- , il- , il- , interferon (ifn)-γ, and lymphotoxin alpha (tnf superfamily member ; poltorak et al, , levine et al, shelton et al, ) . perhaps suggesting a link between inflammation and symptom acuity, and/or reflecting the association between inflammation and heightened anxiety (a known suicide risk), a recent meta-analysis found that levels of il- -β and il- were significantly increased in blood and postmortem brain samples of patients with suicidality compared with both non-suicidal depressed patients and healthy control subjects (black and miller, ) . given their centrality in activating innate immune responses to pathogens, it is striking that no adaptive relationship exists between immune changes and depressive symptoms. immune changes observed in major depressive disorder (mdd) may be beneficial or detrimental to pathogen-host defense and adaptive benefits of depression exist in non-pathogen/immune-related domains (multiple adaptive theories, eg, social navigation hypothesis) depressive symptoms extract a cost to survival and reproduction but this cost is offset by the direct anti-pathogen benefits of heightened inflammation/immune activation (antagonistic pleiotropy) in individuals with reduced immune competence for any number of reasons, depressive symptoms serve pathogen-host defense by inducing social avoidance/energy conservation and behaviors that provide protection from becoming infected and/or energy for immune activity once infection has occurred (kinney and tanaka) genetic alleles that promote an inflammatory bias have undergone positive selection because in ancestral environments they provided direct pathogen defense and because they promoted the development of depressive symptoms in response to immune activation. like sickness behavior, depression in response to immune activation aided in host defense both directly (ie, raised body temperature and energy conservation behaviors) and indirectly (social avoidance, energy conservation, and hypervigilance; raison and miller, pathogen- host defense theory of depression [pathos-d]) adaptive explanations for associations between mdd and altered immune functioning are not considered (frequent unexamined assumption of researchers working on proximal mechanisms) toll-like receptor (tlr) mrna and protein have been reported to be elevated in both the periphery and cns of individuals with mdd (hung et al, (hung et al, , keri et al, ; van dooren et al, ) , with some evidence suggesting that successful pharmaco-or psychotherapy reduces peripheral tlr activity (keri et al, ; hung et al, ) . of these various findings, meta-analyses suggest that the best replicated associations between depression and inflammatory biomarkers are increases in circulating levels of c-reactive protein (crp) and il- (howren et al, ; dowlati et al, ; liu et al, b; valkanova et al, ; black and miller, ; haapakoski et al, ) . taken as a whole, the meta-analytic literature also supports an association with circulating levels of tumor necrosis factor (tnf) and il- -β, although with more variability. because il- -β features so prominently in both the inflammatory response and in animal studies of stress, and depression-like behavior, it is curious that meta-analyses split on whether it is elevated in the blood of patients with mdd (dowlati et al, ; black and miller, ) . one possible explanation that would be consistent with the animal literature is that il- -β has a larger role in the cns than the periphery in terms of depressogenesis. consistent with this, some evidence suggests that il- -β is elevated in the csf of depressed individuals (levine et al, ) . alternately, evidence linking il- -β to mdd may not be robust because of limitations in assay methods in detecting levels of il- in human studies. although increases in inflammatory biomarkers in depressed patients compared with controls are modest when compared with acute infections or autoimmune conditions, recent animal data demonstrate that these types of low-level inflammatory stimulation are sufficient to induce depressive-like behavior and associated brain changes when such stimulation is chronic (tarr et al, a) , such as appears to be the case in many individuals with mdd. figure . man meets microbes. in ancestral environments, heavy pathogen loads applied significant evolutionary pressure on human survival that ultimately entailed a host of adaptations that, according to the pathogen-host theory of depression, shaped interactions between the immune system (inflammation in particular) and the brain, leading to a unique set of behaviors. these behaviors, including anhedonia, fatigue, and psychomotor slowing as well as anxiety, arousal and alarm supported energy conservation for fighting infection and wound healing and hypervigilance to prevent future attack. in addition, these evolutionary forces instantiated a coupling of these behavioral responses with the acute phase response including not only increases in acute phase reactants such as c-reactive protein but also hypoferremia and zinc deficiency, as well as fever, which are better suited for interactions with pathogens than people. in addition, evolutionary pressures from human interactions with the microbial world can explain risk alleles that are specific to the pathogens to which given populations were exposed as well as epidemiologic features of depression such as female sex preponderance, an early age of onset, and occurrence in the postpartum period that supported reproductive success. finally, the pathogen-host theory of depression is consistent with modern risk factors for depressive symptoms, including obesity, processed-food-based diets, sedentary lifestyle, and sleep deprivation, all of which serve to exacerbate the inflammatory bias that is the legacy of our evolutionary past. nonetheless, cross-sectional associations between mdd and inflammation do not establish that immune system activity is capable of producing depression, as would be predicted by pathos-d theory. it might well be that depression is associated with brain states that through their impact on neuroendocrine function secondarily increase inflammatory biomarkers that themselves have no impact on the disorder. however, a wealth of data demonstrate that although it is true that mental states can increase inflammation, it is equally true that activation of the body's inflammatory response system can induce depression, as well as many of the cns and neuroendocrine changes that have been repeatedly associated with mdd. this has been shown in studies examining the acute behavioral and cns responses to a single exposure to endotoxin or typhoid vaccine and even more convincingly by the many studies examining the biological and behavioral effects of chronic immune activation secondary to treatment with the cytokine ifn-α for either cancer or hepatitis c virus infection (capuron et al, (capuron et al, , a (capuron et al, , b (capuron et al, , (capuron et al, , (capuron et al, , wichers and maes, ; wichers et al, b wichers et al, , wichers et al, , majer et al, ; harrison et al, a harrison et al, , b lotrich, ; lotrich et al, ; prather et al, ; raison et al, raison et al, , a raison et al, , b raison et al, , d eisenberger et al, a, b; franzen et al, ; felger et al, felger et al, , . importantly, the induction of depressed mood in response to both acute (ie, typhoid vaccine) and chronic (ie, ifn-α) immune stimulation has been associated with increased concentrations of circulating inflammatory cytokines (wright et al, ; raison et al, a) . also consistent with the ability of immune activation/inflammation to negatively impact mood is the fact that the risk of developing mdd, increases following significant infections (benros et al, ; gunaratne et al, ; bornand et al, ) . finally, a recent meta-analysis confirms findings from a number of individual studies that elevated levels of circulating inflammatory biomarkers predict the future development of depression in currently non-depressed individuals, even when controlling for other depressive risk factors (valkanova et al, ) . increased inflammatory activity has been the most replicated immune change associated with mdd, but there is no a priori reason why this should be the only adaptive immune pattern associated with depression that enhanced pathogen-host defense. a clear prediction of pathos-d theory is that as novel innate immune antipathogenic mechanisms are identified, they will be found to be enhanced in depressed individuals. for example, recent studies indicate that antimicrobial peptides upregulated by inflammatory cytokines (eg, ribonuclease , psoriasin, and human β-defensin- and ; harder et al, ) are increased in the skin of patients with inflammatory skin conditions, such as atopic dermatitis and psoriasis (harder and schroder, ; harder et al, ) , that are comorbid with mdd (gili et al, ; yang et al, ) . one would expect a similar increase in these peptides-although perhaps to a lesser degree-in medically healthy individuals with mdd. the best replicated risk factors for mdd in the modern world have been repeatedly associated with the same changes in immune functioning that are observed in mdd (raison, et al, c) . this is especially apparent in relation to associations between these risk factors and increased inflammation; however, stress-which has been the most extensively studied-has also been repeatedly associated with other immune changes seen in mdd (herbert and cohen, a, b) . taken at face value, these associations appear to provide strong support for pathos-d. however, it is important to note that a number of these factors-such as obesity, sedentary lifestyle and processed foods-are artifacts of modern lifestyles, and therefore are unlikely to have evolved to activate depression as a result of being 'early warning signs' of impending infectious morbidity/mortality in the environment of evolutionary adaptiveness (eea), as would be predicted by pathos-d. however, at least two proinflammatory risk factors for mdd-medical illness and psychosocial stress-are certainly ancient and may well have evolved an association with depression as a result of inducing immune changes that enhanced survival in the eea. pathos-d proposes that depression evolved out of sickness, so it is no surprise that robust associations have been seen between medical illness and increased rates of depression in all cultures examined (moussavi et al, ) . because cytokines are primary drivers of sickness, it is also no surprise that medical illnesses are typically characterized by increased inflammation, nor that increased levels of inflammatory cytokines in medically ill patients are associated with an increased risk for depression (musselman, ) . on the other hand, it is not immediately apparent why psychosocial stressors should so reliably activate inflammatory pathways in animals and humans when these types of adversities are not in and of themselves associated with either an infectious challenge or an immune stimulus. indeed, it has become so commonplace to say that stress increases inflammation that it is easy to lose track of what a remarkable thing this is. consider the trier social stress test (tsst) in which study participants are charged with giving a personally meaningful speech and doing mental arithmetic in the presence of a judgmental panel. predictably, the threatened subject experiences a classic fight or flight response characterized by an increased heart rate and blood pressure and an increased release of cortisol and catecholamines. however, the tsst also activates key intracellular inflammatory transcription pathways (eg, nuclear factor-kappa β (nf-κb)) within peripheral blood mononuclear cells and dramatically increases circulating levels of inflammatory cytokines, such as interleukin (il)- (bierhaus et al, ; pace et al, a; carpenter et al, ) . moreover, individuals with known risk factors for developing depression (eg, early-life adversity) show larger inflammatory responses to laboratory psychosocial stressors than do others (pace et al, a; carpenter et al, ) , and the larger the inflammatory response to the stressor, the more likely the subject is to develop depression over the ensuing months (aschbacher et al, ) . in essence, the body mounts an immune response, not against a pathogen, but against a threat to the subject's self-esteem. theories that view depression as having evolved to serve adaptive social purposes do not yield easy explanations for why non-infectious stressors should activate peripheral inflammatory pathways. on the other hand, pathos-d suggests a straightforward and parsimonious explanation. because the vast majority of stressors in mammals over evolutionary time boiled down to risks inherent in hunting, being hunted or fighting conspecifics in dominance hierarchies for reproductive access/status, it is not surprising that these states are also circumstances in which the risk of pathogen invasion-and subsequent death from infectionwas greatly increased as a result of traumatic opening of the protective skin barrier from wounding (dipietro, ) . such wounding is common in social species and was a significant source of morbidity and mortality among humans in the ancestral environment, and indeed well into the historical period (ross et al, ; eshed et al, ; pinker, ) . in ancestral environments, the association between stress perception and risk of subsequent wounding was reliable enough that evolution, operating by the so-called 'smoke detector' principle, favored organisms that prepotently activated inflammatory systems in response to a wide array of environmental threats and challenges (including psychosocial stressors), even if this activation was often a 'false alarm.' as noted by dhabhar-'stress perception by the brain may serve as an early warning signal to activate the immune system in preparation for a markedly increased likelihood of subsequent infection' (dhabhar, ) . and while chronic stress is best known to suppress certain aspects of immune function (ie, natural killer cell activity, circulating t-cell subsets; herbert and cohen, c) , the types of acute and/ or psychosocial stressors most likely to be associated with immediate risk of wounding and hence infection activate both innate and adaptive immunity (quan et al, ; bierhaus et al, ; avitsur et al, ; viswanathan et al, ; pace et al, b; steptoe et al, ; joachim et al, ; bailey et al, ; rosenberger et al, ; mays et al, ; powell et al, ) . that non-immune stressors can activate inflammationand do so via multiple mechanisms including activation of the intracellular inflammasomes-is now firmly established (miller and raison, ) . however, pathos-d also predicts that stress-induced immune changes should produce an overall enhancement of pathogen-host defense and especially anti-bacterial defense given that these organisms are the chief cause of wound-related infections. this has yet to be proven or disproven, but significant animal data suggest that psychosocial stressors-even when recurrentcan enhance host defense mechanisms and improve immune function. using a repeated social defeat stress paradigm (sdr), sheridan and colleagues have shown that stress primes multiple aspects of the rodent host defense repertoire. sdr increases the bactericidal activity of splenic macrophages through a toll-like receptor-dependent pathway; (bailey et al, ) enhances the innate immune response to a primary hsv- infection in the cornea and trigeminal ganglia; (dong-newsom et al, ) primes dendritic cells and augments clonal expansion of cd (+)t cells during primary influenza a viral infection with a resultant enhancement of adaptive immunity and viral clearance; mays et al, ) and activates natural killer cells (tarr et al, b) . to our knowledge, none of these effects have been directly examined in humans, but it is intriguing to note that improved functional recovery following knee surgery has been associated with an increase in the type of immune cell redistribution effects that are known to be induced by acute stress in animals (rosenberger et al, ) . also relevant to the hypothesis that stress-induced activation of inflammation provides pre-potent protection against pathogens is work by cole and colleagues demonstrating that social isolation is associated with an immune signature well-tailored to the types of infectious challenges that would most likely have resulted from ostracism in ancestral environments. individuals who perceive themselves as chronically socially isolated (ie. lonely) show a pattern of gene transcription in leukocytes characterized by increased expression of inflammatory pathways crucial for protection against bacteria with a concomitant decrease in the expression of genes important for antiviral immunity, especially interferon response genes (slavich and cole, ) . consistent with these findings, individuals with high trait sensitivity to social disconnection produce greater blood levels of proinflammatory cytokines (tnf and il- ) and increased expression of inflammatory genes in response to an endotoxin stimulus (moieni et al, a) . similarly, study participants who responded to an fmri-based social exclusion paradigm with greater activity in the dorsal anterior cingulate cortex and anterior insula-brain regions that have previously been associated with processing rejection-related distress and negative affect-also responded to a laboratory psychosocial stress test with increased salivary production of the inflammatory biomarker soluble tnf-α type ii receptor (slavich et al, ) . as slavich and cole, ( ) have observed, this pattern of findings in response to social isolation (ie, increased inflammation/reduced antiviral immunity) may well have evolved to optimize immune responses to threats from environmental pathogens associated with wounding/starvation that would have been the primary risk for infectious morbidity/mortality in the eea for individuals with diminished human-to-human contact. because ostracism was an almost certain death sentence in ancestral environments, the inflammatory response induced by social isolation/exclusion can be seen as a modern vestige of what once served as an immunological ostracism detection system that worked in concert with other ostracism detection systems described in the literature (spoor and williams, ) to encourage individuals to endure the costs of group existence during the long years of human evolution. this hypothesis might help explain the recent finding that cytokine activation via exposure to endotoxin induces feelings of social disconnection, especially in females. this phenomenon might reflect an evolved and adaptive feedforward mechanism whereby the risk of social exclusion induced a prepotent inflammatory response that then fostered a further sensitivity to the risk of ostracism. and this mechanism may be more apparent in modern females because the offspring of ostracized mothers would have been less likely to survive than those of ostracized fathers (sear and mace, ) . on the other hand, the increased balance of antiviral to anti-bacterial/inflammatory gene expression seen in more socially integrated individuals would have been more optimal for providing protection against more recent viral infections that spread from person to person but that only became widely lethal (and thereby exerted greater selection pressure) with the increased population density that became the norm after the development of agriculture in the first epidemiological transition. if we assume that a chronic inflammatory response is the most common immune profile associated with mdd, then pathos-d predicts that this association should be ancient, because it is this immune response and its associated depressive symptoms that enhanced survival and reproduction via reduced pathogen-related mortality. strong evidence for this comes from innumerable studies in mice and rats showing that inflammatory cytokines and their stimulators induce depressive and anxiety-like behavior (dantzer et al, a) , as well as studies indicating that even when induced by psychological stress, these behavioral responses are dependent upon cytokine activation-especially the induction of il- -β in the cns (goshen and yirmiya, ). the fact that primate and rodent lineages separated from each other in the time of the dinosaurs strongly suggests that the rudiments of the immune-depression link were laid down early in mammalian evolution, if not earlier. however, not all data support an ancient or universal link between inflammation and depression, nor is pathos-d the only evolutionary theory that might account for such a link if it in fact exists. indeed, it is increasingly clear that numerous conditions in the modern world have dysregulated immune functioning in ways that promote autoimmunity and chronic inflammation, while simultaneously reducing any adaptive advantage that might be inherent in either the inflammatory response or mdd (raison et al, c) . this scenario opens the possibility that the association between inflammation and depression is a non-adaptive by-product of first world environmental and social conditions, rather than something inherent in either our genetic or behavioral make-up. said differently, the link between chronic inflammation and mdd may not reflect an evolved adaption, but rather represents an example of non-adaptive environmental mismatch, defined as a state of disequilibrium whereby a trait that enhanced reproductive fitness in the environment in which it evolved becomes maladaptive in another environment. of all the modern environmental conditions that have altered associations between immune function and behavior, none has received more attention than our changed relationships with a huge range of microorganisms that were ubiquitous in the eea, but that are largely missing from the industrialized world. unlike potentially lethal pathogens, most of which produced either immunity or death, many of the now-absent organisms were best dealt with via the development of immune tolerance, either because the organisms were beneficial (eg, the microbiota), harmless (eg, many environmental organisms), infectious but not lethal (eg, hepatitis a virus) or potentially harmful but not easily cleared by the immune system without excessive tissue damage (eg, many helminths). the central insight of recent iterations of the 'hygiene hypothesis' (such as the 'old friends' hypothesis of rook) is that because the immune system had to learn to tolerate these organisms (as opposed to clearing them from the body), they became 'teachers of tolerance,' especially during childhood development when their absence predisposes individuals in industrialized societies to develop a host of allergic, asthmatic, autoimmune, and psychiatric pathologies (raison et al, c) . in a way that seems more than metaphorical, this notion points toward another way that depression may be about relationships. we need to look no further than the impact of bereavement on mood or the strong association between the death of a parent in childhood and the development of mdd in adulthood to see that human relationships characterized by loss can be profoundly depressogenic (berg et al, ) . increasing evidence suggests the same is true of our relationships with the microbial world. via dysregulated inflammatory activity, our moods are haunted by the loss of 'old friend' organisms of which most of us have no conscious knowledge. again, like our relationships with other humans, our relationships with bacteria, viruses, and parasites can be understood as comprising various degrees of competition and cooperation/collaboration. pathos-d suggests that the link between depression and immune activation primarily reflects the competitive element in these relationships. the evolutionary mismatch model suggests the opposite: that the link between depression and immune activation reflects the failure of the modern world to honor many ancient and mutually beneficial collaborations. so, can available data help us determine which theory is better supported? to our minds, the strongest data that support evolutionary mismatch, while simultaneously arguing against pathos-d, come from the ground-breaking work of mcdade and colleagues who have examined associations between stress, mood and inflammation in non-industrialized environments. working in the philippines and lowland ecuador, they have shown that patterns of inflammation observed in the west do not necessarily translate to less-developed environments with significantly higher pathogen exposure and infectious mortality. in industrialized contexts, the concentrations of peripheral inflammatory biomarkers show significant intra-individual stability, with some individuals demonstrating chronically elevated levels of these biomarkers (macy et al, ; ockene et al, ) . moreover, factors other than pathogen exposure (especially obesity) have been repeatedly shown to upregulate inflammation. mcdade et al, ( ) found that neither of these pertain in more traditional societies. in the philippines levels of crp were significantly lower, and levels of the anti-inflammatory cytokine il- significantly higher, than are typically observed in western contexts, and these associations were not accounted for by differences in body mass index between the populations (mcdade et al, ). in lowland ecuador among the indigenous shuar people, levels of crp were found to vary widely within the same individuals, showing a pattern of rapid rise to high levels in response to acute infection and then dropping to often undetectable levels upon infection resolution (mcdade et al, b ). an even more direct challenge to pathos-d predictions came from findings that depression was not associated with elevated crp in the philippines and that associations between both current and childhood stressors (ie, death of a parent) and inflammation were only apparent in the sub-population in the cebu region that had been raised in more hygienic environments (mcdade et al, a) . individuals with high levels of microbial exposure in infancy showed no such associations. taken together, these data suggest that high microbial exposure early in life-such as would have been normative across human evolution-may hone the immune response to be more specific for pathogen exposure and less generalized to the types of psychosocial stress that so reliably induce depression. under these evolutionarily normative environments, the links between inflammation and mdd that are so apparent in the west would not exist. however, these findings must be balanced against more recent work conducted in the lowland jungle of bolivia among the tsimane amerindians, a small-scale society composed of lean forager-horticulturalists with a short average lifespan who live in a high-pathogen environment and thus have significant microbial priming in infancy (stieglitz et al, b) . in all these ways the tsimane represent a reasonable approximation of environmental and social conditions prototypically confronted by humans in the eea. working among these people, stieglitz et al. ( b) examined whether a syndrome recognizable as mdd exists, and if it exists, whether it is associated with increased inflammatory biomarkers. results indicated that a depressive syndrome identical to that seen in the industrialized world exists among the tsimane, and is associated with the same risk factors that have been observed everywhere else in the world: reduced health (indexed as functional disability) and psychosocial stress (indexed by social conflict, especially with non-kin; stieglitz et al, a). when examined as a binary construct based on a median split in symptom scores, depression was also associated with significantly increased serum concentrations of il- , tnf, and crp but not il- -β (stieglitz et al, b) , replicating the overall pattern of metaanalytic findings from studies conducted in industrialized societies. these associations remained after adjustment for age, body mass index, and high leukocyte count (likely indicative of current infection), and associations were observed across a wide range of depressive symptoms. as our discussion of this prediction suggests, currently available data provide a split decision on whether associations between depression and inflammation are ancient and hence potentially adaptive, or are of recent origin and thus likely a result of non-adaptive evolutionary mismatch. one can only mourn the fact that the theoretical perspectives and technology necessary to resolve this issue were not available in the middle years of the th century when a range of hunter-gatherer groups on several continents had yet to be driven to extinction by the inexorable onrush of the modern world. pathos-d theory engenders a sense of urgency to examine this issue in the remnant small-scale societies that remain. two types of studies would be especially relevant. first, it would be of great interest to conduct the equivalent of trier social stress tests in these societies to examine whether objectively induced psychosocial stress activates inflammatory pathways as it does in the west. second, it would be important to examine whether the administration of cytokine activators (ie, endotoxin, typhoid vaccine or longer-term treatment with ifn-α) produce depressive symptoms as they do in the industrialized world. if individuals living in environments more closely aligned with prototypical conditions in the eea showed inflammatory responses to psychosocial stress and if they responded to immune stimulation with depression, strong evidence in support of pathos-d would have been obtained. from a pathos-d perspective there is no a priori reason that mdd should be associated with increased inflammation, only that mdd be associated with genes, physiology and behavior that-on average-decreased infectious mortality across human evolution. the fact that a subset of patients with mdd demonstrate increased inflammation implies the hypothesis that if depression is associated with increased inflammation, and if depression evolved as an adaptive pathogen-host defense strategy, then increased inflammatory activity should have produced an overall survival benefit in high-pathogen ancient environments, if indeed the association between depression and inflammation was adaptive in the eea. for this idea to be credible two things are required: first depression should enhance host defense, and second inflammation should enhance host defense. on the face of it, neither appears to be true, given significant evidence that both depression and inflammation appear to increase-rather than decrease-vulnerability to infection (zorrilla et al, ; evans et al, ; cruess et al, ; raison et al, ; doering et al, ; faulkner and smith, ; leserman, ; leutscher et al, ; andersson et al, ; doran et al, ) . here we consider the question of whether inflammatory activity provided protection against infectious mortality. for at least years it has been recognized that inflammation is essential for human health and a necessary ingredient of our ability to adequately fight infections. we now know that the larger innate immune response-of which inflammation is a core component-provides both direct and rapid protection against all classes of pathogens and has a crucial role in activating slower, but more definitive acquired immune responses. however, studies in recent years have found that the relationship between the innate and acquired immune systems follows the laws of cooperation and competition that define all evolved relationships. especially under conditions of limited energy availability or simultaneous infectious risk from multiple pathogens, immune function can become characterized by trade-offs between these two branches of immunity, such that resources invested in one, mean reduced functionality of the other (mcdade et al, ) . consistent with this notion, chronic inflammation can actually suppress t-and b-cell function through various mechanisms (cope et al, ; moraska et al, ; cope, ; clark et al, ; muller et al, ; vaknin et al, ; eleftheriadis et al, ; blume et al, ) , and rates of infection are often increased -not decreased-in autoimmune conditions characterized by chronic inflammation (doran et al, ) . however, pathos-d theory requires only that across evolutionary time the patterns of enhanced inflammatory activity characteristic of depression provided overall survival benefits that outweighed any costs imposed by associated reductions in other aspects of immune functioning. some evidence for this possibility comes from studies conducted by westendorp and colleagues in ghana and the netherlands. these researchers observed that cytokinestimulated production of tnf-α declines with age in the netherlands, a country with a low infectious burden, but does not decline with age in ghana, a country with high rates of infection (ie, % of ghanan study participants were infected with malaria; may et al, ) , suggesting that increased proinflammatory cytokine production-as observed in mdd-promotes survival under conditions of high-pathogen burden (drenos et al, ) . more definitive evidence for this possibility comes from a second ghanian study that more directly examined the impact of pathogen exposure on the association between inflammation and survival. ghana is a country in which some regions have pure water available from boreholes, whereas other regions must rely on heavily contaminated rivers for drinking water. as would be expected, death rates from infection are far higher in regions that utilize rivers than in areas where borehole-obtained water is available. consistent with the prediction that increased inflammatory signaling is protective in the high-infection environments that were common during human evolution (and especially common since the origin of agriculture and the rise of cities; armelagos et al, ) , a haplotype of the il- gene associated with increased inflammation was found to be significantly more prevalent in populations that relied on river water than in populations that drank from boreholes-suggesting positive selection driven by enhanced pathogen protection (kuningas et al, ) . consistent with this possibility, during a fiveyear follow-up period, the high-inflammation il- haplotype was associated with reduced survival in individuals who drank clean water from boreholes, but increased survival in populations exposed to high levels of pathogens as a result of drinking river water (kuningas et al, ) . we have already noted that depression is associated with increased circulating inflammatory biomarkers in the forager-horticulturist tsimane people of lowland bolivia (see prediction ). researchers also conducted ex vivo stimulation studies with endotoxin and phytohemagglutinin (pha; to assess response capacity of both the innate and acquired immune systems, respectively) and found that tsimane individuals with high levels of depression produced greater levels of il- -β, il- , and tnf in response to both endotoxin and pha, even when excluding individuals with leukocyte counts suggestive of current infection (stieglitz et al, b) . interestingly, this pattern is different from the suppression of mitogen-stimulated immune responses commonly observed in depressed patients in the western world (herbert and cohen, a) , which may point to yet another way in which modern conditions have disabled previously adaptive associations between depression, immune function, and pathogen-host defense. nonetheless, whether the increased mitogen-stimulated inflammatory cytokine response observed among depressed members of tsimane society would translate to enhanced protection against pathogens remains unknown. multiple facets of the modern world, from antibiotics to refrigeration and paved surfaces have profoundly reconfigured our relationship with the microbial world in ways that have reduced the benefits of inflammation and increased its costs (drenos et al, ; raison et al, c) . nonetheless, even in an environment so radically different from that in which humans evolved, data link increased inflammation with improved outcome in the context of at least some infections. for example, in a large study of consecutive patients admitted to hospital with fever, elevations in plasma concentrations of the anti-inflammatory cytokine il- -as well as the ratio of il- /tnf-α-were associated with increased mortality (van dissel et al, ) and increased il- production late in the disease course predicted reduced survival following infection with the pandemic a/h n -/ influenza virus (bermejo-martin et al, ) . as these findings would predict, alleles of the il- gene-such as - g-associated with higher il- production appear to promulgate poor infectious outcomes. for example, the − g allele predicts increased symptom severity and mortality in the context of community acquired pneumonia (gallagher et al, ) and is associated with reduced antibody responses to tetanus, influenza and hepatitis b virus (hbv) vaccines (hohler et al, ; corsini et al, ; li et al, ) . families with a member who died from meningococcal disease were characterized by increased production of il- and reduced production of tnf-α when compared with families with a member who had bacterial meningitis but survived (westendorp et al, ) and elevated crp has been associated with increased survival in children with meningococcal sepsis (joosten et al, ) . the ifn-γ + t allele increases production of this th inflammatory cytokine via enhanced binding of nfkb (pravica et al, (pravica et al, , and protects against mediterranean spotted fever (forte et al, ) . multiple studies and a large meta-analysis find that the t allele also protects against the development of tuberculosis on the individual (pacheco et al, ) and population levels (larcombe et al, ) . in individuals with active tuberculosis, the t allele reduces severity and risk of disseminated disease (ansari et al, ). the t allele also reduces the risk of leprosy (cardoso et al, ) , severe acute respiratory syndrome (sars; chong et al, ) , and chagas disease (torres et al, ) . conversely, the low-producing + a allele predisposes for hepatitis b and c persistence and negatively impacts clinical course of the disease (gao et al, ) . consistent with these findings, reduced peripheral blood mononuclear cell production of ifn-γ and il- is associated with increased severity of respiratory syncytial virus symptoms in infants under one year of age (pinto et al, ) . the association of depression with increased circulating levels of proinflammatory cytokines and crp does not easily lend itself to social explanations for the high prevalence of either mdd or putative depressogenic risk alleles. on the other hand, this association is foundational to the proposal that depression evolved to serve pathogen-host defense functions. however, if this is indeed the case, why should we expect the immune changes seen in depression to be limited to circulating cytokines or crp? in the context of innate immune activation, cytokine stimulation forms only part of a larger phenomenon that comprises the acute phase response (apr). as noted in a recent review (legrand and alcock, ), the apr is a somewhat paradoxical phenomenon, given that many of its features inflict significant energetic costs (eg, fever), hamstring immune responses (eg, iron sequestration, zinc, and tryptophan depletion), or produce behaviors likely to reduce attractiveness to potential mates, induce loss of social status or increase the risk for predation (eg, lethargy, psychomotor slowing, and social withdrawal), all of which would be expected to impair, rather than enhance, host survival, and reproduction. and while fever has been shown to enhance immune function while simultaneously contributing directly to pathogen killing, no direct immune benefits have been shown for many other apr features. a potential resolution to this enigma has been offered recently under the rubric of 'immune brinksmanship', which suggests that many apr features-such as iron sequestration and reduced zinc availability-have evolved to make the host environment less optimal for invading organisms, given that these minerals are essential nutrients for microorganisms, without which they can neither replicate nor survive (legrand and alcock, ) . from this perspective the apr can be seen as being, in part, a strategy that risks damage to the self, but that is likely to kill the offending agent before it kills the host. a comprehensive vision of the apr sees some of its elements as serving direct anti-pathogen functions (eg, leukocytosis), some as serving to reallocate energy to the immune system (eg, psychomotor slowing, somnolence) and some as serving 'immune brinkmanship' functions. studies suggest that, as a group, medically healthy individuals with depression demonstrate multiple biological and behavioral stigmata of an apr (jimenez-fernandez et al, ) . for a full listing of elements of the apr that have been associated with mdd in medically healthy individuals, see supplementary table s . importantly, at least one group has found that the apr in medically healthy adults with mdd is associated with increased levels of il- , suggesting that the apr is indeed part of a larger pathogen-host defense immune response (maes et al, ; sluzewska et al, ) . moreover, if psychosocial stress serves as an 'early warning system' for increased danger of impending wounding and subsequent infection, pathos-d would predict that psychosocial adversity should also be associated with an apr over and above activation of cytokines. consistent with this possibility, stress in animals reduces gastrointestinal absorption and blood levels of iron (zhao et al, ; chen et al, ) and in human males a -day intense psychological stressor ('hell week') reduced blood levels of iron, zinc, and albumin (singh et al, ) , as would be predicted if stress induces an apr. psychological stress has also been reported to elevate core body temperature in animals and humans (soszynski et al, ; kaneda et al, ; hayashida et al, ) , an anti-pathogen defense mechanism that is most effective in conditions of low host iron availability (kluger and rothenburg, ) . that these changes serve immune purposes is suggested by findings that in animals, stressinduced hypoferremia and hyperthermia are associated with stress-induced increases in il- (soszynski et al, ; zhao et al, ) ; however, other mechanisms may also be active, including hypothalamic activation of brown fat (kataoka et al, ) . because at least some biological changes associated with the apr have been shown to have important roles in pathogen-host defense, their presence in depression would be strongly predicted by pathos-d, and their absence would strongly argue against the validity of this approach. on the other hand, their presence in mdd is not parsimoniously explained by theories that focus on potential social benefits of depression. in what ways would elevated body temperature or reduced iron availability aid in negotiating relationships with other humans? similarly, if depression is simply a non-adaptive phenomenon, why would such ancient, highly conserved and highly complex physiological responses be associated with the disorder? inseparable from the physiological aspects of the apr, immune activation also produces a suite of behavioral responses known as sickness behavior that has been retained across vertebrate evolution and that shares many features in common with the symptoms of mdd (dantzer, ; dantzer et al, b) . these symptoms include lethargy/ fatigue, psychomotor slowing, altered sleep, loss of appetite, anhedonia, and social withdrawal. although it is clear that the symptoms of depression can arise from a wide variety of divergent causes-including internally generated thought patterns-pathos-d proposes that mdd initially evolved out of sickness behavior. circumstantial evidence for this type of close relationship between sickness and mdd come from several sources. for example, data suggest that many people cannot accurately identify whether they are developing depression or an infectious illness early in the course of either condition (ratcliffe, ) . similarly, sickness in the first week of treatment with the cytokine ifn-α strongly predicts the development of cognitive/emotional symptoms of depression over the ensuing months of therapy (wichers et al, a) . finally, if inflammation induces sickness and if depression evolved out of sickness, inflammation might be expected to be more strongly associated with the symptoms most often shared by sickness and depression, rather than symptoms such as sadness and guilt/feelings of worthlessness that are more specific to mdd. supporting this possibility, an early report found that the acute phase protein haptoglobin correlated with psychomotor retardation, sleep disturbance, anorexia/weight loss, and anhedonia (maes et al, ) . more recently, a large population-based study (n = ) reported that crp was more strongly associated with sleep abnormalities, fatigue, and appetite changes than with mdd symptoms less common in the context of sickness (jokela et al, ) . in a group of patients with mdd, increased circulating levels of il- and monocyte chemoattractant protein- were positively associated (and levels of il- were negatively associated) with objectively measured psychomotor slowing (goldsmith et al, a) , and elevated plasma crp was associated with reduced functional connectivity between the ventromedial prefrontal cortex and ventral striatum. these changes in functional connectivity mediated associations between increased crp and anhedonia and psychomotor slowing (felger et al, ) . evolutionary processes do not produce perfect products, and all evolved adaptive mechanisms come with associated costs and trade-offs. if indeed mdd is conceptualized as a condition evolved from, and 'designed' to serve the same purposes as, sickness, the obvious question is what pathogen-related survival and reproductive benefits accrue from depressive symptoms that offset their costs. that depression reduces fitness in modern environments has often been remarked upon; however, the costs across evolutionary time of the types of prolonged sickness behaviors that comprise mdd have also likely been substantial. as part of the apr, sickness exacts significant metabolic costs that limit energy available to other tasks essential for survival and reproduction. in many species, including humans, even subtle signs of sickness promote avoidance behavior from conspecifics (schaller, ) . over and above the suppressive effect of inflammation on sexual functioning, individuals who appear sick are judged to be less attractive and hence are less likely to find high-quality mates. in hierarchical social groups the display of sickness risks loss of social status, with associated reductions in survival and reproductive access. finally, sickness increases the risk of predation (schaller, ) . one possibility is that the pathos-d perspective is exactly backwards, and in fact depression is a maladaptive consequence of the benefits of acute sickness. perhaps depression is akin to other chronic conditions, such as hiv and autoimmune diseases, in which ongoing inflammation, rather than providing benefit, actually increases the risk of infection and/or infectious mortality. many studies associating mdd with increased infectious morbidity in the modern world would support this possibility (zorrilla et al, ; evans et al, ; cruess et al, ; raison et al, ; doering et al, ; faulkner and smith, ; leserman, ; leutscher et al, ; andersson et al, ) . if true, this removes pathogen-host defense as an explanation for the conundrum of why genes that contribute to mdd have been retained across hominid evolution. or it may be that by inducing social withdrawal, depression provides an adaptive advantage for individuals at increased risk of infection as a result of immune functioning that for one reason or other is sub-optimal. this is the position taken by kinney and tanaka ( ) in proposing that depression evolved as an adaptive response to cope with pathogens (anders et al, ) . in contradistinction, pathos-d suggests that-whatever its costs in relation to specific pathogens-depression was not primarily a compensation for immune inadequacy, but rather enhanced overall survival in the eea via its association with physiological and behavioral changes that reduced the impact of infectious agents on reproductive success. said differently, in conditions characterized by a heightened risk for infectious mortality, such as occur during active infection or when environmental adversity signals an increased risk for wounding or other processes that increase the risk of infection (ie, starvation secondary to ostracism), the generalized innate immune activation and symptoms associated with depression provided a net survival benefit, whatever their other costs in terms of immune or social trade-offs. importantly, this perspective does not require that depression be the only way for humans to adaptively cope with infectious risk only that it was adaptive enough for its genetic underpinnings to be retained in the human genome at a level significant enough for mdd to be with us today. from a pathos-d perspective, mdd in response to infection or tissue trauma provides the same types of survival benefits as does sickness behavior. in response to stress, depression can be seen as proactive physiological/behavioral sickness behavior-a type of 'just in case' condition that keeps an individual in a state of pathogen defense readiness. proinflammatory cytokine activation in response to either infection or stress induces a behavioral state of conservationwithdrawal (engel and schmale, ; eisenberger et al, b; hannestad et al, ) characterized by depressed mood, anhedonia, psychomotor retardation, fatigue, social avoidance, and anorexia (capuron et al, ; dantzer et al, b; majer et al, ; irwin and cole, ) . this state is an integral component of depressive disorders and has been widely considered to develop in the context of infection and/ or tissue injury as a means of marshalling limited metabolic resources for the expensive tasks of immune activation, fever generation and tissue repair (hanff et al, ) . in addition to energy allocation, conservation-withdrawal symptoms may have also proved adaptive by reducing interpersonal contact and thereby limiting infectious exposure (kinney and tanaka, ). in the paradigm proposed by kinney and tanaka ( ) this is seen as an evolved compensatory mechanism to cope with immunodeficiency. however, it is equally possible that the infectionavoidance benefits of social withdrawal evolved as a complement to its metabolically valuable infection-fighting benefits. indeed, the more survival and reproductive benefits accrue for any one trait the more likely its genetic antecedents are to be retained and to spread in the population. because humans in the eea lived in small coalitional groups of genetically related individuals, the logic of inclusive fitness suggests that social withdrawal might have been adaptive for an individual's genes by reducing the risk of infection in kin, even if such withdrawal limited the provision of much needed care from others and thus reduced individual survival (cole, ; schaller and murray, ) . indeed, given evidence that merely viewing pictures of a sick individual activates the viewer's innate immune system (schaller et al, ) , it may be that by prepotently activating the innate immune responses in one's kin, depression served as an early warning system for increased infectious risk. in this regard, it is interesting to note that associating with depressed individuals increases the risk of developing depression oneself (fowler and christakis, ) , exactly as would be predicted if depression served as an early warning signal to others of infectious risk and if depression enhanced host pathogen defense by priming the innate immune system. a clear prediction of this line of reasoning that awaits testing is that viewing images of depressed individuals should activate the immune system. in addition to potential inclusive fitness benefits, any decrement in survival from depression-induced loss of social aid might have been more than offset by reduced risk of exposure to other pathogens while in a vulnerable state due to a pre-existing infection, given that viral infections promote aggressive immune responses to bacterial superinfections that can greatly increase mortality (degre and glasgow, ; jakab and dick, ; jones et al, ; nguyen and biron, ; molyneux et al, ; beadling and slifka, ; mccullers et al, ) . moreover, social withdrawal and reduced environmental exploration might also have promoted individual survival by limiting a depressed individual's contact with immunologically dissimilar out-group members who potentially harbored pathogens against which the depressed person would have had reduced immunity compared to pathogens endemic in the home group thornhill et al, ) . this idea suggests a hypothesis that to our knowledge has never been tested that mdd should be associated with increased xenophobia. it is intriguing to note, however, that endotoxin administration increased the desire of healthy volunteers to be around individuals they had previously identified as 'support figures' (inagaki et al, ) , consistent with the possibility that immune changes associated with depression might have promoted an in-group bias that reduced exposure to more potentially lethal pathogens harbored by members of other social groups. how such an increased desire to associate with in-group members would square with the potential selective advantages of withdrawing from kin to prevent the spread of infection is unknown. moreover, given that sterile psychosocial stressors also activate inflammation, it is intriguing to speculate that the benefits of seeking support/proximity with kin/in-group members in response to psychosocial dangers may have offset the potential risks of contagion spread as a result of inflammation enhancing prosocial behavior with trusted ingroup members. with the exception of asthma and allergies, inflammatory conditions typically strike after the age of reproduction, and thus alleles that promote them are subject to minimal selective pressure, even in modern environments (beekman et al, ) . almost certainly, the association between chronic inflammation and these disease states (eg, dementia and cardiovascular disease) serves no adaptive purpose and likely results in large measure from environmental conditions unique to the modern world. however, the relationship between inflammation and depression is quite different, given that the incidence of mdd peaks in the - s (eaton et al, ; patten et al, ; kessler et al, ) , an age of primary reproductive/childrearing responsibilities. in this, mdd is not alone. schizophrenia and bipolar disorder -conditions with which mdd is genetically linked-are also characterized by increased inflammation and by an average age of onset early in life when the costs of these disease states to survival and reproduction are (and were) likely to be maximal. although the current article focuses on unipolar depression, there is no reason to think that depressive symptoms occurring in the context of bipolar disorder would be any less efficacious for pathogen-host defense than the same symptoms when they occur in other contexts, and indeed multiple studies demonstrate that bipolar depression is associated with the same types of inflammatory activation that are observed in unipolar mdd (goldstein et al, ) . however, what should we make of the fact that multiple studies show that inflammatory markers are at least as elevated-and maybe more elevated-in manic episodes as they are during bipolar depressions (goldstein et al, ) ? a proximal answer might point to the fact that mania is associated with many of the same neuroendocrine abnormalities as depression, especially glucocorticoid resistance (schmider et al, ) , which is known to release inflammatory pathways from inhibitory control and which has been associated with increased proinflammatory cytokine levels in mdd (maes, ) . moreover, although far less common than depression, full manic episodes have been reported during ifn-α therapy, which produces chronic immune activation, and combinations of depressive and manic symptoms during ifn-α treatment are common (constant et al, ) . however, this type of answer provides no insight into why mania should be associated with increased inflammation. might the association serve adaptive purposes or is it better considered as an example of antagonistic pleiotropy? here pathos-d offers a novel perspective. until it becomes disabling, mania increases the types of social and sexual extroversion that may provide direct darwinian benefits as a result of increased opportunities for reproduction (berry and miller, ) . however, increased social and sexual activity also increase the risk for reduced survival as a result of increased pathogen exposure (hamrick et al, ) . in this context, might genes linking inflammation with mania have undergone positive selection by conferring increased protection against the wide range of infections to which manic behavior would have exposed an individual in the eea? although highly speculative, this hypothesis produces the clear prediction that the increased inflammation observed in mania should be associated with the symptom of hypersexuality, especially in males, who across evolutionary time would have been more likely than females to benefit in terms of reproductive success from sexual activity with multiple partners. interestingly, animal data provide some evidence for this possibility, given that cytokine activation suppresses sexual activity in female, but not male, rats (avitsur and yirmiya, ) . however, this hypothesis would be strengthened by evidence that manic behavior itself can elevate inflammation and to our knowledge this type of causal connection has not been investigated. in a previous publication we provided an exhaustive enumeration of known immune and/or pathogen defense functions for the best-supported risk alleles for mdd derived from genome-wide association studies (gwas) and meta-analyses of candidate gene studies (raison and miller, ) . these gwas findings are presented in supplementary table s . in the time since our prior publication, an additional study utilized sparse wholegenome sequencing in a population of han chinese females to identify and replicate two novel depression risk loci on chromosome , one ′ to the suirtuin (sirt ) gene (rs ) and one in an intron of the phospholysine phospho-histadine inorganic pyrophosphate phosphatase (lhpp) gene (consortium c ( )). as with previously identified 'genes of interest,' pathos-d predicts that these genes should impact immune function in ways that provided adaptive advantages for pathogen-host defense. in this regard, nothing is known regarding lhpp, but an extensive database attests to multiple immune functions of sirtuin , a class iii histone deacetylase enzyme that serves as an important sensor of cellular energy and redox states (supplementary table s ; michan and sinclair, ) . in particular, sirtuin has been shown via epigenetic mechanisms to have a key role in limiting the extent and duration of acute inflammation by promoting a shift from inflammation-driven glycolytic activity to 'adaptation-phase' fatty acid metabolism. sirtuin suppresses nuclear factorkappa b rela/p activity, represses cyclooxygenase gene expression (zhang et al, b) , and induces gene silencing facultative heterochomatin formation at the promotors of proinflammatory genes, including tnf and il- -β, as well as the promotor for hypoxia-inducible factor- -α, a signaling factor important for maintenance of proinflammatory glycolytic activity (vachharajani et al, ) . in addition, sirtuin , in dependence on nad+, increases levels of peroxisome proliferator-activated receptor gamma coactivator (pcg- )-α and β, which promotes the fatty acid metabolism essential for resolution of the inflammatory response (fernandez-marcos and auwerx, ). the end result of these activities is that sirtuin inhibits the transformation of monocytes to macrophages and promotes anti-inflammatory m macrophages and regulatory t cells (treg) at the expense of proinflammatory m -type macrophages and effector t cells (liu et al, a; park et al, ) . however, conflicting data suggest that in at least some contexts sirtuin may have inflammatory and adaptive immune stimulating effects. for example, sirtuin inhibition has been reported to stimulate foxp expression in treg (akimova et al, ) . despite these complexities, evidence of diminished sirtuin activity in medical conditions characterized by chronic inflammation suggest that its function might also be reduced in mdd, especially in patients with elevated peripheral inflammatory biomarkers. however, to our knowledge, no data are available to support or disprove this idea. similarly, given the association of mdd with chronic inflammation, one would predict that the depression risk allele near the sirt gene should reduce sirtuin activity, if it is found to have a functional effect, which is currently unknown. however, these predictions should be tempered by findings suggesting a complex and contradictory role for sirtuin in pathogen defense. in certain contexts, such as the hypoinflammatory phase of sepsis, sirtuin blockade has been reported to reduce bacterial load and enhance survival. sirtuin blockade has also been shown to inhibit hepatitis b replication in hepatocytes (ren et al, ) . similarly, sirtuin-deficient mice have been shown to demonstrate improved intestinal anti-bacterial defense mechanisms (lo sasso et al, ) . on the other hand, the use of celecoxib to stimulate sirtuin within macrophages resulted in an enhanced ability of ampicillin to clear staphylococcus aureus from these cells (annamanedi and kalle, ) , consistent with the observation that low levels of sirtuin within macrophages associates with bacterial infection in these cells (zhang et al, a (zhang et al, , b . sirtuin also appears to be essential for optimal immune clearance of respiratory syncytial virus and has been shown to suppress human t-cell leukemia virus type transcription (owczarczyk et al, ; tang et al, ) . although a perusal of supplementary table s highlights the remarkable degree to which sirt and many other potential depression risk alleles impact both immune function and pathogen defense, it should be noted that there is another pathway whereby these risk alleles might enhance pathogen-host defense without directly modulating immune function. if the pathos-d proposal that depressive symptoms themselves serve pathogen defense functions is correct, genes that promote these symptoms might have served adaptive purposes in fighting infection via this mechanism alone. for example, in addition to multiple immune effects, sirtuin signaling in the hippocampus appears to have antidepressant effects, given animal data that pharmacologic and genetic inhibition of hippocampal sirt function increases depression-like behaviors in response to stress, whereas sirt activation blocks both the development of depression-related phenotypes and aberrant dendritic structures elicited by chronic stress exposure (abe-higuchi et al, ) . cacna c is another putative depression risk gene known to both enhance and impair pathogen-host defense depending on the microorganisms involved (raison and miller, ) . carriers of the rs a risk allele have also been shown in several studies to demonstrate changes in brain function and morphology relevant to mdd (bigos et al, ; erk et al, ; perrier et al, ) , again raising the possibility that even genes with known immune effects might enhance pathogen defense either primarily, or synergistically, via the direct induction of depression in response to environmental adversities known to increase the risk for pathogen-induced morbidity/ mortality (ie, stress signaling an increased risk of wounding, active infection; bigos et al, ; erk et al, ; perrier et al, ) . understanding the impact of putative risk genes like sirt or cacna c on inflammatory and behavioral responses to psychosocial stress would provide invaluable insight into the question of whether they promote depression and pathogen-host defense via the type of inflammatory activation that is a hallmark of mdd in the modern world. pathos-d proposes that depression risk alleles have been maintained in the human genome primarily because, by enhancing pathogen-host defense, they provided a net survival and reproductive advantage. a primary problem that this theory shares with other adaptive explanations for depression is that to date no incontrovertible risk alleles for mdd have been identified. indeed, most large gwas have failed to find snps that meet genome-wide significance and when such snps are found (ie, rs in dcnp and rs in tnf) they are not replicated in gwas conducted in other populations (raison and miller, ) . multiple explanations have been offered for this case of 'missing depression genes,' most often that mdd is heterogeneous in relation to its environmental antecedents, the nature and severity of its symptoms and its disease course; and that therefore it is likely that the condition is a concatenation of numerous as-yet-unidentified more etiologically homogeneous sub-conditions that are likely to be more genetically consistent. although not dismissing disease heterogeneity as a cause for failure to replicate depressive risk alleles, pathos-d offers an additional novel explanation for at least some cases of replication failure. because pathogens can develop resistance to host immune defenses, or in many cases actually evolve to benefit from these defenses via pathogen manipulation strategies, not all genetically programmed physiological or behavioral pathogen-host defense strategies will be equally effective against all microbes. because of this, in cultures/societies exposed to different mixtures of pathogens across time, different immune mechanisms will be adaptive, and therefore likely to become linked to depression. said differently, because of geographic and historical differences in pathogen exposure, we should not expect all human ethnic/racial groups to have benefitted from the same genetically driven pathogen defense strategies and therefore to show the same risk alleles for mdd. thus, a pathos-d perspective suggests that understanding the specific histories of shared and unique co-evolved pathogen-host interactions within and across human societies will be essential for enhancing our understanding of why genotype-phenotype associations observed in one population do or do not replicate across other populations, as well as why certain genetic variants drive physiological processes that induce depression in certain individuals but not in others. these insights may help account for the intriguing finding that the association between sir nor lhpp snps and mdd identified in han chinese females was not replicated in european populations (consortium c ( ) ). pathos-d would predict that this finding might be accounted for by the fact that sirt and lhpp conferred specific anti-pathogen defenses in the chinese environment that were not relevant in europe. if so, one would expect the prevalence of the sirt and lhpp risk alleles to be significantly higher in han populations than in europeans as a result of positive selection, and this is indeed the case (consortium c ( ) . many genes have been replicated across a range of ethnic/ racial groups as risk factors for schizophrenia, suggesting that at least certain pathways for the disorder are ancient and arose prior to the first migrations of modern humans out of africa. from a pathos-d perspective, the fact that no such replicated risk alleles have been found for mdd may suggest that the condition is more environmentally based than are conditions with more universally established genetic risk genes, but not in the way that this environmental primacy is usually understood. instead, mdd may have no universal risk genes because pathogen pressures in local environments over the last years have been pre-eminent in determining which specific immune mechanisms were most adaptively linked to the generation of depressive symptoms in any given environment. if it turns out that the association between depression and increased inflammation is a human universal (not universal in terms of all depressed individuals showing increased inflammation, but rather that groups of depressed individuals demonstrate increased inflammation across all ethnicities/cultures), then the clear prediction of pathos-d would be that alleles that confer depression risk in one population but not another should either ( ) increase inflammatory signaling more in the population in which they confer risk; or ( ) more powerfully increase the prevalence/ severity of depressive symptoms in response to any given degree of inflammatory activity in the population in which they confer risk. similarly, because present-day human societies/cultural groups have historically experienced widely different degrees of exposure to bacterial and viral pandemics that exerted extreme selective pressure (fincher et al, ) , pathos-d predicts that the types of immunity that best cope with these different types of pathogens should have different relationships with depression across human cultures. so, for example, because interferon signaling is especially essential for host defense against viruses, a pathos-d perspective would predict that individuals from cultures with higher rates of historical exposure to lethal viral pandemics should respond to treatment with ifn-α with increased rates of depression development, if indeed depressive symptoms aid in pathogen defense. were it possible to assess, this effect should be largest in hunter-gatherer populations that have evolved in the types of low population density environments that work against widespread viral infections. in such populations, viruses provided little positive selection pressure that would link antiviral defenses with depression, whereas such populations would have benefitted from optimal protection against environmental pathogens (eg, protozoa, bacteria, and fungi) that would have increased mortality under these conditions. were such a population available, the prediction is that they would show enhanced depressive symptoms in response to a bacterial stimulant such as endotoxin, while showing a muted depressive response to ifn-α. alas, given the escalating loss of isolated indigenous cultures, the time for such a definitive study may have passed. adaptive introgression provides another fascinating example of why pathogen-host defense processes may ensure that no universal genes for mdd will ever be found. research over the last several years indicates that~ % of the dna of non-african humans derives from multiple bouts of interbreeding with neanderthals, with current melanesian populations also showing a denisovan genetic inheritance from ancestral interbreeding with that group of now-extinct archaic humans . in general, the results of this interbreeding were deleterious, as attested to by multiple 'desert' regions of human dna depleted of archaic genetic material. against this backdrop, it is striking that other neanderthal and denisovan sequences appear to have undergone positive selection. from a pathos-d perspective it is even more striking that these archaic alleles are associated with both immune function and with an increased risk of depression (mendez et al, ; segurel and quintana-murci, ; simonti et al, ) . although it does not yet appear that the same neanderthal allelic variants code for both immunity and mood, anyone concerned with adaptive explanations for mdd cannot help but be struck by this association, especially given that negative selection appears to have removed archaic human dna from areas of the genome involved with other aspects of cns morphology and function. researchers tend to assume that retained archaic immune alleles provided an adaptive benefit for coping with novel eurasian pathogens; pathos-d would suggest that their proclivity to induce depression provided additional advantages in our endless struggles with microbes and parasites. here we consider ways in which pathos-d might provide adaptive explanations (as opposed to mere proximal/ mechanistic explanations) for three epidemiological features of mdd that are striking characteristics of the disorder and relate directly to sex: ( ) the : female preponderance of the disorder; ( ) the impact of age and sex on the disorder's phenotypic presentation; and ( ) the significant risk of the postpartum period for mdd development. females are approximately twice as likely as males to suffer from mdd (seedat et al, ) , with this imbalance being most pronounced during the reproductive years, exactly when the manifold negative social and biological effects of depression would be expected to impact evolutionary fitness most adversely (fiske et al, ) . while many answers for the female preponderance of depression have been proposed over the years (eg, females are more sensitive than males to the social environment), none have successfully identified an adaptive advantage that would outweigh the costs of female depression to survival and reproduction. in contrast, pathos-d suggests a testable explanation for why depression is so common in females of reproductive age. females are more likely to develop depression during the reproductive years because across evolutionary time depressive symptoms-having evolved out of sickness-promoted behaviors that decreased the risk of pathogen exposure and provided increased protection from pathogens for any given level of inflammatory activation once an infection had occurred (eg, by increasing energy available to immune processes by inducing conservation-withdrawal behavior, maintaining elevated body temperature, and so on). said differently, by inducing social and biological behaviors that promoted host defense against pathogens, depressive symptoms allowed females of reproductive age to 'get by' with less inflammation. the unique importance of this for reproductive success in females is highlighted by the fact that inflammation reduces fertility, impairs lactation and directs metabolic resources away from the multiple energetically-costly aspects of female biology that were required for successful reproduction in ancestral environments (van bodegom et al, ; schaller, ; kobayashi et al, ) . interestingly mdd has also been associated with reduced fertility (williams et al, ; nillni et al, ) . pathos-d theory predicts that this association should less prominent in females who have effectively 'replaced' inflammation with depression-based behavioral pathogen avoidance in the absence of elevated levels of inflammatory biomarkers. although most implications of this idea remain to be tested, some data support the first prediction of this hypothesis, which is that females should develop increased levels of depression for any given amount of inflammatory exposure. indeed, when compared with men, females respond to an injection of lipopolysaccharide (lps) with an increase in depression and feelings of social isolation, both of which are correlated with amount of cytokine response to the lps in females, but not males (moieni et al, b) . similarly, females are more likely than males to develop depression in response to chronic inflammatory stimulation resulting from treatment with the cytokine ifn-α (udina et al, ) . finally, given the pre-eminent role of psychosocial stress as an environmental risk factor for mdd, as well as evidence that hyperthermia enhances host defense, it is intriguing that reproductive aged females appear to be most vulnerable to developing psychogenic fever (kaneda et al, ). however, these findings need to be balanced against results from a small recent study indicating that females mounted an enhanced proinflammatory cytokine response to low-dose endotoxin when compared with men, while showing no difference in inflammation-induced mood or anxiety symptoms-a pattern of findings opposite to this pathos-d prediction (engler et al, ) . if confirmed in larger studies, these findings might support an alternative immune-based mechanism for the female/male preponderance of depression and anxiety, specifically that females respond to immune challenges with greater immune activation which subsequently increases the risk for depression/anxiety. mdd is most commonly characterized by impaired sleep and reduced appetite , but a significant minority of depressed individuals manifest hypersomnia and hyperphagia instead (thase, ). these symptoms are most frequent in females between adolescence and middleage (carter et al, ; posternak and zimmerman, ; matza et al, ; blanco et al, ) . how might pathos-d theory seek to account for the existence of these reversed neurovegative symptoms, as well as their age and sex distribution? a first step is to establish that cytokine pathways known to be activated by infection are capable of producing both hypersomnia and hyperphagia. hypersomnia has been recognized for years as a primary behavioral manifestation of proinflammatory cytokines (krueger and majde, ; krueger and toth, ; krueger and majde, ; opp, opp, , , and studies in healthy adolescents and adults indicate that chronically increased sleep is associated with increased saliva and blood concentrations of il- and crp (el-sheikh et al, ; patel et al, ; prather et al, ) . in terms of feeding behavior, animal models demonstrate that inflammation promotes carbohydrate preference (aubert et al, ) , such as is typical in depressive hyperphagia. less well known are data that highfat diets induce leptin and insulin resistance, as well as obesity, dependent upon activation of inflammatory mediators in the hypothalamus (zhang et al, ; kleinridders et al, ) . conversely, blocking hypothalamic inflammation prevents obesity and other stigmata of the metabolic syndrome, even in the context of high-fat consumption (zhang et al, ; milanski et al, ; posey et al, ). in rodents, exposure to either tnf or il- in utero results in adulthood obesity (dahlgren et al, ) , again suggesting that cytokines can induce hyperphagia/weight gain under certain circumstances. although we know of no data showing that females are more likely than males to respond to inflammatory signaling with hyperphagia, female rodents have less anorexia than males in response to influenza infection (avitsur et al, ) . because the presence of widespread obesity is recent, the question arises as to whether more ancient signals for inflammatory activation might also promote hyperphagia, instead of anorexia. we know of no data that directly address this issue in terms of infection, but note that certain adenoviruses have been associated with weight gain (atkinson, ; mitra and clarke, ) . in terms of psychosocial factors, preclinical studies suggest that stressors associated with a high degree of wounding-such as chronic social defeat-induce hypothalamic resistance to leptin (kleinridders et al, ; chuang et al, ) , consistent with their known ability to activate inflammatory pathways (avitsur et al, ; bailey et al, bailey et al, , powell et al, ) . as in depression, where hyperphagia and weight gain are associated with disease chronicity (posternak and zimmerman, ) , chronic social defeat leads initially to weight loss but to weight gain over time (chuang et al, ) . given the role of leptin resistance in hyperphagia, it is intriguing that increased peripheral leptin levels (consistent with leptin resistance) have been observed more consistently in females than in males with mdd (rubin et al, ; yang et al, ; pasco et al, ; zeman et al, ; cizza et al, ) , and are more common in atypical than non-atypical depression (gecici et al, ) . why might younger individuals in general, and females in particular, be more likely to develop depressive conditions characterized by hypersomnia and hyperphagia? from a pathos-d perspective, the simplest answer may be: because they can. in hunter-gatherer societies youth and females are frequently more protected from predation and to have food supplied to them by others (kaplan and gurven, ) , raising the possibility that in ancestral environments these individuals enjoyed sufficient security to partake of the recuperative effects of sleep and to eat without having to expend energy searching for food. given evidence that anorexia may confer anti-pathogen effects (raison and miller, ) , might similar benefits accrue to hyperphagia, especially in youth and females, under at least some evolutionarily relevant situations? although admittedly speculative, several lines of evidence suggest that by promoting hypothalamic resistance to leptin, genes associated with increased inflammatory signaling might enhance host defense by driving ongoing leptin production despite malnutrition in conditions of food scarcity. this increased leptin production would both augment the drive to seek and consume food and would lower the set point at which food consumption initiated the types of inflammatory responses that are all too common in the obese, but that appear to be life-saving in the face of an infection such as tuberculosis, that was (and is) most lethal for females of reproductive age and for which low body weight and reduced leptin are deadly (van crevel et al, ; wieland et al, ; buyukoglan et al, ; leung et al, ; pednekar et al, ; roth, ) . depression and anxiety are highly comorbid, and in both animals and humans, activation of the inflammatory response produces both types of symptoms. in a previous publication, we devoted significant space to providing a pathos-d perspective on why inflammation produces changes in brain function that subserve not just conservation-withdrawal symptoms likely to aid in defense against pathogens, but that also have been associated with hypervigilance symptoms (eg, anxiety/agitation, insomnia, anger/irritability (association ap, )) that across human evolution would have presumably siphoned metabolic resources away from the life-or-death task of immune protection (raison and miller, ) . we suggested that sickness behavior-while of benefit for surviving infectioncarries its own survival and reproduction costs as a result of increased risk for predation and reduced ability to care for one's young, as well as potential loss of status in a social species and/or loss of breeding territory (miller and cohen, ) , all of which were relevant to humans in the eea. given these competing challenges to survival and reproduction, evolutionary logic may have dictated that inflammatory processes-especially when chronic-might have promoted hypervigilant behavior, which, while shunting energy away from fighting infection, would nonetheless have served adaptive purposes by protecting against environmental dangers engendered by sickness. given that human females and youth likely received more protection in the eea than did adult males (who therefore on average would have more to lose from sickness behavior), a clear prediction from pathos-d is that males should respond to immune challenges/inflammatory activation with an increased ratio of hypervigilant to conservationwithdrawal symptoms, as well as an increase in cytokine effects on brain areas such as the dorsal anterior cingulate that subserve hypervigilance when compared with cytokine effects on areas such as the ventral striatum, which have been linked to inflammatory effects on anhedonia (felger et al, ) , which would be expected to promote social withdrawal. available data provide mixed support for this hypothesis. on the one hand. a recent study found no difference between males and females in the severity of anxiety symptoms induced by low-dose endotoxin (engler et al, ) . on the other hand, a recent large cohort study reported that peripheral inflammatory biomarkers were more strongly associated with anxiety symptoms and anxiety disorders in males than in females (duivis et al, . more definitive confirmation or refutation of the possibility that males respond to inflammation with increased hypervigilance await studies specifically designed to assess the modulating effect of sex on the impact of cytokines on hypervigilant-specific behaviors and on neural pathways known to mediate this type of behavior. given significant evidence that maternal depression in the postpartum period is associated with numerous short-and long-term adverse consequences for the affected offspring (ghodsian et al, ; stein et al, ; beardslee et al, ; beck, ; halligan et al, ; kersten-alvarez et al, ) , the question of why the condition is so common is especially pressing. as with all evolutionary considerations, the first question to consider is whether risk genes that promote postpartum depression (ppd) confer an adaptive benefit, at least in part, via benefits resulting from the condition itself or whether ppd is pleiotropically linked to non-related effects of these genes that benefitted survival and/or reproduction across evolution in ways that outweighed the cost of ppd. from a pathos-d perspective, the question might be framed by asking whether ppd enhanced maternal survival and/or reproduction via an overall net benefit in pathogen-host defense. in terms of maternal survival, ppd would be expected to serve the same pathogen defense purposes as mdd in general. however, what about the survival of the newborn, which reflects a significant investment on the mother's part in her long-term reproductive success? might maternal depression contribute to the child's pathogen-host defense prospects? an interesting twist on this theme might be provided by parental investment theory, which postulates that parents reduce their care for, and involvement with, offspring when the costs of such care and involvement outweigh the benefits (trivers, ) . building on this theory, it has been argued that ppd is an evolved mechanism that aids mothers in disengaging from infants with a low chance of surviving (hagen, ; bottino et al, ) . because infection was the primary cause of infant mortality across human evolution, one might predict that ppd would be more likely to develop in response to giving birth to an unhealthy infant, and data from hunter-horticulturalists in the amazon basin are consistent with this prediction (hagen and barrett, ) . under this scenario, ppd, rather than helping the infant survive pathogen challenge, would actually serve as a 'warning signal' to the mother that the infant's likelihood of surviving postnatal pathogen exposure was not high enough to warrant significant resource investments. although consistent with a pathos-d focus on the primacy of pathogen-host interactions in selecting for depression-promoting genetic variants, the notion that ppd functions as a type of immunological abandonment strategy goes against the general tenor of the theory, which sees the link between depression and immunity as providing overall advantages in the face of infectious morbidity and mortality, whatever their costs in other domains. whether ppd is associated with behavioral or immune changes that might actually help infant survival-especially in conditions such as high environmental pathogen exposure, reduced metabolic resources or increased infant vulnerability to infection-is a question that to our knowledge has never been examined. certainly it is possible that ppd-induced maternal withdrawal might have decreased the risk of infant infection in circumstances when the maternal depression resulted from pathogen-induced cytokine activation. it is also intriguing to speculate that ppd might influence the composition of breast milk in ways that might aid nursing infants in avoiding or surviving infection. intriguingly, a japanese study found ppd to be associated with increased breast milk concentrations of the cytokine transforming growth factor (tgf)-β . tgf-β has complex pro-and antiinflammatory effects (kondo et al, ) , but in the context of the newborn immune system, it has been shown to have a key role in establishing the mucosal immune response, including production of secretory immunoglobulin a (ogawa et al, ; oddy and rosales, ) . also of potential relevance, reduced levels of tgf-β have been associated with increased symptom severity in children infected with plasmodium falciparum (chaiyaroj et al, ; rovira-vallbona et al, ) , suggesting that the provision of increased tgf-β in breast milk from depressed mothers might confer some protection against malaria, which has been a primary source of pathogen-induced mortality across human evolution. importantly, tgf-β and other immune factors in breast milk remain biologically active after passage through the infant stomach as a result of its higher ph (hennet and borsig, ) . in essence, pathos-d does nothing more than attempt to instantiate and provide specifics to the observation from the quote that started this article, that 'the only…compensating fitness benefit that has been documented for major human genetic diseases is resistance to infection' (cochran et al, ) . we have argued that depression and its relationship with inflammation and in particular the acute phase response is yet another example of this evolutionary reality, providing insights into the puzzling lack of consistency of risk alleles across populations as well as the epidemiology of the disorder including its risk factors in ancient and modern times and its age of onset and female preponderance. funding for this study was provided for clr by mary sue and mike shannon, the usona institute and the university of wisconsin-madison and for ahm by r mh ; r mh ; supported in part by the national center for advancing translational sciences of the national institutes of health under award number ul tr . the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. study funders had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. dr raison serves on the scientific advisory board for usona institute. dr miller reports no conflicts of interest. hippocampal sirtuin signaling mediates depression-like behavior targeting sirtuin- alleviates 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to clinical progression of viral illness key: cord- -wtu r j authors: goddard, frederick g. b.; ban, radu; barr, dana boyd; brown, joe; cannon, jennifer; colford, john m.; eisenberg, joseph n. s.; ercumen, ayse; petach, helen; freeman, matthew c.; levy, karen; luby, stephen p.; moe, christine; pickering, amy j.; sarnat, jeremy a.; stewart, jill; thomas, evan; taniuchi, mami; clasen, thomas title: measuring environmental exposure to enteric pathogens in low-income settings: review and recommendations of an interdisciplinary working group date: - - journal: environ sci technol doi: . /acs.est. c sha: doc_id: cord_uid: wtu r j [image: see text] infections with enteric pathogens impose a heavy disease burden, especially among young children in low-income countries. recent findings from randomized controlled trials of water, sanitation, and hygiene interventions have raised questions about current methods for assessing environmental exposure to enteric pathogens. approaches for estimating sources and doses of exposure suffer from a number of shortcomings, including reliance on imperfect indicators of fecal contamination instead of actual pathogens and estimating exposure indirectly from imprecise measurements of pathogens in the environment and human interaction therewith. these shortcomings limit the potential for effective surveillance of exposures, identification of important sources and modes of transmission, and evaluation of the effectiveness of interventions. in this review, we summarize current and emerging approaches used to characterize enteric pathogen hazards in different environmental media as well as human interaction with those media (external measures of exposure), and review methods that measure human infection with enteric pathogens as a proxy for past exposure (internal measures of exposure). we draw from lessons learned in other areas of environmental health to highlight how external and internal measures of exposure can be used to more comprehensively assess exposure. we conclude by recommending strategies for advancing enteric pathogen exposure assessments. exposure to enteric pathogens is associated with a heavy disease burden, largely born by young children living in lowincome settings. globally, enteric infections represent the third leading cause of death among children under five, accounting for approximately deaths in . methods to characterize human exposure to enteric pathogens have not been advanced in the same way as they have in other areas of environmental health, such as exposure to air pollution or industrial chemicals, perhaps in part because the resources are not available in the settings where enteric diseases carry a disproportionally high burden of disease. exposure science was born out of a need to measure exposures to industrial and occupational air pollution and chemical toxicants. this need arose largely in high-income countries following the industrial revolution, which ushered in a period of rapid urbanization and proliferation of factories in urban centers exploiting processes involving fuel combustion. perhaps one of the most notable examples of this sudden increase of population exposure to high levels of air pollution is the london smog incident, which caused an estimated casualties in . thereafter, air pollution exposure scientists developed methods that advanced exposure assessments, ranging from stationary equipment to measure aggregate population-level exposures and pollutant trends, to portable air pollution monitors that provide personal data in real time. chemical exposure science has since evolved to include both external and internal biological measures of several hundred toxicants in large, diverse populations. there are a number of factors that contribute to the difficulty of characterizing exposure to enteric pathogens; some of these factors are associated with the microbial agent, others with the environment and yet others with the host response. first there are technological difficulties of detecting and quantifying a wide range of microbes whose pathogenicity, transmissibility, and fate and transport can vary under different environmental conditions. , even identifying the agents is challenging, as there is still considerable uncertainty about whether and under what conditions detectable microbes are pathogenic, specific pathogens can be present at low concentrations in the environment, and due to frequent discoveries of new and emerging pathogens or known organisms that have acquired disease causing genes. while the source of enteric pathogens is mainly from fecal contamination, there are mixed correlations between the presence of indicators of fecal contamination and enteric pathogens in the environment. , second, environmental enteric pathogen exposure assessments need to consider multiple potential fecal−oral transmission pathways, commonly represented by the f-diagram: fingers, flies, food, fluids (water sources), fields (soil), and fomites. − of increasing interest is the role of zoonotic transmission of enteric pathogens via animal excreta. third, the biological relevance of enteric exposures is complicated by responses to exposure, which is affected by various host susceptibility factors that mediate dose−response relationships, including those engendered by vaccines or previous exposures. response to exposure may also be influenced by gastrointestinal health, such as the diversity of the gut microbiome or increased risk due to enteropathies such as environmental enteric dysfunction. external versus internal measures of exposure to enteric pathogens. exposure to an environmental contaminant is conditional on a complex source-to-host pathway that includes ( ) the release of a contaminant from its source; ( ) the fate and transport of the contaminant in the environment leading to a specific concentration in different environmental media; ( ) the host interaction with those media, ( ) a portal of entry into the human body (i.e., an exposure route), and ( ) uptake of the contaminant into the body. traditional exposure science uses numerous approaches for measuring human exposure along this pathway continuum. a recent national research council report, exposure science in the st century, presented a summary of these approaches, ranging from those that measure environmental concentrations of contaminants to predict exposures before the contaminant reaches the human boundary, to those that estimate a dose after the contaminant has been taken up into the body. here, we propose a similar approach to defining exposure to enteric pathogens, by differentiating between external and internal exposure assessments. we define enteric pathogens as microorganisms transmitted via the fecal−oral route that can cause gastrointestinal infections, leading to acute (e.g., diarrheal disease, gastroenteritis, enteric fevers) and chronic infectious disease outcomes (e.g., environmental enteric dysfunction, growth faltering, impaired cognitive ability). enteric pathogens include bacterial, viral and protozoan pathogens, with fungi and helminths increasingly receiving more attention as causes of neglected tropical infectious disease. , a common approach for external exposure assessments is to detect indicators of fecal contamination or specific pathogens in a known size of environmental sample. environmental measures such as these can act as proxies for external exposure as they assess specific fecal−oral transmission pathways. measuring host-specific microbial source tracking markers in environmental samples can also provide information on the source of fecal contamination (i.e., animal versus human). however, environmental measures on their own do not provide precise measures of the magnitude of exposure (i.e., how much contaminated water one actually ingests) which must be estimated or imputed, so can only be considered as an indirect proxy for actual ingestion of pathogens. augmenting environmental data with data on human interactions with their environment, such as those published by the united states environmental protection agency in the exposure factors handbook, can help provide actual estimates of exposure (i.e., pathogen ingestion). while the handbook is focused on chemical exposures in the united states, it demonstrates the types of frameworks that can be employed to inform assumptions for external exposure assessments, such as ingestion rates (drinking water, soil, and food) and object mouthing. in contrast, internal enteric pathogen exposure assessments using human biological specimens may estimate the actual exposure to enteric pathogens after crossing the human body envelope, typically via oral ingestion. in this respect, they address the main shortcoming of the external exposure assessment, i.e., whether a pathogen has actually been ingested. on the other hand, they provide little information about the source or transmission pathway that external exposure assessments offer. they can, however, provide some information on the presence, types, and frequency of past exposure to enteric pathogens as well as indications of potential health impacts. while the ingested dose of enteric pathogens is not typically measured, past exposure events can be inferred from serology, detection of pathogens in feces, and other biomarkers as proxies of internal exposure. the need for improved exposure methods: the wash case. systematic reviews of water, sanitation, and hygiene (wash) evaluations, conducted to identify the health effects of interventions designed to reduce enteric pathogen exposure, have generally found improved wash to be protective against diarrhea, soil-transmitted helminthiasis, and malnutrition. , however, much of the evidence is from observational studies or short, smaller-scale trials. recent experimental field evaluations of some of these interventions found either no evidence of health benefits, − a reduction in diarrhea but no improvement in child growth, or improved growth but no impact on diarrhea. , these mixed results have focused greater attention on the need for more rigorous exposure assessment, in part to explain why effects of wash interventions are realized in some trials and not others. a recent consensus statement recommends that interventions need to radically reduce fecal contamination in the environment to achieve more consistent child health benefits, though which pathogens and pathways are most important and the necessary reductions needed to achieve health impactsmay be highly context-specific. filling these gaps requires greater attention on more rigorous exposure assessment, as few studies have attempted to directly measure exposure along the various transmission pathways, and those that do normally rely on quantifying indicators of fecal contamination in the environment as a proxy for pathogens. moreover, these approaches are largely confined to assessments of external exposure, sometimes combined with observations or modeling to estimate ingested dose of enteric pathogens. the theory of change underlying the impact of a wash intervention on enteric health outcomes is that an intervention will prevent disease if the intervention (a) is capable of but only a few have assessed the impact of an intervention on exposure to fecal contamination along the transmission pathways targeted by the interventions, in part because there is no consensus methodology for how to measure exposure. some of the null findings from recent wash interventions are consistent with the wash theory of change, reporting null effects from potentially effective interventions delivered to a vulnerable population when coverage and uptake were low. , , , however, other studies have reported null effects on diarrhea , and/or stunting , , even with higher levels of coverage and use; others have reported protective effects on stunting (but not diarrhea) with high levels of coverage and use, especially from reductions in open defecation. , , some wash evaluations, identified from a systematic review investigating the relationship between indicators of fecal contamination and child health outcomes, included exposure assessments that raised questions about the manner in which they characterized exposure (figure ). in a recent trial of individual and combined wash interventions, luby and colleagues reported reduced diarrhea from wash interventions, except for water treatment using chlorine, even though reductions in fecal indicator bacteria were found in both stored drinking water and food in households that received the water treatment intervention compared to control households. this suggests perhaps that the chlorine-susceptible indicator bacteria used to estimate fecal contamination were not representative of chlorine-resistant pathogens, such as protozoa that may have been contributing to diarrhea symptoms. this is consistent with the trial's findings of reductions in giardia infection in all wash arms except for the water treatment arm. the same evaluation also reported reduced diarrhea and prevalence of infections with protozoa and soil-transmitted helminths in the sanitation-only arm , even though there was no evidence of a change in fecal indicator bacteria in water, food, soil, on hands, or a change in fly density in this arm compared to controls, suggesting reductions in disease transmission not captured by the fecal indicator bacteria measurements. reese and colleagues reported no impact on diarrhea but reduced stunting from a water supply and sanitation intervention despite no evidence of a reduction in fecal contamination of drinking water or hands. pickering and colleagues also found no effect of a community-led sanitation intervention on diarrhea but an improvement in child growth, despite no reduction in fecal contamination in drinking water; however, latrine fly presence and observed human and animal feces did significantly decrease in the treatment group. collectively, these studies illustrate the need for improved characterization of external exposures to enteric pathogens. similarly, limited tools currently exist for assessing internal exposures. as described below, some studies combine external assessment with behavioral observations to estimate actual ingestion (e.g., measuring pathogens in soil and frequency of geophagia, or measuring fecal indicators deposited by flies when alighting on food and the number of fly landings). however, these methods rely heavily on assumptions about conditions and behaviors that vary significantly within and between individuals, and over time. others have begun using proxies of internal exposure as an indicator of past exposure, such as measuring enteric pathogen shedding in child stool or serology from dried blood spots as indicators of past exposure. , while investigators have sought to reconcile the inconsistent relationship between exposure measurements and health with accepted theories of change, the lack of a clear and consistent progression between these proxies of exposure and health outcomes raises fundamental questions about current exposure assessment methods. in addition, they raise questions concerning "how clean is clean enough" for realizing reductions in rates of infection and disease. for program implementers to optimize interventions that improve health, rapidly identifying the leading sources of exposure in a community can help develop and test context-specific interventions that address them. epidemiologists are seeking reliable data on which to build models to ascertain determinants of disease risk and prioritize disease control strategies. public health officials are seeking data on exposure to implement cost-effective population-based strategies to mitigate health burdens. all of these demands rest on our ability to generate better, cheaper, faster, field-deployable approaches that can be used in low-resource settings with minimal training. with this as background, an interdisciplinary group of environmental health researchers convened at emory university in atlanta, georgia (u.s.a.) in september for a workshop aimed at identifying priorities for improved approaches to measuring enteric pathogen exposure. while much of the discussion focused on exposure assessment in household-level wash studies, the group agreed that the challenges extended to exposure to enteric pathogens in lowand middle-income countries more generally that are transmitted via the fecal−oral pathway, including exposures from agricultural, commercial and recreational activities. the objectives of the workshop were to (a) identify applications and criteria under which to compare exposure assessment approaches, (b) explore potential lessons in exposure science from other areas of environmental health, (c) critically review current and emerging enteric pathogen exposure assessment practices across a range of applications, and (d) define research priorities to help move the field of enteric pathogen exposure science forward. participants in the workshop were u.s.-based experts purposely selected from a variety of disciplines who were available and willing to meet and explore the issues presented. such expert gatherings present the potential for selection bias in the opinions expressed. we identified four primary applications that could benefit from further resources dedicated to improving enteric pathogen exposure methods. . identifying primary sources and modes of transmission: improved measures to identify the leading sources and modes of transmission of enteric pathogens in a given setting could inform programs and investments designed to improve health by reducing fecal exposure. specific pathogens are differentially mediated by various fecal−oral transmission pathways, and the importance of these pathways may differ between settings (e.g., urban vs rural), subpopulations (e.g., children in different age groups) or time points (e.g., season). improved external exposure measures would contribute to a better understanding of what exposure pathways need to be interrupted in a given setting to achieve consistent child health benefits. . evaluating interventions: better tools to estimate enteric pathogen exposure could be leveraged to evaluate the extent to which interventions are able to reduce exposure, and could have implications for the sample sizes needed to detect differences between groups. these tools could include measuring pathogens or proxies along specific pathways that interventions are designed to interrupt, and measuring pathogens in human biological samples to characterize past exposure. using exposure assessments to evaluate interventions would potentially enable a faster and more objective evaluation of interventions designed to improve health by reducing exposure compared to studies that focus on health end points. . surveillance: novel approaches to characterizing exposure to enteric pathogenssuch as monitoring emerging agents in wastewatercould be applied for surveillance purposes. these purposes could range from monitoring human infection prevalence, rapid assessments of environmental threats, such as potential infectious disease outbreaks or bioterrorism, to monitoring the progress of regional or national efforts toward reducing infections. . exploratory: there are broader research applications that could benefit from improved measures of enteric pathogen exposure. examples include previously understudied links between enteric pathogen exposure and its effects on the gut microbiome and malnutrition, as well as its relationship with comorbidities such as respiratory and vector-borne diseases. improved exposure assessments can also be linked to important subclinical conditions, as well as to a better understanding of inflammatory responses that are increasingly associated with a wide range of health effects. there is also a need to better understand the underlying mechanisms of transmission and pathogenesis associated with environmental exposures to enteric pathogens. when considering different approaches to measuring exposure to enteric pathogens for each of these applications, there are a number of criteria to consider. these include the following: • external vs internal: is exposure characterized in the environment as a proxy for external exposure and does it provide data on the source of exposure, or is it measured after the contaminant has crossed the human boundary (i.e., internal exposure)? for external exposure assessments, is exposure characterized proximal to the human boundary or is it a more distal measure that requires modeling to estimate more proximal exposure? for internal exposure assessments, are the measures mediated by host susceptibility to infection? • pathway-specific: can the exposure assessment quantify the relative contribution to total exposure by different transmission pathways? deployment in the field in low-resource or emergency settings? does the assay require cold-chain transport or a consistent energy source? are the required materials bulky or dangerous to handle? how much training and material is required to collect samples, conduct analyses, and interpret results? is it fast enough to provide actionable feedback to reduce exposures in a population of interest? • ethics: are exposure assessment methods potentially burdensome on the communities and individuals where they are conducted? do they require respondents to provide a substantial amount of resources (e.g., large volume water samples) and time? do they involve an invasion of the respondents' privacy? do they provide interpretable information for end users and how do users respond to that information? ■ lessons from other areas of environmental health air quality. in other areas of environmental health, such as air and chemical pollution epidemiology, moving from ecologic or population-level measures of exposure to measures that better reflect exposure at the individual level has been a focus of research for the past two decades. in , for example, the national research council committee on research priorities for airborne particulate matter outlined the quantification of the difference between proxy and personal measures of exposure as a key research priority for better understanding differences in observed health risk estimates across individuals and locations. since then, new approaches have been designed to measure an individual's inhalation exposure to particulate matter in air, using personal breathing zone samplers. for characterizing exposures to cookstove emissions, in particular, new devices, such as the exposure child monitor (ecm) and the ultrasonic personal air sampler (upas), provide more precise measurements for use in public health research applications. personal samplers for enteric exposure would face significant additional barriers given the diverse agents, the fate and transport of those agents in the environment, their infectious doses, and the myriad of exposure pathways. measuring long-term exposures to air pollution, a known driver for a range of chronic adverse health effects, necessitates alternative approaches for characterizing exposure, and often employ hybrid methods that combine both modeling and personal monitoring. a promising, and increasingly common approach for estimating spatiotemporally resolved long-term exposures to particulate matter and several gaseous pollutants comes from satellite remote sensing. these methods have the ability to use satellite optical instrumentation, calibrated with ground-level ambient monitoring data, to create long-term global exposure surfaces. some water quality parameters can be measured via remote sensing (e.g., chlorophyll-a), but these methods apply more to large water bodies than specific volumes of water consumed by humans. other methods integrate human activity patterns, questionnaires related to sources of exposure, and measurements conducted within defined settings (i.e., microenvironments) to predict individual level air pollution exposures over short-and long-term periods. the air pollution exposure model (apex) is an example of this class of air pollution exposure model, which was developed in response to prior limitations related to air pollution exposure and which may offer insights for novel, combined approaches for characterizing exposures to enteric pathogens as well. similarly, there are analogous hydrological water quality models that have been developed for estimating exposures to water pollution for surface water bodies. chemical toxicants. chemical toxicant exposure assessments, the measurement of a chemical, or its metabolite, degradate, reaction product or surrogate, include external and internal exposure assessments. external chemical exposure assessments can be pathway-specific (e.g., water, air) and route-specific (e.g., ingestion, inhalation) while internal assessments integrate all pathways and routes of exposure through which a chemical has entered the body. for example, assessment of dermal chemical exposures includes the use of hand wipes, patches, and body suits as dosimeters of exposure. the dosimeters are removed after exposure and chemical concentrations are measured in them, estimating dermal exposure. similarly, personal air space pumps or patches are used to estimate inhalational exposures and duplicate diet or water measurements may be used to estimate ingestion exposures. emerging techniques such as the use of silicon wristbands to absorb airborne contaminants have also been used as efficient means to capture exposure to up to air contaminants including polychlorinated biphenyls, pesticides, flame retardants, polycyclic aromatic hydrocarbons and volatile organic chemicals. internal chemical exposure assessments (i.e., biomonitoring) have burgeoned over the last two decades and are often the "gold standard" methods for assessing exposures in relation to adverse health outcomes. biomonitoring of chemical contaminants in urine and serum with mass spectrometry-based methods have resulted in many population-based data reports on human internal exposure in the united states, canada, and korea. − advances in exposomics using high-resolution metabolomics allow for the detection of numerous endogenous and exogenous chemical metabolites simultaneously. biomonitoring is also seeing application in air pollution exposure assessments in low-income settings, for example with the detection of polycyclic aromatic hydrocarbons in urine to quantify household air pollution exposure. in comparison, internal enteric pathogen exposure assessments face the difficulties of pathogens as dynamic biological organisms that can amplify and die-off inside the host as well as acquired immunity and other modifiers of dose−response relationships. measurement error. analytical frameworks have been introduced to assess the impact of exposure measurement error, i.e., the difference between the measured and the true exposure, in health effects models. they have shown that some forms of error may lead to biases and greater uncertainties estimating exposure-outcome relationships. although the nature of measurement error in enteric pathogen exposure assessments may be complex and multifactorial, quantifying sources of error can enable prioritization of how to eliminate the greatest sources of error and better capture biologically relevant exposures. prior research on the effects of measurement error on waterborne disease epidemiology has shown that enumeration methods for indicators of fecal contamination can attenuate the relationship between indicator levels and diarrhea by up to %. another source of error occurs when true exposure variability is not sufficiently reflected when using proxy indicators. for example, spatiotemporal rainfall variability can attenuate associations between heavy rainfall and diarrhea by single measures of water quality can attenuate the relationship between fecal indicator concentrations in water and child linear growth by up to % compared to repeated measures that account for temporal variability in water quality. the same study also reported that neglecting exposures to water sources outside of the household can attenuate the water quality-diarrhea associations by up to %. other examples of sources of measurement error include sampling protocol design introducing data collector error (i.e., through nonspecific sampling instructions), assigning household-or community-level exposure measures to individuals, and environmental sample processing errors (i.e., during sample collection, transport, or analyses) that can lead to under-or overestimates of concentrations of enteric pathogens or fecal indicator organisms in environmental media or to false negative or false positive results. as part of this workshop, participants collectively identified a set of current and emerging methods, some of which have been widely used in other environmental health sectors, that may contribute to improved measures of enteric pathogen exposure. we describe those methods here and summarize them against the criteria listed above in supporting information tables s and s . measuring enteric pathogens in the environment. studies investigating the association between enteric pathogen exposure and disease, or evaluations assessing the effectiveness of wash interventions that include an exposure assessment, have mainly attempted to characterize fecal contamination in different environmental media as a proxy for enteric pathogen exposure, neglecting to characterize human interaction with those media. a systematic review of the effects of sanitation interventions on fecal−oral transmission pathways identified the following approaches used: enteric pathogens or indicator bacteria in environmental samples (drinking water, hands, sentinel toys, food, household and latrine surfaces, and soil); the presence or abundance of flies; and observations of human and animal feces. there are a number of factors to consider when measuring enteric pathogen prevalence in the environment, including environmental sampling strategies, the use of indicators as proxies for enteric pathogens, differentiating between human and animal sources of contamination, detection limits, and selecting which specific pathogens to target. sampling strategies. the vast majority of studies that have measured environmental fecal contamination coupled with child health outcome data have focused on drinking water and, to a smaller extent, fly density, hands, and fomites, with limited data on food and soil contamination. drinking water samples can be collected directly from the source, from the household storage container or from the utensil used to drink water, each progressively capturing additional pathways of contamination between the source and the point of consumption. often a choice has to be made between multiple sources of water used by the household, only some of which are dedicated for drinking. hand contamination has been assessed through rinsing hands in sterile water and analyzing the rinsewater. food samples can include items prepared and stored at home or bought outside the home (e.g., produce typically eaten raw or streetfood). , surfaces can be sampled by swabbing, and soil by scraping topsoil from a designated area. the site of collection of surface swabs or soil is a key decision in sampling protocols. a tool for capturing overall contamination in the domestic environment is sentinel toys, a nonporous object (e.g., plastic ball) that is left in the household for a prespecified amount of time for household members to interact with and then rinsed. fecal contamination in all of these domains is highly variable temporally, − seasonally, , and spatially, , and additional variability can be introduced by the methods used to analyze samples. some strategies to address this variability include selecting sampling locations relevant for exposure for the target population (e.g., soil in areas where children spend time), focusing on key times of exposure (e.g., measuring hand cleanliness before eating) and collecting longitudinal samples. additionally, when the goal of sampling is to assess the relationship between pathogen exposure and health outcomes, environmental samples can be collected prior to ascertaining health end points, allowing an appropriate incubation period before onset of health outcomes. − the common practice of collecting samples and health data during the same household visit, typically for logistical convenience, risks capturing contamination levels at a time that is not relevant for disease transmission. as a result, this practice is vulnerable to introducing reverse causation (e.g., water boiled in response to illness, fecal contamination in the environment due to diarrhea), obscuring the true relationship between exposure and disease. indicators of fecal contamination. fecal contamination in the environment is commonly estimated by using indicators of fecal contamination. these indicators have the advantage that they are easier and less expensive to measure compared to multiple specific pathogens and they can be indicative of a range of enteric pathogens. they provide an indication of the presence of fecal matter in a sample, but do not confirm the presence or absence of pathogens, nor do they provide any indication on the infectivity or diversity of enteric pathogens in a sample. there are a number of indicators of fecal contamination, including chemical indicators (e.g., fecal sterols, caffeine, estrogen hormones) , as well as microbial indicators. fecal indicator bacteria are often grouped into the coliform and streptococcal bacterial groups. total coliforms include a broad spectrum of bacteria occurring in feces, but can also be found in nonfecal matter. fecal coliforms or thermotolerant coliforms are a group of bacteria that are more specific to fecal contamination, with the exception of klebsiella. escherichia coli (e. coli) is the most commonly found fecal coliform bacteria and is more specific to human and animal fecal matter, though genome comparisons suggest that some clades of e. coli primarily originate from nonhost natural environments, and there are reports of e. coli detection in pristine areas of tropical and even temperate environments. − in recent years, several laboratory-grade products have been validated in the field as predictive of e. coli. , however, these instruments require frequent cleaning, are not intended for long-term autonomous operation, are subject to the drawbacks of methods measuring indicators of fecal contamination and are expensive. recent efforts are focused on reducing these product costs and enabling continuous in situ water quality monitoring. fecal streptococci were identified as an alternative to total coliform in the s when it became clear that total coliform was a nonspecific indicator for fecal contamination, but the use of this indicator diminished once more specific methods for e. coli culturing were established. enterococci are a subset of environmental science & technology pubs.acs.org/est critical review species of the fecal streptococci group, more specific to fecal contamination than fecal streptococci and more persistent in the environment. coliphages and crassphages, types of bacteriophages (viruses that infect bacteria), are also used as indicators of fecal contamination because of their similar morphological characteristics and ability to mimic the persistence of viral pathogens in the environment. , the use of fecal indicators was historically established to measure fecal contamination in drinking water and recreational waters and to monitor the performance of water treatment processes. in the united states, total coliform is still used to monitor treatment efficacy and post-treatment contamination of drinking water supplies in accordance with the total coliform rule, which requires the monitoring for the presence of total coliform in public water systems at a frequency proportional to the number of people those systems serve. e. coli and fecal coliform are presently the most commonly used fecal indicator bacteria for monitoring fecal contamination in water. the world health organization (who) uses levels of e. coli and fecal coliform to define microbial quality of drinking water. meta-analyses of the association of drinking water quality measured using fecal indicator bacteria and diarrheal disease in low-income settings have presented mixed results. gundry et al. found no significant association between e. coli or fecal coliform in household water and diarrhea, gruber et al. found a significant association between levels of e. coli in water and diarrhea but not for fecal coliform, whereas hodge et al., using an individual participant data (ipd) meta-analysis, showed a higher risk of diarrhea with increasing levels of fecal coliform in water. a recent ipd meta-analysis found evidence of association between both e. coli and fecal coliform levels in household drinking water and diarrhea and impaired linear growth. fecal indicator bacteria are increasingly used to characterize fecal contamination along other fecal−oral transmission pathways. the shortcomings related to indicators of fecal contamination, when used for purposes other than their original intended use of monitoring water supply systems, have been well documented, including that e. coli has been found in environments where fecal contamination is improbable. these shortcomings are amplified by the limited association between indicators and the presence of enteric pathogens. , however, measurement of fecal indicator bacteria may be valuable as an indication of "total fecal load" in environmental samples and to provide a common metric for comparison with previous studies. source tracking. fib used in solitude and some other indicators of fecal contamination do not distinguish between different sources of contamination. fecal source tracking aims to provide data on the source of fecal contamination by detecting signatures of specific sources (e.g., particular animals or particular geographies). in addition to humans as a source of fecal loading in the environment, animal fecal contamination is of particular interest in low-income settings where households often cohabitate with animals in confined spaces. a recent systematic review outlined the importance of animals as a source of fecal contamination to human health, suggesting an association between animal feces exposure and diarrhea, soil-transmitted helminth infection, environmental enteric dysfunction, growth faltering, and trachoma. a variety of source tracking methods have been described in the literature, ranging from fecal coliform/fecal streptococcus ratios to host-specific molecular markers. host-specific bacteroidales are commonly used in high-income countries to complement the use of fecal indicators, and are seeing increased use in low-income settings to distinguish between human and animal sources of fecal contamination. − bacteroidales can distinguish between sources of fecal contamination, because they adapt to their host differentially, allowing for the identification of host-specific fecal contamination. however, fecal source tracking assays developed in one geographic location must be validated to be used in additional locations, due to geographic variation in human and animal fecal microbiomes, and the molecular methods used to detect bacteroidales provide no viability or infectivity data. host-specific bacteroidales have been measured in tanzania to characterize human-specific fecal contamination on hands and in drinking water, and test the relationship between humanspecific fecal contamination and diarrhea. a nested study within a cluster-randomized controlled sanitation trial in odisha, india utilized fecal source tracking to discern the effectiveness of a sanitation intervention on the reduction of human-specific bacteroidales compared to animal-specific bacteroidales. phage-based methods are a rapid and low-cost approach for distinguishing between human and animal feces. gb- phages that infect human bacteroides fragilis have been used in a number of countries to identify human fecal contamination. − like the molecular bacteroidales method, these phage-based methods also need to be validated in each geographic location. other methods use next generation sequencing data to distinguish between different host microbial communities within a given household or community, , and use that information to attribute sources of contamination. , however, these library-dependent methods are still under development, can be expensive and difficult to standardize, and require shipping samples to sequencing facilities. currently, reference databases used to ascribe taxonomy in these studies are biased toward bacterial communities associated with persons living in high-income communities, but there are continuing efforts to expand these reference databases. enteric pathogen detection with traditional methods. specific pathogen occurrence or concentration, instead of the use of indicators as proxies, are less commonly measured in the environment although they may be more representative of the actual health risk associated with enteric pathogen exposure. exposure assessments that measure specific pathogens need to consider not only the diversity of potential pathogens occurring in the environment but relevance of each included pathogen for health outcomes of interest, which is highly context specific. the possibility for improved specificity from measuring specific pathogens instead of indicators of fecal contamination may come at a loss of sensitivity, since selected pathogens may not be representative of all possible pathogens in the environment and are typically present in lower concentrations in the environment. monitoring water supplies for pathogens can be prohibitively resource intensive due to the array of pathogens and the low concentrations of specific pathogens in water, requiring sensitive assays. the who estimates that concentrations of pathogens in water corresponding to − disability-adjusted life years (dalys) per person per year are typically less than organism per − liters. testing for pathogens in water is achieved by concentrating the water sample, such as filtering large quantities of water, on the order of − l, environmental science & technology pubs.acs.org/est critical review followed by using culture-dependent or culture-independent methods to enumerate the density of pathogens. however, ultrafiltration using kidney dialysis filters is a low-cost method to efficiently and simultaneously concentrate viruses, bacteria, and protozoa from large volumes of water and wastewater. − culture-dependent methods are also limited by their low sensitivity and their resource intensity. furthermore, some enteric pathogens (salmonella typhi, vibrio cholerae, campylobacter spp., and others) can enter a viable but nonculturable state in the environment that may require special resuscitation steps or molecular methods to detect. while resuscitation or enrichment steps increase the probability of pathogen detection from an environmental sample, they only confirm the presence of the pathogen and do not provide quantitative data. enteric pathogen detection with molecular methods. culture-independent molecular methods have been developed for many enteric pathogens, but these have traditionally required extensive laboratory equipment and highly skilled technical staff. as molecular methods, such as as polymerase chain reaction (pcr) based assays have become lower-cost, easier to multiplex, and more robust to inhibitors in environmental samples, a number of studies have successfully detected bacterial, viral, helminth, and protozoan pathogens in drinking water, on hands, and in soil in low-resource settings. , , − liu et al. developed the customizable taqman array card (tac), an emerging method for quantitative detection of multiple enteric pathogens encompassing viral, bacterial, protozoal, and helminth targets for enteric infections. tac is a taqman probe based real-time pcr assay which can test up to eight samples with individual singleplex (could be expanded to duplex) reaction wells per sample in a -well format. these tac assays were validated and field tested in many low-and middle-income country settings. examples of recent environmental applications of the tac method include the simultaneous detection of a number of enteric pathogens in surface water, soil, and infant weaning food in kenya. , pholwat et al. developed a custom environmental surveillance tac to identify antimicrobial resistance genes, poliovirus, and enteric pathogens in environmental samples such as fecal sludge, food, sewage, soil, and water in low resource settings. advantages of tac are the ability to test + targets/pathogens simultaneously, to obtain quantitative results relatively quickly, and to utilize the same method for both environmental and human samples. an important step in detecting a broad spectrum of targets is to extract high quality total nucleic acid (both dna and rna) from the sample. unlike culture-based methods where pathogen growth is dependent on many factors such as antibiotic history, culture media, and temperature, high throughput molecular diagnostics such as tac can detect many pathogens quantitatively and be used across wideranging sample types. however, molecular methods cannot be used to establish viability of the organism and can be prohibitively costly. resource-intensive field collection and lab processes mean these methods are challenging to deploy in low-income settings where resources are limited. environmental metagenomics. metagenomics, the sequencing and analysis of all dna in environmental samples, circumvents the problem that many enteric pathogens cannot be easily cultured and metagenomic data can provide information on the abundance and diversity of microorganisms in environmental samples. unlike many molecular methods, it does not require prespecification of targets, allowing the user to probe for all potential enteric pathogens present in a sample. while it has been historically difficult to identify specific enteric pathogens with amplicon sequencing or low coverage metagenomic approaches (i.e., capturing s rrna genes), shotgun metagenomic approaches (i.e., capturing all metagenomic dna) can be used to distinguish between specific enteric pathogen strains. reduced sequencing costs over time as well as recent advances in sequencing technologies and bioinformatics pipelines will continue to open up opportunities for enteric pathogen detection. long read sequencing technologies, such as oxford nanopore technologies (ont), enable pathogen identification through assembly or direct alignment of long dna reads to published pathogen genomes. metagenomics can be used to characterize both pathogens in environmental samples and in human stool samples. environmental metagenomics has recently been used to profile viral pathogen diversity in environmental waters, examine antibiotic resistance diversity in wastewater, and to demonstrate exchange of antibiotic resistance genes between soil bacteria and clinical pathogens. limitations of metagenomics include poor sensitivity if enteric pathogens are at low prevalence in the microbial community or when sequencing depth is low, it does not confirm viability or infectivity of the pathogens, high cost, required bioinformatics expertise, and the need for improved analysis pipelines for identifying pathogens. wastewater surveillance. wastewater monitoring is an emerging approach to surveillance and has the potential to deepen our understanding of community health. wastewater has been shown to provide useful community-level information regarding illicit drug use, antimicrobial resistance, , signals of chronic disease, and advanced warning of viral outbreaks, , including recent efforts to detect sars-cov- in wastewater. most prominently, wastewater is used for poliovirus detection in global eradication efforts. identification of enteric pathogens in wastewater can indicate downstream exposure potential, as well as suggest past exposure to that pathogen among those contributing waste. however, there are challenges with wastewater surveillance: ( ) large dilution from other households in the catchment area of pathogens shed by a small number of infected people; ( ) need for concentration methods and sensitive detection methods compared to stool samples; ( ) need to understand the wastewater collection network, flow, and catchment area in order to identify strategic sample collection points, and ( ) differential pathogen transport, die-off or regrowth by wastewater network need to be considered. dynamic models can be valuable for optimizing the development of a sensitive environmental surveillance system. , wastewater surveillance may not capture feces from the poorest populations who are most likely to have the highest burden of infection and exposure because these populations may practice open defecation or use on-site sanitation, and as a population-level approach limits individual-level scientific inference. biosensors. instead of using laboratory intensive approaches to estimating pathogen prevalence in the environment, there is room for innovation to detect enteric pathogens on-site in environmental media by using biosensing technologies. biosensors isolate dna encoded biological responses to an electrical signal that can be logged digitally. such methods have the advantage that they have the potential to be deployed in resource-constrained and remote settings, because they environmental science & technology pubs.acs.org/est critical review remove the need for sample collection, transport, and intensive laboratory processing. bioreceptors can include tissues, microorganisms, enzymes, antibodies, and other biologically derived elements, although most current applications are limited to indicators of fecal contamination rather than specific pathogens. a recent application of biosensors to detect fecal contamination in drinking water used an odorant-binding protein derived from mosquitos to test for the presence of coliform bacteria. bacteriophage with a broad host range specificity for e. coli have been combined with magnetic beads to capture and then separate e. coli in drinking water. measuring enteric pathogens in humans. as a proxy measure of internal exposure, infection with enteric pathogens can provide confirmation of actual exposure following ingestion, thus potentially providing important data on past exposure for the evaluation of interventions and for surveillance. findings from these measures are complicated by host immunity, and absence of infection or serological indication of prior exposure cannot confirm that exposure did not occur. more specifically, these data can be employed to evaluate how well interventions reduce exposure to specific pathogens and which pathogens humans are exposed to in their community. data generated from surveillance and epidemiological studies has benefited from advances in surveillance capacity and detection methodologies for estimating the burden of disease, disease severity, attributing health outcomes to pathogens, and pathogens to exposure pathways. these data facilitate hazard characterization, the first step of risk assessment. dose response data are highly variable by pathogen and are limited because human challenge studies are difficult, expensive, usually single-pathogen focused, primarily performed among adults in high-income countries, cannot control for previous exposures and differences in immune responses to infection, and may be at odds with acceptable ethical standards. furthermore, little data is available on how much of an ingested dose passes through specific and nonspecific host defense barriers and reaches the target cells and is capable of inducing infection. animal models often do not exist for enteric pathogens or do not cause the same health outcomes as in humans. exposure assessments can take into account the hazard characterization information derived from epidemiologic and dose−response studies to focus on pathogens that cause the greatest disease burden in the region of interest, taking a more narrowed approach to exposure assessments by focusing on specific pathogen-source pairs. pathogen shedding in stool. methods to detect enteric pathogens in stool samples range from using microscopy or enzyme-linked immunosorbent assays to detect single pathogens, to using molecular or metagenomics methods to characterize multiple pathogens in a sample. multiplex pcr is a technique that has been widely employed in enteric disease surveillance (sporadic and outbreak) and epidemiological studies such as in multicountry case-control and longitudinal birth cohort studies, as well as in recent studies measuring health impacts of wash interventions. , shotgun metagenomic approaches have also been employed, for example to distinguish between foodborne disease outbreak strains of salmonella, and to identify the likely causes of diarrheagenic e. coli in ecuador. the limitation of these techniques is not only the intensive resources they require, but it can also be difficult to attribute a specific pathogen to a disease outcome when multiple enteric pathogens are detected in stool simultaneously or when asymptomatic infections are common. additionally, the sensitivity of these methods can vary. these methods identify infections by detecting pathogens in human stool samples, and pathogens may only be shed by an infected person for a short period of time (days, weeks) or shed intermittently, so infections between sampling events may be missed. furthermore, the duration of shedding after infection is highly pathogen-specific, so these methods can be biased toward persistent pathogens that are shed for a longer period of time compared to more transient pathogens. pathogen-specific immunoassays. another way of estimating past exposure to enteric pathogens is through immunological assays detecting pathogen-specific antibodies in serum or saliva, which can be multiplexed to detect exposure to multiple enteric pathogens. for these immunological methods, the timeline of exposure can be difficult to ascribe as low levels of pathogen-specific antibodies (immunoglobulin (ig)a, and particularly igg) can be present in saliva and serum for weeks to years after infection. this can also be an advantage, as the methods can be used to integrate prior exposure over longer periods of time, rather than relying on pathogen shedding in stool. exposure data without regard to history of infection can be useful for some applications, such as to determine if a population has been exposed to a rare or emerging pathogen or particular microbial strain, which could be important for focusing exposure assessment approaches. sero-epidemiology is a promising approach to measurement of force of infection of enteropathogens across entire populations. − however, one's immune response depends on a number of host-specific factors including history of previous exposure (acquired immunity ), nutritional status, genetics, composition of the gut microbiome, underlying disease such as hiv infection, and age (antibodies can appear in low concentrations in young children, particularly in saliva). measuring interaction with the environment. exposure to enteric pathogens is not only conditional on pathogen presence in the environment, but also on host interaction with that environment. collecting these data can complement exposure assessments and the analysis of exposure data, as has been done in estimating human exposure to other environmental pollutants. survey data on self-reported behaviors or observational data on practices can enable the targeting of environmental media and locations where the study population is predominantly exposed. quantitative observational data can be combined with environmental measurements of enteric pathogens to estimate pathogen ingestion rates. the sanipath tool, an example of such an approach implemented in nine countries to date, combines environmental sample collection and analyses for e. coli with surveys of behavior to estimate exposure. , here we describe current methods that have been used to characterize host interaction with the environment and summarize those methods in supporting information table s . when using these methods, it is important to consider the collection of behavior data disaggregated by gender and age group because of the differences in behavior between men and women, boys and girls, and adults and children. surveys and self-reports. surveys have been used as a rapid and cost-effective tool to collect information on a range of self-reported behaviors that serve as proxies for exposure patterns. while surveys carry the risk of various types of bias, such as recall bias, courtesy bias, and reporting bias associated with self-report of socially desirable behaviors, , surveys are nonetheless a useful tool to obtain information on neutral behaviors that do not trigger these biases. there are multiple approaches for collecting self-reported data. many studies have used household surveys with the head of household or primary caregiver for young children. one major limitation of this approach is that the reported behavior for the respondent is often incorrectly seen as a proxy for behaviors for the entire household. community participatory surveys and surveys in school classrooms, that combine some discussion of the behavior with a method for the participants in the group to confidentially report their own behavior, such as pocket voting, have also been used to identify high-risk behaviors. surveys and self-reports can also be used to inform sampling locations (e.g., where a household obtains their drinking water, prepare their food etc.). observations. spot-check observations can capture wash infrastructure and behaviors that result in high risk of exposure (e.g., latrine cleanliness, presence of a handwashing station, handwashing at key moments, washing raw produce before consumption) that can be difficult to elicit by self-report due to biased reporting. structured observations, , including the use of videography, , offer an opportunity to gather information on complex behaviors, including recording the frequency, duration, and type of interaction with the environment, which could subsequently be used to estimate ingestion rates. these tools are resource intensive at scale due to high heterogeneity of behavior within-host, i.e., observations of individuals, and between-hosts, i.e., observations of public domains. observations can also cause reactivity in participants where the presence of an outside observer leads individuals to alter their behaviors while observed. sensors have been used to compare observed behaviors to reported behaviors with some studies indicating that reported behaviors are inconsistent with sensor measured use. − sanitary surveyssurvey-based inspections of water systems or sanitation facilitieshave been designed and promoted, often in combination with periodic water quality testing, as screening and risk assessment tools for fecal contamination exposure. however, recent studies have shown a poor correlation between sanitary inspection scores and fecal contamination in drinking water supplies. − location tracking. location tracking, for example using personal global positioning system (gps) tracking, can inform where a host is spending time and can thus provide data on where to collect environmental samples, for example by identifying potential hot spots in communities where community members may be experiencing frequent exposures. the use of gps devices has broadly been validated for exposure assessments, and has been used to inform air pollution and chemical exposure assessments. a study in brazil found that gps tracking was an effective tool to quantify personal movements of urban slum residents and evaluate exposure sources of environmental leptospirosis transmission. tracers. tracers, substances introduced into the environment so that their distribution can be detected from their distinctive properties, can provide data on where and how hosts are interacting with their environment. they have been used in air pollution epidemiology to differentiate between indoor and outdoor exposures and a study in china estimated child soil ingestion by measuring concentrations of tracer elements in soil. challenges with using tracers include that seeding several common fecal−oral transmission pathways simultaneously to quantify relative exposure contributions from different pathways may be impractical beyond certain microenvironments, and tracers are needed that do not degrade in the environment and pose no risk to human or environmental health. modeling tools can complement emerging pathogen diagnostics to provide a more nuanced understanding of both pathogen dynamics in the environment as well as infection transmission dynamics. these tools are used to interpret data, are an inherent part of scientific inquiry and can take on many forms. statistical models describe the data in the context of sampling variation and to examine associations that inform environmental determinants of disease risk. for example, they are used to estimate pathogen prevalence, persistence and how that may vary in time and space. conceptual models, sometimes implicit and sometimes explicit, are distinct from statistical models because they represent a hypothesis of the causal relationship between exposure and disease outcome, and are an important driver of how we conduct studies and interpret data. these conceptual models can be codified using mathematics, allowing simulation of different intervention scenarios to conduct thought experiments. such models are complementary to epidemiological analysis. their benefits include lower cost than conducting epidemiology studies that involve recruitment of human subjects. they are also mechanistic, reflecting hypothesized or measured quantitative relationships between key variables that describe complex transport (in the environment) and transmission dynamics (from and between individuals, including via animals and the environment), and have clearly articulated assumptions about these relationships that can be evaluated with real-world data. to study enteric pathogen exposures and risk, there are various types of mathematical models described in the literature. these models allow for in silico experiments and we mention two here. first, quantitative microbial risk assessment (qmra) models estimate the infection or disease risk from a single pathway as a function of pathogen exposure measurements in the environment. for example, by measuring the contamination in surface water, qmra can be used to estimate the risk associated with the drinking water pathway and examine the potential for disease reduction given different mediation efforts. − importantly, the uncertainty in the exposure measurements, or any other data measurement used in the model, can be propagated and reflected in the risk estimate. whether the variance of the risk estimate is too high depends on how this estimate is used; in any case, the variance can be lowered through collection of more precise data. second, infection transmission models dynamically estimate the infection risk associated with multiple pathways of transmission. these models can test scenarios, for example, to understand causes of disease outbreaks from contaminated drinking water, or to explain the interdependency of water, sanitation, and hygiene pathways. , in general, these pathways are often interdependent; i.e., an increase in soil contamination due to poor sanitation can result in increases in contamination of drinking water sources due to runoff events, or poor hygiene can increase the person-to-person transmission rates that result in more people contaminating water sources used for bathing and washing clothes. dynamic infection transmission models can explicitly account for these environmental science & technology pubs.acs.org/est critical review interdependencies to gain an understanding of how mediation in one or more pathways may affect overall infection rates. the power of these tools is that they can generate hypotheses without the need for collection of empirical data, and can then be tested through empirical studies. thus, empirical study design can be driven by model simulation results, and models can be further refined by better parametrization with empirical data in an iterative process. a number of challenges remain in effectively applying these modeling approaches to the study of enteric pathogen exposures. first, modeling results are only as good as the data used to inform parameter estimates. for example, enteric pathogen transmission dynamics are complex and influenced by a range of variables not always measured in field studies. second, modeling results assume that the model structure is correct. for example, dose−response models are highly uncertain and variable, given the limited data available for deriving these relationships, the artificial setting in which dosing trials are conducted and the many variables that may influence the dose−response relationship. more broadly, a transmission system is complex and assumptions are implicit. for example, our models often assume that humans are the most important source of enteric pathogens, that we have accurately identified all relevant pathways of potential exposure, or that the data we have available for use are representative over space and time. many of these limitations apply to science in general and reflect the limitations of the scientific process. modeling approaches bring a number of advantages to understanding enteric exposures. models yield data that may be compared with empirical observationincluding epidemiological data and can therefore help elucidate important mechanistic processes at play, potentially explaining the "why" questions that contextualize epidemiological findings. modeling approaches allow us to interrogate theories of change and identify which interventions have the potential to reduce risks of exposure, and under what conditions. such methods may allow for more rapid development of technologies and approaches that have the greatest potential for improving public health. we have presented the need for new approaches to improve measures of enteric pathogen exposure, identified principal applications that would benefit from better measures, and have considered the merit of current and emerging methods to be employed for these applications as well as lessons learned from exposure assessments for other environmental contaminants. based on this review, we have identified a suite of recommendations that we believe are critical to moving enteric pathogen exposure method development forward: . outline the benefits and potential health impacts of improved enteric pathogen exposure assessments. identify the entities that may harness improved exposure assessments to positively impact health (e.g., governments, international organizations, donors, and funders). emphasize the role of exposure monitoring with respect to other threats to public health, including climate change and antimicrobial resistance. catalog opportunities to link improved exposure assessments with other strategies and campaigns, including sustainable develop-ment goal (sdg) six targets on safely managed drinking water and sanitation services. . create a path forward for exposure assessment methods across the principal applications. develop a vision and the innovations that meet a broad range of applications and criteria necessary to radically improve measures of enteric exposure. consider novel technologies for use in the field and laboratory. collaborate across sectors to comprehensively measure exposure by combining modeling, observational, microbiological, epidemiological, and statistical tools. explore opportunities for integration with one health, planetary health, climate action, nutrition programs, and other initiatives. . dedicate research to defining biologically relevant exposure assessments. consider biological relevance in exposure assessments as it pertains to health outcomes. use archived specimens from previous studies to test new methods, where possible, and implement longitudinal studies to provide rigorous evidence on relative contributions from specific transmission pathways and pathogens to adverse health outcomes. leverage external and internal measures of exposure as complementary approaches for more holistic assessments of exposure. consider how varying approaches reduce differences between measured and true exposure and their subsequent cost-effectiveness in reducing exposure measurement error to design more biologically relevant exposure assessments. . compile exposure data and fill evidence gaps. define and standardize best practices for microbiological and observational methods used for exposure assessments and develop a database framework with uniform data reporting standards to enable better comparisons across different studies and settings. promote the archiving of data and banking of samples for further analysis. identify and fill evidence gaps, including generating data on the temporal and spatial variability of enteric pathogens in environmental matrices, and neglected fecal−oral transmission pathways such as food and soil, to provide a more complete picture of exposure. . demonstrate the potential market and economic drivers of improved exposure methods. model the size of the market across various sectors, and describe how exposure monitoring products could be commercialized. describe the value of these improved methods in higher income settings where there is greater potential to attract investment into research and development while delineating the benefits for improving health in resource-challenged environments. explore the potential use of exposure data in results-based financing, i.e., payfor-performance by defining performance as reducing exposure. in this review, we outlined a number of approaches used to characterize exposure to enteric pathogens. exposure to enteric pathogens is highly context-specific, the density and diversity of pathogens in the environment as well as human interaction with that environment is highly variable over space and time. we presented emerging methods and through lessons learnt from other areas of environmental health distinguished between external and internal measures of exposure. we provided a set of recommendations to narrow the gap between environmental science & technology pubs.acs.org/est critical review the measured and the true exposure, including more holistic exposure assessments that consider approaches across sectors to provide more complete measures of exposure. ■ associated content the supporting information is available free of charge at https://pubs.acs.org/doi/ . /acs.est. c . summary of methods to measure enteric pathogens and indicators of fecal contamination (table s ); summary of methods to characterize interaction with the environment (table s ) (pdf) the global burden of diarrhoeal disease, as estimated from studies published between global burden of disease collaborative network. global burden of disease study exposure science: a view of the past and milestones for the future a retrospective assessment of mortality from 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