key: cord- -s zk l a authors: mihaylova-garnizova, raynichka; garnizov, vasil title: refugee crisis as a potential threat to public health date: - - journal: defence against bioterrorism doi: . / - - - - _ sha: doc_id: cord_uid: s zk l a the refugee crisis in europe continues to persist despite recent data, showing a drop in the number of refugees seeking asylum. the eu has called this as “an unprecedented displacement crisis” and has aimed at devising a comprehensive approach to tackle it, which has been widely criticized. concerns about public healthcare aspects of the crisis have permanently entered the media and policy discourse even though no systematic association between migration and the importation of infectious diseases has been recorded. in this context, the literature has not filled the existing gap between discourse and evidence, and almost no publications with reliable empirical data exist, both thematic (epidemiology) and geographical (eastern europe and bulgaria). among the existing publications, the focus has been on tb and hiv (odone et al., euro j public health ( ): – , ). in light of this, the aim of this research is to contribute to the debate by providing an overview of the refugee situation in bulgaria, as a primary entry-point for refugees entering the eu. in order to achieve this, the article analyses the case of the refugee camp in city of harmanly, close to the bulgarian-turkish border, and assesses the public health risks related to this specific situation. based on a study of patients with different symptoms we aim to draw wider implications about the linkages between public health and migration. the in-depth review of this specific case shows that both the probability and impact of migration on public health increases when the hosting country is relatively poor, the domestic public healthcare system is not efficient, and there is lack of trust in the government and public services. the study contributes to understanding better these risks in order to identify potential mitigation strategies in the region and the eu as a whole. the european migrant/refugee crisis in - deeply challenged the political, economic and healthcare systems in the european union (eu). looking at these developments, we are faced with the key problem of lack of data on the health profiles of migrants, in particular -of refugees. immediately after the crisis began there were many publications that insist, without extensive evidence, that migrants do not pose a health threat to the host population. almost no publications could be found with reliable empirical data, especially epidemiological, including for bulgaria. the absence of reliable and robust data is even more problematic at times of crisis since public health systems cannot prepare adequately. the lack of objective information also opens up opportunities for the emergence of public myths and psychoses, including disinformation that can lead to political destabilization, which could in turn affect national security. the purpose of our article is to collect, summarize and present epidemiological data related to migrants in bulgaria and, on the basis of this information, to analyze the potential risks to public health (including risks to migrants) and to assess the capacity of bulgaria's healthcare system to cope with the refugee crisis. migration is a phenomenon known since antiquity. thanks to it, new countries have emerged and new communities have formed. but what is happening today is extraordinary because millions of people constantly change their place of residence for various economic and non-economic reasons, going beyond the scope of our research. the bottom line is that at the end of , almost million individuals were forcibly displaced worldwide. twenty million of them are refugees. for the first time, turkey has become the country with the largest number of refugees ( fig. . ) . as far as europe is concerned, with the exception of certain high-income member states, most countries did not have serious difficulties with migrants. this was fundamentally altered after the series of events in north africa and the middle east, known as the arab spring, as well as the civil war in libya, and especially since the syrian conflict began in . based on regular easo annual reports, since one can see the changing structure of migration trends altogether. until then migration was mainly from lower income countries to countries with a higher income and the greatest percentage of migrants represented the share of those arriving from western balkan countries. after , migration started affecting not only highincome countries but also all european countries with the highest percentage of asylum seekers being refugees from syria ( fig. . ) . as a result, europe has faced a unique influx of refugees, asylum seekers and other migrants since the establishment of the eu: . million people arrived in the eu only in . since the beginning of , , , applications have been recorded in the eu+ (eu plus norway and switzerland). even though this is % less than in the same period of , when , , applications were lodged, this highlights the extent of the challenge [ ] . this is a not an easy situation to be rationally understood since it is sensitive to strong public opinion and intense debate. even more, from the point of view of public health 'this refugee situation is unparalleled since the end of ww ' [ ] . in this context, often the public healthcare aspects of migrant crises enter the broader public discourse, regardless of expert opinion. the diverse discourses have been addressed by multiple international organisations. in dr. zsuzsanna jakab, who regional director for europe, clearly persons applied for international protection in the eu highlighted that there is no systematic association between migration and infectious diseases stating that: 'communicable diseases are primarily associated with poverty. refugees and migrants are exposed mainly to the infectious diseases that are common in europe, independently of migration. the risk that exotic infectious agents, such as ebola virus or middle east respiratory coronavirus (mers-cov), will be imported into europe is extremely low. experience has shown that, when it occurs, it affects regular travellers, tourists or health care workers rather than refugees or migrants' [ ] . in the european commission also addressed the healthcare aspects of migrant crises, declaring that refugees are actually the ones at risk, rather than a burden on health systems. dg sante made it explicit that measures to protect refugees' health are being taken 'not out of unfounded fears that they might spread infectious diseases' or 'place a burden on health systems. their [refugees'] health is at risk, not the health of eu citizens' ([ ] , emphasis added). who and ec's stance on public health challenges of large-scale migration and preparedness of countries in the european region could be seen as too optimistic. who insisted that 'the health systems in the countries receiving migrants are well equipped and experienced to diagnose and treat common infectious diseases; they should also be prepared to provide such health care to refugees and migrants. should a rare exotic infectious agent be imported, europe is well prepared to respond, as shown over the past years in responses to imported cases of lassa fever, ebola virus disease, marburg virus disease and mers' [ ] . at the same time, events in germany on the weekend of - september demonstrated the opposite: large-scale migration provoked serious malfunctioning of both political and administrative systems including police, social services, housing, public healthcare [ ] . after these events, reported in depth by robin alexander, the responses across europe quickly escalated. once the eu-turkey deal was agreed, balkan border closures organised by austria followed by hungary, serbia and macedonia left some - , migrants blocked in the informal camp in idomeni on the greek-macedonian border [ ] . from the beginning of march to the end of may asylum seekers there survived in poor sanitary conditions with very limited running water, no lavatories, and insufficient food, despite efforts by ngos such as 'hot food idomeni' and medical care from 'médecins sans frontière'. the unhealthy and overcrowded conditions at the camp have given rise to infections and in march some cases of hepatitis a have been reported [ ] . posters with the message 'greece will offer you accommodation, food and healthcare' (written in arabic, farsi and pashtun) were posted in march but the final decision was taken only on th of may. in reality, initial reactions and follow up to the idomeni situation were limited since there was no confirmed information on the epidemiological situation in idomeni, no risk assessment and no further public health measures were taken. in contrast to who and ec statements, bulgarian authorities never declared their readiness to manage a large-scale migrant influx. until bulgaria had not been a country of interest to migrants and, as a result, there was scarce experience of responding to a migrant crisis. rather, the country was a source of migrants towards western europe. bulgarian membership of the eu on january , led to an increase in the flow of migrants to bulgaria and a slow but sustainable decrease of bulgarian emigrants. this situation has changed radically since the start of the civil war in syria in . the geographical position of the country, which shares a km border with turkey, makes it a natural primary point of entry for refugees entering the eu ( fig. . ). moreover, turkey is the largest host country to syrian refugees. the dramatic increase of refugees in bulgaria led to a peak in . compared to , when the number of people legally seeking asylum in bulgaria was , saw an increase to , a % increase from , with the largest share of the migrants, arriving from syria - %. indeed, migrants account for only . % of migrants in the eu , but the ratio is approximately migrants per million people ( fig. . ) . at the same time, the majority of migrants did not intend to stay long in bulgaria, which they saw only as a stage of their journey to western europe. many of the migrants entering bulgaria in moved to france, germany and sweden, probably due to the lower incomes, but also the lack of previous settlers and migrant communities [ ] . the total number of those remaining in bulgaria is relatively low, compared to greece, italy, and germany ( fig. . ). it should be noted that for the period from to , a major share of refugees originated from syria, while in , the ratio changed, and most of those arriving, were fleeing afghanistan and iraq (fig. . ) . in terms of the demographic profile of those arriving, migrants in bulgaria are relatively less educated and the share of men is approximately equal to that of women and children figs. . and . . following the - crisis, in february a who assessment mission to bulgaria took place to access the country's capacity to address the public health implications of sudden large-scale influxes of migrants [ ] . the mission concluded that the level of assistance provided to the migrants by the bulgarian government has clearly improved but the medical response system is very fragile and not fully prepared to respond to a possible new and larger influx of migrants. the main weakness of the response system is the weak provision of primary health care through clinics in migrant centres or the assignment of general practitioners (gp) to migrants due to the fact that clinics are understaffed and interpretation services are not always available. as a consequence, the first recommendation of who is the revision of the national plan for crisis management with a focus on reorganizing primary health care services and rationalizing the use of available resources [ , p. ]. to put this into context, in the following section we provide an overview of the institutions and personnel involved in refugee and migrant management. upon arrival in bulgaria, the asylum seekers first face the authorities of the ministry of interior. by law, migrants can be held for up to hours at a location, where basic health screening is conducted, and from there they are directed to the registration and reception centre (rrc), the transit centre (tc) or detention centres where primary care is carried out. primary healthcare in detention centres is the responsibility of the medical institute of the ministry of interior, situated in sofia, while medical staff in rrc and tc should be provided by the state agency for refugees ( fig. . ) . according to bulgarian legislation, access to public health services requires payment of health insurance contributions. asylum seekers and persons applying for refugee status are funded by the state agency for refugees. in case they have acquired refugee status or the right to asylum, they are obliged to pay health insurance themselves to access the basic package of health care to which bulgarian citizens are entitled under the insurance system. however, the vast majority of refugees cannot cover their health insurance. for this reason, in march the bulgarian government passed a normative act creating an obligation for municipalities to cover health insurance expenses of migrants with official refugee status already granted. after strong public outcry the government withdrew the proposed act and this issue remains open. moreover, even when migrants fulfil their financial contribution, the number of refugees with an assigned gp is limited due to language barriers and the reluctance of many gps to take on patients whose residence is likely to be temporary [ , p. ] . the other important recommendation of the who assessment mission to bulgaria is the revision of the disease surveillance early warning and response system, introducing syndromic surveillance to increase early detection of outbreaks and effectively monitoring selected disease trends [ , p. ] . such a syndromic surveillance system has not been created yet and it is unknown whether the government plans to implement one. at the moment bulgaria only conducts surveillance and control of communicable diseases (cd) under the authority of the ministry of health (fig. . ). however, this information system does not include relevant information on the spread of infectious diseases among migrants/refugees. in the current paper we manage to fill the gap with operational reports provided by the chief state health inspector, angel kunchev from regional health inspections (rhi). these reports include epidemiological data from rhi-haskovo and rhi-sofia for . actually, these two administrations cover the two territories of the country in which almost all migrants are situated and thus the data can be seen as representative for the country. during based on these tests on the territory of rhi-sofia the following communicable diseases have been diagnosed (table . ). despite their limited capacity, authorities conduct basic health screening at the border, along with health services in the migrant reception and detention centres. screening at rhi-haskovo and rhi-sofia showed prevalence of intestinal parasites (giardiasis, ascaridiasis, blastocystosis) and our data indicates that the majority of refugees posed very limited infectious risk. therefore, the findings confirm who at the same time, contrary to analysis by experts, public opinion reacted to the migrants situation as high-risk, demonstrated by the conflict in harmanli migration rrs. residents of the town of harmanli protested on th november, demanding the camp's closure after local media reported on the suspected existence of communicable diseases on site: 'an artificially created tension led to this, following misleading reports that the centre is a hearth of infection,' petya parvanova, head of the bulgarian refugee agency, was quoted by reuters as saying. the government declared that no medical reason for quarantine existed but the authorities took the decision to temporarily close the camp and restricted the free movement of migrants with the aim to calm down town citizens. in response, on th of november some - migrants (from the officially announced registered in the camp) clashed with the police and the gendarmerie. as a result, most of asylum seekers have been moved to other centres. meanwhile, on - th november , immediately after the first articles in the local media and first signs of discontent, a special medical mission from the military medical academy was sent to verify the health status of the migrants concerned. the main findings of the military medical team study were as follows [ ] : • very high share of patients with skin and infectious diseases: patients with different symptoms; as a result of the inspection, the military medical team has diagnosed the following diseases, ordered according to incidence (table . ). the conflict in harmanly town illustrates some of the weaknesses reported in who's assessment of bulgaria's capacity to manage a large migrant influx, notably risk communications and work with local media. even though a special highly qualified medical team was sent to check the medical status of refugees and eventually to refute speculations of an epidemic, the conflict escalated instead of calming down. one explanation for this is the low levels of trust in the authorities and institutions in bulgaria, which also includes lack of trust in healthcare professionals. in such circumstances, both clear risk communication and medical expertise are not sufficient to resolve a growing crisis. in discussing the risks to public health, five topics are commonly considered: infectious diseases, vaccination, antimicrobial resistance, noncommunicable disease and bioterrorism (fig. . ) , as well as the linkages between them. with few exceptions, migrants are not at increased risk of transmitting communicable diseases [ ] . however, infectious diseases can spread when new migrants live together in communal, close-quarter settings [ ] . according to a recent survey in eu/eea countries, screening for infectious diseases among migrants is currently directed towards predominantly human immunodeficiency virus (hiv), tuberculosis (tb), hepatitis b, hepatitis c, gonorrhoea, syphilis, measles and rubella, malaria and chagas disease. these diseases were selected because the european surveillance system (tessy) collects data disaggregated by migrant status or because evidence suggests that they may disproportionately affect migrants in the eu/eea [ ] . other diseases that could be considered include vaccine-preventable diseases, cholera, malaria, helminths and intestinal protozoa (semenza et al. ) [ ] . epidemiological data from bulgaria do not confirm the severity of tb, hiv and sexually transmitted diseases, but confirm the importance of the screening of parasitological diseases. a potential risk to public health is the possibility of outbreaks of vaccine-preventable diseases (vpd) in a population coming from countries where immunization coverage is low [ ] . vaccinations to consider among migrants include: measles, poliomyelitis, meningococcal disease, and diphtheria/tetanus/pertussis. the risk of the spreading of vaccine-preventable diseases among the local population should not be underestimated too. the varicella vaccination, which is not included in bulgarian routine vaccination programs because the majority of individuals in temperate climates develop natural immunity from previous infection before adolescence, is a good example, especially when taking into account cases registered in the harmanli refugee camp. other possible threats could be recognized in the cholera epidemic in iraq [ ] and poliomyelitis in the syrian/lebanon border refugee camp [ ] . population displacement can also threaten global vpd eradication and elimination efforts [ ] . antimicrobial resistance (amr) is not a disease in itself but a complication of the treatment of a disease. in situations such as the crowded settings with poor hygienic conditions found in refugee camps, infections can easily occur and spread; whether they are caused by resistant pathogens depends on their origin, which can be the environment, animals, food or humans [ ] . amr is becoming a global concern with amr strains associated with new resistance mechanisms emerging and spreading worldwide [ ] . the journey the refugees undertook, crowded conditions in refugee camps or settlements, and the lack of regular medical care, are prime drivers of the spread of amr among this vulnerable group especially in multidrug resistance tb cases among refugees [ , ] . the range of potential risks that can be associated with refugees is inevitably wideranging. it includes communicable diseases, trauma, dermatological disease injuries associated with the journey, environment, changes in climate, especially during the winter, and last but not least, mental health and psychological problems [ ] . who assessment in bulgaria reported on physical and psychological trauma, the consequences of post-traumatic stress disorder; dehydration, nutrition disorders and hypothermia; absence or interruption of treatment for chronic diseases. particularly high health risks are faced by vulnerable groups of migrants, including the elderly, people with disabilities, pregnant women and young children [ ] . last but not least, migrant's ncd treatment requires substantial resources that create additional economic pressure on the health systems of affected countries. there are multiple causal relations between (forced/irregular) migration and terrorism -but these are generally complex [ ] . while the link between terrorism and migration is widely discussed, where in the context of the migration inflow hypothesis immigrants are an important vehicle for the diffusion of terrorism from one country to another [ ] , the link between bioterrorism and migration is hardly found in official and scientific publications. however, bioterrorism as a risk and policy measures to address it is discussed in official documents of the bulgarian government, particularly in the annual report of the ministry of health, which names as one of its aims to 'protect the country from importation and distribution of infections with high epidemic risk by creating and maintaining mechanisms for timely and adequate response to health threats of a biological nature, including bioterrorism'. the eu also includes bioterrorism in the list of new threats but not specifically in the framework of migration but in the general context of cross-border threats: 'under eu law on cross-border health threats, existing mechanisms coordinate preparedness for serious cross-border threats to health, linking member states, eu agencies and scientific committees through the early warning and response system. the health security committee, which coordinates member states' responses to threats, may act as a focal point on vulnerabilities in public health, to enshrine hybrid threats, in particular bioterrorism [ ] . therefore, there is a wider recognition that migration and bioterrorism have to be dealt with in a broader institutional and policy context. despite these general considerations of bulgarian authorities and the eu health security committee, none of our past experience, including the data from our research, convinces us that there is a clear link between bioterrorism and migration processes. therefore, in the risk assessment, the likelihood of migrants being instrumental in a bioterrorist attack was assessed as very low (see table . ). our findings confirm who and dg sante expectations that migrants do not pose a significant risk to the local population. at the same time some specific, health risks should be taken into consideration and categorised by varying degrees of probability and impact. the risk assessment should be made by a wide range of specialists based on detailed logitudinal data. our assessment, based on the public data at our disposal, published and analysed in our article, is summarised in table . . the assessment of these risks may seem overwhelming or unrealistic to the external observer, but it seems realistic when considering the geographical location of the country, the institutional weakness and the fragile public health capacity. die getriebenen: merkel und die flüchtlingspolitik: report aus dem innern der macht cholera in iraq strains the fragile state does immigration induce terrorism? migration, refugees, and health risks refugees and antimicrobial resistance: a systematic review annual report on the situation of asylum in the european union annual report on the situation of asylum in the european union european asylum support office. annual report on the situation of asylum in the european union annual report on the situation of asylum in the european union latest asylum trends european centre for disease prevention and control. assessing the burden of key infectious diseases among migrant populations in the eu/eea. stockholm: ecdc joint framework on countering hybrid threats a european union response population movement is a challenge for refugees and migrants as well as for the receiving population vaccine-preventable diseases in humanitarian emergencies among refugee and internally-displaced populations health case study -bulgaria military medical academy the refugee crisis in the middle east and public health europe's migration challenges: mounting an effective health system response state agency for refugees with the council of ministers links between terrorism and migration: an exploration, icct research paper public health needs of migrants, refugees and asylum seekers in europe, : infectious disease aspects european parliament, briefing. martin seychell, deputy director general of the european commission directorate for health and food safety, the public health dimension of the european migrant crisis war and infectious diseases: challenges of the syrian civil war unhcr ( a) un agency for refugees. winter operations cell -daily report asylum trends : levels and trends in industrialized countries. office of the united nations high commissioner for refugees joint report on a mission of the ministry of health of bulgaria and the who regional office for europe migration and health: key issues antimicrobial resistance key: cord- -r en s authors: watanabe, chiho title: health impact of urban physicochemical environment considering the mobility of the people date: - - journal: health in ecological perspectives in the anthropocene doi: . / - - - - _ sha: doc_id: cord_uid: r en s most of the current environmental health researches assumes that exposure to the environmental agents occurs either in the residence or workplace, neglecting the mobility of the people due to commuting and daily activities. mobility of the people varies in terms of spatial and temporal range, that is, from momentary short ones to generation-scale long ones. focusing on the daily movement of the people, various methods for grasping the mobility, which also range from simple observational methods like time allocation to methods with advanced technology like global navigation satellite systems, will be reviewed. referring environmental health studies examining the health effects of either air pollution or heat, importance of the mobility of the people is discussed. assessing the mobility will open a new research avenue for the study of infectious diseases as well as noncommunicable diseases. depending on the media (most of the case, relevant media should be food, water, air, soil, etc.) as well as the agent of concern, a variety of the methods exists to evaluate the exposure, and exposure evaluation has been recognized as an independent research field. the procedure for exposure evaluation can be largely classified into two categories, that is, measuring environment and measuring organism; these are the procedures called as environmental monitoring and biological monitoring. depending on the media and the agent, one or both of these methods are utilized. for example, both (organic) mercury from fish and cadmium from rice are heavy metals exposed through food consumption. therefore, by monitoring the concentration of these metals in respective major food source, combined with food consumption data, exposure (ingested amount of mercury/cadmium) could be estimated. biological monitoring for these metals is also possible by measuring the concentration of mercury in the hair (which reflects the concentration in the blood, and approximates the concentration in the brain) or cadmium in urine (which reflects the concentration in renal cortex). detailed discussions about these two categories of monitoring are beyond the scope of this book, and interested readers should refer to existing textbooks. unlike the case for arsenic in water or mercury in the fish, many of the major "classical" air pollutants like nox, sox, co, pms (either . or ) do not have appropriate biological exposure markers, and we need to rely on the environmental monitoring. this is also true for the exposure to temperature or to noise, both of which are associated with atmospheric exposure. in conventional environmental health study or environmental epidemiology, environmental monitoring of the outdoor atmosphere at the residential area of the participants has been used as surrogates for individual/population exposure to the agents of concern. implicit assumption here is that people would stay in their residence, and variation of the exposure due to their daily mobility is, if any, negligible. although this assumption is not realistic at all, this approach has been successful in a sense evidenced by the existence of numerous epidemiological findings in this area in the past. in fact, exposure to pollutants or physical factors associated with air/atmosphere is heavily influenced by the location of the individual. assume we would like to estimate an individual's exposure to nitric oxides, most of the case, individuals would not stay in the home whole day, particularly in urban settings, commuting into city center or business districts for workplace or for schooling, thereby exposing themselves to environments that are different from their own residential neighborhoods. in this sense, kwan [ ] has suggested that research involving geographical components should reconsider conventional methods to estimate the exposure, referring to environmental health/epidemiology (together with the research on segregation or on the issue of accessibility). richardson et al. [ ] pointed out that due to the accumulation of highly sophisticated spatial and spatiotemporal technology like gis, gps, remote sensing, and computer cartography, collectively termed as geographic information science, it becomes possible to model the disease process involving multiple spatiotemporal data obtained in different disciplines. likewise, the exposure to environmental factors could be evaluated using such spatiotemporal data. spatial resolution of various types of environmental data becomes so high that heterogeneity within the area of commuting distance could be documented. in addition, recent technical progress in downscaling of the climate models (see [ ] ) enables the researchers to predict differential impact of climate change within relatively small areas. health? apart from the environmental data, to consider the mobility of the people, we need to collect the information on the mobility of people with enough spatiotemporal resolution. mobility of people can mean wide variety of phenomenon in terms of time, space, and context as listed in fig. . . time scale of the mobility can range from moment to moment as exemplified by the ecological momentary assessment (later in this chapter) to years, even including hundreds of centuries (like out of africa, the expansion of our ancestors)! duration of the sojourn time should be considered as an independent factor and can also range from few seconds to generations; for example, workplace exposure to hazardous substances (including radioactive materials) would be evaluated in terms of minutes, while the effects of regional migration fig. . schematic classification of various type of human mobility by "relevant" spatial and temporal scales. for example, "migration' takes place with relatively long time, ranging from months to multiple generations and traveling relatively long distance, while "outdoor/indoor" discrimination is needed even a couple of meters apart. 'ema' stands for ecological momentary assessment (see text) may emerge after several generations. accordingly, spatial scale could range from a few meters to thousand kilometers. a few meter matters when micro-scale environments such as indoor (including air-conditioning) vs. outdoor, inside and outside of vehicle are considered, for example. at larger scale, proximity to major roads or any combustion facility could exert significant influences on exposure to noise or air pollutants. far larger scale can change the environment as a whole; an important aspect in today's environmental health is that people can make a global scale travel within h. mobility of people also entails the change in the context; by changing the location, the socioeconomic and cultural aspects of the neighborhood would change, which may affect the meaning of material environment and, in turn, health status of the moving individuals. at the same time, change in location means the change in activities of the individuals; for example, individuals working at outside road construction site might have much higher physical activity levels (and increased ventilation rate) and higher exposure to heat, dust, and noise than staying at home. while it is fairly difficult to consider all of these potential factors, in case of atmospheric exposure, geographical location of the individual should be the factor to consider in the first place, since it does provide the basis of the air which she or he inhales in every moment. in this chapter, main focus will be on daily mobility of people for commuting and for other daily life activities. geographical location is crucial for some physicochemical environmental factors other than air pollutants. one such example is heat environment, which consists of temperature, humidity, air flow, and radiation, and varies even with a very small scale, reflecting the variation of land use or land cover, local topography (layout of surrounding buildings, for example), or elevation. in addition, numerous heat sources are found in human-built environment, including exhaust gases, waste heat, etc. as a result of combined effects of these factors, most of the urban centers have warmer environment, compared to the surrounding areas, termed as heat island. mobility of individuals has been dealt with in many research areas. in urban planning, grasping mobility of individuals is crucial to create an appropriate layout for transportation, public space/facilities, and private houses. in public health, mobility of individuals sometimes play key role in the spread of diseases (described later). also, mobility has been one of a classical topic in the area of human ecology since it is associated with the question of how a population utilizes the environment spatially as well as temporally (time allocation studies). mobility is also associated with energetics (utilization of somatic energy) as a part of physical activities in general. as such, many methods to grasp the mobility of individuals have been developed (table . ), which will be discussed below. the simplest method is field observation. time allocation studies observe the individuals in the targeted field and record the location and type of activity for a given period, which is useful to answer some of the basic questions in human unlikely large ecology or other related fields as noted above. while this method is in a sense "perfect" since the observer can obtain whatever details she or he wants, obvious disadvantages include time-labor intensiveness, biased behavior due to the presence of observing researcher. a simplified variation of the time allocation is the spot-check method, in which researcher will observe the people's activity in certain fixed location(s) (e.g., see [ ] ). despite its simplicity and easiness, spot-check method could provide valuable quantitative information. activity diary is another classical method, which relies on the self-recorded diary. this would solve the issue of laborintensiveness for researcher and could cover much larger number of individuals, but like other self-reporting methods, reliability and accuracy of the record are the main problems to be considered. activity diary is useful when researchers are interested in qualitative aspects of the moving behavior. person-trip is another method, which has long been utilized especially urban planning as well as urban studies (e.g., see ( [ , ] )). person-trip uses a predetermined, formatted questionnaire to be either self-recorded or recorded by interviewers, through which location of the individual, purpose of travel (translocation), and method of travel (either walk, bicycle, private car, public transportations) for a given period will be obtained. while this method has problem of bias and/or inaccuracy due to recall, brevity and simplicity made this method popular, particularly when a large population needs to be covered. many governmental (both national and local levels) surveys utilize this method to quantify the volume of mobility, for example, traffic volume of vehicles. gps (global positioning system) has been used as if it was a generic term, but actually gps is a name of a system developed in usa. generic name for the systems is global navigation satellite systems (gnss), which is referring to any system that locates specific targets by use of the combination of signal-detecting device and a group of satellites. in this book, the "targets" are basically human individuals, but they could be animals (so-called activity logger) or traffic vehicles. spatial resolution of contemporary gps is fine enough to locate individuals for many study fields, and it solves the issues of time-labor intensiveness and false or biased report. thus, this has been used in various research areas including anthropology, human ecology, and presumably sociology, and urban studies (see [ , ] ). another advantage is that gps uses electrical data processing, which also alleviates the risk/errors associated with data transfer. on the other hand, in some area like inside building or underground malls the signal for gps are generally weak and difficult to detect. although the gps could provide very rich and useful information regarding the mobility, individuals rarely own the device, and researchers need to provide and distribute it to the participants. this has been overcome by the recent propagation of gpsequipped mobile phones and alike. mobile phone per se can be also utilized as a tool in capturing the location of individuals without using gps device since every call made by mobile phone is registered by nearby relay station, hence generating a record regarding approximate location of the mobile phone users at the time of the call. by collecting such records of local calls, researcher can trace or reconstruct the translocation of individuals. major advantage of this method is the fact nowadays a large proportion of populations own the mobile phone even in developing countries and in very remote areas. on the hand, researchers need to negotiate with mobile phone company to obtain such record, and the major barriers for such negotiation are as following: ( ) conflict with the protection of privacy information (even when the information is provided in anonymous manner) may arise, ( ) often there are two or more mobile phone companies operating in a given area, and ( ) since the information is provided in anonymous manner, demographic parameters of the mobile owner are not known to the researcher. effort has been made to overcome the last point, in which an algorism has been developed to estimate the demographic attributes of a mobile phone owner through the pattern of mobility, although the feasibility as well as the accuracy of estimated attributes needs further investigation (arai, shibasaki, in preparation). wesolowski et al. [ ] compared the mobility data obtained by a person-trip type survey with the one obtained by mobile phone; both conducted in kenya over the same period. reflecting the nature of the methods, numbers of the participants were and , in the survey and mobile phone analyses, respectively. while the person-trip type survey was cross-sectional in nature, the mobile phone records followed the movement of the people for months. while the resultant two data sets agreed in some aspects of the travels such as ( ) most visited areas (in terms of districts) or ( ) overall relative frequency of individuals with different number of travels, they disagree other aspects such as the number of mobile phone subscribers in the area as much as ten times. in this section, a couple of examples will be presented that incorporates the mobility of the individuals/groups in estimating exposure to physicochemical factors in the air. as the physicochemical factors, air pollutant and heat will be discussed. in the final part, a rather classical, different approach to trace individual exposure will be also introduced. a relatively large spatial scale study has been conducted covering approximately × km area in belgium [ ] , which compared regional exposure estimates for two representative air pollutants, nox and ozone, under two alternative assumptions. first, exposure assumed to occur in the residential place of the participants (static exposure), that is, the mobility of the participants is neglected. second, mobility of the people was taken into account in estimating exposure (dynamic exposure). information on mobility was obtained through activity diaries collected from residents, which is then extrapolated to a synthetic (but reflecting the demographic structure of actual) population of approximately millions. the target geographical region consisted of zones ( municipalities) whose average area was km , and the location of each individual has been predicted for week ( days) by -hr interval. pollutant concentrations are estimated for no and ozone using air pollution models (i.e., an emission model combined with a dispersion model) for a year by h interval and × km resolution with finer resolution along major roads. based on the location data and pollution data, time-weighted exposure estimate was calculated under two conditions: with and without taking peoples' mobility into account. to estimate the municipality-wise health impacts of the exposure to these air pollutants, the time-weighted exposure estimate was converted into the respiratory mortality using the information of existing epidemiological data, which is then converted into years of life lost (yll) following the burden of diseases framework by who. the analyses revealed the pollutant-specific regional difference in the pollutant concentration; that is, for no , urban zones had higher concentrations than rural zones regardless the age and gender, and for ozone, it was vice versa. as expected, urban and industrial zones have much larger population in daytime, which was in contrast with the surrounding zones. as the results, dynamic exposure for no for the whole population was slightly higher than static exposure, while for ozone it was vice versa. while the difference between static and dynamic exposure were statistically significant, the difference was small and reached only up to %. in terms of health impact, again the difference between the two methods was statistically significant but small ( . % increase for no and . % decrease for ozone). at the municipality level, the maximum difference between the two approaches reached as large as %, where dynamic was higher than static, and larger differences were usually observed in rural areas. for ozone, maximum difference was only % (dynamic was lower than static). while the extent of impacts shown in this study might not be so remarkable, the results demonstrated that mobility of people could have significant impact on the estimate of health impact by air pollution and that such an impact could be more remarkable at smaller scale. as pointed out by the authors, considering the mobility of people in air pollution issue inevitably connects the issue of transportation (and urban planning) with health issues, which is also commended from the viewpoint of eco-health. similar dynamic-vs-static comparison was conducted with much smaller sample size in western new york, and as naturally expected the difference between the two approaches depended on the pattern of spatiotemporal pattern of air pollutants, pm . , as well as on that of behaviors [ ] . the human early-life exposome (helix) is a multi-country (eight countries), multi-cohort project in europe to characterize early life exposure to various chemical and physical environmental factors and to associate them with health consequences in early life ( [ ] project url: http://www.projecthelix.eu/en). involving , mother-child pairs, the project would try to grasp the whole picture about the exposure as much as possible, and the use of time-space activity information is planned, which will be utilized to estimate the participants' exposure to not only air pollutants but also noise, uv radiation, temperature, and built environment/green space etc. basically, the environmental data is collected from (ground) monitoring stations and/or remote sensing. in addition, smartphone-based "personal exposure monitoring kit" has been developed that enables to capture not only the location of mothers and children but also their physical activities and air pollution by built-in accelerometer and sensors for uv and pm . . recent progress of this project can be found at the following url: https://www.isglobal.org/en/web/guest/healthisglobal/-/custom-blog-portlet/prova/ / . helix is an ambitious attempt, which needs large amount of budget ( . m euro, according to the web page), time, and manpower. considering the nature of current "environmental exposure," that is, long term, multiple species, and mild to moderate (rather than severe) level, probably such an extensive effort is required to elucidate the relationship between environmental agents and health consequences. heat is another environmental factor, which might have some relevance to the mobility issue, since urban heat, or heat island, is a ubiquitous phenomena common to most of the big urban areas, which would pose additional heat burden to urban dwellers as well urban commuters under the influence of climate change (global warming). usually, effects of heat are considered to be immediate or short, which is different from those of air pollutants, whose effects can be both short term and long term. although not involving mobility assessment, laaidi et al.'s study on the heatmortality relationship [ ] is worth to be discussed here. this study analyzes the relationship between all-cause mortality among the elderlies living in paris, france, or nearby area and land-surface temperature captured by satellites for a period of a heat wave occurred in august . based on a case-control study of pairs of mortal-alive elderly people (age > ), they found elevated odds ratio of mortality with increased land surface temperature (lst) of the residence area. of noteworthy, the elevated odds ratio was associated with minimum (night time) lst averaged over either days (whole observation period) or days preceding the reported deaths but not with any lsts averaged over days preceding deaths or the day of death. this result suggested that the effect of heat might not be limited to immediate effects but might be "cumulative" to some extent. also, approximately . °c of increase in temperature was associated with a significantly elevated odds ratio more than two, showing relatively potent effect of temperature on mortality. in this study, spatial resolution of the lst was km , and the case and control are matched, in addition to age and sex, for residential area, which contains - pixels, allowing the temperature comparison between the case and control. targeting the elderlies, mobility would be less important than in younger generations. we have conducted a study in which mobility of the people is considered in heat exposure issue in a subtropical urban area. this particular issue will be discussed in the next chapter. in the area of industrial health, exposures of the factory workers to air-borne chemicals peculiar to the factory are monitored with device, which is "worn" by each worker. many types of such devices have been developed for various solvents or gaseous pollutants, among which the γ-radiation monitor is the best known. cumulative exposure of each individual to the target chemical/radiation is quantified by analyzing the amount of chemical collected/absorbed by the device. such personal device has been used for appropriate control of worker's exposure to hazardous chemicals, but which could be extended into surveys in general population. while this method provides the estimate of individual exposure, if used in general population, distribution and retrieval of the device could be labor taking, and the quantified results would not give any hint of the potential sources of the exposure, since it only provides the cumulative exposure rather than temporally tracing the individual exposure. potential use of mobility information would not be confined to the issues that have been discussed in this chapter so far. these examples will be discussed in this section. many infectious diseases are transmitted through direct or indirect human-to-human contact. mobility information has been utilized in the development of the models for propagation of some of the infectious diseases like influenza (direct) or malaria (indirect). in developing models, however, most of the attempts have based on simulations under plausible assumptions about the parameters, and not so much have been done using actual mobility data. malaria is one of the diseases that will be propagated by mosquitoes (indirect human contact). propagation of the malaria agent (plasmodium) occurs when a mosquito (anopheles) sucked blood from an infected human individual and then bites an intact individual. since the range of area that traveled by a mosquito is relatively limited (approximately - km/day for anopheles) [ ] , mobility of infected human individuals should play some roles in the propagation of the agent, particularly for long distance propagation. wesolowski [ ] tried to elucidate the role of human mobility in the propagation of malaria in kenya. based on mobile phone records (either call or text) of about million people for year, they reconstructed the mobility patterns of the people and combined this information with spatial prevalence data of malaria cases. location of the people (mobile users) is followed based on approximately , cell towers located in settlements in kenya. travels (change of the location) beyond the border of each participant's "primary settlement" (presumably where the residence is located) was counted and used for data analyses. a malaria prevalence map with km resolution for has been used to classify the settlements according to their prevalence, then, combining the two types of information, that is, travels and prevalence observed in various settlements, they have estimated the proportion of the infected travels, which actually transported the malaria from one settlement to another. in this way, they could identify the source areas and the sink areas; the former supply the malaria, while the latter accept it. with such an analyses, they could show there were several distinct sources and sinks for malaria in kenya. nairobi, the capital, and the area around the victoria lake were serving as the most remarkable sink and the source, respectively. also, they observed that travel of people reflected the regional population density and regular travel, which is different from the travel of the parasites, where the lake regions serve as the primary source of the parasites, which flew into its surrounding areas and the capital area. based on these observations, the authors suggested that the elimination program must take the imported case into account for the program to be successful. in addition, they demonstrated that this method could identify the "hot spot" (settlement), which shows distinct export and import of malaria incidence compared to adjacent settlements and that it can also provide useful information for elucidating the mechanisms of smaller scale transmission. such an analysis provides a good example demonstrating the huge potential of using spatial analyses in the area of disease propagation. they applied the same approach to model and predict the dengue epidemics in pakistan [ ] . mobility of approximately million subscribers was followed for months in across tehsils (small politically defined areas in pakistan), and analyzed with more than , reported cases observed in tehsils over months in . focus of the analyses was the spread of the disease from southern region, where the warm climate supports the existence of vector mosquito throughout the year, to the northern regions with greater seasonality that limits the transmission. the authors calculated the "dengue suitability" of each region mainly based on daily temperature, which was combined with the probability of importing the infection by travelers from epidemic area to estimate the regional (spatiotemporal) risk of dengue epidemic. the results considering the mobile phone data are compared with those obtained from conventional "gravity" model, in which the travel volume of the people depends on the population sizes of and distance between the two regions (beginning and end of the travel). the results of the two models sometimes differ widely; among the two regions that experienced real dengue epidemics in , import of infection could not be predicted by the gravity model, while the model with mobile phone information could. also, the risk map generated for entire pakistan showed substantially different pictures between the two models. part of the reason of such differences is related with the observation that contrary to conventional mobility model, the mobile phone data showed no decay of travel volume with increasing distance of the regions. overall, this research demonstrated the importance of grasping mobility based on real observational data to understand the spread of infectious disease at the level of a country. for the diseases that are propagated through direct human-to-human contact, mobility of individuals among the population at stake should be much more crucial than the case of malaria as described above. for example, in the outbreaks of sars and mers, identifying the "index case" would be important. many quantitative models have been proposed to explain the spread of disease, but many of them have not taken the spatial information into account. as explained in this section, modeling with peoples' mobility for infectious diseases has not been explored so much, while it is a promising field for the future. at a larger spatial scale, spread of infectious diseases is associated with international travels. using the data for international airline travelers, potentially "hot" areas for the spread of zika virus have been identified. potential threat of importation of the virus from americas to africa and asian countries was demonstrated [ ] . relative importance of noncommunicable diseases (ncds) has been increasing both in developed and developing countries; to name a few, ischemic heart diseases, stroke, diabetes mellitus, and various types of cancer are the big ones in this category. it has been quite well established that obesity and hypertension are associated with higher risk of these diseases, which in turn are associated with imbalance in the energetics. numerous reports have been published regarding metabolic aspects of individuals at high risks for the ncds. in these reports, activities are mainly evaluated with activity diary, pedometer, or accelerometer wore by the subjects. while these lines of information would provide valuable data to demonstrate the association between inactivity and risk factors of ncds, a major defect is that it would not easily identify where in the daily life of the individual potential problem lies (i.e., leverage point). gps information or mobile phone call record might be useful in reconstructing daily activity of individuals, since they can provide the information regarding the speed of the translocation, by which researcher can make a reasonable guess if the individual moved actively (i.e., walking or bicycling) or passively (i.e., driving a car, using public transportations). this is a relatively unexplored area, which might bear potential public health importance both in developed and developing countries. ecological momentary assessment (ema) refers to the methods of collecting data from individuals, who are in their daily lives (thus, ecological), providing real-time data (thus, momentary) repeatedly [ ] . this is often enabled by using ict devices that can prompt a series of questions to participating individuals to report their physical and/or mental conditions to the researchers. the devices can be also connected with sensors for physiological or clinical information like heart rate, blood pressure, or body temperature, blood glucose, or blood oxygenation [ ] , thereby health "events" like arrhythmia, asthma attack, and panic episodes can be recorded. in addition, ema device has been connected with physical sensors to air pollution. with this kind of device, chronological data, which can be associated with special health events, can be collected, accumulated, and later be related with physiological and environmental conditions (like air pollution) where the individual was in, revealing hidden association between the health event and certain patterns of preceding environmental and/or behavioral conditions. this methodology has been successful in clinical settings, particularly highlighted in clinical psychology, and its application to environmental health may generate a unique opportunity to grasp individual's "dose" and "response" simultaneously. by combining with environmental monitoring information provided by satellites as well as ground monitoring stations, potential of using ema will be greatly enhanced in the area of environmental health. as noted in the beginning of this chapter, environmental health concerns the relationship between environmental condition and health consequences, spatial information is an indispensable component of this field. conventional environmental health studies have dealt with the spatial aspects as represented by administrative units, which is basically a qualitative variable and black box so to speak, in nature. more quantitative aspect of the spatial distribution is worth to be focused in environmental health, and recent progress in information and communication technology including data processing enables us to develop a new type of research. in this way, spatial information becomes much more manipulative in the analyses with its implication being much clearer. refining spatial information is only a method to improve the accuracy of exposure estimate, hence, associated with other progresses in this field. for example, conventional environmental health study only focused on a very limited number of environmental factors, most often only a single factor, and the dose-response relationship was evolved around this single factor. this is most likely due to the fact that problems in the past were mostly associated with a single environmental agent like one chemical species. contemporary issues, however, involve multiple factors that are converged on a single endpoint. in such a case, approach adopted in the helix study might be useful, although we yet to know what can be obtained with this approach. refining exposure estimate should be considered in such a context to characterize comprehensive exposure. while potential of this field is enormous, especially to be combined with other relevant techniques like ema, there are a couple of issues that needs constant attention. first, as the technology (both hard and soft) advances, more attention should be paid for the importance of the issue of privacy. this is not only saying that full attention should be paid to protection of privacy, but also ( ) considering the benefit for the people obtained through such information and ( ) letting people know both aspects (goods and bads) of mobility information, thereby enabling them to choose appropriate reaction towards such investigation. finally, it should be emphasized that mobility information obtained in the ways described in this chapter might evoke a new discussion about what "true" exposure is and to what extent we need to know about the exposure. as partially discussed before, if you would like to quantify the exposure of an individual as much as possible, you need to actually chase this individual to see how the individual and the environment at a given moment is faced with each other. for example, merely wearing a fine-pore mask would substantially change the exposure to certain air pollutants, which could not be picked up by the approaches discussed in this chapter. after all, required fineness of the quantitative evaluation totally depends on the objectives of the specific research. potential for zika virus introduction and transmission in resource-limited countries in africa and the asia-pacific region: a modelling study downscaling climate models health impact assessment of air pollution using a dynamic exposure profile: implications for exposure and health impact estimates issues of commuter transport in developing countres flight performance of the malaria vectors anopheles gambiae and anopheles atroparvus beyond space (as we knew it): toward temporally integrated geographies of segregation, health, and accessibility the impact of heat islands on mortality in paris during the august heat wave urban form and the ecological footprint of commuting. the case of barcelona gender difference in daily time and space use among bangladeshi villagers under arsenic hazard: application of the compact spot-check method health and the mobile phone spatial turn in health research ecological momentary assessment the human early-life exposome (helix): project rationale and design quantifying the impact of human mobility on malaria quantifying travel behavior for infectious disease research: a comparison of data from surveys and mobile phones impact of human mobility on the emergence of dengue epidemics in pakistan geospatial estimation of individual exposure ot air pollutants: moving form stati monitoing to activity-based dynamic exposure assessment key: cord- - vkhptas authors: wu, tong; perrings, charles title: the live poultry trade and the spread of highly pathogenic avian influenza: regional differences between europe, west africa, and southeast asia date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: vkhptas in the past two decades, avian influenzas have posed an increasing international threat to human and livestock health. in particular, highly pathogenic avian influenza h n has spread across asia, africa, and europe, leading to the deaths of millions of poultry and hundreds of people. the two main means of international spread are through migratory birds and the live poultry trade. we focus on the role played by the live poultry trade in the spread of h n across three regions widely infected by the disease, which also correspond to three major trade blocs: the european union (eu), the economic community of west african states (ecowas), and the association of southeast asian nations (asean). across all three regions, we found per-capita gdp (a proxy for modernization, general biosecurity, and value-at-risk) to be risk reducing. a more specific biosecurity measure–general surveillance–was also found to be mitigating at the all-regions level. however, there were important inter-regional differences. for the eu and asean, intra-bloc live poultry imports were risk reducing while extra-bloc imports were risk increasing; for ecowas the reverse was true. this is likely due to the fact that while the eu and asean have long-standing biosecurity standards and stringent enforcement (pursuant to the world trade organization’s agreement on the application of sanitary and phytosanitary measures), ecowas suffered from a lack of uniform standards and lax enforcement. highly pathogenic avian influenzas have become a major threat to human and livestock health in the last two decades. the h n panzootic ( ongoing) has been one the most geographically widespread and costly, resulting in the loss of hundreds of millions of poultry in countries [ ] and over human deaths worldwide-a mortality rate of percent [ , ] . for h n , and other h subtypes, most countries reporting poultry outbreaks also report evidence of the disease in wild bird populations, and the mechanisms for the spread of h n have been identified as a combination of wild bird transmission and the live poultry trade [ , ] . plos risk factors. our primary interest is in the role of live poultry imports as a source of traderelated avian influenza risk at the regional level. we note that other poultry products, such as packaged meat and eggs, do pose a risk, but it is significantly lower. although avian influenza can persist in frozen meat, contact with that meat is unlikely to cause infection [ ] . furthermore, since hpais are lethal to egg embryos, eggs are not a potential source of transmission [ ] . the data comprise an unbalanced panel covering countries over years; the lack of balance is due to the fact that membership of the eu changed over the timeframe. the response variable in all models estimated was a log transformation of the number of h n poultry outbreaks in a given country in a given year, obtained from the emergency prevention system for animal health (empres), a joint project of the fao and oie [ ] . the log transformation was applied to account for the wide disparities in the numbers of the outbreaks across countries. in , for example, indonesia recorded outbreaks while romania, the only eu country to be infected that year, had only . in addition to reflecting the differing directions and intensities of risk factors, this also reflects differences in reporting conventions for h n at the international level [ ] . a series of outbreaks may be reported separately in one country, but be treated as a single event in another. data on trade in live poultry were obtained from the united nations' comtrade database (comtrade.un.org) and resourcetrade.earth, a project of the royal institute of international affairs (www.chathamhouse.org). these report the total imports of live poultry into a given country in a given year by weight (kg). the data on trade in live poultry did not distinguish between different types of domestic birds, such as chickens, duck, and geese, but grouped them under a single commodity category of "live poultry." with respect to wild bird migration as a pathway for h n spread, we used the density of wild bird habitat as a proxy for the presence and scale of migratory bird populations, and the likelihood that wild and domestic birds will mix. lakes, wetlands, and (irrigated) agricultural areas have been consistently identified as wintering and breeding grounds for migratory birds, and as places where wild birds may come into contact with free-ranging poultry [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the indicator for wild bird habitat used in this study was the set of "important bird and biodiversity areas" (ibas) for "migratory and congregatory waterbirds" identified by birdlife the live poultry trade poses different avian influenza risks in different regions of the world table . the distribution of h n poultry outbreaks between - across the member states of asean, ecowas, and eu. "-" signifies that the country was not a member of its associated trade bloc in that given year . brunei cambodia indonesia laos international (datazone.birdlife.org). in their analysis of h n spread, kilpatrick, chmura ( ) also identified ibas as a proxy for migratory birds and the infection risks they pose. country-level statistics on socioeconomic and agro-ecological conditions were taken from the united nations' food and agriculture organization (www.fao.org/faostat/en/) and the world bank (data.worldbank.org). agricultural land cover was reported as a percentage of total land area of the country. per-capita gdp was reported in purchasing power parity terms as current international dollars. data for for these two variables were missing for certain countries. in these cases, the gaps were filled by extrapolating the missing data as a linear trend of the preceding years. we assume that agricultural land-where free-ranging chickens, ducks, and geese are commonly raised in all three regions-also acts as a relevant proxy for susceptible poultry. data on the biosecurity measures targeting avian influenza undertaken by each country were obtained from the world organisation for animal health (oie) (www.oie.int). these report a standardized series of biosecurity controls targeting wildlife and livestock diseases, including those related to surveillance, vaccination, border checks, and management of wild disease reservoirs, and whether or not a given country undertook them in a given year. we chose a subset of these biosecurity measures we considered most relevant to h n avian influenza risks for inclusion in our model. additionally, in any given year, there were to countries that did not provide a report of biosecurity measures to the oie; we assumed that this indicates an absence of action, and the dataset records these cases as zeroes. our modeling approach relied on generalized linear models (glm) to analyze a panel of data on disease outbreaks and associated risk factors. in this we follow others who have sought to predict the spread of h n at both national and international levels [ ] [ ] [ ] or h n [ ] [ ] [ ] . glms are well suited to epidemiological studies because of their flexibility regarding data type and the distribution of response variables, their simplicity of application, and their frequency of use [ ] . our identification strategy involved the selection of three specifications for each of two estimators. we adopted both random and fixed effects estimators. hausman tests conducted at the all-regions level favored a random effects estimator, as the p-value exceeded the % threshold below which fixed-effects regression is conventionally considered necessary. some factors that influence the likelihood and number of outbreaks in a given country or region are not likely to change significantly over the course of several years, or even a decade. in our dataset, netherlands for example, the amount of land covered by wild bird habitat is time-variant, while agricultural land and even per-capita gdp for many countries experienced relatively modest variations over the timeframe of the study. in this case, and as the hausman diagnostics indicate, a random effects estimator is more appropriate. nevertheless, since we wished to control for timeinvariant characteristics of regions and countries we also implemented fixed effects estimators at both the aggregate and trading bloc levels, implicitly assuming no changes in the trade or biosecurity environment at the bloc level that we are unable to control for. our first specification (model ) included a number of factors related to disease risk but excluded both live poultry imports and biosecurity measures. included predictors were land area, human population, per-capita gdp in purchasing power terms, agricultural area, wild bird habitat area, and the live chicken population. our second specification (model ) added intra-regional trade bloc and extra-bloc imports of live poultry. our third specification (model ) added four main biosecurity measures: border precautions, general surveillance, vaccination prohibition, and wild disease reservoir management. all are categories of oie-reported biosecurity measures taken against avian influenza. the general forms of the estimated random and fixed effects models were: where y it denotes the number of poultry outbreaks in country i in year t, x includes the predictors for model , z includes the additional predictors for model , u includes the additional predictors for model , ecowas and asean are dummy variables for the two titular regional trade blocs (the eu is the reference group), and u it and ε it are the "between" and "within" errors respectively. to account for heteroskedasticity, we used robust standard errors. finally, since the data used in this analysis are reported annually, and h n has been a conspicuous and fast-moving epidemic (meaning the effects of an outbreak are unlikely to persist over a long period of time) among poultry, we did not use a lag structure in our statistical analysis. therefore, we assumed that the factors driving an outbreak in a given year are contemporaneous with it (e.g., an outbreak that occurred in were modelled using trade volumes from ). we were also constrained by data availability in our use of annual increments: although monthly data exist for outbreaks, they do not for important predictor variables such as percapita gdp, human and poultry populations, the volume of live poultry traded, and biosecurity. regressions results from all models, including both random and fixed effects, are reported in tables - . at the all-regions level, the results for the random-and fixed-effects models were very similar, with the same set of predictor variables being statistically significant (i.e., p-values below the % or %) and the same direction of impact on the response variable. this set of predictors was human population (positive direction), per-capita gdp (negative direction), intra-trade bloc live poultry imports (negative direction), extra-trade bloc live poultry imports (positive direction), and the biosecurity measure of surveillance (negative direction). additionally, although the coefficient values for the same predictor differed between the two estimators, all pairs were within the same order of magnitude. the only exception to this was migratory waterbird habitat variable-the percent of land area covered by ibas for migratory and congregatory waterbirds. this was statistically significant and negative (i.e., had a mitigating impact on h n poultry outbreaks) for the fixed-effects model but was not significant for the random-effects model. the overall r-squared for the random-effects model was significantly higher than that for the fixed-effects model ( . vs. . ). the "between rsquared" value was particularly high ( . ) in the random effects model, signaling the importance of variation among countries (as opposed to "within r-squared," which measures the variation within countries over time). as we had expected, we found significant differences across trade regions. in the randomeffects model, ecowas diverged from all-regions conditions and from asean with respect to per-capita gdp and extra-bloc imports: while the two predictors were, respectively, riskdecreasing and risk-increasing at the all-regions level and in asean, they had the opposite impacts in ecowas. furthermore, ecowas differed from the all-regions level and from the eu in terms of intra-bloc imports: while this was risk-decreasing for the former two, it was risk-increasing for ecowas. finally, there were predictors that were statistically insignificant at the all-regions level but had a significant effect within different regions. for asean, agricultural land cover was a mitigating factor for outbreaks while wild disease reservoir management showed a strong positive relation with outbreaks. for ecowas, wild waterbird habitats and border precautions had a mitigating effect on outbreaks while vaccination prohibition and wild reservoir management had a positive effect. in the eu, the population of live chickens had a strong negative relation table . results from the regression models of h n outbreak risk factors for member states in all three regions; regressor coefficients are reported and statistically-significant factors are marked by asterisks. a blank space signifies that the variable was not included in the given model. units model model model with outbreaks, while vaccination prohibition, similar to the case with ecowas, was positively related. following liang, xu ( ), there is a perception that the long distance transmission of highly pathogenic avian influenza h n was largely due to wild bird migration, with the live poultry trade playing a minor and more localized role in some cases. our concern here has been to identify the nature of the risk posed by the live poultry trade in different regions of the world, and the conditions affecting that risk. our measure of development status, per-capita gdp, is simultaneously a proxy for modernization, biosecurity, consumption, and value-at-risk. as a proxy for modernization, it reflects risk-reducing differences in production methods. industrial livestock production methods typically include on-farm biosecurity measures that protect poultry from contact with disease-carrying wild birds. unlike traditional methods of free-range or "backyard" husbandry, factory production minimizes the likelihood of poultry intermingling with wild birds or being exposed to environmental pathogen pollution. for all its epidemiological, ecological, and ethical problems, industrial livestock production allows for more timely and widespread disease surveillance and vaccination, and for greater compliance with animal health regulations [ ] . at the same time, per-capita gdp growth is also associated with risk-increasing changes in meat consumption, and hence poultry production. indeed, the highest income elasticity of demand for meat and fish has been found in the poorest households and the poorest countries [ ] . in developing countries, % of the additions to meat consumption are from pork and poultry, with poultry dominating pork [ ] . absent changes in on-farm biosecurity, increased table . results from the regression models of h n poultry outbreak risk factors for the association of southeast asian nations (asean); regressor coefficients are reported and statistically-significant factors are marked by. a blank space signifies that the variable was not included in the given model. units model model model production implies increased risk. across all regions, the net effect of income growth is to reduce risk, dominating risk-increasing changes. in the ecowas region-the lowest income region-the effect is the opposite. the risk-increasing effects of income growth dominate the risk reducing effects (table ) . amongst the landscape variables-land area, the proportion in agriculture, and the proportion in ibas-our results reveal no uniform relation to h n outbreaks. at the all-regions level we found a weakly negative relation between outbreaks and the proportion of the land area in ibas (table ). this was driven by the european union, which includes the highest proportion of land area in ibas, but also the most industrialized forms of poultry production. the degree to which poultry production is industrialized also shows up in the coefficients on poultry numbers, which are negative and significant only for the eu (table ). while spatial heterogeneity at the landscape scale is important in terms of avian ecology, we were unable to take explicit account of these more detailed considerations in a country-scale analysis. the impacts of regional differences in biophysical conditions that are not directly controlled for are, however, included in bloc-level fixed effects. our primary concern is with the role of the live poultry trade, and how that differs between regions. across all regions we find that live poultry imports into a trade bloc are risk increasing. this is consistent with past studies that have shown that extra-bloc live poultry imports may be a significant source of additional avian influenza risk where they do not meet bloc sanitary and phytosanitary standards. the eu's common market and the asean free trade regime in particular have long-standing and standardized protocols, in accordance with the world trade organization's agreement on the application of sanitary and phytosanitary measures. but the two blocs have quite different exposures to external risk. a study of highly pathogenic avian influenza introductions to vietnam, for example, found that extra-asean imports of table . results from the regression models of h n poultry outbreak risk factors for the economic community of west african states (ecowas); regressor coefficients are reported and statistically-significant factors are marked by asterisks. a blank space signifies that the variable was not included in the given model. units model model model population # people . x - . x - �� . x - . x - �� . live poultry increased the risk of introduction [ ] . this is also what our study finds for the asean region (table ) . we do not see an equivalent effect for the eu (table ), reflecting differences in both import volumes and the biosecurity measures applied to imports. the eu imports less and applies stricter biosecurity measures to those imports. the ecowas story is different. extra-bloc live poultry imports are risk reducing, not risk increasing (table ). it is likely that imports from outside the bloc reduce avian influenza risk in the region in part because they meet biosecurity standards that are more stringent than the standards applied in the region. the effects of intra-bloc trade in live poultry mirror the effects of extra-bloc trade. in the eu and asean, intra-bloc trade is risk reducing (tables and ). this may reflect a "substitution effect" in which imports of safer intra-bloc poultry crowds out riskier extra-bloc imports. other studies have come to similar conclusions. eu-derived live poultry imports to spain, for example, were found to pose no threat of avian influenza introduction [ ] . once again, eco-was is the exception. extra-ecowas imports of live poultry are risk reducing while intrabloc imports are risk increasing (table ). this is likely due to poor internal biosecurity, such as lax standards and inconsistent execution of inspections. regulatory standards within the ecowas trade bloc have been weak for the whole of the study period [ ] . while harmonized sanitary and phytosanitary standards for the member states of ecowas were in principle adopted in , most ecowas states had yet to submit legislation for international certification by [ ] . failure to adopt and enforce unified standards may be partly due to income constraints in ecowas countries. in ppp terms, the bloc's per-capita gdp in was less than half that of asean and approximately / th that of the eu, meaning it had less resources available for biosecurity policies and institutions. political instability may be another important obstacle: a table . results from the regression models of h n poultry outbreak risk factors for the european union (eu); regressor coefficients are reported and statistically-significant factors are marked by asterisks. a blank space signifies that the variable was not included in the given model. units model model model the live poultry trade poses different avian influenza risks in different regions of the world number of ecowas member states, including nigeria, niger, sierra leone, mali, liberia, and cote d'ivoire have suffered from civil wars and armed insurgencies over the past two decades. such fraught geopolitical conditions are not conducive to the establishment and enforcement of cross-border regulations. it goes without saying, though, that certification of sanitary and phytosanitary legislation in ecowas states, and the establishment of enforcement agencies to bring states into compliance with the sps agreement and codex alimentarius is a necessary condition of improving regional trade-related biosecurity. in terms of biosecurity measures more specifically, we did not have direct measures of onfarm biosecurity (but conjecture that biosecurity is increasing in per-capita gdp), but we did have measures of four biosecurity policies at the national level. these include: ( ) border precautions (measures applied at airports, ports, railway stations or road check-points open to international movement of animal, animal products and other related commodities, where import inspections are performed to prevent the introduction of the disease, infection or infestation); ( ) general surveillance (surveillance not targeted at a specific disease, infection or infestation); ( ) prohibition of vaccination (prohibition of the use of a vaccine to control or prevent the infection or infestation); and ( ) management of wildlife reservoirs (measures to reduce the potential for wildlife to transmit the disease to domestic animals and human beings). the management of wild disease reservoirs differs widely across countries, but techniques include vaccination, treatment of infections with drugs, isolation of infected populations, population translocation, reproduction reduction, culling, and control (draining, flooding, or burning) of wild disease reservoir habitat [ ] . of these measures, only general surveillance was significant at the all-regions level, while at the bloc level the effects of the different measures were frequently ambiguous. in the eu, for example, only the prohibition of vaccination was significant, and then in positive relation to outbreaks. for poultry, vaccination may be prohibited because the practice makes it difficult to distinguish infected from vaccinated flocks. this makes it a concomitant of policies centered on livestock culling as the primary response to outbreak risk [ ] . no other biosecurity policy was found to have a statistically significant relation to outbreaks in the region. the same set of policies had opposite effects in asean and ecowas. the prohibition of vaccination and the management of wild reservoirs were positively related to outbreaks in ecowas but negatively related to outbreaks in asean, while border protection measures were negatively related to outbreaks in ecowas but positively related to outbreaks in asean. this may reflect regional disparities in the quality of implementation not captured in the data. but it may also reflect the greater importance of trade in the transmission of the disease in ecowas. in their survey of the international spread of h n in the early years of the global epidemic, kilpatrick, chmura ( ) found that transmission into europe was by wild birds, that transmission into southeast asia was by the poultry trade, and transmission into africa by a balance of both. our results suggest that after introduction, inter-country spread had differing dynamics in each region. while intra-bloc trade facilitated h n spread among west african countries, it did not in either europe or southeast asia. in these areas, greater risk was posed by out-ofregion live poultry imports. in recent decades, avian influenzas have emerged as a major threat to human and animal health across the world. in particular, hpai h n , which was first isolated in , has been the most widespread and among the most devastating in terms of livestock and human mortality. it has inflicted severe losses to poultry stocks and caused hundreds of human deaths. even today, as other avian influenzas have become epidemic, h n remains in circulation among wildlife and livestock. identifying and quantifying the mechanisms of its international spread can help lay the groundwork for prediction and mitigation. it may also provide an instructive framework for the management of other avian influenzas. in this study, we considered the risk posed by the international trade in live poultry and the effects of associated biosecurity measures. differing agro-ecological and socioeconomic conditions across the trade regions were shown to influence epidemic dynamics in different ways, with certain factors being risk-enhancing or risk-decreasing in one region but having the opposite effect, or no significant effect, in another. in policy terms, there is no one-size-fits-all solution to mitigating avian influenza spread. the particular conditions, including those related to the trade agreements and associated regulatory standards, of a given region need to be carefully considered. but overall, biosecurity measures are potentially effective at controlling h n risks, and should be undertaken as a means to forestall spread-in general, mitigation of epidemics is significantly more cost-efficient than suppression [ ] . on-farm and other forms of domestic biosecurity may be more important than trade-related measures, but where the protection of trade pathways is weak, the risk of avian influenza spread is clearly higher. supporting information s file. detailed information on data sources. the public sources of the data used in this study, and how they were acquired, are described. (docx) world organization for animal health. oie situation report for highly pathogenic avian influenza: update: / world health organization. cumulative number of confirmed human cases for avian influenza a(h n ) reported to who food and agriculture organization. h n situation update predicting the global spread of h n avian influenza combining spatial-temporal and phylogenetic analysis approaches for improved understanding on global h n transmission exotic effects of capital accumulation options for managing the infectious animal and plant disease risks of international trade forecasting biological invasions with increasing international trade risk of importing zoonotic diseases through wildlife trade, united states globalization and livestock biosecurity global traffic and disease vector dispersal influences on the transport and establishment of exotic bird species: an analysis of the parrots (psittaciformes) of the world economic factors affecting vulnerability to biological invasions. the economics of biological invasions structural change in the international horticultural industry: some implications for plant health the emergence and evolution of swine viral diseases: to what extent have husbandry systems and global trade contributed to their distribution and diversity? animal movements and the spread of infectious diseases wildlife trade and global disease emergence. emerging infectious diseases ecology of zoonoses: natural and unnatural histories bats are natural reservoirs of sars-like coronaviruses global perspective for foot and mouth disease control a hotspot of non-native marine fishes: evidence for the aquarium trade as an invasion pathway reducing the risks of the wildlife trade the worldwide airline network and the dispersal of exotic species land-use and socio-economic correlates of plant invasions in european and north african countries global transport networks and infectious disease spread epidemiologic clues to sars origin in china. emerging infectious diseases influenza a h n immigration is filtered out at some international borders a statistical phylogeography of influenza a h n . proceedings of the national academy of transboundary animal diseases: assessment of socio-economic impacts and institutional responses the spread of pathogens through trade in poultry meat: overview and recent developments the spread of pathogens through trade in poultry hatching eggs: overview and recent developments. revue scientifique et technique de l'office international des epizooties emergency prevention system (empres) for transboundary animal and plant pests and diseases. the empres-livestock: an fao initiative persistence of highly pathogenic avian influenza h n virus defined by agro-ecological niche mapping h n highly pathogenic avian influenza risk in southeast asia first introduction of highly pathogenic h n avian influenza a viruses in wild and domestic birds in denmark, northern europe environmental factors influencing the spread of the highly pathogenic avian influenza h n virus in wild birds in dynamic patterns of avian and human influenza in east and southeast asia avian influenza viruses in water birds agro-ecological features of the introduction and spread of the highly pathogenic avian influenza (hpai) h n in northern nigeria multiple introductions of h n in nigeria avian influenza h n viral and bird migration networks in asia environmental factors contributing to the spread of h n avian influenza in mainland china spatial distribution and risk factors of highly pathogenic avian influenza (hpai) h n in china different environmental drivers of highly pathogenic avian influenza h n outbreaks in poultry and wild birds mapping spread and risk of avian influenza a (h n ) in china predicting the risk of avian influenza a h n infection in live-poultry markets across asia potential geographic distribution of the novel avian-origin influenza a (h n ) virus animal disease and the industrialization of agriculture assessing current and future meat and fish consumption in sub-sahara africa: learnings from fao food balance sheets and lsms household survey data. global food security rising consumption of meat and milk in developing countries has created a new food revolution. the journal of nutrition risk of introduction in northern vietnam of hpai viruses from china: description, patterns and drivers of illegal poultry trade. transboundary and emerging diseases a quantitative assessment of the risk for highly pathogenic avian influenza introduction into spain via legal trade of live poultry mycotoxins: detection methods, management, public health and agricultural trade: cabi regulation status of quarantine pests of rice seeds in the economic community of west african states (ecowas) training manual on wildlife diseases and surveillance. paris: world organization for animal health emerging biological threats: a reference guide: a reference guide: abc-clio economic optimization of a global strategy to address the pandemic threat we would like to thank ann kinzig, jim collins, ben minteer, and peter daszak for their insightful comments and discussions on the research presented here. conceptualization: tong wu, charles perrings. key: cord- -fie ns authors: white, michael; freistaedter, andrew; jones, gwendolyn j. b.; zervos, emmanuel; roper, rachel l. title: development of improved therapeutic mesothelin-based vaccines for pancreatic cancer date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: fie ns pancreatic cancer is the (th) leading cause of cancer deaths, and there are no effective treatments. we developed a poxvirus platform vaccine with improved immunogenicity and inserted the mesothelin gene to create an anti-mesothelin cancer vaccine. mesothelin expression is mostly restricted to tumors in adult mammals and thus may be a good target for cancer treatment. we show here that the modified vaccinia virus ankara (mva) virus expressing mesothelin and the enhanced mva virus missing the immunosuppressive a gene and expressing mesothelin were both safe in mice and were able to induce ifn-gamma secreting t cells in response to mesothelin expressing tumor cells. in addition, the mva virus has oncolytic properties in vitro as it can replicate in and kill panc pancreatic adenocarcinoma cell line tumor cells, even though it is unable to replicate in most mammalian cells. deletion of the a gene in mva improved t cell responses as expected. however, we were unable to demonstrate inhibition of panc tumor growth in immunocompetent mice with pre-vaccination of mice, boosts, or even intratumoral injections of the recombinant viruses. vaccine efficacy may be limited by shedding of mesothelin from tumor cells thus creating a protective screen from the immune system. pancreatic cancer is the th leading cause of cancer deaths in the united states [ ] and has the lowest -year survival rate ( %) for solid tumors [ ] largely owing to the fact that radiation, surgery, and current chemotherapy options are ineffective. new treatment strategies are essential. human mesothelin is normally expressed only in mesothelial cells of the pleura, peritoneum, and pericardium. however, mesothelin is overexpressed in a high percentage of ovarian cancers, pancreatic cancers, non-small lung cancers, and mesotheliomas, making it a potential target for anti-cancer treatments [ ] [ ] [ ] [ ] [ ] . human and mouse mesothelin share sequence similarity, expression patterns, and biochemical characteristics, [ ] , and the homeostatic function of mesothelin in mammals is unknown: the gene can be deleted without apparent effect in mice plos c bl mice and thus can be grown in syngeneic mice to allow for study of an anti-tumor immune response in an immunocompetent mouse model. this has a great advantage over tumor studies performed with xeno-or allografts in immunocompromised mice. to determine whether we can improve vaccine immunogenicity and efficacy, we test the parental normal mvameso virus vaccine against the mva mesothelin expressing virus that has had the immunosuppressive a gene removed (mvamesoa del). all animal experiments were conducted with the approval of the east carolina university animal care and use committee (k ) in an aaalac accredited animal facility. isoflurane anesthesia was used for tumor injections and for euthanasia. the c bl/ chemically-induced pancreatic adenocarcinoma cell line, panc , was a kind gift from dr. keping xie (md anderson cancer center, houston, tx). panc cells were grown in rpmi, with % fetal bovine serum, mm glutamine, u/ml of penicillin and streptomycin. mesothelin over expressing panc cell lines are described in zervos, et al [ ] . cells were incubated at ˚c with % co . vaccinia virus, mva, and the mva a deletion mutant viruses were previously described [ , ] . baby hamster kidney (bhk) cells were employed for growth of mva and were cultured using dulbecco's modified eagle medium (dmem), supplemented with % fetal bovine serum, mm glutamine, u/ml of penicillin and streptomycin. in order to create a therapeutic anti-mesothelin vaccine, we inserted mouse mesothelin into the mva viral genome. we used the normal wild type mva or the mva in which the a virulence gene had been deleted [ ] to create the mvameso virus and the mvamesoa del recombinant viruses respectively. in both cases, mesothelin was cloned into a vector (plw , a gift from l. wyatt, national institutes of health) designed for insertion into an area of the mva genome where genes had been naturally deleted during passage of the virus [ ] . recombinant viruses expressing the gfp marker protein were selected, purified, and analyzed to confirm the genotype by raid prep pcr analysis [ ] . the presence of the mesothelin gene was confirmed using mesothelin-specific primers (ggctggctatggctgtaagac and agcagtgt tggatgagtccatcgt) with an annealing temperature of ˚c. in order to assess the presence of the a gene, recombinant viruses were analyzed using a specific primers (tgacta taacagatacatt and cagattatctgaaggattgat) at an annealing temperature of ˚c. to confirm the insertion sites of the mesothelin, we used primers in the mva flanking regions, (cataagtataaagtccgactattgttc and gataaagttgcatcatcacctat). the pcr amplicons were run on an . % agarose gel. peptides representing murine mesothelin coding sequence, gvygfqvseadvralgglac and cppgkepykvdedlifyqn, were synthesized and conjugated to klh carrier proteins via the terminal cysteine residues (underlined, genemed synthesis), and used for immunization of two rabbits [ ] . sera were confirmed for reactivity with the immunizing peptides by elisa. bhk cells ( x cells/well) were uninfected or infected with the mva viruses at a multiplicity of infection (moi) of plaque forming units (pfu, infectious virus particles) of virus per cell for h in % co atmosphere. cells were lysed with disruption buffer and heat inactivated at ˚c for five minutes. dna was sheered using a -gauge needle and samples were loaded onto a pre-cast - % gradient gel (thermo scientific) for sds-page and transferred overnight. membrane was blocked for minutes at room temperature with % fat free milk in tbs, and incubated with rabbit anti-mesothelin antibody ( : , ) for h at room temperature, washed, and then incubated in anti-rabbit igg (fc) ap conjugate (promega), : at room temperature for one h, washed and developed with western blue stabilized substrate (promega). hek transected cells, previously described [ ] , were used as control. for analysis of released mesothelin, supernatants were collected from wells containing the normal or mesothelin over-expressing panc cells [ ] , uninfected bhk cells or cells infected with mva viruses. the cell supernatants were run on an sds-page gel, transferred to a pvdf membrane and probed with a rabbit anti-mesothelin antibody and an alkaline phosphatase conjugated ˚antibody as described above. female c bl/ mice (charles river, - weeks of age) were used for experiments. all mice were housed in the east carolina university department of comparative medicine aaalac accredited animal facility and kept in conventional conditions with full access to food and water throughout the study. all procedures were approved by the institutional animal care and use committee and in accordance with recommendations for proper care and use of laboratory animals. in order to assess the safety of injecting mva virus expressing mesothelin, we injected mice i.m. (n = ) with pbs, normal wild type mva, mvameso, or mvamesoa del ( . x pfu/mouse i.m. in ul, and boosted month later with . x pfu/mouse) and monitored mouse health daily by body score condition and weight up to days, similar to previous work [ ] . numbers of gamma interferon (ifn-gamma)-secreting spleen cells were enumerated similarly to methods described previously [ , ] . ninety-six-well plates (immulon h b; thermo electron) were coated overnight with purified rat anti-mouse ifn-gamma ( mg/ml; pharmingen) at ˚c. plates were washed with blocking buffer before adding a titration of murine splenocytes in rpmi medium. splenocytes were harvested from pbs, mva, mvameso, or mvame-soa del infected mice month after vaccination. the in vitro stimulation of splenocytes was achieved either by the addition of panc cells, wild type vaccinia virus (moi = ), or lewis lung cells, which do not express mouse mesothelin [ ] , followed by incubation for h at ˚c. plates were then washed (pbs with . % tween , . % sodium azide) and incubated with biotinylated rat anti-mouse ifn-gamma ( . mg/ml; pharmingen) for h at ˚c. plates were washed and incubated with streptavidin-ap for h at ˚c. plates were developed with an agarose-bcip ( -bromo- -chloro- -indolylphosphate)-amp mixture, and spots were counted by using a dissection microscope. to assess viral replication over time [ , ] , , cells were plated in a -well plate. bhk cells were used as a positive control since mva is known to replicate in them. bhk or panc cells were infected with mva at an moi of pfu/cell for h, then inocula were removed and the cells were washed once. supernatants and cells were harvested separately at six time points: , , , , , and h post-infection. these samples were frozen and thawed three times before being titered in bhk cells in -well plates [ ] . panc cells ( , cells/well) were plated in triplicate in a -well plate. mva was added to the wells at a multiplicity of infection (moi) of . and . pfu/cell. after h, ul -( , -dimethylthiazol- -yl)- -( carboxymethoxyphenyl)- -( -sulphophenyl)- h-tetrazolium, inner salt/phenazine methosulphate (mts/pms) ( . mg/ml mts, promega and . mg/ml pms, sigma) was added to each well, and the absorbance was read at nm [ ] . media only was used to define the background control level. we used the syngeneic heterotopic tumor model employing the murine pancreatic adenocarcinoma panc cell line [ ] . female c bl/ mice (charles river, - weeks of age, n = - per group) were injected s.c. in the right flank with panc cells ( . x ), one injection per mouse, under isoflurane ( %) inhalation anesthesia [ ] . tumor size was measured three times per week by a digital caliper, and volume was calculated as [(smallest diameter ) x largest diameter]/ . mice were humanely euthanized if they reach endpoints of body condition score, tumor size, ulceration, or impairment. for analysis of safety and immunogenicity, mice were injected with . x pfu/mouse i.m. in ul, and boosted month later with . x pfu/ mouse. for treatment of tumor bearing mice, mice were injected s.c. in the right flank with panc cells ( . x ), and then injected i.m. contralaterally with . x pfu/mouse ul times, on days , , after tumor cell injections. for pre-vaccination of mice, mice were injected i.m. with . x pfu/mouse, and then boosted with . x pfu/mouse, and challenged with panc tumor cells as above. for viral injections into tumors, tumors were allowed to grow, and viral injections ( . x pfu/mouse in ul) into tumors began when tumors were mm . experiments were repeated at least three times, and representative data are shown. a twotailed student's t test was used to compare groups. p values < . were considered significant. while tumor associated antigens, such as mesothelin, often do not induce a robust immune response, evidence suggests it is possible to generate an anti-tumor response by presenting the tumor antigen in an immunogenic context, such as by expressing the protein in a viral infection [ , ] . in order to create a putative therapeutic anti-mesothelin vaccine, we inserted the mouse mesothelin gene into the poxvirus mva genome under a viral promoter so that mesothelin would be expressed in any cells infected with the recombinant virus. we constructed both a normal wild type mva virus expressing mesothelin (mvameso) and a mesothelin-expressing mva virus in which the viral a immunosuppressive virulence gene had been deleted (mva-mesoa del) [ ] . the purified viruses were analyzed by pcr to confirm the presence of the mesothelin gene in the expected loci in the genomes and the presence or absence of the a gene. next, we wanted to confirm the expression of mesothelin protein from the recombinant viral genomes in infected cells. as shown in fig , mesothelin protein was detected as a broad band~ kda. mesothelin is glycosylated, so differences in glycosylation can be seen as different mw bands, and glycosylated forms may vary in different cell types [ ] . mesothelin protein was strongly expressed in both mva mesothelin virus-infected bhk cells, with monomers and apparent dimers (~ kda), but not in cells infected with mva parental wild type virus or uninfected bhk cells (fig ) . this indicated that the recombinant mesothelin expressing viruses were able to express significant quantities of mesothelin protein in infected cells. cells were lysed and samples were loaded onto a pre-cast - % gradient gel. rabbit anti-mesothelin antibody and anti-rabbit igg (fc) ap conjugate (promega) detected mesothelin protein (arrow). hek transected cells, previously described [ ] , were used as control. https://doi.org/ . /journal.pone. .g as a control, mesothelin was detected by western blot in hek cells transfected with a mesothelin expressing plasmid with a strong promoter [ ] , but not in hek cells transfected with the control plasmid vector. mesothelin could also be detected in the panc murine pancreatic adenocarcinoma cell line as we have shown previously by western blot and flow cytometry [ ] . while mesothelin is a tumor associated antigen that can be detected in panc cells grown in vitro or as a tumor grown in the mouse, it is not a highly abundant protein [ ] . multiple forms and sizes of cleaved mesothelin proteins have been reported to be released from cells expressing it [ , ] . we have previously shown that forms of the mesothelin protein are found in supernatants of panc cells, a carboxy terminal region released by furin cleavage and one derived from the amino terminus [ ] . we assessed what forms of mesothelin proteins are present in the supernatants of bhk cells infected with the mva viruses (wild type mva, mvameso, and mvamesoa del) and for comparison cultured panc cells overexpressing mesothelin [ ] . as seen in fig , several protein bands that react with rabbit anti mesothelin antisera were detected by western blot, with large quantities of released mesothelin apparent in the cells infected with the recombinant viruses encoding mesothelin. in order to assess the safety of injecting mva viruses expressing mesothelin, we injected mice i.m. (n = ) with pbs, wild type mva, mvameso, and mvamesoa del. mice were boosted month later. we monitored mouse health by body score condition daily and weight for days as shown in fig . there were no significant differences between the groups. we observed no adverse effects after the vaccination. this suggests that no significant anti-mesothelin autoimmune disease was induced in the mice. to determine whether the viruses expressing mesothelin protein were able to induce an immune response in mice, we first attempted to measure anti-mesothelin antibody in vaccinated mouse sera. we attempted to measure reactivity to panc cells, which express mesothelin on the surface by flow cytometry as we have done previously [ ] , however we got very low responses, possibly due to released mesothelin (fig ) binding the anti mesothelin antibodies in vivo. we then measured the frequency of anti-mesothelin ifn-gamma secreting t lymphocytes by elispot assay. mice vaccinated with pbs or mva had very few (< ) splenocytes that secreted ifn-gamma in response to panc cells (fig ) . mvameso vaccinated mice had an average of ifn-gamma secreting t cells per splenocytes, and mvamesoa del had an average of , significantly more (p< . ) than mva and pbs treated mice. in comparison, there were very few spots ( - ) in response to stimulation with lewis lung cells that do not express mouse mesothelin, and mice vaccinated with mva, mvameso and mvamesoa del viruses all had good responses to restimulation with vaccinia virus ( , , and spots respectively). these results indicate that while all mice responded to the virus infection, and the mesothelin expressing viruses induced an immune response in mice that reactivated in response to mesothelin expressing panc cells. additionally, vaccination with the mvame-soa del virus induced % more responding t lymphocytes compared to the mvameso virus, suggesting that removal of the a induced a stronger immune response, but the difference did not reach statistical significance. some oncolytic poxviruses have been shown to specifically target and replicate in tumor tissues in vivo [ ] because the tumor or microenvironment supports viral replication. we therefore wanted to assess if mva virus was able to infect and replicate in panc cells by a one-step viral growth curve [ ] . mva is unable to replicate in almost all mammalian cells: baby hamster kidney (bhk) cells are one known exception in which mva can replicate. bhk and panc cells were infected at an moi of pfu/cell so that each cell would be infected, and the supernatants and cells were harvested separately and titered for virus quantity over time. as shown in fig , over h the number of infectious mva virions (plaque forming units, pfu) increased in bhk cells more than logs. mva replication in panc cells also increased by more than logs, suggesting that the mva viruses might be effective oncolytic viruses in vivo since they can infect and replicate selectively in the panc tumor cells, while unable to replicate in normal mammalian tissues. viruses released into the supernatants also increased during mva infection of both cell lines. these results were promising, because they indicated that the virus could potentially amplify in situ and spread to cells that were not initially infected at the time of virus inoculation. since mva was able to replicate in panc cells, we wanted to assess whether mva was able to kill panc cells in vitro. panc cells were uninfected or infected with an moi of . or pfu/cell and compared to media with no cells. the infection was allowed to proceed for h, and mts reagent was added to measure cell metabolism as a measure of viability. to test the hypothesis that administration of mesothelin expressing viruses could induce an immune response and reduce mesothelin expressing tumor growth, we established tumors in mice [ ] and then treated with the mva mesothelin viruses. to test if the removal of the immunosuppressive a gene improved this therapeutic vaccine efficacy, we assessed the mvameso virus compared to the mvamesoa del virus. tumors were established by s.c. injection of tumors in one flank of the mice. at , , and days post tumor injection, mice , and survival (b) was not improved. we have previously described the construction of stable mesothelin over expressing panc cells and shown that tumors formed by these cells were significantly smaller than the tumors expressing wild type levels of mesothelin [ ] . we also tested whether the mesothelin expressing viruses might have greater efficacy against panc cells that expressed higher levels of the panc target. however, there was also no efficacy against the panc tumor cells expressing increased levels of panc protein, and the tumors continued to grow rapidly. since the panc tumors grow rapidly, we tested whether inducing an immune response to mesothelin prior to tumor induction would be protective against tumor growth. mice were injected i.m. with pbs, wild type mva, mvameso, or mvamesoa del, and boosted weeks later. two weeks after the boost, tumor cells were injected s.c. contralaterally in the flank. as shown in fig a, the prior vaccination with mvameso or mvamesoa del did not significantly decrease tumor growth or increase survival times. next, we attempted direct intratumoral injections since mva is able to replicate in and kill panc cells in vitro. we injected tumor cells s.c. and when tumors reached mm , we injected pbs, wild type mva, mvameso, or mvamesoa del directly into tumors times, days apart, however, we were unable to detect a benefit (fig b) . we also attempted to treat stable mesothelin overexpressing panc [ ] tumors by intratumoral injection, however there was no apparent effect, as tumors continued to grow and mice had to be euthanized. we have shown that murine mesothelin can be expressed from the mva virus genome and detected as a~ kda monomer protein on sds-page immunoblot, and a~ kda presumed dimer. mesothelin is released from cells and can be detected in the supernatants of cultured cells [ ] and in much higher quantities in cells infected with mva viruses that expresses mesothelin under a viral promoter. injection of the mesothelin expressing viruses into animals caused no apparent ill effect to at least months after vaccination, suggesting that mvameso vaccine is safe and does not initiate a deleterious autoimmune response. the mvameso vaccine induced a strong response to the mesothelin expressing panc tumor cell line, only slightly smaller than the amount of anti-vaccinia virus t lymphocytes induced by injection of vaccinia virus [ ] . these results indicate that the mesothelin protein was expressed in an immunogenic form by the virus in the infected mice. furthermore, mva virus (with and without mesothelin expression) was able to replicate in and kill panc tumor cells grown in vitro, suggesting that it would be an effective oncolytic virus in vivo. our previous results suggested that mesothelin might be a good target for control by an immune response [ ] , as mesothelin overexpression reliably caused a decrease in a heterotopic tumor growth in an immunocompetent syngeneic mouse model while in vitro proliferation was not inhibited [ ] . together, these results suggested that mvameso might be an effective treatment for pancc tumors, and that the mvamesoa del might be more effective given its improved immunogenic properties [ ] . however, we were unable to demonstrate any efficacy of the mesothelin mva vaccines prophylactically or therapeutically, even with multiple boosts or when injected directly into the tumors. one possibility is that the panc tumor cell line is simply too aggressive for control in an animal model where the tumor microenvironment may act to protect and promote tumor cell growth [ ] . while we had hoped that intratumoral injection would allow the virus to replicate in, spread, and kill tumor cells, we did not see a potent oncolytic effect in animals. perhaps while the mva virus can kill panc tumor cells in vitro, the anti-viral immune response in vivo limits the replication and spread of the virus. an ineffective anti-tumor immune response may also be caused by the shedding of mesothelin by the tumor in vivo (decoy mesothelin) blocking the immune effector antibodies and t cells. mesothelin is known to be detectable in supernatants of panc cells [ ] and in sera from patients with mesothelin expressing tumors [ , , ] . this shed soluble mesothelin may be taken up by other cells and bind anti-mesothelin antibodies rendering them ineffective. these problems may be worsened by administration of these mesothelin-expressing viruses because they also release soluble mesothelin at high levels. another possibility is that the shed mesothelin induces a state of anergy/tolerance in the animal, but our data show that mesothelin specific t lymphocytes are generated in these animals even though there is soluble circulating mesothelin shed from the mesothelin virus infected cells. perhaps new developments with cart cells [ ] may be able to more effectively facilitate t cell targeting and killing of cancerous cells. our data showed that both mvameso and mvamesoa del vaccines were able to generate a t lymphocyte response that was activated by the panc cells, and the mvamesoa del induced a stronger response, however it may be possible that the panc cells have developed mutations that protect them from effective cytotoxic t lymphocyte killing, even if t lymphocytes are appropriately expanded and targeted to recognize them. tumors that develop in humans and animals have already been highly selected in vivo to avoid the immune responses that control cancers. further improvements in these oncolytic therapeutic vaccines may be made by including cytokines such as il- and gmcsf, which have been reported to improve oncolytic potential of vaccinia virus and ultimately host survival [ , ] . in addition, depletion of t regulatory cells may counteract homeostatic or cancer cell driven immunosuppression and enhance the development of mesothelin-specific cd t cells capable of killing tumor cells [ , ] . although some results suggest that immune responses can be counter-productive: the anti mesothelin immune response may induce autoantibodies to the target antigen as well as other proteins by epitope spreading, and these antigen-spreading autoantibodies may cause increased damage to the animal. it was previously shown that antigen spreading was associated with a trend toward decreased survival in patients with prostate cancer treated with a recombinant viral vaccine [ ] . finally, given the difficulties with targeting an antigen like mesothelin that is shed, it may be more effective to target an antigen that is not shed. recent results targeting a different pancreatic tumor associated antigen, the oncofetal fucose-rich glycovariants of the pathological bile salt-dependent lipase, suggest it may be a superior tumor target antigen [ ] . united states cancer statistics: - incidence and mortality web-based report cancer statistics mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (sage) localization of mesothelin in epithelial ovarian cancer mesothelin is a malignant factor and therapeutic vaccine target for pancreatic cancer mesothelin expression in human lung cancer murine mesothelin: characterization, expression, and analysis of growth and tumorigenic effects in a murine model of pancreatic cancer mesothelin is not required for normal mouse development or reproduction sirna-mediated erc gene silencing suppresses tumor growth in tsc mutant renal carcinoma model mesothelin immunotherapy for cancer: ready for prime time? control of human mesothelin-expressing tumors by dna vaccines analysis of cloned fvs from a phage display library indicates that dna immunization can mimic antibody response generated by cell immunizations control of large, established tumor xenografts with genetically retargeted human t cells containing cd and cd domains identification of novel human ctl epitopes and their agonist epitopes of mesothelin mesothelin-specific cd (+) t cell responses provide evidence of in vivo cross-priming by antigen-presenting cells in vaccinated pancreatic cancer patients mesothelin-targeted immunotherapies for malignant pleural mesothelioma phase i study of ss p, a recombinant anti-mesothelin immunotoxin given as a bolus i.v. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers new high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma replicating poxviruses for human cancer therapy efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing ny-eso- antigen in ovarian cancer and melanoma patients raccoonpoxvirus safety in immunocompromised and pregnant mouse models the evolution of poxvirus vaccines serum antibodies to blood group a predict survival on prostvac-vf the poxvirus vectors mva and nyvac as gene delivery systems for vaccination against infectious diseases and cancer phase i trial of recombinant modified vaccinia ankara encoding epstein-barr viral tumor antigens in nasopharyngeal carcinoma patients genome sequence and comparative virulence of raccoonpox virus: the first north american poxvirus sequence vaccination with alvac and aidsvax to prevent hiv- infection in thailand identification of poxvirus cd + t cell determinants to enable rational design and characterization of smallpox vaccines the poxvirus a protein is an immunoregulator poxvirus safety analysis in the pregnant mouse model, vaccinia and raccoonpox viruses deletion of the a gene from modified vaccinia virus ankara increases immunogenicity and isotype switching clinical development of modified vaccinia virus ankara vaccines modified vaccinia virus ankara-based vaccine vectors induce apoptosis in dendritic cells draining from the skin via both the extrinsic and intrinsic caspase pathways, preventing efficient antigen presentation assessment of the protective effect of imvamune and acam vaccines against aerosolized monkeypox virus in cynomolgus macaques clonal vaccinia virus grown in cell culture as a new smallpox vaccine epithelial immunization induces polyfunctional cd + t cells and optimal mousepox protection immunogenicity of a highly attenuated mva smallpox vaccine and protection against monkeypox shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses the evolving role of immunotherapy in prostate cancer prostvac-vf: a vector-based vaccine targeting psa in prostate cancer vaccinia virus a r inhibits mhc class ii antigen presentation vaccinia virus decreases mhc class ii antigen presentation, t cell priming, and peptide association with mhc class ii antigen presentation assays to investigate uncharacterized immunoregulatory genes severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice rapid preparation of vaccinia virus dna template for analysis and cloning by pcr clin cancer res. a viral vaccine encoding prostate-specific antigen induces antigen spreading to a common set of self-proteins in prostate cancer patients characterization of the vaccinia virus a r protein and its role in virulence establishment of the enzymelinked immunosorbent assay system to detect the amino terminal secretory form of rat erc/mesothelin establishment of a novel specific elisa system for rat n-and c-erc/mesothelin. rat erc/mesothelin in the body fluids of mice bearing mesothelioma gmcsf-armed vaccinia virus induces an antitumor immune response effective depletion of regulatory t cells allows the recruitment of mesothelin-specific cd t cells to the antitumor immune response against a mesothelinexpressing mouse pancreatic adenocarcinoma an iscom vaccine combined with a tlr agonist breaks immune evasion mediated by regulatory t cells in an orthotopic model of pancreatic carcinoma a pancreatic tumorspecific biomarker characterized in humans and mice as an immunogenic onco-glycoprotein is efficient in dendritic cell vaccination the authors wish to acknowledge the expert technical help of melinda carver. key: cord- -u apzw authors: michael, edwin; sharma, swarnali; smith, morgan e.; touloupou, panayiota; giardina, federica; prada, joaquin m.; stolk, wilma a.; hollingsworth, deirdre; de vlas, sake j. title: quantifying the value of surveillance data for improving model predictions of lymphatic filariasis elimination date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: u apzw background: mathematical models are increasingly being used to evaluate strategies aiming to achieve the control or elimination of parasitic diseases. recently, owing to growing realization that process-oriented models are useful for ecological forecasts only if the biological processes are well defined, attention has focused on data assimilation as a means to improve the predictive performance of these models. methodology and principal findings: we report on the development of an analytical framework to quantify the relative values of various longitudinal infection surveillance data collected in field sites undergoing mass drug administrations (mdas) for calibrating three lymphatic filariasis (lf) models (epifil, lymfasim, and transfil), and for improving their predictions of the required durations of drug interventions to achieve parasite elimination in endemic populations. the relative information contribution of site-specific data collected at the time points proposed by the who monitoring framework was evaluated using model-data updating procedures, and via calculations of the shannon information index and weighted variances from the probability distributions of the estimated timelines to parasite extinction made by each model. results show that data-informed models provided more precise forecasts of elimination timelines in each site compared to model-only simulations. data streams that included year post-mda microfilariae (mf) survey data, however, reduced each model’s uncertainty most compared to data streams containing only baseline and/or post-mda or longer-term mf survey data irrespective of mda coverage, suggesting that data up to this monitoring point may be optimal for informing the present lf models. we show that the improvements observed in the predictive performance of the best data-informed models may be a function of temporal changes in inter-parameter interactions. such best data-informed models may also produce more accurate predictions of the durations of drug interventions required to achieve parasite elimination. significance: knowledge of relative information contributions of model only versus data-informed models is valuable for improving the usefulness of lf model predictions in management decision making, learning system dynamics, and for supporting the design of parasite monitoring programmes. the present results further pinpoint the crucial need for longitudinal infection surveillance data for enhancing the precision and accuracy of model predictions of the intervention durations required to achieve parasite elimination in an endemic location. we report on the development of an analytical framework to quantify the relative values of various longitudinal infection surveillance data collected in field sites undergoing mass drug administrations (mdas) for calibrating three lymphatic filariasis (lf) models (epifil, lym-fasim, and transfil), and for improving their predictions of the required durations of drug interventions to achieve parasite elimination in endemic populations. the relative information contribution of site-specific data collected at the time points proposed by the who monitoring framework was evaluated using model-data updating procedures, and via calculations of the shannon information index and weighted variances from the probability distributions of the estimated timelines to parasite extinction made by each model. results show that data-informed models provided more precise forecasts of elimination timelines in each site compared to model-only simulations. data streams that included year post-mda microfilariae (mf) survey data, however, reduced each model's uncertainty most compared to data streams containing only baseline and/or post-mda or longer-term mf survey data irrespective of mda coverage, suggesting that data up to this monitoring point may be optimal for informing the present lf models. we show that the improvements observed in the predictive performance of the best data-informed models may be a function of temporal changes in inter-parameter interactions. such best data-informed models may also produce plos mathematical models of parasite transmission, via their capacity for producing dynamical forecasts or predictions of the likely future states of an infection system, offer an important tool for guiding the development and evaluation of strategies aiming to control or eliminate infectious diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the power of these numerical simulation tools is based uniquely on their ability to appropriately incorporate the underlying nonlinear and multivariate processes of pathogen transmission in order to facilitate plausible predictions outside the range of conditions at which these processes are either directly observed or quantified [ ] [ ] [ ] [ ] . the value of these tools for guiding policy and management decisions by providing comparative predictions of the outcomes of various strategies for achieving the control or elimination of the major neglected tropical diseases (ntds) has been highlighted in a series of recent publications [ , , ] , demonstrating the crucial role these quantitative tools are beginning to play in advancing policy options for these diseases. while these developments underscore the utility of transmission models for supporting policy development in parasite control, a growing realization is that these models can be useful for this purpose only if the biological processes are well defined and demographic and environmental stochasticity are either well-characterized or unimportant for meeting the goal of the policy modelling exercise [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this is because the realized predictability of any model for a system depends on the initial conditions, parameterizations and process equations that are utilized in its simulation such that model outcomes are strongly sensitive to the choice of values used for these variables [ ] . any misspecification of these system attributes will lead to failure in accurately forecasting the future behaviour of a system, with predictions of actual future states becoming highly uncertain even when the exact representation of the underlying deterministic process is well established but precise specification of initial conditions or forcing and/or parameter values is difficult to achieve [ , ] . this problem becomes even more intractable when theoretical models depend on parameter estimates taken from other studies [ , , ] . both these challenges, viz. sensitivity to forcing conditions and use of parameter estimates from settings that are different from the dynamical environment in which a model will be used for simulation, imply that strong limits will be imposed on the realized predictability of any given model for an application [ , , ] . as we have shown recently, if such uncertainties are ignored, the ability of parasite transmission models to form the scientific basis for management decisions can be severely undermined, especially when predictions are required over long time frames and across heterogeneous geographic locations [ , , ] . these inherent difficulties with using an idealized model for producing predictions to guide management have led to consideration of data-driven modelling procedures that allow the use of information contained within observations to improve specification and hence the predictive performance of process-based models [ , , , [ ] [ ] [ ] . such approaches, termed model-data fusion or data assimilation methods, act by combining models with various data streams (including observations made at different spatial or temporal scales) in a statistically rigorous way to inform initial conditions, constrain model parameters and system states, and quantify model errors. the result is the discovery of models that can more adequately capture the prevailing system dynamics in a site, an outcome which in turn has been shown to result in the making of significantly improved predictions for management decision making [ , , , ] . initially used in geophysics and weather forecasting, these methods are also beginning to be applied in ecological modelling, including more recently in the case of infectious disease modelling [ , ] . in the latter case, the approach has shown that it can reliably constrain a disease transmission model during simulation to yield results that approximate epidemiological reality as closely as possible, and as a consequence improve the accuracy of forecasts of the response of a pathogen system exposed to various control efforts [ - , , - ] . more recently, attention has also focused on the notion that a model essentially represents a conditional proposition, i.e. that running a model in a predictive mode presupposes that the driving forces of the system will remain within the bounds of the model conceptualization or specification [ ] . if these driving forces were to change, then it follows that even a model well-calibrated to a given historical dataset will fail. new developments in longitudinal data assimilation can mitigate this problem of potential time variation of parameters via the recursive adjustment of the model by assimilation of data obtained through time [ , , ] . apart from allowing assessment of whether stasis bias may occur in model predictions, such sequential model calibration with time-varying data can also be useful for quantifying the utility of the next measurement in maximizing the information gained from all measurements together [ ] . carrying out such longitudinal model-data analysis has thus the potential for providing information to improve the efficiency and cost-effectiveness of data monitoring campaigns [ , [ ] [ ] [ ] , along with facilitating more reliable model forecasts. a key question, however, is evaluating which longitudinal data streams provide the most information to improve model performance [ ] . indeed, it is possible that from a modelling perspective using more data may not always lead to a better-constrained model [ ] . this suggests that addressing this question is not only relevant to model developers, who need observational data to improve, constrain, and test models, but also for disease managers working on the design of disease surveillance plans. at a more philosophical level, we contend that these questions have implications for how current longitudinal monitoring data from parasite control programmes can best be exploited both scientifically and in management [ ] . specifically, we suggest that these surveillance data need to be analysed using models in a manner that allows the extraction of maximal information about the monitored dynamical systems so that this can be used to better guide both the collection of such data as well as the provision of more precise estimates of the system state for use in making state-dependent decisions [ , [ ] [ ] [ ] . currently, parasite control programmes use infection monitoring data largely from sentinel sites primarily to determine if an often arbitrarily set target is met [ ] . little consideration is given to whether these data could also be used to learn about the underlying transmission dynamics of the parasitic system, or how such learning can be effectively used by management to make better decisions regarding the interventions required in a setting to meet stated goals [ , ] . here, we develop an analytical framework to investigate the value of using longitudinal lf infection data for improving predictions of the durations of drug interventions required for achieving lf elimination by coupling data collected during mass drug interventions (mdas) carried out in three example field sites to three existing state-of-the-art lymphatic filariasis (lf) models [ , , , [ ] [ ] [ ] [ ] [ ] [ ] . to be managerially relevant to current who-specified lf intervention surveillance efforts, we evaluated the usefulness of infection data collected in these sites at the time points proposed by the who monitoring framework in carrying out the present assessment [ ] . this was specifically performed by ranking these different infection surveillance data streams according to the incremental information gain that each stream provided for reducing the prediction uncertainty of each model. longitudinal pre-and post-infection and mda data from representative sites located in each of the three major regions endemic for lf (africa, india, and papua new guinea (png)) were assembled from the published literature for use in constraining the lf models employed in this study. the three sites (kirare, tanzania, alagramam, india, and peneng, png) were selected on the basis that each represents the average endemic transmission conditions (average level of infection, transmitting mosquito genus) of each of these three major extant lf regions, while providing details on the required model inputs and data for conducting this study. these data inputs encompassed information on the annual biting rate (abr) and dominant mosquito genus, as well as mda intervention details, including the relevant drug regimen, time and population coverage of mda, and times and results of the conducted microfilaria (mf) prevalence surveys (table ) . note each site also provided these infection and mda data at the time points pertinent to the existing who guidelines for conducting lf monitoring surveys during a mda programme [ ] , which additionally, as pointed out above, allowed the assessment of the value of such infection data both for supporting effective model calibration and for producing more reliable intervention forecasts. the three existing lf models employed for this study included epifil, a deterministic monte carlo population-based model, and lymfasim and transfil, which are both stochastic, individual-based models. all three models simulate lf transmission in a population by accounting for key biological and intervention processes such as impacts of vector density, the life cycle of the parasite, age-dependent exposure, density-dependent transmission processes, infection aggregation, and the effects of drug treatments as well as vector control [ , , - , , , ] . although the three models structurally follow a basic coupled immigration-death model formulation, they differ in implementation (e.g. from individual to population-based), the total number of parameters included, and the way biological and intervention processes are mathematically incorporated and parameterized. the three models have been compared in recent work [ , ] , with full details of the implementation and simulation procedures for each individual model also described [ , , , , , , , , ] . individual model parameters and fitting procedures specific to this work are given in detail in s supplementary information. we used longitudinal data assimilation methods to sequentially calibrate the three lf models with the investigated surveillance data such that parameter estimates and model predictions reflect not only the information contained in the baseline but also follow-up data points. the available mf prevalence data from each site were arranged into four different temporal data streams to imitate the current who guidelines regarding the time points for conducting monitoring surveys during an mda programme. this protocol proposes that infection data be collected in sentinel sites before the first round of mda to establish baseline conditions, no sooner than months following the third round of mda, and no sooner than months following the fifth mda to assess whether transmission has been interrupted (defined as reduction of mf prevalence to below % in a population) [ , ] . thus, the four data streams considered for investigating the value of information gained from each survey were respectively: scenario -baseline mf prevalence data only, scenario -baseline and post-mda mf prevalence data, scenario -baseline, post-mda , and post-mda mf prevalence data, and scenario -baseline and post-mda mf prevalence data. in addition to these four data streams, a fifth model-only scenario (scenario ) was also considered where no site-specific data was introduced. in this case, simulations of interventions were performed using only model-specific parameter and abr priors estimated for each region. the first step for all models during the data assimilation exercises reported here was to initially simulate the baseline infection conditions in each site using a large number of samples ( , for epifil and transfil, and , - , for lymfasim) randomly selected from the parameter priors deployed by each model. the number of parameters which were left free to be fitted to these data by each model range from (lymfasim and transfil) to (epifil). the abr, a key transmission parameter in all three models, was also left as a free parameter whose distribution was influenced by the observed abr (table ) and/or by fits to previous region-specific datasets (see s supplementary information for model-specific implementations). the subsequent steps used to incorporate longitudinal infection data into the model calibration procedure varied among the models, but in all cases the goodness-of-fit of the model outputs for the site-specific mf prevalence data was assessed using the chi-square metric (α = . ) [ ] . epifil used a sequential model updating procedure to iteratively modify the parameters with the introduction of each subsequent follow up data point through time [ ] . this process uses parameter estimates from model fits to previous data as priors for the simulation of the next data which are successively updated with the introduction of each new observation, thus providing a flexible framework by which to constrain a model using newly available data. fig summarizes the iterative algorithm used for conducting this sequential model-data assimilation exercise [ ] . lymfasim and transfil, by contrast, included all the data in each investigated stream together for selecting the best-fitting models for each time series-i.e. model selection for each data series was based on using all relevant observations simultaneously in the fitting process [ , , ] . although a limitation of this batch estimation approach is that the posterior probability of each model is fixed for the whole simulation period, unlike the case in sequential data assimilation where a restricted set of parameters is exposed to each observation (as a result of parameter constraining by data used in the previous time step)which thereby yields models that give better predictions for different portions of the underlying temporal process-here we use both methods to include and assess the impact that this implementation difference may have on the results presented below. for all models, the final updated parameter estimates from each data stream were used to simulate the impact of observed mda rounds and for predicting the impact of continued mda to estimate how many years were required to achieve % mf prevalence. interventions were modelled by using the updated parameter vectors or models selected from each scenario for simulating the impact of the reported as well as hypothetical future mda rounds on the number of years required to reduce the observed baseline lf prevalence in each site to below the who transmission threshold of % mf prevalence [ ] . when simulating these interventions, the observed mda times, regimens, and coverages followed in each site were used (table ) , while mda was assumed to target all residents aged years and above. for making mf prevalence forecasts beyond the observations made in each site, mda simulations were extended for a total of annual rounds in each site at an assumed coverage of %. while the drug-induced mf kill rate and the duration of adult worm sterilization were fixed among the models (table ) , the worm kill rate was left as a free parameter to be estimated from post-intervention data to account for the uncertainty in this drug efficacy parameter [ , , ] . the number of years of mda required to achieve the threshold of % mf prevalence was calculated from model forecasts of changes in mf prevalence due to mda for each modeldata fusion scenario. the predictions from each model regarding timelines to achieve % mf for each fitting scenario were used to determine the information gained from each data stream compared to the in all scenarios, the initial epifil models were initialized with parameter priors and a chi-square fitting criterion was applied to select those models which represent the baseline mf prevalence data sufficiently well (α = . ). the accepted models were then used to simulate the impact of interventions on mf prevalence. the chi-square fitting criterion was sequentially applied to refine the selection of models according to the post-mda mf prevalence data included in the fitting scenario. the fitted parameters from selection of acceptable models at each data point were used to predict timelines to achieve % mf prevalence. the scenarios noted in the blue boxes indicate the final relevant updating step before using the fitted parameters to predict timelines to achieve % mf in that data fitting scenario. information attributable to the model itself [ , , ] . the relative information gained from a particular data stream was calculated as i d = h m -h md where h measures the entropy or uncertainty associated with a random variable, h m denotes predictions from the model-only scenario (scenario ) which essentially represents the impact of prior knowledge of the system, and h md signifies predictions from each of the four model-data scenarios (i.e. scenarios [ ] [ ] [ ] [ ] . the values of i d for each data scenario or stream were compared in a site to infer which survey data are most useful for reducing model uncertainty. the shannon information index was used to measure entropy, h, as follows: is the discrete probability density function (pdf) of the number of years of mda predicted by each fitted model to reach % mf, and is estimated from a histogram of the respective model predictions for m bins (of equal width in the range between the minimum and maximum values of the pdfs) [ , ] . to statistically compare two entropy values, a permutation test using the differential shannon entropy (dse) was performed [ ] . dse is defined as |h -h | where h was calculated from the distribution of timelines to achieve % mf for a given scenario, y , and h was calculated from the distribution of timelines to achieve % mf for a different scenario, y . the list of elements in y and y were combined into a single list of size y + y and the list was permuted , times. dse was then recalculated each time by calculating a new h from the first y elements and a new h from the last y elements from each permutation, from which p-values may be quantified as the proportion of all recalculated dses that were greater than the original dse. model predictions of the mean and variance in timelines to lf elimination were weighted according to the frequencies by which predictions occurred in a group of simulations. in general, if d , d ,. . .,d n are data points (model predictions in the present case) that occur in an ensemble of simulations with different weights or frequencies w ,w ,. . .,w n , then the weighted mean, here, n is the number of data points and n is the number of non-zero weights. in this study, the weighted variance of the distributions of predicted timelines to achieve % mf prevalence was calculated to provide a measure of the precision of model predictions in addition to the entropy measure, h. a similar weighting scheme was also used to pool the timeline predictions of all three models. here, predictions made by each of the three models for each data scenario were weighted as above, and a composite weighted % percentile interval for the pooled predictions was calculated for each data stream. this was done by first computing the weighted percentiles for the combined model simulations from which the pooled . th and . th percentile values were quantified. the matlab function, wprctile, was used to carry out this calculation. the extent by which parameter constraints are achieved through the coupling of models with data was evaluated to determine if improvements in such constraints by the use of additional data may lead to reduced model prediction uncertainty [ ] . parameter constraint was calculated as the ratio of the mean standard deviation of all fitted parameter distributions to the mean standard deviation of all prior parameter distributions. a ratio of less than one indicates the fitted parameter space is more constrained than the prior parameter space [ ] . this assessment was carried out using the epifil model only. in addition, pairwise parameter correlations were also evaluated to assess whether the sign, magnitude, and significance of these correlations changed by scenario to determine if using additional data might alter these interactions to better constrain a model. for this assessment, spearman's correlation coefficients and p-values testing the hypothesis of no correlation against the alternative of correlation were calculated, and the exercise was run using the estimated parameters from the epifil model. epifil was used to conduct a sensitivity analysis investigating whether the trend in relative information gained by coupling the model with longitudinal data was dependent on the interventions simulated. the same series of simulations (for three lf endemic sites and five fitting scenarios) were completed with the extended mda coverage beyond the observations given in table set here at % instead of % to represent an optimal control strategy. as before, the timelines to reach % mf prevalence in each fitting scenario were calculated and used to determine which data stream provided the model with the greatest gain of information. the results were compared to the original series of simulations to assess whether the trends are robust to changes in the intervention coverages simulated. epifil was also used to perform another sensitivity analysis expanding the number of data streams to investigate if the who monitoring scheme is adequate for informing the making of reliable model-based predictions of timelines for achieving lf elimination. to perform this sensitivity analysis, pre-and post-mda data from villupuram district, india that provide extended data points (viz. scenario - as previously defined, plus scenario -baseline, post-mda , post-mda , and post-mda mf prevalence data, and scenario -baseline, post-mda , post-mda , post-mda , and post-mda mf prevalence) were assembled from the published literature [ , ] . the timelines to reach % mf prevalence and the entropy for each of these additional scenarios were calculated to determine whether additional data streams over those recommended by who are required for achieving more reliable model constraints, which among these data might be considered as compulsory, and which might be optional for supporting predictions of elimination. differences in predicted medians, weighted variances and entropy values between data scenarios, models and sites were statistically evaluated using kruskall-wallis tests for equal medians, f-tests for equality of variance, and dse permutation tests, respectively. p-values for assessing significance for all pairwise tests were obtained using the benjamini-hochberg procedure for controlling the false discovery rate, i.e. for protecting against the likelihood of obtaining false positive results when carrying out multiple testing [ ] . here, our goal was twofold. first, to determine if data are required to improve the predictability of intervention forecasts by the present lf models in comparison with the use of theoretical models only, and second, to evaluate the benefit of using different longitudinal streams of mf survey data for calibrating the three models in order to determine which data stream was most informative for reducing the uncertainty in model predictions in a site. table summarises the key results from our investigation of these questions: these are the number of accepted best-fitting models for each data stream or scenario in the three study sites (table ) , the predicted median and range ( . th - . th percentiles) in years to achieve the mf threshold of % mf prevalence, the weighted variance and entropy values based on these predictions, and the relative information gained (in terms of reduced prediction uncertainty) by the use of longitudinal data for constraining the projections of each of the three lf models investigated. even though the number of selected best-fit models based on the chi-square criterion (see methods) differed for each site and model, these results indicate unequivocally that models constrained by data provided significantly more precise intervention predictions compared to model-only predictions ( table ). note that this was also irrespective of the two types of longitudinal data assimilation procedures (sequential vs. simultaneous) used by the different models in this study. thus, for all models and sites, model-only predictions made in the absence of data (scenario ) showed the highest prediction uncertainty, highlighting the need for data to improve the predictive performance of the present models. the relative information gained by using each data stream in comparison to model-only predictions further support this finding, with the best gains in reducing model prediction uncertainty provided by those data constraining scenarios that gave the lowest weighted variance and entropy values; as much as % to % reductions in prediction variance were achieved by these scenarios in comparison to modelonly predictions between the three models ( table ). the results also show, however, that data streams including post-mda mf survey data (scenarios and ) reduced model uncertainty (based on both the variance and entropy measures) most compared to data streams containing only baseline and/or post-mda mf survey data (scenarios and ) ( table ) . although there were differences between the three models (due to implementation differences either in how the models are run (monte carlo deterministic vs. individual-based) or in relation to how the present data were assimilated (see above)), overall, scenario , which includes baseline, post-mda , and post-mda data, was shown to most often reduce model uncertainty the greatest. additionally, there was no statistical difference between the performances of scenarios and in those cases where scenario resulted in the greatest gain of information (table ) . it is also noticeable that the best constraining data stream for each combination of site and model also produced as expected the lowest range in predictions of the numbers of years of annual mda required to achieve the % mf prevalence in each site, with the widest ranges estimated for model-only predictions (scenario ) and the shorter data streams (scenario ). in general, this constriction in predictions also led to lower estimates of the median times to achieve lf elimination, although this varied between models and sites ( table ) . the change in the distributions of predicted timelines to lf elimination without and with model constraining by the different longitudinal data streams is illustrated in fig for the kirare site (see s supplementary information for results obtained for the other two study villages investigated here). the results illustrate that both the location and length of the tail of the prediction distributions can change as models are constrained with increasing lengths of longitudinal data, with inclusion of post-mda mf survey data consistently producing a narrower or sharper range of predictions compared to when this survey point is excluded. fig compares the uncertainty in predictions of timelines to achieve elimination made by each of the three models without (scenario ) and via their constraining by the data streams providing the lowest prediction entropy for each of the models per site. note that variations in scenario predictions among the three models directly reflect the different model structures, parameterizations, and the presence (or absence) of stochastic elements. the boxplots in the figure, however, show that for all three sites and models, calibration of each model by data the lowest entropy scenario for each site is bolded and shaded grey. additional scenarios shaded grey are not significantly different from the lowest entropy scenario. data assimilation in filarial model predictions greatly reduces the uncertainty in predictions of the years of annual mda required to eliminate lf compared to model-only predictions, with the data streams producing the lowest entropy for simulations in each site significantly improving the precision of these predictions ( table ). this gain in precision, and thus the information gained using these data streams, is, as expected, greater for the stochastic lymfasim and transfil models compared to the deterministic epifil model. note also that even though the ranges in predictions of the annual mda years required to eliminate lf by the data streams providing the lowest prediction entropy differed statistically between the three models, the values overlapped markedly (e.g. for kirare the ranges are - , - , - for epifil, lymfasim and transfil data assimilation in filarial model predictions respectively), suggesting the occurrence of a similar constraining of predictive behaviour among the three models. to investigate this potential for a differential model effect, we further pooled the predictions from all three models for all the data scenarios and evaluated the value of each investigated data stream for improving their combined predictive ability. the weighted % percentile intervals from the pooled predictions were used for carrying out this assessment. the results are depicted in fig and indicate that, as for the individual model predictions, uncertainty in the collective predictions by the three lf models for the required number of years to eliminate lf using annual mda in each site may be reduced by model calibration to data, with the longitudinal mf prevalence data collected during the later monitoring periods (scenarios and ) contributing most to improving the multi-model predictions for each site. the boxplots show that by calibrating the models to data streams, more precise predictions are able to be made regarding timelines to achieve % mf prevalence across all models and sites. the results of pairwise f-tests for variance, performed to compare the weighted variance in timelines to achieve % mf prevalence between model-only simulations (scenario ) and the lowest entropy simulations (best scenario) (see table ), show that the predictions for the best scenarios are significantly different from the predictions for the model-only simulations. significance was determined using the benjamini-hochberg procedure for controlling the false discovery rate (q = . ). for epifil, lymfasim and transfil, the best scenarios are scenarios , , and for kirare, scenarios , , and for alagramam, and scenarios , , and for peneng, respectively. we attempted to investigate if model uncertainty in predictions by the use of longitudinal data was a direct function of parameter constraining by the addition of data. given the similarity in outcomes of each model, we remark on the results from the fits of the technically easier to run epifil model to evaluate this possibility here. the assessment of the parameter space constraint achieved through the inclusion of data was made by determining if the fitted parameter distributions for the model became reduced in comparison with priors as data streams were added to the system [ ] . the exercise showed that the size of the estimated parameter distributions reduced with addition of data, with even scenario data producing reductions for kirare and peneng (fig ) . in the case of alagramam, however, there was very little, if any, constraint in the fitted parameter space compared to the prior parameter space. this result, together with the fact that even using all the data in kirare and peneng produced up to only between . to % reductions in fitted parameter distributions when compared to the priors, indicate that the observed model prediction uncertainty in this study may be due to other complex factors connected with model parameterization. table provides the results of an analysis of pairwise parameter correlations of the selected best-fitting models for data scenario compared to those selected by the data stream that gave the best reduction in epifil prediction uncertainty for alagramam (scenario ). these results show that while the parameter space was not being constrained with the addition of more data, the pattern of parameter correlations changed in a complex manner between the two constraining data sets. for example, although the number of significantly correlated parameters did not differ, the magnitude and direction of parameter correlations were shown to change between the two data scenarios ( table ) . the corresponding results for kirare and peneng are shown in s supplementary information , and indicate that a broadly similar pattern of changes in parameter associations also occurred as a result of model calibration to the sequential data measured from those sites. this suggests that this outcome may constitute a general phenomenon at least with regards to the sequential constraining of epifil using longitudinal mf prevalence data. an intriguing finding (from all three data settings) is that the most sensitive parameters in this regard, i.e. with respect to altered strengths in pairwise parameter correlations, may be those representing the relationship of various components of host immunity with different transmission processes, including with adult worm mortality, rates of production and survival of mf, larval development rates in the mosquito vector and infection aggregation (table ) . this suggests that, as more constraining data are added, changes in the multidimensional parameter relationships related to host immunity could contribute to the sequential reductions in the lf model predictive uncertainty observed in this study. the lf elimination timeline predictions used above were based on modelling the impacts of annual mda given the reported coverages in each site followed by an assumed standard coverage for making longer term predictions (see methods). this raises the question as to whether the differences detected in the case of the best constraining data stream between the present study sites and between models ( table ) could be a function of the simulated mda coverages in each site. to investigate this possibility, we used epifil to model the outcome of changing the assumed mda coverage in each site on the corresponding entropy and information gain trends in elimination predictions made from the models calibrated to each of the site-specific data scenarios/streams investigated here. the results of increasing the assumed coverage of mda to % for each site are shown in fig and indicate that the choice of mda coverage in this study are unlikely to have significantly influenced the conclusion made above that the best performing data streams for reducing model uncertainty for predicting lf elimination pertains to data scenarios and . however, while the model-predicted timelines to achieve the % mf prevalence threshold using the observed mda coverage followed by % mda coverage showed that the data stream which most reduced uncertainty did not change from the impact of using the observed mda coverage followed by % mda coverage modelled for kirare and peneng (table , fig ) , this was not the case for alagramam, where data from scenario with a % coverage resulted in the greatest reduction in entropy compared to the original results using % coverage which indicated that scenario data performed best (table , fig ) . notably, though, the entropy values of predictions using the data scenario and constraints were not statistically different for this site (p-value < . ) (fig ) . epifil was also used to expand the number of calibration scenarios using a dataset with longer term post-mda data from villupuram district, india. this dataset contained two addition data streams: scenario which included baseline, post-mda , post-mda , and post-mda mf data, and scenario , which included baseline, post-mda , post-mda , post-mda , and post-mda mf data. scenario thus contained the most post-mda data and was demonstrated to be the most effective for reducing model uncertainty, but this effect was not statistically significantly different from the reductions produced by assimilating data contained in table . spearman parameter correlations for scenarios (lower left triangle) and (upper right triangle) for alagramam, india. data assimilation in filarial model predictions scenarios and ( table ). the inclusion of more data than are considered in scenario therefore did not result in any significant additional reduction in model uncertainty. epifil was used to evaluate the accuracy of the data-driven predictions of the timelines required to meet the goal of lf elimination based on breaching the who-set target of % mf for all sites, either scenario or had the lowest entropies, and scenario was not significantly different from scenario for kirare and alagramam. these results were not statistically different from the results given % coverage (see table ), suggesting that the data stream associated with the lowest entropy is robust to changes in the interventions simulated. scenarios where the weighted variance or entropy were not significantly different from the lowest entropy scenario are noted with the abbreviation ns. significance was determined using the benjamini-hochberg procedure for controlling the false discovery rate (q = . ). https://doi.org/ . /journal.pntd. .g table . predictions of timelines to achieve % mf in villupuram district, india, considering extended post-mda data. reporting those scenarios which are statistically significantly different from each other by numbers ( - ) as superscripts. for example, the weighted variance for scenario has the superscript numbers ( ) ( ) ( ) ( ) ( ) ( ) to indicate that the weighted variance for scenario is significantly different from the weighted variance for scenarios - . significance was determined using the benjamini-hochberg procedure for controlling the false discovery rate (q = . ) in all pairwise statistical tests. + information gained by each data stream (scenario - ) are presented in comparison to the information contained in the model-only simulation (scenario ) https://doi.org/ . /journal.pntd. .t data assimilation in filarial model predictions prevalence. this analysis was performed by using the longitudinal pre and post-infection and mda data reported for the nigerian site, dokan tofa, where elimination was achieved according to who recommended criteria after seven rounds of mda (table ). the data from this site comprised information on the abr and dominant mosquito genus, as well as details of the mda intervention carried out, including the relevant drug regimen applied, time and population coverage of mda, and outcomes from the mf prevalence surveys conducted at baseline and at multiple time points during mda [ ] . the results of model predictions of the timelines to reach below % mf prevalence as a result of sequential fitting to the mf prevalence data from this site pertaining to scenarios - (as defined above) are shown in table . note that in the post mda , and surveys, as no lf positive individuals were detected among the sample populations, we used a one-sided % clopper-pearson interval to determine the expected upper one-sided % confidence limits for these sequentially observed zero infection values data assimilation in filarial model predictions using the "rule of three" approximation after k empty samples formula [ ] . the results show that model constraining by scenario , which includes baseline and post-mda data, and scenario , which includes baseline, post-mda , and post-mda data, resulted in both the least entropy values and the shortest predicted times, i.e., from as low as to as high as years, required for achieving lf elimination in this site ( table ). the data in table show that the first instance the calculated one-sided upper % confidence limit in this setting fell below % mf prevalence also occurred post mda (i.e after years of mda). this is a significant result, and indicates that apart from being able to reduce prediction uncertainty, the best data-constrained models are also able to more accurately predict the maximal time ( years) by which lf elimination occurred in this site. our major goal in this study was to compare the reliability of forecasts of timelines required for achieving parasite elimination made by generic lf models versus models constrained by sequential mf prevalence surveillance data obtained from field sites undergoing mda. a secondary aim was to evaluate the relative value of data obtained at each of the sampling time points proposed by the who for monitoring the effects of lf interventions in informing these model predictions. this assessment allowed us to investigate the role of these data for learning system dynamics and measure their value for guiding the design of surveillance programmes in order to support better predictions of the outcomes of applied interventions. fundamentally, however, this work addresses the question of how best to use predictive parasite transmission models for guiding management decision making, i.e. whether this should be based on the use of ideal models which incorporate generalized parameter values or on models with parameters informed by local data [ ] . if we find that data-informed models can reduce prediction uncertainty significantly compared to the use of theoretical models unconstrained by data, then it is clear that to be useful for management decision making we require the application of model-data assimilation frameworks that can effectively incorporate information from appropriate data into models for producing reliable intervention projections. antithetically, such a finding implies that using unconstrained ideal models in these circumstances will provide only approximate predictions characterized by a degree of uncertainty that might be too large to be useful for reliable decision making [ , , ] . here, we have used three state-of-the-art lf models calibrated to longitudinal human mf prevalence data obtained from three representative lf study sites to carry out a systematic analysis of these questions in parasite intervention modelling (see also walker et al [ ] for a recent study highlighting the importance of using longitudinal sentinel site data for improving the prediction performances of the closely-related onchocerciasis models). further, by iteratively testing the reduction in the uncertainty of the projections of timelines required to achieve lf elimination in a site made by the models matching each observed data point, we have also quantified the relative values of temporal data streams, including assessing optimal record lengths, for informing the current lf models. our results provide important insights as to how best to use process models for understanding and generating predictions of parasite dynamics. they also highlight how site-specific longitudinal surveillance data coupled with models can be useful for providing information about system dynamics and hence for improving predictions of relevance to management decision-making. the first result of major importance from our work is that models informed by data can significantly reduce predictive uncertainty and hence improve performance of the present lf models for guiding policy and management decision-making. our results show that these improvements in predictive precision were consistent between the three models and across all three of our study sites, and can be very substantive with up to as much as % to % reductions in prediction variance obtained by the best data-constrained models in a site compared to the use of model-only predictions ( table ). the practical policy implications of this finding can also be gleaned from appraising the actual numerical ranges in the predictions made by each individual model for each of the modelling scenarios investigated here. in the case of epi-fil, the best data-informed model (scenario in peneng) gave an elimination prediction range of - years, while the corresponding model-only predictions for this site indicated a need for between - years of annual mda (table ). these gains in information from using data to inform model parameters and hence predictions were even larger for the two stochastic models investigated here, viz. lymfasim and transfil, where ranges as wide as - years predicted by model-only scenarios were reduced to - years for the best data-informed models in the case of lymfasim for kirare village, and from as broad as - years to - years respectively in the case of transfil for peneng (table ). these results unequivocally indicate that if parasite transmission models are used unconstrained by data, i.e. based on general parameter values uninformed by local data, it would lead to the making of predictions that would be marked by uncertainties that are likely to be far too large to be meaningful for practical policy making. if managers are risk averse, this outcome will also mean their need to plan interventions for substantially much longer than necessary, with major implications for the ultimate cost of the programme. note also that although statistically significant changes in the median years of mda required to achieve lf elimination were observed for the best datainformed models for all the three lf model types in each site, these were relatively small compared to the large reductions seen in each model's predictive uncertainly (table , fig ) . this result highlights that the major gains from constraining the present models by data lies in improving their predictive certainty rather than in advancing their average behaviour. however, our preliminary analysis of model predictive accuracy suggests that the best data-constrained models may also be able to generate more accurate predictions of the impact of control ( table ), indicating that, apart from simply reducing predictive uncertainty, such models could additionally have improved capability for producing more reliable predictions of the outcomes of interventions carried out in a setting. the iterative testing of the reduction in forecast uncertainty using mf surveillance data measured at time points proposed by the who (to support assessment of whether the threshold of % mf prevalence has been reached before implementation units can move to post-treatment surveillance [ ]) has provided further insights into the relative value of these data for improving the predictive performance of each of the present lf models. our critical finding here is that parameter uncertainty in all three lf models was similarly reduced by the assimilation of a few additional longitudinal data records (table ). in particular, we show that data streams comprising baseline + post-mda + post-mda (scenario ) and those comprising baseline + post-mda data (scenario ) best reduced parameter-based uncertainty in model projections of the impact of mdas carried out in each study site irrespective of the models used. although preliminary, a potential key finding is that the use of longer-term data additional to the data measured at the who proposed monitoring time points did not lead to a significant further reduction in parameter uncertainty (table ) . also, the finding that the who data scenarios and were adequate for constraining the present lf models appears not to be an artefact of variations in the mda coverages observed between the three study sites (fig ) . these results suggest that up to years of post-mda mf prevalence data are sufficient to constrain model predictions of the impact of lf interventions at a time scale that can go up to as high as to years depending on the site and model, and that precision may not improve any further if more new data are added ( table , table ). given that the who post-mda lf infection monitoring protocol was developed for the purpose solely focussed on supporting the meeting of set targets (e.g. the % mf prevalence threshold) and not on a priori hypotheses regarding how surveillance data could be used also to understand the evolution and hence prediction of the dynamical parasitic system in response to management action, our results are entirely fortuitous with respect to the value of the current lf monitoring data for learning about the lf system and its extinction dynamics in different settings [ ] . they do, nonetheless, hint at the value that coupling models to data may offer to inform general theory for guiding the collection and use of monitoring data in parasite surveillance programmes in a manner that could help extract maximal information about the underlying parasite system of interest. our assessment of whether the incremental increase in model predictive performance observed as a result of assimilating longitudinal data may be due to parameter constraining by the addition of data has shed intriguing new light on the impact that qualitative changes in dynamical system behaviour may have on parameter estimates and structure, and hence on the nature of the future projections of system change we can make from models. our major finding in this regard is that even though the parameter space itself may not be overly constrained by the best data stream (scenario in this case for alagramam village), the magnitude and direction of parameter correlations, particularly those representing the relationship of different components of host immunity with various transmission processes, changed markedly between the shorter (scenario ) and seemingly optimal data streams (scenario ). this qualitative change in system behaviour induced by alteration in parameter interactions in response to perturbations has been shown to represent a characteristic feature of complex adaptive ecological systems, particularly when these systems approach a critical boundary [ ] [ ] [ ] . this underscores yet another important reason to incorporate parameter information from data for generating sound system forecasts [ ] . the finding that additional data beyond years post-mda did not appear to significantly improve model predictive performance in this regard suggests that pronounced change in lf parameter interactions in response to mda interventions may occur generally around this time point for this parasitic disease, and that once in this parameter regime further change appears to be unlikely. this is an interesting finding, which not only indicates that coupling models to at least years post-mda will allow detection of the boundaries delimiting the primary lf parameter regions with different qualitative behaviour, but also that the current who monitoring protocol might be sufficient to allow this discovery of system change. although our principal focus in this study was in investigating the value of longitudinal data for informing the predictive performance of the current lf models, the results presented here have also underscored the existence of significant spatial heterogeneity in the dynamics of parasite extinction between the present sites ( table , fig ) . in line with our previous findings, this observed conditional dependency of systems dynamics on local transmission conditions means that timelines or durations of interventions required to break lf transmission (as depicted in table ) will also vary from site to site even under similar control conditions [ ] [ ] [ ] ] . as we indicated before, this outcome implies that we vitally require the application of models to detailed spatio-temporal infection surveillance data, such as that exemplified by the data collected by countries in sentinel sites as part of their who-directed monitoring and evaluation activities, if we are to use the present models to make more reliable intervention predictions to drive policy and management decisions (particularly with respect to the durations of interventions required, need for switching to more intensified or new mda regimens, and need for enhanced supplementary vector control) in a given endemic setting [ ] . as we have previously pointed out, the development of such spatially adaptive intervention plans will require the development and use of spatially-explicit data assimilation modelling platforms that can couple geostatistical interpolation of model inputs (eg. abr and/or sentinel site mf/ antigen prevalence data) with discovery of localized models from such data in order to produce the required regional or national intervention forecasts [ ] . the estimated parameter and prediction uncertainties presented here are clearly dependent on the model-data fusion methodology and its implementation, and the cost function used to discover the appropriate models for a data stream [ ] . while we have attempted to evaluate differences in individual model structures, their computer implementation, and the data assimilation procedures followed (e.g. sequential vs. simultaneous data assimilation), via comparing the collective predictions of the three models versus the predictions provided by each model singly, and show that these factors are unlikely to play a major role in influencing the current results, we indicate that future work must address these issues adequately to improve the initial methods we have employed in this work. currently, we are examining the development of sequential bayesian-based multi-model ensemble approaches that will allow better integration of each model's behaviour as well as better calculation of each model's transient parameter space at each time a new observation becomes available [ ] . this work also involves the development of a method to fuse information from several indicators of infection (e.g. mf, antigenemia, antibody responses [ ] ) together to achieve a more robust constraining of the present models. as different types of data can act as mutual constraints on a model, we also expect that such multiindicator model-data fusion methods will additionally address the problem of equifinality, which is known to complicate the parameterization of complex dynamical models [ , ] . of course, the ultimate test of the results reported here, viz. that lf models constrained by coupling to year post-mda data can provide the best predictions of timelines for meeting the % mf prevalence threshold in a site, is by carrying out the direct validation of our results against independent observations (as demonstrated by the preliminary validation study carried out here using the dokan tofa data (tables and )). we expect that data useful for performing these studies at scale may be available at the sentinel site level in the countries carrying out the current who-led monitoring programme. the present results indicate that access to such data, and to post-treatment surveillance data which are beginning to be assembled by many countries, is now a major need if the present lf models are to provide maximal information about parasite system responses to management and thus generate better predictions of system states for use in policy making and in judging management effectiveness in different spatiotemporal settings [ , ] . given that previous modelling work has indicated that if the globally fixed who-proposed % mf prevalence threshold is insufficient to break lf transmission in every setting (and thus conversely leading to significant infection recrudescence [ ] ), the modelling of such spatio-temporal surveillance data will additionally allow testing if meeting this recommended threshold will indeed result in successfully achieving the interruption of lf transmission everywhere. the epidemiology of filariasis control. the filaria epidemiological modelling for monitoring and evaluation of lymphatic filariasis control mathematical modelling and the control of lymphatic filariasis. the lancet infectious diseases heterogeneous dynamics, robustness/fragility trade-offs, and the eradication of the macroparasitic disease, lymphatic filariasis continental-scale, data-driven predictive assessment of eliminating the vector-borne disease, lymphatic filariasis, in sub-saharan africa by sequential modelling of the effects of mass drug treatments on anopheline-mediated lymphatic filariasis infection in papua new guinea assessing endgame strategies for the elimination of lymphatic filariasis: a model-based evaluation of the impact of dec-medicated salt predicting lymphatic filariasis transmission and elimination dynamics using a multi-model ensemble framework data-model fusion to better understand emerging pathogens and improve infectious disease forecasting ecological forecasting and data assimilation in a data-rich era the role of data assimilation in predictive ecology effectiveness of a triple-drug regimen for global elimination of lymphatic filariasis: a modelling study. the lancet infectious diseases epidemic modelling: aspects where stochasticity matters. mathematical biosciences relative information contributions of model vs. data to short-and long-term forecasts of forest carbon dynamics inference in disease transmission experiments by using stochastic epidemic models plug-and-play inference for disease dynamics: measles in large and small populations as a case study the limits to prediction in ecological systems big data need big theory too hierarchical modelling for the environmental sciences: statistical methods and applications parameter and prediction uncertainty in an optimized terrestrial carbon cycle model: effects of constraining variables and data record length bayesian calibration of simulation models for supporting management of the elimination of the macroparasitic disease, lymphatic filariasis an improved state-parameter analysis of ecosystem models using data assimilation. ecological modelling bayesian calibration of mechanistic aquatic biogeochemical models and benefits for environmental management the model-data fusion pitfall: assuming certainty in an uncertain world transmission dynamics and control of severe acute respiratory syndrome curtailing transmission of severe acute respiratory syndrome within a community and its hospital transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions philosophical issues in model assessment. model validation: perspectives in hydrological science a sequential monte carlo approach for marine ecological prediction a sequential bayesian approach for hydrologic model selection and prediction inferences about coupling from ecological surveillance monitoring: approaches based on nonlinear dynamics and information theory. towards an information theory of complex networks using model-data fusion to interpret past trends, and quantify uncertainties in future projections, of terrestrial ecosystem carbon cycling rate my data: quantifying the value of ecological data for the development of models of the terrestrial carbon cycle estimating parameters of a forest ecosystem c model with measurements of stocks and fluxes as joint constraints. oecologia global mapping of lymphatic filariasis adaptive management and the value of information: learning via intervention in epidemiology general rules for managing and surveying networks of pests, diseases, and endangered species geographic and ecologic heterogeneity in elimination thresholds for the major vector-borne helminthic disease, lymphatic filariasis modelling strategies to break transmission of lymphatic filariasis-aggregation, adherence and vector competence greatly alter elimination mathematical modelling of lymphatic filariasis elimination programmes in india: required duration of mass drug administration and post-treatment level of infection indicators mathematical models for lymphatic filariasis transmission and control: challenges and prospects. parasite vector the lymfasim simulation program for modeling lymphatic filariasis and its control the dynamics of wuchereria bancrofti infection: a model-based analysis of longitudinal data from pondicherry parameter sampling capabilities of sequential and simultaneous data assimilation: ii. statistical analysis of numerical results an evaluation of models for partitioning eddy covariance-measured net ecosystem exchange into photosynthesis and respiration normalized measures of entropy entropyexplorer: an r package for computing and comparing differential shannon entropy, differential coefficient of variation and differential expression impact of years of diethylcarbamazine and ivermectin mass administration on infection and transmission of lymphatic filariasis controlling the false discovery rate-a practical and powerful approach to multiple testing epidemiological and entomological evaluations after six years or more of mass drug administration for lymphatic filariasis elimination in nigeria the study of plant disease epidemics bayesian data assimilation provides rapid decision support for vector-borne diseases modelling the elimination of river blindness using long-term epidemiological and programmatic data from mali and senegal. epidemics socio-ecological dynamics and challenges to the governance of neglected tropical disease control. infectious diseases of poverty early-warning signals for critical transitions early warning signals of extinction in deteriorating environments practical limits for reverse engineering of dynamical systems: a statistical analysis of sensitivity and parameter inferability in systems biology models multi-sensor model-data fusion for estimation of hydrologic and energy flux parameters. remote sensing of environment key: cord- - mrzaxi authors: kim, byunghyun; kim, kyuseok; lee, jieun; kim, joonghee; jo, yoo hwan; lee, jae hyuk; hwang, ji eun title: impact of bacteremia prediction rule in cap: before and after study date: - - journal: the american journal of emergency medicine doi: . /j.ajem. . . sha: doc_id: cord_uid: mrzaxi abstract objective in cases of community acquired pneumonia (cap), it has been known that blood cultures have low yields and rarely affect clinical outcomes. despite many studies predicting the likelihood of bacteremia in cap patients, those results have been rarely implemented in clinical practice, and use of blood culture in cap is still increasing. this study evaluated impact of implementing a previously derived and validated bacteremia prediction rule. methods in this registry-based before and after study, we used piecewise regression analysis to compare the blood culture rate before and after implementation of the prediction rule. we also compared -day mortality, emergency department (ed) length of stay, time-interval to initial antibiotics after ed arrival, and any changes to the antibiotics regimen as results of the blood cultures. in subgroup analysis, we compared two groups (with or without the use of the prediction rule) after implementation period, using propensity score matching. results following the implementation, the blood culture rate declined from . % to . % (p = . ) without significant changes in -day mortality and antibiotics regimen. the interval to initial antibiotics ( min vs. min, p = . ) and length of stay ( min vs. min, p = . ) were not significantly changed. in subgroup analysis, the group that use the prediction rule showed min faster antibiotics initiation (p = . ) and min shorter length of stay (p = . ) than the group that did not use the rule. conclusion implementation of the bacteremia prediction rule in cap patients reduced the blood culture rate without affecting the -day mortality and antibiotics regimen. blood culture before antibiotics treatment has been a routine diagnostic test in patients presumed to be infectious [ ] . however, in patients with community acquired pneumonia (cap), it has been known that the rate of true positive blood cultures is b % [ , ] . in emergency departments (ed), the true positive results from blood cultures rarely affected patient management [ , ] . additionally, ed overcrowding may increase the risk of blood culture contamination [ , ] . as part of an effort to reduce unnecessary blood culture and improve the yield of blood culture in cap, there have been many studies describing bacteremia prediction rules [ ] [ ] [ ] . despite those studies, in a recent study, it has been reported that the blood culture rate in cap patients is still increasing [ ] . therefore, whether those decision rules clinically impacted the reduction of blood cultures is questionable. for clinical decision rules to have an impact on standard treatment, the clinical decision rules should pass three stages: derivation, validation, and implementation [ , ] . in our previous studies, we derived the bacteremia prediction model in cap patients and tested the generalizability of the rule by a multicenter external validation [ , ] . here, we evaluated the impact of implementing the bacteremia prediction model on the rate of blood cultures. we hypothesized that implementation may reduce the blood culture rate without changing the mortality rate. also without blood culture, the time interval from patient presentation in the ed to the first intra-venous antibiotics administration could be shortened as well as the length of ed stay. this study is an uncontrolled before and after study based upon a retrospective review of a patient registry database. before implementation of the bacteremia prediction rule, a prospective registry of patients diagnosed with pneumonia in the ed had been in place since . the registry includes the patient's baseline characteristics and co-morbidities. time variables such as ed arrival time and ed discharge time were included in the registry as well as the initial antibiotics administration time. the registry also provided the patient's initial vital signs and laboratory findings including the blood culture results if a blood culture had been performed. finally, the patient's dispositions from the ed including admission to the ward or icu as well as the -day mortality were included in the registry. this study was performed at a -bed tertiary academic hospital with an annual ed census of , patients and was approved by the institutional review board of the hospital. the implementation of the bacteremia prediction rule was initiated in january of . we retrospectively analyzed the patient registry data from january to january . exclusion criteria were the same as in previous studies [ , ] . in short, we excluded patients who were diagnosed with hospital-acquired pneumonia (hap), health care-associated pneumonia (hcap), and ventilator-associated pneumonia (vap). we also excluded patients who were transferred from our hospital because of difficulty in follow-up and limited information on the changes of antibiotics. for ease of use of the bacteremia prediction rule, we created an excel file in which the recommendations for blood culture appeared with risk-stratified scores, calculated automatically by entry of variables ( supplementary fig. ). because the resident physicians in the ed changed monthly according to a rotation system with other hospitals, the instructions for the excel file and the explanations of the prediction rule were introduced on the first wednesday of every month. all emergency physicians were encouraged to record the score calculated with the excel file on electronic medical record (emr) of patients suspected of pneumonia regardless of the performance of blood cultures. the prediction rule use records for each emergency physician were reported as a feedback three times a week. the primary outcome was the blood culture rate before and after implementation of the bacteremia prediction rule. the secondary outcomes included the -day mortality rate, the ed length of stay, the time-interval to initial antibiotics after ed arrival, and any changes to the antibiotics regimen as a result of the blood culture findings. for the cost analysis of reduced blood culture usage due to this intervention, we compared the mean cost for blood cultures per patient before and after implementation of the prediction rule. for any positive blood culture results, we thoroughly reviewed the results, including preliminary reporting and final reporting times. in preliminary results, only gram staining results of the species growing in culture bottle were reported. in the final report, the exact names of the identified pathogens were reported with the antibiotics susceptibility test results. for changes to the antibiotics regimen following the blood culture results, we reviewed the daily antibiotics administration with the exact times until h after the final result report time. for adverse events due to contaminations, we defined the unnecessary administration of vancomycin as the start of administration after the preliminary reports and its discontinuation following the identification of contaminants in the final reports. additionally, unnecessary followup blood cultures were defined as blood cultures performed after preliminary reports that were confirmed as contaminants in the final reports and the follow-up results were negative. the following isolates were identified as contaminants: coagulase-negative staphylococcus, bacillus species, micrococcus species, corynebacterium species, and propionibacterium species. continuous variables were presented as the means and standard deviations. binomial variables were presented as the frequency of occurrence. wilcoxon's rank-sum test, the chi-square test, or fisher's exact test was performed, as appropriate, for comparisons before and after intervention. all p values were -tailed, and p values b . were considered statistically significant. we adopted a piecewise regression discontinuity approach using generalized linear models (using odds for binary results) to evaluate the impact of the intervention. for adjusted intervention effects, we constructed multivariable linear regression models using variables with statistical significance (p value b . ) from comparisons before and after the intervention. additionally, we performed box-pierce tests to identify statistically significant autocorrelations in time series models we used (p value b . ). because irrespective of prediction rule, the blood culture rates could be reduced only by education introducing the low yield of blood culture in cap, we performed subgroup analysis only using the patients after intervention. for the subgroup analysis, we used propensity score matched analysis to compare the groups that use the prediction rule and did not. we compared the blood culture rate, time-interval to initial antibiotics, and ed length of stay of both groups. all analyses were performed using stata (version ; statacorp, college station, tx) and any patients with missing variables were not included in analyses. of the registered patients during study period, a total of patients were included in the study after patients were excluded (fig. ). among the eligible patients, and patients were assigned to the before and after intervention groups, respectively. among the patients after intervention, patients ( . %) had the bacteremia score record on their emr. table summarizes the baseline characteristics of the two groups. after implementation of the bacteremia prediction rule, the blood culture rate decreased from . % to . %, representing . % reduction ( % confidence interval [ci] = . to . , p = . ) ( table ) . additionally, the monthly trend of the blood culture rate changed from increasing to decreasing (fig. ) . after model adjustment, the blood culture rate decreased from . % to . %, which represents . % reduction ( % ci = . to . , p = . ) reduction. the monthly trend of the blood culture rate still appeared to decrease after implementation of the intervention ( table ). after the implementation, the -day mortality rate decreased from . % ( % ci = . to . ) to . % ( % ci = . to . , p b . ). however, after model adjustment, this difference was not statistically significant (p = . ) ( table ) . the time-interval to initial antibiotics after ed arrival decreased from min to min but did not reach statistical significance (p = . ). after model adjustment, no statistically significant difference was found between the two groups (table ) . after implementation, the ed length of stay also decreased from min to min but the difference between the two groups was not statistically significant (table ) . table shows the blood culture results before and after intervention. the overall blood culture contamination rate was . % ( / ). after the implementation, the blood culture contamination rate was not changed (p = . ). antibiotics regimen changes based on positive blood culture results were not significantly different between the two groups ( . %; / before implementation vs. . %; / after implementation, p = . ). among the patients with contaminations, . % ( / ) had to undergo unnecessary phlebotomy for follow-up blood cultures and . % ( / ) were administered unnecessary vancomycin treatment. after score implementation, the total adverse event rates decreased from . % ( / ) to . % ( / ). however, this difference did not reach statistical significance (p = . ). in the propensity score matched subgroup analysis, we compared two groups that used the prediction rule and did not, during intervention period (supplementary table ). the baseline characteristics of the two groups were similar but, the group with the bacteremia score had the lower blood culture rate than the group that did not use the bacteremia score ( . % vs. . %, p b . ). additionally, the group that use the prediction rule had shorter time-interval to initial antibiotics ( min vs. min, p = . ) and reduced ed length of stay ( min vs. min, p = . ). in our time series analysis study, we reported that the implementation of the bacteremia prediction rule successfully reduced the blood culture rate without any significant -day mortality and antibiotics regimen changes. blood culture technique is time-consuming and resource-consuming considering the need for an aseptic technique and venous access to more than two sites [ , ] . especially in the ed, it is well known that blood culture contamination is common, and many eds have higher contamination rates than clinical and laboratory standards institute (clsi) recommendation of % [ ] [ ] [ ] . false positive results from blood cultures are associated with increased medical cost and hospital length of stay [ , ] . clinically, blood culture contaminations result in unnecessary antibiotics administration [ ] . in one previous study, a % of additional unnecessary glycopeptide administration in pseudobacteremia cases was observed, which is consistent with our results (table ) [ ] . as multidrug resistance pathogens in pneumonia have been increasing, concerns exist about treatment failure without initial blood cultures [ ] . however, in a recent study, multidrug resistance pathogens were uncommon in cap patients, which accounted for . % [ ] . according to our bacteremia score system, blood cultures could be omitted in patients in the low-risk group, of which the true bacteremia rate was b % [ ] . in this study, the true bacteremia in the low-risk group was . % ( / ) and antibiotics step up due to multidrug resistance organisms isolated from blood cultures was . % ( / ). concerning the adverse event rate due to false positive blood cultures of . % ( / ), it seems reasonable to omit blood culture in the low-risk group (table ) . contrary to our hypothesis, the time to initial antibiotics and ed length of stay tended to decrease, but no statistically significant differences were measured between the before and after intervention groups (table ). according to the sepsis guideline, initial antibiotics should be administered within h after recognition of sepsis [ ] . for patients with severe initial presentation, ed physicians may urge the initiation of iv antibiotics and blood cultures without adherence to the bacteremia score. calculation of the bacteremia score takes n . h because the scoring system includes albumin and c-reactive protein results. this fact may be why the bacteremia score adherence group had mild presentations and lower pneumonia severity index (psi) scores (supplementary table ). for patients with mild symptoms and stable initial vital signs, ed physicians may wait for the laboratory tests before the initiation of iv antibiotics. those patients may be stratified into the low-risk group for bacteremia and could have more chances to avoid unnecessary blood cultures of low yield. therefore, by the time a decision had to be made regarding initiating iv antibiotics without blood culture, n min had already passed, and the time-saving effect of omitting blood cultures may have been diminished. this study has several limitations. first because this study was a time series before and after study based on a retrospective review of a registry. missing not at random (mnar) which occupied a small fraction ( / ; . %) but not included in analysis, may have created a bias. also there could be many confounders influencing the results besides the intervention. second, the baseline characteristics of the two groups before and after intervention were significantly different. the middle east respiratory syndrome coronavirus (mers-cov) outbreak in may have increased the sensitivity of the ed visit population to respiratory symptoms, which may be one of the reasons accounting for the difference [ ] . third, the adherence rate for the bacteremia score was . % which may be too low to expect clinical and statistical significance. generally, evidence-based changes in the clinical practice of physicians take a substantial amount of time and effort [ , ] . in addition to those periods of adaptation, the time needed for calculation of the score make a decrease of adherence inevitable in severe patients for whom blood cultures and iv antibiotics were urgent. additionally, the calculation of the score with multiplication and addition is difficult and the use of excel file could be another cumbersome step to emergency physicians in a crowded ed. finally the target population only included patients with cap and this would limit the broad application of the table blood culture results with contamination and antibiotics regimen changes before and after implementation of bacteremia prediction model. ⁎ adjusted by variables with statistical significance (p value b . ): age, sex, history of diabetes mellitus, hypertension, heart failure, cerebrovascular disease, renal disease, liver disease, and known neoplasm, systolic blood pressure, respiratory rate, pulse rate, body temperature, white blood cell count, hematocrit, platelet count, glucose, albumin, bun, psi, admission rate. a the numbers of patients with any missing variables used in analysis was and those patients were not included in analyses. rule in the clinical field. despite those limitations, we could observed a statistically significant reduction in blood cultures after intervention without affecting -day mortality as well as decreasing trends in the initial antibiotics administration time and ed length of stay. considering the low adherence rate to the score system, a more thorough education and feedback is needed to achieve statistically significant clinical impacts on the time of initial antibiotics administration and ed length of stay. in conclusion, the implementation of the bacteremia prediction rule in cap patients reduced the blood culture rate without affecting the day mortality and antibiotics regimen. and bacteremia prediction score was independently associated with a reduction in initial antibiotics administration time and ed length of stay. further prospective multicenter studies should be performed to evaluate the impact of wide implementation of the clinical prediction rule. none. the clinical significance of positive blood cultures: a comprehensive analysis of episodes of bacteremia and fungemia in adults. i. laboratory and epidemiologic observations do emergency department blood cultures change practice in patients with pneumonia? selective use of blood cultures in emergency department pneumonia patients clinical impact of blood cultures taken in the emergency department do peripheral blood cultures taken in the emergency department influence clinical management the impact of overcrowding on the bacterial contamination of blood cultures in the ed reducing blood culture contamination in community hospital emergency departments: a multicenter evaluation of a quality improvement intervention predicting bacteremia in patients with community-acquired pneumonia a prediction rule for estimating the risk of bacteremia in patients with community-acquired pneumonia bacteremia prediction model using a common clinical test in patients with community-acquired pneumonia blood culture use in the emergency department in patients hospitalized for community-acquired pneumonia implementation of clinical decision rules in the emergency department framework for the impact analysis and implementation of clinical prediction rules (cprs) bacteremia prediction model for community-acquired pneumonia: external validation in a multicenter retrospective cohort contaminant blood cultures and resource utilization. the true consequences of false-positive results guidelines on blood cultures reducing blood culture contamination in the emergency department: an interrupted time series quality improvement study reducing blood culture contamination rates in the emergency department clinical and economic impact of contaminated blood cultures within the hospital setting impact of blood cultures drawn by phlebotomy on contamination rates and health care costs in a hospital emergency department clinical significance of potential contaminants in blood cultures among patients in a medical center risk factors for drug-resistant pathogens in community-acquired and healthcare-associated pneumonia epidemiology and predictors of multidrug-resistant community-acquired and health care-associated pneumonia the surviving sepsis campaign sepsis guideline preventive behaviors by the level of perceived infection sensitivity during the korea outbreak of middle east respiratory syndrome in from best evidence to best practice: effective implementation of change in patients' care what drives change? barriers to and incentives for achieving evidence-based practice none. supplementary data to this article can be found online at https://doi. org/ . /j.ajem. . . . key: cord- -y pzsoqa authors: adalja, amesh a. title: biothreat agents and emerging infectious disease in the emergency department date: - - journal: emerg med clin north am doi: . /j.emc. . . sha: doc_id: cord_uid: y pzsoqa the challenges faced by the emergency physician with recognizing and treating category a biothreat agents and emerging infectious disease are summarized and reviewed. emergency physicians in every location in the world, in developed and developing countries alike, will undoubtedly be confronted with the possibility of an emerging infectious disease in their career. a subset of these physicians may be faced with a patient who has potentially been exposed to biological weapons. of the myriad infectious disease emergencies an emergency physician contends with, these possibilities are the gravest and most impactful. in such scenarios, the emergency department (ed) clinician can be the key in recognizing or containing an outbreak. the challenge inherent with emerging infectious diseases presenting in the ed is that such cases can be camouflaged, lurking amongst innumerable infectious disease clinical syndromes, from common colds to viral rashes. this article provides guidance to emergency physicians as to how to approach this challenging problem as well as familiarizing readers with specific microbial threats of high consequence. a key method for detecting the presence of an emerging infectious disease syndrome or a biological weapons exposure in an ed patient is to develop a general approach that seeks out key historical and physical examination clues. this approach is not different from what is included in a full history and physical examination but requires meticulous attention to certain aspects of the history. travel history becomes a key focus of the history because many infectious diseases, especially of the emerging variety, have delimited borders in which they are prevalent. the travel history must be coupled, however, with situational awareness as to what infections are known to be present in specific parts of the world. such a task is daunting for most physicians and, therefore, it is important that they know where such resources can be found. both the centers for disease control and prevention (cdc) (www.cdc.gov/travel) and promed (program for monitoring emerging diseases) (www.promedmail.org) are such resources that are easy to access and continually updated. using these resources, a busy provider can quickly assess which specific infection risks any given country might confer. an important component of the travel history is understanding the dates of travel and how they relate to the incubation period of specific infections. travel must be contextualized and integrated with incubation period, because domestic infections acquired before or after travel might be mistaken for a travel-related infection. additionally, eds in a given geographic locale (eg, metropolitan area, county, or state) should develop a mechanism to have insight into changes in ed volume, chief complaint mix, and unusual diagnoses at other eds in the region. much of this can be accomplished through leveraging emergency health care coalitions and local or state health departments to develop tools to enhance insight into the vicissitudes of a given region's ed-relevant infectious disease problems through syndromic surveillance programs. an important component of an individual's risk for particular infections is related to exposures. attention must be paid to animal exposures (domestic and wild), eating habits, occupation, and hobbies. additionally, it is essential to determine if a person has had any sick contacts or has attended a mass gathering, because an ed physician might be seeing one of the first formal presentations of a wider outbreak. of the specific biological agents, the category a agents (anthrax, plague, tularemia, and botulism), and certain viral hemorrhagic fevers (vhfs) (eg, ebola, marburg, machupo, and lassa fever) are of the highest priority. table provides salient points regarding the treatment of the category a biothreat agents. in all cases of uncertainty, prompt consultation with an infectious disease physician is recommended. anthrax is caused by the gram-positive bacillus, bacillus anthracis. it is a ubiquitous spore-forming gram-positive bacterium that is found naturally in the soil worldwide. it is a disease of herbivores. humans can contract of forms of the infection: cutaneous, inhalational, injectional, and gastrointestinal. , of these forms, cutaneous is by far the most common and represents a majority of cases. an intentional release of anthrax is expected to result in primarily inhalational cases. anthrax is not contagious from person to person and no special precautions are required. adalja cutaneous anthrax is characterized by a painless black ulceration ( fig. ) that occurs on the site of exposure. infection is more common in those exposed to animal products contaminated with spores, such as meat, drum skins, or wool. after an incubation period of approximately days, the lesion characteristically begins as a papule and progresses to a black eschar. diagnosis is often clinical, although culture, biopsy, polymerase chain reaction (pcr), and serology confirm the diagnosis. mortality is low if the disease is recognized and treated with appropriate antimicrobials. treatment regimens include oral ciprofloxacin or doxycycline (although penicillin may be used if susceptibility is known) for days. if exposure was through a biological attack, treatment duration is extended to days to cover incubating spores that may have been inhaled. injectional anthrax has been exclusively linked to use of contaminated illicit drugs whereas gastrointestinal anthrax is due to ingestion of contaminated food. , inhalational anthrax is the deadliest form of anthrax and occurs on inhalation of as little as spore. anthrax was historically known as wool sorter's disease because of its linkage with the occupation of wool sorting, in which spores on sheep's wool became aerosolized. the disease is characterized not by pneumonia but by mediastinal widening (fig. ) that can progress rapidly to shock. toxin-laden pleural effusions may be present. the disease begins after a week-long incubation period and is typically biphasic with flulike symptoms (with the notable exception of rhinorrhea) occurring before a terminal phase. when anthrax of any form progresses, the grave complication of hemorrhagic meningitis can occur. the treatment of systemic anthrax syndromes (inhalational, gastrointestinal, and injectional) involves first ruling out the presence or absence of meningitis via a lumbar puncture. if meningitis is confirmed or cannot be ruled out, the treatment regimen should include central nervous system penetrating drugs, of which should be a protein synthesis inhibitor (eg, linezolid) and of which should be bactericidal (eg, meropenem). the third drug could be ciprofloxacin. if meningitis has been ruled out, ciprofloxacin and clindamycin or linezolid could be used for treatment (with deescalation of ciprofloxacin to penicillin once drug susceptibility is known). treatment is for weeks to weeks. adjunctive antibody therapies, available from the cdc, such as anthrax immune globulin, raxibacumab, and obiltoxaximab, also should be given. additionally, if present, pleural effusions, pericardial effusions, and ascites should be drained, a factor that has likely improved survival rates from inhalational anthrax in the modern era. postexposure prophylaxis, for those exposed to anthrax spores, includes both an abbreviated -dose regimen of the vaccine coupled with days of oral ciprofloxacin or doxycycline (antibody therapies can be used in this manner when no other prophylaxis method can be used). in a mass event the post-exposure prophylaxis regimen for adults can be shortened to days after the first vaccine dose or weeks after the last vaccine dose, which ever comes later. plague is caused by the gram-negative bacillus yesinia pestis and is endemic in many parts of the world, including the western united states. this zoonotic infection is naturally spread from rodents, such as prairie dogs, to humans via the bite of a flea. there are forms of plague: bubonic (the most common), pneumonic, and septicemic. if used as a bioweapon, plague is expected to present in its pneumonic form. bubonic plague is characterized by marked painful lymphadenopathy (fig. ) that develops after a -day to -day incubation period whereas pneumonic plague (fig. ) may be indistinguishable from ordinary community-acquired pneumonia but has a mortality rate that can reach %. pneumonic plague has a -day to -day incubation period. pneumonic plague is transmissible from person to person through respiratory droplets and requires patients be placed in droplet isolation. , diagnosis of pneumonic plague in an intentional attack requires a high index of suspicion and can be made through pcr, serology, and/or culture. , the treatment of plague is with aminoglycoside antibiotics, such as gentamicin or streptomycin, for days to days, whereas postexposure prophylaxis of those exposed to an aerosol in a bioweapon attack consists of oral ciprofloxacin or doxycycline. , tularemia tularemia is caused by the gram-negative bacillus francisella tularenesis and is naturally a zoonotic infection that is common in many parts of the united states. naturally, tularemia may occur through tick, fly, and mosquito bites or through contact with reservoir animals (eg, rabbits). contaminated uncooked food or water can also be a vehicle for spread. the most common presentation of tularemia is the ulceroglandular cutaneous form whereas a biologic attack likely results in pneumonic tularemia. tularemia is not contagious between humans. tularemia is notable for its low infectious dose in which inhalation of just a small number of bacilli can result in disease. because of this, it is important to notify laboratory personnel about the possibility of tularemia, so they are able to don appropriate personal protective equipment when working with clinical specimens. pneumonic tularemia occurs after a -day to -day incubation period and is essentially indistinguishable from community-acquired pneumonia. even physicians who live in endemic areas often miss the diagnosis of tularemia in its various form, highlighting the need for tularemia to be in the differential diagnosis of compatible syndromes in endemic areas. temperature-pulse disassociation may be present and can serve as a clue to diagnosis. the treatment of tularemia is aminoglycoside antibiotics, such as gentamicin or streptomycin, for days to days. postexposure prophylaxis, to those exposed to an aerosol in a biological weapons attack, is with oral doxycycline or ciprofloxacin. , botulism botulism is caused by the acetylcholine release-blocking neurotoxin released by clostridium botulinum, a ubiquitous spore-forming gram-positive rod. there are several forms of naturally occurring botulism: infant, wound, and gastrointestinal. these forms result from exposure to spores of the bacteria, which then germinate and elaborate toxin. in a biological attack, inhalational botulism is expected, and it manifests similar to gastrointestinal botulism. botulism is not contagious. , after hours to hours postexposure to spores, clinical botulism occurs. it is characterized by a symmetric, flaccid, descending paralysis without sensory symptoms. patients are afebrile and not tachycardic. cranial neuropathies are common. paralysis progresses to involve respiratory muscles and can be prolonged, requiring long durations of mechanical ventilation. diagnosis is largely clinical. confirmatory mouse bioassay testing is used to determine which of the several botulinum toxinotypes is responsible. , heptavalent antitoxin, which neutralizes toxinotypes a to g, is obtainable from the cdc and can neutralize toxin. antibiotics and babybig (california department of public health, a bivalent botulinum antitoxin used in infant botulism) are not indicated. , there was controversy over the existence of an eighth toxinotype (h) but it has been shown to be a hybrid toxin and is neutralized by a-type antitoxin. more recently, a toxinotype x has been described and is unable to be neutralized by any available antitoxin. smallpox is the only human infectious disease that has been eradicated from the planet. as such, there is little current clinical experience with this disease. smallpox is a significantly contagious disease that is spread via airborne, respiratory droplet, or direct contact route. fomites are also known to spread the virus. , the clinical presentation of smallpox begins with flulike symptoms after a -day incubation period which is followed by the characteristic rash. the rash begins in a papular form and then progresses to umbilicated lesions and finally to pustules that crust and scab. a person with smallpox is contagious only from the appearance of the rash until the rash is scabbed, a key factor that led to its control. , the rash of smallpox (fig. ) must be distinguished from similar rashes that can be seen with varicella. several points of distinction are important. the rash of smallpox is centrifugal with more lesions on the face and extremities while the varicella rash is centripetal. the lesions of the smallpox rash are all at identical stages with identical appearances whereas the rash of varicella may have lesions of different stages. the case fatality rate of smallpox was historically %. , a diagnosis of smallpox would be a national security emergency of the highest order because even case represents either a laboratory accident or a biological attack. because smallpox vaccination is no longer routine, there is a sizable amount of the world population that lacks immunity. any suspicion of smallpox should prompt infectious disease consultation, airborne isolation procedures, and notification of local, state, and national public health authorities. the cdc has a telephone consultation service in place to discuss potential cases with experts. a diagnosis of smallpox initially is based on clinical suspicion while confirmatory testing by pcr, viral culture, or electron microscopy is performed under appropriate biosafety conditions. , there is no food and drug administration-approved treatment of smallpox currently, although several experimental antiviral compounds are in late stages of clinical development and might be accessible. the smallpox vaccine is effective as postexposure prophylaxis, even during the incubation period, and should be given to all patient contacts, who also will be placed under public health surveillance. the vaccine is contraindicated in the immunosuppressed and pregnant and those with eczema. experimental attenuated vaccines may be more suitable for these individuals. [ ] [ ] [ ] additionally, the smallpox vaccine carries a risk of myocarditis. vhfs are caused by a diverse group of viruses, each with its own unique microbiological, epidemiologic, and clinical features. of this group, which ranges from yellow fever to ebola, certain are more important as potential biological weapons than others. in the biological weapons context, it is the filoviruses (ebola and marburg) as well as the arenaviruses (lassa fever, machupo, and others) that merit concern. despite their differences, this group of viruses is characterized by a clinical presentation that often includes general malaise, fever, rash, prostration, pharyngitis, nausea, vomiting, and diarrhea. disease can rapidly progress to shock and multiple organ dysfunction syndrome with disseminated intravascular coagulation and hemorrhagic manifestations. diagnosis can be made using molecular tests, but a high index of suspicion is needed to differentiate these infections from ordinary septic shock. in the ed patient, travel to endemic areas, exotic animal exposure, or laboratory work with vhfs might be the only clue to the etiology. in a biological attack, a cluster of patients with similar symptoms may present to several eds in a given region. any suspicion of a vhf should prompt immediate consultation with an infectious disease physician and state and local health authorities. although these viruses are spread via blood and body fluid exposure and do not spread between humans via the airborne route, the experience of the united states during the west africa ebola outbreak has influenced infection control recommendations. the nosocomial infections at a hospital in texas have led to recommendations for strict airborne and body fluid isolation for patients suspected of having a vhf, with transfer to definitive care at specialized units for confirmed cases. treatment is generally supportive and has proved life-saving in the case of ebola. the recent experience with ebola highlighted the fact that simple supportive care with fluids and electrolytes brought fatality rates down from % to less than %. there are several experimental treatments and vaccines (which can be used for postexposure prophylaxis) that are available for filovirus infections and arenavirus infections that would likely be used in any domestic vhf cases caused by these groups of viruses. for ebola exposures, the experimental vaccine would be indicated for postexposure prophylaxis whereas a combination of experimental antiviral agents (eg, favipiravir) and antibody-based therapies, such as zmapp, might be indicated after consultation with cdc. lassa fever can be treated with intravenous ribavirin, which is available via cdc. the possibility of vhf infection should be considered in those with severe illness and travel to areas in which these infections are endemic, such as parts of africa (eg, democratic republic of congo, uganda, and nigeria) or south america (eg, brazil and argentina). consultation of the cdc travel web site (www.cdc.gov/travel) is advised to determine specific vhf travel risks. coronaviruses (covs) are major causes of the common cold and rarely cause severe disease in immunocompetent hosts. there are, however, covs that have the capacity to cause severe disease: severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov. although both sars and mers present to the ed as ordinary upper or lower respiratory tract infections, they have distinct geographic and epidemiologic features that should alert an ed physician to the possibility of their presence. sars, which emerged in china in , was a worldwide infectious disease emergency that led to more than cases worldwide, with approximately % of cases fatal-including in the united states. the virus was zoonotic in origin and linked to human consumption of palm civet cats. the spread of the virus was abetted by the presence of superspreading events in which certain individuals infected a disproportionate number of others. the epidemic extinguished once infection control measures were instituted in health care settings and the consumption of palm civet cats ceased. mers is also a zoonotic respiratory cov that emerged in the arabian peninsula in and has been linked to contact with both bats and camels. all cases have an epidemiologic link to the arabian peninsula, including a multiple-ed superspreading event that occurred in south korea. mortality rates are approximately %. in the united states, imported mild cases have been diagnosed in travelers returning from the middle east. mers should be suspected in individuals with upper or lower respiratory infection after travel to the middle east in the prior weeks, and confirmatory molecular testing can be done in conjunction with state and local health authorities. many respiratory viral panels have the capacity to identify the presence of a cov and may be helpful in the work-up. infectious disease consultation and institution of droplet or airborne precautions are advised. there are no antivirals or vaccines available for any cov. influenza is often considered one of the highest pandemic threats. prior influenza pandemics have killed millions and have caused severe societal disruption. each modern pandemic ( , , , and ) has been linked to the emergence of a novel influenza a variant of zoonotic (avian, swine, or a combination) origin. zoonotic influenza viruses, in their first forays into humans, can cause a range of illness, ranging from ordinary influenza to fulminant disease, including pneumonia and acute respiratory distress syndrome. poor to limited nonsustained human-tohuman transmission characterizes these viruses in the prepandemic stage, with most cases linked directly or indirectly to poultry exposure. it is when sustained human-to-human transmission occurs that a pandemic is eminent. because of this threat, monitoring and surveillance efforts exist for avian influenza infections in humans and poultry. currently, of the myriad zoonotic influenza infections, the h n strain of influenza a has been deemed the highest threat amongst these viruses currently, although others (such as h n ) are also important to track. the ed physician should suspect avian influenza in travelers from china and other areas in which avian influenza is known to circulate, who present within approximately week after travel with upper or lower respiratory tract infection. additionally, domestic agricultural workers or those with agricultural contact with flulike symptoms (eg, children at fairs) also may harbor zoonotic influenza infections. diagnosis is similar to ordinary influenza, but a rapid or standard molecular test may or may not be able to detect influenza. confirmatory testing is via health authorities. treatment involves supportive care coupled to antiviral therapy with either oral oseltamivir or, if disease severity is high, intravenous peramivir. infectious disease consultation and institution of droplet precautions is advised. mosquito-borne arboviruses have increasingly taken on importance in the field of emerging infectious disease with the explosion of cases of west nile, chikungunya, and zika in the western hemisphere. additionally, local transmission of dengue fever has occurred in florida, texas, hawaii, and new york. , chikungunya, dengue fever, and zika are all spread by the aedes species of mosquitoes, which have habitats both within and outside the united states and cause clinically indistinguishable syndromes. these syndromes all involve fevers, rash, myalgias, and arthralgias. conjunctivitis has been noted with zika. prolonged debilitating arthralgias can occur with chikungunya whereas severe illness, including shock and hemorrhagic manifestations, can occur with dengue fever (especially with repeat infection with disparate strains). , zika has been linked to guillain-barré syndrome and requires special counseling regarding sexual transmission and pregnancy given its ability to cause a devastating congenital syndrome. , travel history and residence in an endemic area (eg, key west, hawaii, and texas) are important elements of the history. diagnostic testing is commercially available for each infection. consultation with the cdc travel web site is advised to assess specific risks for individual patients' travel history. no vaccines or antiviral therapies are available for these infections. emergency physicians play a crucial and unique role in the defense against emerging infectious disease and are most likely to encounter the first cases of any new infectious disease syndrome. the challenge that the emergency physician faces is that these cases do not announce themselves and are hidden among the sea of chief complaints that any ed sees on a given day. with many clinical scenarios, a busy physician may not be inclined to pursue a specific diagnosis if it "doesn't change treatment," may lengthen ed length of stay, and will not produce a result while a patient is in the ed, creating follow-up logistical problems. failing to diagnose an epidemiologically important emerging infectious disease or biological attack, however, can have tremendous cascading effects involving all sectors of society (health care, government, and economy) that can be minimized or averted with a proactive approach. in the current era, there are several molecular multianalyte tests available, some of which are clinical laboratory improvement amendments (clia) waived and available at point of care for specific infectious disease syndromes, such as gastrointestinal infections, meningitis, and respiratory infections, that can be used to increase the rate of microbial specific diagnoses in ed settings. biodefense cartridges, which probe for select bioagents, are also available. these tests, in the hands of an astute physician, can improve the nation's resiliency to infectious disease emergencies, and negative results in the right clinical context may prompt further investigation for a specific etiology. by having a working knowledge of emerging infectious disease and biothreat landscape, an emergency physician becomes a key component of the infectious disease emergency system. injectional anthrax in heroin users clinical management of potential bioterrorismrelated conditions mandell, douglas, and bennett's principles and practice of infectious diseases centers for disease control and prevention expert panel meetings on prevention and treatment of anthrax in adults systematic review: a century of inhalational anthrax cases from to mandell, douglas, and bennett's principles and practice of infectious diseases s principles and practice of infectious diseases clinical recognition and management of tularemia in missouri: a retrospective records review of cases mandell, douglas, and bennett's principles and practice of infectious diseases a novel botulinum neurotoxin, previously reported as serotype h, has a hybrid-like structure with regions of similarity to the structures of serotypes a and f and is neutralized with serotype a antitoxin identification and characterization of a novel botulinum neurotoxin orthopoxviruses: vaccinia (smallpox vaccine), variola (smallpox), monkeypox, and cowpox acam : the new smallpox vaccine for united states strategic national stockpile safety and immunogenicity of imvamune smallpox vaccine using different strategies for a post event scenario safety and immunogenicity of lc m , an attenuated smallpox vaccine in vaccinia-naive adults acam prescribing information mandell, douglas, and bennett's principles and practice of infectious diseases systems for rapidly detecting and treating persons with ebola virus disease -united states evidence-based guidelines for supportive care of patients with ebola virus disease experimental therapies for ebola virus disease: what have we learned? including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) summary of influenza risk assessment tool (irat) results outbreak of influenza a (h n ) variant virus infections among persons attending agricultural fairs housing infected swine -michigan and ohio lessons learned during dengue outbreaks in the united states health commissioner reports dengue virus case japanese encephalitis, west nile encephalitis, st. louis encephalitis, tick-borne encephalitis, kyasanur forest disease, alkhurma hemorrhagic fever, zika) mandell, douglas, and bennett's principles and practice of infectious diseases guillain-barre syndrome associated with zika virus infection in columbia zika virus and birth defects-reviewing the evidence for causality key: cord- -cob ur authors: sharma, vaneet kumar; sharma, ity; glick, james title: the expanding role of mass spectrometry in the field of vaccine development date: - - journal: mass spectrom rev doi: . /mas. sha: doc_id: cord_uid: cob ur biological mass spectrometry has evolved as a core analytical technology in the last decade mainly because of its unparalleled ability to perform qualitative as well as quantitative profiling of enormously complex biological samples with high mass accuracy, sensitivity, selectivity and specificity. mass spectrometry‐based techniques are also routinely used to assess glycosylation and other post‐translational modifications, disulfide bond linkage, and scrambling as well as for the detection of host cell protein contaminants in the field of biopharmaceuticals. the role of mass spectrometry in vaccine development has been very limited but is now expanding as the landscape of global vaccine development is shifting towards the development of recombinant vaccines. in this review, the role of mass spectrometry in vaccine development is presented, some of the ongoing efforts to develop vaccines for diseases with global unmet medical need are discussed and the regulatory challenges of implementing mass spectrometry techniques in a quality control laboratory setting are highlighted. as the target populations for the vaccines are healthy individuals, pregnant women, or infants, vaccine safety is of paramount importance. appropriately, the vaccine landscape is changing from traditional vaccine approaches to cost-effective, highly scalable, and safe recombinant vaccines. , using recombinant dna technology, antigens are expressed in yeast, escherichia coli, baculovirus expression vector system (bevs), or mammalian cell lines. [ ] [ ] [ ] recombinant antigens are engineered to mimic the first step of virus attachment to the cell surface which is mediated by specific glycoproteins. the expressed recombinant antigens undergo multiple purification cycles to produce highly purified vaccines. , in order for recombinant vaccines to be acceptable to regulatory authorities, in-depth analytical characterization needs to be performed in this review, we focus on the expanding role of mass spectrometry in vaccine development, irrespective of the route of production. we also highlight the regulatory challenges and limitations of mass spectrometry-based techniques which constrain its further implementation as a quality control batch release assay in cgmp manufacturing. the role of mass spectrometry ( alternatively, an o-linked glycan can be formed by linking the oligosaccharide to a serine or threonine residue. glycosylation play a fundamental role in antigen conformation, folding, stability, solubility, and importantly, immune response. , plants, yeasts, and non-human cell lines generate glycans that are not compatible and bioactive within human hosts. thus, about % of all recombinant glycoproteins are produced in mammalian-based expression systems such as chinese hamster ovary (cho) cells. analytical characterization of a glycoprotein is challenging as there is inherent unpredictability associated with the glycans; they are either macroheterogeneous (potential glycan site in the protein not glycosylated) or microheterogeneous (different glycan structures found on the same site in the expressed protein). glycosylation analysis is performed to understand the nature of structural heterogeneity of glycans, quantify them, and more importantly to determine where glycosylation occurs (site specific analysis). mass spectrometry based techniques are valuable tool for detecting and investigating glycosylation ( figure ). although ms-based glycosylation analysis is not without limitations, the advances in ms instrumentation and glycan analysis software have led to increased resolution, automated identification, quantitative determination, and accurate structural characterization. , , another critical quality attribute (cqa) of glycoprotein is intramolecular disulfide bonds (s-s linkages); they ensure correct folding, functional activity and stability. incorrect formation of disulfide bonds can cause protein misfolding which tends to promote aggregation which could result in an unwarranted immune response. confirmation of correct disulfide bond formation in the recombinant first study analyzing the site-specific nglycosylation of gp . this report describes the structural characterization of the expressed gp . reversed phase hplc of the tryptic digest. peptides collected from rp-hplc were further identified by amino acid analysis (aaa) or nterminal sequencing analysis. leonard et al hiv- gp expressed in cho cells. mass spectrometric characterization of the glycosylation pattern. of hiv- gp . maldi and nanoesi-lc-ms/ms using a hybrid quadrupole-time-of-flight tandem mass spectrometer (q-tof) was used to assign glycosylation sites. zhu et al hiv gp jr-fl and con-s env expressed in cho and hek cells. a glycopeptide-based mass mapping approach was used to characterize the glycosylation of two env protein vaccine candidates in a glycosylation site-specific fashion. mass spectrometry based approach was used to map the complete glycosylation profile at every site in eleven hiv- env trimers. high-resolution lc-ms/ms was performed using an orbitrap velos pro hybrid mass spectrometer (thermo scientific) equipped with etd and coupled to an acquity uplc system (waters). go et al hiv gp monomers of the bg . sosip. global n-glycan site occupancy of hiv- gp by metabolic engineering and high-resolution intact mass spectrometry was performed. released n-glycan analysis was carried out using synapt g si ion mobility mass spectrometer. native highresolution mass spectrometry was performed on q exactive hybrid quadrupole-orbitrap mass spectrometer. as illustrated in the following section and in table , mass spectrometry-based techniques have been used to perform the structural characterization, glycosylation profiling and antigen quantitation during the development of the hiv, influenza, dengue, ebola, meningococcal, and other vaccines. the review also highlights that mass spectrometry-based methods such as glycan analysis has been used to analyze a specific envelope glycoproteins (env) and has broad applicability to any other glycoprotein-based vaccines. the quest for a safe and effective vaccine to protect against human one of the hypotheses being explored for protective immunity against hiv is to induce broadly neutralizing antibodies (bnabs) that target the highly glycosylated hiv- virion-associated envelop. a number of approaches are being pursued in the hiv- vaccine development field to establish protective immunity, including monomeric gp subunits and oligomeric gp and trimeric envelope glycoprotein to elicit bnabs. [ ] [ ] [ ] each of these envelop (env) immunogens have multiple exposed epitopes and are heterogeneously glycosylated molecules, with more than % of their mass consisting of glycans (∼ n-linked glycans). it is critically important to monitor this hiv env glycan shield during vaccine development. crispin and collaborators also extensively investigated the native glycosylation profile of envs, in particularly, trimeric immunogen bg sosip. . [ ] [ ] [ ] [ ] their studies led to an increased understanding of the glycan shield of the env and it is now largely accepted that to induce bnabs, the hiv vaccine candidate should have a glycan profile similar to the one present on the native env trimers. [ ] [ ] [ ] in a thorough study on trimeric env glycosylation, behrens et al influenza virus is a segmented, enveloped rna virus and is among the ic-idms-mrm method is truly an alternative to the approved srid method as it has dual purpose "potency and content determination", was found to be equivalent to the srid method and can also be used in response to a pandemic influenza threat. , additionally, a direct ultra-performance liquid chromatography (uplc)-idms method was reported for the rapid and accurate quantification of influenza na. label-free ms-based methods have also been reported for the simultaneous identification and quantification of ha and na in influenza vaccine with samples analyzed by lc-ms e on a waters synapt g mass spectrometer. quantification of proteins by labelfree lc-ms e is a powerful tool, in this method, alternating scans of low collision energy and elevated collision energy during lc-ms analysis to obtain both protein identity and quantity in a single experiment. quantification based on the experimental data showed that the signal intensity was proportional to concentration which allowed for the amount of any protein in the mixture to be estimated. lc-ms e utilizes parallel, multiplex fragmentation where all peptide precursors are simultaneously fragmented throughout the chromatographic the srid assay has been used for over years as a quantitative and potency method throughout the world despite issues regarding variability and availability of standard reagents. thus, even though new methods like hplc and mass spectrometry are being developed, it will take some time for these methods to be adopted worldwide. as the next influenza pandemic cannot be predicted, the health authorities' pandemic preparedness efforts include efforts to ensure expedited availability of pandemic vaccines. methods such as hplc and ms, with their ability to quantitate antigens without standard reference reagents, can become a cornerstone of pandemic influenza preparedness. the viral genus flavivirus, includes dengue virus, yellow fever virus, and and were glycosylated and, predominately, the n-glycan at asn was a high mannose-type and at asn was mainly a combination of complex-and hybrid-type glycans. this study provided important new insights for the role of glycans in the dengue virus-host cell interactions. in a separate study, accurate quantitation of the expressed four viral particles in the tetravalent dengue vaccine (cyd) was performed using targeted ms in selected reaction monitoring (srm) mode. the study described an orthogonal quantitation strategy (targeted ms in srm mode) and demonstrated that the variability of the ms method was low (between % and %) and the assay was linear between . and nmol/l. based on the reported method performance, it could be used to release future batches of the tetravalent dengue vaccine. ebola the data presented demonstrated that the n-glycan patterns were similar between gp , but o-glycan patterns were remarkably different on gp , the five ebola viruses. this study should serve as the foundation for future ebola viral entry and immunogenicity studies. to improve on the conventional approaches for absolute quantitation of gp in ebola virus-like particles (evlps), an isotope dilution full-scan liquid chromatography-high-resolution mass spectrometry method was developed using an ultimate hplc and an orbitrap elite hybrid ion trap-orbitrap mass spectrometer. the reported ms quantitation method provided not only a means to rapidly determine evlp batch quality based upon quantitation of antigenic gp but also ensured adequate preclinical/clinical dosing. chikungunya is a mosquito-borne viral disease, endemic in africa and southeast asia and has also recently emerged in the caribbean. currently there are no drugs or vaccines available for treatment or prevention. the name "chikungunya" derives from a makonde word meaning "to become contorted," and describes the stooped appearance of sufferers with joint pain (arthralgia ms-based approaches have been used to perform the physicochemical characterization for all of these vaccines. in one of the studies, researchers used lc-ms to characterize glycosylated lysine residues in menveo®. in another study a lc-ms method was used to determine the relative reactivity of lysine residues in crm to determine which of these amino acids were more susceptible to conjugation. lc-ms was also used to quantify the bexsero® vaccine which was the first vaccine developed by reverse vaccinology; a genome-based approach to vaccine development. in the field of vaccines, vera-velasco et al not only quantified the viral f, g and m proteins present in the viral particles but also analyzed the cellular proteomic composition in the niv vaccine candidate. traditionally, viral particles have been described as pure entities carrying only viral-derived proteins but the authors successfully analyzed the ratio between cellular and viral proteins in the niv vaccine candidate using lc-ms/ms. west nile fever is a flavivirus that causes a viral infection typically spread by mosquitoes. to date, no vaccine is available to prevent the west nile infection. partially purified virus-like particles were resolved by sds-page, and the coomassie blue-stained band corresponding to env protein was excised from the gel, destained, and analyzed by ms. microcapillary lc-ms was performed using a lcq deca ion-trap mass spectrometer (thermo finnigan) to identify the presence of env protein in virus-like particles to help in developing a potential vaccine. in should be a supplemental batch release test to ensure that the product is within specifications and without any unwarranted modifications. using ms techniques will ensure improved quality and increased safety for clinical trial participants. in conclusion, the evidence suggests that the emergence of ms-based techniques has advanced the field of vaccine development. history of vaccination vaccines, new opportunities for a new society research and development of new vaccines against infectious diseases recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production. hum vaccines immunother the challenge of emerging and reemerging infectious diseases emerging infectious diseases: threats to human health and global stability establishing a global vaccinedevelopment fund new vaccines against epidemic infectious diseases recombinant subunit vaccines: potentials and constraints recombinant therapeutic protein vaccines virus-like particles as vaccine viral vaccines and their manufacturing cell substrates: new trends and designs in modern vaccinology cell culture technology protein subunit vaccine purification cgmp production and analysis of bg sosip. , an extensively glycosylated, trimeric hiv- envelope glycoprotein vaccine candidate a systematic approach to protein glycosylation analysis: a path through the maze structural mass spectrometry in biologics discovery: advances and future trends impact of host cell line choice on glycan profile therapeutic glycoprotein production in mammalian cells analysis of protein glycosylation by mass spectrometry carbohydrates on proteins: site-specific glycosylation analysis by mass spectrometry recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry the quest for an antibody-based hiv vaccine developing an hiv vaccine vaccination with alvac and aidsvax to prevent hiv- infection in thailand new approaches to hiv vaccine development antigenicity and immunogenicity of rv vaccine aidsvax clade e envelope immunogen is enhanced by a gp n-terminal deletion comparable antigenicity and immunogenicity of oligomeric forms of a novel, acute hiv- subtype c gp envelope for use in preclinical and clinical vaccine research hiv- envelope glycoprotein immunogens to induce broadly neutralizing antibodies the hiv glycan shield as a target for broadly neutralizing antibodies assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type recombinant human immunodeficiency virus envelope glycoprotein (gp ) expressed in chinese hamster ovary cells comparison of hplc/esi-fticr ms versus maldi-tof/tof ms for glycopeptide analysis of a highly glycosylated hiv envelope glycoprotein glycosylation site-specific analysis of hiv envelope proteins (jr-fl and con-s) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopesʼ accessibility glycosylation site-specific analysis of clade c hiv- envelope proteins characterization of glycosylation profiles of hiv- transmitted/founder envelopes by mass spectrometry methods development for analysis of partially deglycosylated proteins and application to an hiv envelope protein vaccine candidate analysis of the disulfide bond arrangement of the hiv- envelope protein con-s gp Δcfi shows variability in the v and v regions characterization of host-cell line specific glycosylation profiles of early transmitted/founder hiv- gp envelope proteins glycosylation and disulfide bond analysis of transiently and stably expressed clade c hiv- gp trimers in t cells identifies disulfide heterogeneity present in both proteins and differences in o-linked glycosylation influences on the design and purification of soluble, recombinant native-like hiv- envelope glycoprotein trimers generation and characterization of a bivalent hiv- subtype c gp protein boost for proof-ofconcept hiv vaccine efficacy trials in southern africa glycosylation benchmark profile for hiv- envelope glycoprotein production based on eleven env trimers cell-and protein-directed glycosylation of native cleaved hiv- envelope composition and antigenic effects of individual glycan sites of a trimeric hiv- envelope glycoprotein molecular architecture of the cleavage-dependent mannose patch on a soluble hiv- envelope glycoprotein trimer glycosylation profiling to evaluate glycoprotein immunogens against hiv- envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens the glycan shield of hiv is predominantly oligomannose independently of production system or viral clade structural constraints determine the glycosylation of hiv- envelope trimers ion mobility mass spectrometry for extracting spectra of n-glycans directly from incubation mixtures following glycan release: application to glycans from engineered glycoforms of intact, folded hiv gp maldi-ms/ms with traveling wave ion mobility for the structural analysis of n-linked glycans ion mobility mass spectrometry for ion recovery and clean-up of ms and ms/ms spectra obtained from low abundance viral samples global nglycan site occupancy of hiv- gp by metabolic engineering and high-resolution intact mass spectrometry structural principles controlling hiv envelope glycosylation global site-specific nglycosylation analysis of hiv envelope glycoprotein comprehensive characterization of reference standard lots of hiv- subtype c gp proteins for clinical trials in southern african regions cell culture as a substrate for the production of influenza vaccines: memorandum from a who meeting current and emerging cell culture manufacturing technologies for influenza vaccines critical review of current and emerging quantification methods for the development of influenza vaccine candidates influenza vaccine: the challenge of antigenic drift confronting the next pandemic-workshop on lessons learned from potency testing of pandemic (h n ) influenza vaccines and considerations for future potency tests application of deglycosylation and electrophoresis to the quantification of influenza viral hemagglutinins facilitating the production of pandemic influenza (h n ) vaccines at multiple manufacturing sites in china quantification of influenza virus hemagglutinins in complex mixtures using isotope dilution tandem mass spectrometry optimization of digestion parameters for protein quantification simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry quantification of viral proteins of the avian h subtype of influenza virus: an isotope dilution mass spectrometry method applicable for producing more rapid vaccines in the case of an influenza pandemic development of influenza a (h n ) candidate vaccine viruses with improved hemagglutinin antigen yield in eggs quantification of immunoreactive viral influenza proteins by immunoaffinity capture and isotope-dilution liquid chromatography-tandem mass spectrometry immunocapture isotope dilution mass spectrometry in response to a pandemic influenza threat quantification of influenza neuraminidase activity by ultra-high performance liquid chromatography and isotope dilution mass spectrometry label-free mass spectrometry-based quantification of hemagglutinin and neuraminidase in influenza virus preparations and vaccines quantification of proteins by label-free lc-mse absolute quantification of proteins by lcms e a virtue of parallel ms acquisition selective and quantitative detection of influenza virus proteins in commercial vaccines using two-dimensional high-performance liquid chromatography and fluorescence detection approach to the profiling and characterization of influenza vaccine constituents by the combined use of size-exclusion chromatography, gel electrophoresis and mass spectrometry strain identification of commercial influenza vaccines by mass spectrometry simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by lc-ms e fast and highly selective determination of hemagglutinin content in quadrivalent influenza vaccine by reversed-phase high-performance liquid chromatography method advancing dengue vaccine development site-specific characterization of envelope protein n-glycosylation on sanofi pasteur's tetravalent cyd dengue vaccine absolute quantification of dengue virus serotype chimera vaccine candidate in vero cell culture by targeted mass spectrometry after ebola in west africaunpredictable risks, preventable epidemics identification of n-glycans from ebola virus glycoproteins by matrix-assisted laser desorption/ ionisation time-of-flight and negative ion electrospray tandem mass spectrometry comparison of n-and o-linked glycosylation patterns of ebolavirus glycoproteins development of a liquid chromatography high resolution mass spectrometry method for the quantitation of viral envelope glycoprotein in ebola virus-like particle vaccine preparations chikungunya virus: pathophysiology, mechanism, and modeling development and application of a reversed-phase high-performance liquid chromatographic method for quantitation and characterization of a chikungunya virus-like particle vaccine characterization of nglycosylation profiles from mammalian and insect cell derived chikungunya vlp label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group b outer membrane vesicle vaccine physicochemical characterisation of glycoconjugate vaccines for prevention of meningococcal diseases defined conjugation of glycans to the lysines of crm guided by their reactivity mapping quantification by lc-ms e of outer membrane vesicle proteins of the bexsero ® vaccine the dual role of lipids of the lipoproteins in trumenba, a self-adjuvanting vaccine against meningococcal meningitis b disease robust manufacturing and comprehensive characterization of recombinant hepatitis e viruslike particles in hecolin ® hepatitis e virus: a renewed hope with hecolin molecular and structural characterization of the l virus-like particles that are used as vaccine antigens in cervarix™, the as -adjuvanted hpv- and- cervical cancer vaccine viruslike particles in vaccine development virus-like particle-based human vaccines: quality assessment based on structural and functional properties status of vaccine research and development of vaccines for nipah virus proteomic composition of nipah virus-like particles production of immunogenic west nile virus-like particles using a herpes simplex virus recombinant vector characterization of human enterovirus virus-like particles used for vaccine antigens mass spectrometric characterization of the glycosylation pattern of hiv-gp expressed in cho cells glycan profiles of the n-glycosylation sites of the hiv envelope protein cn gp comparative analysis of the glycosylation profiles of membrane-anchored hiv- envelope glycoprotein trimers and soluble gp mapping the complete glycoproteome of virion-derived hiv- gp provides insights into broadly neutralizing antibody binding haemagglutinin quantification and identification of influenza a&b strains propagated in per. c ® cells: a novel rp-hplc method hplc-based quantification of haemagglutinin in the production of egg-and mdck cell-derived influenza virus seasonal and pandemic vaccines optimization and qualification of a quantitative reversed-phase hplc method for hemagglutinin in influenza preparations and its comparative evaluation with biochemical assays characterization of a recombinant influenza vaccine candidate using complementary lc-ms methods comparative glycomics analysis of influenza hemagglutinin (h n ) produced in vaccine relevant cell platforms glycosylation analysis of engineered h n influenza a virus hemagglutinins with sequentially added historically relevant glycosylation sites particle quantification of influenza viruses by high performance liquid chromatography design and expression of a qconcat protein to validate hi protein quantification of influenza vaccine antigens evaluation of haemagglutinin content by rp-hplc to generate pandemic influenza vaccine topological n-glycosylation and site-specific n-glycan sulfation of influenza proteins in the highly expressed h n candidate vaccines glycosylation characterization of an influenza h n hemagglutinin series with engineered glycosylation patterns: implications for structurefunction relationships cross reactive material glycoconjugate vaccines contain privileged conjugation sites quality by design approach in the development of an ultra-high-performance liquid chromatography method for bexsero meningococcal group b vaccine proteomics-driven antigen discovery for development of vaccines against gonorrhea production and purification of filovirus glycoproteins in insect and mammalian cell lines key: cord- -uo hdrgc authors: de vries, rory d.; rennick, linda j.; duprex, w. paul; de swart, rik l. title: paramyxovirus infections in ex vivo lung slice cultures of different host species date: - - journal: methods protoc doi: . /mps sha: doc_id: cord_uid: uo hdrgc in vivo experiments in animal models of disease are of crucial importance for viral tropism and pathogenesis studies. however, these experiments must be complemented with in vitro and ex vivo experiments. here, we describe a protocol for the preparation and ex vivo infection of lung slices from different mammalian host species with various respiratory paramyxoviruses expressing fluorescent reporter proteins, and suggest follow-up experiments including immunohistochemistry, flow cytometry and confocal microscopy. ex vivo models provide an important bridge between in vitro and in vivo experiments. the use of agarose-inflated lung slices for respiratory virus pathogenesis studies has been described previously [ ] [ ] [ ] [ ] [ ] [ ] . here, we describe a protocol in which agarose-inflated lung slices can be kept viable in culture for at least seven days post-necropsy of an experimental animal. the combination of these viable lung slices with recombinant viruses expressing fluorescent reporter proteins [ ] [ ] [ ] allows for accurate, sensitive and reproducible assessment of respiratory virus infection and dissemination over time. furthermore, use of these recombinant viruses allows for real time monitoring of infection processes, using multiple methods for measurement of fluorescence (e.g., flow cytometry and confocal laser scanning microscopy). lung slices are also suitable for analysis by immunohistochemistry, thereby visualizing virus cell tropism and spatial localization of infected cells within the tissue. we have validated this technique by infecting lung slices of multiple host species (cotton rats, ferrets, dogs and macaques) with various paramyxoviruses expressing fluorescent reporter proteins (measles virus (mv), canine distemper virus (cdv), human respiratory syncytial virus (hrsv) and human metapneumovirus (hmpv)) [ ] . this technique, however, is directly transferable to different host species and different viruses [ ] . this protocol describes the ex vivo agarose inflation of lungs and generation of slices for virus infection. lungs should be taken from the experimental animal during necropsy, keeping the material as intact as possible and attached to the primary bronchus and trachea. using a (blunt-end) needle or flexible catheter, the fresh lungs are inflated through the trachea (or primary bronchus, if inflation of a half lung or single lobe is desired) with low-melting point agarose mixed with culture medium. after solidification on ice, slices can be prepared by hand using microtome blades and cultured in -or -well plates with culture medium, dependent on the size of the slices. these lung slices are subsequently inoculated with infectious virus (or could be used for any other experimental purpose) and can be followed in time by phase-contrast inverted light microscopy or fluorescence, when reporter viruses expressing fluorescent proteins are used (see figure ). in addition, emigrant cells in the culture medium can be harvested in time and analyzed by flow cytometry. although these emigrant cells could be present due to tissue degradation, they can be detected at early time-points after refreshing the culture medium, suggesting they are mobile emigrant cells. we have detected cells from lymphoid, myeloid and epithelial origin using this method. at the end of the experiment, slices can be used as desired; potential applications include fixation followed by paraffin embedding and immunohistochemistry, or generation of single cell suspensions to be analyzed via flow cytometry. . after solidification on ice, thin slices of approximately -mm thick can be cut by hand (c) and are transferred to -or -well plates pre-filled with culture medium (d). slices can subsequently be inoculated with the desired virus (e) and infection of the slices is followed in time (f). in this example in panel f, a macaque lung slice infected with recombinant measles virus is shown. all animals used in these studies were housed and experiments were conducted in strict compliance with european guidelines (eu directive on animal testing / /eec) and dutch legislation (experiments on animals act, ). protocols were approved by the independent animal experimentation ethical review committee dcc in driebergen, the netherlands. animal welfare was observed on a daily basis. critical step prepare % (w/v) low-melting point agarose by dissolving agarose in phosphate-buffered saline (pbs) and boil for several minutes (e.g., using a microwave oven, be careful for boiling over). as an alternative, agarose can be prepared by autoclaving. be sure to allow agarose to cool down to • c (e.g., in a water bath) before inflating the lungs. the volume of low-melting point agarose should be adjusted according to the size of the lungs. resect the lungs, including the distal part of the trachea, from the experimental animal. critical step resection should be performed within h after euthanasia, preferably sooner. critical step be sure not to damage the lungs while resecting, as this will interfere with the inflation process. locate the trachea (for small species) or primary bronchus (for larger species) through which you wish to inflate. for smaller mammals, inflation through the trachea works best, but for larger animals (body weight > - kg) inflation through the primary bronchus is more efficient. fill a syringe with a • c pre-warmed : mixture of % (w/v) low-melting point agarose and lung slice medium (dmem/ham's f medium supplemented with % (v/v) fbs, penicillin, streptomycin, l-glutamine and amphotericin b). final agarose percentage is % (w/v). critical step avoid the formation of air bubbles in the agarose. decide on the size of the syringe dependent on the size of the trachea or bronchus through which you will inflate. either the pipette tip, blunt-end needles, catheters, cannulas the end of the syringe should be a tight fit into the trachea or bronchus. insert the end of the syringe into the trachea or primary bronchus, enforce placement inside the trachea or bronchus by clamping (or use sutures as an alternative) the trachea or bronchus around the needle. critical step be careful not to damage the trachea or bronchus while inserting the needle into the trachea or bronchus. we advise the use of pipette tips, blunt-end needles, catheters or cannulas instead of sharp needles at the end of the syringe. . inject the proper amount of agarose into the lungs. check inflation while injecting; keep adding agarose until the lungs are completely inflated (see video s ). critical step it is not always easy to determine the best level of inflation. in our experience, the volume used for inflation should be slightly higher than the tidal volume of the animal, which is usually around ml per kg body weight. if you are inflating just one lobe of the lung, the volume should be adapted accordingly. remove needle but keep the clamp positioned on the trachea or bronchus and allow the inflated lungs to solidify on ice ( min for smaller species and - min for larger species). transfer lungs to biosafety cabinet, remove the clamp, prepare a surface for cutting (cutting board or petri dish) and microtome blade. prepare the lungs for slicing by finding anatomically interesting locations. critical step dependent on the focus of the experiment, you have to decide which part is anatomically interesting. decide on whether you want to use for example the left or right side, and either the upper or lower parts of available lobes. to check viability, we advise to include a cut of the primary bronchus in the slice, to determine the beating of cilia indicating viable epithelial cells. start with an initial cut to ensure that a straight edge is available for cutting. now manually prepare slices of approximately mm thickness. if desired, the lungs can be attached to the cutting board by needles (see video s ). as a rule of thumb, slices are thin enough for culture if you can see through the slice while cutting it and actually see the microtome blade. gently transfer cut slices into -, -, -, or -well plates (dependent on size of the slices) that were pre-filled with lung slice culture medium. infection of slices can be performed immediately. pause step slices can also be infected after h of culture at • c in % (v/v) co . we have never observed discrepancies between obtained results after either direct infection or after overnight culture. transfer the lung slices into empty -, -, -, or -well plates (dependent on size of the slices). gently add virus (preferably a recombinant virus expressing a fluorescent reporter protein) in a drop wise fashion onto the lung slice. typically, we used an inoculation volume of µl; always include a mock infection as negative control. we typically use - × tissue culture infectious dose- (tcid ) per slice; variations are possible. . incubate at • c in % (v/v) co for h. after h, add an appropriate volume of lung slice medium (inoculum can be washed away) and return plates to • c in % (v/v) co . . in our experience, slices can be held viable (based on beating cilia in epithelium) and followed in time for a maximum of days. culture medium has to be replaced to ensure viability every other day (i.e., day , and ). critical step when replacing culture medium, medium should be taken off and directly replaced, not allowing the lung slices to dehydrate. beating of cilia is the best measure for viability, however when beating of cilia is no longer observed slices start disintegrating. depending on the goal of the experiment, non-viable slices should be removed or kept in culture. slices should be checked for viability on daily basis: find a bronchus with cilia under a normal light microscope. movement (see video s ) of cilia is indicative of viable epithelial cells, suggesting there are live cells in culture. if fluorescent reporter viruses were used, slices should be checked for fluorescence on a daily basis (see video s and figure ). use a macroscopic imaging lamp as described previously [ ] in combination with an emission filter to visualize fluorescence macroscopically. shine lamp directly on lung slices and find fluorescent spots. photographs can be acquired if desired. use an inverted uv microscope or confocal laser scanning microscope to identify fluorescent-positive cells. during infection and culture, effluent cells can be harvested and checked for expression of fluorescent proteins by flow cytometry if desired. harvest supernatants from slices (e.g., on day , and while replacing culture medium), transfer supernatant to tubes and centrifuge ( - min, × g). transfer pellets to -wells v-bottom plate, wash once in facs buffer (pbs + . % (v/v) bsa + mm edta). optional step to determine which subsets of cells are infected, a fluorescence activated cell sorter (facs) staining for specific cell types can be performed at this point. cells that migrate out of the lung slices are mainly lymphoid, myeloid or epithelial in origin. to study the tropism of infection you can selectively stain for example t-cells, b-cells, natural killer (nk) cells, neutrophils, granulocytes, dendritic cells, macrophages or epithelial cells, or combinations thereof. acquire cells on flow cytometer to determine number of virus-positive cells. multiple experiments can be performed at the end of culture or at desired time points. slices can be directly stained for confocal laser scanning microscopy (section . . ), fixed for immunohistochemistry (section . . ) or used to generate a single cell suspension for flow cytometry (section . . ). critical step not all fluorophores are well-preserved by pfa fixation. sensitivity of fluorophores to fixation should be tested beforehand, and the fixative of mounting medium can be adjusted accordingly. wash slices after min and permeabilize in . % (v/v) triton x- for min. wash slices and subsequently stain with fluorescent antibodies of choice. critical step to understand tissue morphology, it is important to counterstain nuclei (for example with to-pro- or nucblue) and add a marker of choice. we found it useful to stain cilia using an antibody against class iv β-tubulin. transfer slices to thin-bottom/glass-bottom dishes. view fluorescence by confocal laser scanning microscopy (using an inverted microscope, see figure ). since slices are approximately -mm thick, z-stacks can be generated and from those d images can be rendered. in addition to using lung slices as an ex vivo culture and infection system, the method can also be used to screen the lungs of in vivo virus-infected animals for infection and determine the viral tropism (e.g., see reference [ ] ). two approaches are possible: instead of inflating lungs with the : mixture of % (w/v) low-melting point agarose and lung slice medium, prepare a : mixture of % (w/v) low-melting point agarose and % (w/v) pfa in pbs. the rest of the protocol is identical, after preparing lung slices these can be screened by fluorescence microscopy directly, virus-positive slices can be processed further as desired (immunohistochemistry, staining for confocal laser scanning microscopy, preparation of single cell suspensions for flow cytometry). as an alternative, slices can be inflated with : mixture of % (w/v) low-melting point agarose and lung slice medium and kept in culture for a period of time after necropsy, to evaluate viral tropism and dissemination [ ] . with the protocol described here, dependent on the size of the species used in the experiment, viable lung slices from potentially any species can be obtained for ex vivo experiments. we have observed viability of these slices for up to seven days post resection; however, this could be dependent on the species and culture conditions. infections with respiratory viruses of lung slices are normally relatively successful: figures and show examples of ex vivo paramyxovirus-infected lung slices. in these experiments, macaque lungs were inflated and infected with mv, which is clearly able to infect the macaque lung slices. of course, viruses corresponding to the target species should be chosen to obtain positive results. in the family of paramyxoviridae for example, viruses exclusively infecting primates or carnivores exist. measles virus, a virus of primates, successfully infected non-human primate but not dog lungs, whereas cdv, a virus of carnivores, behaved vice versa. using viruses expressing fluorescent reporter proteins allows for sensitive detection of virus-infected cells in these experiments; however, these experiments can also be performed with non-fluorescent viruses. staining should still make infected cells visible in immunohistochemistry, confocal laser scanning microscopy or flow cytometry. the model described here of course has some limitations, as a single region of the lung is directly infected, often with a relatively high inoculum. therefore, in vivo experiments in animal models of disease are of crucial importance for viral tropism and pathogenesis studies. however, these experiments must be complemented with proper in vitro and ex vivo experiments, indicating the potential of ex vivo experiments in these cultured lung slices. % agarose: prepare correct weight of hydroxyethylagarose in dpbs; • lung slice medium: dmem/ham's nutrient mixture f powder, glutamax, fbs, penicillin/streptomycin, amphotericin; • fluorescence activated cell sorter buffer: pbs + . % (v/v) bsa + mm edta (use stock of . m in distilled water); • paraformaldehyde: dissolve appropriate weight in pbs with high ph by initially supplementing with naoh. after pfa has completely dissolved, ph should be re-adjusted to . . always prepare fresh or store aliquoted at − • c. do not use buffered formalin when directly screening fluorescence, as this will disrupt the fluorescence of most reporter proteins. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . video s : inflation of cotton rat lungs. syringe is filled with agarose mixed with lung slice medium and inserted into the trachea, kept in place by a suture. lungs are slowly inflated by injecting agarose and medium into the trachea; video s : slicing of cotton rat lungs. solidified cotton rat lungs are places on petri-dish and subsequently sliced. an initial cut is made for a straight edge followed by the preparation of a single lung slice; video s : viability screening of lung slice. a bronchus consisting of viable epithelial cells with beating cilia; video s : fluorescence screening of live lung slice infected with a paramyxovirus expressing a fluorescent reporter protein (green). the movie initially shows a combination of normal light and fluorescent light, followed by a shutdown of the normal light, indicating the identical area but now with fluorescence light only. time for completion: h . wash slices twice in hank's balanced salt solution (hbss) transfer slices into petri dishes and manually cut in small pieces transfer pieces into -well plate pre-filled with hbss + collagenase ( units/ml) + dnase ( . mg/ml) incubate for h on rocking platform at • c in % (v/v) co prepare single cell suspension by straining small pieces over µm cell strainer centrifuge and lyse red blood cells by adding ml red blood cell lysis buffer to pellet, incubate • c in % (v/v) co for three minutes centrifuge, resuspend pellet in facs buffer and perform facs staining of choice acetylcholine-induced calcium signaling and contraction of airway smooth muscle cells in lung slices responsiveness of individual airways to methacholine in adult rat lung explants early target cells of measles virus after aerosol infection of non-human primates a prominent role for dc-sign + dendritic cells in initiation and dissemination of measles virus infection in non-human primates pb -f modulates early host responses but does not affect the pathogenesis of h n seasonal influenza virus long-term maintenance of mature pulmonary parenchyma cultured in serum-free conditions predominant infection of cd + lymphocytes and dendritic cells during measles virus infection of macaques an improved plaque reduction virus neutralization assay for human metapneumovirus observation of measles virus cell-to-cell spread in astrocytoma cells by using a green fluorescent protein-expressing recombinant virus paramyxovirus infections in ex vivo lung slice cultures of different host species asymptomatic middle east respiratory syndrome coronavirus infection in rabbits the authors wish to thank geert van amerongen for providing excellent technical assistance during the establishment of this technique. we acknowledge nih for their ongoing funding of respiratory virus pathogenesis studies, specifically grant ai to w.p.d. the authors declare no conflict of interest. key: cord- -q exf s authors: toosy, arshad haroon; o'sullivan, sean title: an overview of middle east respiratory syndrome in the middle east date: - - journal: fowler's zoo and wild animal medicine current therapy, volume doi: . /b - - - - . - sha: doc_id: cord_uid: q exf s nan middle east respiratory syndrome (mers) is an emerging infectious zoonotic disease caused by a novel coronavirus (cov). mers was first reported in in jeddah, kingdom of saudi arabia (ksa), and in jordan, respectively. the disease was considered a potential pandemic threat to public health in the persian gulf region. (see also chapter .) most known covs infect and circulate in animals, mainly bats, but a number of covs are known to cause human disease (see also chapter ). [ ] [ ] [ ] the rapid emergence of mers-cov coupled with its limited geographic distribution has led to the suspicion that this is a zoonotic disease with an animal reservoir, and the evidence supports the hypothesis that dromedary camels (dcs) are the reservoir host. in dcs mers-cov causes a mild, transient upper respiratory tract (urt) infection. [ ] [ ] [ ] a mild or asymptomatic disease has also been reported in humans, but this is not always the case. mers-cov infection in humans often results in a severe, life-threatening disease of the lower respiratory tract (lrt), with high mortality. , , immunocompromised, elderly people and those with comorbidities, usually with a history of close contact with infected dcs, are particularly susceptible. is a positive-sense enveloped single-stranded rna virus and is the first lineage of c betacoronavirus known to infect humans. , it is more closely related to bat covs hku and hku (lineage c) than to the severe acute respiratory syndrome cov (sars-cov, (lineage b). , recent genome sequencing analysis reported the genomic evolution rate ( . × substitutions per site), suggesting that mers-cov diverged from its viral ancestor in march . analysis of human mers-cov sequences has identified several circulating genotypes. these distinct genotypes are phylogenetically classified into clades a, b, and, most recently, c, which correlate with outbreaks of mers among humans. , , , the emergence of divergent mers-cov clades in humans since is consistent with several independent sporadic introductions into the human population from an animal reservoir, of which the camel was unquestionably the source. , , , pathogenesis host cell entry of mers-cov is mediated by the binding of mers spike (s) proteins to a specific cellular receptor known as dipeptidyl peptidase (dpp ). dpp is expressed on the epithelial and endothelial cells of most human organ tissues in ex vivo studies using human tissue culture lines; this may account for the multisystem clinical spectrum of the mers-cov infection. , a strain cultured from a fatal human case was experimentally inoculated into three dcs using intratracheal, intranasal, and conjunctival routes. a mild transient disease resulted in submucosal inflammation and necrosis in the urt and lrt, but the alveoli remained unaffected. experimental inoculation of rhesus macaques (macaca mulatta) and common marmosets (callithrix jacchus) resulted in mild to severe lrt disease causing multifocal interstitial pneumonia in the macaques and extensive fatal pneumonia in the marmosets. , , since at least . several mers-cov serologic surveys confirm that the disease is not present in domesticated livestock (namely, horses, sheep, goats, and cattle) and is enzootic in the dc population across the arabian peninsula as well as in north and east africa. , , , , [ ] [ ] [ ] [ ] [ ] historically, the camel was the mainstay of land-based trade transportation and was used extensively as a food source across the entire region prior to industrialization during the latter part of the th century. , the free movement of humans and animals across the region supports the widespread prevalence and genetic diversity of mers-cov in the dc populations of arabia and east africa today. , the temporal dynamics of mers infection in dcs in al-ahsa, ksa, was examined by collecting nasal swabs and lung tissue during postmortem examination from two independent groups of animals over the course of a year and testing these for mers-cov rna by real-time reverse-transcriptase polymerase chain reaction (rt-rtpcr). positive samples were typically associated with young immunologically naive animals (< years of age) rather than adults (> years of age). seasonal peaks were detected during the winter months and coincided with the calving season, less extreme environmental conditions, cooler ambient temperatures, and higher relative humidity, for the transmission of infection amongst susceptible individuals. this seasonal peak has also been described in epidemic nosocomial outbreaks in humans that occur more frequently during the winter months. , extensive virologic evidence has been accumulated since supporting the epidemiologic link between dcs and humans in the transmission of mers-cov, although epidemiology mers-cov belongs to a lineage commonly associated with bats, the closest relatives of which lineage were recently identified in vesper bats (i.e., various species of the family vespertilionidae) from europe, asia, and south africa. initial research efforts have focused on establishing an epidemiologic link between bats and humans. , , , there is no conclusive evidence to support the theory that bats are the source of human infection, although there is consensus that bats are the ancestral hosts of the disease. , , a related mers-like cov virus, isolated from an african pipistrelle bat (pipistrellus hesperidus) in uganda, has shown high divergence of the s protein nucleotide sequence compared with an index mers-cov s protein sequence ( % amino acid identity divergence). this suggests that the two viruses differ significantly in receptor binding properties, implying that the mers-like cov virus is not a zoonotic threat and supporting the theory of a common ancestry. to date the only direct link between bats and human disease was a single instance when an rna sequence from a fatal human mers index case showed a % nucleotide match of a polymerase chain reaction (pcr)-amplified sequence of a fecal pellet from an egyptian tomb bat (taphozous perforates) collected in the same area of bisha, ksa. the human fatality was an owner of four dcs, which also tested positive for the same strain of mers-cov. at present, bat-to-human infection by mers-cov is considered to be purely speculative. surveillance of dcs in ksa has shown that mers-cov clade b has been enzootic in the camel population in arabia genetic deep sequencing methods (i.e., high-throughput sequencing) have been readily available to researchers since the disease was first reported. sequenced data have been used in these cases to successfully construct the phylogenetic tree between related viruses and hosts. , , , direct mers-cov antigen detection is possible but has been rarely performed. immunochromatography assays and monoclonal antibody-based capture elisas targeting the mers-cov nucleocapsid protein have been described. since the virus was first reported in , a range of comprehensive laboratory tests has been developed. , to better understand the disease, it has been important to collate sampling methodology data, laboratory results, and analyses in combination with clinical and epidemiologic data. until laboratory assays are fully validated, a combination of molecular and serologic laboratory tests is required to improve confidence in laboratory diagnosis during outbreaks. in cases of mild or asymptomatic infection, full validation of serologic assays is required to rule out false-negative results. validation is also required to successfully apply newly developed diagnostic serology algorithms to inform public health decisions. , , treatment therapeutic options for mers-cov are limited. supportive treatment is indicated for hospitalized patients, but vigilance for complications is essential. empirical use of antimicrobial agents or steroids has not succeeded in reversing the progression of severe disease. , , no specific drug or vaccine is currently available to treat mers. indeed, it has been stated that the complexity and time required for the development and registration process of drugs for human use impedes the ability to counter the rapid threat against an emerging infectious agent. for example, there is no vaccine available against sars-cov because of the brevity of the threat to the public health. , it is likely that a mers-cov vaccine for human use may not be developed due to a lack of commercial interest, or if the threat posed by mers-cov declines in the meantime. nevertheless, given the prevalence of mers-cov infection in the middle east's dc population and due to the potential for spillover to the human population in direct contact with dcs, the development of a vaccine for use in dcs may be feasible. , , a recent successful trial of a mers orthopoxvirus vaccine has conferred mucosal immunity in the urts of dcs. eradication of mers-cov from herds may be possible, if vaccines are administered to young, immunologically naive camels prior to exposure. , , identification of the zoonotic source of mers guides control strategies at the human-animal interface. , by preventing spillover of mers-cov from animals to humans, the risk of nosocomial and familial outbreaks in the middle east could be eliminated. strategic serosurveys of humans using samples collected after have been infrequent. , , there is a paucity of baseline data to describe the proportion of the potentially infected human population for much of the arabian peninsula and all of east africa, including the horn of africa. the exact mechanism of transmission from camels to human remains uncertain. sustained close contact is most probably necessary for transmission by aerosolized droplets, as mers-cov viral rna has been detected in air samples from a barn housing infected dcs in qatar, and the virus may remain viable in aerosol for up to minutes. , [ ] [ ] [ ] the potential public health risk resulting from aerosol-generating activities ranges from contamination of a room occupied by a symptomatic patient to slaughter practices. , , aerosolized transmission of mers-cov has been attributed to hospital outbreaks in ksa and south korea. , , mers-cov spreads inefficiently from human to human, but transmission is effective in a hospital environment, where susceptible individuals are concentrated and the risks are amplified by poor infection prevention and control (ipc) protocols. in some reported cases of mers, direct contact with camels was not apparent. , camel-to-human transmission through other routes is, however, possible owing to the consumption of unpasteurized camel milk or raw camel meat and in traditional medicine, when camel urine is consumed as a natural remedy for a variety of ailments. , a recent survey has found that infected camels may shed mers-cov virus in milk and urine, and the virus has been shown to remain infectious for days in milk stored at °c. , the transmission risks associated with the handling of camel products, raw milk, urine, and meat during animal slaughter are yet to be fully elucidated. further studies are needed to demonstrate the potential of camel-to-human transmission. serologic methods with high sensitivity and specificity to detect mers-cov antibodies have been developed for use in seroepidemiologic studies. methods include indirect immunofluorescence assays, enzyme-linked immunosorbent assays (elisas), protein microarray technology, and microneutralization (mn) assays. , [ ] [ ] [ ] pseudoparticle virus neutralization tests (ppnts) and conventional mn assays have also been used to detect neutralizing antibodies to mers-cov. validated molecular assays have been developed. , , rt-rtpcr is the preferred diagnostic method for the detection of mers-cov and has been endorsed by the who. confirmation of mers in suspected cases requires the screening of samples targeting a number of genes specific to mers-cov, namely up e, orf a, orf b, and n genes. , , , its infancy. aside from bats, the role that other wildlife may play in the ecology of mers-cov in east africa and arabia is yet to be elucidated. at present the implementation of intensive ipc measures in human health care is vital, including improving education and awareness among healthcare workers. , most human cases have been linked to lapses in ipc, as one-fifth of viral infections have been reported among healthcare workers. , stringent precautions while handling suspected mers-cov patients include the use of personal protective equipment (ppe) (i.e., disposable gloves, gowns, respiratory protection, and eye protection). , , immunocompromised individuals and those with preexisting medical conditions should avoid close contact with dcs. , public health authorities should adopt a standardized public health response protocol to include standardized case reporting methodology as defined by the who. , standardization of case definitions aids accurate calculation of a case fatality ratio by including mild or asymptomatic cases. the health authority of abu dhabi in the united arab emirates recently implemented a standardized reporting option for mers, successfully incorporating it into existing epidemiologic surveillance systems with the aim of enhancing surveillance, educating healthcare workers, and ensuring laboratory capacity. in countries where mers-cov is enzootic in dcs, mers control at the animal-human interface is unlikely to succeed unless appropriate preventive strategies are implemented. these should include the following: • strict regulation of camel movement with imposition of a requirement for mers clearance prior to the importation and transport of camels, including animals presented for slaughter. • camels with detectable mers-cov rna should be quarantined and tested at regular intervals. • use of appropriate ppes while handling dcs. • increased awareness among camel owners and the general public of the risks of consuming unpasteurized camel milk and urine. this may prove challenging given the depth of customs and beliefs in some areas. • accelerated development of safe and effective mers vaccines for animal and human use. , conclusions mers-cov has been observed for only years, and vigilance is vital for the containment of the disease due to the high case fatality rate in humans and possible genetic instability of the virus. continued laboratory testing, genetic sequencing, analysis, timely data sharing, and clear communication are essential if such vigilance is to be effective. nonetheless, despite the potential for a pandemic outbreak at multiple mass gatherings during the islamic calendar (hajj, eid, and umrah) there were no reported outbreaks of mers during or immediately after these events. as such mers-cov is not a virus of pandemic concern. since our understanding of mers has increased greatly although gaps in knowledge still exist. the understanding of the disease's ecology-especially the interplay between camels, humans, and the environment-is still in who mers-cov global summary and risk assessment middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps evidence for zoonotic origins of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd who emergencies: mers-cov. available at mers coronavirus: diagnostics, epidemiology and transmission dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia pathogenesis of middle east respiratory syndrome coronavirus replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels response to emergence of middle east respiratory syndrome coronavirus transmission of middle east respiratory syndrome coronavirus infections in healthcare settings hospital outbreak of middle east respiratory syndrome coronavirus detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient mers-cov outbreak in jeddah-a link to health care facilities mers coronavirus: data gaps for laboratory preparedness asymptomatic mers-cov infection in humans possibly linked to infected camels imported from oman to united arab emirates an orthopoxvirusbased vaccine reduces virus excretion after mers-cov infection in dromedary camels centers for disease control and prevention: interim prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov). available at an animal model of mers produced by infection of rhesus macaques with mers coronavirus further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronaviruses in dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia antibodies against mers coronavirus in dromedaries middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan isolation of mers coronavirus from a dromedary camel mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia the authors wish to thank the following: h. e. ghanim mubarak al hajeri and the senior management of al ain zoo for their encouragement and support, dr. ahsan ul haq of dubai camel hospital for his technical insight, and dr. andrew higgins, fellow of the zoological society of london and honorary editor in chief of the veterinary journal, who reviewed the manuscript. key: cord- -bvh o r authors: galasiti kankanamalage, anushka c.; kim, yunjeong; damalanka, vishnu c.; rathnayake, athri d.; fehr, anthony r.; mehzabeen, nurjahan; battaile, kevin p.; lovell, scott; lushington, gerald h.; perlman, stanley; chang, kyeong-ok; groutas, william c. title: structure-guided design of potent and permeable inhibitors of mers coronavirus cl protease that utilize a piperidine moiety as a novel design element date: - - journal: european journal of medicinal chemistry doi: . /j.ejmech. . . sha: doc_id: cord_uid: bvh o r abstract there are currently no approved vaccines or small molecule therapeutics available for the prophylaxis or treatment of middle east respiratory syndrome coronavirus (mers-cov) infections. mers-cov cl protease is essential for viral replication; consequently, it is an attractive target that provides a potentially effective means of developing small molecule therapeutics for combatting mers-cov. we describe herein the structure-guided design and evaluation of a novel class of inhibitors of mers-cov cl protease that embody a piperidine moiety as a design element that is well-suited to exploiting favorable subsite binding interactions to attain optimal pharmacological activity and pk properties. the mechanism of action of the compounds and the structural determinants associated with binding were illuminated using x-ray crystallography. proteins, s (spike glycoprotein), e (envelope protein), m (membrane glycoprotein), and n (nucleocapsid protein), which play a critical role in virion-cell receptor binding, replication and virion assembly, are located at the end of the genome [ , ] . coronavirus entry is initiated by the binding of the spike protein (s) to cell receptors, specifically, dipeptidyl peptidase (ddp ) and angiotensin converting enzyme (ace ) for mers-cov and sars-cov, respectively [ e ] . entry into cells requires host proteases for cleavage at two sites in the s protein, in the case of most cov [ , ] translation of the genomic mrna of orf a yields polyprotein pp a, while a second polyprotein (pp b) is the product of a ribosomal frame shift that joins orf a together with orf b. orf a encodes a papain-like cysteine protease (plpro) and a c-like cysteine protease ( clpro). polyproteins pp a and pp b are processed by clpro ( cleavage sites) and plpro ( cleavage sites) resulting in sixteen mature nonstructural proteins, including rna-dependent rna polymerase (rdrp) and helicase, which play important roles in the transcription and replication of coronaviruses (fig. ). both proteases are essential for viral replication, making them attractive targets for drug development [ , , e ] . mers-cov clpro is a chymotrypsin-like cysteine protease having a catalytic cys -his dyad and an extended binding site [ e ] . the protease displays a stringent primary substrate specificity for a p gln residue [ ] and has a strong preference for a p leu residue. the p residue side chain is oriented toward the solvent while the s subsite is shallow, preferring a small hydrophobic p residue (ala). functional and structural studies have delineated the similarities between the clpro of coronaviruses that can be exploited in the design of broad-spectrum inhibitors [ ] . we have recently reported the first demonstration of clinical efficacy of a coronavirus protease inhibitor (a dipeptidyl aldehyde bisulfite adduct inhibitor designated gc ) [ , ] . specifically, administration of gc to cats infected with fipv, a coronavirus that is % fatal in cats, reversed the progression of fatal fip and resulted in clinical remission in a majority of animals (> %). since fip disease progression is quite rapid and its pathogenesis primarily immune-mediated, features shared by mers-cov, we hypothesized that a viral protease inhibitor could reverse the pathogenesis of mers-cov in affected hosts. interrogation of this hypothesis entailed, as a first step, the design of a new and versatile class of peptidomimetic inhibitors of mers-cov cl protease. we describe herein the structure-guided design of inhibitors of mers-cov clpro that embody a piperidine moiety as a novel design element, as well as pertinent structural and biochemical studies. these inhibitors were also examined against other coronaviruses, including sars-cov, fipv and mhv to evaluate the spectrum of activity against multiple coronaviruses. the structure-guided design of inhibitor (i) encompassed the following steps: (a) we first determined a high resolution x-ray crystal structure of mers-cov clpro in complex with gc ( fig. /panel a) . examination of the active site of the complex revealed that the aldehyde bisulfite adduct had reverted to the precursor peptidyl aldehyde, which subsequently formed a tetrahedral hemi-thioacetal upon reaction with the active site cys . notably, the electron density at this stereocenter was consistent with the formation of both r and s enantiomers at the covalent binding site (also observed for the other structures described in the following sections). the structure reveals a network of backbone hydrogen bonds which ensure correct positioning of the inhibitor to the active site, as well as two critical hydrogen bonds with the p gln surrogate [ ] side chain. the inhibitor p leu side chain is ensconced in the hydrophobic s subsite of the enzyme. importantly, the structure shows a hydrophobic-driven interaction between the benzyl group of the inhibitor and the g-lactam ring of the gln surrogate side chain; (b) based on the forgoing, we reasoned that extending the "cap" would allow the inhibitor to assume an extended conformation and orient the phenyl ring toward the hydrophobic s pocket of the enzyme. validation of this idea was obtained by synthesizing extended inhibitor gc and determining a high resolution x-ray crystal structure of the mers-cov clpro:gc complex ( fig. /panel b) . the m-cl phenethyl side chain is clearly shown to occupy the hydrophobic s subsite. in addition to an array of h-bonds with gln , glu , and gln and the backbone of the inhibitor, which serve to correctly position the inhibitor at the active site, the inhibitor interacts with the s , s and s subsites, but not the s subsite; (c) we hypothesized that the attachment of a piperidine ring to the peptidyl component would yield a structurally novel peptidomimetic (i) capable of ( ) orienting recognition elements r and r in a correct vector relationship for optimal interactions with the s and s subsites, ( ) rendering a dipeptidyl inhibitor equivalent to a tetrapeptidyl inhibitor with potentially diminished pk liabilities and, ( ) providing a flexible means for the structure-guided parallel optimization of admet/pk and physicochemical properties using diversity sites r and r in inhibitor (i) (fig. ) . in summary, the piperidine-based design strategy is a hitherto unrecognized effective means of rendering a dipeptidyl inhibitor equivalent to a tetrapeptidyl inhibitor capable of engaging in optimal binding interactions with all four s -s subsites but which, however, is anticipated to display diminished pk liabilities due to its reduced peptidyl character. furthermore, the aforementioned piperidine-based design strategy has wide applicability and can be extended to any protease with an extended binding site. preliminary evidence in support of this approach is provided by the results of enzyme and cell-based screening of derivatives of (i) (tables and ) , as well as the results of structural studies (vide infra) (see table ). the synthesis of final compounds (a-f) and (a-f) is outlined in scheme . -boc- -piperidinone was reacted with different grignard reagents to yield the corresponding -boc- -piperidinol derivatives ( c and e). refluxing (l) leucine methyl ester hydrochloride with trichloromethyl chloroformate yielded the isocyanate which was reacted with ( c and e, or commercially-available nsubstituted -piperidinol a) to form the corresponding carbamate adducts ( a, c and e) that were hydrolyzed to the corresponding acids ( a, c and e) with lithium hydroxide in aqueous thf. subsequent coupling with glutamine surrogate methyl ester hydrochloride afforded the desired dipeptidyl esters ( a, c and e) which were either treated with lithium borohydride directly or were first treated with dry hcl in dioxane followed by reaction with an alkyl sulfonyl chloride or alkyl chloroformate, to yield esters ( b, d and f) prior to reduction with lithium borohydride, to yield alcohols (a-f). dess-martin oxidation, followed by flash chromatography, yielded pure aldehydes (a-f). the enantiomeric purity of the aldehydes was consistently high, with the amount of epimerized aldehyde ranging between and %. the corresponding bisulfite adducts (a-f) were readily obtained as white solids by stirring the aldehydes with sodium bisulfite in an ethyl acetate/ water mixture. the synthesized compounds are listed in table . the inhibitory activity of the synthesized compounds against clpro of mers-cov, sars-cov or fipv, and the antiviral activity of two representative compounds (compounds a and c) in a cellbased system including mers-cov, fipv and mhv were evaluated as described in the experimental section. the ic , ec , and cc values, are listed in tables and these are the average of at least two determinations. it is evident that derivatives of (i) function as highly potent inhibitors of all tested coronaviruses in enzyme (table ) and cell based assays (table and fig. ). more importantly, representative aldehyde bisulfite adduct compounds a and c display potent inhibition toward mers-cov in both enzyme and cell-based systems, with low cytotoxicity (cc > mm) ( table and fig. ). for example, compound a has a selectivity index (si ¼ cc /ec ) of > . with the exception of compounds e- e, the aldehyde and aldehyde bisulfite adducts were found to have comparable in vitro potency toward mers-cov clpro. furthermore, pharmacological activity was found to be dependent on the nature of the r group (compounds e- e are -fold less active toward mers-cov clpro than compounds a-d, a-d and f- f). in order to establish the mechanism of action of (i), as well as obtain structural information that can be used to guide the optimization of pharmacological activity, the high resolution x-ray crystal structures of several derivatives of (i) bound to mers-cov clpro were determined, including the cocrystal structure of the mers-cov clpro:inhibitor c complex (fig. a) . the formation of a tetrahedral adduct via the reaction of the aldehyde, generated from aldehyde bisulfite adduct c under the crystallization conditions used [ , ] , with the active site cysteine (cys ) is clearly evident, confirming the mechanism of action of (i). inspection of the structure reveals the presence of prominent electron density consistent with the structure of inhibitor c; however, the n-bocpiperidinyl moiety was disordered. the position and orientation of the benzyl group suggest that the piperidine ring is likely projecting toward the s subsite. inhibitor c is bound to the active site of the enzyme via a network of backbone h-bonds with gln , gln , and glu (fig. b ). additionally, a h-bond with his serves to stabilize the hemi-thioacetal tetrahedral adduct. also clearly evident are three critical h-bonds involving the p gln surrogate ring oxygen and nitrogen with glu , his and phe . the h-bonding interactions are near identical to those of inhibitor gc (fig. /panel b) . the structural complementarity of inhibitors c and gc is also evident in the electrostatic surface representation of the enzyme with the two inhibitors nestled in the active site (fig. ) . the cocrystal structure of the mers-cov clpro:aldehyde bisulfite adduct e complex also showed that, under the crystallization conditions used, the aldehyde bisulfite adduct reverted to the precursor aldehyde, which subsequently formed a tetrahedral adduct with the active site cysteine (cys ) (fig. a ). the piperidinyl moiety was disordered and consequently its precise location could not be discerned. however, inhibitor e is engaged in the same h-bonding interactions as inhibitor c (fig. b ). mers-cov constitutes a global public health concern. there are currently no licensed vaccines or antiviral drugs for the prevention and treatment of coronavirus infections. we disclose herein for the first time the design and utilization of a general class of piperidinebased peptidomimetic inhibitors of coronavirus cl proteases. attachment of the piperidine moiety to a dipeptidyl component permits the resultant hybrid inhibitor to engage in favorable binding interactions with the s and s subsites of the enzyme. more importantly, the approach disclosed herein can be extended to other proteases of medical relevance. finally, the disclosed compounds potently inhibit mers-cov, and their mechanism of action and mode of binding to mers-cov cl protease have been illuminated using x-ray crystallography. reagents and dry solvents were purchased from various chemical suppliers (aldrich, acros organics, chem-impex, tci america, and bachem) and were used as obtained. silica gel ( e mesh) used for flash chromatography was purchased from sorbent technologies (atlanta, ga). thin layer chromatography was performed using analtech silica gel plates. visualization was accomplished dropwise under a n atmosphere to a solution of -boc- piperidinone ( mmol) in dry thf ( ml) in an ice bath kept at c. the reaction mixture was stirred for h at room temperature under a n atmosphere while monitoring completion of the reaction by tlc. the reaction mixture was diluted with water ( ml) and the solution was acidified to ph~ using % hydrochloric acid. the solvent was removed on the rotary evaporator and the residue was extracted with ethyl acetate ( ml) and the layers separated. the organic layer then washed with brine ( ml) and dried over anhydrous sodium sulfate, filtered and concentrated to yield a colorless oily product which was purified by flash chromatography to yield c and e. . . . synthesis of (l) leucine methyl ester isocyanate (l) leucine methyl ester hydrochloride ( mmol) was placed in a dry -ml rb flask and then dried overnight on the vacuum pump. the flask was flushed with nitrogen and dry dioxane ( ml) was added followed by trichloromethyl chloroformate ( . g, mmol), and the reaction mixture was refluxed for h. the solvent was removed on the rotary evaporator and the residue was vacuum distilled to yield pure isocyanate as a colorless oil [ , ] . table crystallographic data for mers-cov clpro in complex with compounds gc , gc , c and e. mers-cov clpro: gc mers-cov clpro: compound c mers-cov clpro: compound e hkl. c r factor ¼ Ʃ hkl jjf obs (hkl) j -jf calc (hkl) jj/Ʃ hkl jf obs (hkl)j; rfree is calculated in an identical manner using % of randomly selected reflections that were not included in the refinement. d r meas ¼ redundancy-independent (multiplicity-weighted) r merge. [ , ] r pim ¼ precision-indicating (multiplicity-weighted) r merge. [ , ] . e cc / is the correlation coefficient of the mean intensities between two random half-sets of data [ , ] . . . . synthesis of substituted piperidine-derived carbamates a, c and e. general procedure a solution of substituted or unsubstituted -boc- -piperidinol ( c, e or a) ( mmol) in dry acetonitrile ( ml) was treated with triethylamine ( . g, mmol) followed by the amino acid methyl ester isocyanate ( mmol). the resulting solution was refluxed for h and then allowed to cool to room temperature. the solution was concentrated and the residue was taken up in ethyl acetate ( ml). the organic layer was washed with % hcl (  ml) and brine ( ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, leaving compounds a, c and e as colorless oils. . . . synthesis of acids a, c and e. general procedure a solution of ester ( a, c or e) ( mmol) in tetrahydrofuran ( ml) was treated with m lioh ( ml). the reaction mixture was stirred for h at room temperature and the disappearance of the ester was monitored by tlc. most of the solvent was evaporated off and the residue was diluted with water ( ml). the solution was acidified to ph~ using % hydrochloride acid ( ml) and the aqueous layer was extracted with ethyl acetate (  ml). the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to yield the corresponding compounds a, c and e as colorless oils. . . . synthesis of compounds a, c and e. general procedure edci ( . g, . mmol, . eq) and hobt ( . g, . mmol, . eq) were added to a solution of compound ( a, c or e) ( mmol) in dry dmf ( ml) and the mixture was stirred for min at room temperature. in a separate flask, a solution of deprotected glutamine surrogate ( . g, mmol) in dmf ( ml) cooled to e c was treated with diisopropylethylamine (diea) ( . g, mmol, eq), stirred for min, and then added to the reaction mixture containing the acid. the reaction mixture was stirred for h while monitoring the reaction by tlc. the solvent was removed and the residue was partitioned between ethyl acetate ( ml) and % citric acid (  ml). the layers were separated and the ethyl acetate layer was further washed with saturated aqueous nahco ( ml), followed by saturated nacl ( ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to yield a yellow-colored oily product. purification by flash chromatography yielded esters a, c and e as white solids. . . . synthesis of compounds b, d and f. general procedure m hcl in dioxane ( ml) was added to a solution of compound ( a, c and e) ( mmol) in dry dcm ( ml) and the mixture was stirred for h at room temperature. the solvent was removed and the residue was dried under high vacuum for h before the product was dissolved in dry thf ( ml). an appropriate alkyl sulfonyl chloride or alkyl chloroformate derivative ( mmol/ . eq) was added to the solution with stirring. the reaction mixture was stirred for h at room temperature and the residue was dissolved in ethyl acetate ( ml) and washed with % hcl (  ml). the ethyl acetate layer was further washed with saturated nacl ( ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to yield a crude product. purification by flash chromatography yielded the corresponding esters b, d and f as white solids. lithium borohydride ( m in thf, . ml, mmol) was added dropwise to a solution of ester ( or ) ( mmol) in anhydrous thf ( ml), followed by absolute ethyl alcohol ( ml) and the reaction mixture was stirred at room temperature overnight. the reaction mixture was then acidified by adding % hcl and the ph adjusted tõ . removal of the solvent left a residue which was taken up in ethyl acetate ( ml). the organic layer was washed with brine ( ml), dried over anhydrous sodium sulfate, filtered, and concentrated to yield compounds (a-f) as white solids. compound (a-f) ( mmol) was dissolved in anhydrous dichloromethane ( ml) under a nitrogen atmosphere and cooled to c. dess-martin periodinane reagent ( . g, . mmol, . eq) was added to the reaction mixture with stirring. the ice bath was removed and the reaction mixture was stirred at room temperature for h (monitoring by tlc indicated complete disappearance of the starting material). a solution of % aqueous sodium thiosulfate ( ml) was added and the solution was stirred for another min. the aqueous layer was removed and the organic layer was washed with % aqueous sodium thiosulfate ( ml), followed by saturated aqueous sodium bicarbonate (  ml), water (  ml) and brine ( ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. the yellow residue was purified by flash chromatography (silica gel/methylene chloride/ ethyl acetate/methanol) to yield a white solid (a-f). absolute ethanol ( ml) was added to a solution of aldehyde (a-f) ( mmol) in dry ethyl acetate ( ml) with stirring, followed by a solution of sodium bisulfite ( mg; mmol) in water ( ml) and the reaction mixture was stirred for h at c. the reaction mixture was allowed to cool to room temperature and then vacuum filtered. the solid was thoroughly washed with absolute ethanol and the filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to yield a yellowish oil. the oily product was treated with ethyl ether (  ml) to form a white solid. the white solid was stirred with ethyl ether ( ml) and ethyl acetate ( ml) for min. careful removal of the solvent using a pipette left the corresponding aldehyde bisulfite adducts (a-f) as white solids. the fret protease assay was performed by preparing stock solutions of the substrate (dabcyl-ktsavlq/sgfrkme-edans derived from the cleavage sites on the viral polyproteins of sars-cov) and inhibitor in dmso and diluting into assay buffer which was comprised of mm hepes buffer, ph , containing nacl ( mm), edta ( . mm), glycerol ( %), and mm dithiothreitol (dtt). the expression and purification of the clpro of mers-cov, sars-cov or fipv was performed by a standard method described previously by our lab [ , ] . the protease ( clpro of mers-cov, sars-cov or fipv) was mixed with serial dilutions of each compound or with dmso in ml of assay buffer and incubated at c for min, followed by the addition of ml of assay buffer containing substrate. fluorescence readings were obtained using an excitation wavelength of nm and an emission wavelength of nm on a fluorescence microplate reader (flx ; biotec, winoosk, vt) h following the addition of substrate. relative fluorescence units (rfu) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values, as described previously [ ] . the dosedependent fret inhibition curves were fitted with a variable slope by using graphpad prism software (graphpad, la jolla, ca) in order to determine the ic values of the inhibitors. the effects of compounds a and c on the replication of mers-cov, fipv or mhv-a were examined in vero , crfk or ccl . cells, respectively [ ] . briefly, confluent and semi-confluent cells were infected at an moi of . pfu/cell. following adsorption, cells were incubated with medium containing dmso (< . %) or each compound (up to mm) for h. after incubation, viral titers were determined with a tcid (fipv or mhv) or plaque assay (mers-cov). ec values were determined using graphpadprism software [ ]. the cytotoxic dose for % cell death (cc ) for compounds a and c was determined in vero , crfk or ccl . cells. confluent cells grown in -well plates were treated with various concentrations ( e mm) of each compound for h. cell cytotoxicity was measured by a cytotox nonradioactive cytotoxicity assay kit (promega, madison, wi). the in vitro therapeutic index was calculated by dividing the cc by the ec . purified mers-cov clpro, in mm nacl, mm tris ph . , was concentrated to mg/ml ( . mm). stock solutions of mm gc , gc , compound c or compound e were prepared in dmso and the complex with mers clpro was prepared by mixing the concentrated protein supplemented with mm compound and incubating overnight at c. all crystallization experiments were conducted using compact (rigaku reagents) sitting drop vapor diffusion plates at c using equal volumes of protein and crystallization solution equilibrated against ml of the latter. crystals of mers clpro in complex with gc , compound c and compound e that displayed a prismatic morphology were obtained from the index ht screen (hampton research) condition g ( % (w/v) peg , mm bis-tris ph . , mm mgcl ) in e days. crystals of the gc complex were obtained from the index ht screen (hampton research) condition e ( % (v/v) peg mme, mm bis-tris ph . , mm cacl ). samples were transferred to a fresh drop containing % crystallant and % (v/v) peg before storing in liquid nitrogen. x-ray diffraction data were collected at the advanced photon source beamline -id using a dectris pilatus m pixel array detector. intensities were integrated using xds [ , ] using autoproc [ ] and the laue class analysis and data scaling were performed with aimless [ ] , which suggested that the highest probability laue class was /m and space group c . structure solution was conducted by molecular replacement with phaser [ ] using a previously determined isomorphous structure of mers clpro (pdb: rsp [ ] ) as the search model. structure refinement and manual model building were conducted with phenix [ ] and coot [ ] , respectively. disordered side chains were truncated to the point for which electron density could be observed. structure validation was conducted with molprobity [ ] and figures were prepared using the ccp mg package [ ] . coordinates and structure factors for the mers clpro inhibitor complexes were deposited to the worldwide protein data bank (wwpdb) with the accession codes: wkj (gc ), wkk (gc ), wkl (inhibitor c) and wkm (inhibitor e). coronaviridae in field's virology coronaviruses: important emerging human pathogens sars and mers: recent insights into emerging coronaviruses update on human rhinovirus and coronavirus infections middle east respiratory syndrome and severe acute respiratory syndrome pathogenesis of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease middle east respiratory syndrome coronaviruses: drug discovery and therapeutic options antiviral drugs specific for coronaviruses in preclinical development mers-cov vaccine candidates in development: the current landscape role of the spike glycoprotein of human middle east syndrome coronavirus (mers-cov) in virus and syncytia formation middle east respiratory syndrome infection mediated by the transmembrane serine protease tmprss host cell entry of middle east respiratory distress syndrome coronavirus after two-step, furin-mediated activation of the spike protein an overview of severe acute respiratory distress syndrome-coronavirus (sars-cov) cl protease inhibitors: peptidomimetics and small molecule chemotherapy the sars-coronavirus papainlike protease: structure, function and inhibition by designed antiviral compounds identification, synthesis, and evaluation of sars-cov and sars-cov c-like protease inhibitors structures of the middle east respiratory syndrome coronavirus c-like protease reveal insights into substrate specificity from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design prediction and biochemical analysis of putative cleavage sites of the c-like protease of middle east respiratory syndrome coronavirus inhibitor recognition specificity for mers-cov papain-like protease may differ from that of sars-cov e where s , s , s , …. s n and s ', s ', s ', …. s n ' correspond to the enzyme subsites on the n-terminus and c-terminus side, respectively, of the scissile bond structure of main protease from human coronavirus nl : insights for wide spectrum anti-coronavirus drug design reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor efficacy of a c-like protease inhibitor in treating various forms of acquired feline infectious peritonitis tripeptide aldehyde inhibitors of human rhinovirus c protease: design, synthesis, biological evaluation, and cocrystal structure solution of p glutamine isosteric replacements structure-guided design and optimization of dipeptidyl inhibitors of norovirus cl protease. structure-activity relationships and biochemical, x-ray crystallographic, cell-based, and in vivo studies inhibition of norovirus cl protease by bisulfite adducts of transition state inhibitors broad-spectrum antivirals against c or c-like proteases of picornaviruses, noroviruses and coronaviruses alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sarscoronavirus infection in a mouse model automatic indexing of rotation diffraction patterns data processing and analysis with the autoproc toolbox an introduction to data reduction: space-group determination, scaling and intensity statistics phaser crystallographic software ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp protease ( clpro): implications for nsp regulation and the development of antivirals phenix: a comprehensive python-based system for macromolecular structure solution features and development of coot molprobity: all-atom structure validation for macromolecular crystallography developments in the ccp molecular graphics project scaling and assessment of data quality improved r-factors for diffraction data analysis in macromolecular crystallography global indicators of x-ray data quality linking crystallographic model and data quality resolving some old problems in protein crystallography towards automated crystallographic structure refinement with phenix.refine supplementary data related to this article can be found at https://doi.org/ . /j.ejmech. . . . key: cord- -sfa d ux authors: lei, h.; li, y.; xiao, s.; lin, c.‐h.; norris, s. l.; wei, d.; hu, z.; ji, s. title: routes of transmission of influenza a h n , sars cov, and norovirus in air cabin: comparative analyses date: - - journal: indoor air doi: . /ina. sha: doc_id: cord_uid: sfa d ux identifying the exact transmission route(s) of infectious diseases in indoor environments is a crucial step in developing effective intervention strategies. in this study, we proposed a comparative analysis approach and built a model to simulate outbreaks of different in‐flight infections in a similar cabin environment, that is, influenza a h n , severe acute respiratory syndrome (sars) coronavirus (cov), and norovirus. the simulation results seemed to suggest that the close contact route was probably the most significant route (contributes %, % confidence interval [ci]: %‐ %) in the in‐flight transmission of influenza a h n transmission; as a result, passengers within rows of the index case had a significantly higher infection risk than others in the outbreak (relative risk [rr]: . , % ci: . ‐ . , p = . ). for sars cov, the airborne, close contact, and fomite routes contributed % ( % ci: %‐ %), % ( % ci: %‐ %), and % ( % ci: %‐ %), respectively. for norovirus, the simulation results suggested that the fomite route played the dominant role (contributes %, % ci: %‐ %) in most cases; as a result, passengers in aisle seats had a significantly higher infection risk than others (rr: . , % ci: . ‐ . , p = . ). this work highlighted a method for using observed outbreak data to analyze the roles of different infection transmission routes. knowledge about the relative importance of different transmission route(s) is fundamental to developing effective intervention strategies for infectious respiratory and enteric diseases in indoor environments. epidemiological analysis together with in-depth environmental investigations often provides useful insights, and meta-analysis may also be carried out for a particular disease. in this study, we proposed an alternative comparative analysis approach in which we studied outbreaks of different diseases in the same environment using the same approach, by examining differences in the spatial infection patterns. this approach could partly overcome the limitation of traditional individual outbreak analysis that outbreak cannot be repeatedly observed, because the comparative analysis of different diseases in the same environment is like that one disease happened several times. our hypothesis is that the different transmission routes of infection lead to different spatial patterns of secondary cases. for example, close contact transmission always happens with - m of the source, which means that secondary cases infected via close contact route would be close to the index case(s). the airborne transmission may occur over long distance, and the secondary cases infected via airborne route would distribute uniformly in a space when the air is fully mixed. aircraft cabins were selected as the context for our study. the more or less fixed seating arrangement in aircraft cabins permits a spatial pattern of the secondary cases to be identified in some outbreaks. the norovirus is the leading cause of nonbacterial gastroenteritis in humans. , the major possible routes in aircraft cabins are close contact, airborne, and fomite. in this study, a mathematical model was built to study the in-flight infection transmission process, based on the studies by atkinson and wein and nicas and jones. this technique enables detailed physical and biological processes to be modeled and the impact of environmental parameters to be easily integrated. we compared the simulated relative importance of different transmission routes in in-flight outbreaks with the reported spatial distribution of the secondary cases. we performed a literature search for in-flight outbreaks of influenza a h n , sars cov, and norovirus in appendix s . all chosen outbreaks occurred in boeing aircraft cabins with flight duration . or hours. the main criteria for identifying suitable outbreaks include the availability of detailed seating information for both the infected and noninfected, airplane type and flight duration. figure illustrates the detailed spatial distribution of the secondary cases in the chosen outbreaks. the definitions for the relevant transmission routes ( figure ) in our multiroute model are as follows. the airborne route refers to direct inhalation of an infectious agent through small droplet nuclei, that is, the residue of large droplets containing microorganisms that have evaporated to an aerodynamic diameter of less than microns (termed respirable). these respirable droplets can deposit in the respiratory tract. close contact route includes direct contact and large droplet transmission. direct contact refers to infection through person-to-person contact with the index source passenger, such as handshaking. we assume there is no body-to-body contact between index source passenger(s) and other passengers during the flight. we only consider large droplet transmission in the model, which refers to the inhalation of the virus carried in respirable airborne particles with a diameter between and microns (termed inspirable), and the droplet spray of large droplets (> microns in diameter) onto facial target membranes. the fomite route refers to infection by touching objects or surfaces that have earlier been contaminated by hands or by direct deposition • our identification of the dominated routes, that is the close contact route (large droplet) for influenza, the fomite route for norovirus, and all routes for sars cov, suggested the relative importance of different environment intervention for different infectious diseases in air cabins and probably also in other indoor environments. for minimizing in-flight fomite transmission, the aisle seatbacks and toilets should be cleaned and disinfected effectively. f i g u r e spatial distribution for in-flight infection outbreaks, (a) norovirus, (b) sars cov, and (c) influenza a h n of infectious pathogens from the index source passenger, which is also sometimes termed indirect contact route. for respiratory disease, coughing is used as surrogate to model the virus-containing droplets from all respiratory activities such as breathing, talking, and sneezing, as the size distribution of the droplets from coughing, talking, and sneezing is similar, and the amount of droplets generated due to breathing is negligible. assume that cough frequency for infector is f c per hour and that one cough can produce n c droplets with the size distribution f c r . then, the generation rate (number/h) of droplets with radius r (μm) from individual i is given by: for enteric disease, such as norovirus, virus-containing droplets are emitted from the infector in vomit and/or diarrhea. a study by simulated vomiting device showed that the volume of the aerosolized droplets ranged from . to ml, with a mean value of . ml. to the best of our knowledge, there is no study on the size distribution of the droplets from vomit, and we assume that these droplets have same size distribution as those from coughing. for respirable droplets with aerodynamic diameter of less than microns, they could move a long distance with the airflow and dis- for inspirable droplets with a diameter between and microns, we assumed that the maximum horizontal distance they could move was m because of gravity and the relatively high deposition rate on environmental surfaces. they distributed within m of the source, and the volume of this space was denoted to be a rapid death rate of pathogens atomized into air had been observed, , and evaporation of droplets was believed to play an important role. xie et al found that there was a fast viability decline stage when the droplets completely evaporated, when viability decreased to about % and then slowly declined. here, the survival ratio due to evaporation was defined as e r ,s = l r ,t ∕l r , where l r ,t is the concentration of viable viruses (tcid /ml or genome copies/ml) in droplets with initial radius r (μm) at the time t (s) after being exhaled; t e r is the evaporation time (s) for the drop- illustration of different transmission routes considered in this study. note that all sizes of droplets are involved in the fomite route assume there is no resuspension of droplets from environmental surfaces into the air. in the air cabin, on the one hand, viable virus is generated from index case(s) at rate ( ) was the exposure through each route was modeled separately, and then the dose-response model was used to assess an integrated risk. the dose to individual i via the airborne route in the lower and upper respiratory tracts is denoted as d i al and d i au (tcid or genome copies), and for a flight duration t, they can estimated as follows: where r a is the largest radius for airborne droplets and r a = μm ; p is the pulmonary ventilation rate and p = . m /h ; r is the droplets' initial radius; and r is the final radius after complete evaporation. here, we assume that r = r / ; l (r) and u (r) are the deposition fraction of droplets with radius r in the lower and upper respiratory tracts, respectively. the model from icrp was used in this study. transmission by close contact refers to either inhalation of the virus carried in airborne particles with a diameter between and microns, or the spray of large droplets on the susceptible individuals' mucous membranes. for norovirus transmission, it is difficult for the "large" droplets generated from vomiting to move to the inhale air of the seated susceptible passengers, which is always more than one meter above the ground, because of the high downward velocity, gravity, and the relatively high deposition rate. therefore, the close contact route was not considered in the norovirus transmission. then the dose via inhalation of inspirable droplets in upper respiratory tract (d i cr (tcid or genome copies)) was where r b was radius of the maximum inspirable droplets and for the spray of large droplets on mucous membranes, because of the seating arrangement we assumed that there was no face-to- the negative exponential dose-response model was used to estimate the infection risk, which implies that a single particle can start an infection, all single particles are independent of each other. the infection risk of individual i during the flight can be calculated according to the following equation where η l η l , η u , and η m are the dose-response rates (/tcid or/genome copy) in lower/upper respiratory tracts and on mucous membranes, respectively. here it is also assumed that η u = η m . table ( ) d i cm = t ∫ o ∞ ∫ r a a m a v c i c (r) kr v t πr l r ,t drdt ( ) c j s � k + � = � c j s (k) + ∑n p i= (c i h (k)a hs τ hs −c j s (k)a hs τ sh )ps i,j (k) a s � e −b s ×Δt ( ) c i h � k + � = � c i h (k) − ∑n p i= (c i h (k)a hs τ hs −c j s (k)a hs τ sh )ps i,j (k) a h − pm i (k)a m h τ hm c i h (k) a h � e −b h ×Δt ( ) d i fm = n t ∑ k= pm i (k) a m h τ hm c i h (k) ( ) p i = − e −(η l d i al +η u d i au )−(ηud i cr +η m d i cm )−ηmd i fm in this study, we built a mathematical model to study the inflight transmission of different viruses, using a novel comparative analysis approach. the results suggested that the dominant transmission routes in air cabins are probably the close contact route for influenza, the fomite route for norovirus, and all routes (airborne, close contact, and fomite routes) for sars cov. the dominant transmission routes vary for the viruses, mainly depending on the pathogen-specific t a b l e infection risks of passengers within rows of the index case(s) and others from the simulation results and reported outbreak data, respectively (with the tuned range of the virus shedding magnitudes) outbreak data within rows (others) as far as we are aware there are no data on the dose-response rate of sars cov either on mucous membranes or in the respiratory tracts of humans. we assume that the dose-response rate on human mucous membranes is the same as that on mice mucous membranes, and the dose-response rate in the human respiratory tract is times that of mucous membranes (similar to the influenza a h n data). we also performed the sensitivity analysis of the dose-response rates, with different ratios of median dose-response rate, that is (in lower respiratory tract)/(on mucous membranes) (see detail in appendix s ), and we found that all routes were important in in-flight sars cov transmission for different ratios of median dose-response rate. the fecal-oral spread is known to be the primary mode of transmission of norovirus. the fomite route transmission of norovirus is well supported by the reported widespread environmental contamination with norovirus. , our simulation of a norovirus outbreak confirms that the fomite route is dominant in transmission. it is also suggested that vomiting can produce aerosol droplets containing norovirus particles, and inhaled by exposed susceptible individuals, depositing in the upper respiratory tract and subsequently swallowed along with the respiratory mucus. airborne norovirus was detected from an air sample in one outbreak. a study of a norovirus outbreak in a hotel found an inverse relationship between the infection risk and the distance from the person who vomited when a food source was not implicated. this study simulated both the airborne and fomite route transmission of norovirus. and the results showed that in most cases, the fomite route plays the dominant role. the predicted infection risk from the fomite route for aisle seat passengers is . times higher than that for nonaisle seat passengers. the aisle passenger-tonon-aisle passenger relative risk in the outbreak ( . ) is much higher than . , and may be attributable to a small sample size of secondary cases ( ). in conclusion, a mathematical model was built to simulate the physi- additionally, this study highlights a way to use observation outbreak data to analyze the relative importance of different routes in infection transmission. evidence of airborne transmission of the severe acute respiratory syndrome virus spread of a novel influenza a(h n ) virus via global airline transportation respiratory infections during air travel transmissibility of pandemic influenza norovirus gastroenteritis human susceptibility and resistance to norwalk virus infection transmission of infectious diseases during commercial air travel quantifying the routes of transmission for pandemic influenza relative contributions of four exposure pathways to influenza infection risk aerosolization of a human norovirus surrogate, bacteriophage ms , during simulated vomiting short-range airborne transmission of expiratory droplets between two people aerosol survival of pasteurella tularensis disseminated from the wet and dry states effect of relative humidity on the survival of airborne unicellular algae airborne infection: transmission and control bacterial survival in evaporating deposited droplets on a teflon-coated surface how far droplets can move in indoor environments -revisiting the wells evaporationfalling curve inflight cabin smoke control accurate and high-resolution boundary conditions and flow fields in the first-class cabin of an md- commercial airliner cabin air quality: indoor pollutants and climate during intercontinental flights with and without tobacco smoking characterizing the risk of infection from mycobacterium tuberculosis in commercial passenger aircraft using quantitative microbial risk assessment predictive models of control strategies involved in containing indoor airborne infections human respiratory tract model for radiological protection. a report of a task group of the international commission on radiological protection the fraction of total hand surface area involved in young children's outdoor hand-to-object contacts determination of hand surface area by sex and body shape using alginate informing optimal environmental influenza interventions: how the host, agent, and environment alter dominant routes of transmission likely transmission of norovirus on an airplane transmission of the severe acute respiratory syndrome on aircraft influenza a(h n )pdm during air travel who technical advice for case management of influenza a(h n ) in air transport high infectivity and pathogenicity of influenza a virus via aerosol and droplet transmission transmission of influenza a in human beings an outbreak of influenza aboard a commercial airliner role of air distribution in sars transmission during the largest nosocomial outbreak in hong kong experimental study of dispersion and deposition of expiratory aerosols in aircraft cabins and impact on infectious disease transmission severe acute respiratory syndrome coronavirus on hospital surfaces widespread environmental contamination with norwalk-like viruses (nlv) detected in a prolonged hotel outbreak of gastroenteritis a norovirus outbreak at a longterm-care facility: the role of environmental surface contamination norovirus, gastroenteritis, and indoor environmental quality detection and quantification of airborne norovirus during outbreaks in healthcare facilities evidence for airborne transmission of norwalk-like virus (nlv) in a hotel restaurant additional supporting information may be found online in the supporting information tab for this article. key: cord- -v n ff authors: ahn, inkyung; heo, seongman; ji, seunghyun; kim, kyung hyun; kim, taehwan; lee, eun joo; park, jooyoung; sung, keehoon title: investigation of nonlinear epidemiological models for analyzing and controlling the mers outbreak in korea date: - - journal: journal of theoretical biology doi: . /j.jtbi. . . sha: doc_id: cord_uid: v n ff abstract much concern has arisen regarding serious epidemics due to the middle east respiratory syndrome (mers) coronavirus. the first mers case of korea was reported on may , and since then, the mers outbreak in korea has resulted in hundreds of confirmed cases and tens of deaths. deadly infectious diseases such as mers have significant direct and indirect social impacts, which include disease-induced mortality and economic losses. also, a delayed response to the outbreak and underestimating its danger can further aggravate the situation. hence, an analysis and establishing efficient strategies for preventing the propagation of mers is a very important and urgent issue. in this paper, we propose a class of nonlinear susceptible-infectious-quarantined (siq) models for analyzing and controlling the mers outbreak in korea. for the siq based ordinary differential equation (ode) model, we perform the task of parameter estimation, and apply optimal control theory to the controlled siq model, with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. simulation results show that the proposed siq model can explain the observed data for the confirmed cases and the quarantined cases in the mers outbreak very well, and the number of the mers cases can be controlled reasonably well via the optimal control approach. first confirmed on may , the latest outbreak of middle east respiratory syndrome coronavirus (mers-cov) infections in korea accounted for laboratory-confirmed cases, including deaths, recovered individuals discharged from hospitals, and remaining in hospitals up to july , a de facto end date of the outbreak ( ministry of health and welfare , moh ). it spread remarkably fast in hospitals, which has caused the largest mers outbreak outside the middle east. the case fatality rate of . % is, however, much lower than the reported rate of . % prior to the outbreak in korea, according to the world health organization (who). the mers-cov is a novel betacoronavirus which was first identified from the sputum of a -year-old man in fall ( zaki et al., ) . clinical features of mers range from mild illness to fatal conditions such as acute respiratory distress syndrome * corresponding authors. e-mail addresses: ahnik@korea.ac.kr (i. ahn), parkj@korea.ac.kr (j. park). and multi-organ failure resulting in death, especially in patients with underlying comorbidities ( zumla et al., ) . although it is initially known as a zoonotic disease, human-to-human transmission occurs in health care settings and now is linked with significant morbidity ( oboho et al., ) . this outbreak of the mers-cov infection, including the index cases roommates, their caregiver, and even the healthcare workers, in what is called as a super-spreading event ( kupferschmidt, ) , raised several important issues for global public health surveillance and raised several issues regarding infection control policies, including the control of nosocomial transmission to avoid a repeated outbreak ( keeling and rohani, ) . in this study, we carry out an epidemiological assessment of the mers-cov outbreak in korea in order to provide a mathematical framework for understanding the complex dynamics of the pathogen spread and establishing efficient guidelines for implementing quarantine and isolation strategies. more specifically, we propose a class of nonlinear susceptible-infectious-quarantined (siq) models for analyzing and controlling the mers outbreak in korea. the proposed siq model is innovative, in that the mers https://doi.org/ . /j.jtbi. . . - /© elsevier ltd. all rights reserved. transmission probability is time-dependent, monotone decreasing, and squashing-type. more specifically, it is initially almost flat, then decreasing rapidly, and finally gradually reaching a saturation point, which is reasonable because this can reflect the change in individuals' hygienic behaviour with time. recently, there has been much interest in investigating some aspects of the time-dependent nature of the disease transmission probability. in particular, several methods have been considered to model the time-dependence due to the impact of media coverage ( cui et al., ; liu et al., ; sun et al., ; xiao et al., ) . however, these studies have mostly focused on only a single factor under consideration. in this paper, we consider all relevant factors that can affect the mers transmission probability ( e.g. , media coverage, increased awareness, etc.) collectively, and try to model the resultant time-dependent transmission probability using a sigmoidal function. note that the sigmoidal functions are quite popular in the field of machine learning when one needs to address monotone and squashing-type phenomena. for the proposed siq model, we perform the task of parameter estimation, and apply optimal control theory to the controlled siq model, with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. simulation results show that the proposed siq model can explain the observed data for the confirmed cases and the quarantined cases in the mers outbreak very well, and the number of the mers cases can be controlled reasonably well via the optimal control approach. finally in the last two sections of this paper, discussion and concluding remarks are given along with brief descriptions of data treatment. in this section, we will propose a nonlinear susceptibleinfectious-quarantined (siq) model for analyzing the mers outbreak in korea. the siq model is a generalization of an epidemiological population model involving susceptible, infectious, and quarantined compartments ( hethcote et al., ; keeling and rohani, ; lenhart and workman, ; xiao et al., ) . in the siq model, the four compartment populations are used as the model's state variables: s ( t ), the number of individuals that are susceptible to the mers disease at time t; i ( t ), the number of individuals that are actively infectious at time t; s q ( t ), the number of quarantined susceptible individuals at time t ; and c q ( t ), the number of confirmed cases at time t . note that in the siq model, q stands for the super-compartment comprising s q and c q (see fig. ). also, note that c q ( t ) is a collective sink-type compartment, which includes the number of the mers patients under treatment, the recovered cases, and the dead cases altogether at time t . as shown in fig. , the siq model considers two kinds of non-pharmaceutical interventions: quarantine of the susceptible and infected individuals, and isolation of the infectious individuals following contact tracing. as a result of a contact tracing, a proportion, q , of individuals who are contacted in connection with a mers patient is quarantined. the quarantined individuals can move to compartments c q or s q , depending on whether they are exposed to the mers coronavirus or not. hence, the quarantined individuals, if uninfected, move to the compartment s q at a rate of c( − β(t)) q, where c is the contact rate, i.e. , the average number of contacts of the whole population per unit time, and β( t ) is the probability of the mers transmission per contact at time t . note that in the siq model, the mers transmission probability, β( t ), is time-dependent, monotone decreasing, and squashing-type. obviously, using timedependent transmission probability is more reasonable than using the constant one, because it can reflect the change of individuals' hygienic behavior with time. as shown in fig. , if infected, the quarantined individuals move to the compartment c q at a rate of c β( t ) q . also, the remaining proportion ( i.e. , the proportion missed from the contact tracing), − q, can move to compartment i or stay in compartment s , depending on whether they are exposed to the mers coronavirus or not. the transmission dynamics of the siq model is illustrated in fig. , and its state equations are as follows: the first equation of ( ) describes the rate of change of the susceptible compartment population, with four terms on its right-hand side. its first term concerns the transition from s to s q due to quarantine of susceptible individuals. this term can be explained in terms of a bilinear incidence law having a contact rate c together with β( t ), the probability of the mers transmission per contact at time t , and q , the probability of quarantine per contact. the second and third terms model the transition from s to c q and the transition from s to i , respectively. the fourth term represents the transition from s q to s , and this transition means the release from quarantine into the wider community. in the second equation of ( ) , the rate of change of the infectious compartment population is described by two terms. the first represents the transition from the susceptible state to the infectious state, and the second term denotes the transition from i to c q due to detection and isolation of the infectious patients. the remaining equations of ( ) describe the rates of change of s q and c q in the super-compartment q , respectively, and the exact meaning of their terms can be explained similarly. the natural birth and death rates are not considered in the siq model, and this omission allows us to focus on the core theme of the paper. note that consideration of these additional aspects is relatively straightforward, and would lead us to some further related observations. for example, if the natural birth rate of a susceptible population, , and the natural death rate, d , are consid- ered, the resultant reduced siq system with a constant transmission probability, β , would have the disease-free equilibrium point as previously mentioned, the mers transmission probability, β( t ), of our siq model, is time-dependent, monotone decreasing, and squashing-type. since many factors ( e.g. , media coverage, increased awareness, etc.) can alter individuals' hygienic behavior, we employ the squashing-type function of fig. for β( t ). more specifically, we utilize where σ ( ·) is the logistic sigmoidal function ( bishop, ) defined as σ (x ) = / ( + exp (−x )) ; t β is the inflection point of β( t ); s β is the parameter determining the slope of β( t ) at its inflection point. note that the use of logistic sigmoidal functions is quite popular in the field of machine learning ( bishop, ) when one needs to represent monotone and squashing-type phenomena. also, note that using the logistic sigmoidal function of ( ) lead to the following simplification when we compute its derivative: this property can be utilized effectively in further studies on the qualitative properties of the siq model ( e.g. , study of the global stability of the disease-free and endemic equilibrium points of the siq model). an explanation of the siq parameters is given in table . by fitting the siq model to the reported data for the confirmed cases and the quarantined cases, we obtain the following tant approach that can be addressed along these lines is the event-dependent approach, where the transmission probability could be dependent on the number of the newly-added or accumulated mers cases. parameters: we performed simulations based on the estimated parameters. simulation results ( fig. ) show that the proposed siq model can explain the observed data ( fig. ) for the confirmed cases and the quarantined cases in the mers outbreak very well. our parameter estimation results show that the mers transmission probability, β( t ), is initially almost flat, then decreasing rapidly, and finally gradually reaching a saturation point (see the solid line of fig. ). this has a natural interpretation, in that as information on the mers outbreak becomes more widely known with the passage of time, health authorities' and individuals' effort s against the epidemic intensify accordingly, which results in the mers transmission probability decreasing in the squashing-type fashion, as in fig. . in retrospect, if the initial effort to reduce the mers transmission probability were more effective, the magnitude of β could be decreased further. in order to investigate this aspect, we additionally performed simulations for a scenario with β reduced to % of its estimated value (see the dashed line of fig. ). fig. shows that the infectious population could be reduced significantly if the aforementioned effort were successful. in order to clarify why it is important to consider the incidence rate of the type shown in ( ) , we also considered the constant beta case, and provided the corresponding simulation results ( fig. ) . comparing figs. and shows the superiority of using the time-dependent β. since quarantine and isolation strategies are the most important and effective measures against the outbreaks of disease when one does not have valid medicines and vaccine (see e.g. , castillo-chavez et al., ; day et al., ; yan and zou, ; yan et al., ) , one can view the effort s of implementing quarantine and isolation strategies as the actions that control the entire model. in this paper, we utilize optimal control theory ( lenhart and workman, ; lewis and syrmos, ) for the possibility of improving our control effort s. note that recent applications of optimal control theory are increasingly used in communities of biological systems ( e.g. ( buonomo and messina, ; joshi et al., ; jung et al., ; kirschner et al., ; de pillis et al., ) ), and in particular, they have been widely used and discussed in the control of epidemics ( e.g. caetano and yoneyama, ; feng et al., ; gupta and rink, ; joshi et al., ; lenhart and workman, ) . by incorporating the control inputs, q * ( t ) and θ * ( t ) into our siq model ( ) , one can obtain the following state equations for the controlled model: note that for the optimal control of ( ) , it is enough to consider the first three variables only. hence, we consider the following controlled siq (c-siq) model: from the data fitting based on the siq model, we have already obtained the estimation results for the quarantine probability, q , and the isolation rate, θ . in retrospect, however, control-theoretic investigation is desirable for the purpose of improving our response to the outbreak. in the following, we consider the problem of improving the quarantine probability and isolation rate further with additional effort s ( q * ( t ) and θ * ( t )) by minimizing an objective function in the form of j = t f g(i(t ) , q * (t) , θ * (t)) dt. note that in this problem, the quarantine probability and isolation rate at time t are represented by and the control inputs to be determined via optimal control theory are the additional effort s described by q * ( t ) and θ * ( t ). here g ( · ) should be reasonably chosen to reflect the relative importance of the quarantine and isolation effort s over the infection. more precisely, in order to minimize an objective function comprising the infection cost ( i.e. , the infectious compartment population) and the cost of implementing quarantine and isolation strategies, we consider the following optimal control problem: subject to the c-siq state equations ( ) . in the cost rate of this problem, c q and c θ are the trade-off constants defining the relative importance of the implementation costs over the infection cost. note that the cost rate considers quadratic cost terms for the control inputs, which is a commonly used strategy in related control problems dealing with epidemicmodel-based systems ( e.g. , lenhart and workman, ) . also, note that the existence of an optimal control and corresponding optimal state trajectory comes from the convexity of the integrand of the objective function with respect to the control and the lipschitz property of the state system with respect to the state variables (see, e.g. , fleming and rishel, ) , and based on the existence, we can now rely on the pontryagin maximum principle (pmp) ( pontryagin et al., ) for an optimal solution. as is well known, the necessary conditions for an optimal solution of ( ) can be obtained via the pontryagin maximum principle. for this, the hamiltonian h of the optimal control problem ( ) is defined as and its costate equations can be obtained via - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - t s q (t) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - from the optimality conditions, ∂h ∂ q * = and ∂h ∂ θ * = , we can also obtain the following condition for optimal control: also, if upper bounds for the nonnegative control inputs are forced, then by confining the control input to be nonnegative and subject to a positive upper bound q max and θ max , the optimal control of ( ) can be written in the following form: from the above steps, we can conclude that any solution to the optimal control problem ( ) must satisfy the following: s( ) = s , by solving this boundary value ordinary differential equation (ode) problem numerically, we can obtain optimal control inputs for problem ( ) . in order to illustrate the optimal control policy, we simulated the optimally controlled c-siq system. the parameters considered for the simulations are set to be the same as the estimation results except the control inputs, q * ( t ) and θ * ( t ). for the trade- off constants of ( ) , we used c θ = , , c q = . the initial conditions for the simulations were also taken from the estimation results, i.e. , s = , , i = , and s q = . the simulations considered two scenarios. the first scenario does not have bounds on the control inputs, while the second scenario has the bounds of q max = . and θ max = . . with the goal of keeping the infection level low with reasonable control effort s, we solved the boundary value ode problem ( ) using bvp c , the matlab function to solve boundary value problems for ordinary differen-tial equations). figs. - show the simulation results for the first scenario. figs. and show that under the optimal control of the first scenario, the best method of fighting the infection is to initially enter large amounts of q ( t ) and θ ( t ), and later after days to slowly reduce them to zero. the resultant state trajectory of i ( t ) (the dashed line of fig. ) shows that, with the optimal control strategy, the infectious compartment population can be reduced significantly compared to the original case (the solid line of fig. ) . to consider the robustness of the quarantine and isola- tion control, we also conducted sensitivity analysis for the following cases: ( ) when c is % lower and % higher; ( ) when λ is % lower and % higher. for each case we simulated the resultant controlled system, and the results on the number of infectious individuals are shown in figs. and , respectively, for cases ( ) and ( ). comparing figs. and shows that c is more important for the quarantine and isolation control. simulation results for the second scenario, which deals with the bounded input case, are shown in show that under the optimal control of the second scenario, the best method of fighting the infection is to apply the maximum amounts of the control inputs from the start, and then to slowly reduce them after days to zero. the state trajectory of i ( t ) resulting from the optimal control is shown in fig. . note that the infectious compartment population can be reduced significantly even with the bounds on control inputs. from the simulation results ( figs. - ), we can conclude that the mers disease spread can be properly handled by the optimal control approach, and we can obtain a practical guideline, whereby quarantine and isolation effort s in the early stage are critically important in effectively controlling the mers outbreak. in recent years, global pandemic viral infections, including the severe acute respiratory syndrome, the h n influenza, and the ebola outbreak, have been devastating but provided valuable experience in outbreak responses. for public health control, increased vigilance by health professionals and voluntary compliance by the public are essential in implementing rapid effective response interventions. in this study, we carried out an epidemiological assessment of the mers-cov outbreak in korea in order to provide a mathematical framework for understanding the complex dynamics of the pathogen spread and establishing efficient guidelines for implementing quarantine and isolation strategies. the following have been observed by the assessment: • by fitting the siq model, which employs a squashing-type function of fig. for β( t ) , to the real data on the confirmed cases and the quarantined cases, we obtained reasonable performance, as shown in fig. . also, it turned out that the resultant estimated parameters belonged to plausible ranges. by comparison, the conventional siq model utilizing a constant transmission probability could not explain the observed data well. • our nonlinear epidemiological models showed that the mers transmission probability decreased in the squashing-type fashion and then approached a saturation point in a timedependent manner. as information on the mers outbreak be-came widely known in the nation, effort s against this epidemic, including individuals hygienic behavior, and interventions by health care facilities and by authorities were accordingly strengthened. in our siq-based analysis, the inflection point for transmission probability was found to be t β = . , corresponding to a couple of days after june . interest-ingly, june was the day when the korean government revealed the names of mers-affected hospitals to the public. after releasing the names of affected medical facilities, the rate of increase in new confirmed cases abated. as a practical guideline to avoid another similar unexpected outbreak, we draw the conclusion that combined effort s in the early stage are critically important, and sharing information including the names of affected hospitals or countries, clinical situations, and prevention methods might be important for global public health control. • we applied optimal control theory to the controlled siq model with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. simulation results show that the number of the mers cases can be controlled reasonably well via the optimal control approach. in conclusion, this paper proposes a nonlinear epidemiological ode for the mers outbreak in korea, the siq model, in which the state variables are defined as the populations of four compartments ( s ( t ), i ( t ), s q ( t ), and c q ( t )), and the mers transmission probability, β( t ), is modelled by the time-dependent sigmoidal function. we performed the task of parameter estimation for the siq model, and the data fitting results explained the observed data for the confirmed cases and the quarantined cases in the mers outbreak very well. we also applied optimal control theory to the controlled siq model with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. our simulation results show that mers propagation was controlled reasonably well via the optimal control approach. in future work, we will conduct further simulation studies, with the aim of revealing the strengths and weaknesses of the proposed method, and investigate stability and control issues for its various extensions, including a stochastic differential equation (sde) approach. we retrieved publicly available data ( ministry of health and welfare , moh ) from the centers for disease control and prevention and the ministry of health and welfare in the republic of korea. the data included information on the cumulative number of reported cases. the first mers case in korea was confirmed on may , and the numbers of newly confirmed cases and suspected patients who had been quarantined to prevent possible spread of the mers have reached cases and , cases, respectively, as of july . the data also included a brief description of each confirmed case with exposure date and onset of symptoms, and they were sufficient to estimate our siq model for the mers outbreak epidemiology. in this model, we assumed mers is unlikely to spread to another region. we implemented the parameter estimation procedure as a matlab program.we used fminsearch , a matlab function for unconstrained nonlinear optimization, along with some changes of variables in order to carry out the data fitting procedure for the siq model ( ) with the time-dependent variable β( t ) specified in ( ) . the performance index (pi) used in the optimization for parameter estimation was defined as follows: p i = w × ( square d error of c q ) + w × ( square d error of s q ) . the objective function of ( ) , pi , was based on the numbers of the confirmed mers cases and the quarantined cases between may and july. the weight values ( w = and w = ) were obtained empirically via a tuning process based on the training data. simulation results show that the above set of weight values yielded reasonably good fitting results. finally, note that a more sophisticated markov chain monte carlo (mcmc) algorithm ( e.g. , rasmussen et al., ) could be used for parameter estimation of the siq model. however, the use of fminsearch , which was simpler and more transparent, was sufficient for the purposes of this paper. pattern recognition and machine learning impact of vaccine arrival on the optimal control of a newly emerging infectious disease: a theoretical study optimal and sub-optimal control in dengue epidemics mathematical models of isolation and quarantine the impact of media on the control of infectious diseases when is quarantine a useful control strategy for emerging infectious diseases? timely identification of optimal control strategies for emerging infectious diseases deterministic and stochastic optimal control optimal control of epidemics effects of quarantine in six endemic models for infectious diseases optimal control methods applied to disease models optimal control of treatments in a two-strain tuberculosis model modeling infectious diseases in humans and animals optimal control of the chemotherapy of hiv amid panic, a chance to learn about mers optimal control applied to biological models optimal control media/psychological impact on multiple outbreaks of emerging infectious diseases ministry of health and welfare (moh) and center for disease control and prevention mers-cov outbreak in jeddah-a link to health care facilities chemotherapy for tumors: an analysis of the dynamics and a study of quadratic and linear optimal controls the mathematical theory of optimal processes inference for nonlinear epidemiological models using genealogies and time series effect of media-induced social distancing on disease transmission in a two patch setting media impact switching surface during an infectious disease outbreak control of epidemics by quarantine and isolation strategies in highly mobile populations optimal quarantine and isolation strategies in epidemics control isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome this work was supported by a korea university grant. key: cord- - zlu g y authors: nan, yuchen; zhang, yan-jin title: antisense phosphorodiamidate morpholino oligomers as novel antiviral compounds date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: zlu g y phosphorodiamidate morpholino oligomers (pmo) are short single-stranded dna analogs that are built upon a backbone of morpholine rings connected by phosphorodiamidate linkages. as uncharged nucleic acid analogs, pmo bind to complementary sequences of target mrna by watson–crick base pairing to block protein translation through steric blockade. pmo interference of viral protein translation operates independently of rnase h. meanwhile, pmo are resistant to a variety of enzymes present in biologic fluids, a characteristic that makes them highly suitable for in vivo applications. notably, pmo-based therapy for duchenne muscular dystrophy (dmd) has been approved by the united states food and drug administration which is now a hallmark for pmo-based antisense therapy. in this review, the development history of pmo, delivery methods for improving cellular uptake of neutrally charged pmo molecules, past studies of pmo antagonism against rna and dna viruses, pmo target selection, and remaining questions of pmo antiviral strategies are discussed in detail and new insights are provided. researchers realized decades ago that antisense nucleic acids could be used to treat diseases. since then, antisense therapies using a variety of nucleic acids or nucleic acid analogs have been evaluated for use as therapeutic compounds in numerous applications. among these candidate treatments, morpholino oligos, also known as phosphorodiamidate morpholino oligomers (pmo), have demonstrated promising effectiveness in developmental biology research involving gene knockdown as well as clinical trials focusing on treatments of genetic disorders (summerton, ) . specifically, pmos are short single-stranded dna analogs that contain a backbone of morpholine rings connected by phosphorodiamidate linkages (summerton, ) . due to the neutral charged property, they are less likely to interact with proteins while maintain the binding to nucleic acids (moulton and jiang, ). more specifically, pmo bind to complementary sequences of target mrna by watson-crick base pairing and block mrna translation through sequence-specific steric blockade. this process is distinct from the rnase h-dependent mechanism for protein translation inhibition, as induced by other antisense compounds such as phosphorothioate dna (summerton, ) . importantly, pmo are resistant to a variety of enzymes in biologic fluids, which makes them highly suitable for in vivo applications (hudziak et al., ) . to date, pmo-based therapy for duchenne muscular dystrophy (dmd) has shown great success by bypassing effects of a gene mutation underlying a human disease. specifically, this pmo-based therapy restores production of functional dystrophin by altering rna splicing to remove the mutated dystrophin exon that disrupts downstream full-length dystrophin protein translation from the mutated mrna. this drug has been approved by the u.s. food and drug administration and is available in the market under the trade name exondys tm (eteplirsen). approval of exondys tm is a hallmark of pmo-based antisense therapy that demonstrates the potential safety and effectiveness of pmo technology, is driving future development of such therapies for treatment of other diseases. aside from genetic disorders, pmo therapies have also been evaluated as treatments for other broad categories of untreatable diseases, including viral infections, antibiotic-resistant bacterial infections and cancers (enterlein et al., ; warfield et al., ; cansizoglu and toprak, ; chen et al., ) . in this review, pmo background, as well as research focusing on intracellular delivery of pmo, pmo efficacy against both dna and rna viruses, and pmo target selection are outlined. finally, current challenges for development of successful pmo antiviral strategies are discussed in detail and new insights are provided. the concept of an antisense oligonucleotide (on) as a potential therapeutic agent was first demonstrated experimentally through on targeting of a translation initiation site within rous sarcoma virus rna. the on mechanism was initially explained by its participation in formation of an rna-dna duplex with viral rna that sterically blocks viral gene expression and ultimately prevents viral replication (zamecnik and stephenson, ) . subsequent studies revealed that on also participated in a second inhibitory mechanism involving rna:dna duplex recognition by the cellular enzyme rnase h, resulting in rna cleavage and abrogation of virus gene expression as well (tadokoro and kanaya, ). more recently, rnase h-mediated degradation of target rna has been shown to be the main rnainterference mechanism responsible for microrna (mirna), small interference rna (sirna), and dna-directed rna interference (saurabh et al., ) . due to their effectiveness and specificity, such technologies have subsequently attracted the attention of global researchers as tools for therapeutic purposes (davis et al., ; millington-ward et al., ; wittrup and lieberman, ) . the first antisense on tested in clinical trials beginning in was designed to target p for treatment of acute myelogenous leukemia (bayever et al., ) . as a medical milestone for the application of antisense technology, the first antisense drug (fomivirsen) was approved by the fda in for treatment of cytomegalovirus retinitis in immunodeficient patients (marwick, ; wikipedia, ) . since then, numerous antisense drugs have been tested in clinical trials for a variety of other human diseases. to date, five antisense compounds have received marketing authorization from the fda, including eteplirsen. currently, more than ongoing clinical trials of antisense compounds are listed on the website clinicaltrials.gov (godfrey et al., ) . however, compared with traditional drug development, industrial development of therapeutic sirna, dna-based on, or on analogs for control of gene expression have been less successful, as exemplified by the removal of the first commercially available antisense drug, fomivirsen, from the market. a key feature underlying the effectiveness of antisense on is that their nuclease resistance helps them to remain intact for hours in extracellular medium or within cells (summerton, ) . methylphosphonate-linked dna analogs developed in the late s constituted a major advance in the emerging antisense field, resulting in production of the first antisense drug exhibiting acceptable stability in biological systems (hudziak et al., ) . however, in addition to stability, the specificity of rnase-hbased antisense therapy is an important concern, since rnase-h cleaves dna/rna duplexes as short as -or -base pairs in length and is highly active against duplexes that are only - base pairs in length (monia et al., ; summerton, ) . therefore, rnase-h-independent steric blockage of antisense on may be a safer strategy. notably, the effective target region of the steric blocking agent on for inhibiting translation is generally limited to the -utr (untranslated region) and start codon regions of mrna, achieving a good specificity with fewer adverse off-target effects. furthermore, the binding of an rnase h-independent antisense on to a partially matched rna sequence is unlikely to have biological consequences (lebleu et al., ) . oligomers possessing a morpholino phosphorodiamidate backbone, also called pmo (figure ) , constitute a novel type of on analog that is synthesized from ribosides. the ribose ring is opened by oxidation, re-closed using ammonia, which forms a substituted morpholine moiety (summerton and weller, ) . next, the phosphodiester intersubunit bonds are replaced with phosphorodiamidate linkages (summerton and weller, ) . pmo demonstrate excellent resistance to nucleases, figure | comparison of chemical structures of pmo and dna. illustration of phosphorodiamidate morpholino oligomer (pmo) contains backbone of morpholine rings connected by phosphorodiamidate linkages. the ribose ring is opened by oxidation, re-closed using ammonia to form a substituted morpholine moiety. the phosphodiester intersubunit bonds are replaced with phosphorodiamidate linkages. proteases, esterases, and a variety of other enzymes present in biologic fluids (hudziak et al., ) . matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof ms) analysis has demonstrated that pmo are completely resistant to different hydrolases in serum and plasma (hudziak et al., ) , bolstering pmo suitability for in vivo applications (hudziak et al., ) . furthermore, as uncharged molecules, pmo do not interact strongly with proteins, which minimizes hybridization-independent protein interactions since decreased effectiveness of on is likely due to their charged phosphorothioate backbone (moulton and jiang, ; hagedorn et al., ) . pmos bind to complementary sequence in target mrna by watson-crick base pairing to block translation through rnase h-independent steric blockade (figure ) (lebleu et al., ) . moreover, pmo targeting intron-exon junction sequence is capable to modulate pre-mrna splicing as well (figure ) (havens and hastings, ) . for above reasons, antisense pmos have been a revolutionary tool in developmental biology (eisen and smith, ) . by microinjecting pmos into eggs or single or pauci-cellular zygotes, pmos are apportioned into daughter cells during cell division to ensure their delivery to each cell during subsequent cell proliferation (heasman et al., ) . in addition to their use for studies of gene function during embryonic development, pmo have been also tested as treatments for a broad range of diseases in numerous clinical trials (mainly promoted by avi biopharma, inc., now sarepta biopharma inc.). however, application of unmodified pmo is greatly limited by inefficient in vivo delivery unless a relatively high doses are administrated (moulton and jiang, ). in fact, in the animal dmd model, high doses of unmodified pmo injection in dystrophic muscle were needed to induce functional dystrophin expression in skeletal muscle (moulton and jiang, ). thus, two forms of pmos with distinct chemical modifications were developed to facilitate intracellular delivery of pmos, including peptide-conjugated pmo(ppmo) and vivo-pmo. moreover, pmoplus, another novel form of positively charged pmo with a morpholino oligomer-based backbone, has been recently developed as the latest version of pmo. these novel pmo forms are discussed in detail below. initial techniques to deliver pmo into cultured cells were based on mechanical delivery methods such as microinjection or scraping (moulton et al., ) , with associated limitations. more recently, additional mechanical scraping methodologies, electroporation, or use of endosomal escape reagents have been evaluated to improve delivery of on into cytosolic or nuclear compartments in tissue culture (partridge et al., ; moulton and jiang, ) . currently, the most intensively developed and widely used in vivo pmo delivery strategy is based on the use of arginine-rich cell penetrating peptide (cpp) (lebleu et al., ; morcos et al., ) . cell penetrating peptide, also known as protein transduction domain (ptd) or tat-cpp (dietz and bahr, ) , was originally identified after discovery of an unexpected property of the human immunodeficiency virus (hiv) trans-activator of transcription (tat) protein (dietz and bahr, ) . the novel activity was revealed by observations that tat could transactivate a hiv- ltr promoter after crossing cellular and nuclear membranes (dietz and bahr, ) . subsequent structural analysis pinpointed a short peptide located in aa - of tat (sequence grkkrrqrrrppq) that is responsible for the membrane-crossing activity of the parent tat protein (vives et al., ; abes et al., a) . this novel peptide sequence, later named as cpp, was then evaluated for its ability to confer membrane-crossing abilities to other proteins and compounds, including pmo. after fluorescence microscopy or flow cytometry analysis of cells treated with fluoresceintagged pmo, cpp conjugation remarkably enhances cellular uptake of pmo by -to -fold compared with non-conjugated pmo (alonso et al., ) . moreover, other cationic peptides conjugated pmo were shown to be much less effective than pmo conjugated to tat-cpp, while tat-cpp significantly enhanced delivery of pmo to nearly all cells assayed (moulton et al., ) . furthermore, cpp-mediated delivery is a much simpler procedure to conduct than mechanical delivery methods. however, tat-cpp mediated pmos delivery required high pmos concentrations (above µm) to achieve therapeutic antisense activity with cytotoxicity observed. meanwhile, tat-cpp pmos conjugate studies established that the conjugate associated with cell membranes and that internalized conjugate localized to vesicles, cytosol, and nucleus (moulton et al., ) . therefore, cpp sequence optimization, to reduce cytotoxicity and increase uptake efficiency, should enhance pmo effectiveness. this concept has prompted a comparison of two types of cpp (rxr peptide and r pen peptide) (lebleu et al., ) . the most efficient cpp in this study was (r-ahx-r) r, in which ahx represents a -aminohexanoic acid spacer lebleu et al., ) . importantly, (r-ahx-r) r-pmo conjugates were shown to be effective in several murine viral infection models , as well as in treatment of duchenne muscular dystrophy. unfortunately, in some applications, effective doses have approached cytotoxic levels. this obstacle has limited their use in clinical settings (abes et al., b) . vivo-pmo exploits a non-peptide-based transporter to deliver pmo into cultured cells or tissues (morcos et al., ) . currently, gene tools llc (philomath, or, united states) is the major supplier of vivo-pmo for research and development applications. unlike cpp-conjugated pmos, vivo-pmo are covalently linked to a molecular scaffold that carries a dendritic structure assembled around a triazine core that holds eight guanidinium head groups optimally oriented for cell membrane penetration (morcos et al., ; ferguson et al., ) . vivo-pmo effectively entered in vitro cultured cells as well as a wide variety of mouse tissues in vivo to induce correction of a pre-mrna splicing error, as detected by using an experimental test system designed to detect such an event in cells and tissues (morcos et al., ) . compared with cpp-pmos, vivo-pmo have been less frequently investigated for inhibitory effects against target genes. however, available data suggest that at least a % knockdown of target genes can be achieved using vivo-pmo, with no adverse side effects both in vitro and in vivo (guo et al., ; kang et al., ; nazmi et al., ; reissner et al., ) . meanwhile, mouse model studies have also shown that intravenous (iv) and intraperitoneal (ip) administration of vivo-pmo were equally efficacious (reissner et al., ; sartor and aston-jones, ) . furthermore, it appears that vivo-pmo is less cytotoxic than cpp-pmo, since only one report has demonstrated cytotoxicity of vivo-pmo (ferguson et al., ) . it appears that the dendrimer of vivo-pmo is capable to induce red blood cell sedimentation, prompting ferguson et al. to recommend that oligonucleotide analogs should be analyzed for potential to base pair hybridization that may induce dendrimer clustering. moreover, supplementation of vivo-pmo with physiological saline or anticoagulation therapy holds promise for counteracting vivo-pmo toxicity (ferguson et al., ) . compared to cpp-conjugated pmo and vivo-pmo, pmoplus tm is newer type of charged pmo that contain positively charged piperazine groups within its molecular backbone . pmoplus is the most recently developed form of pmo and studies have demonstrated this type of pmo is well tolerated and exhibits improved efficacy in numerous in in vivo viral infection models relative to other pmo therapies (swenson et al., ; warren et al., warren et al., , warren et al., , meng et al., ) . however, pmoplus is still unavailable to most researchers due to its proprietary status as technology solely owned by avi. however, there are reports available to date suggest that pmoplus may be less cytotoxic than cpp-pmo. in a phase i clinical trial to evaluate pmoplus compounds against ebola virus, healthy male and female subjects between and years of age in six dose-expansion cohorts of subjects per dosage group, each received a single i.v. infusion of the active study drug ( . , . , . , . , , or . mg/kg pmoplus). results demonstrated that pmoplus treatments were safe and well tolerated at these doses studied (heald et al., ) , with safety superior to that of cpp-pmo or vivo-pmo. however, a systematic investigation is still needed to compare vivo-pmo and pmoplus with regard to efficiency, stability, and cytotoxicity. an illustration of chemical structures of cpp-pmo, vivo-pmo and pmoplus tm was listed as pmo have been explored as antiviral compounds against rna viruses, including ebola virus, flavivirus, coronavirus, picornavirus, and others. in this section, major advances in this research field are presented. the filoviruses, including marburg virus and ebola virus (ebov), are negative-sense, single-stranded rna viruses that are highly pathogenic, causing human outbreaks of viral figure | chemical structures of cpp-pmo, vivo-pmo, and pmoplus tm . pmo conjugated with cell penetrated peptide (r-ahx-r) r, in which ahx represents a -aminohexanoic acid spacer. vivo-pmo are covalently linked to a molecular scaffold that carries a dendritic structure assembled around a triazine core that holds eight guanidinium head groups optimally oriented for cell membrane penetration. pmoplus tm is charged pmo that contains positively charged piperazine groups within its molecular backbone. hemorrhagic fever with up to % fatality. the - epidemic of ebola virus disease caused , deaths of , total cases in west africa (fischer et al., ) . because currently no commercial vaccines or effective therapeutics are yet available for filovirus infections (fischer et al., ; reynolds and marzi, ; trad et al., ) , antiviral drugs are urgently needed. thus, both peptide-conjugated pmo (ppmo) and non-conjugated pmos have been tested against ebola virus infection in cultured cells and animal models. an earlier study showed that a mer ppmo targeting the translation start site region of ebov vp positive-sense rna exhibited sequence-specific, time-and dose-dependent inhibition of ebov replication in cultured cells (enterlein et al., ) . moreover, this ppmo provided complete protection of mice when administered before or after challenge with a lethal dose of ebov. interestingly, a corresponding non-conjugated pmo also provided protection of mice when administered prophylactically as well. meanwhile, another report in same year demonstrated that a combination of ebovspecific pmos targeting viral mrnas for vp , vp , and rna polymerase l protected rodents against ebov challenge when administered before and after exposure . in the same study, non-conjugated pmo were also tested in a prophylactic proof-of-principal trial in rhesus macaques whereby the same pmo formulation protected % of macaques from lethal ebov infection. more recently, pmoplus has also been shown to be effective against ebola infection in monkeys. when delivered - min after infection, pmoplus avi- (composed of avi- and avi- that target ebov vp and vp , respectively) (iversen et al., ) protected over % of rhesus monkeys against lethal infection with zaire ebola virus (zebov) (warren et al., ) . similarly, pmoplus avi- (composed of avi- and avi- that target vp and vp of marburg virus, respectively) protected % of cynomolgus monkeys against infection with lake victoria marburg virus (marv) when delivered after infection (iversen et al., ) . therefore, pmoplus holds great promise for treatment of patients infected with these highly pathogenic viruses. in another study, the same research group showed that pmoplus avi- targeting vp alone was sufficient to protect monkeys against lethal ebov infection, whereas pmoplus avi- , targeting vp alone, failed to do so (warren et al., ) . these results thus confirm that vp may be a key ebov virulence factor and may serve as a promising target for further development of effective anti-ebov treatment strategies. picornaviruses are non-enveloped positive-sense, single-stranded rna viruses belong to the family picornaviridae. this family includes more than genera and species (zell et al., ) , with rna genomes length ranging from . to . kb. many picornaviruses are important human and animal pathogens, including poliovirus, coxsackievirus b (cvb ), enterovirus (ev- ), and foot-and-mouth disease virus (fmdv). no vaccine yet exists for picornaviruses other than for poliovirus and fmdv. moreover, no effective antiviral therapy yet exists for treatment of infections caused by any pathogenic picornavirus. however, ppmo targeting conserved internal ribosome entry site (ires) sequences have been shown to be highly effective in protecting cultured cells against infection by human rhinovirus type , coxsackievirus type b , and poliovirus type (pv ) (stone et al., ) , with reduction of pv titers by up to log . this mer ppmo (enterox) targets an ires sequence that is identical for > % of all human enteroviruses and rhinoviruses which has been successfully used to treat poliovirus receptor (pvr) transgenic mice to prevent pv infection after challenge with three times the % lethal dose (ld ). this result also showed that mice receiving ppmo treatment exhibited an approximately % higher survival rate than controls, with significant reduction of viral titers in small intestine, spinal cord, and brain (stone et al., ) . coxsackievirus b (cvb ) is a primary cause of viral myocarditis, without any effective therapy. in study tested eight cvb -specific ppmo in cultured hela cells and hl- cardiomyocytes, as well as in a murine infection model (yuan et al., ). among eight ppmos tested, ppmo- , designed to target the portion of the cvb ires, was especially potent against cvb replication in cultured cells. when cells were treated prior to or shortly after cvb infection, virus proliferation was significantly inhibited, with approximate log decrease of viral titers. in a/j mice, ppmo- intravenous administration once prior to and once after cvb infection significantly reduced cardiac tissue damage, with notable decreases in myocardium virus titers than control (yuan et al., ) . enterovirus (ev- ) generally causes mild hand-foot-andmouth disease, but severe neurological complications with high mortality rates have been reported. in one study, testing of vivo-pmo designed to target the ev- ires and the rnadependent rna polymerase (rdrp) was performed (tan et al., ) . vivo-pmo targeting ev- ires significantly reduced ev- replication in human embryonal rhabdomyosarcoma rd cells. in contrast, vivo-pmo targeting ev- rdrp was less effective. the results suggest that ires-targeting vivo-pmo are potential antiviral candidates that can abrogate early ev- infection (tan et al., ) . fmdv causes a highly contagious viral disease of clovenhoofed animals that can lead to severe economic losses to the livestock industry. six ppmos were designed to target and utrs of the fmdv genome (strain a( ) cruzeiro/brazil/ [a( )cru]) and were evaluated in cultured cells (vagnozzi et al., ) . three of the ppmos, targeting domains including the portion of the ires and the two translational start codoncontaining regions, were highly effective in inhibiting fmdv replication. at low micromolar concentrations, ppmo led to a dose-dependent and sequence-specific virus titer reduction of over log , while three other ppmo that targeted other genome regions were less effective (vagnozzi et al., ) . the order nidovirales includes the families coronaviridae, arteriviridae, roniviridae, and mesoniviridae (cong et al., ) . the members of this group are positive-sense, single-stranded rna viruses. the coronaviridae and arteriviridae include groups of viruses infecting vertebrates (mainly mammalian species), whereas the other two families include viruses infecting invertebrates. pmo have been tested as antivirals against members of both coronaviridae and arteriviridae. members of coronaviridae include severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), which are recently identified as potent human pathogens (cong et al., ) . mouse hepatitis virus (mhv), also belongs to this family, which has long served as a model for understanding viral hepatitis in humans and to assess pmo antiviral compounds (neuman et al., ; burrer et al., ) . ppmo targeting the mhv replicase exhibited low toxicity in dbt astrocytoma cells, a cell line for studying mhv infection (neuman et al., ) . a later study tested ppmos against several mhv strains in cell culture and in vivo using mouse models (burrer et al., ; moulton et al., ) . among ten ppmos against various viral genome target sites, ppmo term, which targeted the terminus of the rna genome, was found to be highly effective in inhibiting six different mhv strains. in mice, term ppmo treatment led to prevention of virus-induced tissue damage. prophylactic treatment with term ppmo also decreased mhv-induced weight loss and prolonged post-challenge survival. this study also showed that no weight loss or detectable histopathologic changes were observed after prolonged ppmo treatment of uninfected mice (burrer et al., ) . besides, ppmo have also been shown to inhibit sars-cov replication as well (neuman et al., ). among all ppmo tested, two ppmo targeting the viral transcription-regulatory sequence (trs) within the utr brought about the most significant inhibition of cpe and reduced cell-to-cell spread when administered prior to peak viral proliferation in cultured cells (neuman et al., ) . members of the arteriviridae include an economically important virus, porcine reproductive and respiratory syndrome virus (prrsv). prrsv causes a contagious swine disease characterized by reproductive failure in sows and respiratory disease in pigs of all ages. the disease has plagued the global swine industry since it was first reported in and current strategies used for prrs control are still inadequate (du et al., ) . research in our laboratory has focused on development of ppmos against prrsv. based on the genome sequence and viral protein function, a series of ppmos with various targets were designed that included three ppmo targeting the end utr of the prrsv genome, namely up , up , and hp, as well as six ppmo targeting the translation initiation regions of orfs - , namely p , p , p , p , p , and p han et al., ) . ppmo targeting the utr were highly effective for inhibiting prrsv replication in cell culture in a dosedependent and sequence-specific manner. specifically, ppmo up or hp caused a . log reduction of prrsv yield. moreover, up and hp also demonstrated broad inhibition of heterogeneous prrsv isolates . in addition, if ppmo up targeting the utr of prrsv genome was paired with ppmo p and p , which targeted translation initiation regions of orfs and , respectively, the combination enhanced inhibition of heterologous strains of the north american prrsv genotype more effectively than individually in vitro (han et al., ) . inhibition was verified at both prrsv rna and protein expression levels. since ppmo up , which targets a highly conserved sequence within the -terminal region of prrsv genome, effectively induces a multi-log inhibition of prrsv replication in vitro, we also conducted an in vivo evaluation of this ppmo (opriessnig et al., ) . prrsv-negative -week-old piglets received ppmo intranasally at h before infection as well as and h after prrsv infection. ppmo treatment was well tolerated in piglets, with no weight change observed across all piglet groups and ppmo administration significantly reduced prrsv viremia and interstitial pneumonia. moreover, in alveolar macrophages isolated at days post-infection, elevated expression of antiviral genes in ppmo-treated piglets was observed as well (opriessnig et al., ) . besides prrsv, antiviral effects of pmo were also evaluated against equine arteritis virus (eav), another member of the family arteriviridae (van den born et al., ) . similar to prrsv studies above, ppmo designed to target the terminal utr of the eav genome remarkably reduced virus replication in a sequence-specific and dose-responsive manner. however, ppmo that targeted -terminal regions of the viral genome or its anti-genome only resulted in moderate reduction of eav replication when relatively high concentrations of the ppmos were applied. moreover, ppmo targeting the eav trs, which is essential for subgenomic rna synthesis, were ineffective to achieve transcription interference (van den born et al., ) . however, ppmo targeting the utr of eav were able to cure viral infection of persistently infected hela cells (zhang et al., ) . flavivirus is a viral genus within the family flaviviridae. this genus includes west nile virus (wnv), dengue virus (denv), yellow fever virus, zika virus, and several other viruses which may cause encephalitis (coffey et al., ; musso and gubler, ) . except for yellow fever virus, no effective vaccine or antiviral drug exists against flaviviruses, prompting evaluation of pmo against several members of flavivirus. among a panel of ppmo against wnv, ppmo targeting the -and -termini of the wnv genome, designated end or csi, exhibited the greatest potency in blocking wnv replication (deas et al., ) . moreover, treatment of wnv-infected cells with either end or csi pmo led to a significant reduction of virus titers by approximately - log without apparent cytotoxicity. ppmo end inhibited wnv translation, whereas ppmo csi suppressed wnv rna replication. meanwhile, ppmo csi also inhibited other mosquito-borne flaviviruses when the targeted csi-like sequences which were relatively conserved to the respective wnv csi target sequence. therefore, ppmo targeting conserved cis-acting elements of flavivirus genomes should be explored as anti-flavivirus therapeutics (deas et al., ) . more recently, both ppmo end or csi were also tested in a mouse model of wnv infection and provided partial protection when administered at or µg/day. moreover, minimal to no ppmo-mediated toxicity observed, while toxicity was observed at a larger dosage of µg/day (deas et al., ) . a panel of ppmo was also tested against denv (kinney et al., ) whereby ppmo targeting -terminal nucleotides of the serotype denv (den- ) virus genome exhibited relatively poor suppression of den- virus titer. however, moderate reduction of titer was observed for ppmo targeting either the aug translation start site region of the single open reading frame or the cyclization sequence region. the most highly effective ppmo were sl and cs (targeting the -terminal nucleotides and the cyclization sequence region, respectively), which reduced viral titer by greater than . log compared to controls at days post-infection with den- virus. notably, treatment with µm cs inhibited replication of all four den virus serotypes by over log , in most cases to below detectable limits (kinney et al., ) . a third ppmo that was designed to target the top of the stem-loop ( slt) inhibited den replication in bhk cells (holden et al., ) . the inhibitory mechanism was studied using a novel den reporter replicon and a den reporter mrna. the results demonstrated that sl inhibited viral translation and cs blocked viral rna synthesis but not viral translation, whereas the slt inhibited both viral translation and rna synthesis (holden et al., ) . more recently, anti-denv ppmo sl and cs were also tested in ag mice before and/or after infection with denv- . intraperitoneal (ip) infection of ag mice with - pfu of denv- (strain new guinea c) shortened survival to - days. when sl or cs were administered before and after denv infection, the average survival time was extended by up to more days. this study also included pharmacokinetic and toxicology analysis of non-infected animals. the results showed that the mice had high concentrations of ppmo in liver following nine consecutive once-daily ip treatments of mg/kg ppmo, with little impact on overall mouse health . pmo and ppmo have also been studied for their ability to prevent or treat japanese encephalitis virus (jev) infection as well. a ppmo (p ) targeting the cyclization sequence ( csi) of jev exhibited significant antiviral activity in vero (epithelial), neuro a (neuronal), and j e (macrophage) cells at non-toxic concentrations (anantpadma et al., ) . addition of p to cells before infection decreased jev replication to undetectable levels in vero cells and resulted in a and % reduction in jev titer in j e and neuro a cells, respectively. in this study, antiviral effects of p were also assessed in vivo. when treated intracerebrally with a mg/kg dose of p every h for days, - % of -week-old mice were protected from a lethal dose of jev (anantpadma et al., ) . meanwhile, testing of vivo-pmo targeting the and utr of the jev genome have also been conducted in mice (nazmi et al., ) . administration of intraperitoneal injections of vivo-pmo ( mg/kg body weight) daily for up to days immediately after jev infection of mice prolonged survival, with reduced viral load and viral protein expression in brain. moreover, proinflammatory cytokine levels in brain, which normally increase after jev infection, were reduced following pmo treatment and align with observations of reduced microglial activation in brain as well (nazmi et al., ) . chikungunya virus (chikv) causes infection in humans that is associated with debilitating and persistent arthralgia and arthritis. two ppmo were designed to target highly conserved sequences present in chikv non-structural and structural polyproteins (lam et al., ) . cpmo , a ppmo that targets the orf aug region, significantly suppressed chikv replication in hela cells when administered before infection. notably, in neonatal mice, administration of µg/g cpmo before infection conferred % survival against chikv disease (lam et al., ) . ppmo have also been tested for antiviral effects toward group v (-)ssrna virus families that include pneumoviridae, paramyxoviridae, orthomyxoviridae, arenaviridae, and others. respiratory syncytial virus (rsv), a member of the family pneumoviridae, is a major cause of lower respiratory tract infections in infants, young children, and high-risk adults. currently, no vaccine exists to prevent rsv infection. however, two antisense ppmos designed to target the -terminal region and the translational start site of rsv l mrna have been tested and exhibited minimal cytotoxicity (lai et al., ) . ppmo aug- inhibited rsv replication by reducing viral titers by over log . when administered before rsv intranasal inoculation, ppmo aug- protected balb/c mice from infection, with reduced viral titers in lung tissue and attenuation of pulmonary inflammation (lai et al., ) . measles virus (mev) is a member of the family paramyxoviridae. mev is a highly contagious human pathogen that can be treated effectively with available antiviral compounds and prevented using a vaccine. five ppmos targeting mev genomic rna or mrna were tested in cultured cells (sleeman et al., ) . ppmo , targeting a conserved sequence in the translation start site of the mrna coding for viral nucleocapsid protein, was highly effective against multiple genotypes of mev (sleeman et al., ). influenza a virus, a member of the family orthomyxoviridae, is a relentless ongoing global public health concern. when delivered by intranasal administration, ppmo inhibited replication of equine influenza a virus fluav a/eq/miami/ / (h n ) in mice and exhibited no toxicity at effective antiviral concentrations in vivo (lupfer et al., ) . meanwhile, a ppmo panel was developed to target rna genome segments encoding polymerase subunits of a highly pathogenic mouse-adapted influenza a virus strain (sc m; h n ) (gabriel et al., ) . in this study, virus replication in mdck cells was significantly inhibited by three ppmo targeting either the translation start site region of pb or np mrna or the -terminal region of np viral rna (vrna). in a mouse model, when ppmo targeting the pb -aug region or np vrna were administered intranasally once h before and once days after intranasal infection with a lethal dose of sc m, treated mice exhibited significantly lower viral titers in lungs and % greater survival versus untreated controls over the -day duration of the experiment (gabriel et al., ) . besides targeting viruses, ppmo have also been tested for efficacy against host mrna encoding proteases crucial for viral infectivity. such ppmo can block host protease cleavage of the influenza virus hemagglutinin (ha) to inhibit viral infectivity. treatment of human calu- airway epithelial cells with a ppmo t-ex , designed to interfere with splicing of hacleaving protease tmprss pre-mrna, resulted in production of tmprss mrna lacking exon that resulted in production of an enzymatically inactive form of tmprss (bottcher-friebertshauser et al., ) . ultimately, t-ex ppmo was shown to prevent cleavage of has of various human seasonal and pandemic influenza a viruses, leading to significant reduction of viral titers by to log (bottcher-friebertshauser et al., ) . junín virus, a threat to human health and a member of the arenaviridae family, can cause meningitis and hemorrhagic fever. ppmo designed to interfere with translation have been shown to be effective in reducing junín virus replication (neuman et al., ) . in cultured cells, ppmo target sequences located at the termini of both genomic segments are highly conserved across the arenaviruses. consequently, these ppmo are effective against junín virus, tacaribe virus, pichinde virus, and also against lymphocytic choriomeningitis virus (lcmv) whereby they suppress viral titers in livers of lcmv-infected mice (neuman et al., ) . the genus alphavirus includes positive-sense rna viruses that threaten human health (paessler et al., ) . sindbis virus (sinv) is a member of the family togaviridae. ppmo targeting both the -terminus and aug translation start site of the sinv genome significantly suppressed sinv replication in tissue culture (paessler et al., ) . venezuelan equine encephalitis virus (veev) is another member of the togaviridae. ppmo targeting veev regions corresponding to sinv regions mentioned above inhibit several strains of veev in vitro. notably, mice pre-treated with pmo were protected from lethal veev infection, while only partial protection was observed for mice receiving only post-infection pmo treatment (paessler et al., ) . noroviruses, which belong to the caliciviridae family, are non-enveloped, positive-sense, single-stranded rna viruses with genomes of approximately . kb in length that encode three orfs. noroviruses cause non-bacterial epidemic gastroenteritis (bok et al., ) . ppmo targeting the first aug region of the orf near the -end of the murine norovirus (mnv) genome effectively inhibited mnv replication in cultured cells (bok et al., ) . moreover, a consensus ppmo targeting the corresponding -end of the genome of several diverse human norovirus genotypes also inhibited norwalk virus protein expression (a species of norovirus) in replicon-bearing cells in a cell-free luciferase reporter assay (bok et al., ) . similar to noroviruses, hepatitis e virus (hev) is a positivesense, single-stranded rna virus containing three orfs and is currently classified within the family hepeviridae (nan and zhang, ) . hev shares a similar genome structure with members of caliciviridae and was previously classified in that family (nan and zhang, ) . our study demonstrated that ppmo targeting the terminal of the hev genome at the start codon of orf are highly effective against both hev genotypes and infection in vitro (nan et al., ) . moreover, ppmo targeting the utr of the hev genome or the terminus of antisense hev rna can also block hev replication, but to a lesser extent than achieved by ppmo targeting the terminus (nan et al., ) . since the utr and terminus of antisense hev rna are generally considered binding sites for hev rnadependent rna polymerase (rdrp), it appears that ppmomediated steric blockade also applies to rdrp as well (nan et al., ) . as compared with pmos' applications against rna viruses, pmos use against dna viruses has been much less studied. to date, only members of herpesviridae have been tested for pmo-mediated inhibition. research from our lab evaluated pmos against kaposi's sarcoma-associated herpesvirus (kshv). kshv, also known as human herpesvirus (hhv- ), is a human oncovirus belonging to the gamma herpesvirus subfamily. kshv is associated with kaposi's sarcoma (ks) and two b-cell lymphoproliferative diseases: primary effusion lymphoma (pel) and multicentric castleman's disease (mcd) (purushothaman et al., ) . these diseases are aids-related malignancies in hiv-infected patients. kshv infection in humans exhibits either a lifelong immunologically silent and latent infection or a transient lytic infection with distinct viral gene-expression profiles (lagunoff et al., ; bechtel et al., ) . during the predominantly latent state of kshv infection, the kshv genome is maintained as circular, extra-chromosomal dna that replicates inside host cells in a cell cycle-dependent manner. expression of a few key viral regulators, such as latency-associated nuclear antigen (lana) encoded by orf , viral cyclin (vcyclin) encoded by orf , and viral flip (vflip) encoded by orf , are observed during latency (uppal et al., ) . upon reactivation to assume a lytic infection state, a full repertoire of lytic viral genes, including orf (transcription activator, rta), orf , orf , orf , orf , orf , orf-k , orf-k (virf- ), orf-k (viral interleukin- , vil- ), orf (viral g protein-coupled receptor vgpcr), and viral chemokines (orf-k /vccl-i and orf-k /vccl-ii) are expressed in a temporally-regulated manner (purushothaman et al., ) . based on the functions of kshv latent and lytic genes, a panel of ppmos was designed against a set of genes including lana, vil- , rta, and virf- (zhang et al., (zhang et al., , (zhang et al., , . treatment of kshv-positive pel cells with an rta-specific ppmo rp not only reduced rta expression but also caused down-regulation of several other early and late kshv gene products, including vil- , virf- , and orf-k . a. moreover, kshv dna copy numbers both in ppmo rp -treated pel cells and culture supernatants were reduced, demonstrating inhibition of kshv lytic replication (zhang et al., ) . furthermore, treatment of bcbl- cells with ppmo against lana reduced lana expression (zhang et al., ) . meanwhile, ppmo against vil- and virf- were also evaluated. the viral homologue of the proinflammatory cytokine il- , vil- , is believed to contribute to vascular permeability and formation of pel effusions (sakakibara and tosato, ) . this cytokine shares low but significant homology to human irf family members and acts as an oncogene to inhibit interferon induction (gao et al., ) . treatment of pel cells with ppmo designed against vil- mrna led to marked reduction in the proportion of vil- -positive pel cells and reduced both the growth of pel cells and kshv dna levels (zhang et al., ) . meanwhile, ppmo targeting virf- have also been shown to inhibit viral dna replication in addition to blocking virf- expression in bcbl- cells (zhang et al., ) . interestingly, reduction of virf- expression in kshv-infected cells resulted in higher expression levels of cellular irf- and of the signal transducer and activator of transcription (stat ) (zhang et al., ) . encouraged by these in vitro data, an in vivo evaluation of ppmo effects on expression of multiple viral genes was conducted (zhang et al., ) . however, in vivo results defied expectations drawn from in vitro studies. specifically, only ppmo against vil- demonstrated promising in vivo inhibition whereby scid mice treated with this ppmo exhibited no engraftment of kshv-infected pel cells and remained healthy throughout the -day study (zhang et al., ) . conversely, in scid mice receiving a combination of pmo against two virf- , there was a trend of less engraftment of kshv-infected pel cells, but the difference in results was not statistically significant between treated and control mice. therefore, ppmo targets selected using in vitro assessments may not achieve expected inhibition in vivo, emphasizing the fact that careful validation using adequate animal models is necessary. acyclovir (acv) is a nucleic acid analog of guanosine that is used to treat hsv. viral resistance to acv has become a recent concern. as a potential treatment for acv-resistant hsv, ppmo against resistant hsv- and hsv were evaluated both in vitro and in a mouse model as well. for hsv- , icp and icp were selected as virus ppmo targets. icp from both hsv- and hsv- acts as an e ubiquitin ligase to antagonize host innate immunity (halford et al., ; lanfranca et al., ) . icp is a multiple function regulator involved in pre-mrna splicing and the host innate immune response (christensen et al., ; tang et al., ) . when ppmo targeting translation start site regions of hsv- icp or icp mrna were applied before or soon after hsv- infection of cultured cells, a - % reduction of hsv- yield was observed, as assessed by reduced plaque formation. moreover, icp -specific ppmo also inhibited acvresistant hsv- plaque formation by - %, while an equivalent dose of acv only led to a - % plaque reduction (moerdyk-schauwecker et al., ) . in vivo data suggest that ppmo are well-tolerated in uninfected mice after days of administration of µg/day (moerdyk-schauwecker et al., ) . topical application of µg icp -specific ppmo into the eyes of hsv- -infected mice reduced the incidence of eye disease by . - % compared to controls. therefore, ppmo holds promise as an antiviral drug for use in treating hsv- ocular infection (moerdyk-schauwecker et al., ) . with regard to hsv- , ppmo targeting icp or icp mrna were also highly effective against either non-acvresistant or acv-resistant hsv- strains. in one in vivo study, ppmo were well-tolerated in balb/c mice and cotton rats. cotton rats receiving icp -specific ppmo h after hsv- inoculation showed a reduction in genital lesions and a . % reduction in mortality at days post-infection. mice receiving a combined regimen of µm of icp -and icp -specific ppmo before hsv- inoculation were completely free from genital viral infection at - days post-inoculation (eide et al., ) . a variety of studies suggest that pmo would be good candidate for antiviral therapeutics against emerging or reemerging viruses in the absence of other effective therapies. however, effective target sequences for pmo design need to be carefully validated. a list of previously published effective virus target regions used for pmo designed against rna viruses are briefly summarized in table . to date, for most pmos evaluated against positivesense rna viruses, targeting sequences of pmo are mainly located in the terminal ends of viral genomes (table ) . notably, it appears mrna-like properties of positive-sense rna virus genomes make them highly susceptible to pmo-mediated translation inhibition if the pmos pairing sequence are located at the terminal end of the viral genomes, with few exceptions. moreover, utr at either end of viral genomes are generally conserved among viral strains. conversely, for some positivesense rna viruses, pmo targeting of the ' terminal ends of the viral genome were tested as well. however, data appears to be mixed for terminal-targeting pmos. on the one hand, for members of flaviviridae, pmos targeting the ' cyclization sequence located within the terminal end are highly effective in inhibiting virus replication (deas et al., (deas et al., , kinney et al., ; holden et al., ; stein et al., ; anantpadma et al., ; nazmi et al., ) . on the other hand, the use of pmo targeting either the -terminal regions of the viral genome or of the negative genomic strand only resulted in moderate reduction of eav (van den born et al., ) . this result suggests that pmo targeting the terminal end are less effective for inhibiting eav replication compared to pmo targeting the terminal utr of the eav genome. therefore, the viral genome end is the preferred target region for antiviral pmo design against positive-sense rna viruses. in addition to pmo targets within the terminal end of positive-sense rna virus genomes, pmo targeting rna secondary structures should also be considered, especially for common conserved elements among rna viruses, such as ires sequences. generally, unstructured regions are more accessible to oligonucleotide binding than are structured regions, since internal structures within target rna can impede pmo binding (childs-disney and disney, ) . however, in some cases, direct targeting of ires sequences could inhibit replication of some viruses, including members of the order picornavirales (yuan et al., ; stone et al., ; tan et al., ) . therefore, a deep analysis of the secondary structures of target rna may aid pmo target selection. for pmos evaluated for antiviral activity against negativesense rna viruses, current reports mainly focus on blocking translation of individual viral genes rather than direct targeting the rna genome. for example, pmo targeting ebov vp alone was sufficient to protect monkeys against lethal ebov infection, whereas a pmoplus formulation designated avi- that targeted vp failed to do so (warren et al., ) . therefore, screening to find effective pmo targets of negative-sense rna viruses may require more careful consideration than needed for positive-sense rna viruses. although not yet extensively studied, pmo target selection appears to be more complicated for large dna viruses such as herpesviruses. due to their large genome size, such dna viruses encode many genes, including indispensable and dispensable genes. on the one hand, some indispensable genes studied so far do not appear to be good pmo targets. for example, hsv- early genes ul and ul encode the viral dna polymerase and the large subunit of ribonucleotide reductase, respectively, which are essential for viral dna replication (eide et al., ) . however, blocking hsv- ul and ul mrna translation by pmo did not significantly reduce viral replication or transmission from infected cells (eide et al., ) . on the other hand, dna viruses encode many dispensable accessory proteins with multiple functions, such as antagonist to host innate immunity. such genes need to be carefully validated both in vitro and in vivo before use as pmo targets. for example, pmo targeting of virf- of kshv demonstrated little protection against kshv-infected pel engrafts in scid mice, in spite of its effectiveness as a pmo target in vitro (zhang et al., ) . aside from challenges regarding target selection, the emergence of resistant virus after sequence-specific therapy is another common challenge faced by antivirals. indeed, a hcmv mutant with sequence-dependent resistance to the phosphorothioate oligonucleotide fomivirsen was discovered almost simultaneously with market approval of the drug (mulamba et al., ) . because pmo-based antiviral therapy is sequence-specific, pmo-resistant target mutations would abolish pmo inhibition, as has been already observed (neuman et al., ) . for west nile virus, the sequencing of ppmo-resistant wnv mutants has demonstrated that viruses resistant to -end ppmo treatments contained two to three mismatches within the ppmo-binding site, whereas csi ppmo-resistant viruses accumulated mutations outside the ppmo-targeted region (deas et al., ) . meanwhile, ppmo-resistant-wnv infection of mice was shown to antagonize pmo-mediated protection against virus (deas et al., ) . similar pmo-resistance has been reported for ebola virus after treatment with pmo targeting ebola virus vp and vp as well (kugelman et al., ) . therefore, mutations within or outside of pmo-targeting sequences might lead to pmo resistance and will be a future challenge. in our research of ppmo inhibition of prrsv, a multiple log reduction of viral rna copies was achieved when pmo sequence was complementary to a conserved region within the utr of the prrsv genome, although low level viral rna replication was still observed. this suggests that pmoresistant mutants might have arisen or that the effective one possible strategy to encounter pmo-resistance mutants would be incorporating of promiscuous bases such as inosine to compensate for predicted base-pair mismatches . however, it is a challenge to accurately predict the potential mutation nucleotides within a pmo-targeting sequence. therefore, the pmo target sequence against any specific rna virus must be carefully selected to minimize generation of mutants. moreover, use of multiple pmos against different targets of the same virus may help to avoid mutant virus generation. an alternative strategy to prevent pmo-resistant virus is indirect inhibition of a host factor essential for virus replication. currently, only one of such study has been conducted on influenza virus and exploits the fact that cleavage of viral hemagglutinin (ha ) by host proteases is crucial for viral infectivity. in this study, treatment of human calu- airway epithelial cells with a ppmo designed to interfere with pre-mrna splicing of tmprss (the host protease responsible for ha cleavage) resulted in tmprss mrna lacking exon , finally led to expression of an enzymatically inactive form of tmprss (bottcher-friebertshauser et al., ) . therefore, blocking of ha cleavage by this ppmo was confirmed in different human seasonal and pandemic influenza a viruses and resulted a significant reduction of viral titers (bottcher-friebertshauser et al., ) . in addition to above investigation, variety host proteins have been identified as essential factors required for virus replication, host defense and viral pathogenesis in recent years (rajsbaum et al., ; tripathi et al., ) . the putative ubiquitin ligase ubr was identified as a novel host factor involved in the budding of influenza virion (tripathi et al., ) , targeting such factor could block the release of influenza virus. although it is unknown if ubr could be employed by other enveloped viruses for budding as well, it is possible that targeting a commonly used host factor which is required for viral replication could broaden the antiviral spectrum of pmo. therefore, application of pmo to target host factor as antiviral strategy may not only avoid generation of pmo-resistant virus, but also expand the antiviral spectrum as long as the targeted host factor is employed by different virus for replication. it has been two decades since the first approval of an antisense-based therapy. pmos have become important on analogs that have driven development of antisense therapies with efficacy and safety demonstrated by the recent fda approval of eteplirsen. indeed, biological stability, neutral charge, and rnase h-independent mechanism of action are all unique pmo features. moreover, besides their development as antiviral compounds, pmo evaluation as anti-cancer or antibacterial agents is ongoing (enterlein et al., ; warfield et al., ; cansizoglu and toprak, ; chen et al., ; summerton, ) . since pmo have a good track record as useful tools in developmental biology and as therapeutic agents, three generations of pmo have been developed (unmodified pmo, conjugated pmo, and pmoplus) to improve intracellular delivery (daly et al., ) . however, several issues remain unresolved before pmo are adopted for widespread use. on the one hand, more studies are needed to establish a convenient route for pmo administration in vivo. in most in vivo studies, pmo are administrated via either intravenous or intramuscular injection. although reports have demonstrated that pmo administered via intranasal delivery can inhibit replication of viruses with a respiratory tract tropism (opriessnig et al., ; rajsbaum, ) , it is notable that in these in vivo studies employed pmos for as antiviral agents against two respiratory viruses, influenza virus and prrsv (opriessnig et al., ; rajsbaum, ) . since there is no investigation conducted to see if intravenous or intramuscular administration of pmo also effectively against influenza virus and prrsv, it is possible intranasal delivery of pmo is preferable for virus causes respiratory infection. moreover, it is also possible that virus-specific delivery routes based on the initial infection sites may offer a better antiviral efficiency rather than using intravenous route as universal way for pmo administration. on the other hand, although pmo is a highly adaptive platform for delivery of nucleic acid sequence-specific drugs, selection of an effective target and avoidance of the emergence of mutant virus also require more investigation. if these issues could be properly addressed, pmo should serve as a promising strategy for treatments of a variety of diseases, including difficult-totreat viral infections. as is generally true for antiviral drug development, high efficacy, low toxicity, good pharmacokinetics, good bioavailability, and low cost are all characteristics sought in a pmo compound. results discussed in this review show great promise and warrant further research to develop safe and effective antisense treatments for a variety of human diseases. yn and y-jz designed this manuscript. yn prepared the main body of the manuscript. y-jz prepared the figure and revised the manuscript. all authors approved it for publication. arginine-rich cell penetrating peptides: design, structure-activity, and applications to alter pre-mrna splicing by steric-block oligonucleotides delivery of steric block morpholino oligomers by (r-x-r) peptides: structure-activity studies inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers inhibition of japanese encephalitis virus replication in cultured cells and mice by a peptide-conjugated morpholino oligomer systemic administration of a phosphorothioate oligonucleotide with a sequence complementary to p for acute myelogenous leukemia and myelodysplastic syndrome: initial results of a phase i trial host range of kaposi's sarcoma-associated herpesvirus in cultured cells inhibition of norovirus replication by morpholino oligomers targeting the -end of the genome inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease tmprss antiviral effects of antisense morpholino oligomers in murine coronavirus infection models fighting against evolution of antibiotic resistance by utilizing evolvable antimicrobial drugs modulation of nuclear rest by alternative splicing: a potential therapeutic target for huntington's disease approaches to validate and manipulate rna targets with small molecules in cells hsv- icp targets the tbk -activated sting signalsome to inhibit virus-induced type i ifn expression chikungunya virus-vector interactions the interaction between nidovirales and autophagy components inhibition of bacterial growth by peptide-conjugated morpholino oligomers evidence of rnai in humans from systemically administered sirna via targeted nanoparticles in vitro resistance selection and in vivo efficacy of morpholino oligomers against west nile virus inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and rna replication delivery of bioactive molecules into the cell: the trojan horse approach antiviral strategies against prrsv infection reduction of herpes simplex virus type- replication in cell cultures and in rodent models with peptide-conjugated morpholino oligomers controlling morpholino experiments: don't stop making antisense vp knockdown inhibits ebola virus amplification and protects against lethal infection in mice lessons learned from vivo-morpholinos: how to avoid vivo-morpholino toxicity ebola virus disease: an update on post-exposure prophylaxis morpholino oligomers targeting the pb and np genes enhance the survival of mice infected with highly pathogenic influenza a h n virus kshv orf k (virf) is an oncogene which inhibits the interferon signaling pathway delivery is key: lessons learnt from developing spliceswitching antisense therapies transcription factor sox b is involved in spinal cord regeneration in adult zebrafish managing the sequence-specificity of antisense oligonucleotides in drug discovery a live-attenuated hsv- icp virus elicits to times greater protection against genital herpes than a glycoprotein d subunit vaccine enhanced inhibition of porcine reproductive and respiratory syndrome virus replication by combination of morpholino oligomers splice-switching antisense oligonucleotides as therapeutic drugs safety and pharmacokinetic profiles of phosphorodiamidate morpholino oligomers with activity against ebola virus and marburg virus: results of two single-ascending-dose studies beta-catenin signaling activity dissected in the early xenopus embryo: a novel antisense approach inhibition of dengue virus translation and rna synthesis by a morpholino oligomer targeted to the top of the terminal stem-loop structure resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation discovery and early development of avi- and avi- for the treatment of ebola virus and marburg virus infections antisense-induced myostatin exon skipping leads to muscle hypertrophy in mice following octa-guanidine morpholino oligomer treatment inhibition of dengue virus serotypes to in vero cell cultures with morpholino oligomers evaluation of the potential impact of ebola virus genomic drift on the efficacy of sequence-based candidate therapeutics de novo infection and serial transmission of kaposi's sarcomaassociated herpesvirus in cultured endothelial cells inhibition of respiratory syncytial virus infections with morpholino oligomers in cell cultures and in mice antiviral phosphorodiamidate morpholino oligomers are protective against chikungunya virus infection on cell-based and murine models hsv- icp : an e ubiquitin ligase that counteracts host intrinsic and innate immunity cell penetrating peptide conjugates of steric block oligonucleotides inhibition of influenza a h n virus infections in mice by morpholino oligomers first "antisense" drug will treat cmv retinitis quantitative determination of avi- (radavirsen), a phosphorodiamidate morpholino oligomer (pmoplus((r)) ), in human plasma using lc-ms/ms suppression and replacement gene therapy for autosomal dominant disease in a murine model of dominant retinitis pigmentosa inhibition of hsv- ocular infection with morpholino oligomers targeting icp and icp evaluation of -modified oligonucleotides containing '-deoxy gaps as antisense inhibitors of gene expression vivo-morpholinos: a non-peptide transporter delivers morpholinos into a wide array of mouse tissues cell-penetrating peptide-morpholino conjugates alter pre-mrna splicing of dmd (duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo hiv tat peptide enhances cellular delivery of antisense morpholino oligomers gene knockdowns in adult animals: ppmos and vivo-morpholinos human cytomegalovirus mutant with sequence-dependent resistance to the phosphorothioate oligonucleotide fomivirsen (isis ) zika virus inhibition of hepatitis e virus replication by peptide-conjugated morpholino oligomers molecular biology and infection of hepatitis e virus antiviral and neuroprotective role of octaguanidinium dendrimer-conjugated morpholino oligomers in japanese encephalitis sting mediates neuronal innate immune response following japanese encephalitis virus infection development of peptide-conjugated morpholino oligomers as pan-arenavirus inhibitors inhibition, escape, and attenuated growth of severe acute respiratory syndrome coronavirus treated with antisense morpholino oligomers inhibition and escape of sars-cov treated with antisense morpholino oligomers antisense morpholino-oligomers directed against the end of the genome inhibit coronavirus proliferation and growth inhibition of porcine reproductive and respiratory syndrome virus infection in piglets by a peptide-conjugated morpholino oligomer inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oligomers a simple method for delivering morpholino antisense oligos into the cytoplasm of cells peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication kshv-mediated angiogenesis in tumor progression intranasal delivery of peptide-morpholinos to knockdown influenza host factors in mice unanchored k -linked polyubiquitin synthesized by the e -ubiquitin ligase trim stimulates the interferon-ikkepsilon kinase-mediated antiviral response use of vivo-morpholinos for control of protein expression in the adult rat brain ebola and marburg virus vaccines viral interleukin- : role in kaposi's sarcomaassociated herpesvirus: associated malignancies a septal-hypothalamic pathway drives orexin neurons rna interference: concept to reality in crop improvement inhibition of measles virus infections in cell cultures by peptideconjugated morpholino oligomers treatment of ag mice with antisense morpholino oligomers increases survival time following challenge with dengue virus a morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus morpholino antisense oligomers: the case for an rnase h-independent structural type morpholino antisense oligomers: design, preparation, and properties invention and early history of morpholinos: from pipe dream to practical products chemical modifications of antisense morpholino oligomers enhance their efficacy against ebola virus infection ribonuclease h: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes herpes simplex virus icp regulates alternative pre-mrna polyadenylation and splicing in a sequence-dependent manner ebola virus disease: an update on current prevention and management strategies meta-and orthogonal integration of influenza "omics" data defines a role for ubr in virus budding kshv lana-the master regulator of kshv latency antiviral activity of morpholino oligomers designed to block various aspects of equine arteritis virus amplification in cell culture structure-activity relationship study of the plasma membrane translocating potential of a short peptide from hiv- tat protein gene-specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers advanced morpholino oligomers: a novel approach to antiviral therapy advanced antisense therapies for postexposure protection against lethal filovirus infections delayed time-to-treatment of an antisense morpholino oligomer is effective against lethal marburg virus infection in cynomolgus macaques a single phosphorodiamidate morpholino oligomer targeting vp protects rhesus monkeys against lethal ebola virus infection knocking down disease: a progress report on sirna therapeutics inhibition of coxsackievirus b in cell cultures and in mice by peptide-conjugated morpholino oligomers targeting the internal ribosome entry site inhibition of rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide ictv virus taxonomy profile: picornaviridae curing of hela cells persistently infected with equine arteritis virus by a peptide-conjugated morpholino oligomer blockade of viral interleukin- expression of kaposi's sarcoma-associated herpesvirus inhibition of primary effusion lymphoma engraftment in scid mice by morpholino oligomers against early lytic genes of kaposi's sarcoma-associated herpesvirus inhibition of replication and transcription activator and latency-associated nuclear antigen of kaposi's sarcomaassociated herpesvirus by morpholino oligomers key: cord- -yl jdarm authors: le, aurora b.; brooks, erin g.; mcnulty, lily a.; gill, james r.; herstein, jocelyn j.; rios, janelle; patlovich, scott j.; jelden, katelyn c.; schmid, kendra k.; lowe, john j.; gibbs, shawn g. title: u.s. medical examiner/coroner capability to handle highly infectious decedents date: - - journal: forensic sci med pathol doi: . /s - - - sha: doc_id: cord_uid: yl jdarm in the united states of america, medical examiners and coroners (me/cs) investigate approximately % of all deaths. unexpected deaths, such as those occurring due to a deceased person under investigation for a highly infectious disease, are likely to fall under me/c jurisdiction, thereby placing the me/c and other morgue personnel at increased risk of contracting an occupationally acquired infection. this survey of u.s. me/cs′ capabilities to address highly infectious decedents aimed to determine opportunities for improvement at me/c facilities serving a state or metropolitan area. data for this study was gathered via an electronic survey. of the electronic surveys that were distributed, the overall response rate was n = ( %), with of those respondents completing all the questions within the survey. at least one me/c responded from of states, and the district of columbia. select results were: less than half of respondents ( %) stated that their office had been involved in handling a suspected or confirmed highly infectious remains case and responses indicated medical examiners. additionally, me/c altered their personal protective equipment based on suspected versus confirmed highly infectious remains rather than taking an all-hazards approach. standard operating procedures or guidelines should be updated to take an all-hazards approach, best-practices on handling highly infectious remains could be integrated into a standardized education, and evidence-based information on appropriate personal protective equipment selection could be incorporated into a widely disseminated learning module for addressing suspected or confirmed highly infectious remains, as those areas were revealed to be currently lacking. increased international travel and exchange are factors that escalate the risk for rapid transmission of emerging and reemerging infectious diseases, and highly infectious diseases (hids). while neither the centers for disease control and prevention (cdc) nor the world health organization (who) has formally published a current standard list of pathogens deemed to be highly infectious, multiple 'priority' pathogens (e.g. coronaviruses, viral hemorrhagic fever viruses, bacillus anthracis, yersinia pestis) are frequently cited as requiring advanced resources, protocols, and training to minimize risk of disease transmission and mortality [ ] [ ] [ ] . the - west africa ebola virus disease (evd) outbreak, for example, challenged the capabilities, capacities, and efficacy of healthcare facilities in caring for patients with a highly infectious pathogen both abroad and in the united states [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition to the direct clinical care provided to patients with confirmed and suspected evd, public health departments and other affiliated sectors including emergency management, clinical and research laboratories, medical waste management, and mortuary services collaborated with medical providers to optimize and support patient care while reducing the risks to environmental and public safety [ ] [ ] [ ] [ ] [ ] . due to the highly infectious nature of evd, comprehensive aspects of infection prevention-including appropriate handling of highly infectious remains-had to be carefully considered and planned to contain disease spread. careful handling of highly infectious remains was particularly imperative given that viral loads were found to be high immediately after an evd-infected individual died, thereby posing hazardous to pathology and mortuary personnel [ , , ] . the risk of infection for death care sector workers posed by hids was exemplified by the infection of a german mortuary worker with lassa fever following the processing of remains previously unknown to be infected with lassa virus [ ] . at the height of the - outbreak, the cdc published guidelines for handling evd-infected human remains informed by evidence-based best practices to address the transmission risk posed to workers [ ] ; however, a recent gap analysis survey of the u.s. death care sector-specifically of funeral homes and crematories-revealed that most were unaware of these guidelines. overall, the study found a lack of up-to-date education, training and resources provided to this industry to safely manage potential or confirmed highly infectious remains scenarios [ ] . as evd is now re-emerging in other regions of africa [ ] and incidence and mortality of other infectious diseases is globally on the rise, the concerns raised in this study remain ever relevant. thus, it is imperative for those working with potentially infected human remains to receive the requisite training and resources to enable effective disease containment and prevent secondary transmission. in the united states of america (u.s.), medical examiners and coroners (me/cs) investigate approximately % of all deaths. typically, mes are physicians, usually pathologists specializing in forensic pathology, who are appointed government officials. they are charged with investigating unexpected, suspicious, and unnatural deaths in order to determine cause and manner of death and perform autopsies as needed. coroners, conversely, could be non-physicians or nonpathologist physicians who are elected or appointed at the county level; they look into deaths similar to those investigated by mes. coroners largely rely upon pathologists to perform the autopsies. unexpected deaths, such as those occurring due to a deceased person under investigation (pui) for a hid, are likely to fall under me/c jurisdiction, thereby placing the me/c and other morgue personnel at increased risk of contracting an occupationally acquired infection [ , ] . historically, me/cs have often been among the first to encounter infectious disease outbreaks. for instance, me/c offices were instrumental in recognizing outbreaks of diseases such as hantavirus pulmonary syndrome, west nile encephalitis, and novel severe acute respiratory syndrome coronavirus (sars-cov) [ ] [ ] [ ] . while the cdc recommends that no autopsy be performed for a confirmed patient with ebola virus disease, it is likely that if a patient were to die from an unidentified hid that an autopsy would be conducted [ ] . additionally, me/c offices play a critical role in discovering the pathogenesis of infectious diseases as well as providing a means of disease surveillance on a global level [ , ] . in puerto rico, me/c autopsy samples have been used to track dengue virus fatalities, while in south asia they have diagnosed deaths due to emergent nipah virus. me/c offices have also helped demonstrate the lethality of pediatric influenza and confirmed deaths due to creutzfeldt-jakob disease (cjd) [ ] [ ] [ ] . despite the essential role me/c offices play in public health, there have been multiple published reports of considerable obstacles to effective infectious disease and mortality surveillance including the following: inadequate morgue biosafety infrastructure, lack of appropriate staff training/ educational updates, and critical shortages in the numbers of forensic pathologists [ ] [ ] [ ] . uncertainties persist on the capabilities of me/c offices to address increasing baseline case volumes, of which the majority are lower risk infectious disease scenarios. disease containment, in this setting, including prevention of secondary transmission, is critical for the benefit of public health, emergency management, medicine, and the general public. given the critical role me/cs play in this endeavor, and the apparent lack of resources dedicated to this sector of the workforce to protect themselves from potential occupational exposures, this study was conducted to evaluate what protocols are in place for suspected or confirmed highly infectious remains, as well as determining levels of training among u.s. me/cs to handle highly infectious remains. total population purposive sampling was utilized for this nonexperimental design, as each state has its own unique death investigation system [ ] . a contact list of me/c offices serving populations of , or greater was compiled for each state by the national association of medical examiners (name) ad hoc committee for bioterrorism and infectious disease. this minimum population limit was selected in an effort to avoid duplication of survey results, as geographic areas with smaller populations often outsource to larger me/ c offices. an electronic survey with questions created by the authors was distributed via qualtrics© (software version . , provo, ut) through a link in an email solicitation (indiana university institutional review board exemption protocol # ). survey questions included: demographic information (e.g. title, population served, state), personal protective equipment (ppe) worn in different infectious scenarios, procedures performed in different infectious scenarios, duration of training received, biosafety level (bsl) capabilities, and jurisdictional handling of highly infectious remains. the name ad hoc committee for bioterrorism and infectious disease sent email solicitations from december , to february , to encourage responses from me/c offices nationwide, ensuring a comprehensive view of u.s. me/c practices. the survey was closed after weeks. data from qualtrics© was exported and data was analyzed utilizing sas version . (copyright (c) - by sas institute inc., cary, nc, us). frequencies and percentages were used to summarize question responses and chi-square tests were performed to investigate associations between variables; only significant findings were reported and individual states were not named to protect the identity of the state me/c. all responses to questions were voluntary so response rates between questions varied. of the electronic surveys that were distributed, the overall response rate was n = ( %), with of those completing all the questions within the survey. at least one me/c responded from of states, and the district of columbia; three states were excluded because their largest me/c office did not serve a population size of , or greater. medical examiners represented the majority of respondents ( %), followed by coroners ( %) and 'other' titles ( %) (e.g. forensic pathologist, deputy coroner, sheriff-coroner). there appeared to be a difference in distribution of professions across the region, with mes being more evenly distributed than other titles. each u.s. region, as delineated by the department of health and human services, had at least medical examiners, while several regions had zero or one respondent who selected 'coroner" or 'other'. for coroners, % were from the midwest (il, in, mi, mn, oh, and wi) and % of those who selected 'other' were from the west coast (az, ca, hi, and nv) [ ] . twenty-five percent of respondents worked in an office that served a population size between , - , ; % served , - , ; % served , up to million and % of respondents came from an office that served a population greater than million people. when asked which entity was responsible for their office's oversight, % stated a government agency, % public safety or law enforcement, % 'other' (academic medical center or university, city or county health department, political subdivision, or self), % state health department, and % a forensic laboratory. respondents were asked to select all ppe worn when performing standard duties, i.e. when no known infectious disease outbreak was occurring locally or regionally or was reported to the me/c (table ) . 'other' optional items of ppe listed were: a plastic apron over the surgical gown (n = ), waterproof sleeve covers (n = ), hair nets/bonnets (n = ), dedicated autopsy socks (n = ), and one respondent noted use of a tyvek suit for standard duties. for comparison, respondents were asked to select what ppe they would wear when performing duties on suspected or confirmed highly infectious remains (table ) . 'other' optional items of ppe listed were: disposable apron (n = ), waterproof sleeves (n = ), hair net/bonnet (n = ), self-contained breathing apparatus (scba) (n = ), hazardous waste and emergency response standard (hazwoper) gear (n = ), tyvek suit (n = ), and two layers of clothes (cloth and plastic) (n = ). four respondents stated their office would not perform autopsies on such cases. slightly more than half of respondents ( %; / ) stated their office staff had received training on donning and doffing ppe in suspected or confirmed cases of highly infectious remains; nearly one-third ( %) ( / ) reported the amount of cumulative training in hours per person, on average per year, was h or less while % ( / ) spent between and h of training. the entity that provided the ppe training varied widely among respondents. common responses included: in-house staff (n = ), state or local health department (n = ), an affiliated university (n = ), occupational health or a safety and compliance coordinator external to the me/c office (n = ), online-based training (n = ), risk or emergency management (n = ), an infectious disease or infection control specialist (n = ), or individuals highly trained in hazardous materials (hazmat) external to the me/c office (n = ). in the event of suspected highly infectious remains, respondents were asked what procedures would be permissible and performed by their office ( table ). the most frequent responses for 'other' were: dependent on a case-by-case basis/contingent upon suspected pathogen (n = ), sending the remains to the appropriate biocontainment facility (n = ), and one noted a written policy for handling highly infectious remains does not exist and would require discussion with multiple stakeholders, including the safety committee to evaluate risk. for comparison, respondents were asked which procedures or tasks would be performed in the event of confirmed highly infectious remains (table ) . 'other' responses echoed those in a suspected case where it was dependent on the circumstances and suspected pathogen, or only as required by the cdc or local health department (n = ). one respondent stated they would decline jurisdiction if no circumstance beyond the confirmed infectious disease made it reportable. less than half of respondents ( %; / ) stated that their office had been involved in handling a suspected or confirmed highly infectious remains case. the most commonly encountered highly infectious pathogens were: cjd and unclassified prions (n = ); forms of tuberculosis (including extremely drug-resistant [xdr-tb] and multiple drug resistant [mdr-tb]) (n = ); forms of meningitis (streptococcus pneumoniae, meningococcal) (n = ); anthrax (bacillus anthracis) (n = ); suspected cases of ebola and other hemorrhagic fevers (n = ); human immunodeficiency virus (hiv) a percentages add up to more than % because this question was multiple-select a percentages add up to more than % because this question was multiple-select (n = ); h n influenza (swine strain) (n = ); and sars (n = ). while just under half of the respondents stated their office had been involved in handling such remains ( %; / ), those cases do not appear equally distributed across regions. no respondents from upper west coast states (al, id, or, wa) reported office involvement, while all respondents from east coast states (ct, me, ma, nh, ri, vt) reported office involvement. other regions had between % to % of respondents reporting office involvement. eighty-one percent ( / ) of respondents did not have a biosafety level (bsl- ) facility within their office to conduct examinations in suspected highly infectious cases; some did have bsl- capabilities ( %; / ) and bsl- capabilities ( %; / ). table provides definitions of the bsl levels [ ] . in regard to the location at which autopsies were performed in a suspected highly infectious remains case, % ( / ) stated there was a separate autopsy area where no other autopsies were being performed at the same time; % ( / ) stated they do not examine suspected highly infectious remains at their facility; and % indicated 'other', which include not having a separate room/in the regular autopsy area (n = ), altering protocol to limit staff and only have that single case autopsied at the time if there was not a separate room available (n = ), performing autopsy in a negative pressure disaster portable morgue unit (n = ), and that autopsy was contingent on the case load and space available (n = ). one open-ended comment emphasized that it was case dependent, and that "tb gets autopsied, ebola straight to the funeral home" . this statement raises concern as the study by le et al. that surveyed the level of education and training received by u.s. funeral home and crematory personnel on highly infectious disease mitigation and management revealed large gaps in knowledge, including incorrectly marking routes of exposure for evd [ ] . more than half ( %; / ) indicated that their staff was not trained to carry out specialized decontamination procedures following autopsy of suspected or confirmed highly infectious remains. of those who had been trained on decontamination procedures, the most frequent response for the average cumulative length of training in hours per year per person was h or less ( %; / ), followed by h ( %; / ). additionally, a little over one-third of respondents ( %; / bsl- builds upon bsl- but includes additional precautions and facility features which are appropriate for work with moderate-risk microorganisms that are associated with human disease of varying severity. laboratory access is restricted when work is conducted. enhanced engineering controls and personal protection is needed. ppe typically includes lab coats and gloves; eye protection and face shields as needed. in addition to the sink for handwashing, there should also be an eyewash station. all aerosol or splash-generating procedures should be performed in a biological safety cabinet (bsc). there must be an autoclave or alternate method of decontamination for proper waste disposal, and the facility must have self-closing, lockable doors. an example of an organism appropriate for use in a bsl- laboratory is human immunodeficiency virus (hiv). biosafety level- (bsl- ) bsl- builds upon the requirements of bsl- but includes additional precautions and facility features which are appropriate for work with microorganisms which cause serious or potentially fatal disease through respiratory transmission. access to the facility is restricted and controlled at all times. in addition to all the aforementioned ppe, respirators may be worn and are required when experimentally infected animals are present. all microorganisms must be handled within a bsc. a hands-free sink and eyewash station must be available near an exit, exhaust air cannot be recirculated and the facility must have sustained directional airflow from clean areas to more contaminated areas. lastly, entrance into the facility is through two sets of self-closing, locking doors. an example of an organism appropriate for use in a bsl- laboratory is severe acute respiratory syndrome (sars) coronavirus. a these definitions are paraphrased from those provided by the centers for disease control and prevention [ ] ) indicated staff were trained to handle and transport (i.e. package and ship) specimens for suspected highly infectious cases. if staff were trained, specimens were sent most frequently to one or two of the following locations: the cdc (n = ), state reference laboratory (n = ), the national prion disease pathology surveillance center (npdpsc) at case western reserve university for prion diseases (n = ), or an academic medical center/hospital laboratory (n = ). of those that had received training on handling and transporting specimens, the most frequent response for the average cumulative length of training in hours per year per person was h ( %; / ), followed by less than h ( %; / ). there was a statistically significant relationship determined between those answering "no" to their office being involved in handling highly infectious remains and those answering "no" to receiving training to safely handle/transport the specimens. for those who answered "no" to involvement, % had no training for transporting specimens and % did; however, for those who answered "yes" to office involvement, only % had training for transporting specimens and % did not. when asked what their jurisdiction permitted for highly infectious remains, % ( / ) stated embalming was permitted, % ( / ) traditional burial practices, % ( / ) cremation and % ( / ) were unsure. survey respondents also had the opportunity to provide open-ended comments at the end of the survey; respondents did. comments included: a desire for formalized or more frequent training in the area of handling highly infectious remains (n = ); a need for more resources or a lack of preparedness or appropriate facilities to address highly infectious remains (n = ); the difficulty of answering questions pertaining to newly emerging and re-remerging highly infectious diseases because policies had not been written or revised (n = ); a need to formalize and update protocols (n = ); and a need for better funding to attract more prospective forensic pathologists to practice and to purchase greater stocks of ppe since what was available was expired or on back-order (n = ). select direct quotations and themes included that the, "national infrastructure for autopsy biosafety is woefully inadequate" and a perception of being overlooked/neglected in infection control training but still an office "they hand bodies off to" without regard for the limited training and resources. as sudden unexpected deaths fall under medical examiner/ coroner jurisdiction, they may play a fundamental role in the response to infectious disease deaths. if communication between various health sectors is unclear or protocols have not been established by the local health department, there is a risk for occupational exposure for all parties involved, and the potential for a me/c to be exposed to a highly infectious death that has yet to be confirmed. the logistical challenges associated with the response to highly infectious pathogens is demanding for public health sectors focused on the treatment and management of living patients. the role of the death care sector in effective disease surveillance and containment of infectious diseases is often overlooked; including the fundamental role of me/cs. me/cs frequently investigate deaths with little clinical information on the circumstances preceding death. hence, it is crucial to for me/cs to have robust, up-todate education and training in potential highly infectious remains handling, ppe donning and doffing, and clear protocols used when handling human remains that stress universal precautions. to determine what training areas are insufficient or need to be supplemented, this survey evaluated current me/c office capability to handle highly infectious remains. this survey provided a national view of the handling of highly infectious remains by capturing a sample of me/cs from nearly every state and washington d.c. medical examiners comprised the majority of the survey respondents and were more evenly geographically distributed than coroners. nearly half of the respondents served large counties or metropolitan areas with populations of greater than one million people, highlighting the large populations that may be covered by a single me/c office. additionally, most me/c offices, including the body storage areas (morgues), are under government oversight. in order to gauge circumstance-dependent ppe use among me/cs, respondents were asked for standard ppe ensembles worn during routine autopsies and those worn for autopsies on suspected or confirmed highly infectious remains (table ) . slightly more than half ( %) reported wearing an n respirator during routine autopsies and this increased to only % for autopsies on suspected or confirmed highly infectious remains. other higher level ppe such as a powered-air purifying respirator (increased by over %), tyvek suit, hazwoper gear and scba also showed an increase. a surgical mask was worn by % in standard autopsies and by % of me/cs for a suspected or confirmed highly infectious case. typically, at minimum an n is recommended for protection against aerosolized particles arising such as tb, monkeypox, sars and others, rather than a surgical mask [ ] . additionally, an autopsy is inherently an aerosol-generating procedure, even organisms that might normally require only large droplet precautions (i.e. surgical mask) can be aerosolized at autopsy due to oscillating saws, aspirating hoses, etc. and thus require added respiratory precautions (i.e. n respirator or papr) [ , , ] . use of a face shield rather than glasses/goggles also has been shown to reduce contamination of respirators by particles but only % of me/c respondents routinely wear them [ ] . the following ppe changes occurred for suspected or highly infectious remains: the use of inner gloves, a face shield, and boot/shoe cover wear increased by %, %, and %, respectively, while donning eye protection decreased by %. usage of a n respirator increased by more than % and the use of a powered-air purifying respirator notably increased by nearly %. higher level ppe, such as a tyvek suit, hazwoper gear and scba also were used when autopsies were performed on suspected or confirmed highly infectious remains. of concern, these results indicate me/c alter their ppe based on suspected versus confirmed highly infectious remains rather than taking an all-hazards approach. despite some improvements in more protective ensembles in the suspected increased risk cases, the amount of training received by respondents was lacking. little more than half ( %) of respondents had received training on donning and doffing ppe in such scenarios, with the % of those who did have ppe training having spent an average of only h or less per person per year on the topic. additionally, the entity that provided ppe training widely varied (e.g. in-house staff, affiliated university, safety and compliance departments), and no information was collected on the survey on the expertise level of those delivering trainings. the lack of reproducible training time and variability of training entity suggest that more standardized training might be of benefit. designating a knowledgeable public organization to offer standardized training modules could lead to the following: ( ) standardization of the organizational source of training; ( ) content of training materials and modules based on reproducible, evidence-based best practices commonly found in the me/c field; and ( ) subscription to online training as it will likely be the most cost-effective and convenient means of training, as was proven successful in healthcare [ ] . moreover, best practices and evidence-based studies have demonstrated that regular training for donning and doffing high level ppe in highly infectious scenarios provide substantially better occupational safety and health outcomes for the employee [ ] . in the event of suspected or confirmed highly infectious remains, most me/c offices stated that the situation was handled on a case-by-case basis, depending on the pathogen that was suspected and required detailed conversations with all stakeholders. as shown in table , procedures did vary between what would be performed with a suspected highly infectious body versus a confirmed infectious body. in a confirmed case, all but one of the listed procedures as decreased compared to a suspected case (e.g. complete autopsy [ % decrease], washing or cleaning of the body [ % decrease], body storage in freezer [ % decrease]). the only increase was, "bypass office and have body directly transported to funeral home/crematorium" by % which, as previously mentioned, may result in funeral home and crematory personnel being placed at risk. fewer than % of me/c offices having been involved with handling a suspected or confirmed case, demonstrating a lack experience in handling highly infectious remains. when asked which suspected or confirmed pathogens were encountered, however, many noted category a or b pathogens (table ) [ , ] that require specific deactivation and decontamination procedures-of which only approximately onethird ( %) of respondents had received training in. it is possible that after such an event that the me/c office would hire an appropriate contractor to conduct the appropriate deactivation and decontamination; however, the possibility remains [ , ] that the task could go to individuals within the me/c offices without proper training. while most offices did not have a bsl- facility, nearly twothirds ( %) of those without a bsl- did have bsl- capabilities. however, if % have only bsl- capability, then these morgues would essentially be considered appropriate for work only with agents not known to consistently cause disease in healthy human adults per cdc guidelines [ ] . in essence, a sizeable percentage of morgues in the u.s. are not equipped to safely perform autopsies on human remains with a large number of infections, especially those highly infectious disease autopsies. in addition to improved training, more investment in morgue infrastructure would be necessary to enhance their capabilities. anecdotally, some larger me/c offices have a computerized tomography (ct) scanner in which triple bagged sealed infectious remains can undergo virtual autopsy. these bags can be constructed with portals to collect needed specimens for microbiologic/virologic studies. the triple bagging prevents leaks and contamination and the remains can safely be sent to funeral home. it would also be beneficial to have list of pathologists and support personnel in each me/c office who could volunteer to take vaccines to handle certain cases with suspected contagious diseases (i.e. smallpox, etc.). approximately % of respondents reported that they did not examine suspected or confirmed highly infectious remains at their facility. given the lack of proper bsl facilities, this would be appropriate. slightly more than % noted the lack of space and/or a lack of staff in their offices as a limitation for being able to perform autopsies of suspected or confirmed highly infectious remains in a separate room or alone. for biosafety, it is recommended that autopsy facilities should have a minimum of air exchanges per hour, be negatively pressurized relative to surrounding office spaces, and exhaust air outside of the facility and away from areas of high pedestrian traffic. morgue laminar air flow should travel from clean to progressively less clean areas with downdraft table ventilation to decrease personnel exposure to aerosolized pathogens [ , ] . it is likely that significant financial investment would be required to retrofit many existing morgues to meet these standards. another option would be to have jurisdictional planning to transport suspected or confirmed cases to known centers that currently have the necessary bsl capability; again, body transportation would incur costs but likely lower costs than that associated with retrofitting many existing morgues. in addition to improved morgue biosafety, it would also benefit me/c facilities to have better publicized, easily accessed, and clearly laid-out protocols for various infectious scenarios in which limited autopsy (e.g. brain-only in suspected cjd cases) or no autopsy (e.g. evd cases) is currently recommended. when asked about level of training to handle and transport specimens for suspected highly infectious cases, only onethird of respondents had received this training with % spending on average less than h per year per person on the topic. nearly half of respondents ( %) were unsure of what their jurisdiction permitted in the case of highly infectious remains for ultimate disposal, and alarmingly, % of respondents stated their jurisdiction permitted embalming and % traditional burial. for evd, for example, the recommended procedure is cremation to ensure complete deactivation of the virus in order to prevent spread to workers and the environment; those who were killed by the disease will have high viral loads present in their body post-mortem [ ] . while needs for funding, resources, supplies and appropriate capabilities may be universal across the death care sector, this survey's results strongly suggest that it would benefit state or regional-specific me/cs to have standardized education and training throughout the u.s [ ] . likewise, open-ended comments from respondents indicated a need for augmented up-to-date formalized trainings, as well as revised written policies and procedures, and enhanced resources (including facilities and funding). there were general perceptions of unpreparedness to address highly infectious remains, budgetary constraints and a weak national structure regarding autopsy biosafety, and a lack of incorporation of me/c offices into infection control planning despite me/c office involvement with highly infectious remains. there were limitations to this study. because of the study's exploratory nature, the survey was not validated beyond subject matter expert vetting. additionally, the survey only included me/c offices that served larger populations; smaller offices may still encounter hid cases if they do not outsource larger nearby offices. therefore, this study may not be generalizable to smaller offices (i.e. those serving populations < , ). also, the survey instrument was designed to allow respondents to check multiple boxes when asked about the use of ppe. the results, therefore, were not clear whether the respondent meant the ppe would be used simultaneously or one instead of the other. for example, a face shield and respirator may be used simultaneously or a face shield may be used instead of a respirator. additionally, a limitation related to potential response bias may exist. although this study was not funded, there could have been sponsor bias on behalf of the respondents, as the survey was distributed by members of name, thereby potentially affecting the candidness of their responses. lastly, non-responses may have arisen because it would not appeal to prospective participants to take a survey about a topic for which they are not trained out of concern their answers may not be "correct." nevertheless, this study addresses a critical gap about what is known and unknown about u.s. me/c capabilities to handle highly infectious remains. in conclusion, this survey of u.s. medical examiners and coroners' capabilities to address highly infectious decedents presents opportunities for improvement at me/c facilities serving their state or metropolitan area. standard operating procedures or guidelines (sops or sogs) should be updated to take an all-hazards approach, best-practices on handling highly infectious remains could be integrated into a standardized education, evidence-based information on appropriate ppe selection could be integrated into a widely disseminated learning module, and existing relationships with the local health department, funeral homes and crematories could be bolstered to develop a multi-sectoral concept of operations for addressing suspected highly infectious remains. while some issues will require greater capital and resources to address-such as retrofitting facilities to meet better biosafety recommendations, or more financial resources to enhance operation-the hope is that this study will draw attention to these more systemic issues and stimulate a call to action from the appropriate entities. . u.s. medical examiners/coroners play a critical role in death investigation, yet their capabilities to address highly infectious remains are unknown. occupationally-acquired infection. . this survey, with respondents from nearly every u.s. state, revealed current levels of medical examiner/ coroner training and education to address suspected or confirmed highly infectious remains. . questions, and thereby results, focus on permissible autopsy procedures, personal protective equipment, and biosafety-level facility capabilities. . medical examiners/coroners could benefit from updates to standard operating procedures and standardized education on handling suspected or highly infectious remains that taken an all-hazards approach. niaid emerging infectious diseases/pathogens list of blueprint priority diseases infection control in the management of highly pathogenic infectious diseases: consensus of the european network of infectious disease clinical care of two patients with ebola virus disease in the united states ebola virus disease cluster in the united states ebola virus disease: preparedness and infection control lessons learned from two biocontainment units caring for patients with ebola: a challenge in any care facility lessons learned: critical care management of patients with ebola in the united states reflections on interprofessional team-based clinical care in the ebola epidemic: the nebraska medicine experience current capabilities and capacity of ebola treatment centers in the united states safe management of patients with serious communicable diseases: recent experience with ebola virus clinical management of ebola virus disease in the united states and europe nebraska biocontainment unit perspective on disposal of ebola medical waste considerations for safe ems transport of patients infected with ebola virus transport and management of patients with confirmed or suspected ebola virus disease us ebola treatment center clinical laboratory support nebraska biocontainment unit patient discharge and environmental decontamination after ebola care guidance for safe handling of human remains of ebola patients in u. s. hospitals and mortuaries the contribution of ebola viral load at admission and other patient characteristics to mortality in a medecins sans frontieres ebola case management centre control measures following a case of imported lassa fever from togo a gap analysis of the united states death care sector to determine training and education needs pertaining to highly infectious disease mitigation and management ebola situation reports: democractic republic of the congo infectious disease surveillance by medical examiners and coroners overview of medical examiner/coroner systems in the united states: development, current status, issues and needs the pathology of human west nile virus infection hantavirus pulmonary syndrome in the united states: a pathological description of a disease caused by a new agent biosafety level laboratory for autopsies of patients with severe acute respiratory syndrome: principles, practices, and prospects three decades of responding to infectious disease outbreaks operational research during the ebola emergency role of the medical examiner in zika virus and other emerging infections cdc grand rounds: discovering new diseases via enhanced partnership between public health and pathology experts emerging infectious diseases and the medical examiner medical examiners, coroners, and biologic terrorism death investigation systems department of health & human services. regional offices hospital respiratory protection program toolkit: resources for respirator program administrators aerosol generation during bone-sawing procedures in veterinary autopsies biosafety considerations for autopsy efficacy of face shields against cough aerosol droplets from a cough simulator personal protective equipment in health care: can online infection control courses transfer knowledge and improve proper selection and use? guidelines for safe work practices in human and animal medical diagnostic laboratories age and ebola viral load correlate with mortality and survival time in ebola virus disease patients acknowledgments we would like to extend our gratitude to the members of the national association of medical examiners (name) ad hoc committee for bioterrorism and infectious disease for their support of this research and distribution of the survey; these members include: second author erin brooks (chair), paul chui, karen kelly, john matthew lacy, micheline lubin, lakshmanan sathyavagiswaran, leah schuppener, suzanne utley-bobak, and steven white. additionally, we acknowledge the national institute of environmental health sciences (niehs) worker training program (wtp) ebola biosafety and infectious disease response training uh information, grant number uh es . while the grant funding did not contribute to the development and distribution of this gap analysis survey, the program did highlight the need to explore research in this area. lastly, we also thank from the university of nebraska medical center: elizabeth beam and at harvard t.h. chan school of public health: paul biddinger for their partnership and support. conflict of interest none of the authors have any conflicts of interest to disclose.ethical approval this study was deemed exempt by indiana university institutional review board (protocol # ).informed consent survey participants were informed of potential risks and benefits prior to taking the voluntary survey. this informed consent survey was reviewed by the institutional review board and approved as part of the exemption in the aforementioned protocol number. key: cord- -otf ruvj authors: prohaska, stefanie; schirner, andrea; bashota, albina; körner, andreas; blumenstock, gunnar; haeberle, helene a. title: intravenous immunoglobulin fails to improve ards in patients undergoing ecmo therapy date: - - journal: j intensive care doi: . /s - - - sha: doc_id: cord_uid: otf ruvj background: acute respiratory distress syndrome (ards) is associated with high mortality rates. ards patients suffer from severe hypoxemia, and extracorporeal membrane oxygenation (ecmo) therapy may be necessary to ensure oxygenation. ards has various etiologies, including trauma, ischemia-reperfusion injury or infections of various origins, and the associated immunological responses may vary. to support the immunological response in this patient collective, we used intravenous igm immunoglobulin therapy to enhance the likelihood of pulmonary recovery. methods: ards patients admitted to the intensive care unit (icu) who were placed on ecmo and treated with (ivig group; n = ) or without (control group; n = ) intravenous igm-enriched immunoglobulins for days in the initial stages of ards were analyzed retrospectively. results: the baseline characteristics did not differ between the groups, although the ivig group showed a significantly reduced oxygenation index compared to the control group. we found no differences in the length of icu stay or ventilation parameters. we did not find a significant difference between the groups for the extent of inflammation or for overall survival. conclusion: we conclude that administration of igm-enriched immunoglobulins as an additional therapy did not have a beneficial effect in patients with severe ards requiring ecmo support. trial registration: clinical trials: nct ; retrospectively registered. acute respiratory distress syndrome (ards) is characterized by pulmonary inflammation that can be caused by pulmonary and extrapulmonary origins. sepsis, bacterial pneumonia, polytrauma, and aspiration pneumonia are the most common causes of ards [ ] . predictors of survival include age, the type of underlying medical condition, the severity of pulmonary injury, the presence of extrapulmonary organ dysfunction, and ongoing sepsis [ ] . currently, clinical attempts to rescue ards patients include individualized ventilation and fluid management, adequate infection control, including early application of broad-spectrum anti-infectives, neuromuscular blockade using cisatracurium, sedation strategies, prone positioning, and finally extracorporeal membrane oxygenation (ecmo) to ensure oxygenation [ ] [ ] [ ] . although the incidence of ards is relatively high, with five to eight cases per , european inhabitants and even more in the usa, the various pathomechanisms are only partially understood, resulting in different experimental approaches to understand immune responses during early ards [ ] . one well-described issue is decreased immunoglobulin levels in patients with severe infection [ ] as an element of the immunological response in the initial phase of inflammation in response to sepsis [ ] . therefore, one approach to support critically ill patients is intravenous administration of igm-enriched immunoglobulins since this could potentially decrease the severity of inflammation. although this treatment was omitted in recent sepsis guidelines due to a lack of supporting evidence in high-quality trials [ ] , several studies, including one meta-analysis, describe beneficial effects of immunoglobulins in acute pneumonia induced by drug-resistant bacterial infections [ ] [ ] [ ] . furthermore, several case reports describe beneficial effects of antiviral therapy in combination with intravenous immunoglobulin therapy in immune-compromised patients [ ] [ ] [ ] . based on these data, we treated patients with ards requiring ecmo therapy with igm-enriched immunoglobulins immediately after intensive care unit (icu) admission. the objective of this study was to investigate whether intravenous immunoglobulin administration could improve the clinical course of ards in patients treated with ecmo. therefore, mortality, the duration of ecmo therapy, the incidence of renal replacement therapy, the duration of vasopressor and anti-infective therapy, length of stay in the icu, and length of stay in the hospital were analyzed retrospectively in ards patients requiring ecmo therapy. the study was approved by the local research ethics committee of the university hospital and the eberhard-karls university tübingen, germany. patients with severe ards treated with ecmo therapy between january and january at our institution were analyzed retrospectively. in all patients, ecmo therapy was required due to hypoxia and/or increased pulmonary resistance precluding protective lung ventilation. ecmo therapy was performed according to the guidelines of the extracorporeal life support organization (elso) using ila-active systems (novalung, stolberg, germany [ ] . fifty-seven patients were analyzed. twenty-eight patients were treated with igm-enriched immunoglobulins (pentaglobin®; biotest; dreieich, germany) (ivic-group). pentaglobin is an igm-enriched polyvalent immunoglobulin preparation derived from a plasma pool. it contains mg of igm, mg of iga, and mg of igg ( % igg , % igg , % igg , and % igg ) per millilitre. the indication for igm-enriched immunoglobulin (ivig) treatment was determined at the discretion of the treating intensivist. ivig was applied if a viral infection or an infection due to multi-resistant gram-negative bacteria (mrgn) was suspected. ivig dosing was performed according to the instruction of the manufacturer: . ml/kg/h (up to ml) as the initial dose, followed by . ml/kg/h for h until a total dose of mg/kg was achieved. twenty-nine patients did not receive immunoglobulins (control group). vasopressors (norepinephrine; arterenol® sanofi-aventis; germany) were used after volume resuscitation according to the sepsis guideline ( ) . renal replacement therapy was performed with citrate anticoagulation (multifiltrate, fresenius medical care, bad homburg v.d.h. germany) in patients with acute renal failure. data were retrospectively extracted from an ardsspecific database at our institution. since the electronic patient management system was changed during this time frame, not all required data were available for all ards patients. mrsa and vre screening was performed routinely in all patients admitted to the icu. to identify causal infections, bronchoalveolar lavage (bal) samples, blood cultures, urinary samples, and perioperative samples were analyzed. in patients at risk, pcr for atypical pathogens was also performed in bal samples. infection was defined as > colony-forming units (cfu) in bal and/or urinary cultures. for blood cultures, any replicate of bacterial growth was defined as infection. during flu season, influenza infection was detected by pcr. bal samples were also analyzed for other viral pathogens, such as cytomegalovirus (cmv) and herpes simplex virus (hsv). positive findings for these viruses were confirmed by cell cultures. patients with unknown pathogens and immune suppression were additionally screened for other pathogens. anti-infective therapy was applied according to the local guideline considering local resistance patterns. categorical data are reported as numbers and percentages, and continuous data are summarized with the median and range (i.e., the minimum and maximum values) unless otherwise indicated. to compare categorical variables or outcomes, such as the occurrence of infection or in-hospital death, between the ivig group (n = patients) and the control group, which included patients who did not receive ivig therapy, the chisquared test was used. for inter-group comparisons of continuous data, the two-tailed two-sample t test was generally performed. if the original data exhibited a lognormal distribution, e.g., icu length of stay (los), then raw data were log-transformed prior to analysis with the t test. a p value < . was assumed to indicate a statistically significant difference between the groups. the data analysis was performed using jmp® . statistical software (sas institute inc., cary, nc, usa). a total of patients with ards requiring ecmo therapy between and were analyzed retrospectively. twenty-eight patients were treated with igm-enriched immunoglobulins (ivig) for days according to the manufacturer's instruction (ivig group), and patients did not receive ivig therapy (control group). no adverse events were reported after ivig application. the median age of both groups was years, ranging from to years in the control group and from to years in the ivig group. the median simplified acute physiology score (saps) and acute physiology and chronic health evaluation (apache) score were comparable between both groups ( table ). the median apache score in the ivig group was vs. in the control group. in % ( / ) of the ivigtreated patients and % ( / ) of the control patients, the apache score was equal to or greater than . regarding preexisting disease, diabetes mellitus, cardiac disease, immune suppression, and malignancy were more common in the ivig group (table ) . five patients in the control group suffered ards of extrapulmonary origin (three patients with pancreatitis and two with polytrauma). the initial pao /fio ratio in the ivig group was significantly lower than that in the control group (ivig, median vs control, ; t test, log-transformed data, p = . ). seventy-five percent of the ivig-treated patients had severe ards, % had moderate ards, and no patient showed mild ards. in the control group, % of patients had severe ards, % had moderate ards, and % had mild ards. the ecmo duration was shorter in the control group ( - days; median days) compared to that in the ivig group ( - days; median days) and could be reduced earlier in the control group (median days) than in the ivig group (median days). the duration of ventilator support was longer in the ivig group ( - h; median h) compared to that in the control group ( - h; median h; t test, logtransformed data, p = . ). five patients in the control group and three patients in the ivig group were extubated before ecmo was discontinued. ten patients in the control group and seven patients in the ivig group suffered anemia due to bleeding complications, including cerebral hemorrhage (control: n = ; ivig: n = ) ( table ) . all patients suffered from septic shock requiring vasopressor therapy (control, - days vs. ivig, - days). renal replacement therapy was more often required in the control group ( % vs. %; p = . ; pearson's chi-squared test). hepatobiliary dysfunction was comparable between both groups (control, %; ivig, %). in % of the control patients and % of the ivig-treated patients, one or more pathogens could be identified as the cause of pulmonary inflammation. in ( %) patients in the control group, bacterial pathogens such as legionella (n = ; %), streptococcus pneumoniae (n = ; %), pneumocystis jirovecii (n = ; %), and e. coli (n = ; %) were identified in bal samples. none of the control patients showed multidrug-resistant bacteria in any samples. in % ( of ) of the control patients, a viral pathogen was detected in bal samples: influenza (n = ; %) and herpes virus (n = ; %). herpes virus infection included hsv (n = ; %) and cmv (n = ; %). herpes viral infection was confirmed by cell-based culture. in five of the control patients, fungal infection was diagnosed via blood culture (n = ), abdominal sample (n = ), and bal (aspergillus, n = ). in nine control patients, no causal pathogen could be identified ( %). two of the ivig patients were infected with resistant bacteria ( mrgn stenotrophomonas maltophilia and mrsa). in ivig patients, bacterial pathogens such as legionella (n = ; %), streptococcus pneumoniae (n = ; %), pneumocystis jirovecii (n = ; %), and mycoplasma (n = ) were identified in bal samples. viral infections were more frequent in the ivig group (p = . ; pearson's chi-squared test), especially influenza infection (p < . ). in more than half of the ivig patients, viral pneumonia was diagnosed according to the results of bal samples, radiological findings, and clinical symptoms. one or more of the following viruses could be detected in bal samples: influenza (n = ; %) and herpes virus (n = ; %). herpes virus infection included hsv (n = ; %), cmv (n = ; %), and hhv (in bal and blood; n = ). in of the ivig-treated patients, fungal infection was diagnosed in urinary tract (n = ) and abdominal samples (n = ). in addition, one patient suffered aspergillosis pneumonia. in three ivigpatients, no causal pathogen could be identified ( %). the duration of anti-infective treatment was significantly longer in the ivig group than that in the control group (control, median days; - days; ivig, median days; - days; t test, log-transformed data, p = . ). six patients in the control group and four patients in the ivig group underwent anti-infective treatment before admission to the icu. excluding these patients, the duration of anti-infective therapy relative to los in the icu was not significantly different between the groups (control median, % fraction of los icu; ivig, % of los icu). patients treated with ivig stayed for a median of . days in the icu ( to days) and . days in the fig. ). forty-three percent ( out of ) of these patients died; three patients died within the first week due to cerebral hemorrhage (n = ) or multiorgan failure due to septic shock (n = ). the control patients spent a median of days in the icu ( to days) and days in the hospital ( to days); % ( out of ) of these patients died. five patients died during within first week due to cerebral hemorrhage, heart failure, subarachnoidal bleeding (polytrauma), and hypoxic brain injury h after admission, and others died of multiorgan failure due to acute pancreatitis. lymphocyte levels were analyzed retrospectively when available. initially after admission, lymphocyte levels were not different between the groups (control alive . % ± . ; control dead . % ± . ; ivig alive . % ± . ; ivig dead . % ± . ). however, lymphocyte levels were higher in survivors compared to those in non-survivors (fig. ) . the increase in lymphocytes was more prominent in survivors compared to that in non-survivors (control alive . ± . ; ivig alive . ± . ; control dead . ± . ; ivig dead . ± . ; fig. ). in addition, in patients treated with ivig, this increase during treatment was more prominent than that in the control group in the first days (fig. ). since we did not evaluate differential blood counts routinely, limited data were available for determining significant difference. the purpose of this analysis was to systematically investigate the potential effect of igm-enriched immunoglobulins on the outcomes of ards patients requiring ecmo therapy. we analyzed patients; patients were treated with igm-enriched ivig, and patients did not receive ivig therapy. of all patients suffering from septic shock, % of the ivig-treated patients and % of the control patients had severe ards of various origins. however, although the ivig group had a lower pao /fio ratio and required longer ventilation support, longer vasopressor therapy, and longer anti-infective therapy and included mrgn-infected patients, outcome parameters such as los in the icu, los in the hospital, and mortality were not significantly different between the groups. in several studies, hypogammaglobulinemia and overexpression of genes encoding immunoglobulin segments were identified in patients with septic shock [ , , ] . therefore, ivig treatment could be a logical requirement in these patients. we included ivig therapy in our ards patients based on these considerations, although we did not measure immunoglobulin levels before treatment. indeed, in a recent retrospective analysis of patients, high igg levels were associated with high mortality in sepsis patients [ ] . pcr analysis showed an early molecular response to igm in the blood of patients with sepsis [ ] . igm levels remain low in non-surviving sepsis patients, whereas igm levels increase transiently in surviving patients [ ] . single studies and metaanalyses have shown beneficial effects of ivig therapy in patients with sepsis [ ] [ ] [ ] . however, ivig treatment remains controversial. randomized studies have not shown any beneficial effect of this intervention in patients with sepsis or sepsis-associated conditions [ ] . therefore, ivig therapy is not currently recommended in the latest sepsis guidelines [ ] . immunoglobulins interact with cd t-lymphocytes during bacterial eradication. in severe sepsis, b and t cells are depleted due to apoptosis [ ] . since our analysis is retrospective, we could not measure hla-dr expression or cytokine profiles in these patients. however, we found a trend of increased lymphocytes in non-survivors compared to survivors, which may reflect why we did not see any beneficial effect of ivig therapy. although immunoglobulins may play an important role in the innate immune response during ards, nonspecific immunoglobulins may not be as effective in a lymphopenia environment. lymphopenia has been described in ards patients and in animal models after viral infection or mycoplasma infection and affects all lymphoid tissue [ ] [ ] [ ] . however, in our small group of [ , ] , but whether this variability in response is due to genetic alterations [ ] , immune evasion by bacteria [ ] , or different stages of sepsis is unknown. this study has some limitations. it is a retrospective analysis of a limited number of ards patients. since we changed our ecmo system during the study period, we only included patients treated with the one system to exclude any impact of system differences. furthermore, since ivig was ordered by a physician primarily when a viral infection or mrgn infection was suspected, the ivig group included more patients with these infections. however, this may reflect true clinical circumstances in that the cause of disease is typically not determined at admission. ards is a multifactorial disease with a heterogeneous pathogenesis and variable timing and clinical presentations. therefore, one specific therapy is unlikely to improve outcomes. rather than excluding single therapeutic options, identifying patients' risk factors and the individual ards stage may be more important for successful outcomes. we report here in our retrospective analysis that intravenous igm administration in the initial stages of severe ards did not improve overall outcomes or the severity of disease. the acute respiratory distress syndrome the alien study: incidence and outcome of acute respiratory distress syndrome in the era of lung protective ventilation the standard of care of patients with ards: ventilatory settings and rescue therapies for refractory hypoxemia neuromuscular blockers in early acute respiratory distress syndrome prone positioning in severe acute respiratory distress syndrome transcriptomic evidence of impaired immunoglobulin g production in fatal septic shock immunologic alterations and the pathogenesis of organ failure in the icu treatment of neonatal sepsis with intravenous immune globulin protective effects of intravenous immunoglobulin and antimicrobial agents on acute pneumonia in leukopenic mice iv immunoglobulin for acute lung injury and bacteremia in pseudomonas aeruginosa pneumonia mantaring jb rd. intravenous immunoglobulin for treating sepsis, severe sepsis and septic shock respiratory syncytial virus upper respiratory tract illnesses in adult blood and marrow transplant recipients: combination therapy with aerosolized ribavirin and intravenous immunoglobulin first report of severe parainfluenza virus b and rhinovirus c coinfection in a liver transplant recipient treated with immunoglobulin the human metapneumovirus: a case series and review of the literature early levels in blood of immunoglobulin m and natural killer cells predict outcome in nonseptic critically ill patients assessment of plasmatic immunoglobulin g, a and m levels in septic shock patients serum igg levels and mortality in patients with severe sepsis and septic shock : the sbits data quantification of igm molecular response by droplet digital pcr as a potential tool for the early diagnosis of sepsis kinetics of circulating immunoglobulin m in sepsis: relationship with final outcome use of polyclonal immunoglobulins as adjunctive therapy for sepsis or septic shock a case of acute respiratory distress syndrome associated with novel h n treated with intravenous immunoglobulin g early therapy with igm-enriched polyclonal immunoglobulin in patients with septic shock surviving sepsis campaign: international guidelines for management of sepsis and septic shock sepsis-induced apoptosis causes progressive profound depletion of b and cd + t lymphocytes in humans clinical diagnosis of pandemic a(h n ) influenza in children with negative rapid influenza diagnostic test by lymphopenia and lower c-reactive protein levels. influenza other respir viruses clinical features and outcomes of severe acute respiratory syndrome and predictive factors for acute respiratory distress syndrome depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h n ) virus isolated from humans subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials acute respiratory distress syndrome subphenotypes respond differently to randomized fluid management strategy the role of genetics and antibodies in sepsis immunoglobulins and their receptors, and subversion of their protective roles by bacterial pathogens none. this work was supported by a grant of the deutsche forschungsgemeinschaft to peter rosenberger dfg-ro / - . the datasets obtained and/or analyzed during the current study are available from the corresponding author upon reasonable request. authors' contributions sp prepared the manuscript and analyzed the data. as and ab are responsible for the data acquisition. ak is responsible for the data acquisition and analysis. gb is responsible for the data analysis. hah is accountable for all the aspects of the work and for ensuring that the questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. all authors read and approved the final manuscript. the ethics committee of the university hospital tuebingen (eberhard-karls universität tübingen; ethik-kommission; gartenstraße ; tübingen, germany) approved the study. the project number is / /bo . informed consent was not required from the patients involved into this retrospective analysis. this proceeding was approved by the local ethics committee (project number / /bo ) of eberhard karls university tuebingen since extraction of pseudonymous medical records provides structured data and therefore legally permits evaluation of the data for research. not applicable. the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -ntpm cg authors: otašević, s.; momčilović, s.; stojanović, n.m.; skvarč, m.; rajković, k.; arsić-arsenijević, v. title: non-culture based assays for the detection of fungal pathogens date: - - journal: j mycol med doi: . /j.mycmed. . . sha: doc_id: cord_uid: ntpm cg traditional, culture based methods for the diagnosis of fungal infections are still considered as gold standard, but they are time consuming and low sensitive. therefore, in order to overcome the limitations, many researchers have focused on the development of new immunological and molecular based rapid assays that could enable early diagnosis of infection and accurate identification of fungal pathogens causing superficial and invasive infection. in this brief review, we highlighted the advantages and disadvantages of conventional diagnostic methods and possibility of non-culture based assays in diagnosis of superficial fungal infections and presented the overview on currently available immunochromatographic assays as well as availability of biomarkers detection by immunodiagnostic procedures in prompt and accurate diagnosis of invasive fungal infections. in addition, we presented diagnostic efficiency of currently available molecular panels and researches in this area. the primary task of microbiological/mycological diagnostics is to provide adequate information of the infection cause and its antimicrobial susceptibility in optimal period of time. any delay in accurate pathogen identification and appropriate treatment initiation undoubtedly affect disease outcome. despite significant advances in medical technology in terms of rapid accurate diagnostic laboratory tests following by new therapeutics that have had a major effect in decreasing of morbidity and mortality of many fatal diseases, we are the witnesses that invasive fungal infections (ifis) are still one of the biggest problems in medicine [ ] [ ] [ ] . increasing the number of immunocompromised patients with t-cell mediated immunity deficiencies, mucosal-cutaneous barrier or metabolic dysfunction, neutropenia, aging, excessive use of antibiotics, cytotoxic therapy and transplantation, as predispose risk factors and conditions, significantly influence the higher incidence of ifis [ ] . moreover, the incidence and prevalence of nosocomial ifi are still high and candida species represent the third most common blood isolate from hospitalized patients. out of all ifi, % of invasive candidosis are from intensive care units (icu) . candida bloodstream infection is prolonging stay in the icu with mortality ranges from to %. also, it can cause complications during the clinical course of primary disease and increases treatment costs [ ] . as opposed to ifi, superficial fungal infections (sfi) of the keratin rich structures are rare systemic and serious. these infections are caused commonly by dermatophytes, yeasts or non-dermatophytic molds and represent one of the dominant infections worldwide with global prevalence ranges from - % [ , ] . the lack of rapid diagnostic methods for sfi consequently influence that only % of physicians in general practice and % dermatologists require mycological analyses before prescription of empiric treatment with systemic antifungals [ ] . additionally, these infections can affect the patient's quality of life as a potential cause of social, professional and emotional problems [ ] . thus, traditional, culture based methods for the diagnosis of fungal infections are still considered as gold standard, but they are time consuming and low sensitive. therefore, in order to overcome the limitations, many researchers have focused on the development of new immunological and molecular based rapid assays that could enable early diagnosis of infection and accurate identification of fungal pathogens causing superficial and invasive infection. in this brief review, we highlighted the advantages and disadvantages of conventional diagnostic methods and possibility of non-culture based assays in diagnosis of superficial fungal infections and presented the overview on currently available immunochromatographic assays as well as availability of biomarkers detection by immunodiagnostic procedures in prompt and accurate diagnosis of invasive fungal infections. in addition, we presented diagnostic efficiency of currently available molecular panels and researches in this area. c elsevier masson sas. all rights reserved. fungal infections of the skin, nail or hair could not be considered as a cosmetic problem of relatively minor significance. given the fact that deep infections and dissemination of pathogen can be expected in immunodeficiency, some genetic abnormalities such as card mutation, and leukemia [ , ] , the prompt diagnosis and treatment of sfis could prevent complications and unwanted clinical outcomes. so far, in the group of sfi, mucosal (oropharyngeal, vulvovaginal, intestinal) fungal infections have also been included. today, the prevalence of oropharyngeal and vulvovaginal mucosal candidosis and candida-overgrowth in intestines significantly rises up and the treatment of chronic/recurrent form is a big challenge for physicians. also, frequent changes of local therapy by systemic, with the goal to improve the treatment, probably influences the emergence of resistant candida species to azoles [ ] [ ] [ ] [ ] [ ] . new, highly specific and sensitive rapid tests will disable misdiagnosis and unnecessary proscribing of antifungals, which will be in accordance with recently initiate appeal that systemic antifungals have to be kept for ifi [ ] . historically, it is very often highlighted that traditional/ conventional methods for diagnosis of sfi and ifi are time consuming and low sensitive. the propagating/cultivating the causative agent is method with only deficiency in required time for isolation, but when we can conduct it, it is the unique analysis that provide antigen (ag) detection, molecular, biochemical identification of etiological agents and antimicrobial susceptibility testing. on the other hand, histopathological examination of the tissue samples that is considered as a ''gold standard'' for ifi has a major shortcoming that lays in the complexity of the invasive biopsy procedure [ ] [ ] [ ] . this review discusses currently available data on the advantages and disadvantages of some rapid assays for fungal detection, identification and the diagnosis of sfis and ifis. . rapid assays for diagnosis of superficial fungal infections (sfis) conventional microscopic examination (me) is cheap, easy-to use and fast method for detection of fungi in a patient's sample. although staining procedures offer additional information concerning morphology of microorganisms, me in a form of wet mount technique provides rapid detection of fungal blastoconidiayeast and pseudohyphal-hyphal forms in patient's material ( fig. ) [ ] . wet mount with chlorlactophenol or koh as reagents is still irreplaceable technique for screening of patients with suspected sfi of the skin, hair and nail. additionally, diagnosis of vaginal discharge by wet mount microscopy is useful for the diagnosis of candida vaginitis -one of the most prevalent genital infections in women [ , ] . nowadays, specific staining with chemicals or fluorescent dyes (acridin-orange, calcofluor wight, blankoflor) can improve visualization and detection of fungi [ , , ] . currently, there are efforts for establishing the imunochromatographic (ic) assays for the determination of dermatophyte [ ] and candida ag [ ] , which will represent significant progress in the diagnosis of sfis and genital candidosis, respectively. fast, easy to perform and easy to interpret, these assays can be a satisfactory replacement of me. so far, there have been reports of ic assays in which trichophyton antibodies (ab) have been used with colloidal gold particles to capture ag of different dermatophyte species such as t. rubrum, t. mentagrophytes, t. violaceum, t. tonsurans, microsporum canis, m gypseum, epidermophyton floccosum. comparing the diagnostic performances of ic for dermatophytes detection in skin and nail samples by me, higashi et al. reported that ic sensitivity and specificity were . % and . % for all specimens, . % and . % for scarified skin and . % and . % for samples taken from the nail plate, respectively [ ] . based on these results, authors recommended ic assay, as easy to use and rapid tool, for screening of dermatophyte infections, but for definitive diagnosis, me and culture are still recommended methods. in the study conducted by noriki and ishida, in-house developed ic assays showed very good diagnostic performances for dermatophyte detection in nail samples [ ] . similarly, another ic assay which used the colloidal gold-antimannan igg conjugate for candida detection in vaginal swabs was reported recently. advantage of this newly developed ic assay is the possibility of use in clinics and laboratories and detection of candida spp. in the concentration of cfu/ml of patients sample in min without expensive equipments. however, in comparison with culture, ic candida assay in vaginal swabs showed the high specificity . %, positive predictive value . , followed by lower sensitivity . %, and negative predictive value % for c. albicans. for the second most prevalent species c. glabrata (causing more-intensive and chronic form of genital candidosis), concentration has to be more then cfu/ml in patients sample [ ] . despite the few data in reference literature and the lack of commercial ic assays for sfi, further research, design and establishment of these assays for sfi will be of great significance for supporting conventional methods and facilitating the diagnosis of dermatophytosis and genital candidosis in women. during the last forty years, a number of molecular techniques have been developed for the identification of dermatophytes at species or strain level. these include fingerprinting methods, conventional pcr (employing selected genetic markers e.g. internal transcribed spacers (its), chitin synthase gene (chs ), topoisomerase ii gene, small and large ribosomal rna subunit and non-transcribed spacer/nts region), its restriction fragment length polymorphism (rflp) and total mitochondrial dna (mtdna) analyses which change the view to the taxonomy of dermatophytes [ ] [ ] [ ] [ ] . molecular diagnosis provides specieslevel identification of dermatophytes more accurately regarding their development in human, soil or animals. recent studies showed that the conventional pcr of its and chs regions might be helpful tool for a better understanding of dermatophyte identification, taxonomy, ecology and epidemiology [ ] . these findings revolutionized the diagnosis of sfi caused by dermatophytes and the use of ''in house'' or rare commercial molecular tools enable their rapid determination directly in patients sample within h. as for ''in house'' molecular tools, various types of pcr techniques have been developed in the last decade. these assays had evolution from conventional pcr, nested pcr, pan-nested pcr which was recommended as gold standard with higher sensitivity compared to conventional methods [ ] , followed by multiplex real-time pcr which provides detection of t. rubrum, t. interdigitale, t. violaceum and m. audouinii [ ] and later developed single-tube dermatophyte qpcr tool based on its sequences which design allows determination of species of three dermatophyte genera (trichophyton, microsporum and epidermophyton) directly in patients sample [ ] . besides, reported multiplex pcr for detection of t. rubrum and t. mentagrophytes in nail samples, which is based on chitin synthase i and its region, was proved as molecular tool with excellent diagnostic performance ( % sensitivity and % specificity) [ ] . few commercial assays have also been designed and some of them with high diagnostic performance such as duplex pcr which combines pan-dermatophyte pcr with a trichophyton rubrum-specific pcr. this assay provides rapid detection and identification of trichophyton rubrum in nail specimens and has shown specificity and sensitivity of % and %, respectively, in comparison with conventional methods [ ] . on the other hand, some multiplex pcr methods for dermathophytes or dermatophytes/candida species in clinical samples were also developed and clinical evaluation is still in progress [ ] . recently, published results [ ] showed that real-time pcr with specific pan-dermatophyte primer for detection of most agents from this group of fungi in clinical samples detected more infected patients compared with me. however, with unsatisfactory sensitivity of . % and positive predicative value of . %, this method cannot be suggested yet as sufficient replacement of conventional diagnosis. in addition, new data of tinea capitis in sub-saharan africa highlighted the importance of early detection of causative agents. the use of the pcr-elisa and rapid determination of antropophylic species (t. violaceum, m. audouinii, t. soudanense, t. rubrum) allow the effective and appropriate therapy, monitoring of possible source of infection and implementation of preventive measures with the goal of preventing the spread of causative agents and infection [ ] . by all means, the development, standardization, as well as the commercialization of molecular tools are of great importance for the perspective in the rapid diagnostics of sfi. prolong survival of high-risk patients (transplantation, immunodeficiency, immunomodulatory regimens, malignancy, longer stay in icu, etc.) with ifi primarily depends on timely, accurate diagnosis and antifungal treatment [ ] . rapid advances in the field of diagnostic methods for prompt diagnosis of ifi caused by candida spp., aspergillus spp., pneumocystis jirovecii (p. jirovecii) have been achieved in recent years [ ] . however, the standardization of currently available immunological, pcr and fluorescence in situ hybridization assays is still ongoing worldwide. besides, the development and utilization of highly specific and sensitive diagnostic tests for detection of ifi caused by other fungal species is also required [ ] . conventional methods and designed rapid assays for the diagnosis of ifi were summarized in the table . conventional me can be used in diagnosis of ifi but require a high level of expert knowledge, which represents the main disadvantage of this procedure [ ] [ ] [ ] [ ] [ ] . detection of invasive aspergillus hyphae by direct microscopic examination of samples obtained from primarily sterile regions is equated as proven invasive fungal disease [ ] . on the other hand, this infection can be ''probable'' in patients with positive microscopic finding of sputum or broncho alveolar lavage (bal) fluid in combination with the appropriate clinical criteria and predisposing factors. also, cryptococal meningitis can be rapidly diagnosed if encapsulated cryptococcus neoformans (cry. neoformans) yeast cells are visible by me of cerebrospinal fluid (csf) [ ] . adding of indian ink in wet mounts of csf sediment can improve cryptococcus detection and the diagnostic efficacy. specific staining of patients samples with giemsa, wright, wright-giemsa, fontana-masson, grocott-methenamins silver (gms) or fluorescent dyes (acridine orange, calcofluor whight or blankofor) can improve visualization of fungi and their detection by me [ , , ] . specific staining methods are most commonly used for the detection of p. jiroveci (carinii) in lower respiratory tract specimens [ ] or to identify the presence of intracellular forms of histoplasma capsulatum in bal, bone marrow, peripheral blood smears or touch preparations of lymph nodes and other tissues [ ] . acridine orange allows the detection of p. jiroveci in purulent specimens [ ] . these fluorescent stains can bind to the cellulose and chitin in the fungal cell wall [ ] and have become very useful tool in laboratory mycology. also, any fungi can be detected by this methods but their identification is difficult or impossible in many cases. the most important advantage of immunoassay is the possibility of performing directly on a clinical sample obtained from patients. ic assays for fungal ags are low-cost, easy to use, and applicable at the patient's bedside, and can be performed easy to a number of samples without special equipment (even without electricity). however, currently, there are only few commercially available immunochromatographic assays that enable the detection of surface or soluble yeasts or aspergillus ag in clinical samples. their detailed characteristics are presented in table . one of the most significant achievements in mycology is the development, evaluation and manufacture of immunoassays for diagnosis of ifi. with the introduction of immunoassays for ag detection in patients biological fluids such as serum, bal or csf, the diagnosis of ifis, previously diagnosed only by histopathological examination of tissue samples after biopsy, have become more rapid and non invasive. latex agglutination tests for cryptococcal gxm are chip, sensitive, specific and easy to perform. the polysaccharide capsular gxm that is produced in large quantities by the crytococcus can be detected in both csf and blood samples [ ] . also, detection of candida mn, aspergillus gm, , b-d-glucan (bdg) in serum, bal or other fluids significantly improved the diagnosis of ifi [ , ] . in the diagnosis of candidemia, mn and bdg are currently available. mn is a major component of candida cell wall, accounting for up to % of total dry cell weight, which is released in bloodstream during candida replication during infection. the platelia candida ag test (bio-rad laboratories, marnes-la-coquette, france) enables the detection of mn in blood (serum) samples in elisa format [ ] . several retrospective and prospective studies have evaluated the utility of mn detection for the diagnosis of invasive candidosis in haematological and icu patients with an overall sensitivity (sn) of % and specificity (sp) of % [ ] [ ] [ ] [ ] . the sn of the test seems to vary with the infecting candida species, being the highest in the case of c. albicans [ ] . a positive mn test occurs several days before radiological detection of hepatosplenic candidosis or positive blood cultures (bc) [ ] . in high-risk patients, the test should be performed two to three times per week since the presence of mn ag in blood is short-lived due to rapid clearance during each episode of candidemia with the concomitant appearance of anti-mn ab [ ] . another ag-based test detects the presence of bdg, the important component of the cell wall of most fungi, in sera samples and is approved by food and drug administration of the united states of america [ , ] . the test has been evaluated, mostly in icu patients, with an overall sn of % and sp of % for subjects with proven or probable ifi [ , ] . however, bdg is not species specific; therefore it is a pan fungal assay. results of many large studies have shown that with higher values of sn ( . %) and sp ( . %), extremely low value of diagnostic odds ratio ( . %) have been obtained and bdg concentration vary in ifi due to different fungal species. white et al. proved that bdg concentration for proven/probable ifi ( pg/ml) is significantly higher then in possible/suspeced cases and control population ( pg/ml) and it is different regarding the cause of ifi. bdg is useful for diagnosis of invasive aspergillosis, invasive candidosis and p. jirovecii pneumonia, but bdg concentration vary based on causative agents [ ] . additionally, positive bdg in the cases of p. jirovecii pneumonia cannot distinguish infection from coloniza- table summary of rapid assays for fungi identification and invasive fungal infections (ifis) diagnosis. tion, to promote the higher sensitivity bdg concentration of pg/ ml has to be considered as positive, but only concentration threshold of pg/ml increased specificity. the interpretation could be only with additional clinical, radiologic examination, immunoor molecular assays [ , ] . the test should be applied twice weekly and a single positive test is indicative of infection. true positive cases usually show falling bdg titter that eventually becomes negative in patients responding to antifungal treatment, a trend that cannot be observed in patients who does not respond to therapy. false positive bdg results may occur due to several reasons such as haemodialysis, abdominal surgery, treatment with blactam antibiotics and concomitant presence of lipopolysaccharide originationg from gram-negative bacteremia, which makes its application rather difficult in clinical settings [ , ] . however, based on excellent negative predictive value of this test (nearly %), lack of bdg detection is most useful for exclusion of ifi [ , ] . also, the colonization of individuals with candida sp. has no apparent effect on the bdg test outcome [ , , ] . as for the use of gm in diagnosis of invasive aspergilosis, recently published data suggest that detection of this aspergillus ag in blood and parallel pcr diagnostics provide very high sensitivity of % with specificity of % which influence % negative predictive value in high risk patients and enable the consideration of no-existing invasive aspergillosis in these patients and no need for antifungal therapy. contrary, positivity of both tests has positive predictive value of % that suggest probably occurrence of invasive aspergillosis [ ] . however, the cross-reactivity of fusarium spp. in the aspergillus gm elisa assay has been observed and needs to be taken into consideration in some patients, contraries or settings. the most significant progress in the diagnosis of infectious diseases, fungal infections included, has been made with the introduction of molecular biology. molecular detection and identification as well as nucleic acid testing (nat) have a great importance in the diagnosis of infectious diseases, especially in the presence of a suggestive patient history and clinical manifestations of the infection. these methods can be used as a sensitive and specific tool for the detection and identification of numerous microorganisms in patient specimens. the application of nat can enhance the speed of diagnosis. some of these tests can be completed in a few hours and have high sn and sp [ ] [ ] [ ] . however, the disadvantages include the high cost of reagents and instruments, appropriately trained staff and well-equipped laboratories that are able to fulfil the highest standards. additionally, possibility of samples or reagents contamination could be also the lack of these assays [ , , , ] . peptide nucleic acid fluorescent in situ hybridization (pna-fish) is a brand new, rapid, molecular diagnostic platform developed by advandx (woburn, massachusetts) based on proprietary of pna probe technology. the pna-fish uses a variety of currently approved probes, including ones for staphylococcus aureus, coagulase-negative staphylococci (cons), enterococcus faecalis and other enterococci, escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, c. albicans, c. glabrata, c. parapsilosis, c. krusei, and c. tropicalis (advandx) [ ] [ ] [ ] [ ] [ ] . the pna-fish uses fluorescent-labelled probes to target species-specific rrna sequences in a high sn and sp fluorescence in situ hybridization assay. the probes bind to the target rna tightly, without electrostatic repulsion from the charged rna backbone and since rrna is amplified in viable cells of growing cultures, there is no need for its further amplification [ , , ] . cell lyses, usually used for isolation of genetic material, are also not necessary since the pna probes hybridize to rrna inside the bacteria or yeast enabling the whole cell analysis. the assay requires only limited sample preparation as cells do not need to be lysed to isolate genetic material, allowing a simple test procedure with visual results that match gram-stain morphology [ , ] . the major characteristics of pna-fish in a routine monitoring of analyses of sepsis are safety, sp, sn, the potential for highthroughput testing and economical feasibility. the pna-fish is easy to perform in clinical laboratory and does not require a significant capital equipment costs unlike microarrays or matrixassisted laser desorption ionization-time of flight mass spectroscopy (maldi-tof) [ , , ] . the pna-fish method requires only microscope equipped with a fluorescent lamp and dual band filters for results interpretation. the accuracy and sp of pna-fish can significantly affect antibiotic and antifungal utilization, allowing more targeted therapy, reduction of treatment duration, with an overall reduction in healthcare costs and increased benefit for patients [ , ] . recently, this methodology is additionally improved by introducing new quickfish, developed by advandx, that enables very fast ( minutes) identification of bacteria or yeasts from positive blood samples. . . molecular diagnosis of p. jirovecii pneumonia p. jirovecii is an opportunistic unicellular fungal pathogen that causes an acute and life-threatening pneumocystis pneumonia in immunocompromised hosts [ , ] , such as hiv-infected patients (especially those who do not know that they are hiv positive, do not comply with or respond to antiretroviral therapy or pneumocystis prophylaxis) [ ] , solid organ transplant and haematopoietic stem cell transplant recipients, patients with malignant diseases, non-hiv-infected patients receiving immunosuppressive medications (e.g. corticosteroids, monoclonal antibodies or cytokine inhibitors) for autoimmune or inflammatory diseases and subjects with congenital immunodeficiencies [ ] [ ] [ ] . the clinical course of infection is more acute and severe in hiv-negative immunocompromised patients than in hivinfected hosts with significantly higher rates of mortality ( - % vs. - %) [ ] . in some cases, however, patients may become colonized with p. jirovecii without signs or symptoms of acute disease [ ] . according to the results of the conducted studies, it has been shown that even - % of hiv patients and - % of non-hiv immunosuppressed patients are asymptomatic carriers of p. jirovecii [ ] . in patients with pneumocystis pneumonia, rapid and accurate establishment of the diagnosis and prompt treatment initiation are critical determinants for favorable clinical outcomes [ ] . considering the fact that p. jirovecii cannot be cultured in vitro, microscopic visualization of p. jirovecii cysts and/or trophozoites in lower respiratory samples such as induced sputum and bal fluids still remains the gold standard for the diagnosis of pneumocystis pneumonia [ ] . however, this method is largely subjective, nonspecific, low sensitive, and depends on the skill and experience of the observer, and the type of sample [ ] . despite all disadvantages, me is still frequently used in resource-limited laboratories for the detection of p. jirovecii in clinical specimens. this applies in particular to immunofluorescent assay (ifa) which is, compared with calcofluor white, grocott-gomori methenamine silver, and diff-quik staining method, significantly more sensitive ( . % vs. . %, . % and . %) but less specific ( . % vs. . %, . % and . %) [ , ] . in recent two decades, introduction of molecular detection methods into the clinical laboratory practice has led to significant progress in the diagnosis of pneumocystis pneumonia [ ] . nowadays, these methods have greatly expanded their scope of application and are also successfully used for investigation of potential outbreak scenarios, determination of epidemiology and transmission of pneumocystis pneumonia, and, as opposed to conventional microscopy, offer a high degree of objectivity [ , ] . pcr, as one of the most popular molecular diagnostic techniques in clinical microbiology at this moment, has been reported as a valuable tool for detection of p. jirovecii in different non-invasively and invasively collected clinical samples from the respiratory tract [ ] . in addition, a newly developed, improved version of the pcr assay -real-time pcr has contributed to a significant reduction in turnaround time (results are available after . h including dna extraction) and an overall increase in diagnostic sensitivity compared to ifa [ ] . moreover, as opposed to nested pcr, such one-step assays are easier to perform and decrease the risk of carry-over contamination [ ] . until today, various samples have been used for pcr detection of p. jirovecii pneumonia. pcr on bal sample has higher diagnostic sensitivity ( . - %) [ , ] in comparison with non-invasive samples such as sputum ( . %) [ ] or oropharyngeal wash fluid ( %)( ), but is difficult to obtain in some critically ill patients due to the increased risk of respiratory deterioration [ ] . also, in patients with non-productive cough suspected of having p. jirovecii pneumonia, induced sputum is not easy to obtain [ ] . it is important to emphasize that, although highly sensitive, negative pcr results of bal fluid do not provide reliable exclusion of p. jirovecii pneumonia given that the false negative results can occur when mutation at position (c t) of the mitochondrial large subunit ribosomal rna (mtlsurrna) gene of p. jirovecii is present [ ] . on the other hand, studies have shown that pcr analysis of oropharyngeal wash fluid is more specific ( %) than bal ( . - %) and sputum ( . %), which indicates that the pcr detection of p. jirovecii in the upper part of respiratory tract is a good indicator of p. jirovecii pneumonia [ , ] . in order to overcome insufficient specificity of pcr on bal fluid and sputum, a relatively new concept in the diagnosis of p. jirovecii pneumonia has been developed. this method is characterized by minimal invasiveness and includes pcr detection of cell-free dna, fragments of dna which are present extra cellular in different body fluids, in serum samples [ ] . the pioneering experimental studies have shown that diagnostic sensitivity of pcr detection of p. jirovecii dna in sera may vary from - % [ , ] . however, the most recent study has shown that, when compared with gms staining as the gold standard, the sensitivity of cell-free dna pcr is the same as bal fluid pcr and sputum pcr, while the specificity is much higher [ ] . when above mentioned studies are analyzed together, it can be concluded that diagnostic performances of pcr on serum dna depend largely on the amount of sample used for dna extraction, dna extraction method (magnetic bead methods are more efficient in serum cell free-dna extraction than proteinase k-phenol-chloroform) and the type of primers used for detection of pneumocystis [pneumocystis heat shock protein (hsp ) gene has higher sensitivity and specificity in pneumocystis detection than pneumocystis mt lsu rrna gene] [ , , ] . invasive pulmonary aspergillosis is a devastating opportunistic infection caused by molds belonging to the genus aspergillus. this infection mainly affects immunocompromized hosts, such as patients with cancer or hematologic malignancies, allogeneic hematopoietic stem cell and solid organ transplant recipients (especially lung transplant recipients), patients with advanced aids, and those presenting with chronic granulomatous disease [ , ] . in recent years, increase in incidence of invasive pulmonary aspergillosis has been noted in patients with lympho proliferative syndromes and in medical icu patients, particularly in those treated with corticosteroids. nowadays, despite the advances in understanding the disease and development of highly effective antifungals such as voriconazole and posaconazole, global mortality and morbidity rates are still high [ ] . timely and accurate diagnosis and early initiation of appropriate antifungal treatment significantly improve disease prognosis [ ] . diagnosis of invasive pulmonary aspergillosis is traditionally based on the isolation of fungi of the genus aspergillus in culture in combination with cme or radiographic findings [ ] . however, the sensitivity of culture is low and may take a long time to results. besides, cultures are incapable of differentiating between infection, colonization, or contamination. on the other hand, me, as inexpensive and easy-to-perform complementary diagnostic method, improves positive predictive value by confirming positive culture results and provides information about the infecting cell morphology, the state of infected tissues, and biofilm formation, but is not organism specific [ ] . immuno assays which are based on the detection of fungal cell wall biomarkers, such as bdg or aspergillus gm, have significantly reduced turn-around times compared with me, but it has been shown that they can be low sensitive and specific in certain patient groups [ ] . molecular techniques represent a novel approach to the diagnosis of invasive aspergillosis. currently, pcr is the most widely applied molecular diagnostic test, which enables amplification and detection of extremely low concentrations (from to pg) of aspergillus dna in various clinical specimens such as bal fluid, serum, plasma, whole blood and biopsy material [ , ] . also, pcr assays can quickly identify species within the genus aspergillus from cultures and non-cultured samples which is of great significance for epidemiologic studies and clinical practice given the fact that newly described species such as aspergillus lentulus and aspergillus calidoustus have been shown to have elevated minimum inhibitory concentrations (mics) to several antifungal drugs, including azoles [ ] . due to the lack of standardization and potential false-positive results, pcr was in excluded from european organization for the research and treatment of cancer/ mycoses study group (eortc/msg) definitions [ ] . in , a group of experts conducted an investigation in which aspergillus pcr was compared with gm and bdg and, based on obtained results; they concluded that ''pcr is now mature enough for inclusion in the eortc/msg definitions'' [ ] . studies have shown that performance of molecular tests varies depending on the specimen type and patient population [ , ] . pcr analysis of blood fractions is the most desired testing modality given that it requires minimally invasive sampling procedure [ ] . also, it is important to emphasize that this method is less exposed to accidental contamination and, in comparison with pcr on bal samples, offers higher diagnostic sensitivity when screening [ ] . moreover, it has been noted that the diagnosis of invasive aspergillosis can be excluded if consistently negative results are obtained during the testing of multiple blood samples [ ] . conducted meta-analyses have shown that aspergillus pcr on blood samples achieves sn ranging from to % and sp from to . % [ , , ] . when different fractions of blood are compared, meta-analysis showed that pcr assays of whole blood are more sn ( % vs. %), but less sp ( % vs. %) than those of sera. however, this difference is not statistically significant [ ] . on the other hand, another study which included hematology patients at risk of invasive aspergillosis reported the highest diagnostic sn ( %) of aspergillus pcr testing of plasma in comparison with pcr testing of serum ( %) and whole blood ( %) [ ] . this disagreement in the results of two above mentioned studies can be due to the differences in sample processing and prolonged specimen storage methods for the whole-blood specimens used in the second study [ ] . in high-risk patients, at least two positive whole-blood pcr specimens per patient may dramatically increase sp to % with positive predictive value of %, which is very indicative for invasive aspergillosis. however, this approach significantly decreases sn to %, which indicates that this diagnostic strategy is more valuable as a confirmatory tool than as a screening tool [ ] . another factor that may also affect the sn of pcr testing is current immunological status of the patient. in the study, which examined diagnostic performance of pcr analysis of sera obtained from neutropenic and non-neutropenic patients, it was shown that this testing modality has higher sn in neutropenic patients ( . %) than in nonneutropenic patients ( . %), but without statistical significance. likewise, the same study noted that the initial level of aspergillus dna is highly predictive of the -day mortality rate (below copies/ml-low probability; above copies/ml-high probability), which is of great significance for identification of patients who may benefit from more intensive care [ ] . finally, based on all of the above facts, it can be concluded that pcr analysis of serum or plasma seems to be diagnostic modality with acceptable sn and sp. also, these sample types, as opposed to whole blood, do not require preanalytical cell lysis steps and allow other biomarker diagnostic tests, such as gm, to be run [ , ] . bal fluid represents another commonly used sample for the diagnosis of invasive aspergillosis. compared with blood samples, the sp of aspergillus pcr bal testing is significantly higher [ ] . based on the results of conducted meta-analysis, the pooled sp of pcr on bal fluid specimens was even % for proven/ probable invasive aspergillosis, while sn was % [ ] . however, one of the major limitations of pcr testing of respiratory samples is the inability to differentiate airway colonization from invasive disease. also, the risk of sample contamination at the patient's bedside or in the laboratory by airborne spores is high which may lead to the occurrence of false positive results. furthermore, it is estimated that even % of bal samples from healthy subjects would be falsely positive after molecular testing which is significantly higher than in other diagnostic modalities (e.g. detection of gm) [ ] . quantitative pcr (qpcr) detection of high fungal burdens may be indicative of invasive aspergillosis, but this diagnostic modality has still many limitations [ ] . in recent years, resistance to azoles in a. fumigatus has emerged as a global health problem [ ] . based on published results of conducted studies, the resistance occurs as a result of tr /l h and tr /y f/t a mutations in the cyp a gene and its promoter region [ ] . these mutations were first found in the netherlands in (tr /l h) and (tr /y f/t a) [ ] and have since been reported in multiple european countries, middle east, asia, africa, australia and the united states [ ] . azole-resistant invasive aspergillosis is difficult to diagnose using traditionally culture-based methods given that aspergillus cultures are negative in the majority of patients. on the other hand, immunoassays, which provide detection of fungal cell wall biomarkers, are unable to identify patients with azole-resistant disease [ ] . consequently, two commercial multiplex real-time pcr assays (mycogenie (ademtech, pessac, france) and asper-genius (pathonostics, maastricht, the netherlands)) have been developed to enable the detection of both a. fumigatus dna and the most prevalent cyp a mutations responsible for azole resistance in clinical samples. these assays have been evaluated using bal fluid and serum samples. the aspergenius multiplex real-time pcr test includes two pcrs -the species pcr and the resistance pcr. in respiratory samples, the species pcr generated thesn and sp of . % and . %, respectively, in patients with hematological disorders, while the resistance pcr was able to detect resistanceassociated mutations in a culture-negative patient with invasive aspergillosis. as for the serum samples, species assay showed the sn and sp of . % and %, respectively in patients in whom fungal disease status had been previously defined using the revised eortc criteria [ ] [ ] [ ] . on the other hand, mycogenie showed thesn of . % and sp of . % for detection of aspergillus dna in respiratory samples, while in serum samples, the sn and sp were % and . %, respectively. it is important to emphasize that all isolates harboring the tr /l h mutations were accurately detected [ ] . recently, multiplex pcr and real-time mpcr molecular tests have been developed for detection and identification of various closely related pathogens causing respiratory, gastrointestinal and sexually transmitted infections as well as meningitis and sepsis. because of similar clinical symptoms, signs and possible simultaneous presence of several different agents, the differential diagnosis of these infections includes a large number of potential agents, which are clinically indistinguishable. use of molecular panels allows timely detection and discrimination of the infecting pathogens, which is important to optimize medication, prevent secondary spread of infection and unnecessary antibiotic use in viral infections, and may help in decisions regarding hospitalization and infection control measures. also, these panels include internal control for monitoring of every procedure step [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in medical mycology, the use of multiplex pcr for pathogens detection implies panels for detection of yeasts in blood of patients with sepsis and panels for cry. neoformans detection in csf of patients with meningitis/encephalitis. characteristics of these molecular panels are shown in the table . in recent years, different rapid molecular methods have been developed to speed up the identification of the microorganisms that grow in blood-culture (bc) bottles. on the first place, maldi-tof ms sepsityper kit tm can be used to directly analyze positive bc in real time and to provide definitive species identification within to minutes, with the identification rate of . % [ ] . the maldi-tof has the advantage of rapid turn-around time and ability to identify a large number of microorganisms directly. disadvantages include the high start-up costs associated with the purchase of mass-spectrometry equipment and training of personnel. as an alternative to maldi-tof, film array blood culture identification (bcid) panel (bio fire, usa), a multiplexed pcr based diagnostic test, can be used for positive bc. the bcid can detect bacteria, five candida spp. and three antimicrobial resistance determinants (mec a, van a/b, bla-kpc) in h from the time of bc positivity [ ] . this method has a several advantages: the large number of targets can be evaluated in a single test and all steps of the assay, from nucleic acid extraction to interpretation of amplification data, are performed in closed system and using a single pouch on a minimally processed clinical sample. the required laboratory procedures are not technologically complex and can be performed by persons who do not have training in molecular techniques [ ] . other methods such as nano particle probe technology (nucleic acid extraction and pcr amplification) and nano sphere's verigene can be used for identification. the platform consists of two assay panels: the bc-gp assay and the bc-gn assay. also it can detect drug resistance markers such as: mec a, van a/b, kpc, ndm, ctx-m, vim, imp and oxa genes [ ] . t magnetic resonance (mr) assay for the rapid diagnosis of candidemia in whole blood is one of tests in the new era of molecular diagnostics which can provide fast and species -specific (c. albicans, c. tropicalis, c. glabrata, c. krusei, c. parapsilosis) detection directly in the clinical samples. regarding the fact that t mr has % sensitivity and > % specificity, does not require cultivation as well as sample purification or preparation, it can be highlighted that this test represents example of point-of-care diagnostic test which can ensure the prompt and accurate results in . ae . hours with the possibility of treatment modification in cases of candidemia. this novel technology has . % negative predictive value in population of patients with candida invasive infection, disease with estimated prevalence of %, which will decrease the number of patients on empiric therapy and resistant strains occurrence [ ] . regarding the candidemia caused by c. glabrata, so far, it was considered that fluconazole (flc) resistance is common in c. glabrata and echinocandins are often used as first-line therapy. one of the major advantages of t mr is a possibility of rapid detection of c. glabrata in blood of patients with higher sensitivity (to > %) than culture (se = %) [ ] and its determination on low level of detection of cfu/ml. this is very important since that early initiation of appropriate treatment can improve prognosis and significantly decreases mortality. however, further improvement of diagnostic performances of t mr is required given that nearly % of patients had indeterminate t dx results (n = / ). also, the price of this method is high and does not enable day-to-day monitoring, contrarily to bc. in addition, only species of candida are detected by this assay [ ] [ ] [ ] . however, the resistance to echinocandin therapy associated with fks and fks gene alterations of c. glabrata has been noticed in recent years. these findings influence the establishment of a novel (allele-specific molecular beacon and dna melt analysis followed by asymmetric pcr) and highly accurate diagnostic assay for rapid identification of fks mutations associated with echinocandin resistance in this candida species. this platform can be diagnostic method for rapid detection of infections caused by c. glabrata resistant strains to echinocandin and will timely provide the choice of efficient treatment [ , ] . currently, there are efforts for development of mass spectrometryy (ms) procedure for fungal disaccharide (ds) detection in patient's sera for rapid diagnosis of ifi. applaing of maldi-tof ms for fungal molecules detection have shown very good diagnostic efficacy for early diagnosis of ic, ia and more important invasive mucor infection (imi). the first researches in this field have pointed out the fact that the sensitivity of ms-ds (with application of the higher cutoff) is higher than that of mn or bdg detection. in addition, neither neutropenia nor bacteriemia have been shown to influence the detection of fungal disaccharide dd in patients with ic. also, this method has provided earlier positive results than other tests on the first serum samples in the cases of ia. moreover, results of evaluation of this method suggest the possibility of panfungal analyses and diagnosis of imi for which currently there are no available commercial serological tests that are efficient for the diagnosis. however, it has to be highlighted that this biological test requires further evaluation and validation in multicenter studies [ ] . delays in the diagnosis of ifi and appropriate therapy application clearly affect the infectious diseases outcome in a negative fashion. on the other hand, non-established accurate identification of fungal pathogens that cause sfi can influence failure in implementation of appropriate anti-epidemic prevention measures. in the era of large number of commercial rapid tests, clinicians must decide which of them should be introduced to the routine work, based on an individual experience, consideration of the disease incidence in the population, the cost-effectiveness and finally adequate assessment and selection of patients who would have the most benefit from rapid treatment. the study was supported by the serbian ministry of education and science [the grant no and grant no iii ]. the authors declare that they have no competing interest. diagnostic performance of ! -b-d-glucan in neonatal and pediatric patients with candidemia candida bloodstream infections in serbia: first multicentre report of a national prospective observational survey in intensive care units medical mycology and parasitology epidemiology of invasive candidiasis: a persistent public health problem hospital epidemiology and infection control in acute-care settings the prevalence of candida onychomycosis in southeastern serbia from superficial mycoses in the niš region, southeast-serbia dermatophytosis, trends in epidemiology and diagnostic approach the effect of qswitched nd:yag nm/ nm laser in the treatment of onychomycosis in vivo the diagnostic yield of skin biopsy in patients with leukemia and suspected infection deep dermatophytosis and inherited card deficiency vulvovaginal candidosis recurrent genital candidosis-diagnostic and epidemiological aspects arsic-arsenijevic v. modelling of antifungal treatment with azoles and essential oils for non-albicans candida spp. causing vulvo-vaginal infections a retrospective study of cases of nasopharyngeal carcinoma with or without oropharyngeal candidiasis during radiotherapy therapeutic alternatives of natural compounds in treatment of candida-associated denture stomatitis optimizing antifungal strategies to improve patient survival koneman's colour atlas and textbook of diagnostic microbiology using rapid diagnostic tests to optimize antimicrobial selection in antimicrobial stewardship programs use of cell culture and a rapid diagnostic assay for chlamydia trachomatis screening melnick & adelberg's medical microbiology how to teach the potassium hydroxide preparation: a disappearing clinical art form diagnosis of vaginal discharge by wet mount microscopy: a simple and underrated method impact of reporting gram stain results from blood culture bottles on the selection of antimicrobial agents is gram staining a diagnostic tool or a guide for optimal empirical therapy? evaluation of a newly-developed immunochromatography strip test for diagnosing dermatophytosis rapid detection of vaginal candida species by newly developed immunochromatography production of an anti-dermatophyte monoclonal antibody and its application: immunochromatographic detection of dermatophytes molecular characterization of selected dermatophytes and their identification by electrophoretic mutation scanning molecular epidemiology, phylogeny and evolution of dermatophytes mega : molecular evolutionary genetics analysis (mega) software version . molecular analysis and mating behaviour of the trichophyton mentagrophytes species complex rapid detection of dermatophytes from skin and hair diagnosis of common dermatophyte infections by a novel multiplex real-time polymerase chain reaction detection/ identification scheme evaluation of a single-tube real-time pcr for detection and identification of dermatophyte species in clinical material multiplex pcr assay for the detection of common dermatophyte nail infections comparison of a new commercial test, dermatophyte-pcr kit, with conventional methods for rapid detection and identification of trichophyton rubrum in nail specimens development of a new commercial qpcr assay to detect and differentiate dermatophyte infections of the skin, nails and hair clinical evaluation of b-tubulin real-time pcr for rapid diagnosis of dermatophytosis, a comparison with mycological methods identification of the causative dermatophyte of tinea capitis in children attending mbarara regional referral hospital in uganda by pcr-elisa and comparison with conventional mycological diagnostic methods molecular and nonmolecular diagnostic methods for invasive fungal infections utility of pcr in diagnosis of invasive fungal infections: real-life data from a multicenter study district laboratory practice in tropical countries part district laboratory practice in tropical countries part detection of infection or infectious agents by use of cytologic and histologic stains clinical infectious diseases: a practical approach practical handbook of microbiology multicenter evaluation of a lateral-flow device test for diagnosing invasive pulmonary aspergillosis in icu patients methods of rapid diagnosis for the etiology of meningitis in adults development of a dual path platform (dpp ) immunoassay for rapid detection of candida albicans in human whole blood and serum comparison of four assays for the detection of cryptococcal antigen alternaria-associated fungus ball of orbit nose and paranasal sinuses: case report of a rare clinical entity new enzyme immunoassays for sensitive detection of circulating candida albicans mannan and antimannan antibodies: useful combined test for diagnosis of systemic candidiasis comparative evaluation of ( , )-b-d-glucan, mannan and anti-mannan antibodies, and candida species-specific snpcr in patients with candidaemia early diagnosis of invasive candidiasis with mannan antigenemia and antimannan antibodies value of ( - )-b-d-glucan, candida mannan and candida dna detection in the diagnosis of candidaemia revised definitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group b-dglucan as a diagnostic adjunct for invasive fungal infections: validation, cutoff development and performance in patients with acute myelogenous leukemia and myelodysplastic syndrome diagnostic performance of the ( -> )-b-d-glucan assay for invasive fungal disease an evaluation of the performance of the dynamiker fungus ( - )-b-d-glucan assay to assist in the diagnosis of invasive aspergillosis, invasive candidosis and pneumocystis pneumonia an evaluation of the performance of the dynamiker fungus ( - )-b-d-glucan assay to assist in the diagnosis of pneumocystis pneumonia diagnostic value of ( - )-ß-d-glucan guided real-time pcr diagnostics of pneumocystis jirovecii pneumonia (pcp) for non-hiv immune-compromised patients galactomannan and polymerase chain reaction-based screening for invasive aspergillosis among high-risk hematology patients: a diagnostic meta-analysis nosocomial bloodstream infections in us hospitals: analysis of , cases from a prospective nationwide surveillance study application of molecular diagnostic techniques for viral testing nucleic acid testing for human disease microbial dna typing by automated repetitive-sequence-based pcr real-time pcr in clinical microbiology: applications for routine laboratory testing review of rapid diagnostic tests used by antimicrobial stewardship programs use of peptide nucleic acid-fluorescence in situ hybridization for definitive, rapid identification of five common candida species multicenter evaluation of a new shortened peptide nucleic acid fluorescence in situ hybridization procedure for species identification of select gram-negative bacilli from blood cultures peptide nucleic acid fluorescent in situ hybridization for hospitalacquired enterococcal bacteremia: delivering earlier effective antimicrobial therapy impact of rapid in situ hybridization testing on coagulase-negative staphylococci positive blood cultures peptide nucleic acid fluorescence in situ hybridization for rapid detection of klebsiella pneumoniae from positive blood cultures evaluation of the yeast traffic light pna fish probes for identification of candida species from positive blood cultures pneumocystis jiroveciifrom a commensal to pathogen: clinical and diagnostic review detection of pneumocystis jirovecii by quantitative pcr to differentiate colonization and pneumonia in immunocompromised hiv-positive and hiv-negative patients colonization by pneumocystis jirovecii and its role in disease diagnosis of pneumocystis jirovecii pneumonia with serum cell-free dna in non-hiv-infected immunocompromised patients detection of pneumocystis jirovecii in oral wash from immunosuppressed patients as a diagnostic tool diagnosis and management of pneumocystis jirovecii infection real-time pcr assay-based strategy for differentiation between active pneumocystis jirovecii pneumonia and colonization in immunocompromised patients development and validation of a pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of pneumocystis pneumonia pneumocystis jirovecii pneumonia in rheumatoid arthritis patients: risks and prophylaxis recommendations cost-effectiveness analysis of diagnostic options for pneumocystis pneumonia (pcp) comparison of quantitative real-time pcr and direct immunofluorescence for the detection of pneumocystis jirovecii detection of pneumocystis jiroveci in respiratory specimens by four staining methods performance of a pcr assay for detection of pneumocystis carinii from respiratory specimens a mis-leading false negative result of pneumocystis real-time pcr assay due to a rare punctual mutation: a french multicenter study comparison of different blood compartments for the detection of circulating dna using a rat model of pneumocystis pneumonia short communication: diagnosis of pneumocystis jirovecii pneumonia by detection of dna in blood and oropharyngeal wash, compared with sputum pulmonary aspergillosis: clinical presentation, diagnostic tests, management and complications pcr in diagnosis of invasive aspergillosis: a meta-analysis of diagnostic performance diagnosis of invasive pulmonary aspergillosis: updates and recommendations molecular diagnostic testing for aspergillus aspergillus pcr in serum for the diagnosis, follow-up and prognosis of invasive aspergillosis in neutropenic and nonneutropenic patients defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus aspergillus polymerase chain reaction: systematic review of evidence for clinical use in comparison with antigen testing use of pcr for diagnosis of invasive aspergillosis: systematic review and meta-analysis comparison of performance characteristics of aspergillus pcr in testing a range of blood-based samples in accordance with international methodological recommendations a systematic literature review on the diagnosis of invasive aspergillosis using polymerase chain reaction (pcr) from bronchoalveolar lavage clinical samples azole resistance in aspergillus fumigatus: can we retain the clinical use of mold-active antifungal azoles? azoleresistant aspergillus fumigatus harboring tr /l h, tr /y f/t a and tr mutations related to flower fields in colombia first detection of tr l h and tr y f t a cyp mutations in aspergillus fumigatus isolates in the united states analytical and clinical evaluation of the pathonostics aspergenius assay for detection of invasive aspergillosis and resistance to azole antifungal drugs directly from plasma samples pcr-based detection of aspergillus fumigatus cyp a mutations on bronchoalveolar lavage: a multicentre validation of the aspergenius assay in patients with haematological disease suspected for invasive aspergillosis validation of a new aspergillus real-time pcr assay for direct detection of aspergillus and azole resistance of aspergillus fumigatus on bronchoalveolar lavage fluid molecular diagnosis of invasive aspergillosis and detection of azole resistance by a newly commercialized pcr kit comparison of the filmarray rp, verigene rv+, and prodesse proflu+/ fast+ multiplex platforms for detection of influenza viruses in clinical samples from the - influenza season in belgium the increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections commercial multiplex technologies for the microbiological diagnosis of sepsis evaluation of the seeplex meningitis ace detection kit for the detection of common bacterial and viral pathogens of acute meningitis diagnostic performance of a multiplex pcr assay for meningitis in an hivinfected population in uganda direct simultaneous detection of sexually transmitted pathogens from clinical specimens by multiplex polymerase chain reaction and auto-capillary electrophoresis performance of anyplex tm ii multiplex real-time pcr for the diagnosis of seven sexually transmitted infections: comparison with currently available methods an automated nested multiplex pcr system for multipathogen detection: development and application to respiratory tract infection clinical evaluation of the filmarray blood culture identification panel in identification of bacteria and yeasts from positive blood culture bottles system for rapid detection of pathogens in patients with presumed sepsis -a systemic review and meta-analysis evaluation of the light-cycler septifast test in newborns with suspicion of nosocomial sepsis accurate and rapid identification of bacterial species from positive blood cultures with a dna-based microarray platform: an observational study accurate and rapid identification of candida spp. frequently associated with fungemia by using pcr and the microarray-based prove-it sepsis assay biomarkers and molecular analysis to improve bloodstream infection diagnostics in an emergency care unit clinical evaluation of commercial nucleic acid amplification tests in patients with suspected sepsis dnaemia detection by multiplex pcr and biomarkers for infection in systemic inflammatory response syndrome patients multicenter evaluation of biofire filmarray meningitis/encephalitis panel for detection of bacteria, viruses, and yeast in cerebrospinal fluid specimens evaluation of four commercial multiplex molecular tests for the diagnosis of acute respiratory infections detection of respiratory viruses using a multiplex real-time pcr a multiplex real-time pcr assay for identification of pneumocystis jirovecii, histoplasma capsulatum and cryptococcus neoformans/cryptococcus gattii in samples from aids patients with opportunistic pneumonia comparison of the maldi biotyper system using sepsityper specimen processing to routine microbiological methods for identification of bacteria from positive blood culture bottles enhancing pathogen identification in patients with meningitis and a negative gram stain using the biofire filmarray meningitis/encephalitis panel t magnetic resonance assay for the rapid diagnosis of candidemia in whole blood: a clinical trial rapid detection of fks-associated echinocandin resistance in candida glabrata increasing echinocandin resistance in candida glabrata: clinical failure correlates with presence of fks mutations and elevated minimum inhibitory concentrations application of mass spectrometry technology to early diagnosis of invasive fungal infections key: cord- - xk gln authors: baier, claas; linderkamp, christin; beilken, andreas; thol, felicitas; heuser, michael; ebadi, ella; ganzenmueller, tina; heim, albert; bange, franz-christoph title: influenza and respiratory syncytial virus screening for the detection of asymptomatically infected patients in hematology and oncology date: - - journal: gms hyg infect control doi: . /dgkh sha: doc_id: cord_uid: xk gln introduction: respiratory syncytial virus (rsv) and influenza virus infections are a significant healthcare risk for immunocompromised patients. in addition to community onset, nosocomial acquisition and transmission may also occur. detection of asymptomatic shedders (e.g., patients in the incubation period or immunosuppressed long term shedders) facilitates control of nosocomial transmission. methods: to strengthen the existing infection control concept, a pcr-based screening for rsv and influenza virus was implemented for all patients lacking respiratory symptoms (asymptomatic patients) who were hospitalized on an adult and a pediatric hemato-oncological ward. laboratory results of this screening were analyzed retrospectively. results: respiratory specimens were obtained for screening from patients ( % were years and younger) from december to april . in patients without respiratory symptoms, either influenza virus or rsv infection was found, resulting in a detection rate of about %. in patients, the infection was presumably detected during the incubation period, because an increase of viral load was observed in subsequent specimens. positive screening results facilitated timely implementation of adequate infection control precautions. nosocomial clusters of rsv or influenza were not detected during the screening period on the two wards. conclusion: the seasonal screening program expanded our existing infection control concept in terms of patients lacking respiratory symptoms who shed influenza virus or rsv. it enabled us to identify rsv or influenza infections in patients lacking respiratory symptoms in a -month period and thus to rapidly take isolation precautions. influenza virus (family orthomyxoviridae) and respiratory syncytial virus (rsv; family pneumoviridae) are both single-strand rna viruses and cause upper and lower respiratory tract infections in children and adults [ ] , [ ] , [ ] , [ ] . influenza virus and rsv are spread by droplet and contact transmission. therefore, nosocomial acquisition may occur due to exposure to another infected patient, visitor or healthcare workers (hcws), as well as by contact with contaminated, inanimate surfaces. immunocompromised patients with underlying hemato-oncological disorders or cancer are at risk for increased mortality, especially in the case of lower respiratory tract infection due to these viruses [ ] , [ ] , [ ] . both community and nosocomial acquisition of influenza and rsv are possible in this patient group. nosocomial outbreaks in oncologic care facilities have been described [ ] , [ ] in spite of multiple infection control measures to prevent nosocomial transmission. these measures include stringent hand hygiene, patient isolation, and use of barrier precautions such as surgical masks or vaccination (influenza) [ ] , [ ] , [ ] , [ ] . during a localized nosocomial cluster with rsv at our clinic in the winter season / , we introduced a rt-pcr-based screening of asymptomatic patients for rsv and influenza virus which contributed to the successful control of the rsv cluster [ ] . to strengthen our existing infection control measures in hematology and oncology, we subsequently implemented a systematic influenza and rsv screening of patients lacking respiratory symptoms on a pediatric and an adult hemato-oncological ward as a prophylactic infection control measure in the following winter ( / ). this screening intended to identify patients shedding rsv or influenza virus without signs of disease and -in case of a positive screening test -to establish infection control measures similar to those used for typical symptomatic (e.g. cough, sneezing, fever, respiratory distress) patients with rsv or influenza. in this paper, we describe the laboratory results of this prophylactic screening program for asymptomatic patients (season / ) and discuss its value for infection control in hematology and oncology. the screening program for rsv and influenza was implemented on two wards located in the department of pediatric hematology and oncology and in the department of hematology, hemostasis, oncology and stem cell transplantation at hannover medical school. these departments are tertiary referral centers for children and adults, respectively, with hematologic and solid neoplasia. the pediatric ward contains single (one-bed) and twobed rooms. the adult ward has single (one-bed) rooms, two-bed rooms, and four-bed rooms. all single rooms on the pediatric ward and single rooms on the adult ward have an anteroom and high-efficiency particulate air filtration with the air flow directed to the hallway. single rooms are usually reserved for patients with severe immunosuppression or palliative settings. the wards are serviced by permanent healthcare workers (hcws) and housekeeping staff. parents are allowed to stay overnight with their children on the pediatric ward. during their stay on the ward, parents are permitted to wear street clothes. however, a regular change of their clothing is recommended. the pcr-based seasonal screening for rsv and influenza started in the second half of december and ended at the beginning of april . the start and finish of the screening period were set jointly by the infection control staff, virologists and the clinicians involved based on available epidemiological data. on the adult ward, the screening was performed upon admission (admission screening) and once weekly (prevalence screening). on the pediatric ward, the pcr-based screening was performed once weekly. respiratory material was obtained from all patients lacking respiratory symptoms (asymptomatic patients). prophylactic prevalence screening was continued as long as patients stayed on the wards. patients with any symptoms of respiratory disease, e.g., cough, sneeze, fever or oxygen need (symptomatic patients), received a full diagnostic virology panel including influenza virus and rsv, and other respiratory viruses such as human metapneumovirus, adenovirus, parainfluenza virus, coronavirus and rhino-/enterovirus. in order to guarantee high adherence to the screening program, repeated audits and feedback talks took place. specimens were collected as a combined nasopharyngeal swab or pharyngeal lavage (oral wash) with sterile saline and were processed at the institute of virology. for realtime rt-pcr, rna was extracted using a qiaamp viral rna mini kit in a qiacube according to the manufacturer's instruction (qiagen, hilden, germany). cdna synthesis, amplification and detection of nucleic acid were performed in an applied biosystems ® real-time pcr system (life technologies, carlsbad, california, usa) using a commercially available one-step real-time rt-pcr kit (rsv/hmpv r-gene ® pcr kit, biomérieux, nürtingen, germany) according to the manufacturer's instructions, and an influenza a/b multiplex real time rt-pcr [ ] . the influenza rt-pcr differentiates between types a and b. for semi-quantitative comparison of viral loads with follow-up specimens, the cycle threshold (ct) value of the real time pcr was used. a decrease of the ct by ≥ values (usually equivalent to a more than -fold increase of viral load) was considered as a significant increase of viral load. infection control interventions for asymptomatic patients with rsv or influenza virus, detected by the screening infection control recommendations for asymptomatically infected patients with rsv and influenza virus were the same as for symptomatic patients. they were labeled in an electronic alert system, and it was recommended that the patients each be placed in a single (one-bed) room. each positive pcr test was electronically communicated to the infection control staff. contact and droplet precautions (surgical mask, gown, gloves) were mandatory for hcws and all visitors entering the room of affected patients. the patients were restricted to their single (onebed) room and were trained in hand hygiene. if they had to leave the room, they wore a surgical mask. two negative rt-pcr results at a minimum -day interval were required to stop all isolation precautions. hcws were strongly encouraged to let themselves be vaccinated against influenza before and during the winter season / . the vaccination rate among hcws for influenza was about % on the pediatric ward and about % percent on the adult ward. from december to april , asymptomatic patients were screened. ( %) of these patients were aged or younger and were screened on the pediatric ward. of the remaining adult patients, ( . %) received swabs on the emergency ward, whereas patients were screened exclusively on the adult hematooncological ward. overall, screening samples were obtained, % of which (n= ) were from pediatric patients and % from adults (n= ). the mean screening sample number per patient was . for pediatric patients and . for adults. the majority ( %) of the screening samples were nasopharyngeal swabs (n= ). samples were pharyngeal washes and were exclusively obtained from adult patients. altogether, asymptomatic patients tested positive either for influenza or rsv in a screening sample, i.e., a combined detection rate of %. patients ( adults and one child) tested positive for influenza type a. patients ( adults and children) were tested positive for rsv. co-infections with influenza virus and rsv were not detected. an overview is shown in table . interestingly, in patients ( influenza virus patients and rsv patient), a significant increase of the viral load in samples was observed following the positive screening specimen ( table ) . course of viral load in patients who showed an increase in viral load (presumed detection during the incubation period). the patients showed an increase in viral load during their clinical course and were therefore presumably detected during the incubation period. of the ( %) asymptomatic patients were in twoand four-bed rooms at the time the screening test was reported positive ( positive for rsv, positive for influenza virus). of these patients in shared rooms were immediately moved to separate single rooms, following the infection control recommendations. patients with influenza were kept together, as they occupied the same -bed room when they simultaneously tested positive. patients with rsv and patient with influenza stayed in the shared rooms, and patients had already been discharged when the test result became available. of the asymptomatic patients were already located in single (one-bed) rooms (for reasons other than infection control) and remained in this room after virus detection. for all patients identified (whether located in private or shared rooms after positive test results were available), barrier precautions (droplet and contact) were implemented as described above (see "materials and methods" section). on both wards, all patients presenting with symptoms of acute respiratory infections were routinely tested with a broad panel for respiratory viruses (including rsv and influenza). in these patients with symptoms of acute respiratory infection, we found influenza case and rsv case on the pediatric ward and influenza case and rsv cases on the adult ward from december to april . prevalence of other respiratory viruses was low in this season on these two wards. parainfluenza case, human metapneumovirus cases and rhinovirus cases were found on the pediatric ward. on the adult ward parainfluenza case was detected. clusters of nosocomial transmission of rsv or influenza were not observed during the screening program period. respiratory tract infections caused by rsv and influenza virus are a significant healthcare risk for hospitalized and non-hospitalized patients with underlying hemato-oncological disease. during the winter season, acquisition of these viruses typically occurs in the community, and patients may be admitted with symptoms of infection or infection to a hospital. nosocomial acquisition due to transmission occurs as well [ ] , [ ] , [ ] , [ ] . recom-mendations and suggestions for controlling the spread of influenza and rsv in healthcare settings in general and especially in hemato-oncological patients exist (e.g. [ ] , [ ] ), but should be adapted to each hospital's specific needs. to extend our existing standard infection control measures, we introduced a prophylactic screening program for asymptomatic patients targeting rsv and influenza on an adult and a pediatric hemato-oncological ward. the screening was intended to detect patients that shed the virus without having signs or symptoms of disease. shedding my occur before the onset of disease (incubation period), during an asymptomatic clinical course, or following disease as a result of prolonged viral shedding, particularly in the immunocompromised host [ ] . these groups of asymptomatic patients, who often remain unnoticed, can contribute to nosocomial spread. of the asymptomatic shedders, we detected an increased viral load in patients in follow-up specimens (see table ). it is likely that these patients were in the incubation period. this group of patients is of particular interest for infection control strategies, since with increasing viral load, the risk of transmission also rises. we identified of a total of asymptomatic patients with an rsv or influenza virus infection (detection rate of about %).this means that about asymptomatic patients needed to be screened to detect one shedder of either rsv or influenza virus. other studies found a similar prevalence in asymptomatic individuals for influenza ( . %) and rsv ( . %) using rt-pcr [ ] , [ ] . campbell et al. examined samples from asymptomatic and symptomatic patients prior to hematopoietic stemcell transplantation (hsct) in a study to evaluate the clinical outcomes associated with respiratory virus detection before hsct [ ] . during a -year period, asymptomatic adult and pediatric patients were screened, but only one patient with rsv and none with influenza virus were identified. interestingly, this study found other viruses (e.g., human rhinovirus or coronavirus) in patients. these respiratory viruses were not included in our screening program, as they rarely cause severe lower respiratory tract disease. out of asymptomatic patients who tested positive for rsv or influenza virus in our study occupied shared patient rooms at the time of virus detection. the majority were moved to single rooms (one-bed rooms) or cohorting was used. droplet and contact precautions were used for all affected patients. isolation and barrier precautions regarding virus shedders is important, as transmission to roommates has been described [ ] . the transfer to single (one-bed) rooms was challenging in some cases, due to the lack of single-room availability. nonetheless, because of the high mortality rate of rsv and influenza infection in hemato-oncological patients, isolation and barrier precautions were enforced. with these measures, we aimed to lower the risk of patient-to-patient transmission by reducing the exposure of contact patients. the usage of droplet and contact precautions (such as surgical mask and gown) was also intended to reduce exposure of hcws. all hcws, whether vaccinated against influenza or not, wore masks when they performed care in patients with rsv or influenza virus. a further benefit of early detection of influenza virus is the possibility of early antiviral therapy for infected patients and prophylaxis with antiviral agents for contact patients [ ] . an additional effect of the screening program and the associated audits and feedback talks was the heightened awareness among hcws and visitors for respiratory viral infections in this highly susceptible, immunocompromised patient population. during the screening period, no nosocomial cluster of influenza virus or rsv was observed on the two wards. our investigation has limitations. first, it relied on virological laboratory data analyzed retrospectively. further information, such as the clinical course and outcome of the patients identified by screening, was not accessible for this study. we therefore could not correlate the laboratory results with the clinical course of the patients. viral loads were estimated semi-quantitatively by comparing ct values of real-time pcr between positive screening and follow-up specimens. however, this approach has been established for respiratory specimens by previous studies on influenza and various other pathogens [ ] , [ ] , [ ] , [ ] . multifaceted approaches are necessary to help to control the nosocomial spread of rsv and influenza in healthcare settings. infection control is based on standard precautions (e.g., compliance with hand hygiene, cough etiquette, etc.) together with rsv and influenza pathogenspecific precautionary measures (e.g. droplet precautions, isolation). in special high-risk settings such as hematology and oncology units, additional measures can be useful to lower the risk of hospital acquisition of rsv and influenza. therefore, we implemented a prophylactic rt-pcrbased rsv and influenza screening program targeting asymptomatic patients. this seasonal screening program enabled us to identify patients who lacked symptoms for respiratory disease, but were infected with either rsv or influenza during a period of about months. for these patients the same infection control practices were implemented as for symptomatic patients with rsv and influ-enza infection. in our view, this screening program proved useful for identifying asymptomatically infected patients with viral shedding, thus reducing the risk of transmission and potential nosocomial clusters of rsv and influenza virus on hemato-oncological wards. nevertheless, future studies on larger cohorts, including the analyses of clinical data, are necessary to further validate the use of a seasonal screening program as suggested here. competing interests respiratory syncytial virus -a comprehensive review systematic review of respiratory viral pathogens identified in adults with community-acquired pneumonia in europe community acquired respiratory virus infections in cancer patients -guideline on diagnosis and management by the infectious diseases working party of the german society for haematology and medical oncology respiratory viruses in transplant recipients: more than just a cold. clinical syndromes and infection prevention principles influenza virus infections in patients with malignancies --characteristics and outcome of the season / . a survey conducted by the infectious diseases working party (agiho) of the german society of haematology and medical oncology (dgho) consecutive yearly outbreaks of respiratory syncytial virus in a haemato-oncology ward and efficacy of infection control measures nosocomial outbreak of the pandemic influenza a (h n ) in critical hematologic patients during seasonal influenza - : detection of oseltamivir resistant variant viruses what transmission precautions best control influenza spread in a hospital? fourth european conference on infections in leukaemia (ecil- ): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus effect of two-step hygiene management on the prevention of nosocomial influenza in a season with high influenza activity molecular characteristics and successful management of a respiratory syncytial virus outbreak among pediatric patients with hemato-oncological disease diagnostic approach for the differentiation of the pandemic influenza a(h n )v virus from recent human influenza viruses by real-time pcr combining highresolution contact data with virological data to investigate influenza transmission in a tertiary care hospital active surveillance for influenza reduces but does not eliminate hospital exposure to patients with influenza laboratory-based surveillance of hospital-acquired respiratory virus infection in a tertiary care hospital practical prevention of nosocomial influenza transmission, "a hierarchical control" issue the natural history of influenza infection in the severely immunocompromised vs nonimmunocompromised hosts influenza virus prevalence in asymptomatic and symptomatic subjects during pandemic and postpandemic periods respiratory syncytial virus evaluation among asymptomatic and symptomatic subjects in a university hospital in sao paulo, brazil in the period of - . influenza other respi viruses clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant comparison of simplexa hsv & pcr with culture, immunofluorescence, and laboratorydeveloped taqman pcr for detection of herpes simplex virus in swab specimens a comparison of nasopharyngeal and oropharyngeal swabbing for the detection of influenza virus by real-time pcr assessment of the quantitative ability of advansure tb/ntm real-time pcr in respiratory specimens by comparison with phenotypic methods comparison of real-time pcr assays for diagnosis of pneumocystis jirovecii pneumonia in human immunodeficiency virus (hiv) and non-hiv immunocompromised patients influenza and respiratory syncytial virus screening for the detection of asymptomatically infected patients in hematology and oncology the authors declare that they have no competing interests. we obtained ethical approval for this study from the ethics committee of the hannover medical school (number - ). this research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. we acknowledge the technical laboratory staff for the processing of screening specimens. all authors contributed to the manuscript according to the icmje (international committee of medical journal editors) recommendations: ah and cb were responsible for data acquisition. all authors were involved in analysis and interpretation of the data. tg and ah supervised laboratory diagnostics. cb and fcb prepared the manuscript. cb organized the drafting process. cl, ab, mh and ft implemented and supervised the screening program on the wards. all authors critically revised the manuscript and are accountable for accuracy and correctness. all authors have read and agreed to the final draft before submission. key: cord- -sqetz h authors: hou, yixuan; meulia, tea; gao, xiang; saif, linda j.; wang, qiuhong title: deletion of both the tyrosine-based endocytosis signal and the endoplasmic reticulum retrieval signal in the cytoplasmic tail of spike protein attenuates porcine epidemic diarrhea virus in pigs date: - - journal: journal of virology doi: . /jvi. - sha: doc_id: cord_uid: sqetz h porcine epidemic diarrhea virus (pedv) causes high mortality in neonatal piglets. the pedv spike (s) protein contains two intracellular sorting motifs, yxxΦ (tyrosine-based motif yevf or yeaf) and kvhvq at the cytoplasmic tail, yet their functions have not been fully elucidated. some vero cell-adapted and/or attenuated pedv variants contain ablations in these two motifs. we hypothesized that these motifs contribute to viral pathogenicity. by transiently expressing pedv s proteins with mutations in the motifs, we confirmed that the motif kvhvq is involved in retention of the s proteins in the endoplasmic reticulum (er)-golgi intermediate compartment (ergic). in addition, we showed that the yxxΦ motif triggers endocytosis of s proteins. these two motifs synergistically regulate the level of s expressed on the cell surface. to investigate their role in viral pathogenicity, we generated three recombinant pedvs by introducing deletions or a mutation in the two motifs of the infectious clone of pedv pc a strain (icpc a): (i) icΔ aa (ΔyxxΦekvhvq), (ii) icΔ aa (Δkvhvq), and (iii) icya (y a, to an inactivated motif, aevf). infection of vero cells with icΔ aa resulted in larger syncytia and more virions, with reduced numbers of s protein projections on the surface compared with icpc a. furthermore, we orally inoculated five groups of -day-old gnotobiotic piglets with the three mutants, icpc a, or a mock treatment. mutant icΔ aa caused less severe diarrhea rate and significantly milder intestinal lesions than icpc a, icΔ aa, and icya. these data suggest that the deletion of both motifs can reduce the virulence of pedv in piglets. importance many coronaviruses (covs) possess conserved motifs yxxΦ and/or kxhxx/kkxx in the cytoplasmic tail of the s protein. the kxhxx/kkxx motif has been identified as the er retrieval signal, but the function of the yxxΦ motif in the intracellular sorting of cov s proteins remains controversial. in this study, we showed that the yxxΦ of pedv s protein is an endocytosis signal. furthermore, using reverse genetics technology, we evaluated its role in pedv pathogenicity in neonatal piglets. our results explain one attenuation mechanism of vero cell-adapted pedv variants lacking functional yxxΦ and kvhvq motifs. knowledge from this study may aid in the design of efficacious live attenuated vaccines against pedv, as well as other covs bearing the same motif in their s protein. taxonomically, pedv is a member of the alphacoronavirus genus within the coronaviridae family. the mature pedv virion consists of four structural proteins: spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. as the major glycoprotein on the pedv envelope, s proteins form trimers, which appear as projections on the surface of a virion using an electron microscope, and bind to cellular receptors and mediate virus-host membrane fusion. proteolytic cleavage of s proteins expressed on the cell surface triggers syncytium formation ( , ) . like those of other coronaviruses (covs), pedv virions assemble at the endoplasmic reticulum (er)-golgi intermediate compartments (ergic) ( ) ( ) ( ) . the amounts of pedv s proteins in the ergic, in other organelles, or on the cell surface are likely regulated by two nearby motifs in its cytoplasmic tail (ct): a tyrosine-based motif, yxx⌽ (x is any residue and ⌽ is a bulky hydrophobic residue: f, m, i, l, or v), and an er retrieval signal (errs), kvhvq ( ) ( ) ( ) ( ) , as well as other viral and cellular proteins. the cov errs, either in the dilysine or the dibasic form (kxkxx, kkxx, or kxhxx), is a weak ergic retention signal ( , ) . it interacts with coatomer complex i (copi), a cellular protein involved in cargo transportation from the golgi to er, and prevents large amounts of the s proteins from being transported to the cell surface through the canonical secretory pathway ( , ) . in addition, the errs in the s protein of severe acute respiratory syndrome cov (sars-cov) promotes the interaction between s and m proteins in the golgi region ( ) . inactivation of the errs in the sars-cov s protein impaired its incorporation into virus-like particles when coexpressed with the m in the cells ( ) . for pedv, the amino acid sequence of the errs is kvhvq, which is highly conserved among different genotypes. one study demonstrated that a single amino acid substitution in this motif (kvhvq to kvrvq) weakens the intracellular retention function of the s proteins of the th passage of a murine-adapted pedv variant, mk-p ( ) , resulting in enhanced syncytium formation in vero cells. however, this impaired kvrvq motif does not alter the incorporation of s into the mk-p virions ( ) . although the yxx⌽ motif is a well-studied, clathrin-dependent endocytosis signal among numerous viral and host cellular transmembrane proteins ( ) ( ) ( ) ( ) ( ) ( ) ( ) , its function in cov s proteins has not been fully understood. most s proteins of alphacoronaviruses, such as transmissible gastroenteritis virus (tgev), and gammacoronaviruses, such as infectious bronchitis virus (ibv), contain this motif in their cts (fig. a) . a previous study demonstrated that the yxx⌽ motif is responsible for intracellular retention but not endocytosis of tgev s proteins into cells ( ) . interestingly, this retention signal could be transformed into a strong endocytosis signal when the first lysine residue of the tgev kxhxx motif was replaced by methionine (yepiekvhvh to yepiemvhvh; change is in bold type and yxx⌽ and kxhxx motifs are underlined) ( ) . for ibv, the role of the yxx⌽ motif in s protein intracellular transport is controversial. one group reported that this motif is not an endocytosis signal ( ) . the other group demonstrated that the yxx⌽ motif functioned as an endocytosis signal in a chimeric protein composed of the vesicular stomatitis virus (vsv) g protein ectodomain and the ct of the ibv s protein ( ) . it has also been demonstrated that a recombinant ibv harboring a mutated yxx⌽ (yttf to ytta) in the ct of the s protein could not be recovered in cell culture, suggesting the essential role of this motif in the ibv life cycle. moreover, since the =end of the ibv s gene overlaps with the transcription regulatory sequence (trs) of accessory gene , mutagenesis of the ibv errs (kksv to aasv) impaired the synthesis of subgenomic rna (sgrna ), resulting in decreased expression of e proteins in the ibv-infected cells ( ) . previously, we generated an attenuated pedv strain by serially passaging a highly virulent strain, pc a, in vero cells. at the th (p ) and later passages, we identified a premature stop codon in the =end of the s gene ( ) . this stop codon truncates the s protein by amino acids (aa) (evfekvhvq) . interestingly, at least five pedv variants (fl , sm- , chm , avct , and knu- ) containing a premature terminated s gene have been reported (table ) ( ) ( ) ( ) ( ) . all these pedv variants are either cell culture attenuated or clinically mild in pigs, except for avct , whose pathogenicity is unknown. these variants share the same s truncation pattern as pc a-p : partially ablated yxx⌽ and completely deleted kvhvq. it is possible that the loss of kvhvq may enhance the surface s protein level and increase its ability to induce syncytia in cell culture. however, the function of the incomplete yxx⌽ motif in viral replication and pathogenicity is unknown. notably, two types of yxx⌽ motif exist in the cts of s proteins among pedv genotypes: classical strains (g a) and some highly virulent asian strains (g a) contain a yeaf motif, whereas highly virulent north american strains (g b), s insertion and deletion (indel) strains (g b), and some g a strains harbor a yevf motif. we hypothesized that the yxx⌽ motif of pedv, regardless of its amino acid sequence, is an endocytosis signal and determines viral virulence in pigs. in this study, we evaluated the phenotypes of transiently expressed s mutants containing mutations or deletions in these two motifs in mammalian cells and the virulence of three representative recombinant pedvs in gnotobiotic piglets. motifs yxx⌽ and kvhvq regulate the levels of pedv s proteins on the cell surface. to determine the roles of the two motifs in the intracellular sorting of s proteins, we constructed plasmids bearing a wild-type s gene (of pc a) or an s gene with different mutations in the cts ( fig. b and c) . also, the dispensable -aa region (residues to ) in domain of each s protein was replaced by fluorescent protein mcherry ( aa), as we demonstrated previously that this -aa region does not interfere with the formation of s protein trimers ( ) . wt and wt contain the yevf and yeaf motifs, respectively. using these two wild-type constructs as templates, we generated additional mcherry-tagged s mutants, including mutants bearing point mutations and the ct-truncated variants ( fig. ; table ). the Δ aa mutant lacks both motifs. the Δ aa and hd mutants contain the cts of pedv strains pc a-p and knu- , respectively. the Δ aa mutant bears the same ct as four s-truncated pedv variants, fl , sm- , avct , and chm . mk-p contains the identical ct to that of the pedv strain mk-p . to evaluate the difference in functions between yevf and yeaf, three pairs of mutants (ya and ya-a, Δ aa and Δ aa-a, and mk-p and mk-p -v) were generated. to confirm the role of the kvhvq motif in ergic retention, transiently transfected vero cells were fixed with methanol at h posttransfection (hpt) and stained with an antibody against ergic marker ergic- ( fig. a) . we observed distinct sorting patterns among the recombinant s proteins. quantification of the colocalization between the s protein and ergic signals was indicated as pearson's correlation coefficient (pcc) value ( , ) , by measuring to individual cells from each sample (fig. b) . the higher pcc values indicate the higher levels of colocalization between the two fluorescent signals. for the four s proteins (wt , wt , ya, and ya-a) with an intact kvhvq motif in the ct, most intracellular s proteins were concentrated at the ergic region, with significantly higher pcc values than for the other s mutants (p Ͻ . ). interestingly, only the six s proteins with an intact yxx⌽ motif in their cts (wt , wt , Δ aa, Δ aa-a, mk-p , and mk-p -v) formed clear puncta in the cells. to determine the steady-state levels of these s proteins, we quantified the fluorescent intensities of the mcherry signals in individual vero cells of each sample at hpt (fig. b) . no statistically significant difference (p Ͼ . ) was observed among samples, indicating that all the recombinant s proteins were expressed at similar levels. next, we measured the levels of s proteins on the cell surface using digital quantitative method and syncytium induction assay. the transfected cells were fixed with % formaldehyde without permeabilization, and the surface s proteins were stained with green fluorescence (fig. a) . total (both surface and intracellular) s proteins were observed for the red fluorescence. since each s protein produces equal intensity of red fluorescent signal, we quantified the levels of surface s proteins by measuring the ratios of gray values of the green fluorescent signal to mcherry signal and designated it the surface/total ratio. fifty individual cells in each sample were measured and are plotted in fig. c . wt did not differ statistically from wt . eight mutants (Δ aa, ya, ya-a, Δ aa, Δ aa, hd, mk-p , and mk-p -v) expressed higher levels of s proteins than the two wild-type s proteins on the cell surface, while Δ aa and Δ aa-a expressed significantly lower levels (p Ͻ . ). because proteolytic cleavage of surface s proteins triggers cell-cell fusion, a syncytium induction assay was performed by culturing the transfected cells in medium containing trypsin. the syncytia induced by the eight mutants (Δ aa, ya, ya-a, Δ aa, Δ aa, hd, mk-p , and mk-p -v) contained significantly more nuclei than those induced by the two wild types (wt and wt ), while almost no syncytia were induced by the Δ aa and Δ aa-a mutants ( fig. d and e). collectively, these data suggest that an intact kvhvq motif (wt , wt , ya, and ya-a) retains s proteins in the ergic, and the two motifs synergistically regulate the level of pedv s protein expressed on the cell surface via different mechanisms. the yxx⌽ motif is an endocytosis signal and regulates surface s protein internalization to early endosomes. to determine whether the yxx⌽ motif functions as an endocytosis signal in pedv s protein, we performed an antibody-antigen (gp -s protein) uptake assay. we evaluated the portions of the gp -bound surface s proteins (green) being endocytosed after incubation at °c. as controls, one set of cells were incubated at °c, whereby the cell endocytosis activities ceased. therefore, both the gp -bound s proteins (green) and the surface s proteins (red), which were labeled after incubation and fixation without permeabilization, were colocalized on the cell surface ( fig. ) . on the other hand, for the cells being incubated at °c, a large portion of the gp -bound s proteins (green) of wt , wt , mk-p , mk-p -v, Δ aa and Δ aa-a formed puncta in the cells and were not colocalized with the surface s proteins (red). this was further confirmed when we compared the pcc values between the gp -bound s protein signal and total surface s protein signal of each s protein at °c and °c (fig. a ). we found that the pcc values of the six s proteins (wt , wt , mk-p , mk-p -v, Δ aa, and Δ aa-a) at °c were significantly lower than those at °c, suggesting that partial gp -bound s proteins of those six samples were internalized from the cell surface after incubation at °c. additionally, we probed the total gp signals on the cell surface without permeabilization in the gp -s uptake assay (fig. b ). we measured the ratios of surface s (gp ) signal to total s (mcherry) signal in the transfected cells incubated at °c and °c. wt , wt , and the mk-p , mk-p -v, Δ aa, and Δ aa-a mutants had significantly lower ratios at °c than at °c. in contrast, the other six mutants (Δ aa, ya, ya-a, Δ aa, Δ aa, and hd) had similar pcc values (fig. a ) and surface/total ratios (fig. b ) at °c and °c. these results indicate that the s mutants without an intact yxx⌽ motif were defective in endocytosis. endocytotic vesicles deliver cell surface proteins to early endosomes in the cytoplasm ( ) . to determine whether the internalized s puncta of wt , wt , mk-p , mk-p -v, Δ aa, and Δ aa-a ( fig. a and fig. ) were located in early endosomes, we performed the antibody-antigen (gp -s protein) uptake assay (fig. ). after performing fixation and permeabilization of cells, we immunostained the gp -bound s proteins (green) and the early endosome marker rab proteins (red). the gp -bound s protein signals in the cells incubated at °c showed a significantly higher percentage ). collectively, these data suggest that an intact yxx⌽ motif in the ct is necessary for endocytosis of the s protein from the cell surface to early endosome. pig anti-s antiserum gp at °c for min. after washing with pbs three times, one set of cells was cultured at °c for min to allow endocytosis. another set of cells was kept at °c for min as the control group. cells were fixed with % formaldehyde without permeabilization. surface s proteins were stained with mouse anti-s monoclonal antibody sd - and goat anti-mouse af -conjugated secondary antibodies (red). then the cells were permeabilized with triton x- and stained with goat anti-guinea pig af -conjugated secondary antibodies (green). nuclei were stained with dapi (blue). cells were randomly selected, and images were taken by using a leica tcs sp confocal microscope. scale bar: m. virulence of the recombinant pedv lacking both the yxx⌽ and kvhvq motifs was reduced in neonatal gnotobiotic pigs. to investigate the function of the two motifs in pedv replication and pathogenesis, we generated three recombinant pedvs by introducing the Δ aa, Δ aa, and ya mutations into the ct of the pedv infectious cdna clone of strain pc a (icpc a) ( ) . we inoculated -day-old gnotobiotic piglets with the three mutant pedvs, icpc a, and phosphate-buffered saline (pbs; mock treatment) to determine the role of the two motifs in viral pathogenicity. clinical signs fig. . to stain the gp bound on the cell surface, cells were fixed with % formaldehyde without permeabilization. gp was probed using af -conjugated secondary antibodies (green). the fluorescent intensities of surface (af ) to total s (mcherry) ratios were measured in to individual cells. values are plotted in a bar chart, and shown as means Ϯ sds. values of the same sample cultured at °c and °c were analyzed by student's t test. ns, p Ͼ . ; **, p Ͻ . ; ***, p Ͻ . ; ****, p Ͻ . . deletion of yxx and kvhvq from s attenuates pedv journal of virology internalized s proteins colocalized with early endosomes. antibody-s protein uptake assays were performed at hpt for the two wild-type s proteins (wt and wt ) and the four mutants, Δ aa, Δ aa-a, mk-p , and mk-p -v, that formed puncta as shown in fig. . cells were incubated with guinea pig anti-s antiserum gp at °c for min. after washing with pbs three times, one set of cells were cultured at °c for min to allow endocytosis and another set of cells were incubated at °c for min. after fixation with methanol, early endosome marker rab proteins were stained with rabbit anti-rab mab (c b ) followed by af -conjugated goat anti-rabbit antibody (red). gp -bound s proteins were stained with goat anti-guinea pig af -conjugated secondary antibodies (green a ). the villous atrophy in the duodenum, jejunum, and ileum was quantified by measuring villus height to crypt depth (vh/cd) ratios of each piglet (fig. d to f). a lower vh/cd value indicates more severe atrophy in the intestines. compared with the virulent icpc a group, the lesions of icΔ aa-infected pigs were the least severe in all three small intestinal regions (duodenum, jejunum, and ileum). although there were no statistically significant differences in duodenum and ileum, the lesions in the jejunum of icya-infected piglets were significantly milder than those of the icpc a-and icΔ aa-infected pigs. we did not observe significant differences in clinical signs, fecal viral shedding titers, or vh/cd ratios between the icΔ aa and icpc a groups. we concluded that the virulence of icΔ aa was milder than that of icpc a, and the icΔ aa maintained virulence similar to that of icpc a. these data suggest that deletion of both the yxx⌽ and kvhvq motifs from the s protein significantly impaired the pathogenicity of the highly virulent pedv icpc a. the yxx⌽ and kvhvq motifs are dispensable for pedv replication, but the lack of either impairs viral replication efficiency in vitro. we measured the one-step (with a multiplicity of infection [moi] of ) growth kinetics of the viruses in vero cells (fig. a) . the three pedv mutants had significantly lower peak titers than icpc a at hpi. subcellular structures of the four recombinant pedv-infected vero cells at hpi were visualized by transmission electronic microscopy (tem) of cellular sections. like other covs, replication of pedv induces dramatic membrane rearrangements ( , ). we did not observe differences in the rearranged membrane structures among cells infected with the four recombinant pedvs. however, in the icpc a-infected cells, golgi vacuoles contained more mature virions, which are smaller particles with denser cores than immature virions ( ), than those in the icΔ aa-and icya-infected cells (fig. f and g) . the icΔ aa-infected cells contained intermediate amounts of mature virions in the vacuoles. trans-golgi vacuoles containing large numbers of mature virions were observed exclusively in icpc a-infected cells. this single-membrane structure contains viral particles prior to their egress. collectively, these data suggest that neither the yxx⌽ nor kvhvq motif is essential for pedv replication, but the absence of either motif impairs the generation of infectious pedv viral particles. recombinant pedvs lacking an intact yxx⌽ motif in the ct of the s protein formed larger syncytia than the wild-type virus. we measured multistep growth kinetics (moi ϭ . ) of the four recombinant pedv in vero cells (fig. b) . we found that the speed of viral replication varied among the four recombinant viruses and the infectious titers gradually decreased after the majority of cells were lysed due to the formation of syncytia. in the growth curve, the peak titers of icΔ aa and icya occurred earlier but were lower than those of icpc a and icΔ aa. replication of icΔ aa in vero cells showed a peak titer similar to that of icpc a, but with a delay. we performed plaque assays and measured the kinetics of the plaque formation of the four viruses ( fig. c and d) . at hpi, the icΔ aa virus had induced the largest plaques, followed by the icya and then by the parental icpc a, whereas the icΔ aa had formed the smallest plaques. we stained the surface s proteins of infected cells at an early time point ( hpi) (fig. e) . the fluorescent signals represented s proteins anchoring either on cell membrane or on the egressing virions on the surface. the s proteins of icpc a-infected cells clustered mainly in a small area on the cell surface, presumably virions clustering at the egress sites, in contrast to the evenly distributed surface s proteins of icΔ aa and icya on the entire cytoplasmic membrane, presumably s proteins anchoring directly on the cell membrane. the surface s proteins of icΔ aainfected cells were distributed in a pattern similar to that of icpc a but showed less intense staining. these data suggest that the yxx⌽ motif decreases the amount of s proteins on the cell surface in pedv-infected cells. to determine whether the attenuation of icΔ aa in vivo was due to the amount of s proteins incorporated into virions, we visualized the s proteins on the purified virion surface using tem. the culture and purification of the four recombinant pedvs were performed simultaneously to minimize the variations due to sample preparation. we observed virions with or without s protein projections on virions in all four samples (fig. a) . the virions without s projections could be those that lack enough s proteins on the surface or whose s projections were lost during sample preparation for tem. the icΔ aa pedv contained the lowest percentage of virions with s protein projections, followed by icya and then by icpc a and icΔ aa. next, we quantified the number of s projections on the individual virions of each pedv sample. among the four mutants, the icΔ aa pedv contained significantly fewer s projections than icpc a, icΔ aa, and icya (p Ͻ . ). these data suggest that the deletion of both the yxx⌽ and kvhvq motifs from the s protein impairs the incorporation of s proteins into pedv virions. previous studies showed that the m protein retained the s protein of sars-cov in the golgi regions when they were coexpressed in the same cell ( , ) . to characterize whether the s proteins of the four recombinant pedvs can still interact with m, we cloned the pedv m gene into the vector puc -sgrna. the s and m proteins were coexpressed in vero cells (fig. a ) or the s protein alone was expressed. when the s and m proteins were coexpressed in cells, we observed that they colocalized, with equal pcc values ( fig. b and c) . we stained the surface s proteins of the cells cotransfected with the s and m plasmids or transfected with the s plasmid alone and measured the ratios of fluorescent intensities of surface s to total s. we found that the expression of m significantly decreased the surface s protein levels in the same cell (fig. c) , as evident by the significantly lower surface s to total s ratios in the m and s plasmidcotransfected cells compared with those in the cells transfected with only s plasmid. in this study, we determined that the yxx⌽ and kvhvq motifs at the ct of pedv s proteins are dispensable for pedv replication. however, the deletion of both motifs reduced the viral virulence in vivo and the incorporation of s proteins into virus journal of virology particles in vitro. the yxx⌽ motif and the kvhvq motif function as an endocytosis signal and an er retrieval signal, respectively. yxx⌽ and kvhvq together regulate surface s protein levels and s proteininduced syncytium formation. the number of nuclei in a pedv s protein-induced syncytium, or the size of a syncytium, correlates with the amount of s proteins on the cell surface ( ) . collectively, our data support the hypothesis that both motifs are involved in the regulation of pedv s proteins on the cell surface: the kvhvq motif retains the s proteins in the ergic, limiting its transport to the cell surface, whereas the yxx⌽ motif decreases the amount of surface s proteins via endocytosis. the mutants Δ aa, Δ aa, Δ aa, and hd, which lack the complete kvhvq motif and complete/partial yxx⌽ motif, had the highest level of surface s proteins ( fig. a and c) , probably due to the lack of both er retrieval and endocytosis. the mutants ya, ya-a, mk-p , and mk-p -v had the second highest level of cell surface s proteins. for the ya and ya-a, this is probably due to the inactivation of the endocytosis signal, while their errs remained intact. for mk-p and mk-p -v, it may be due to the impaired errs (kvrvq; r is the mutation that leads to the impaired errs) while their yxx⌽ motif was intact. surprisingly, the mutants Δ aa and Δ aa-a had significantly lower levels of surface s proteins than wt and wt . the lack of the entire kvhvq motif resulted in significantly lower levels of ergic colocalization of Δ aa and Δ aa-a than of wt and wt (fig. ) , confirming that the kvhvq motif functions as an errs in pedv as described previously ( , ) . why, then, did we not observe an increased level of surface s proteins for these two mutants? we hypothesized that the loss of the entire kvhvq motif in the ct of the s protein altered the intracellular sorting of s proteins. one possibility could be that the potency of their yxx⌽ motifs, as the endocytosis signal, was dramatically enhanced due to the lack of the entire errs motif. a similar endocytosis enhancement effect was reported for the yxx⌽ motif of tgev when its kxhxx motif's conformation was changed due to the k-to-m mutation ( ) . another possibility could be that the deletion of the entire kvhvq motif may cause other deficiencies in s protein sorting, such as impaired export from the er or altered transportation to other organelles. detailed mechanistic studies regarding the sorting of Δ aa and Δ aa-a s proteins are necessary to answer this question. the endocytosis role of the yxx⌽ motif in pedv s intracellular sorting. the conserved location of the yxx⌽ and kxhxx/kkxx motifs in the ct implies that the s protein sorting mechanisms may be similar among different covs (fig. a) . however, previous studies on the yxx⌽ motif of tgev and ibv s proteins did not provide consistent conclusions ( , , , ) . one reason could be that the expression efficiencies of vectors varied in those studies. due to the small amount of s proteins expressed on the cell surface, the endocytosed s proteins might be too subtle to be detected in the antibody-antigen uptake assay. in our study, the plasmid puc -sgrna overexpressed pedv s proteins in vero cells compared to some commercially available vectors (data not shown), probably due to the inclusion of the =leader sequence of subgenomic rna (sgrna) and pedv =untranslated region (utr) that enhanced the translation. although we determined that both yevf and yeaf are endocytosis signals of pedv s proteins, it is possible that their efficiencies may vary. in the future, more advanced quantitative approaches, such as mechanically induced trapping of molecular interactions (mitomi) ( ) , could be used to determine their affinities for binding to cellular factors. additionally, one limitation of this study is that we did not applied approaches to monitor the kinetic trafficking of the endocytosis of s proteins from the cell surface. future studies to address these points are necessary. in this study, we did not detect the colocalization of endocytosed s protein puncta and lysosomal marker lamp in wt -, wt -, Δ aa-, Δ aa-a, mk-p -and mk-p -v-puc -sgrna transfected cells (data not shown). also, the total s protein levels (mcherry signals) of those mutants did not decrease during up to days' culture. we hypothesize that the internalized pedv s proteins are transported back to the virion assembly sites rather than being degraded in the lysosome. for pedv, this endocytosis may be a strategy to efficiently use the expressed s proteins for the generation of infectious viral particles. this hypothesis may be supported by the quantitative data on s proteins incorporated into virions (fig. ) , because the icΔ aa pedv with an intact yxx⌽ motif had a number of s projections similar to that of icpc a. this endocytosis signal yxx⌽ motif may also help pedv evade host immune responses by constantly decreasing the amounts of viral s proteins on the cell surface. previous studies reported that inactivation of the yxx⌽ motif in the envelope proteins of simian immunodeficiency virus (siv) and human immunodeficiency virus (hiv) attenuated the corresponding virus and enhanced the t cell responses in vivo ( , ) . moreover, host antibodies that have already bound to cell surface s proteins would be endocytosed into the cytoplasm, to evade antibody-dependent cell lysis ( , ) . another possible function of this yxx⌽ motif in pedv s protein is that it could downregulate the levels of viral receptors on the cell surface, which may be involved in superinfection exclusion. the role of the yxx⌽ and kvhvq motifs in pedv pathogenicity. in the gnotobiotic piglet study, we observed that the virulence of icΔ aa was significantly reduced compared with that of the parental virus, icpc a. in contrast, no statistically significant differences were observed in the virulence between icpc a and the icΔ aa mutant, which lacks the kvhvq motif. mutant icya caused significantly milder villous atrophy in the jejunum than icpc a. although not significant, it caused a delayed onset of diarrhea and lower peak fecal shedding titers than did icpc a. therefore, icya pedv with an inactivated yxx⌽ motif was slightly attenuated. we investigated why the deletion of both motifs from the s protein (icΔ aa), but not the deletion of errs alone (icΔ aa), significantly attenuates a virulent pedv. tem images showed that icΔ aa-infected vero cells contained significantly higher numbers of mature virions in the golgi vacuoles than those infected with icΔ aa ( fig. f and g) . particularly, icΔ aa had a significantly lower percentage of purified virions with s projections on the surface and lower numbers of s projections on individual virions with surface s projections than the other three viruses (fig. ) . therefore, icΔ aa may generate the highest level of defective virions (without enough s protein projections on the viral surface) among the four pedvs, causing attenuation in vivo. the difference in s incorporation into viral particles among the pedv mutants may be due to three reasons. first, the lack of both sorting signals renders more icΔ aa s proteins to be transported onto the cell surface, decreasing its level in the virus assembly site, ergic. second, the intense and rapid cell-cell fusion induced by surface s proteins in the infected cells causes significant intracellular membrane rearrangements ( ) , which may interfere with the icΔ aa budding and egress process. in contrast, icΔ aa does not cause significant syncytium formation. the third reason may be the altered interaction between s and m proteins in the virion assembly site. although some previous studies revealed that mutation of the errs (klhyt) of sars-cov s protein altered the s-m interaction and s incorporation into virions ( , ) , another study suggested that inactivation of this motif in the s of ibv did not affect the s-m interaction ( ) . for pedv, a previous study of mk-p also showed that the impaired kvrvq motif did not alter the s incorporation ( ) , which is similar to our observation for icΔ aa (fig. ). in our s and m coexpression assay, we found that the four s proteins colocalized with the m proteins in the cytoplasm at the same level, and the coexpression of m protein significantly decreased the transportation of the four s proteins onto the cell surface, implying that those four s mutants may still interact properly with m proteins, although more experiments to evaluate s-m physical interactions are needed. our current data support the first and second possibilities. another reason for the attenuation of icΔ aa pedv in pigs is that mutation of pedv yxx⌽ may also interfere with the synthesis of orf sgrna, since the predicted trs of open reading frame (orf ) sgrna overlaps with the nucleotide sequences encoding the tyrosine of the yxx⌽ motif ( ) . it was reported that a recombinant ibv bearing mutations in its dilysine motif (kkxx) expressed less e protein, due to the decrease of sgrna synthesis ( ) . in this study, the sgrna levels in the icΔ aa-and icya-infected cells were about log and log lower than that in the icpc a-infected cells, as determined by sgrna -specific taqman real-time reverse transcription-qpcr (rt-qpcr) (data not shown). sgrna expresses the accessory protein orf . we do not know whether the e gene in the sgrna can be translated during pedv infection. therefore, it is highly possible that the orf protein level was decreased in the icΔ aa-and icya-infected cells. however, due to the lack of effective antibodies against pedv orf and e proteins, we could not confirm these results at the protein level. the decrease of orf expression may also contribute to the significant attenuation of icΔ aa and the slight attenuation of icya in piglets because we determined previously that a pedv lacking an intact orf gene is partially attenuated in piglets ( ) . the role of s ct truncation in vero cell-adapted pedvs and pedv vaccine development. in our study, the deletion of both the yxx⌽ and kvhvq motifs from the s protein could not completely attenuate a highly virulent pedv strain in piglets, as icΔ aa still caused moderate diarrhea in all the piglets (n ϭ ). therefore, the ablation of the two motifs was probably not the only determinant of the reduced virulence for those attenuated/mild pedv variants (fl , sm- , chm , pc a-p , and knu- ). the attenuation of these strains must be the combined consequence of all the mutations in their genomes. we investigated why some vero cell-adapted pedv variants (pc a-p , sm- , and knu- ) lost function of the yxx⌽ and kvhvq motifs during continuous passaging. we found that the corresponding mcherry-tagged s proteins (Δ aa, Δ aa, and hd) caused larger syncytia than the wild-type s proteins. the loss of the two motifs may facilitate the rapid spread of those pedv variants between cells through enhanced syncytium formation. this idea was supported by a study of the mutant avct . jengarn et al. ( ) demonstrated that recombinant avct was rescued in vero cells only if the infectious cdna clone contained the Δ aa mutation in the s ct. we conclude that inactivation of both the yxx⌽ and kvhvq motifs of the s protein could be applied in the rational design of a pedv vaccine since it results in more s proteins being transported onto cell surface in the infected enterocytes, facilitating antigenic recognition by the host immune system, and may not decrease protective immunogenicity because no neutralizing epitope has been identified in the s ct of pedv. we aim to test this hypothesis in future studies. to achieve complete attenuation and to increase the safety of an attenuated vaccine, the inactivation of the two motifs should be combined with mutations in other key genes in the pedv genome. in conclusion, we showed that the yxx⌽ and kvhvq motifs in the ct of pedv s protein regulate the surface s protein level. the deletion of both motifs significantly reduced the virulence of pedv in pigs. our discovery can aid in the rational design of attenuated vaccines for pedv and other covs containing similar motifs in their s proteins. cells, reagents, and antibodies. vero cells (atcc ccl ) were cultured in dulbecco modified eagle medium (dmem; gibco, carlsbad, ca) supplemented with % fetal bovine serum (fbs) (atlanta biologicals, flowery branch, ga). the guinea pig polyclonal antibody (pab) gp against pedv s was generated in our laboratory as described below. pedv s subunit (aa to ) gene of vero cell-adapted pedv pc strain at passage level (genbank accession no. km ) was cloned into pfuse-higg -fc vector (invivogen, san diego, ca), expressed in hek- t cells, and purified using a protein a affinity column (pierce, waltham, ma). guinea pigs were first immunized with the purified s subunit mixed with freund's complete adjuvant via subcutaneous injection, followed by two booster injections of the same dose in freund's incomplete adjuvant with a -to -week interval between doses. the total igg antibodies in the hyperimmune serum were purified. the production of guinea pig serum was approved by the institutional animal care and use committee (iacuc) of the ohio state university. mouse monoclonal antibodies (mabs) sd - against pedv s (residues to ) ( ) and sd - against pedv n proteins were kindly provided by diego diel and eric nelson at south dakota state university, respectively. rabbit hyperimmune serum against pedv m proteins was kindly provided by ying fang at kansas state university. mouse mab against the ergic- (oti a ) was purchased from enzo life science (farmingdale, ny). rabbit mab against rab (c b ) were purchased from cell signaling technology (danvers, ma). alexa fluor (af )-or af -conjugated secondary goat antibodies against the igg antibodies of different species were all purchased from life technologies (gibco, carlsbad, ca). icΔ aa (n ϭ ), icya (n ϭ ), or pbs (mock treatment; n ϭ ). postinoculation, we observed clinical signs and collected rectal swabs every h. all the piglets were euthanized at hpi to evaluate histopathological lesions at the acute phase of infection. taqman rt-qpcr for the detection of fecal pedv n rna, plaque assay for the detection of fecal infectious pedv titers, and immunohistochemistry staining of pedv antigens using mouse mab against pedv n protein (sd - ) were performed as described previously ( , ) . this animal study was approved by the iacuc of the ohio state university. growth curves of infectious pedvs. the growth curves of pedv replication in vero cells were determined by inoculation of monolayers of cells with each of the four recombinant pedvs with an moi of . or . after h absorption, the inocula were removed and the cell monolayers were washed with pbs three times. maintenance medium containing g/ml of trypsin was added to the cell culture. the total viruses (intracellular and extracellular) in each sample were collected by freezing and thawing the culture at appropriate time points. the infectious titers of viral samples were titrated by plaque assays. plaque assays. plaque assays were performed as described previously ( ) . briefly, monolayers of vero cells were inoculated with appropriate dilutions of recombinant pedvs for h. then the inocula were removed and the cells were washed with pbs three times. the cells were overlaid with an agarose-mem (gibco, carlsbad, ca) mixture containing g/ml of trypsin. the diameters of individual plaques were measured by using an ix- fluorescence microscope (olympus, tokyo, japan) and its software. tem. transmission electron microscopy (tem) of pedv-infected cell sections was performed as described previously ( ) . to visualize the subcellular structure in tem, recombinant pedv-infected vero cells (moi ϭ ) were cultured in trypsin-free medium for h and fixed with fixative ( % glutaraldehyde, % paraformaldehyde in . m potassium phosphate buffer [ph . ]) for h. samples were embedded in agarose, dehydrated, and re-embedded in em bed resin (electron microscopy science, hartfield, pa). ultrathin sections of each sample were prepared and stained. to visualize s protein projections on the virions, vero cells were inoculated with the recombinant pedvs (moi ϭ ) in the presence of trypsin. without freezing and thawing, virions in -ml culture supernatants of each recombinant pedv were purified by using peg- (sigma-aldrich, st. louis, mo) precipitation, followed by % sucrose-cushion ultracentrifugation ( , ϫ g for h), according to a previous protocol ( ) . to maintain consistency, the four pedvs were cultured and prepared simultaneously. samples of subcellular section and purified virions were stained with % uranyl acetate prior to tem imaging. images were visualized using an h- tem (hitachi, japan). statistical analysis. the statistical analyses were performed using graphpad prism . . the data in fig. b , b to e, d to f, g, and b are shown in box-whisker plots. the outliers were the values / greater than the upper quartile or / smaller than the lower quartile ( ) . comparisons of means of groups in these figures and fig. a, d , b, and c were analyzed by one-way analysis of variance (anova) followed by tukey's multiple-comparison test. in the fig. a , b, b, and c, the two groups were analyzed by student's t test. a p value between each two groups less than . was considered significantly different. porcine epidemic diarrhea in china updated estimated economic welfare impacts of porcine epidemic diarrhea virus (pedv) assessment of the economic impacts of porcine epidemic diarrhea virus in the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture mutation in the cytoplasmic retrieval signal of porcine epidemic diarrhea virus spike (s) protein is responsible for enhanced fusion activity coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding coronavirus structural proteins and virus assembly ultrastructural characterization of membrane rearrangements induced by porcine epidemic diarrhea virus infection incorporation of spike and membrane glycoproteins into coronavirus virions the spike protein of infectious bronchitis virus is retained intracellularly by a tyrosine motif contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection a novel sorting signal for intracellular localization is present in the s protein of a porcine coronavirus but absent from severe acute respiratory syndrome-associated coronavirus intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site the contribution of the cytoplasmic retrieval signal of severe acute respiratory syndrome coronavirus to intracellular accumulation of s proteins and incorporation of s protein into virus-like particles the cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds copi and promotes interaction with membrane protein molecular basis for recognition of dilysine trafficking motifs by copi mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo identification and targeting of an interaction between a tyrosine motif within hepatitis c virus core protein and ap m essential for viral assembly molecular architecture and functional model of the endocytic ap complex a structural explanation for the binding of endocytic dileucine motifs by the ap complex an immunoreceptor tyrosine-based inhibition motif in varicellazoster virus glycoprotein b regulates cell fusion and skin pathogenesis vzv gb endocytosis and golgi localization are mediated by yxx motifs in its cytoplasmic domain mutation of the dominant endocytosis motif in human immunodeficiency virus type gp can complement matrix mutations without increasing env incorporation endocytosis of the nipah virus glycoproteins a lysine-methionine exchange in a coronavirus surface protein transforms a retention motif into an endocytosis signal attenuation of an original us porcine epidemic diarrhea virus strain pc a via serial cell culture passage identification and pathogenicity of a variant porcine epidemic diarrhea virus field strain with reduced virulence genetic manipulation of porcine epidemic diarrhoea virus recovered from a full-length infectious cdna clone development of the full-length cdna clones of two porcine epidemic diarrhea disease virus isolates with different virulence genetic characteristics, pathogenicity, and immunogenicity associated with cell adaptation of a virulent genotype b porcine epidemic diarrhea virus deletion of a -amino-acid region in the n-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets dynamics of three-dimensional replication patterns during the s-phase, analysed by double labelling of dna and confocal microscopy a practical guide to evaluating colocalization in biological microscopy endosome maturation structural maturation of the transmissible gastroenteritis coronavirus in vivo attenuation of simian immunodeficiency virus by disruption of a tyrosine-dependent sorting signal in the envelope glycoprotein cytoplasmic tail enhancement of immunogenicity of an hiv env dna vaccine by mutation of the tyr-based endocytosis motif in the cytoplasmic domain porcine reproductive and respiratory syndrome virus-infected alveolar macrophages contain no detectable levels of viral proteins in their plasma membrane and are protected against antibody-dependent, complement-mediated cell lysis antibody-induced internalization of viral glycoproteins and ge-gi fc receptor activity protect pseudorabies virus-infected monocytes from efficient complement-mediated lysis syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus pedv leader sequence and junction sites characterization of a pathogenic full-length cdna clone and transmission model for porcine epidemic diarrhea virus strain pc a the s glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes engineering the largest rna virus genome as an infectious bacterial artificial chromosome measurement of colocalization of objects in dual-colour confocal images the gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses experimental infection of a us spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original us pedv infection pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene purification and electron cryomicroscopy of coronavirus particles some implementations of the boxplot complete genome sequence of novel porcine epidemic diarrhea virus strain gd- in china sequence of the spike protein of the porcine epidemic diarrhoea virus phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china this study was funded by the national institute of food and agriculture, u.s. department of agriculture, under award number - - (q.w., principal investigator [pi]; l.j.s., co-pi).we thank andrea kaszas and xiaohong wang for technical assistance and juliette hanson, ronna wood, megan strother, dennis hartzler, sara tallmadge, and jeff ogg for animal care assistance. we thank chang-won lee and scott kenney for their critical reviews of the manuscript. we are grateful to diego diel and eric nelson at south dakota state university for providing us the mouse mabs against pedv s and n proteins and to ying fang at kansas state university for providing us the rabbit pab against pedv m proteins. key: cord- - gzont l authors: guo, nan; zhang, bingzhou; hu, han; ye, shiyi; chen, fangzhou; li, zhonghua; chen, pin; wang, chunmei; he, qigai title: caerin . suppresses the growth of porcine epidemic diarrhea virus in vitro via direct binding to the virus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gzont l porcine epidemic diarrhea (ped) has re-emerged in recent years and has already caused huge economic losses to the porcine industry all over the world. therefore, it is urgent for us to find out efficient ways to prevent and control this disease. in this study, the antiviral activity of a cationic amphibian antimicrobial peptide caerin . against porcine epidemic diarrhea virus (pedv) was evaluated by an in vitro system using vero cells. we found that even at a very low concentration, caerin . has the ability to destroy the integrity of the virus particles to block the release of the viruses, resulting in a considerable decrease in pedv infections. in addition, caerin . showed powerful antiviral activity without interfering with the binding progress between pedv and the receptor of the cells, therefore, it could be used as a potential antiviral drug or as a microbicide compound for prevention and control of pedv. alphacoronavirus of the family coronaviridae with single-stranded positive-sense rna [ ] . it is the causative agent of an acute infectious enteric disease known as porcine epidemic diarrhea (ped) that is clinically manifested by severe watery diarrhea, vomiting, and dehydration in the suckling piglets [ ] . with the high mortality in the piglets, ped infection finally caused enormous economic losses to the global swine industry, especially after its recent re-emergence caused by variant pedv strain throughout the world [ , ] . however, the classical ped vaccines could not provide appropriate protection against the variant pedv infection. considering this, relevant studies of new antiviral materials are needed to prevent and control emerging or re-emerging infectious diseases such as ped [ ] . antimicrobial peptides (amps) are important components of the nonspecific immune system of animals to eradicate invaders [ ] , and the skin secretion of anuran amphibians are rich sources for collecting amps [ ] [ ] [ ] . amps have a wide spectrum of antimicrobial activity against microorganisms such as bacteria, viruses, fungi, and parasites [ , ] , but are very friendly to host cells [ ] [ ] [ ] . moreover, amps can work as growth and health promoters to improve the performance of pigs by enhancing the immune status, improving the intestinal health, and alleviating the toxic effects of deoxynivalenol in pigs [ ] . there are also research findings that amps can modulate immune responses like chemokines, cytokine production, and pro-inflammatory responses [ , ] . caerin . is a peptide from the granular glands within the skin of the australian green tree frog with -residues (gllsv lgsva khvlp hvvpv iaehlnh ). nuclear magnetic resonance (nmr) of caerin . in the membrane mimetic environments showed that it has two α-helices and a flexible hinge region composed of two prolines [ ] . both prolines are essential for the antimicrobial activities [ ] . some studies have reported that caerin . exhibits antibacterial and antiviral properties by forming pores on the membrane to destroy the integrity of the particles [ , ] . meanwhile, it has been confirmed that caerin . has a complete inhibitory effect against hiv by preventing viral fusion to target cells and disrupting the hiv envelope, and, remarkably, that caerin . is also highly effective in inhibiting the transfer of hiv from dendritic cells (dcs) to t cells even when dcs are continuously exposed to peptides for h after virus capture, and that caerin . has a bacteriostatic activity against escherichia coli and bacillus subtilis [ , ] . although the mechanism that suppresses the activities of some bacteria and viruses has been illustrated clearly, it remains unknown whether caerin . can inhibit the growth of pedv. so, this study is aimed to investigate the function of caerin . . for this purpose, we investigated the inhibitory ability and antiviral mechanisms of caerin . against different pedv strains in vero cells, which will provide an insight into amps' antiviral mechanisms and its application as antiviral drugs or as drug loading compounds. vero cells (african green monkey kidney cell lines) were propagated at • c in a % co humidified incubator using dulbecco's modified eagle medium ((dmem), gibco, langley, va, usa) containing % fetal bovine serum (invitrogen, carlsbad, ca, usa). cells were infected with three different pedv strains: yn (accession no. kf ), cv (accession no. kt ), and dr (accession no. jq ). the yn strain ch/ynkm- / was isolated from a suckling piglet suffering from acute diarrhea. the cells infected with pedv strains were cultured in dmem supplemented with µg/ml trypsin. caerin . and n-terminus fitc labeled caerin . (purity: both > %) were chemically synthesized by bioyeargene (wuhan, china). caerin . was initially dissolved in . % acetic acid to reach the concentrations of mg/ml and mg/ml as stock solutions, respectively and stored at − • c until further use [ ] . the cytotoxicity of caerin . in vero cells grown in -well culture plates was assessed by mtt ( -( , -dimethyl- -yl)- , -diphenyltetrazolium bromide, mg/ml) assay. the cells were incubated with caerin . at different concentrations for h, then µl/well mtt was added and cultured for additional h. the supernatant was then removed and dimethylsulfoxide (dmso) was added to culture plates ( µl per well), then the plates were shaken at room temperature (rt) for min. finally, the optical density (od) value was measured at the wavelength of nm [ ] . the plaque forming assay was performed on vero cells cultured in -well plates. pedv ( pfu/well) was incubated using different concentrations of caerin . for h at • c before infection and then incubated pedv was added on vero cells covering % monolayer. after h of viral adsorption, the supernatant was removed, and the cells were rinsed with pbs and overlaid with µg/ml trypsin supplemented with sodium carboxymethyl cellulose-containing medium. the plates were fixed with % formaldehyde after h infection, and then stained with crystal violet solution [ ] . vero cells cultured in -well plates were washed with pbs for times and inoculated respectively with single medium, or single pedv, or pedv pre-incubated with different concentrations of caerin . . the cells were rinsed with pbs after h infection, the cells were fixed with % formaldehyde for min at rt. then the cells were incubated with an anti-pedv monoclonal antibody (made in our laboratory) for h and fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse antibody ( : dilution) for h at • c. the immunofluorescence images were taken with a nikon eclipse ti microscope (nikon, tokyo, japan) [ ] . vero cells were cultured in -well plates and infected with pedv, and treated with caerin . at different time points. the cells were rinsed three times with pbs at h post infection (hpi), and treated with µl/well lysis solution containing protease inhibitors (pmsf). then µl of sodium dodecyl sulfate (sds) loading buffer was added to the cell extracts and the samples were boiled for min. the proteins were separated by % sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and then transferred into pvdf membranes (millipore, mississauga, on, canada). membranes were blocked with % skimmed milk for h at • c and then incubated with primary antibodies over night at • c. the blots were then incubated with corresponding horseradish peroxidase (hrp)-conjugated secondary antibodies (abclonal, wuhan, china). the membranes were washed times at each step. the protein bands were visualized using the clarity™ western ecl blotting substrate (bio-rad, hercules, ca, usa). the protein blots were quantified by image j software (national institutes of health, bethesda, md, usa). pedv-infected cell culture was prepared and centrifuged at • c for min at × g to get rid of the cell debris. the supernatants were ultracentrifuged at • c for h at , × g. then the purified virus pellets were re-suspended with pbs and treated with caerin . ( µg/ml) for h at • c and the viruses treated in the same manner were used as positive control. afterwards, the cocktail was dripped on the copper grid and negatively stained with phosphotungstic acid (pta). finally, the samples were examined using the electron microscope (hitachi h , tokyo, japan) [ ] . pedv suspensions containing different concentrations of caerin . were pre-incubated for h at • c, and were serially diluted before they were inoculated on the % confluent vero cell monolayers grown in the -well plates, followed by washing times with pbs. about hpi, the inoculates were removed and the cells were washed again with pbs for times and incubated for another days with dmem containing trypsin ( µg/ml). the infectivity was calculated by tcid (tissue culture infectious dose ) following the reed-muench method established by l. j. reed and h. muench [ ] . the vero cell monolayers were infected with pedv ( pfu). at hpi, the cells were washed twice with pbs and then treated with different concentrations of caerin . , and at hpi, the culture supernatants and the cells were collected respectively [ ] . the cell samples were frozen and thawed times to completely release the intracellular virus particles. both the extracellular and intracellular virus yields were determined by tcid . the vero cells in -well plates were inoculated with pedv in the presence or in the absence of caerin . at different time points: a. h before infection (pre); b. incubation of caerin . and pedv for h before infection (co); c. hpi (post). the supernatants and the cells were collected together at different time points and the samples were treated in the same way mentioned above in an infectious virus yield reduction assay. on the other hand, the inhibitory ability of caerin . against pedv was examined using a -fold higher concentration for further understanding antiviral activities in the pre and post treatment situations. finally, the cell lysates were collected in a parallel set for western blot assay. cells were pre-chilled at • c for h before infection and were chilled again after inoculated with pedv for h, then the cells were washed with ice cold pbs times, replenished with dmem containing trypsin, were incubated at • c for h, and the inhibitory effects were determined by tcid . caerin . was separately added at two time points: together with pedv at the time of viral absorption, and after the cells were washed at the time of viral entry to reach a highest final concentration of µg/ml. the cell lysates were processed in the same procedures with western blot analysis [ ] . vero cells were seeded onto some glass coverslips and were separately treated with fitc-caerin . ( µg/ml), pedv ( pfu), and both fitc-caerin . ( µg/ml) and pedv ( pfu), then incubated at • c for h. cells were fixed with % formaldehyde followed by permeabilization for min with . % triton x- , and were blocked with % bsa for h. the cells were washed times with pbs at each step. cell nuclei were counterstained with . % , -diamidino- -phenylindole ((dapi), invitrogen), and the antibodies used here were: pedv-s monoclonal antibody (made in our laboratory) and alexa fluor -conjugated affinity pure donkey anti-mouse igg (h+l) (ant gene, wuhan, china). the samples were examined using a confocal microscope (lsm meta, carl zeiss, munich, germany) [ ] . all experiments were performed with three independent experiments, and the calculated results were presented as mean ± standard deviation (sd). statistical analyses were performed using student's t-test. graph pad prism . was used to analyze the statistics in this study. the statistical significances were defined as p < . (*), and the higher significance was denoted by p < . (**) and p < . (***). the result of cytotoxicity assay indicated that cell viability increased with the decrease of the concentration of caerin . , that the viability of cells remained over % when treated with caerin . at the concentrations not exceeding µg/ml, and that the cell morphology was not affected even at the concentration µg/ml with a cell viability value of %, as seen in figure . thus, µg/ml caerin . was chosen as the highest final concentration in most of the subsequent experiments. we used µg/ml, µg/ml, µg/ml, and . µg/ml of caerin . in the ifa assay and in the addition assay. different incubating conditions of pedv in the presence of caerin . can directly affect the antiviral activity of caerin . . in this experiment, we determined the titer of pedv under different incubating temperatures and time durations. the results showed that the most suitable incubating temperature was °c, shown in figure a , and time length was h, shown in figure b . low temperature and inadequate incubation time would obviously affect the inhibition activity of caerin . against pedv. different incubating conditions of pedv in the presence of caerin . can directly affect the antiviral activity of caerin . . in this experiment, we determined the titer of pedv under different incubating temperatures and time durations. the results showed that the most suitable incubating temperature was °c, shown in figure a the results are presented as the mean ± sd of three independent experiments. the statistical analysis was performed using graph pad prism . . significance was defined as p < . (*), and higher significance was defined as p < . (**). to fully understand the inhibitory effect of caerin . against pedv-yn strain, several experiments were performed including cytopathic effects (cpe) observation, plaque reduction assay, tcid , and ifa. the cpe observation results showed that there were remarkable differences between the cells infected with pedv in the absence or presence of caerin . . caerin . -treated cells were in a relatively normal status, and the cpe appeared in caerin . -treated group about h later than in the pedv control group, as seen in figure a . with the addition of caerin . , the number of plaques declined at a surprising speed and the diameters decreased notably. similar results can be observed even with pfu of pedv treated by caerin . . the cells infected with pfu pedv exactly remained the same as the cells in negative control group, as seen in figure b . caerin . significantly inhibited the multiplication of pedv in a dose-dependent manner, demonstrating that pedv infection to vero cells was blocked to a large extent. the results of tcid , shown in figure c , and ifa, in figure d , indicated that the titer and the fluorescence intensity of pedv decreased significantly with the increase in the concentration of caerin . . to fully understand the inhibitory effect of caerin . against pedv-yn strain, several experiments were performed including cytopathic effects (cpe) observation, plaque reduction assay, tcid , and ifa. the cpe observation results showed that there were remarkable differences between the cells infected with pedv in the absence or presence of caerin . . caerin . -treated cells were in a relatively normal status, and the cpe appeared in caerin . -treated group about h later than in the pedv control group, as seen in figure a . with the addition of caerin . , the number of plaques declined at a surprising speed and the diameters decreased notably. similar results can be observed even with pfu of pedv treated by caerin . . the cells infected with pfu pedv exactly remained the same as the cells in negative control group, as seen in figure b . caerin . significantly inhibited the multiplication of pedv in a dose-dependent manner, demonstrating that pedv infection to vero cells was blocked to a large extent. the results of tcid , shown in figure c , and ifa, in figure d , indicated that the titer and the fluorescence intensity of pedv decreased significantly with the increase in the concentration of caerin . . the inhibitory effects of caerin . against another two different pedv strains were examined to evaluate the anti-pedv potential of caerin . based on ifa. as shown in figure , vero cells were infected with pedv ( pfu) pre-incubated with different concentrations of caerin . . compared with the virus control, treatment group exhibited excellent inhibitory effects in a dose dependent manner even at extremely low concentrations of caerin . . these results revealed that the intracellular viruses evidently reduced in the presence of caerin . in a dose dependent manner. groups (pedv) were not treated by caerin . . the results of three independent experiments are presented as the mean ± sd. the statistical analysis was performed using graph pad prism . . the significance was defined as p < . (*), and the higher significance was defined as p < . (**), p < . (***); (d) fluorescence intensity of pedv in the presence of caerin . at different concentration. the inhibitory effects of caerin . against another two different pedv strains were examined to evaluate the anti-pedv potential of caerin . based on ifa. as shown in figure , vero cells were infected with pedv ( pfu) pre-incubated with different concentrations of caerin . . compared with the virus control, treatment group exhibited excellent inhibitory effects in a dose dependent manner even at extremely low concentrations of caerin . . these results revealed that the intracellular viruses evidently reduced in the presence of caerin . in a dose dependent manner. to better understand the inhibitory effects of caerin . against the progeny virus production of pedv, we added caerin . at hpi to pedv infected cells. and both the intracellular and extracellular virus titers were determined separately by using tcid , as shown in figure a . the progeny virus titer decreased significantly as compared to that of virus control in a dose dependent manner, as seen in figure a . on the other hand, extracellular virus titers were also determined at different time points and different caerin . concentrations. the results showed that the increase of extracellular virus titer slowed down under the treatment of caerin . , as seen in figure b . incubated with caerin . at the concentrations of . (c), (d), (e) and (f) μg/ml for h. scale bar, μm. to better understand the inhibitory effects of caerin . against the progeny virus production of pedv, we added caerin . at hpi to pedv infected cells. and both the intracellular and extracellular virus titers were determined separately by using tcid , as shown in figure a . the progeny virus titer decreased significantly as compared to that of virus control in a dose dependent manner, as seen in figure a . on the other hand, extracellular virus titers were also determined at different time points and different caerin . concentrations. the results showed that the increase of extracellular virus titer slowed down under the treatment of caerin . , as seen in figure b . in the fluorescent confocal experiment, the fluorescence of caerin . and pedv around cell nucleus could be observed when we incubated them with vero cells respectively. additionally, the distribution of caerin . and pedv were almost the same, so the results of confocal laser scanning microscopy verified that caerin . could combine with pedv, and that the antiviral function of caerin . in the cell plasma could be observed especially in the areas showing severe cpe, as seen in figure . in the fluorescent confocal experiment, the fluorescence of caerin . and pedv around cell nucleus could be observed when we incubated them with vero cells respectively. additionally, the distribution of caerin . and pedv were almost the same, so the results of confocal laser scanning microscopy verified that caerin . could combine with pedv, and that the antiviral function of caerin . in the cell plasma could be observed especially in the areas showing severe cpe, as seen in figure . in caerin . -pedv pre-incubation process, the morphological changes of pedv particles were observed clearly. so caerin . could destroy the structure of the virus to block the infection of the host cells, as seen in figure a , resulting in the inhibition of viral release, which in turn could reduce the transmission of the progeny virus among the adjacent cells. as shown in the growth curve, pedv co-incubated with caerin . showed the lowest titer, while pedv was only slightly inhibited in preincubation process of caerin . -cells and the delayed usage of caerin . , as seen in figure b . in a western blot experiment, the protein band could barely be observed in pedv-caerin . co-incubation group. however, no obvious differences in pedv-n protein expression were observed among the cell-caerin . pre-incubation group, the post treatment group, and pedv control group, as seen in figure c . to further explore whether caerin . could bind to some virus receptors in host cells to interfere with the attachment and entry process, we kept the cells at °c to maintain the attaching status. caerin . did not show obvious inhibitory effects during the attachment and entry process. there were no significant differences in virus titers between the virus control groups and caerin . treated groups during both attachment period and entry period, even if the concentration of caerin . was increased tenfold, as seen in figure d . the western blot assay did not exhibit any obvious differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group, shown in figure e , either. in caerin . -pedv pre-incubation process, the morphological changes of pedv particles were observed clearly. so caerin . could destroy the structure of the virus to block the infection of the host cells, as seen in figure a , resulting in the inhibition of viral release, which in turn could reduce the transmission of the progeny virus among the adjacent cells. as shown in the growth curve, pedv co-incubated with caerin . showed the lowest titer, while pedv was only slightly inhibited in pre-incubation process of caerin . -cells and the delayed usage of caerin . , as seen in figure b . in a western blot experiment, the protein band could barely be observed in pedv-caerin . co-incubation group. however, no obvious differences in pedv-n protein expression were observed among the cell-caerin . pre-incubation group, the post treatment group, and pedv control group, as seen in figure c . to further explore whether caerin . could bind to some virus receptors in host cells to interfere with the attachment and entry process, we kept the cells at • c to maintain the attaching status. caerin . did not show obvious inhibitory effects during the attachment and entry process. there were no significant differences in virus titers between the virus control groups and caerin . treated groups during both attachment period and entry period, even if the concentration of caerin . was increased tenfold, as seen in figure d . the western blot assay did not exhibit any obvious differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group, shown in figure e , either. figure b . gapdh (glyceraldehyde- phosphate dehydrogenase) was used as a loading control; (d) tcid showed that caerin . had no obvious inhibitory effects during the attachment and entry process. caerin . was added at pedv attachment and entry periods, then the titers of pedv were detected; (e) western blot analysis of the expression level of pedv-n protein when caerin . was added in different ways as shown in figure d . gapdh was used as a loading control. pedv is one of the most important pathogens leading to diarrhea in pig industry. the aim of this study is to find whether caerin . has the antiviral activity against the three pedv strains and to reveal the antiviral mechanism of caerin . . according to published data, caerin . is a cationic peptide with two α-helices and has proved to be an effective agent against hiv by damaging the virus envelope and stopping the infection of hiv to t cells [ , ] . some α-helical peptides such as melittin and cecropin have been reported to exert antiviral activities by direct disruption of virus membranes or inhibition of the virus replication [ ] . the current study found that caerin . was able to destroy the structure of viral particles and reduce the viruses capable of infecting the cells and decrease their titers almost up to logs. this is the first attempt to reveal that caerin . has strong antiviral activity against pedv infection. figure b . gapdh (glyceraldehyde- -phosphate dehydrogenase) was used as a loading control; (d) tcid showed that caerin . had no obvious inhibitory effects during the attachment and entry process. caerin . was added at pedv attachment and entry periods, then the titers of pedv were detected; (e) western blot analysis of the expression level of pedv-n protein when caerin . was added in different ways as shown in figure d . gapdh was used as a loading control. pedv is one of the most important pathogens leading to diarrhea in pig industry. the aim of this study is to find whether caerin . has the antiviral activity against the three pedv strains and to reveal the antiviral mechanism of caerin . . according to published data, caerin . is a cationic peptide with two α-helices and has proved to be an effective agent against hiv by damaging the virus envelope and stopping the infection of hiv to t cells [ , ] . some α-helical peptides such as melittin and cecropin have been reported to exert antiviral activities by direct disruption of virus membranes or inhibition of the virus replication [ ] . the current study found that caerin . was able to destroy the structure of viral particles and reduce the viruses capable of infecting the cells and decrease their titers almost up to logs. this is the first attempt to reveal that caerin . has strong antiviral activity against pedv infection. some amps including cecropin d (cd-prrsv, salps-aiv) were reported to have blocked viral attachment and invasion by interacting with the receptors of the host cells [ ] . based on these findings, so we tried to keep pedv infection within the periods of the attachment and entry through the temperature control, and the results showed no evident differences in protein expression among attachment period treated group, entry period treated group, and the pedv control group. the number of virus infecting cells did not diminish. this means that caerin . did not interfere with the attachment and entry process. considering the fact that caerin . does not compete with the virus to conjugate with the cells, and the fact that viral attachment-entry processes are very fast and caerin . will not have enough time to act against the viral membrane, it can be concluded that caerin . has less significant antiviral activities in the pre-incubation of caerin . -cells progress. to further understand the mechanism in the post-treatment process, we examined the antiviral activity during the replication period. the results showed that caerin . did not have obvious antiviral effect during viral replication period, but it can control the infection progress by blocking the release of pedv particles to reduce viral transmission among the adjacent cells. antiviral activities of caerin . is expected to be improved through the combination usages with other different amps or other antiviral agents like graphene oxide (go), which could make caerin . a good weapon to deal with the diseases caused by viruses including pedv [ ] . as a matter of fact, we did a combination treatment with piscidin which was also proved to be effective against pedv [ ] , but the results were not as good as expected based on our unpublished work. the mechanisms still remain to be further investigated. although the joint use of piscidin and caerin . did not work as well as expected, other candidates can be explored for the prevention of pedv and other pathogens in drug combination studies. on the other hand, some related research work has been done in our laboratory. our other unpublished work has preliminarily testified the extraordinary ability of caerin . against some other viruses including prv (pseudorabies virus) which is a herpesvirus with double-stranded dna that can infect many mammal species. therefore, many potential functions of caerin . are still to be explored in the future. in this study, we investigated the antiviral activities of caerin . against pedv strains and its antiviral mechanism. the results show that caerin . can maintain the integrity of the host cells since it has very low cytotoxicity, and that it exhibits excellent virucidal activity in a dose-dependent manner even at very low concentrations. caerin . can reduce the viruses infecting the cells by destroying the integrity of the viral membrane, resulting in the decrease in the virus replication and protein expression. caerin . can also interfere with the virus release process essential for the inhibition of intracellular infection. therefore, caerin . is an excellent antiviral material against pedv. in conclusion, our study testifies the outstanding antiviral activities of caerin . and lays a foundation for future research and for the application of amphibian antimicrobial peptides. the authors have declared that no competing financial interests exist. comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus a new coronavirus-like particle associated with diarrhea in swine isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states new variants of porcine epidemic diarrhea virus, china phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china the evolution and genetics of innate immunity antimicrobial peptides from amphibian skin: an expanding scenario: commentary antimicrobial peptides from amphibian skin potently inhibit human immunodeficiency virus infection and transfer of virus from dendritic cells to t cells antimicrobial peptides of multicellular organisms antimicrobial peptide m or m identified from lactobacillus casei atcc can trigger apoptosis in the human colorectal cancer cell line sw high-quality d structures shine light on antibacterial, anti-biofilm and antiviral activities of human cathelicidin ll- and its fragments antimicrobial and host-defense peptides as new anti-infective therapeutic strategies antimicrobial peptides (amps) with dual mechanisms: membrane disruption and apoptosis control of cell selectivity of antimicrobial peptides the application of antimicrobial peptides as growth and health promoters for swine immunomodulatory activities of small host defense peptides host antimicrobial proteins as endogenous immunomodulators solution nmr studies of amphibian antimicrobial peptides: linking structure to function? investigating the importance of the flexible hinge in caerin . : solution structures and activity of two synthetically modified caerin peptides specific and selective peptide-membrane interactions revealed using quartz crystal microbalance inhibition of hiv infection by caerin antimicrobial peptides antimicrobial host defense peptides in an arteriviral infection: differential peptide expression and virus inactivation he, q. the ubiquitin-proteasome system is required for the early stages of porcine circovirus type replication the cytotoxicity to leukemia cells and antiviral effects of isatis indigotica extracts on pseudorabies virus proteome analysis of porcine epidemic diarrhea virus (pedv)-infected vero cells inhibitory effects of silver nanoparticles against adenovirus type in vitro a simple method of estimating fifty per cent endpoints antiherpes simplex virus type activity of the antimicrobial peptide subtilosin inhibitory activity and mechanism of two scorpion venom peptides against herpes simplex virus type porcine epidemic diarrhea virus induces autophagy to benefit its replication antimicrobial peptides as new recognition molecules for screening challenging species inhibition of porcine reproductive and respiratory syndrome virus by cecropin d in vitro antiviral activity of graphene oxide: how sharp edged structure and charge matter comparative pharmacokinetics and preliminary pharmacodynamics evaluation of piscidin against prv and pedv in rats antimicrobial peptide ifa indirect immunofluorescent assay nmr nuclear magnetic resonance mtt -( , -dimethyl- -yl)- , -diphenyltetrazolium bromide ped porcine epidemic diarrhea pedv porcine epidemic diarrhea virus tcid tissue culture infectious dose key: cord- - pmts ui authors: nema, vijay title: microbial forensics: beyond a fascination date: - - journal: dna fingerprinting: advancements and future endeavors doi: . / - - - - _ sha: doc_id: cord_uid: pmts ui microbiology has seen a great transition from culture-based identification of microbes using various biochemical and microscopic observations to identify and functionally characterize the microbes by just collecting the dna and sequencing it. this advancement has not only moved in and around microbiology but has found its applications in fields which were earlier considered to be the remote ones. forensics is one such field, where tracing the leftover evidence on a crime scene can lead to the identification and prosecution of the culprit. when leftover microbes in the biological material or objects used by the culprit or the person in question are used to correlate the identity of the individual, it takes us to the new field of science—“microbial forensics.” technological advances in the field of forensics, molecular biology, and microbiology have all helped to refine the techniques of collecting and processing of the samples for microbiological identification using dna-based methods followed by its inference in the form of evidence. studies have supported the assumption that skin or surface microflora of an individual is somewhat related with the microflora found on the objects used by that individual and efforts are ongoing to see if this is found consistently in various surroundings and with different individuals. once established, this technique would facilitate accurate identification and differentiation of an individual or suspect to guide investigations along with conventional evidence. legal investigations are not only the field where microbial forensic could help. agriculture, defense, public health, tourism, etc. are the fields wherein microbial forensics with different names based on the fields are helping out and have potential to further support other fields. tracing the leftover evidences on a crime scene remained the only way of getting to the culprit. the history and evolution of forensic sciences has been very fascinating and interesting for even those who do not understand the science behind it. the journey started with william james herschel sometimes in when he recognized that the fingerprints remain unique to the individuals. it was used on legal and administrative documents then and was published in with all evidences and their analysis [ ] . it was gradually picked up for all possible uses including crime scene investigations. fingerprints remained the landmark evidence in all forensic investigations and are still playing important roles. additional techniques came in to support forensics in the form of dna fingerprinting, wherein dna matching became useful in various cases. dna fingerprinting is used to establish a link between biological evidence and a suspect in a criminal investigation [ ] [ ] [ ] [ ] [ ] . dnaor genetic-fingerprinting relies heavily on the principle that no two individuals share the same genetic code. recently, there has been a new discipline co-emerged with culture-independent techniques of identifying microbes. this new discipline looks for microbes and tries to co-relate them with individuals as like fingerprints or dna. there is a constant interaction of individuals with microbes in their surrounding, and they leave microbes into their surroundings. whenever there is a physical contact, bacteria hop across from the skin to the material used. the microbial communities attached with an individual's skin or other sites are being explored, and preliminary evidences suggest that they are unique and can identify individuals or the material used by them in a few cases [ ] [ ] [ ] . this became the base for coining of a new term "microbial forensics." however the field has other dimensions too and would be discussed in detail in this chapter. the microbial forensics rely on the inputs from various fields of basic and applied sciences. these include microbiology, genetics, bioinformatics, forensic science, immunology, population genetics, biochemistry, molecular biology, epidemiology, etc. along with the law enforcement, public health, policy, and intelligence communities. bacterial density on the human skin may be as high as cells/cm [ ] and can be freely transferred to surface which comes in contact. evaluating the traces of skin microbiome left on questioned objects may be useful for forensic identification. calculating the distances from samples and their donators, it seems possible to estimate whether items or palm prints belong to one specific person or not. this has become possible because of the culture-independent method of tracing all microbial species present in a given environment. this technique used here is called nextgeneration sequencing, and the population of microbes no matter cultivable or nonculturable is called microbiome. in this process, total dna of the given environment or object is taken out, and the specific gene for prokaryotes, i.e., srrna gene, is amplified. this gene has a property like a clock with fast-moving arm and slowmoving arm. this concept has been explained by the work of karl woose who named this gene as a biological clock [ ] [ ] [ ] [ ] . the gene contains highly conserved regions to identify the microbes with consistency and at the same time some variable regions to differentiate between closely related microbes. the technique has advantages over dna from individuals as the microbiome dna is abundant in touch dna, and these organisms are much more stable because of complicated cell wall structures. hence, it might be easier in certain instances to extract bacterial dna than human dna from surfaces target samples. another good reason for this is the higher abundance of bacterial cells on the skin and shed epidermal cells as compared to human cells. there are some surfaces like fabrics, smudged surfaces, or highly textured surfaces from where obtaining clear fingerprints is very difficult. bacterial dna can still be found there and can help in solving the purpose [ ] . microbiology and forensic science were always considered to be different fields, and establishing a link between the two for better investigative power was not thought of earlier. microbiology is simply defined as study of microorganisms wherein microorganisms are the organisms that exist as single cells or cell clusters and must be viewed individually with the aid of a microscope. microbiology classifies microbes into various groups, and based on their characteristics and physiology, they could be assigned different genera and species. most of the microbes, especially the saprophytic ones, depend on other substrates and interact specifically with their environment and habitat to gain important nutrients for their survival. this requirement remains very specific and unique with respect to one community of microbes and here comes a link with forensic sciences. forensic science explores about those unique things (belongings or body remains) which could be co-related with the career (criminal) of those things, for instance, fingerprints or dna. because microbial communities on a particular individual's body are expected to be unique, identifying that individual with leftover microbial traces on the material used by the individual is a possibility and is being confirmed using various experiments. however, culturing of all those microbes and identifying them is a near impossible task. the reason is that all microbes do not grow on known culture media, and some which can grow are sometimes overtaken by the competition with other microbes in the same community. hence collecting the total dna and amplifying for the microbial signature gene is a good solution ( fig. . ). this technique is called metagenomics. further, a sequencing technique that sequences the metagenome is called next-generation sequencing (ngs). ngs also known as high-throughput sequencing, represents different modern sequencing technologies for sequencing dna and rna much faster than sanger sequencing and with lower economical and technical inputs. identifying individuals is important in forensic science, and various developments have supported it from time to time. however, while making use of dna, its quantity becomes crucial for accurate detection with desired quality for prosecution of crime. a smart offender can be cautious enough to decrease or degrade the traces of biological leftovers like blood, semen, etc. along with the fingerprints from the crime scene, which can complicate offender detection. hence although fingerprints and dna fingerprints remain very precise techniques, the availability of raw material becomes limiting factor in their use. microbes can help in such scenario as their dna is not as easily destroyed as a human dna and remains available on surfaces at crime scene or on objects used. the pattern of bacterial dna is dictated by the surrounding environment and the individual's microbiome [ , ] . it is possible that the different bacterial patterns or the type of bacteria with typical physiology could discriminate individuals with different lifestyles. hence, bacterial dna analysis may serve as a complimentary technique in cases where standard dna identification is partially informative [ ] . along with this, technologies like pcr, real-time pcr, mlst (multilocus sequence typing), mlva (multilocus vntr analysis), fish (fluorescence in situ hybridization), and microarrays are being employed based on the need of the case or the availability of the infrastructure. additional new methodologies like matrix-assisted laser desorption/ionization-time of flight (malditof), gas chromatography-mass spectroscopy (gc-ms), and liquid chromatography-mass spectroscopy (lc-ms) are also well established in resolving minor difference in bacterial strains. investigation of a crime scene is not just potential application wherein microbial forensics can play an important role. bioterrorism, biosecurity, biometry, medical forensics, etc. are the upcoming fields wherein definitive detection of microbes and their correlations may help a lot. research on the transmission of microbes between human and surrounding environments has proved to be a property with potential for microbiome to be used in forensic investigations. in some cases, typical human microbial associations have been used to relate individuals to objects they have used [ ] . pattern of associated microbes with the surfaces at home and the family living and working with them have shown a predictive correlation to an extent that family's home and that individuals within a home can be differentiated [ ] . similar is the case with smartphones which rather remain in contact with specific individual and for longer durations [ ] . interestingly the microbial communities were different on the top and bottom of the phone differentiating the individuals and surface flora. an interesting property of the deposited microbiome is that it changes with a constant rate on a particular surface and can be utilized for forensic calculations. work by metcalf and co-workers have revealed that postmortem, the microbiome of animal hosts changes radically, but the pattern is much more predictable [ ] . this can help tracing the time and direction of the events. lax and co-workers worked to determine if the surface type and individuals have an effect on the microbial communities and have found that microbial community structure was determined both by surface type and participant. another aspect of microbial forensics is its role in bioterrorism. herein the microbial forensics could be defined as "the discipline of applying scientific methods to the analysis of evidence related to bioterrorism, biocrimes, hoaxes, or the accidental release of a biological agent or toxin for attribution purposes" [ ] . microbial forensics, while dealing with bioterrorism, concentrate on identification of the agent or toxin and/or the mode of its production and dissemination. in addition, traditional forensic methods are used in conjunction to reach the goal of identifying the perpetrators of the crime. around , microbial species or strains are listed as dangerous for humans [ ] . building individual diagnostic methods for these many numbers of agents is an impossible task, and hence ngs is the only tool to do massive parallel sequencing and to identify unknown pathogens, microorganisms modified to create panic, and pathogens in complex communities or samples with low abundance. with the global expansion of trade and communication, frequent movement of individuals from one country to other is unavoidable. in such scenario carriage of an endemic pathogen or drug resistance to a new geographical region is a looming threat. recent outbreaks and transmission incidents of sars and ebola have raised an alert. microbial forensic has a crucial role in such cases wherein the status is not declared by individuals, is not detected by routine quarantine, or is a new pathogen altogether. detection of microbial drug resistance or the emerging resistance is becoming increasingly important for human health. similar things are true in the case of plant pathogens and food borne diseases [ , ] . the foodborne diseases have always been a substantial global challenge to public health. a huge population worldwide become sick of foodborne illnesses every year with a substantial burden on public health as well as on economy. addressing this problem has many steps, out of which one age-old problem is the rapid identification of the food source of the contamination. in a classic laboratory study, we could trace back the source of the foodborne outbreak, but the finding could not be utilized in helping the troubled ones immediately [ ] . this is due to the infrastructural limitations and technical challenges in identifying the pathogens. a technology which was considered to be reliable and was used till recent past was pulsed-field gel electrophoresis (pfge). however its resolution in pinpointing the source of the outbreak has not been satisfactory. recent employment of whole-genome sequencing (wgs) in such investigations has shown promise. a retrospective study by us food and drug administration's center for food safety and applied nutrition (fda-cfsan) in could provide a far better resolution of the causal factors. all the isolates were sequenced on the illumina miseq. wgs using illumina could distinguish all of the isolates which looked exactly the same. ultimately they concluded that the isolates from the outbreak were most closely related to a -year-old historical isolate that was linked to a processing facility only km away from the source of the outbreak [ ] . this could not only allow newer findings but also traced back the source of contamination to further allow the rectification. such cases are sometimes accidental but are often criminal too, and tracing back the source would prevent such cases. in bio crimes, serious disease outbreak by natural occurrence or intentional may result in harm or death, causing disruption, creating fear, and affecting economic well-being. microbial forensics thus plays an important role in consumer protection, food security, and even in litigation. agriculture and agricultural goods are also the susceptible area for microbial interventions and hence are important in terms of microbial forensics. there could be deliberate misuse of microbes or their products affecting flora and fauna which is important for agriculture. this could be given a name such as "agroterrorism" [ ] . a field of investigation emerged against this threat for investigating into the violations and used scientific knowledge and technology to do so. this was given the name of bioforensics [ ] . another aspect of microbial forensic application to the foodborne pathogens is to trace pathogens in cash crops especially spices and other costly ones. van doren and co-workers have studied reported illness outbreaks from canada, denmark, england and wales, france, germany, new zealand, norway, serbia, and the united states which occurred due to consumption of pathogen-contaminated spice during - . the outbreaks were reported from a few developed countries only. the reason for not including other countries was that those countries did not have updated technology to investigate and report similar findings. it was reported that these outbreaks resulted in human illnesses, hospitalizations, and deaths. infants/children were the primary population segments impacted by % ( / ) of spice-attributed outbreaks [ ] . the economic aspect associated here is the detection of pathogens after shipment and then the recall of the material. this involved a huge cost. recent development in microbial forensics in agricultural sciences also aids in pest control as well as deliberate introduction of pests along with food imports or use of pathogens as anticrop bioweapons. different companies are coming up with molecular detection tools for rapid detection of specific pathogens in such products. for instance, hu and co-workers have compared and evaluated the effectiveness of the molecular methods ( m molecular detection system (mds) and ansr pathogen detection system (pds)) for the detection of salmonella in egg products and compared the same with culture methods to find that the molecular methods are the superior and faster ones [ ] . budowle and co-workers have established a criterion comprising a foundation for investigators to establish, validate, and implement high-throughput sequencing (hts) as a tool in microbial forensics [ ] . likewise, design principles for an effective microbial forensics program for law enforcement and national security purposes have been provided [ ] . microbial pathogens or toxins can be used to commit acts of terror; they can be used as weapons for execution of a crime. in biological warfare, transmissible lethal agents are used to attack the targeted populations. the impact of the bioterrorism was seriously considered after the anthrax attack in the united states in . this incidence in the united states helped the world understand that bioterrorism can have drastic and global impacts. microbial forensics has a role in such cases by applying scientific methods for the analysis of evidence from such a bioterrorism attack. microbial forensic in conjunction with epidemiology can try to decipher if an outbreak is natural, accidental, or intentional [ , ] . for instance, study by price and co-workers found that the bacillus anthracis injectional anthrax cases were originated from heroin users in scotland [ ] . ou and co-workers used molecular tracking of hiv to report for the first time about the passage of hiv infection from dentist to patient after invasive healthcare procedure [ ] . a spanish anesthetist infected patients with hepatitis c virus which could be found using phylogenetic and molecular clock analysis [ ] . in the ebola outbreak, the origin and transmission could be traced using bioforensic methods [ ] , etc. other important and relatively new aspect of microbial forensics in medicine and medicolegal field is "thanatomicrobiome" (thanatos-death) that studies the microorganisms found in internal organs and cavities upon death. the thanatomicrobiome tries to investigate the total microbial communities including bacterial and fungi from all the body locations of decomposing corpses. these studies are important in providing evidence in medicolegal death investigations [ ] . by doing this, the concept of human postmortem microbiome project (hpmp) has also been introduced which would create a consortium of research projects to identify and characterize the thanatomicrobiome and epinecrotic communities (e.g., epithelial tissues, body cavities, and the alimentary canal), relating to human decomposition with a potential of finding a state-of-the-art, more dependable, and molecular way of determining the time of death [ ] . it has been shown that typical characteristics and pattern of the human microbiome might identify individuals and remain constant in the individual but differ from the others [ , , ] . this means that individuals might be specifically and consistently identified using their microbiome. however microbiome-based identifiability is still a long way to go. franzosa and co-workers have suggested a few means to achieve the target of identifiability using microbes. the identification of a "metagenomic code" that remains unique for an individual for a longer duration of time and stands true for a sizable population is the key [ ] . hence microbiome establishment, structure, personalization, and temporal stability are the standard terms and areas to work upon. there are some untraveled avenues which start from microbial forensics. emergence of antimicrobial resistance, prediction and prevention of future outbreaks, etc. are some of the fields wherein a surveillance system making use of the principles of microbial forensics can help. antimicrobial resistance in microorganisms emerges naturally. however, antimicrobial exposure due to human practices in healthcare, agriculture, sanitation, industrial processes, travel, and other fields contributes significantly. timely detection of pathogens harboring resistance can mitigate the onward transmission among individuals and among different geographical locations. timely detection of pathogens like hiv, severe acute respiratory syndrome (sars) virus, and pandemic influenza could have avoided the big health emergencies which we have witnessed in recent past. similar is the case with the pandemic spread of sars coronavirus in and h n influenza in resulting in substantial economic loss. microbial forensics can play a crucial role in such cases by checking the emergence in real time and suggesting measures to prevent transmission. however, a lot of investment is required to place such services at all vulnerable points or check points. technological advances especially in the field of metagenomics have paved way for identification of potential human pathogens among other species and have attained good predictive power regarding transmissibility and virulence of the novel microbes. the science of microbial forensics is in its infancy and needs much more that what has already been done. a few things which needs to be taken care as preparation before we take this as a routine science are protocols and procedures for collecting specimens at the attack site, recognizing that an attack is occurring and diagnosing the disease, analysis of specimens in contained facilities, quality assurance and control. although microbial forensics involve techniques or methodologies from basic laboratory sciences, the problems in question, processes engaged in and expected outcomes need more than that. there are efforts and continuous need for validating all the tools and techniques involved which should be acceptable to peers and stakeholder from scientific, legal, and policy making side. the optimization of methods to answer key questions pertaining to investigative and legal needs is a must to satisfy the criterion of acceptability. meeting these challenges will allow the establishment of a complementary and reliable method to compensate for the lacuna of dna fingerprinting and fingerprinting as discussed earlier. meeting the challenges needs the consorted efforts from global communities of workers from basic sciences, epidemiologists, forensic experts, medical experts, legal experts, and a big team of technology developers. the most reliable technique till date for microbial forensics is metagenomics-a culture-independent approach for identifying and enumerating microbes. metagenomic have been an outstanding technique for sequencing the genomes of unculturable microbes, which represent the vast majority of microorganisms, particularly from environmental samples. however, technological advancement identifies the rare taxon is awaited. as microbial metagenomics is undergoing a formative phase as a diagnostic technique, optimization of methods and their validation remain a challenge. other aspects of this are the leadership role and generic availability of tools and techniques. countries with major resources would be able to take lead in basic research, while the resource-limited setting may not be able to adapt microbial forensics owing to its monitory needs. availability of equipment and techniques for rapid and precise molecular diagnosis is important for controlling and responding to the needs of microbial forensics. till date, next-generation sequencing is the only technology that seems promising for microbial forensics. but the instrument and the reagents remain very costly as compared to biochemical tests and a few basic molecular assays being used in forensic laboratories. moreover, expert workers and bioinformatics analysis of the huge data generated after massive parallel sequencing or next-generation sequencing require a devoted facility and expertise. microbial forensics may, in most of the cases, be associated with the detection of causal pathogen in cases of biological terrorism, but microbiome associated with individuals and objects used or touched by them at crime scenes may also be used as a tool in providing forensically relevant information. this science has got its diverse utility in the field of medicine, agriculture, trade especially in food articles, etc. as discussed in this chapter. the evidence and the literature available till date indicate a definitive linkage between an individual and microbial communities inhabiting that individual skin or other body parts. the science is progressing toward identifying these communities, next steps of which would be to see if these communities have something in common and that common character has something to do with the individual. later work in this field may identify chemical entities and their microbial connections to land up to the final conclusions about signature communities of microbes and their forensic potential. however, we may have to continue our search to see those days soon. the origin of fingerprinting dna 'fingerprinting' of captive family groups of common marmosets (callithrix jacchus) forensic application of dna 'fingerprints' application of dna "fingerprints" to paternity determinations the man behind the dna fingerprints: an interview with professor sir alec jeffreys demographic study of a wild house sparrow population by dna fingerprinting forensic identification using skin bacterial communities comparison of bacterial dna profiles of footwear insoles and soles of feet for the forensic discrimination of footwear owners forensic analysis of the microbiome of phones and shoes microbial ecology of human skin in health and disease microbiology's scarred revolutionary - ) phylogenetic structure of the prokaryotic domain: the primary kingdoms towards a natural system of organisms: proposal for the domains archaea, bacteria, and eucarya microbial forensic analysis of bacterial fingerprint by sequence comparison of s rrna gene cohabiting family members share microbiota with one another and with their dogs is human dna enough?-potential for bacterial dna longitudinal analysis of microbial interaction between humans and the indoor environment mobile phones carry the personal microbiome of their owners a microbial clock provides an accurate estimate of the postmortem interval in a mouse model system expansion of microbial forensics risk factors for human disease emergence practical value of food pathogen traceability through building a whole-genome sequencing network and database singleton sequence type , an emerging clonal group of listeria monocytogenes associated with three multistate outbreaks linked to contaminated stone fruit, caramel apples, and leafy green salad isolation and characterization of heat resistant enterotoxigenic staphylococcus aureus from a food poisoning outbreak in indian subcontinent tracing origins of the salmonella bareilly strain causing a food-borne outbreak in the united states a methodology for assessing the risk posed by the deliberate and harmful use of plant pathogens in characterization of the threat resulting from plant pathogen use as anticrop bioweapons: an eu perspective on agroterrorism foodborne illness outbreaks from microbial contaminants in spices evaluation of m molecular detection system and ansr pathogen detection system for rapid detection of salmonella from egg products validation of high throughput sequencing and microbial forensics applications designing an effective microbial forensics program for law enforcement and national security purposes microbial forensic investigation of the anthrax-letter attacks review of the scientific approaches used during the fbi's investigation of the anthrax letters molecular epidemiologic investigation of an anthrax outbreak among heroin users economou an ( ) molecular epidemiology of hiv transmission in a dental practice genomic variation landscape of the human gut microbiome the thanatomicrobiome: a missing piece of the microbial puzzle of death human thanatomicrobiome succession and time since death the long-term stability of the human gut microbiota molecular evolution in court: analysis of a large hepatitis c virus outbreak from an evolving source identifying personal microbiomes using metagenomic codes key: cord- -usgpe cz authors: zuwala, kaja; riber, camilla f.; løvschall, kaja borup; andersen, anna h.f.; sørensen, lise; gajda, paulina; tolstrup, martin; zelikin, alexander n. title: macromolecular prodrugs of ribavirin: polymer backbone defines blood safety, drug release, and efficacy of anti-inflammatory effects date: - - journal: j control release doi: . /j.jconrel. . . sha: doc_id: cord_uid: usgpe cz macromolecular (pro)drugs hold much promise as broad-spectrum antiviral agents as either microbicides or carriers for intracellular delivery of antiviral drugs. intriguing opportunity exists in combining the two modes of antiviral activity in the same polymer structure such that the same polymer acts as a microbicide and also serves to deliver the conjugated drug (ribavirin) into the cells. we explore this opportunity in detail and focus on the polymer backbone as a decisive constituent of such formulations. fourteen polyanions (polycarboxylates, polyphosphates and polyphosphonates, and polysulfonates) were analyzed for blood pro/anti coagulation effects, albumin binding and albumin aggregation, inhibitory activity on polymerases, cytotoxicity, and anti-inflammatory activity in stimulated macrophages. ribavirin containing monomers were designed to accommodate the synthesis of macromolecular prodrugs with disulfide-exchange triggered drug release. kinetics of drug release was fast in all cases however enhanced hydrophobicity of the polymer significantly slowed release of ribavirin. results of this study present a comprehensive view on polyanions as backbone for macromolecular prodrugs of ribavirin as broad-spectrum antiviral agents. viruses are fascinating products of evolution and life-threatening pathogens at the same time. indeed, these non-cellular assemblies of macromolecules and lipids are simpler than even minimalistic replicating cells but can infect living organisms in both plant and animal kingdoms. throughout history, viral infections repeatedly had a devastating effect on human society and even in modern times, viral outbreaks create an enormous socio-economic burden [ , ] . recent examples include the outbreaks of coronavirus-associated southeast asian and middle eastern respiratory syndromes (sars, mers) as well as the ebola and zika virus outbreaks. to counter this, interdisciplinary efforts have been made over the past decades in the design and development of antiviral drugs. in large part, this was spurred by the spread of the human immunodeficiency virus (hiv) [ ] . currently, there are nearly one hundred antiviral drugs approved by the us fda [ ] . nevertheless, significant challenges remain. it is now understood that a significant limitation of current strategies is that countermeasures to viral pathogens are most typically developed individually and specifically to each emerging pathogen. an aspect that came into the focus of research attention is the development of "broad-spectrum antiviral agents" [ ] . such agents can constitute prophylactic, [ ] preventative, [ , ] or curative [ , ] measures. prophylaxis (vaccination) holds a great appeal but universal antiviral vaccines are yet to be developed [ ] . in fact, the overall majority of pathogenic viruses have no corresponding vaccination product. in turn, curative measures are highly attractive in their own right. however, while replicational cycles of viruses are similar, there is a great variability between viral enzymes and other proteins that could serve as drug targetsmaking the design of universal antiviral agents challenging [ ] . interferon is an endogenous protein that serves as a potent stimulatory signal to human immunity and acts as a non-specific, broadly acting antiviral agent. however, viruses developed multiple interferon inhibitors which compromise induction or activity of interferon [ ] . moreover, due to its side effects, its clinical use is limited [ ] . several small molecule drugs also serve as broadly acting antivirals. of these, ribavirin (rbv) has a long history of medicinal use and appears on the world health organization list of essential medicines for both adults and children [ , ] . however, this drug also has a dose limiting toxicity. several other experimental drug leads are in different stages of (pre)clinical development [ ] [ ] [ ] [ ] . finally, preventative antivirals that act through neutralization of viruses and/or blocking virus cell entry appear to be highly promising as broad-spectrum antiviral agents [ ] [ ] [ ] . a notable class of such agents is polymers. in the early s, it was first observed that viruses interact with polymers [ ] and inhibit virus infectivity [ ] . over subsequent decades, this effect was shown to be affecting viral pathogens across the viral genus and families, and observed for polymers diverse in their structure, natural and synthetic, positively or negatively charged [ ] [ ] [ ] [ ] [ ] [ ] . de clercq et al. proposed that activity of the negatively charged polymers, at least in vivo, is indirect and proceeds via stimulation of interferon production [ ] . however, antiviral activity proceeds potently and efficaciously in vitro, in absence of interferon, and the direct contact between polymers and the virus with ensuing neutralizing activity is now well-documented. inherent skepticism towards polyanions and other polymers as antivirals [ ] originates in the failures of hallmark clinical trials for these agents both when administered systemically or as topical microbicides [ ] . specifically for systemic administration, it has been concluded that preventative antiviral effect would have to rely on a sustained, high concentration of these agents in blood not achievable without toxic effects [ ] . however, other polymers are successfully commercialized as antivirals, specifically as lubricants in condoms acting as microbicides with activity against hiv and herpes simplex viruses [ ] . nucleic acid based polyanions progress through clinical trials as agents against hepatitis b virus with potential of further development as broad-spectrum antiviral activity [ ] . it is important to note that failed clinical trials are indeed discouraging yet important, game-changing developments have occurred in polymer chemistry in recent decades. specifically, novel polymerization techniques are now available to afford polymers with well-controlled molar mass and architecture and a significantly broader available polymer functionality (side chains) [ , ] . another notable advancement includes the development of polymeric virucidal agents that compromise stability of the viral envelope and exert permanent damage to the virion [ , ] . together, the broad spectrum of activity of polymers against viral pathogens and the chemical versatility of these agents renders this class of antiviral agents unique and attractive for deeper investigation. in our past studies, we developed broad-spectrum antiviral agents based on macromolecular prodrugs (mp) of ribavirin. when conjugated to polymers, ribavirin did not accumulate in the red blood cells which in vivo is the main side effect of the drug [ ] [ ] [ ] . mp of ribavirin broadened the therapeutic window of the drug (in vitro) [ ] and were effective against diverse viruses such as hcv (in a replicon cell culture model), measles, and influenza, in the latter case revealing antiviral effects in chicken embryo model [ , ] . we also experimentally confirmed the link between ribavirin and the synthesis of nitric oxide in macrophages, with relevance to the treatment of hepatitis [ ] . key to successful delivery of ribavirin using mp was the development of a disulfide-containing linker that releases the drug from the prodrug upon cell entry [ ] . from a different perspective, we also focused on preventative measures and conducted a systematic study of polymers with anionic charge as inhibitors of virus cell entry [ ] . our broad study considered polymers, infectious zika virus, hiv, and hsv, as well as pseudotyped viral particles for ebola, lassa, lyssa, sars, and other viruses. the main finding of our work was that anionic charge alone (be it carboxylate, phosphate/phosphonate, or sulfonate) did not endow the polymer with broad-spectrum antiviral activity. decisive factor for such activity was the enhanced (but balanced) hydrophobicity of the macromolecule. identified lead candidate polymers exhibited antiviral activity against all the enveloped viral pathogens, including the zika virus. for the latter, we presented evidence of direct contact between the polymer and the viral particle and concurrent inhibition of viral infectivity [ ] . one intriguing possibility lies in the prospect of combining the curative and the preventative antiviral activity within the same macromolecular antiviral agent [ , ] . such agents represent a combination therapy wherein the carrier exerts extracellular preventative effect [ ] whereas the conjugated drug, once released inside the cell, is an intracellularly active therapeutic [ ] . it is also possible that polyanions have an intracellular antiviral effect of their own, that is the inhibition of viral polymerases, [ , , ] presumably through competitive electrostatic interaction with the protein. in this work, we explore the design of such combination therapy in detail. we focus on the choice of the macromolecular backbone as a carrier for the conjugated drug and analyze blood coagulation, binding to albumin, albumin aggregation, inhibitory activity on polymerases, and cytotoxicity for polymers differed by their anionic charge (carboxylates, phosphates and phosphonates, sulfonates). further, we synthesize ribavirin-containing counterparts to the polyanions, investigate kinetics of drug release for the resulting macromolecular prodrugs, and investigate their use for intracellular drug delivery of ribavirin in an anti-inflammatory model in macrophages. as a result, we identify polymers and macromolecular prodrugs that are devoid of blood anti-coagulation activity but are strong as inhibitors of polymerases and efficacious as delivery vehicles for ribavirinthus being attractive for the development of broad-spectrum antiviral agents. we also identified polymers that are benign and devoid of any activity in these tests thus being attractive as "stealth" materials for diverse biomedical applications [ ] . results of this study would be important for the advancement of biomedical engineering, specifically with regards to development of antiviral therapies and drug delivery techniques. homopolymers of different anionic monomers with variations in the anionic functionality (carboxylic acid, sulfonic acid, phosphonic acid and phosphate, fig. ) were obtained through the reversible addition-fragmentation chain transfer (raft) polymerization [ ] . synthetic approach to the synthesis of the corresponding macromolecular prodrugs was chosen to proceed via copolymerization of the drug containing monomer and that corresponding to the carrier polymer. towards this end, rbv containing monomers were designed to be compatible with polymerization of diverse co-monomers, acrylic and methacrylic, hydrophilic and hydrophobic (fig. ). of these, the synthesis of the hydrophobic methacrylate monomer (monomer in fig. ) was reported in our previous publications [ ] . acrylic counterparts were obtained via similar protocols with minor variations. design consideration also concerned polarity of co-monomers, an aspect that exerts limitations to the choice of the solvent for polymerization. polarity of the applied anionic monomers varied from those with a hydrophilic character (phosphates/phosphonates) to those with well pronounced hydrophobic character (ethylacrylic acid, propylacrylic acid). rbv monomers were therefore designed to mimic the polarity of the anionic comonomers through the use of silyl protecting groups to render the monomer more hydrophobic or using deprotected, hydrophilic ′, ′-hydroxyl containing monomers. in total four different rbv prodrug monomers were applied (fig. ) , allowing for the copolymerization to be performed under hydrophobic and hydrophilic conditions with either acrylate/acrylamide or methacrylate/methacrylamide containing monomers. in each case, the monomer structure also comprised a disulfide bond for intracellular degradation, coupled to a self-immolative linker for drug release [ ] . due to the variations in monomer type and monomer polarity, a unique set of reaction conditions was necessary for each copolymerization including the choice of raft agent, solvent (or solvent mixture), reaction time, initiator, and purification procedure (table , for full details see experimental section). synthesized macromolecular (pro)drugs were analyzed by h nmr during synthesis to assess the conversion of the two monomers and in the purified form to confirm the absence of unreacted monomers. polymers were also characterized through size exclusion chromatography (using an -angle static light scattering detector) to determine the molar mass and dispersity of the polymer samples (table ) . we acknowledge that (co)polymers exhibited variability in their degree of polymerization (molar mass). for this reason, analyses presented below do not take into account the likely, non-negligible influence of molar mass on the properties of the polymers. in doing so, we assume that the polymer structure is the factor determining the polymer properties whereas the molar mass is of secondary importance. indeed, our recent publication on the antiviral activity of the polymers revealed clear structure-activity correlations with regards to the polymer functionality whereas no clear correlation with the polymer molar mass was observed [ ] . blood pro/anticoagulation is the most known side effect of polyanions upon their systemic administration. indeed, depending on structure, polyanions may exhibit anticoagulation effect, [ ] as is wellknown for heparin, [ ] a polysaccharide approved by the us fda specifically for the purpose of blood anticoagulation. other polyanions such as polyphosphates (nucleic acids) are reported to be pro-coagulants [ ] . no interference with blood coagulation is a highly desired safety feature of injectable formulations and identification of coagulation-inert polymers was one of the aims of this study. we investigated activated partial thromboplastin time (aptt) in presence of the synthesized polyanions. the platelet-depleted plasma from healthy donors was incubated with polyanions at concentrations of or mg/l and the aptt was measured (fig. ). polysulfonates and pvbza at mg/l enhanced blood coagulation time to a value over s, similarly to heparin, and this indicates that they are strong blood anti-coagulants. this was also observed with pvpa, which doubled the aptt value. in contrast, other polyphosphates and polyphosphonates papa, pmpa, paep and pmep had minor if any influence on blood coagulation time. of the carboxylates, paa and pmaa exhibited a noticeable anti-coagulation effect. in contrast, peaa and ppaa, two polymers with an enhanced hydrophobicity but decreased anionic character compared to paa and pmaa (higher pka) had no effect on blood coagulation time at mg/l. taken together, results in fig. reveal that six out of the tested polymers (polyphosphates, polyphosphonates, peaa and ppaa) appear to have no inhibitory activity with regards to the blood coagulation times. albumin is the most abundant protein in human plasma. it has numerous physiological functions that include the maintenance of table polymerization conditions and macromolecular characteristics of polyanions and macromolecular prodrugs used in this work. for chemical structures and details of synthesis of the rbv containing monomers through , see fig. and experimental section, respectively. chain transfer agents were cyanomethyl dodecyl trithiocarbonate (cdtc), ( -cyano- -[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid) (cdpa), cyanomethyl methyl(phenyl)carbamodithioate (cmpd), -(dodecylthiocarbonothioylthio)- -methylpropionic acid (dcma) or -cyano- -(phenylcarbonothioylthio)pentanoic acid (cppa); polymerization initiators were azobisisobutyronitrile (aibn), , ′-azobis( -methoxy- . -dimethyl valeronitrile) (v- ) or , ′-azobis( -cyanovaleric acid) (acva). m n : number-average molar mass, sec-mals: size exclusion chromatography with multi-angle light scattering detection, dp: degree of polymerization. values of dn/dc were calculated from sec/mals measurements assuming full mass recovery; dp values for ppaa, ppaa-rbv, and pvbza (marked with a * symbol) are calculated from m n (calc). experimental conditions for anionic homopolymers are taken from ref. osmotic pressure and the transport of hydrophobic solutes through the blood. this protein is safe-guarded by the body and physiological mechanisms exist to ensure that albumin is not degraded but retained in the body. as a result, albumin blood residence time reaches a phenomenal value of weeks [ ] . albumin binding is among the most successful methodologies in biomedicine to extend the circulation times of solutes and this has been achieved to a range of drug molecules, from small molecule anticancer drugs [ ] to peptide hormones and proteins [ ] . recently, we observed that synthetic polymers (peaa) can bind albumin and this afforded hepatic deposition of the polymer [ ] . albumin binding for peaa is not altogether surprising in that this protein is well known to bind hydrophobic solutes and anionic solutes, both features being emphasized in the structure of peaa. herein, we aimed to investigate albumin binding for the library of anionic polymers, with and without conjugation of ribavirin. albumin has several binding sites, most notable of which are the so-called sudlow i and sudlow ii sites [ , ] . binding of solutes to these sites can be interrogated using fluorescent probes that exhibit site-specific biding to albumin, specifically dansyl asparagine (sudlow i) and dansyl sarcosine (sudlow ii) [ ] . fluorescence of these probes is significantly decreased upon displacement from the protein globule, and this presents itself as a facile methodology to quantitate binding of albumin with the polymers. polymers were mixed with albumin at μm ( . g/l) concentration of the protein and molar equivalents of the polymer to albumin. the immediate observation from the results of these experiments ( fig. ) is that polymers differed significantly in their capacity to interact with albumin via the two nominated sites. polyphosphonate polymer papa brought about no decrease in the fluorescence of probes indicating no detectable polymer interaction with albumin. papa also exhibited no anticoagulation activity (fig. ) , and together these data suggest that papa is a blood-safe carrier for diverse drug delivery applications. albumin binding for the structurally similar pmpa as well as the phosphate analogues paep and pmep was also not pronounced and at -fold mole excess of the polymer to albumin, probe fluorescence decreased by no more than %. polysulfonates also exhibited minor association with albumin and afforded no change in fluorescence for the sudlow i binding probe and a change in fluorescence for the sudlow ii probe within %. exception to this was the most hydrophobic of these polymers, styrenic psvbs which showed a % probe displacement. in contrast, polycarboxylates exhibited a strong tendency for binding with albumin and exhibited a highly efficacious probe displacement. binding was not site specific and probes were displaced from both sudlow i and ii sites. in the case of paa, displacement of probes for both sudlow i and ii sites was nearcomplete indicating a strong binding. for this polymer, incorporation of rbv abrogated polymer binding to albumin. this illustrates that paa binding to albumin is structure-programmed and change to this macromolecule such as addition of rbv interferes with albumin binding. pmaa, peaa and ppaa also exhibited strong probe displacement capacity albeit not as pronounced as paa. interestingly, unlike paa-rbv, peaa-rbv and ppaa-rbv were strong albumin binders. albumin binding was also analyzed by sec-mals. this experiment confirmed the difference between the polymers in their capacity to binding albumin and highlighted further changes in the result of the polymer-protein association, fig. . elution of albumin was hardly altered upon its incubation with pamps (polysulfonate) taken at an equimolar concentration to albumin and there was minor protein aggregation in the presence of this polymer. this observation was similar for the protein incubation with papa and all polyphosphonates and polyphosphates as well as for pvsa and pspa. in contrast, for the carboxylates paa, pmaa, ppaa, pvbza and the hydrophobic polysulfonate psvbs, polymer-protein interaction afforded strong aggregation and formation of high molar mass products (mda). finally, upon incubation with peaa, a strong sudlow i/ii probe displacing polymer, elution of albumin was shifted to shorter times indicating increased molar mass of the protein, as would be expected for a polymer-protein adduct, with only minor protein aggregation. peaa is a unique polymer and albumin binding without noticeable aggregation was not observed fig. . polyanions and macromolecular prodrugs exhibit structure-dependent binding to albumin via sudlow i/ii sites. interaction with albumin was quantified using fluorescent probe displacement assay using dansyl asparagine and dansyl sarcosine as probes for sudlow i and sudlow ii sites, respectively. fluorescence intensities of the probe in the presence of polyanions were normalized to that in control experiments. % probe fluorescence indicates no probe displacement and % probe fluorescence implies quantitative displacement and indicates strong interaction between the polymer and albumin. results are presented as average of three independent experiments ± sd. statistical significance compared to the control samples was evaluated via one-way anova with dunnett's multicomparison post-hoc analysis. *p ≤ . ; **p ≤ . ; ***p ≤ . . for other polymers studied in this work. next, we examined macromolecular prodrugs with regards to the kinetics of drug release. rbv conjugation was designed such as to be reversed upon the scission of the disulfide linkage, specifically upon cell entry. intracellular environment is characterized by a relatively high concentration of a thiol containing tripeptide, glutathione (gsh), [ ] and this effectively triggers the degradation of disulfide linkages inside the cells [ ] [ ] [ ] . accessibility of the disulfide within the structure of macromolecular prodrugs can vary due to the difference in the size of the side groups functionalities. polymers may thus exhibit significant differences in the drug release kinetics. we have previously investigated the release of rbv and other drugs conjugated to polymers via the same linkage as used in this study (disulfide trigger paired with a self-immolative linker) via hplc [ , , ] . herein, we established an h nmr-based method to quantify the release of rbv as illustrated in fig. on an example of papa-rbv. macromolecular prodrug was incubated in the presence of gsh. disulfide reshuffling initiates the spontaneous cyclization of the self-immolative linker with ensuing release of the pristine drug, ribavirin (fig. , a) . chemical shift of the ribavirin protons exhibits a well-defined migration in the h nmr spectrum and with time, the signal corresponding to the polymer-bound drug decreases whereas that for the free rbv increases (fig. , b) . integration of the corresponding peaks provides a facile means to quantify rbv bound to the polymer and released from the prodrug. release of rbv from the macromolecular prodrug was fast and within h, over % of the drug was released from the macromolecular carrier (fig. ) . this kinetics profile is highly advantageous, significantly faster than that reported for the peptide-based linkers between the drug and the carrier, [ ] and allows for rapid drug release upon prodrug cell entry. we note that data in fig. are rather similar to the drug release data collected in our prior studies via hplc [ , , ] and this serves to validate the established herein nmr-based approach to monitor drug release. h nmr based method to quantify drug release was applied to the macromolecular prodrugs based on other polyanions as well as a nonionic carrier, phpma, fig. . within h of observations, all mp exhibited a pronounced release of their payload. a significant finding from this experiment was that hydrophobicity of the polymer chain has a pronounced effect on the kinetics of drug release. in contrast, no such correlation appears to exist with regards to the charge of the polymer (anionic vs charge neutral) or the nature of the negative charge on the polymer. indeed, structurally similar phpma and pmaa as well as papa and paep released ribavirin quantitatively within h revealing no difference in drug release kinetics. however, relatively small structural change from pmaa to peaa or from peaa to ppaa afforded a significant decrease in the drug release. similarly, a single methyl group variation from papa to pmpa afforded a pronounced drop in the amount of rbv released within h. a likely explanation to this phenomenon is the more compact, less hydrated and thus less accessible polymer coil for increasingly hydrophobic polymers which hinder gsh from initiating drug release. , as well as a significantly slower release from the methacrylamide based phosphonate relative to the acrylamide based phosphonate. all data are displayed as mean ± standard deviation from three independent experiments. for comparison a oneway anova (analysis of variance) was performed, followed by a tukey's post hoc test. ***: p ≤ . . therapeutic and/or side effects elicited by the polyanionic (pro) drugs investigated herein would likely manifest themselves due to the interaction of the polymers with other macromolecules such as proteins. the spectrum of polyanionic endogenous compounds is rather large, and competition [ ] for electrostatic interactions with these polymeric partners is highly likely. in the context of antiviral performance of the polymers, we [ ] [ ] and others [ ] have shown that polyanions can act as efficient inhibitors of polymerases. charge neutral polymers were devoid of such activity [ ] pointing to the anionic charge of the polymer (that is, electrostatic interaction with the target) as the origin of the observed phenomenon. polymers synthesized in this work were analyzed as inhibitors of two types of polymerase enzymes, namely a dna-dependent dna polymerase (replicase) and an rnadependent dna polymerase (reverse transcriptase), fig. . despite being similar in carrying multiple anionic charges, polymers differed markedly in their ability to interfere with the performance of polymerases. polyphosphates and polyphosphonates were nearly devoid of any inhibitory activity in these assays and regardless of the polymer concentration, polymerases remained uninhibited. together with the data presented above, e.g. papa appears to have unique "stealth-like" properties with no-interference in coagulation assays, albumin binding, and competition for binding with polymerases. in contrast, polycarboxylates and polysulfonates exhibited a dose-dependent polymerase inhibition. a rather surprising finding is that high anionic character alone does not make the polymer a strong inhibitor and in the homologous row of carboxylates, it was peaa that exhibited the strongest inhibitory activity (complete inhibition of reverse transcriptase activity at mg/l polymer concentration). this observation echoes our recent findings on the apparent unique pairing of negative character and hydrophobicity of the polymer backbone that renders peaa an efficacious inhibitor of e.g. hepatitis c virus intracellular replication [ ] and a lead polymer with broad-spectrum antiviral activity [ ] . similar observation can be made for the negatively charged pvbza, also characterized by pronounced hydrophobicity of the chain due to the aromatic ring in each repeat unit of the polymer. finally, as a test for activity of the polymers in cell culture, we evaluated polyanions and their counterparts conjugated to rbv as inhibitors of inflammation in stimulated macrophages. our previous findings revealed that paa-rbv inhibited the synthesis of an inflammatory marker nitric oxide (no) in the lipopolysaccharide (lps)stimulated macrophages whereby both the polymer and the released drug elicited their individual anti-inflammatory activity [ ] . we tested the library of polyanions presented above and quantified inflammatory responses in cells incubated with polymers in a range of concentrations from . to mg/l. independently, metabolic activity of the cells (indicative of cell viability) was measured by the presto blue assay. these experiments therefore provide information on both, toxicity of the polymers to macrophages in cell culture and anti-inflammatory activity of the polymers. none of the tested polymers, with or without conjugated ribavirin, exhibited noticeable toxicity at concentrations up to mg/l, fig. . the overall majority of polymers were also devoid of activity with regards to inflammation. however, several polymers comprised notable exceptions and were efficacious inhibitors of inflammation without associated toxicity to the cells. specifically, among the polyanions, pmaa, peaa, pvbza and psvbs afforded significant decrease in the levels of no produced by macrophages. an apparent unifying structural feature of these four polymers is that these are the most hydrophobic of the polyanions used in this work. decreased inflammatory response by pvbza and psvbs was observed already at mg/l making these polymers most potent of the tested polyanions. it is also striking that fig. . activity of the polymerase enzyme in the presence of polyanions ( or mg/l in the reaction mixture) expressed in % of de novo synthesized nucleic acid relative to the un-inhibited polymerase reaction. statistical significance compared to control was evaluated via a one-way anova with dunnett's multicomparison posthoc analysis. *p ≤ . ; **p ≤ . ; ***p ≤ . . these two polymers, as well as peaa, afforded a highly efficacious decrease in the synthesis of no with a near-complete suppression of inflammation without associated toxicity to the cells. this comes in stark contrast with the rbv based treatment which could only afford ca. % reduction in the levels of no at sub-toxic concentrations of the drug [ ] . it is worthy of note that polyanions are typically deemed to have restricted cell entry [ ] . however, confocal laser scanning microscopy evaluation of the polymer uptake by macrophages revealed pronounced polymer-associated fluorescence inside the cells (fig. ) . this was true for all polymers: polycarboxylates, polyphosphates and polyphosphonates, and polysulfonate. contrary to expectations, cell entry does not appear to be decisive in the observed activity of the polymers. macromolecular prodrugs of rbv were synthesized for the (meth) acrylate/(meth)acrylamide type of anionic monomers thus excluding pvbza, pvpa, psvbs, and pvsa. both pmaa and peaa displayed an inherent, structure-driven effect in the anti-inflammatory assay and in these cases it is therefore not possible to discern the effects elicited by the polymer or the intracellularly released rbv. it is likely that both the polymer and the released drug contribute to the overall anti-inflammatory response in macrophages for pmaa-rbv and peaa-rbv. surprising other leads in this assay were macromolecular prodrugs based on pmpa and pamps. in both cases, statistical significance of anti-inflammatory effects was observed at concentrations at and above mg/l. this effect was efficacious and had minor if any associated toxicity. to further probe the mechanism of activity of the polymers in this assay, we investigated potential antagonism of the polyanions with lps. indeed, the latter has anionic component to it and competion with polyanions for interaction with the receptor is not inconceivable. furthermore, prior reports suggested that sulfated polyaromatic suramin can antagonize lps [ ] . to investigate this on the example of peaa, lps stimulation of macrophages was performed using increasing concentration of lps in the presence or absence of a fixed concentration of the polymer (fig. ) . in this assay, antagonism at the receptor in absence of intracellular effects would manifest itself as a shift in the potency of lps without a change in efficacy. in turn, intracellular effects in absence of receptor antagonism would mean a decreased efficacy of the inflammatory response with no change in the potency of lps. experimental data suggest that both effects are likely observed (fig. ) and peaa exerts its activity through both, receptor antagonism and intracellular effects. indeed, the polymer affords statistically significant decrease in the inflammation and also a change, albeit a very minor one, in the ic value for lps with regards to stimulation of macrophages ( . vs . mg/l in the presence of peaa). results of this study provide a comprehensive view on polyanions as the backbone for macromolecular broad-spectrum antiviral therapeutics. together with the results of the screen of these polyanions for inherent antiviral activity, [ ] presented data provide a suggestive view on the utility of polyanionic macromolecular prodrugs of ribavirin as antiviral agents (summarized in table ). first, we wish to evaluate the validity of our assumption that (table ) can mask or skew the observed effects and lead to false negatives or misinterpretation of data. furthermore, terminal groups arising from the different raft agents used in this study to produce the polymers may also considerably change polymer properties [ ] . with regards to the latter point, we note that in our hands, macromolecular prodrugs of rbv prior to and after removal of their cleavable end-groups revealed no difference in the antiinflammatory activity [ ] . in this work, to accommodate the synthetic diversity of polymers, we used five different raft agents (table ) and we see no correlation of polymer activity (table ) and the raft agent used for the polymer synthesis. similarly, experimental results presented in this work appear to show no correlation with the polymer molar mass. blood anti-coagulation effect of the polymers (fig. ) shows strong dependence on the nature of the polymer charged functionality (sulfonates > carboxylates > phosphates and phosphonates) but not with m n (dp). albumin binding results (probe displacement results, fig. ) illustrate that polycarboxylates are strong albumin binders despite the variation of molar mass while phosphate-containing polyanions are weak binders, even the highest molar mass papa. drug release kinetics for macromolecular prodrugs (fig. ) shows a statistically significant correlation with the polymer hydrophobicity but not with polymer dp. polymers most active in rbv delivery were not those characterized by the lowest or the highest dp (in fact, the lowest and the highest dp polymers were not effective in this screen) suggesting that polymer structure, not the number of repeat units in the structure, is decisive in the utility of the polymer as a carrier for the conjugated drug. without doubt, examples of strong, pronounced effects of molar mass on the biomedical properties of polymers are numerous and importance of this factor should not be overlooked. in our own prior work, with regards to the delivery of rbv, we found that high molar mass restricted the polymer cell entry and in doing so limited the observed therapeutic effects [ , ] . not disregarding the importance of these observations, the data of this study strongly suggest chemical structure of the polymer is a dominant factor in its properties (at least in the experiments presented in this study) whereas polymer chain length is of secondary importance. the first important conclusion with regards to the activity of polymers is that the lead polymer structures with inherent broad-spectrum antiviral activity (peaa, pvbza, psvbs) [ ] appear to also be strong anti-coagulants and strong albumin binders (figs. , ) . in turn, weak anti-coagulants and weak albumin binders are also weak inhibitors of virus infectivity. this conclusion may not be altogether surprising in that both anti-coagulation and antiviral effects are likely due to the interaction between the polymers and a proteinaceous target (for antiviral effectsglycoproteins of the virus capsid). as such, this result implies that polyanionic macromolecular (pro)drugs are not well suited for systemic administration to exert antiviral activity in circulation. from a different perspective, the three leads with broad-spectrum antiviral activity [ ] are also shown in this work to be inhibitory to the activity of polymerases and to possess inherent activity within macrophages, in absence of cytotoxicity. this observation is intriguing in that these three polymers may therefore be inhibitory to viruses both extraand intracellularly, in part fulfilling the desired combination of antiviral effects. in our view, these polymers become lead candidates for optimization and design of microbicides for non-systemic, localized antiviral treatments. regretfully, design of ribavirin mp was only considered in this work for (meth)acrylates and (meth)acrylamides; we are currently investigating the synthesis of mp based on pvbza and psvbs as a next step in the optimization these antiviral (pro)drugs. also worthy of note is the discovery of a family of polyanions that are devoid of any inhibitory activity in the screens we have performed, namely the polyphosphates and polyphosphonates. these polymers appear to have only weak and in most cases minimal interaction with the proteinaceous targets in our assays. due to this, as well as their high solubilizing power (as is well used in the design of low molar mass prodrugs [ ] ) and inherent affinity to bone [ ] , these polymers are highly attractive as carriers for diverse drug delivery applications and specifically for bone targeting. the results are an average of three independent experiments n = ± sd. lower standard error bars were omitted for clarity. the statistical difference in inflammation between peaa and non-peaa treated cells was compared using unpaired student's t-test. p < . **. summary of experimental results of this study and the screen for inherent broad-spectrum antiviral activity (**, taken from ref. [ ] ). results are denoted as "+" if the polymer exhibited the nominated property (being increasingly pronounced on a scale from + to +++), "−" if the polymer is devoid of this property or effect, and "n/a" if the polymer was not analyzed in this test. anti-inflammatory activity is denoted '+' if the effect is observed with minimal cytotoxicity of treatment. all chemicals were purchased from sigma aldrich and used without further purification unless stated otherwise. ribavirin was purchased from apichemistry. h nmr and c nmr were recorded with a bruker biospin gmbh mhz nmr spectrometer. multiplicities are indicated by abbreviations. s = singlet, bs = broad singlet, d = doublet, t = triplet, p = pentet, m = multiplet. hrms was recorded on a bruker maxis impact-tof-ms with electrospray ionization (esi+). size-exclusion chromatography (sec) was performed using a system comprising a lc- ad shimadzu hplc pump, a shimadzu rid- a refractive index detector and a wyatt dawn heleos light scattering detector along with a spd-m a pda detector, equipped with either ) a hema-bio linear column with μm particles, a length of mm and an internal diameter of mm from mz-analysentechnik in series with a ohpak sb- hq shodex column with the dimensions . × mm a particle size of μm or b) mz-gel sdplus linear column with μm particles length of mm and an internal diameter of mm from mz-analysentechnik providing an effective molecular weight range of - . . or c) superose increase / gl from ge healthcare with a pore size of um, a particle size of . um, an inner diameter of mm and a length of mm providing an effective molecular weight range of , - , , da with an exclusion limit > , , da. the solvent used was either a) . m pbs filtered through a . μm filter with ppm sodium azide at . ml/ min at °c or b) dmf with mm libr, at . ml/min at °c or c) . m pbs filtered through a . μm filter with ppm sodium azide, at . ml/min at °c. values of dn/dc used for molar mass calculations were established by wyatt mals/astra software assuming full mass recovery. plate reader experiments were performed in black well optiplates on an enspire multilabel reader (perkin elmer®). monomer ( mg, . mmol, equiv.) was dissolved in dry thf ( ml) under a n atmosphere. tea· hf ( . ml, . mmol, equiv.) was added and the reaction was stirred at room temperature for h. the product was purified by flash column chromatography meoh/dcm : to : . residual triethylamine was removed by dissolving the crude mixture in dcm, washing with nh cl twice, once with water, and once with brine. the organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo yielding the pure product. tea ( . g, . mmol, equiv.) was added over a cold ( °c) solution of -hydroxyethyl disulfide ( . g, . mmol, equiv.) in dcm ( ml) under n atmosphere. acryloyl chloride ( . g, . mmol, equiv.) was added dropwise maintaining the temperature at °c. the reaction mixture was heated slowly to room temperature and stirred for h. the reaction was quenched and washed twice with nh cl and brine. the organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo. the product was purified by flash column chromatography pentane/etoac : to : yielding the pure product as a light yellow liquid ( . g, %). a solution of (( r, r, r, r)- , -bis((tert-butyldimethylsilyl)oxy)- -( -carbamoyl- h- , , -triazol- -yl)tetrahydrofuran- -yl)methyl ( nitrophenyl) carbonate ( . g, . mmol, equiv.) in dry dcm ( ml) was added over a cold solution of -(( -hydroxyethyl)disulfaneyl)ethyl acrylate ( . g, . mmol, equiv.), diea ( . g, . mmol, equiv.) and dmap ( . g, . mmol, . equiv.) in dry dcm ( ml) under n atmosphere. the reaction mixture was stirred at room temperature for h resulting in a conversion of %. the reaction was quenched and washed twice with nh cl and brine. the organic phase was dried over magnesium sulfate, filtered, and concentrated in vacuo. the product was purified by flash column chromatography pentane/etaco : to : . residual p-nitrophenol was removed by basic alumina column chromatography etoac/meoh : yielding the pure product as a colorless solid ( . g, %). monomer was synthesized following the protocol described above with an in situ deprotection performed without purification of the monomer . thus, the crude mixture obtained according to the protocol for the synthesis of monomer was dissolved in thf ( ml) under n atmosphere. tea• hf ( . g, . mmol) was added and the reaction was stirred for h. the solvent was removed in vacuo and the product was purified by flash column chromatography dcm/meoh : to : . residual tea was removed by dissolving the product in chloroform and washing twice with nh cl yielding the pure product ( . g, %). experimental details for the syntheses of ethylacrylic acid, propylacrylic acid, -methacrylamidoethyl phosphate, -acrylamidoethyl phosphate, -methacrylamidoethyl phosphonic acid and -acrylamidoethyl phosphonic acid were performed according the protocols published previously [ ] . anionic homopolymers were synthesized as previously described in ref. dcma ( . mg, . mmol, equiv.) and v- ( . mg, . mmol, . equiv.), were dissolved in methanol ( . ml). monomer was dissolved in dmf ( . ml), amps dissolved in water ( . ml) was added. five rounds of freeze-pump-thaw were performed before the ampule was flame sealed and the reaction performed at °c for h. the reaction mixture was diluted with a ph = . nahco / na co buffer and purified by dialysis followed by lyophilisation to obtain the pure product ( mg recovered). . . . poly( -methacrylamidoethyl phosphonic acid-co-rbv) -methacrylamidoethyl phosphonic acid ( . g, . mmol, equiv.) was dissolved in acetate buffer ( . ml). monomer ( . g, . mmol, equiv.) dissolved in meoh ( . ml) was added together with cdpa ( . mmol, equiv.) and acva ( . mmol, . equiv.) stock solutions. methanol ( . ml) was added giving a slightly unclear solution. five rounds of freeze-pumpthaw were performed before the ampule was flame sealed and the reaction performed at °c for h. the reaction mixture was diluted with a ph = . nahco /na co buffer and purified by dialysis followed by lyophilisation to obtain the pure product ( mg). monomer ( . mmol, equiv.), cdpa ( . g, . mmol, equiv.), and v- ( . mmol, . equiv.) dissolved in methanol ( . ml) was added to -acrylamidoethylphosphonic acid ( . g, . mmol, equiv.) dissolved in water ( . ml). six rounds of freeze pump thaw were performed before the ampule was flame sealed and the reaction was performed at °c for h. the reaction mixture was diluted with a ph = . nahco /na co buffer and purified by dialysis, obtaining the pure product upon lyophilisation. cdpa ( . g, . mmol, equiv.), v- ( . g, . mmol, . equiv.), and monomer ( mg, . mmol, equiv.) were dissolved in methanol ( . ml) and added to -methacrylamidoethyl phosphate ( mg, . mmol, equiv.) dissolved in water ( . ml). the mixture was slightly unclear. five rounds of freeze pump thaw were performed before the ampule was flame sealed and the reaction performed at °c for h. a small amount of precipitate was observed upon reaction, the mixture did not change notably in viscosity. the reaction mixture was diluted with a ph = . nahco /na co buffer and purified by dialysis. a second dialysis was performed in : ethanol/water yielding the pure product which was lyophilized. monomer ( . mmol, equiv.), cdpa ( . g, . mmol, equiv.), and v- ( . mmol, . equiv.) dissolved in methanol ( . ml) was added to -acrylamidoethyl phosphate ( . g, . mmol, equiv.) dissolved in water ( . ml). six rounds of freeze pump thaw were performed before the ampule was flame sealed and the reaction performed at °c for h. the reaction mixture was diluted with a ph = . nahco /na co buffer and purified by dialysis. a second dialysis was performed in : ethanol/ water yielding the pure product upon lyophilization. the competition assay was performed in -well plates, and fluorescence analyzed (excitation wavelength nm, emission wavelength nm) on a plate reader. polymers were pre-dissolved in minimal amounts of dmso and then diluted in . m pbs (ph . ). stock solutions of human serum albumin (hsa) and fluorescent probes (dansyl asparagine/dansyl sarcosine) were prepared in . m pbs (ph . ). the components were mixed and pbs added to obtain a total volume of μl in each well, with the following concentrations: hsa μm, dansyl sarcosine μm or dansyl asparagine μm, and polymer at μm, corresponding to equivalents compared to albumin. upon mixing the plates were incubated at °c for h to allow the system to equilibrate. the data represents three independent experiments, which each included three technical replicates. polymers and albumin ( g/l) were dissolved in . m pbs with ppm nan in equimolar amounts. the samples were incubated for h at room temperature and then directly injected into the sec-mals system equipped with a biocolumn and using pbs as an eluent. results were analyzed using the wyatt software astra . a sample of albumin with no polymer added was used as reference in order to compare the albumin peak both with regards to the determined molecular weight and the elution times observed. ribavirin containing polymers were pre-dissolved in minimal amounts of ddmso (if necessary) and diluted in . m pbs (ph . ) prepared from d o. glutathione (gsh) was also dissolved in . m pbs (ph . ) prepared from d o. an aliquot of the polymer solution was mixed with an aliquot of the gsh solution to obtain a . ml sample containing . g/l of polymer and . mm gsh. the sample was incubated for the described time upon which ellmann's reagent was added in equimolar amounts compared to gsh to quench the reaction. the quenched samples were analyzed by h nmr spectroscopy as described in the main text. time point measurements were repeated at least three times for each polymer. raw . macrophage cell line (tested negative for mycoplasma) was used to determine the anti-inflammatory activity of polyanions. the cells were seeded on -well flat-bottomed plate at initial density of · cells per well in μl of media: high glucose dmem, stable lglutamine, without phenol red (gibco, ) supplemented with % fbs and penicillin/streptomycin. three hours after the cells were seeded, the polymers were added at indicated concentrations in μl of the media and incubated with the cells for h. after h, media was removed and the cells were washed with pbs. subsequently, the cells were stimulated with lps (sigma, l ) for h. the level of nitric oxide was quantified via griess assay. briefly, μl of the cell supernatant was mixed with μl of sulfanilic acid ( g/l, % phosphoric acid). after min of incubation, μl of n- -napthylethylenediamine dihydrochloride ( g/l) was added and absorbance ( nm) was measured using a plate reader (bmg labtech, ortenberg, germany). the level of nitrite was quantified against sodium nitrite standard curve and normalized to the negative control which was lpsstimulated cells only. cell viability was measured using prestoblue viability reagent (invitrogen, a ). briefly, the cells were incubated with medium containing % volume of prestoblue reagent. after h of incubation time the fluorescence ( nmexperimental wavelength, nmreference wavelength) was measured on the plate reader (bmg labtech, ortenberg, germany). the cell viability was normalized to the viability of the cells incubated with pure media. the blood obtained from healthy donors was collected into sodium citrate . % tubes. platelet poor plasma (ppp) was obtained by centrifuging the blood at g for min at °c. the polymers were dissolved in pbs buffer containing % dmso. polymers in the solution were mixed with ppp in volume:volume ratio : resulting in the final polymers concentration of and mg/l. prothrombin time was measured using mrx owren's pt reagent (medirox, ghi - ) and activated partial thromboplastin time was measured using dade actin fs (siemens, b - ). all measurements were performed on sysmex cs- i. pbs and pbs with % dmso were used as a negative control. heparin (sigma, h ) was used as a positive control. to analyze inhibitory effect of polymers on dna-dna polymerase activity, quantity pcr (qpcr) reaction was performed using power sybr green pcr master mix (life technologies) according to the protocol provided by the manufacturer. for one sample μl of green pcr master mix were mixed with forward. ′-ggtctctctggttagaccagat- ′ and reverse primer ′-ctgctagagattttccacactg- ′, . ng of phxb -env plasmid (nibsc, programme eva centre for aids reagents, reference number: arp ) and a polymer diluted in pbs to a desired concentration. primers were complementary to ltr upstream element of gag. the program used was °c for min followed by cycles with °c for s, °c for s and during the last cycle, a melting curve was made. a level of inhibition of taq polymerase was calculated by comparing to a positive control without polymer as % of enzyme activity. influence of polymers on activity of reverse transcriptase was determined using enzchek reverse transcriptase assay kit (life technologies). the reaction mixture was prepared according to the protocol provided by the manufacturer. subsequently polymers diluted in pbs were added at the given concentration, and then u of mulv reverse transcriptase (life technologies, cat. nr. n ). the reaction was performed at °c for h. nucleic acids were stained with picogreen dye and fluorescence was measured on a plate reader (bmg labtech, ortenberg, germany). cellular uptake of polyanions in macrophages was evaluated by confocal laser scanning microscopy. coverslips (thermofisher scientific) placed in well plates were coated with . % poly-l-lysine in water (sigma-aldrich) at °c for h, and washed twice with pbs ph . (gibco). raw . cells (mycoplasma negative) were seeded on each coverslip in the -well plate in μl media (high glucose dmem, stable l-glutamine (gibco) supplemented with % fbs and penicillin/streptomycin). h after seeding, media was removed from wells, and μl of mg/l of fluorescently labelled polyanion diluted in media was added to the well. h after addition of polyanions, cells on coverslips were processed for confocal microscopy: coverslips were washed twice with pbs and fixed with % paraformaldehyde in pbs for min at rt, followed by washing twice with pbs. coverslips were stained with μg/ml dapi in pbs for min, and dapi was removed by washing twice with pbs. cell membrane was stained with μg/ml concanavalin a conjugated to alexa fluor® (lifetechnologies) for min at rt. following this staining step, coverslips were washed twice with pbs and transferred, cells facing down, to a microscopy slide (vwr), and then the cells were submerged in a prolong™ diamond antifade mountant (thermofisher scientific). slides were visualised using a zeiss lsm confocal with ×/ . oil dic objective, using excitation line nm for dapi, nm for fitc and nm for alexa fluor® with no spectral overlap in collected emission. emerging viral diseases: confronting threats with new technologies global and regional mortality from causes of death for age groups in and : a systematic analysis for the global burden of disease study the design of drugs for hiv and hcv approved antiviral drugs over the past years combating emerging viral threats advances in antiviral vaccine development topical microbicides to prevent hiv: clinical drug development challenges microbicides and other topical strategies to prevent vaginal transmission of hiv broad-spectrum antiviral agents broad-spectrum agents for flaviviral infections: dengue, zika and beyond the use of interferon-alpha in virus infections is the -year hepatitis c marathon coming to an end to declare victory? ribavirin -current status of a broad spectrum antiviral agent antiviral agents active against influenza a viruses broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis nitazoxanide: a first-in-class broad-spectrum antiviral agent a novel peptide with potent and broad-spectrum antiviral activities against multiple respiratory viruses broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses broad-spectrum antivirals against viral fusion nucleic acid polymers: broad spectrum antiviral activity, antiviral mechanisms and optimization for the treatment of hepatitis b and hepatitis d infection macromolecular antiviral agents against zika, ebola, sars, and other pathogenic viruses the isolation and crystallization of plant viruses and other protein macro molecules by means of hydrophilic colloids inhibition of mumps virus multiplication by a polysaccharide the combination of lysine polypeptides with tobacco mosaic virus inhibitory effect of synthetic lysine polypeptides on growth of influenza virus in embryonated eggs inhibitory effect of heparin on herpes simplex virus antiviral activity of polyacrylic and polymethacrylic acids. i. mode of action in vitro dextran sulfate suppression of viruses in the hiv family: inhibition of virion binding to cd + cells macromolecular (pro) drugs in antiviral research interferon and its inducers-a never-ending story: "old" and "new" data in a new perspective the rise and fall of polyanionic inhibitors of the human immunodeficiency virus type living radical polymerization by the raft process -a third update atom transfer radical polymerization (atrp): current status and future perspectives a highly lipophilic sulfated tetrasaccharide glycoside related to muparfostat (pi- ) exhibits virucidal activity against herpes simplex virus polymeric coatings that inactivate both influenza virus and pathogenic bacteria macromolecular prodrugs of ribavirin combat side effects and toxicity with no loss of activity of the drug macromolecular prodrugs for controlled delivery of ribavirin macromolecular prodrugs of ribavirin: towards a treatment for co-infection with hiv and hcv polyanionic macromolecular prodrugs of ribavirin: antiviral agents with a broad spectrum of activity macromolecular prodrugs of ribavirin: structure-function correlation as inhibitors of influenza infectivity macromolecular (pro)drugs with concurrent direct activity against the hepatitis c virus and inflammation highly active macromolecular prodrugs inhibit expression of the hepatitis c virus genome in the host cells synthesis of new covalently bound kappa-carrageenan-azt conjugates with improved anti-hiv activities polyanionic (i.e., polysulfonate) dendrimers can inhibit the replication of human immunodeficiency virus by interfering with both virus adsorption and later steps (reverse transcriptase/integrase) in the virus replicative cycle triple activity of lamivudine releasing sulfonated polymers against hiv- overcoming the peg-addiction: well-defined alternatives to peg, from structure-property relationships to better defined therapeutics self-immolative linkers literally bridge disulfide chemistry and the realm of thiol-free drugs overview of anticoagulant activity of sulfated polysaccharides from seaweeds in relation to their structures, focusing on those of green seaweeds guide to anticoagulant therapy: heparin: a statement for healthcare professionals from the extracellular rna constitutes a natural procoagulant cofactor in blood coagulation the neonatal fc receptor, fcrn, as a target for drug delivery and therapy prodrugs in medicinal chemistry and enzyme prodrug therapies materials and methods for delivery of biological drugs synthetic polymer with a structuredriven hepatic deposition and curative pharmacological activity in hepatic cells the interaction of ms- with human serum albumin and its effect on proton relaxation rates crystal structure analysis of warfarin binding to human serum albumin -anatomy of drug site i glutathione homeostasis and functions: potential targets for medical interventions disulfide-cleavage-triggered chemosensors and their biological applications drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities ligand-targeted drug delivery disulfide reshuffling triggers the release of a thiol-free anti-hiv agent to make up fast-acting, potent macromolecular prodrugs prodrug strategies in anticancer chemotherapy competitive reactions in solutions of poly-l-histidine, calf thymus dna, and synthetic polyanions: determining the binding constants of polyelectrolytes macromolecular prodrugs of ribavirin: concerted efforts of the carrier and the drug tools of gene transfer applied to the intracellular delivery of non-nucleic acid polyanionic drugs inhibition of lipopolysaccharide and il- but not of tnf-induced activation of human endothelial-cells by suramin phosphorus-containing polymers: a great opportunity for the biomedical field we wish to acknowledge financial support from the danish council for independent research, technology and production sciences, denmark (anz; project dff - - ). key: cord- - laifjgz authors: anuj, samir a.; gajera, harsukh p.; hirpara, darshna g.; golakiya, baljibhai a. title: bactericidal assessment of nano-silver on emerging and re-emerging human pathogens date: - - journal: j trace elem med biol doi: . /j.jtemb. . . sha: doc_id: cord_uid: laifjgz with the threat of the growing number of bacteria resistant to antibiotics, the re-emergence of previously deadly infections and the emergence of new infections, there is an urgent need for novel therapeutic agent. silver in the nano form, which is being used increasingly as antibacterial agents, may extend its antibacterial application to emerging and re-emerging multidrug-resistant pathogens, the main cause of nosocomial diseases worldwide. in the present study, a completely bottom up method to prepare green nano-silver was used. to explore the action of nano-silver on emerging bacillus megaterium mtcc and re-emerging pseudomonas aeruginosa mtcc pathogenic bacteria, the study includes an analysis of the bacterial membrane damage through scanning electron microscope (sem) as well as alternation of zeta potential and intracellular leakages. in this work, we observed genuine bactericidal property of nano-silver as compare to broad spectrum antibiotics against emerging and re-emerging mode. after being exposed to nano-silver, the membrane becomes scattered from their original ordered arrangement based on sem observation. moreover, our results also suggested that alternation of zeta potential enhanced membrane permeability, and beyond a critical point, it leads to cell death. the leakages of intracellular constituents were confirmed by gas chromatography-mass spectrometry (gc–ms). in conclusion, the combine results suggested that at a specific dose, nano-silver may destroy the structure of bacterial membrane and depress its activity, which causes bacteria to die eventually. antibiotics deserve much of the credit for the dramatic increase in life expectancy around the world in the th century. a famous physician expert once noted that the discovery of penicillin in the early s showed more curative power to a lone provider than the collective talent of all the physicians in new york at that time [ ] . unfortunately, it is inevitable that, after sometime, bacteria develop resistance to existing antibiotics, making infectious disease more difficult to treat [ ] . the resistance profile has probably been smouldering issue for years, but nowadays it's almost like a switch got triggered. infectious disease experts are alarmed by the prospect that effective antibiotics may not be available to control the re-emergence of previously deadly infections and the emergence of new infections caused by drugresistant bacteria in near future. recent outbreaks of pneumonia (pseudomonas aeruginosa) and tuberculosis (mycobacterium tuberculosis), for example, both of which have re-emerged within the region itself, have also focused regional, national and international attention on the threat developed by re-emerging infectious diseases and in particular on southeast asia as the epicentre of these types of diseases. indeed, tuberculosis and pneumonia are re-emerging in a drug-resistant form [ , ] . southeast asia is also a hotspot for emerging infectious disease in particular, zoonotic diseases as a result of many factors including population growth, mobility and environmental changes. severe acute respiratory syndrome (sars), nipah virus and the most recent pandemic influenza a h n are only a few of many examples of emerging infectious diseases in the southeast asia; each of these diseases has caused global societal impact related to unexpected illnesses [ ] . other emerging infectious diseases caused by bacteria are less terrible than these examples; however, they nonetheless may take a significant increase in morbidity as well as cost related to prolong treatments. in recent years, the infection caused by bacteria in man to have probably emerged in china was brain abscess during infection with bacillus megaterium [ ] . to the best of our knowledge, infections caused by these bacteria are rare and have not been reported as the cause of any important clinical diseases [ , ] . while it is not known which new infectious diseases will emerge tomorrow, populations have to be protected by staying one step ahead of the microbes by searching a new antimicrobial drugs. moreover, researchers also discovered some bacterial species that are resistant to different classes of antibiotics, including new synthetic drugs, even though these bacteria have never exposed to antibiotics [ , ] . these studies support a hypothesis that antibiotic resistance is genetically rich natural phenomenon, deeply embedded in the bacterial pan-genome in which bacteria evolve; it can be slowed but not stopped. if these types of non-pathogenic bacteria create any health problems, then it's very difficult to control them by conventional antibiotics. hence, we need new antibiotics to control resistant bacteria, which must act at several cellular and molecular levels. the mode of action of nano-silver is not known for specific at single sight but to influence bacterial membrane permeability and many metabolic pathways at same time. the present study aimed to understand the antibacterial activity and acting mechanism of nano-silver, the mechanism of inhibition against emerging b. megaterium mtcc and re-emerging p. aeruginosa mtcc through alternation of overall surface charges and destroying membranous structure with consequent leakages of important intracellular constituents such as amino acids, fatty acids, organic acids and sugar acids. the study represented a potential template for the design of novel antibacterial agents to overcome the issue of emerging and re-emerging infectious diseases. the medicinal plant, tinospora cordifolia was selected from gir forest region of gujarat, india on the basis of its medicinal property [ ] . silver nitrate, ar grade was procured from thermo fisher scientific, vadodara, india. the two bacterial strains one gram positive (bacillus megaterium mtcc ) and one gram negative (pseudomonas aeruginosa mtcc ) are clinical isolates that were obtained from the microbial type culture collection (mtcc), institute of microbial technology, chandigarh, india. bacterial strains were cultivated on nutrient broth medium (ph . ± . ; himedia, vadodara, india) at °c with shaking at rpm. bacterial cell suspensions were diluted with a sterile milli-q water (ph ) to obtain a final concentration of cfu/ ml. four broad spectrum antibiotics, namely, amoxicillin (sun pharmaceutical, ankleshwar, india), chloramphenicol (alps pharmaceuticals, uttarakhand, india), trimethoprim-sulfamethoxazole (abbott healthcare, mumbai, maharashtra), and linezolid (glenmark pharmaceutical, ankleshwar, india) were used for bactericidal assay. all other chemicals used in this study were analytical grade (sigma aldrich, ahmedabad, india). for the preparation of aqueous extract, g of dry stem powder was boiled in a conical flask with ml of deionised water for min at °c on magnetic stirrer at rpm, then the mixture was filtered through whatmanno. filter paper, and the stem extract was collected and stored at °c for further use in synthesis of nano-silver. aqueous solution ( − m) of silver nitrate (agno ) was prepared and stem extract of t. cordifolia was added ( : ratio) in dark condition for the reduction of silver nitrate at room temperature [ ] . the bioreduction of silver nitrate was confirmed by uv-vis spectrophotometric (agilent technologies, cary ) analysis. dynamic light scattering (dls) technique was used to determine the size and the potential stability of the nano-silver in the suspension using nanotrac wave (s ). the surface morphology of nano-silver along with elemental analysis was determined by scanning electron microscope (zeiss, evo- ) coupled with an energy dispersive x-ray analysis (edx) facility by applying an acceleration voltage of kv. the comparative antibacterial activities of nano-silver and broad spectrum antibiotics was effectively accessed against emerging pathogens bacillus megaterium mtcc and re-emerging pathogens pseudomonas aeruginosa mtcc using agar well diffusion assay method [ ] . the drug of choice for each tested strain was used to prove the unique bactericidal action of nano-silver compared to conventional broad spectrum antibiotics, namely, amoxicillin (inhibits bacterial cell wall synthesis), trimethoprim-sulfamethoxazol (inhibits synthesis of proteins and nucleic acids), chloramphenicol (inhibits bacterial protein synthesis by preventing elongation process) and linezolid (inhibits the initiation process in bacterial protein synthesis at a very earlier stage) [ ] . the concentration of nano-silver and antibiotics was adjusted to μg/ml, and the concentration of bacterial cells was cfu/ml. after incubation at °c for h the zones of inhibition was measured [ ] . the assays were performed with three replications and standard deviation between replication was calculated. samples from bacterial cultures (bacillus megaterium mtcc and pseudomonas aeruginosa mtcc ), mixed with μg/ml of nano-silver, were collected at h and pre-fixed with . % glutaraldehyde for min; these were then washed two times in the same buffer and post-fixed for h in % osmium tetroxide. after washing with buffer, dehydration process was conducted with , , , , and % of ethanol. prior to analysis by sem (zeiss, model evo- ), the samples were dried at °c and subjected to analysis with kv accelerating voltage. control experiment was conducted in absence of nano-silver [ ] . bacterial surfaces are characterised by their surface electric charge, which allows the measurement of zeta potential through nanotrac wave (model s ). the bacterial cultures were harvested by centrifugation at , rpm for min, and the cell pellets washed three times with . mm potassium phosphate buffer solution (ph . ) and finally re-suspended in the same buffer to the final concentration of cfu/ml. the washed bacterial cell suspensions were incubated with μg/ml of nano-silver for h [ ] . zeta potential was also carried out for normal and autoclaved (at °c, psi for min) bacterial cells [ ] . the average bacterial size of the sample was also measured in nanotrac wave (model s ) for autoclaved, non-treated and treated bacterial cells at ambient temperature ( °c). gc-ms profile was carried out to detect the leakages of intracellular constituents from non-treated and nano-silver treated bacterial cells (same as zeta potential). the known volume of bacterial supernatant (water v/v) were dried at °c in nitrogen turbo evaporator (biotage, turbovap®lv); followed by derivatized with μl of a mg/ ml methoxyamine hydrochloride solution in ml of pyridine for min at °c on shaker. subsequently, the samples were silylated for min at °c with μl n,o-bis (trimethylsily) trifluoroacetamide (bstfa) on shaker. the derivatized bacterial supernatants were analyzed with gas chromatograph-mass spectrometer (gcms-qp plus, shimadzu, japan) coupled with shimadzu gcms-qp se mass selective detector. about μl aliquots of the supernatants were injected into a db- ms capillary column ( m × . mm i.d., . μm film thickness) using split less injection ( °c, . min). helium gas was used as a carrier gas at a flow rate of ml/min. leakages of intracellular compounds were identified by mass spectra analysis ( - m/z range) through the national institute standard and technology (nist) library and gcms solution software to calculate the similarity index and above % match was considered acceptable [ ] . the mass spectrum of identified intracellular compounds of treated bacteria was compared with the spectrum of the non-treated bacteria (control) and identified leakage constituents due to nano-silver treatment on bacterial surface. the gc-ms profile of aqueous stem extract of t. cordifolia was also carried out. the common compounds between t. cordifolia aqueous extract and nano-silver treatment of bacterial culture as well as between bacterial suspension without silver and nano-silver treatment were identified and subtracted to identify unique compounds (linkages) release due to nano-silver treatment on bacterial surface. the nano-silver was successfully synthesized from aqueous stem extract of t. cordifolia by mixing with mm silver nitrate solution. additions of plant extract into the silver nitrate solution resulted in the gradual change of the colour of silver nitrate solution from light yellow to dark brown, indicating the formation of nano-silver (fig. a) . similar colour changes were reported in previous studies and hence confirmed the completion of reaction between silver nitrate and stem extract [ ] . the synthesis of nano-silver was further confirmed by uv-vis spectrophotometer. a strong specific peak for the synthesized nano-silver was obtained at nm (fig. a) . dynamic light scattering data showed that the synthesized nano-silver were in the range of . nm + . (fig. b) , which is well in agreement with sem result (fig. c) . the surface zeta potential value of nano-silver was measured to be slightly positive and was found to be . mv (data not shown in fig. ) . the positive charge of nano-silver prevents them from aggregation and increases their stability, as well as help to enhance their antibacterial activity by interacting with negatively charged biomolecules of bacterial membrane [ ] . the sem image of the sample showed the formation of spherical nano-silver in aggregated form with average . nm size, which was confirmed to be silver by edx analysis (fig. c and d) . the edx analysis showed a strong peak at kev, which is typical for the absorption of metallic nano-crystallites silver due to unique surface plasmon resonance (spr), thereby confirming the formation of nano-silver [ ] . the presence of additional peak for oxygen indicates that some nano-silver got oxidized by forming silver oxide [ ] . the comparative antibacterial activities of the nano-silver and broad spectrum antibiotics were assessed against emerging b. megaterium (mtcc ) and re-emerging p. aeruginosa (mtcc ) bacteria. a direct comparison of the nano-silver and the broad spectrum antibiotics under the same dose range ( μg/ μl for cfu/ml inoculum) showed that the tested microorganisms developed resistance to antibiotics belonging to different classes (fig. ) . the mean of three replicates of the diameter of inhibition zone around each well of nanosilver and broad spectrum antibiotics is represented in table . the inhibition zones of nano-silver obtained in this study indicated that a nano-silver has potential to control emerging and re-emerging multidrug-resistant pathogens compared to tested antibiotics. these findings further agree with previous results by other researchers, where it was proven that nano-silver exert the same result on multidrug-resistant bacteria [ ] . our findings showed that b. megaterium mtcc developed resistance to all tested antibiotics, except linezolid ( fig. a and b) . linezolid is a new class of antibiotics called the oxazolidinones that are useful to control the emergence of drug resistance gram-positive organisms [ ] . unfortunately, b. megaterium mtcc developed resistance to trimethoprim-sulfamethoxazol, which has been valuable combinatorial therapy for treating a variety of infections. this emerging resistance to trimethoprim-sulfamethoxazol is disturbing because it is a combination of two broad spectrum antibiotics that act synergistically against a wide variety of urinary and respiratory tract infections [ ] . there are limited data available to the infections caused by b. megaterium, there have been no previous data on resistant to antibiotics, except cloxacillin and streptomycin and thus, careful investigation is required [ , ] . the increasing bacterial resistance among gram-positive species is concerning because they are responsible for / of nosocomial infectious diseases [ ] . on the other hand, p. aeruginosa mtcc developed resistance to all tested broad spectrum antibiotics ( fig. c and d ). our findings of bactericidal activity of nano-silver against multidrug-resistant p. aeruginosa are exactly in accordance with findings shown by lara et al. [ ] . in recent years, p. aeruginosa bacteria that are resistant to different classes of antibacterial agents is one of the most feared bacteria that causes pneumonia [ , ] . the high antibiotic prescribing rate is one of the leading causes for the development of antibiotic resistant microbes in case of re-emergence of infectious diseases. however, the speed of the resistance development varies depending on the antibiotics and microbes. overall, our findings suggests that there was significant difference between the bactericidal efficacy of nano-silver and antibiotics on emerging b. megaterium mtcc and re-emerging p. aeruginosa mtcc , demonstrating that the bactericidal mechanism of nano-silver was not affected by those known resistance mechanisms that differentiate these strains from susceptible strains. the data presented in the study are novel as nano-silver exhibit excellent bactericidal effect towards emerging and re-emerging bacterial infections regardless of their drug resistant mechanisms. to observe the membrane deformities upon the nano-silver treatment, we scanned the nano-silver treated and non-treated bacterial cells using sem. the images indicate clumping of nano-silver on bacterial membrane in treated cells than non-treated cells (fig. ) . upon interaction with nano-silver, the surface potential of bacterial membrane is neutralized, resulting into increased in surface tension. after h of treatment, the interactions result in surface potential changes which lead into the membrane depolarization at the point of clumping. as a result, bacterial membrane become scattered from their original ordered arrangement. the scattered membrane containing cells no longer remain intact, often found in aggregates (fig. b, d) . surface charge neutralizations of the bacterial cell membrane are important target site of the certain membrane acting compounds particularly with antimicrobial properties, which acts on bacterial cell surface [ ] . according to our observations, nano-silver produced alternation of zeta potential in both gram-negative and gram-positive bacteria. as shown in fig. , the initial interaction of nano-silver with a cell surface of b. megaterium mtcc and p. aeruginosa mtcc cells display zeta potential of − . and − . , respectively. however, dead (autoclaved) bacterial cells of b. megaterium mtcc and p. aeruginosa mtcc had a lower zeta potential of . and − . mv compared to normal bacterial cells (data not shown in fig. ) . in our study, it was found that, the zeta potential value moved towards neutral with treatment of nano-silver. the calculated zeta potential values of b. megaterium mtcc and p. aeruginosa mtcc at different time intervals are shown in fig. . there were significant differences in the zeta potential of b. megaterium mtcc compared to p. aeruginosa mtcc , when they were normal or exposed to nano-silver or autoclaved. moreover, it was also found that the magnitude of decrease in the negativity of the zeta potential of bacterial cell surface was found to be greater in b. megaterium mtcc and such alternation was found to be time dependent (studied till h) as well. these low values of the zeta potential indicated the interaction between cationic nano-silver and the negative surface potential of bacterial surface. interestingly, such change in zeta potential through nano-silver can be correlated with the increased bacterial membrane permeability as it was evident from gc-ms analysis (fig. supplementary material). the present findings are in conformity with earlier reports, where it has been stated that surface neutralization of the bacterial membrane leads to increase in cell permeability [ ] . gc-ms based secretome analysis has important applications in discovering the mode of action of nano-silver and helps unravel the effect of nano-silver on membrane permeability of bacteria. in this study, the leakages of the intracellular constituents from bacterial cells were identified from their mass spectra and retention times, as indicated by the chromatogram of the extra cellular components of the bacterial cells after treatment of nano-silver ( fig. ; supplementary material). in case of treated bacterial cells, some constituents were from plant extract, by which we synthesized nano-silver. in general, the total constituents present in extracellular materials in treated cells, nontreated cells as well as plant extract detected in gc-ms were shown in table . an increase in some unique intracellular components in treated cells was observed, compared with those in the plant extract and nontreated cells. the common and unique compounds identified from bacillus megaterium mtcc and pseudomonas aeruginosa mtcc , before and after treatment with nano-silver along with plant extract are depicted in fig. . total compounds were identified common to bm_t, pa_t and pl_e followed by unique compounds in bm_t and in pa_t. moreover, compounds found to be common for pa_t and pl_e. the nature of compounds identified after treatments were sugars, organic acids, amino acids and fatty acids, which showed significant differences with non-treated cells and plant extracts (table ; supplementary material). this may indicate that nano-silver were found to interact with bacterial cell surfaces and results in membrane damage. nano-silver is considered a novel antibacterial agent, which offers several advantages such as broad spectrum antibacterial activity and lower tendency to induce resistance. the present study demonstrating the interaction of green synthesized nano-silver exerted a slight stress on the bacterial cell membrane through alternation of zeta potential. the strong antibacterial activity of nano-silver possesses the potential to serve as a platform for developing nano-silver into a novel antibacterial drug to overcome the problem of emerging b. megaterium mtcc and re-emerging p. aeruginosa mtcc . the antibacterial efficacy of nano-silver against multidrug-resistance indicated that the mode of action of nano-silver is not the same as the mode of action exerted by the antibiotics. hence, with the threat of multidrug-resistant strains of bacteria, nano-silver could be a good alternative to control emerging and re-emerging infectious diseases. the authors declare that they alone are responsible for the writing and content of this paper. the authors report no conflicts of interest in this research work. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. authors do not declare any conflict of interest. as antibiotic discovery stagnates, a public. health crisis brews accelerating resistance, inadequate antibacterial drug pipelines and international responses emerging and re-emerging infections overview of nosocomial infections caused by gram-negative bacilli brain abscess caused by bacillus megaterium in an adult patient bacillus megaterium delayed onset lamellar keratitis after lasik primary cutaneous infection with bacillus megaterium mimicking cutaneous anthrax antibiotic resitance is ancient an antibiotic-resistance enzyme from a deep-sea bacterium tinospora cordifolia; a pharmacological update plant mediated synthesis of nano-silver by using dried stem powder of tinospora cordifolia, its antibacterial activity and comparison with antibiotics preparation of agar wells for antibiotic assay antibiotics: classification and mechanisms of action with emphasis on molecular perspectives biosynthesis of nano-silver from staphylococcus aureus and its antimicrobial activity against mrsa and mrse antibacterial activity and mechanism of nano-silver on escherichia coli alteration of zeta potential and membrane permeability in bacteria: a study with cationic agents surface charge and zeta potential of metabolically active and dead cyanobacteria gc-msstudy of nutritious leaves of ehretia laevis synthesis of nano-silver using azadirachta indica (neem) extract and usage in water purification the effects of interfacial potential on antimicrobial propensity of zno nanoparticle synthesis and effect of nano-silver on the antibacterial activity of different antibiotics against staphylococcus aureus and escherichia coli nanomed synthesis of silver nanoparticles by silver salt reduction and its characterization production of nano-silver with strong and stable antimicrobial activity against highly pathogenic and multidrug resistant bacteria linezolid: it's role in the treatment of grampositive, drug-resistant bacterial infections increases in rates of resistance to trimethoprim bacillus megaterium resistance to cloxacillin accompanied by a compensatory change in penicillin binding proteins streptomycin resistance in bacillus megaterium antibiotic resistance. nosocomial gram-positive infection ixtepan turrent, cristina rodrıguez padilla, bactericidal effect of silver nanoparticles against multidrug-resistant bacteria unique level of multidrug resistance (mdr) in pseudomonas aeruginosa strain deb high prevalence of multidrug resistant pseudomonas aeruginosa recovered from infected burn wounds in children the application of biophysical techniques to study antimicrobial peptides, spectrosc escherichia coli cell surface perturbation and disruption induced by antimicrobial peptides bp and pepr the authors convey their thanks to department of biotechnology, junagadh agricultural university, junagadh, gujarat, india, for providing laboratory facilities to conduct experiment and analysis with sophisticated instruments. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jtemb. . . . key: cord- -vbq izw authors: coban, cevayir; lee, michelle sue jann; ishii, ken j. title: tissue-specific immunopathology during malaria infection date: - - journal: nat rev immunol doi: . /nri. . sha: doc_id: cord_uid: vbq izw systemic inflammation mediated by plasmodium parasites is central to malaria disease and its complications. plasmodium parasites reside in erythrocytes and can theoretically reach all host tissues via the circulation. however, actual interactions between parasitized erythrocytes and host tissues, along with the consequent damage and pathological changes, are limited locally to specific tissue sites. such tissue specificity of the parasite can alter the outcome of malaria disease, determining whether acute or chronic complications occur. here, we give an overview of the recent progress that has been made in understanding tissue-specific immunopathology during plasmodium infection. as knowledge on tissue-specific host–parasite interactions accumulates, better treatment modalities and targets may emerge for intervention in malaria disease. supplementary information: the online version of this article (doi: . /nri. . ) contains supplementary material, which is available to authorized users. activation-induced cytidine deaminase (aid) . an enzyme that is required for two crucial b cell events in the germinal centre: somatic hypermutation and class-switch recombination. by incomplete immunity to malaria causes long-term hidden pathologies . one such pathology is burkitt lymphoma, a cancer that develops in both childhood and adulthood in africa and that is closely related to p. falciparum and epstein-barr virus co-endemicicity . the robust and long-lasting expansion of germinal centre b cell populations that occurs during plasmodium infection may induce increased activation-induced cytidine deaminase (aid) expression, eventually leading to chromosomal translocations [ ] [ ] [ ] that predispose to the development of lymphoma in immune tissues. an increasing recognition of the association between malaria infection and physical growth retardation in children in africa, regardless of nutritional status , , suggests a detrimental effect of chronic malaria infection on growth, possibly via an effect on the bone tissue environ ment . nevertheless, any plasmodium spp. causing infection in humans result in varying degrees of complications, which can not only be severe and life threatening but can also have a long-term impact on patients' quality of life even after recovery. in this review, we focus on how systemic infection by plasmodium parasites causes local but tissue-specific immunopathologies during the blood stage of infection, mainly through the manipulation of inter-tissue interactions between the blood and other host tissues that often result in dysfunction of certain, but not all, organs. acute systemic immune activation, such as pro-inflammatory cytokine production, lymphocyte activation and vessel congestion, is evident; however, how these pathological events affect each tissue or organ is not understood well. current interventions for malaria are based on the administration of anti malarial drugs aimed at killing parasites and on supportive care to reduce systemic symptoms, such as coma, high fever, severe anaemia and acidosis. in this review, we emphasize the need to focus on host interactions with plasmodium parasites at various tissue levels and the importance of targeting local and specific organ failure and/or pathologies during, as well as long after, infection. these pathologies might be critical for determining diagnostic as well as therapeutic targets for the development of novel adjunct therapies to be used in combination with current antimalarials. we describe new evidence and new ways of targeting infected tissues of a few, but not all, unique organs such as the brain, gut and bones. we initially summarize how the blood tissue plays a central role in the initial pathogenesis caused by plasmodium parasites, following which we explore the interaction of infected blood tissue with specific organs composed of multiple, unique interacting tissue layers. largely owing to limitations of space, topics regarding the sporozoite stages of infection in the liver and those on the innate immune recognition of plasmodium para sites , are out of the scope of this review. in fact, plasmodium parasites do possess several ligands used to manipulate host cell invasion that could potentially be exploited as host factor targets for anti-malaria therapy , , a notion close to the topic of this review, but we exclude and leave this to an excellent recent article focusing on this topic . pathology within the blood tissue blood is a tissue (box ) that is formed by the plasma and blood cells (that is, erythrocytes, platelets and leukocytes) and is primarily located in the blood vessels. the infection of erythrocytes by plasmodium parasites results in extensive erythrocyte remodelling and dysfunction followed by cell lysis, which contribute to the pathology of severe anaemia. anaemia. plasmodium parasites (a merozoite form) are released into the bloodstream from liver hepatocytes within specialized vesicles called merosomes and continue a repetitive erythrocyte-invasion cycle in the blood tissue. merozoites released from the liver enter into erythro cytes through a very dynamic and complex process , (fig. ) . once erythrocytic infection is established, continuous destruction of erythrocytes due to schizont rupture contributes to anaemia. a moderate degree of anaemia is caused simply by the naturally occurring life cycle of para sites in erythrocytes. however, studies in both humans and mice have clearly indicated an increased removal of infected and uninfected erythrocytes that plays an essential and major role in severe malarial anaemia [ ] [ ] [ ] . clinical observations and mathematical modelling have suggested that for each infected erythrocyte, approximately uninfected rbcs are removed during malaria . chronic anaemia may also be mediated by the generation of self-reactive anti-phosphatidylserine antibodies . phosphatidylserine on infected erythrocytes is thought to be pathologically exposed to the immune system during malaria, leading to the generation of anti-phosphatidylserine antibodies. under usual circumstances, uninfected erythrocytes, mainly young cells called reticulocytes, express high levels of cd , a 'do not eat me' signal , , which allows them to escape from phagocytosis. however, young erythrocytes generated during malaria expose phosphatidylserine earlier, thus leading anti-phosphatidylserine antibodies to bind to uninfected erythrocytes and facilitate their clearance, contributing to chronic anaemia. one of the outcomes of the destruction of high numbers of erythrocytes during malaria is an increase in intravascular haem release , , which is an important factor for neutrophil activation but in turn could be the reason for increased oxidative damage and thus decreased macrophage function and the neutrophil exhaustion observed during malaria. this implies that not only erythrocytes but also blood tissue components including various leukocytes collectively react to the presence of plasmodium parasites (covered previously by seminal review articles , , ) and their related products in the blood circulation and tissues (fig. ) . erythrocytes undergo extensive deformation and remodelling after invasion by plasmodium parasites. while remodelling allows nutrient acquisition and parasite growth, it leads to changes in erythrocyte structure, with an increase in rigidity and rosetting . hence, the mature schizont stages easily sequester in the host microvasculature, mostly during p. falciparum and p. knowlesi infection and to a lesser extent during p. vivax infection , whereas immature ring stages freely circulate in the vessels. plasmodium berghei anka infection in mice shows a similar sequestration phenotype [ ] [ ] [ ] [ ] , and in this instance, sequestration via cd may be beneficial for the survival of the parasite . it is believed that infected erythrocytes, mostly those in the schizont stages of parasite infection, adhere along with activated leukocytes (mainly cd + t cells) to the endothelial cells of small blood vessels in the brain via binding to cd , intercellular adhesion molecule (icam ), endothelial protein c receptor (epcr) and platelet endothelial cell adhesion molecule (pecam ) [ ] [ ] [ ] [ ] . however, endothelial cell acti vation can occur without direct adhesion of leukocytes to the endothelial cells, possibly as a result of metabolites produced by leukocytes or parasites . indeed, there are unidentified soluble factors released by irbcs that cause endothelial cell pathology . in line with this, endothelial cells, irbcs, platelets, leukocytes and monocytes were shown to increase their release of extracellular vesicles during plasmodium infection, and this contributed to inflammation and correlated with disease severity . extracellular vesicles are mostly released from irbcs during schizogony and contain rbc components, parasite proteins and various rnas that can activate innate immune cells . in addition, endothelial cells have been shown to act as antigen-presenting cells (apcs) through the phagocytosis of merozoites and the presentation of malarial antigens to cd + t cells, which leads to ifnγmediated and perforin-mediated disruption of the blood-brain barrier (bbb) . most of these models of malaria-induced pathology are still under debate, and more evidence from both humans and animal models is clearly needed. overall, while the process of sequestration is not completely understood, it is known to cause obstruction of blood flow in small capillaries and post-capillary venules (pcvs), endothelial cell activation and inflammation and severe pathology in many organs including lung, adipose tissue, spleen and brain , , , (fig. ) . plasmodium-infected erythrocytes, is transported to organs via blood vessels. between large arteries and veins, various smaller sized vessels such as arterioles, capillaries and pcvs are present and have a role in controlling blood pressure and velocity in organs (fig. a) . the speed of blood flow substantially decreases as the blood enters arterioles, drops dramatically again in the capillaries and then slightly increases in the venules and veins (fig. a) . depending on the size and tissue environment, blood vessel structures vary. generally, the smaller the diameter of the vessel, the less smooth muscle it contains. capillaries, which play a major role in exchanging materials in blood, are the smallest vessels and are composed of endothelial cells with firm tight junctions and a basement membrane and do not include a smooth muscle layer (fig. b) . unlike capillaries, pcvs may have a few thin smooth muscle a tissue operates as an orchestration of large numbers of similar cells for a specific function. mammals have four basic types of tissues: epithelial tissue, muscle tissue, nerve tissue and connective tissue. two or more different tissues form and function as an organ. in general, the cells in their related tissue environment and organ are found in harmony, but during infection and inflammation, this harmony is likely to be disrupted. depending on the organ that the tissue or tissues interact with and support, multiple specific layers are present, which gives tissue specificity to healthy and diseased conditions. connective tissue deserves special attention because it includes various subcategories such as blood tissue, which is a major target of plasmodium parasites. this tissue, which mainly connects and supports various other tissues, is composed of various subtypes in addition to blood tissue, such as adipose tissue, areolar tissue, cartilage and bone tissue, lymphatic tissue and fibrous tissue. the main characteristics of connective tissues are that they contain an extracellular matrix composed of various collagen and/or elastin fibres, they contain interstitial fluid and they are vascularized with blood vessels. importantly, blood vessels are lined by endothelial cells, a component of epithelial tissue, and covered by smooth muscles (a muscle tissue); they carry blood cells, infected erythrocytes and various materials to the tissues and organs of the entire body, suggesting that even at the basic blood vessel level, many complex tissues specifically interact with each other. because tissue specificity occurs within an organization of different interacting tissues, it is reasonable to hypothesize that plasmodium parasites will reach different tissues and organ environments and develop unique and specific interactions with other tissues via blood tissue. also known as virchow-robin spaces. spaces located between brain-penetrating pial vessels and the brain tissue grey matter. (isf). the solution present extracellularly, that fills the spaces between cells and tissues. (csf). the solution surrounding the brain and spinal cord, which mainly serves to protect these two important organs. layers (fig. c) . importantly, depending on the organ, capillaries and pcvs interact with additional cells in tissues (fig. d) . for instance, brain capillaries and pcvs are additionally surrounded by pericytes, astrocyte end-feet and microglia (fig. d) . in particular, pcvs differ from capillaries in that they have leakier tight junctions and thus, may create perivascular spaces that apcs can easily home to and that allow for the infiltration of inflammatory cells during inflammatory conditions . moreover, the capillary beds in various organs are mostly in close proximity to lymphatic capillaries, into which drain interstitial fluid (isf; mainly leukocytes) and materials coming from blood vessels. the isf drained into the lymphatic vessels reaches the lymph nodes and finally returns to the final destination veins (fig. a) . therefore, it is reasonable to think that the type and diameter of a blood vessel in a specific organ in addition to the specific connective tissue and smooth muscle that is present to support vessel integrity may determine the degree of sequestration of plasmodium parasites and the outcome of disease. the walls of the smallest capillaries and pcvs are presumably more susceptible to internal changes occurring in the vessels. therefore, in the following sections, we detail the blood vessels within a few organs, such as the brain, gut and bones, and highlight their unique involvement with irbcs during malaria. tissue pathology within the brain the brain is severely affected by p. falciparum infection and to a lesser extent by p. knowlesi and p. vivax infections. this unique brain pathology, known as cerebral malaria, involves convulsions, coma and high fever and develops with the presence of mostly ring-stage infected erythrocytes in the periphery (suggesting a sequestration of late-stage parasites in the organs) [ ] [ ] [ ] . disruption of bbb integrity is an established outcome in cerebral malaria and may cause brain swelling and death in both humans and animals , . the activation of blood tissue components and their effect on brain immune cells (microglia, astrocytes) have been addressed , - , but how an obligate erythrocyte-resident pathogen can cause bbb disruption is not well understood. although the brain is considered an immune-privileged organ that is protected by the presence of the tight and selective bbb and by the lack of lymphatic drainage (a notion that has been disputed recently , ), the loss of bbb integrity due to the direct invasion and inflammation of meninges by various extracellular pathogens is well known . to understand why and how brain tissue is affected during malaria, we should consider recent advances in the neuroscience field, which could help to expand our understanding of cerebral malaria pathogenesis and development. the central nervous system (cns) is composed of the brain and spinal cord, which are protected by the meninges (formed by three layers: dura mater, arachnoid mater and pia mater) and are associated with two types of fluids: cerebrospinal fluid (csf) running through the subarachnoid space and isf in the brain and spinal cord parenchyma (box ) . csf is derived from plasma but has far fewer proteins, no erythrocytes, very nature reviews | immunology cells neutrophils, monocytes, platelets, t and b cells etc. brain, lung, kidney, intestine, bone etc. following the release of plasmodium merozoites from infected hepatocytes into the bloodstream, repetitive erythrocyte-invasion cycles occur in the blood. this leads to the release of several parasite by-products, such as haemozoin, and the parasites themselves into the bloodstream. the blood-stage cycle of plasmodium infection causes various pathologies, such as anaemia, toxic haem release, immune cell activation (modulation of platelets, neutrophils, monocytes, macrophages, t cells and b cells) and can cause a cytokine and chemokine storm. in blood tissue, infected red blood cells (irbcs) and their products may interact with other infected and uninfected rbcs (causing rosetting), or they may interact with immune cell populations (causing a cytokine storm) or with the endothelial cells of blood vessels (causing rbc sequestration and microhaemorrhage). these cell-cell interactions have organ specificity and thus take place in specific tissue environments, resulting in specific immunopathologies. an organ located on the cribriform plate, which functions in smell. the bulb surface is surrounded by complex olfactory nerve structures that originate from the nasal cavity and project to the brain. small trabecular capillary structures are the main vessel structures inside the bulb. few leukocytes and higher sodium, chloride and magnesium contents. isf fills the basement membranes of brain capillaries, where most of the exchange between blood and the cns occurs. the selective bbb inside the brain blood vessels, especially capillaries, is composed of uniquely specialized endothelial cells with intracellular tight junctions, pericytes, astrocyte end-feet and microglia in addition to the basement membrane, suggesting that blood vessel structures in the brain differ from those in other organs of the body, with a unique involvement of brain cell astrocytes and microglia in disease processes (fig. d) . advancing our understanding of brain physiology (box ) clearly has important implications for neurological diseases, such as alzheimer disease and multiple sclerosis , , as well as for cerebral malaria. indeed, recent preclinical studies using cutting-edge imaging technologies, such as ultra-high-field . t magnetic resonance imaging (mri) and multi photon live imaging microscopy, in experimental cerebral malaria models have expanded our understanding of how cerebral malaria develops. below, we consider the roles of the retina, olfactory bulb and the perivascular spaces of the brain during cerebral malaria. arteries supplying blood to the brain divide into smaller arteries on figure | interaction of plasmodium-infected red blood cells with various blood vessels and lymphatics. a | infected red blood cells (irbcs) circulate in blood vessels, which vary in size from large arteries to veins, with blood flow running from arteries, arterioles, capillaries and post-capillary venules (pcvs) to venules and veins, providing controlled blood pressure and velocity in organs. the speed of blood flow is lowest in capillaries and pcvs. in various organs, these capillary beds are mostly near lymphatic vessels, through which interstitial fluid and materials coming from blood vessels drain the lymph nodes and finally return to veins. irbcs and irbc-mediated immune responses, therefore, may have profound effects on these smaller vascular beds in each organ. b | capillaries have only an endothelial cell wall and no smooth muscle; therefore, they are in close contact with irbcs. c | two capillaries fuse and form pcvs, which may have some smooth muscle and where leukocyte rolling can occur. d | capillaries and pcvs in the brain are additionally surrounded by pericytes, astrocyte end-feet and microglia; irbc-related events in capillaries are sensed immediately. a small, perforated bone structure that lies on top of the nasal cavity and supports the olfactory bulb. olfactory nerves running from the nasal cavity to the olfactory bulb pass thorough cribriform plate. the pial surface and penetrate into the brain parenchyma before further dividing into arterioles. arterioles (and venules) are surrounded by perivascular space which is filled with isf drained from the brain parenchyma (fig. a) . a newly defined 'glymphatic system' has been proposed in which the glia limitans, a barrier comprising astrocyte end-feet that surrounds small capillaries and veins, creates perivascular spaces via aquaporin (aqp ) channels. the aqp channels facilitate the flux of isf into perivascular spaces to be mixed with csf , , and this eventually contributes to the clearance of particles from the csf. interestingly, dilatation at perivascular spaces and astroglia and reduced expression of aqp protein have been reported during cerebral malaria in animal models , . furthermore, in the absence of aqp , mice with cerebral malaria succumbed to death more quickly , suggesting aqp has protective roles during cerebral malaria, perhaps in controlling liquid exchange, oedema and cell transmigration at the bbb. apcs with phagocytic properties such as dendritic cells and macrophages located in the pial surfaces and perivascular spaces have been shown to present antigens to t cells in various cns diseases . as visualized by electron microscopy and intravital two-photon microscopy, it seems that leukocytes and cd + t cells, and probably irbcs or their products, increasingly accumulate in perivascular spaces in very deep and branching vessels during cerebral malaria [ ] [ ] [ ] [ ] . these phagocytic apcs and activated endothelial cells in vessels have the capacity to present malarial antigens , . furthermore, all these events that are mediated by ifnγ could be reversed by blocking the adhesion molecules box | the steady state brain drainage system owing to the presence of the highly selective blood-brain-barrier (bbb), the brain has long been considered to have no classical lymphatic system and thus be an immune-privileged organ (see the figure) . in homeostatic conditions, the cerebrospinal fluid (csf) is continuously produced by the choroid plexus and drained into the bloodstream via arachnoid granulations in the venous sinuses or nasal lymphatics under the cribriform plate next to either the olfactory nerves or the spinal and cranial nerve sheaths. immune surveillance via selective epithelial cells and homeostatic leukocyte trafficking in csf have been acknowledged ; however, recent studies by louveau et al. and aspelund et al. have challenged previous knowledge by demonstrating the presence of a classical lymphatic drainage system located in the brain meninges. the authors clearly showed that this lymphatic network works alongside the classical known csf drainage system, with the additional capacity to drain other constituents such as toxic macromolecules and immune cells into the deep cervical lymph nodes, providing evidence of a connection between the csf, the brain and the periphery. essentially, these newly identified brain lymphatic vessels lay in the dura mater and collect csf and interstitial fluid (isf) together with macromolecules such as amyloids and immune cells from perivascular spaces (pvss) of the brain parenchyma. similar lymphatics are also present around another immune-privileged organ, the eye (along with the optic nerve) , and over the olfactory bulb, aligned throughout meninges as simple narrow vessels that become wider in the transverse sinuses, ultimately reaching the cerebellum. all lymphatic vessels finally drain into the deep cervical lymph nodes. it is of note that similar to blood vessels, these lymphatic vessels are different in size depending on their anatomical location in the central nervous system, and owing to that, their interaction with local blood vessels or astrocyte end-feet may be different during homeostasis than in diseased conditions. pcv, post-capillary venule. figure | infected red blood cells in close proximity with brain components. a | small arteries on the pial surface of the brain penetrate into the brain parenchyma and further divide into arterioles; these are surrounded by perivascular space (pvs) which is filled with interstitial fluid (isf) that has drained from the brain parenchyma. arterioles become capillaries, venules then veins, which finally drain into dural venous sinuses, and each of these areas potentially contain abundant infected red blood cells (irbcs), as well as lymphocytes such as cd + t cells. lymphatic vessels located in the dura carry immune cells and cerebrospinal fluid (csf) into deep cervical lymph nodes. b | the retina is rich in photoreceptors (cones, bipolar cells and ganglions), nerve cells and complex blood vessels, especially capillary structures surrounded by müller cells, a retina-specific astrocyte-like cell. the optic nerve serves as a connector between the retinal nervous tissue and the brain, where meningeal membranes, dura lymphatics and csf cover the optic nerve up to the retina border. this area may contain abundant irbcs. c | the olfactory bulb is surrounded by meninges of brain dura and arachnoid and pial layers. csf similarly runs throughout the bulb and reaches the cribriform plate area. olfactory nerves originate from the nasal mucosa and terminate in the olfactory bulb through the cribriform plate, which is also the route of the blood vessels surrounding the olfactory nerves. these olfactory nerves project any signal for smell to the olfactory bulb and the brain. inside the olfactory bulb, very dense blood capillaries are oriented in different directions (radially and tangentially), with thin astrocyte end-feet surrounding the vessels, and sense irbc-related events and secrete several cytokines and/or chemokines including cc-chemokine ligand (ccl ). blood capillaries easily bleed during cerebral malaria. pcv, post-capillary venule. a mechanism explaining the relationship between neuronal activity and the cerebral blood vessels and the blood flow. very late antigen (vla ) and lymphocyte function-associated antigen (lfa ) (ligands for vascular endothelial adhesion protein (vcam ) and icam , respectively), which are expressed on activated cd + t cells during cerebral malaria . one of the important features of inflammation in perivascular spaces is that the parasite and inflammatory products may directly activate astrocytes , , microglia and neurons , and may cause an increase in the levels of protein s b (a biomarker for astrocytes) and in the levels of the microtubule-associated protein tau (a biomarker for degenerated axons) in csf . nevertheless, future studies are needed to provide direct evidence of this. it would also be interesting to investigate in the future whether there are other types of perivascular spaces found at different locations in the brain , , because pia mater also covers the cerebellum and other cns areas, such as the spinal cord. therefore, the interaction of perivascular spaces with small blood vessels may vary depending on the anatomical location and unique architecture of the tissue, for example, in the brain cortex, spinal cord, cerebellum or olfactory bulb. the back of the eye ball is lined by the retina, with the optic nerve in the middle and going through to the brain. the optic nerve serves as a connector between the nervous tissue of the retina and the brain, where brain meninges, dura mater lymphatics and csf cover up the optic nerve up until the retina border. the retina is rich in photo receptors, nerve cells and complex blood vessels, especially capillary structures (fig. b) . retinal changes (such as haemorrhages, multiple peripheral and macular patchy whitening areas and papilloedema) have been commonly observed in children and in adults who develop sudden comatose cerebral malaria symptoms after a few days of fever [ ] [ ] [ ] . these retinal changes occur in areas that show high sequestration of parasites accompanied by thrombi (composed of fibrin and platelets) , , which cause impaired capillary perfusion , and are well correlated with the severity of disease . interestingly, other areas in the eye, such as the optic nerve head, ciliary body and iris, are similarly affected by irbcs , indicating that specific blood vessel similarities may occur within other parts of the brain. retinal homeostasis is maintained through neurovascular coupling involving astrocytes, müller cells (a specific type of glial cell found in the retina) and resident retinal microglia, with müller cells, pericytes and astrocytes mainly ensheathing the retinal blood capillaries . the observation that the accumulation of fluid between processes of müller cells and photoreceptor cells in the macular area produces intra-retinal spaces in human cerebral malaria suggests that these areas represent perivascular-space-like areas in the retina that support immunological events similar to those that occur in the perivascular spaces in deep brain areas. furthermore, high-field mri studies have identified damage to the optic and trigeminal nerves during experimental cerebral malaria even before the development of oedema, which may cause loss of visual acuity . the olfactory bulb and cerebral malaria. the olfactory bulb is situated on the cribriform plate, located on top of the nasal sinus. although the olfactory bulb has several characteristics that distinguish it from the brain, it is covered by similar meningeal structures and by the csf and is thus included in the cns. however, in contrast to other parts of the brain, olfactory nerves initiate from the nasal mucosa and terminate in the olfactory bulb through the cribriform plate, which is also the route of the blood vessels surrounding olfactory nerves. these olfactory nerves project any signals for odour to the olfactory bulb and to the brain. inside the olfactory bulb, very dense blood capillaries are oriented in different directions (radially and tangentially) with thin astrocyte end-feet surrounding the vessels (fig. c) . owing to complex structural interactions of nerves through the cribriform plate, capillaries, microglia and astrocyte endfeet in this region, the olfactory organ is considered to be the gateway of the otherwise well-protected brain to the outside world. it is known that several molecules, cells and even pathogens can gain access to the brain parenchyma through this route . by use of . t mri in mice, it was discovered that the olfactory bulb is the first region during experimental cerebral malaria to show vascular leakage and bleeding , a phenomenon possibly related to the unique small capillary trabecular structure of the olfactory bulb. this was also directly visualized and confirmed by intravital two-photon microscopy during cerebral malaria . these dense and directionally structured olfactory blood capillaries , which are thought to be leakier than those in the brain cortex or pons , are a suitable scaffold for irbcs . sequestration of irbcs in the olfactory bulb capillaries results in astrocyte and microglia activation , , and in the loss of tight junction integrity, and these parasite-mediated events lead to olfactory dysfunction and loss of smell, which could be a valuable early diagnostic marker for cerebral malaria. moreover, astrocytes surrounding small trabecular capillaries in the olfactory bulb were found to sense changes in the vessels and secrete cytokines and chemokines, such as cc-chemokine ligand (ccl ), which are involved in the accumulation of pathological cxcr + cd + t cells in the olfactory bulb . expression of cc-chemokine receptor (ccr ), which is the canonical receptor for ccl and ccl , was shown to be essential for antigen cross-presentation by cd α + dendritic cells and for the activation of cxcr + cd + t cells in the spleen during cerebral malaria . by contrast, ccl secreted from astrocytes played a role in the recruitment of pathological cxcr + cd + t cells in the olfactory bulb via its non-canonical receptor cxcr (ref. ) . a pathological role for several chemokines and chemokine receptors (including cxcchemokine ligand (cxcl ), cxcl and cxcr ) is well recognized in experimental cerebral malaria , but the study discussed above highlights how the specific role for chemokines in cerebral malaria might differ depending on the tissue and organ environment. extended mri studies using sensitive contrast reagents have further confirmed these findings and have shown that micro-haemorrhages originate in the olfactory lacteals small lymphatic capillaries located in the villi of the small intestine. vessels with a structure similar to capillaries but that have larger diameters and porous endothelial cells, which allow for permeability and exchange of materials; they are located in bone marrow, liver and spleen. bulb together with oedema, but that the oedema further spreads along the rostral migratory stream, similar to the migration route of immune cells and neuroblasts , and may reach the brainstem . moreover, the glymphatic system may play a key role in the rapid inflammatory spread of cerebral malaria from the olfactory bulb and rostral migratory stream axis to deeper brain areas such as perivascular spaces . it is also possible that these events that occur in the olfactory bulb during cerebral malaria may block the drainage of csf into nasal lymphatics and aggravate brain oedema (fig. c) . clearly, the role of the olfactory bulb in cerebral malaria in humans warrants further research. tissue pathology within the gut gastrointestinal symptoms such as abdominal pain, vomiting and diarrhoea are common symptoms during malaria infection. autopsies of human patients who died from severe malaria have shown that sequestration of irbcs occurs in the gastrointestinal tract (including in the colon, jejunum, ileum and stomach) , suggesting that gastrointestinal bleeding occurs during malaria. the presence of plasmodium dna in faeces additionally supports the occurrence of intestinal bleeding during infection ; however, how parasites interact with other cells in gut tissue is a research area that has only recently gained attention. recent studies have confirmed that pathological changes (including detachment of epithelia and shortening of villi and the colon) occur in the intestinal tract during malaria ; such changes may cause increased intestinal permeability, ruptures and leakage of infected erythrocytes into the lumen, causing dysbiosis of the intestinal microbiota, which may also contribute to disease severity . moreover, there is a close correlation between malaria infection and increased rates of infection with other gut pathogens (for example, nontyphoidal salmonella and helminths), possibly owing to an impairment of intestinal barrier function during malaria [ ] [ ] [ ] . several factors may have a role in this, such as increased histamine release from mast cells in the gut or the modulation of intestinal inflammation and immune responses via an increase in the number of exhausted neutrophils or an increase in the levels of il- family cytokines, including il- and il- , in the intestinal tissues during malaria infection, as these factors may ameliorate responses to secondary bacterial infections [ ] [ ] [ ] . however, it should be kept in mind that the composition of the gut microbiota has a clear impact on the severity of malaria disease [ ] [ ] [ ] , suggesting that there is a bidirectional relation between the gut and plasmodium parasites that is not well understood and requires further investigation. nevertheless, a close look at the intestinal tissues may give some clues for understanding gut-plasmodium interactions (fig. a) . the blood capillaries carrying irbcs reach the lamina propria of the intestinal mucosa and possibly come into close proximity with lacteals, which are specialized lymphatic vessels of intestinal villi that play a key role in nutrient absorption and in gut immune responses, such as the transport of microbial antigens and apcs to the mesenteric lymph nodes, where antigen presentation by apcs promotes the induction of t cells with gut-homing properties . there is a gut-vascular barrier comprising endothelial cells, enteric pericytes and enteric glial cells that is very similar to the barriers found in other parts of the body (such as the bbb) and that actively blocks bacterial dissemination from the gut epithelium to the blood . therefore, it is reasonable to hypothesize that intestinal bleeding may occur as a result of malaria-mediated immune responses that directly affect lacteals. it is possible that lacteals with disturbed remodelling have impaired survival and villi length , and this may have knock-on effects on immune cell composition and on the microbiota of the gut tissue. tissue pathology within bone bone tissue is a mineralized connective tissue supporting the whole body and it houses the bone marrow, which comprises specialized niches that maintain and regulate haematopoiesis, erythropoiesis and bone remodelling (fig. b) . arteries in the bone marrow unite and become specialized vessels called bone marrow sinusoids, which allow cells to pass between the circulation and bone marrow . without any cyto adherence, plasmodium parasites can invade and multiply in nucleated erythroblasts located in extravascular spaces of the bone marrow [ ] [ ] [ ] . the parasites can survive and hide in these erythroid precursors and develop into gametocyte stages [ ] [ ] [ ] , likely assisting in the continuous success of malaria transmission to mosquitoes. however, it is still not known why plasmodium parasites preferentially differentiate into gametocytes in the bone marrow and whether a specific bone marrow niche is required to support the development of gametocytes. unlike haematopoietic niches, which are located adjacent to sinusoids, erythroid niches are found throughout the bone marrow and are generally located away from the sinusoids, although they move closer to the sinusoids as they mature . this may suggest a major role for the erythroid niche in plasmodium gametocyte development. from an immunological point of view, it has recently been shown that cd + t cells produce ifnγ in response to infected mhc class i-expressing erythroblasts residing in the bone marrow; by contrast, mature erythrocytes residing in the blood circulation lack any mhc molecules and cannot present antigens to cd + t cells , . activated cd + t cells promote the exposure of phosphatidylserine on infected erythro blasts, and this enhances the susceptibility of the infected cells to phagocytosis by macrophages , suggesting a protective role of cd + t cells against malaria. however, whether bone marrow erythroid cells contribute to the expansion of pathological populations of cd + t cells that can drive cerebral malaria has not been investigated. despite the growing body of information on how bone marrow niches are manipulated by plasmodium parasites, very little is known about the potential outcomes of interactions between plasmodium parasites and bone tissue. in bone marrow, haematopoietic stem cells give rise to blood cells and bone-resident osteoclasts, whereas mesenchymal stem cells give rise to osteoblasts, adipocytes and stromal cells. a very recent study has suggested that during plasmodium infection, parasites and their products -mainly haemozoin -continuously accumulate in bone marrow and cause acute as well as chronic bone loss . although it is not precisely known how plasmodium products are retained long term in the bone marrow, it is possible that engulfment by macrophages or other phagocytic cells, such as bone-resident osteoclasts, or trapping by extracellular matrix in the bone marrow contributes to this phenomenon . chronic bone loss during malaria infection is mediated by over-activation of osteoclast resorption activity. the osteoclasts are activated by the key osteoclastogenic cytokine rankl (also known as tnfsf ), which is upregulated in osteoblasts through myd -dependent signalling triggered by the persistence of parasite products in the bone marrow . these findings highlight how, not only during acute malaria infection but also after recovery from infection, plasmodium products continue to interact with tissueresident cells, including bone cells, osteoclasts and osteoblasts, and how they can cause long-term effects in the host, such as bone loss and growth problems. the molecular and immunological pathways underlying the interaction between plasmodium spp. and the niches of various bone marrow cells and the effect of these interactions on the immune system , need to be addressed in the future. malaria is a serious disease with acute life-threatening and long-term complications, all of which can be attributed to local but specific organs in which plasmodium figure | infected red blood cells in gut and bone marrow niches. a | the intestinal tract is made up of several tissue layers. the villous mucosa is covered by mucus that is secreted by the goblet cells of the epithelium. it lies on a connective tissue, the lamina propria, which is an environment rich in blood vessels, lymphatics, nerves and immune cells. it also contains peyer's patches and is lined by smooth muscle tissue (muscularis mucosa). blood capillaries that are carrying infected red blood cells (irbcs) reach the lamina propria of the intestinal tract and can come into close proximity with lacteals at this location. b | bone tissue supports the whole body and is home to bone cells, blood vessels, nerves and endothelium, as well as containing the bone marrow niches. the bone marrow is composed of haematopoetic and mesenchymal stem cells that are involved in both haematopoiesis and bone remodelling, such as osteoclasts and osteoblasts. cells and irbcs pass through the circulation and bone marrow via specialized venules called bone marrow sinusoids. plasmodium parasites can invade erythroblasts, the erythrocyte precursors, and preferentially form gametocytes in the bone marrow erythroblastic niche. released plasmodium products including haemozoin can be engulfed by osteoclasts and activate osteoblasts and osteoclast precursors. hsc, haematopoietic stem cell; msc, mesenchymal stem cell; osb, osteoblast. haemozoin a unique by-product of plasmodium parasites that accumulates in several organs, including spleen, liver and bone marrow, even after the infection resolves. parasites and their products or irbcs interact with the tissue, causing an imbalance in the specific tissue environ ment. here, we have attempted to summarize the recent progress of research focusing on the tissue environment in a few example organs, such as the brain, gut and bone, where plasmodium parasites reach the tissue via the blood. however, the lungs , kidneys and placenta represent other organs that are seriously affected during malaria infection, and these tissues should also be examined more closely in future studies. here, our aim has been to show that the influence of malarial parasites reaches beyond erythrocytes and may vary depending on, and be tightly regulated by, the vessel-specific and/or tissue-specific microenvironment. on the basis of the available data, we suggest that the brain pathology caused by plasmodium parasites is not homogeneously controlled by parasite sequestration but is rather controlled by the local, specific and unique anatomical structure of vessels, parenchyma and lymphatics, which closely interact with each other during both homeostasis and infection. this concept is in accordance with the recent understanding that the vascular and epithelial barriers do cooperate in an organ-specific manner in homeostatic and diseased conditions . we additionally emphasize that parasite-derived products can continue to stay in the body, interact with tissues and cause chronic pathologies even long after recovery from infection, particularly in the case of bone tissue. the gut pathology caused by malaria, which has recently been recognized, was summarized, and the possible anatomical interaction between blood tissues and special gut lymphatics has been introduced. there are multiple benefits of understanding malaria-related pathologies in various tissues. for instance, understanding the reaction of microglia and astrocytes to irbcs will be beneficial for generating intervention strategies that block these interactions to prevent cerebral malaria and/or related long-term sequelae. supporting the gut microbiota during and after plasmodium infection might help to protect against and promote recovery from secondary infections. similarly, additional therapies like vitamin d supplementation to support the bone environment affected during and after malaria might help the growth of children or support bone health in elderly people. the search for a full understanding of malaria-related pathologies is important for ensuring that malaria is one day a curable and/or preventable disease. towards this goal, novel approaches should be investigated to analyse unknown or overlooked anatomical -as well as immunological -host-parasite interactions at various tissue levels of each organ, not only during infection but also long after clearance of the initial infection by the parasite. by doing so, we will be able to prevent the disease, diagnose and treat patients, predict prognoses and allow patients to receive appropriate medical care through the use of effective vaccines, diagnostic tools, adjunct therapies and antimalarials in a cost-effective and timely manner in the near future. global, regional, and national incidence and mortality for hiv, tuberculosis, and malaria during - : a systematic analysis for the global burden of disease study bad air, amulets and mosquitoes: , years of changing perspectives on malaria mapping plasmodium falciparum mortality in africa between prioritising infectious disease mapping is plasmodium vivax malaria a severe malaria?: a systematic review and meta-analysis clinical profile of plasmodium falciparum and plasmodium vivax infections in low and unstable malaria transmission settings of colombia human infections with plasmodium knowlesi -zoonotic malaria severe plasmodium knowlesi infection with multi-organ failure imported to germany from thailand/myanmar pathogenesis of cerebral malaria -inflammation and cytoadherence this is an important human study that systematically identifies the sequestration of parasites in not only the brain pulmonary pathology in pediatric cerebral malaria this is an important study that quantifies multi-organ sequestration of parasites in fatal human malaria cognitive impairment after cerebral malaria in children: a prospective study neurologic sequelae after severe falciparum malaria in adult travelers immune effector mechanisms in malaria chronic exposure to plasmodium falciparum is associated with phenotypic evidence of b and t cell exhaustion malaria-associated atypical memory b cells exhibit markedly reduced b cell receptor signaling and effector function young lives lost as b cells falter: what we are learning about antibody responses in malaria asymptomatic" malaria: a chronic and debilitating infection that should be treated epidemiology: clues to the pathogenesis of burkitt lymphoma the link between plasmodium falciparum malaria and endemic burkitt'slymphoma -new insight into a -year-old enigma a multifactorial role for p. falciparum malaria in endemic burkitt's lymphoma pathogenesis this study shows that murine plasmodium infections can promote the development of mature b cell lymphoma by inducing aid expression in lymphoid tissues delay in skeletal maturity in malawian children parasitism in children aged three years and under: relationship between infection and growth in rural coastal kenya this study shows experimentally for the first time that plasmodium metabolic products innate recognition of malarial parasites by mammalian hosts innate sensing of malaria parasites a forward genetic screen identifies erythrocyte cd as essential for plasmodium falciparum invasion basigin is a druggable target for host-oriented antimalarial interventions expanding the antimalarial toolkit: targeting host-parasite interactions manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids malaria, erythrocytic infection, and anemia malaria biology and disease pathogenesis: insights for new treatments severe malarial anemia of low parasite burden in rodent models results from accelerated clearance of uninfected erythrocytes malarial anemia: of mice and men insights into the immunopathogenesis of malaria using mouse models a retrospective examination of anemia during infection of humans with plasmodium vivax anaemia of acute malaria infections in non-immune patients primarily results from destruction of uninfected erythrocytes malaria parasite clearance anti-self phosphatidylserine antibodies recognize uninfected erythrocytes promoting malarial anemia macrophage engulfment of a cell or nanoparticle is regulated by unavoidable opsonization, a speciesspecific 'marker of self' cd , and target physical properties an apoptotic 'eat me' signal: phosphatidylserine exposure malaria impairs resistance to salmonella through heme-and heme oxygenasedependent dysfunctional granulocyte mobilization a central role for free heme in the pathogenesis of severe malaria: the missing link? lipocalin bolsters innate and adaptive immune responses to blood-stage malaria infection by reinforcing host iron metabolism immunity to malaria: more questions than answers immune mechanisms in malaria: new insights in vaccine development erythrocyte remodeling by plasmodium falciparum gametocytes in the human host interplay rifins are adhesins implicated in severe plasmodium falciparum malaria sequestration and tissue accumulation of human malaria parasites: can we learn anything from rodent models of malaria? olfactory plays a key role in spatiotemporal pathogenesis of cerebral malaria this is the first study to show that the blood-brain barrier is disrupted at the olfactory bulb during experimental cerebral malaria cd + t cells and ifn-γ mediate the time-dependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria targeting the olfactory bulb during experimental cerebral malaria reduced cd -dependent tissue sequestration of plasmodium-infected erythrocytes is detrimental to malaria parasite growth in vivo the neuropathology of fatal cerebral malaria in malawian children severe malaria is associated with parasite binding to endothelial protein c receptor investigating the pathogenesis of severe malaria: a multidisciplinary and crossgeographical approach structure-guided identification of a family of dual receptor-binding pfemp that is associated with cerebral malaria brain microvessel crosspresentation is a hallmark of experimental cerebral malaria plasmodium falciparum adhesion on human brain microvascular endothelial cells involves transmigration-like cup formation and induces opening of intercellular junctions the role of extracellular vesicles in plasmodium and other protozoan parasites malaria-infected erythrocytederived microvesicles mediate cellular communication within the parasite population and with the host immune system a unified hypothesis for the genesis of cerebral malaria: sequestration, inflammation and hemostasis leading to microcirculatory dysfunction immunobiology of plasmodium in liver and brain perivascular spaces and the two steps to neuroinflammation structure and function of the blood-brain barrier differentiating the pathologies of cerebral malaria by postmortem parasite counts pathogenesis, clinical features, and neurological outcome of cerebral malaria cerebral malaria brain swelling and death in children with cerebral malaria this study shows that death occurs in humans during cerebral malaria owing to brain swelling and oedema imaging experimental cerebral malaria in vivo: significant role of ischemic brain edema early activation of microglia in the pathogenesis of fatal murine cerebral malaria pathological role of toll-like receptor signaling in cerebral malaria experimental cerebral malaria spreads along the rostral migratory stream uptake of parasite-derived vesicles by astrocytes and microglial phagocytosis of infected erythrocytes may drive neuroinflammation in cerebral malaria redistribution and degeneration of retinal astrocytes in experimental murine cerebral malaria: relationship to disruption of the blood-retinal barrier axonal injury in cerebral malaria axonal and astrocyte injury markers in the cerebrospinal fluid of kenyan children with severe malaria a dural lymphatic vascular system that drains brain interstitial fluid and macromolecules structural and functional features of central nervous system lymphatic vessels a journey into the brain: insight into how bacterial pathogens cross blood-brain barriers establishment and dysfunction of the blood-brain barrier revisiting the mechanisms of cns immune privilege the anatomical and cellular basis of immune surveillance in the central nervous system implications of the discovery of brain lymphatic pathways brain-wide pathway for waste clearance captured by contrast-enhanced mri electron microscopic features of brain edema in rodent cerebral malaria in relation to glial fibrillary acidic protein expression protective role of brain water channel aqp in murine cerebral malaria effector t cell interactions with meningeal vascular structures in nascent autoimmune cns lesions perivascular arrest of cd + t cells is a signature of experimental cerebral malaria scanning electron microscopy of the neuropathology of murine cerebral malaria quantitation of brain edema and localisation of aquaporin expression in relation to susceptibility to experimental cerebral malaria experimental cerebral malaria pathogenesis -hemodynamics at the blood brain barrier activated brain endothelial cells cross-present malaria antigen measuring antigen presentation in mouse brain endothelial cells ex vivo and in vitro cd + t cells induce fatal brainstem pathology during cerebral malaria via luminal antigen-specific engagement of brain vasculature the crossroads of neuroinflammation in infectious diseases: endothelial cells and astrocytes correlation of hemorrhage, axonal damage, and blood-tissue barrier disruption in brain and retina of malawian children with fatal cerebral malaria histologic characteristics of normal perivascular spaces along the optic tract: new pathogenetic mechanism for edema in tumors in the pituitary region virchow-robin spaces at mr imaging this is the first study to describe malarial retinopathy in human cerebral malaria cerebral malaria: mechanisms of brain injury and strategies for improved neurocognitive outcome magnetic resonance imaging of the brain in adults with severe falciparum malaria platelet accumulation in brain microvessels in fatal pediatric cerebral malaria retinal pathology of pediatric cerebral malaria in malawi redefining cerebral malaria by including malaria retinopathy cerebral malaria in children: using the retina to study the brain severity of retinopathy parallels the degree of parasite sequestration in the eyes and brains of malawian children with fatal cerebral malaria glia-neuron interactions in the mammalian retina in the eye of experimental cerebral malaria intranasal delivery of cells to the brain two-photon imaging of capillary blood flow in olfactory bulb glomeruli immunocytochemical evaluation of the blood-brain barrier to endogenous albumin in the olfactory bulb and pons of senescence-accelerated mice the role of chemokines in severe malaria: more than meets the eye dna from pre-erythrocytic stage malaria parasites is detectable by pcr in the faeces and blood of hosts this study shows that p. berghei parasites may promote gut pathology and dysbiosis of the intestinal microbiota invasive nontyphoidal salmonella disease: an emerging and neglected tropical disease in africa malaria-associated l-arginine deficiency induces mast cell-associated disruption to intestinal barrier defenses against nontyphoidal salmonella bacteremia epidemiology of malaria and helminth interaction: a review from mast cells and histamine alter intestinal permeability during malaria parasite infection il- protects against liver pathology and lethality of an experimental bloodstage malaria infection the mucosal inflammatory response to non-typhoidal salmonella in the intestine is blunted by il- during concurrent malaria parasite infection il- dampens the t cell response in experimental malaria gut microbiota elicits a protective immune response against malaria transmission this study shows that the gut microbiota modulates the severity of malaria stool microbiota composition is associated with the prospective risk of plasmodium falciparum infection dll promotes continuous adult intestinal lacteal regeneration and dietary fat transport organ-specific protection mediated by cooperation between vascular and epithelial barriers the bone marrow niche for haematopoietic stem cells cytotoxic activities of cd + t cells collaborate with macrophages to protect against blood-stage murine malaria production of erythropoietic cells in vitro for continuous culture of plasmodium vivax stage-specific susceptibility of human erythroblasts to plasmodium falciparum malaria infection the extravascular compartment of the bone marrow: a niche for plasmodium falciparum gametocyte maturation? plasmodium falciparum transmission stages accumulate in the human bone marrow molecular evidence for the localization of plasmodium falciparum immature gametocytes in bone marrow erythroblastic islands: niches for erythropoiesis cd + t cell activation by murine erythroblasts infected with malaria parasites the bone marrow at the crossroads of blood and immunity the critical and specific transcriptional regulator of the microenvironmental niche for hematopoietic stem and progenitor cells respiratory manifestations of malaria malaria-induced renal damage: facts and myths the sick placenta -the role of malaria orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates the schlemm's canal is a vegf-c/ vegfr- -responsive lymphatic-like vessel pathophysiology of severe malaria in children increased gastrointestinal permeability in patients with plasmodium falciparum malaria changes in gastric mucosa in acute malaria plasmodium vivax: clinical spectrum, risk factors and pathogenesis plasmodium ovale: parasite and disease plasmodium ovale: a case of not-so-benign tertian malaria plasmodium malariae: parasite and disease human infections and detection of plasmodium knowlesi the authors are supported by grants-in-aid for scientific research (b grant no. h to c.c.) and by the japan agency for medical research and development (amed j-pride fm h to c.c.). c.c. wrote the manuscript; all authors contributed to researching data, discussing the content and reviewing and editing the manuscript. the authors declare no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -v m fzw authors: sun, yan-gang; li, rui; jiang, longguang; qiao, songlin; zhi, yubao; chen, xin-xin; xie, sha; wu, jiawei; li, xuewu; deng, ruiguang; zhang, gaiping title: characterization of the interaction between recombinant porcine aminopeptidase n and spike glycoprotein of porcine epidemic diarrhea virus date: - - journal: int j biol macromol doi: . /j.ijbiomac. . . sha: doc_id: cord_uid: v m fzw porcine epidemic diarrhea (ped) has caused huge economic losses to the global pork industry. infection by its causative agent ped virus (pedv), an alpha-coronavirus, was previously proven to be mediated by its spike (s) glycoprotein and a cellular receptor porcine aminopeptidase n (papn). interestingly, some recent studies have indicated that papn is not a functional receptor for pedv. to date, there is a lack of a direct evidence for the interaction between papn and pedv s protein in vitro. here, we prepared papn ectodomain and the truncated variants of pedv s protein in drosophila s cells. these recombinant proteins were homogeneous after purification by metal-affinity and size-exclusion chromatography. we then assayed the purified target proteins through immunogenicity tests, pedv binding interference assays, circular dichroism (cd) measurements, papn activity assay and structural determination, demonstrating that they were biologically functional. finally, we characterized their interactions by gel filtration chromatography, native-polyacrylamide gel electrophoresis (page) and surface plasmon resonance (spr) analyses. the results showed that their affinities were too low to form complexes, which suggest that papn may be controversial as the genuine receptor for pedv. therefore, further research needs to be carried out to elucidate the interaction between pedv and its genuine receptor. coronaviruses are enveloped single-stranded positive-sense rna viruses, comprising alpha-, beta-, gamma-, and delta-coronaviruses [ ] . they are critical causative agents, whose infections are often associated with respiratory, digestive, and neurological diseases in humans and animals. among these viruses, two beta-coronaviruses severe acute respiratory syndrome coronavirus (sars-cov) [ ] [ ] [ ] [ ] [ ] and middle east respiratory syndrome coronavirus (mers-cov) [ ] [ ] [ ] [ ] are lethal to humans, and an alpha-coronavirus transmissible gastroenteritis virus (tgev) has caused severe intestinal diseases to pigs [ ] . the infection of a coronavirus is mediated by its spike (s) glycoprotein and the s protein is further cleaved into s and s subunits by endogenous and/or exogenous proteases [ ] . the s subunit recognizes and binds to the corresponding host receptor, whereas the s subunit mediates membrane fusion between the virus and host cells [ ] [ ] [ ] . since intervention of s subunit binding to host receptor shows a great antiviral potential, there are increasing studies to elucidate their interaction mechanisms. angiotensin-converting enzyme (ace ) [ , ] and dipeptidyl peptidase (dpp ) [ ] [ ] [ ] have been identified as the receptor for sars-cov and mers-cov, respectively. the viral receptor-binding domains (rbd) are located in the c-terminal domains (ctds) of the s subunits (s -ctds) [ , , ] . human coronavirus e (hcov- e) [ , ] and tgev [ , ] utilize aminopeptidase n (apn) as the host receptor and their rbds are also located in the s -ctds [ , ] . porcine epidemic diarrhea (ped), characterized by vomiting, diarrhea and dehydration, has caused huge economic losses to the global pork industry [ ] [ ] [ ] [ ] [ ] [ ] . its causative agent, ped virus (pedv), was an alpha-coronavirus. previous studies showed that a porcine receptor apn (papn) mediated pedv infection [ ] [ ] [ ] [ ] and its density appeared to be an important factor in contributing to the virus efficient infection [ ] . pedv s -ctd (residues - ) was further demonstrated to interact with papn ectodomain (residues - ) [ ] . interestingly, a few recent studies show that papn is not the functional receptor for pedv infection [ , ] . however, there is a lack of direct demonstration of the interaction between papn and pedv s protein to clarify this discrepancy. in this study, we prepared the extracellular domain of papn, pedv s and its truncated variant (s t) in drosophila schneider (s ) cells. after purification, we assayed the target proteins for their biological functions. finally, we characterized their interactions by three canonical assays. ipec-j cell line (from porcine small intestines) was kindly provided by zhanyong wei of henan agricultural university, china. vero cell line (african green monkey kidney epithelial cell line), drosophila s cell line, pmt/bip/v -his a vector, pcoblast vector [ , ] and positive serum against pedv were kept in our laboratory. peasy-blunt-papn plasmid was constructed and kept in our laboratory. cellfectin ii reagent, blasticidin s, sf- ii serum-free medium (sfm) and trizol reagent were purchased from invitrogen (carlsbad, usa). escherichia coli strain trans α competent cells were purchased from transgen biotech (beijng, china). fetal bovine serum (fbs), dulbecco's modified eagle's medium (dmem), roswell park memorial institute- medium (rpmi- ), and antibiotics were purchased from gibco (grand island, usa). schneider's insect medium, tryptose phosphate broth (tpb), tpck-trypsin, and l-alanine -nitroanilide hydrochloride were purchased from sigma-aldrich (st. louis, usa). the mouse anti-his epitope tag antibody was purchased from proteintech (wuhan, china). horseradish peroxidase (hrp)-conjugated anti-mouse immunoglobulin g (igg) was purchased from santa cruz biotechnology (delaware ave, usa). pre-stained protein ladder ( to kda) was purchased from thermo fisher scientific (waltham, usa). mlu i, bgl ii and peptide-n-glycosidase f (pngase f) enzymes were purchased from new england biolabs (massachusetts, usa). enhanced chemiluminescence (ecl) plus reagent was purchased from solarbio (beijing, china). the cdnas encoding pedv s (residues - ) and its truncated variants (residues - , - and - ) were amplified by pcr using the codon-optimized s gene of the pedv ch/hnxc strain as template. the cdna encoding papn ectodomain (residues - ) was amplified by pcr from the peasy-blunt-papn plasmid. all pcr primers were listed in table . the pcr products were isolated and inserted into the pmt/bip/v -his a expression vector between the bgl ii and mlu i sites. all constructs were transformed into escherichia coli strain trans α competent cells and the recombinant expression vectors were verified by sequencing at shanghai sangon biotech co. ltd. (shanghai, china). the drosophila s cells were kept in schneider's insect medium supplemented with % heat-inactivated fbs at °c in a humidified incubator. the vero cells were grown in dmem supplemented with % heat-inactivated fbs at °c in a humidified incubator with % co . ipec-j cells were grown in rpmi medium with % heat-inactivated fbs at °c with % co . all cell cultures were supplemented with antibiotics ( u/ml penicillin, μg/ml streptomycin). the recombinant expression vectors and the pcoblast vector were co-transfected into drosophila s cells by cellfectin ii reagent according to the manufacturer's instructions. the stably transfected s cells were selected in schneider's insect medium supplemented with % fbs, antibiotics, and μg/ml blasticidin s. the stably transfected cell lines were then grown in sf- ii sfm and induced by . mm cuso for days. the culture supernatants were harvested by centrifugation at rpm at °c for min. expression of recombinant proteins was checked by mouse anti-his epitope tag antibody and hrp-conjugated anti-mouse igg using western blot. the supernatant containing papn ectodomain, pedv s or s t protein was adjusted to ph . by . m tris-hcl ph . , followed by centrifugation at rpm at °c for min. after filtration through . μm filter membranes, the supernatant was applied to histrap excel prepacked column (ge healthcare, fairfield, usa) or/and hitrap q hp prepacked column (ge healthcare) for purification. then, each target protein was subjected to size-exclusion column hiload / superdex pg (ge healthcare) and superdex increase / gl (ge healthcare) for further purification with mm tris-hcl ph . and mm nacl as the elution buffer. the purified proteins were treated with pngase f according to the manufacturer's instructions. the pngase f-treated and -untreated proteins were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) under reducing (with β-mercaptoethanol) or non-reducing condition (without β-mercaptoethanol). . . function analyses of pedv s and s t proteins . . . immunogenicity tests of pedv s and s t proteins purified pedv s and s t proteins were subjected to sds-page and transferred to pvdf (polyvinylidene fluoride) membranes. the immunogenicity was then tested by positive serum against pedv and ecl plus reagent. the pedv ch/hubei/ strain was propagated on vero cells as previously described [ ] . briefly, when the confluence reached %, vero cells were washed three times and inoculated with tcid ( % tissue culture infective dose) pedv at °c for h. after that, the viruses not entering were removed and the cells were cultured with growth medium consisting of . % tpb, . % yeast extract and table primers utilized in this study. sequence a a the boldface letters indicate restriction sites. μg/ml of tpck-trypsin. when obvious cytopathic effects (cpe) appeared, the cells were frozen and thawed three times, and centrifuged at rpm at °c for min. finally, the supernatant was stored at − °c for further use. . . . interference of pedv binding by purified s and s t proteins the ipec-j cells were pre-incubated with indicated concentrations of the purified pedv s or s t protein for h at °c. cells were further inoculated with pedv ch/hubei/ strain at an moi (multiplicity of infection) of . for h at °c. then, the virus and protein mixtures were washed away. total rna was isolated using trizol reagent according to the manufacturer's instructions and the pedv binding was represented by the copies of the pedv nucleocapsid (n) gene using absolute quantitative pcr (the primers were listed in table ) [ ] . . . circular dichroism (cd) measurements of pedv s and s t proteins cd measurements ( - nm) were carried out on aviv sf spectrometer (lakewood, usa) at room temperature (rt) using a mm path-length quartz cell. the instrument was calibrated with mm phosphate buffer (pb) ph . , and freshly purified pedv s and s t proteins were adjusted to . mg/ml and . mg/ml in mm pb ph . , respectively. five scans were accumulated at a scan speed of nm/step with average time of . s. . . . activity assay of papn ectodomain nm purified papn ectodomain was mixed with μm to mm l-alanine -nitroanilide hydrochloride as substrate in μl mm kh po buffer ph . at °c for min. then, the product pnitroanilide was measured using a microplate reader (bmg labtech, offenburg, germany) at nm [ ] . the purified papn ectodomain was concentrated to mg/ml in mm tris-hcl ph . , mm nacl. crystallization of papn ectodomain was carried out by the sitting-drop vapor diffusion method with an equal volume of the target protein and various crystallization reagents from the crystallization screening kits (hampton, usa) at rt. the crystals were acquired with a reservoir solution containing % (wt/vol) peg , . m sodium fluoride and mm hepes ph . . the crystals were flash-frozen in liquid nitrogen using a crystal freezing buffer containing % ethylene glycol, % peg , . m sodium fluoride and mm hepes ph . . x-ray data sets of the crystals were collected at a wavelength of . Å on the beam line bl u at national center for protein sciences shanghai (ncpss) and shanghai synchrotron radiation facility (ssrf). the crystal structure was solved by molecular replacement [ ] using the mammalian apn structure (pdb code: hom) [ ] as the search model. the structure was refined by ccp program package [ ] and manually adjusted by the molecular graphics program coot [ ] until convergence of the refinement. solvent molecules were added using a fo-fc fourier difference map at . σ in the final refinement step. statistics of data collection and final model refinement were summarized in table . the coordinates of papn ectodomain were deposited in the protein data bank (pdb code z , http://www.rcsb.org). the final structures were analyzed by software pymol [ ] . papn ectodomain was mixed with excess pedv s or s t protein for h at rt in mm tris-hcl ph . , mm nacl. then, pedv s or s t protein, papn ectodomain and their mixtures were individually loaded on the superdex increase / gl column (ge healthcare) in the same volume and superposed for uv absorption peaks. the eluted proteins were analyzed by sds-page. the % native-page gel and running buffer were prepared without sds. the papn ectodomain and pedv s or s t protein were mixed in μl mm tris-hcl ph . , mm nacl at a molar ratio of : / : . after incubation at rt for min, pedv s or s t protein, papn ectodomain and their mixtures were analyzed by native-page. spr analyses were performed on biacore s (ge healthcare) to determine the equilibrium binding constants (k d , m), the association rate constants (k on , m − s − ) and the dissociation rate constants (k off , s − ) of papn ectodomain binding to pedv s or s t protein. pedv s or s t protein was coupled to the cm sensor chips (ge healthcare) and papn ectodomain was injected with the indicated concentrations ( . nm, nm, nm, nm and nm). the running buffer was hbs-ep ( mm hepes ph . , mm nacl, mm edta, . % surfactant p ). the binding kinetics were analyzed with the software biacore s evaluation . , and the k on , k off and k d were calculated using a : interaction model as previously described [ ] . the equations used for calculation are represented below with biacore spr terminology: where dr dt (response unit/s, ru/s) is the slope of the curve during association phase, c (m) is the concentration of injected papn ectodomain, rmax (ru) is the signal response for maximum absorption of pedv s or s t protein, and r (ru) is the signal response for papn ectodomain binding to pedv s or s t protein. all experimental data were presented as group means and standard errors of the means (sem). the experimental data were analyzed using the unpaired, -tailed student t-test using graphpad prism software (graphpad). differences at the % confidence level (p b . ) were considered statistically significant. the recombinant papn ectodomain (residues - ), pedv s protein (residues - ) and s truncated variants (residues - , - and - ) were produced in drosophila s cells. western blot analyses verified that papn ectodomain (fig. a) , pedv s protein (fig. b) and a pedv s truncated variant (s t, residues - , fig. c) were highly expressed. the expression of other pedv s truncated variants (residues - and - ) was too low to be detected (data not shown), which were consequently not considered in our subsequent experiments. the target proteins were purified through metal-affinity and sizeexclusion chromatography. after purification, these proteins achieved to a high purity (n %, fig. ). papn ectodomain (fig. , top) , pedv s (fig. , middle) and s t proteins (fig. , bottom) were eluted at . ml, . ml and . ml on the calibrated size-exclusion column, corresponding to approximately kda, kda, kda, respectively. as shown in fig. , there were differences between the bands of each target protein under reducing (lane ) and non-reducing (lane ) conditions, which implied that they contained disulfide bonds. in addition, the bands showed a significant change after de-glycosylation using pngase f (lane and lane ). therefore, the recombinant proteins were heavily glycosylated compared to their calculated molecular masses (papn ectodomain: kda, pedv s protein: . kda, pedv s t protein: . kda). based on the results above, recombinant papn ectodomain was obtained as dimers, and pedv s or s t protein existed as monomers, which showed similar natures of mammalian apn [ ] and other coronavirus s proteins [ ] [ ] [ ] as previously reported. . . immunogenicity tests, interference of pedv binding and cd measurements of purified pedv s and s t proteins we carried out immunogenicity tests and pedv binding interference assays to analyze whether purified pedv s and s t proteins were functional. as shown in fig. a and b, purified pedv s and s t proteins could be recognized by positive serum against pedv, indicating that these recombinant proteins had a comparable immunogenicity to the native virus. since porcine small intestines are the major target organ for pedv infection, ipec-j cells from porcine small intestines were applied in interference of pedv binding by purified pedv s and s t proteins. ipec-j cells incubated with pedv s or s t protein showed the viral rna reduction in a concentration dependent manner (fig. c ). especially at a concentration of μg/ml, pedv s or s t protein pretreatment caused a significant reduction compared to that incubated with control protein buffer (p b . ), demonstrating that purified pedv s and s t proteins were in functional form (fig. c) . we have taken efforts to crystallize functional pedv s or s t protein, and determine their crystal structures. unfortunately, crystallization of these proteins was unsuccessful (data not shown). cd measurement is usual for determination of protein secondary structures [ ] [ ] [ ] [ ] [ ] [ ] and we performed cd measurements to study their secondary structures. as shown in fig. , one minimum at about nm was observed for both pedv s and s t proteins, suggesting that the two proteins had β-sheets. these results were consistent with other coronavirus s proteins, which have a high content of β-sheets [ , , [ ] [ ] [ ] [ ] . mammalian apn is a member of zinc-dependent m metallopeptidases, which is widely distributed on the intestinal epithelia and the nervous system cells [ ] . it plays multifunctional roles in tumor angiogenesis and metastasis, signal pathway, and degradation of enkephalins [ ] . based on these functions, we performed activity assay for purified papn ectodomain. the enzymatic kinetics showed pedv s protein (residues - ). lane - : pedv s protein; lane : mock. (c): pedv s truncated variant (s t, residues - ). lane : pedv s t protein. lane m: thermo pre-stained protein ladder ( to kda). that purified papn ectodomain could degrade the substrate l-alanine nitroanilide hydrochloride into the product p-nitroanilide. the k m and k cat were . ± . mm and . ± . /s, respectively (table ) . furthermore, we determined the crystal structure of papn ectodomain. purified papn ectodomain was crystallized with one molecule in each asymmetric unit and its crystal structure was determined at . Å in a c space group (table ) . as described previously [ , , , , ] , papn ectodomain adopts a hook-like conformation or so-called seahorse shape ( fig. b and c) . it is consisted of four domains (i-iv) and a zinc ion in domain ii, characteristics of m metallopeptidases ( fig. a and b ). in addition, it is heavily glycosylated, and cys and cys in association with cys and cys bridge into two disulfide linkages in domain iv (fig. b) , which are consistent with our results stated above (fig. ) . a close structural comparison of our current papn ectodomain and that determined by others (pdb code: hom) reveals that the root mean square deviation (rmsd) differences are slight ( . Å for matching cα atoms), demonstrating that they share almost identical structural fold (fig. c ). taken together, purified papn ectodomain was shown to be biologically active on the basis of its enzymatic activity and crystal structure. papn was previously demonstrated as a functional receptor for pedv [ ] [ ] [ ] [ ] . however, the vero cell lines, possessing no apn fig. . cd spectra of functional pedv s (a) and s t proteins (b). one minimum at about nm was observed for both pedv s and s t proteins. fig. . immunogenicity tests and interference of pedv binding by purified s and s t proteins. purified pedv s (a) and s t proteins (b) were detected by western blot using the positive antiserum against pedv. (c) pedv binding interference with purified pedv s or s t protein. data represent means ± sem of three independent experiments. *significantly reduced viral rna (p b . ). [ , ] , are efficiently infected by pedv, and widely utilized for isolation and series propagation of the virus [ , [ ] [ ] [ ] [ ] . recently, no effects were reported on the susceptibility to pedv after knocking out apn in porcine swine testis cells and human cell lines (human hepatoma cells, huh cells and human cervical cancer cells, hela cells) by crispr/cas genome editing [ ] . moreover, soluble papn ectodomain neither interacted with pedv nor inhibited its infection [ ] . all these studies infer that papn may be not the receptor for pedv. to date, there are few direct evidences to clarify this discrepancy by characterization of the interaction between papn ectodomain and pedv s protein in vitro. we speculate that the lack of related studies is probably due to the difficulty in preparing the large pedv s protein, which contains many disulfide bonds and glycosylation sites as other coronaviruses [ , ] . in the current study, three canonical assays were carried out to characterize the interaction between papn ectodomain and pedv s or s t protein since these functional target proteins were successfully prepared. according to previous reports, coronavirus s-rbds and their host receptors have high affinities and yield stable complexes in vitro, which can be obtained by gel filtration chromatography [ , , , , , ] . therefore, we firstly characterized the interaction between papn ectodomain and pedv s or s t protein by gel filtration chromatography. the results showed that there were no complexes formed in these protein mixtures as no complex peaks appeared (fig. ) . additionally, we performed native-page to detect whether they formed complexes. as shown in fig. a , there were no bands of the protein complexes compared to single proteins. interestingly, pedv s t protein showed other forms in addition to monomers (figs. b and a) . in order to further corroborate the interaction between the proteins, the kinetics of papn ectodomain binding to pedv s or s t protein were measured by spr using biacore s . the profiles showed that there were no signal responses during their association and dissociation phases, which demonstrated that papn ectodomain neither interacted with pedv s nor s t protein under physiological condition (fig. b ). in contrast, hcov- e [ ] and tgev [ ] rbds had high affinities to their receptor apn. these results were similar to sars-cov rbd not binding to dpp or mers-cov rbd not binding to ace , while the k d of dpp binding to mers-cov rbd was about . nm (k on : . × m − s − , k off : . × − m − s − ) [ ] . in conclusion, our work characterized the interaction between recombinant papn ectodomain and pedv s protein in vitro. the results suggest that papn may be controversial as the genuine receptor for pedv because functional papn ectodomain neither binds to pedv s nor s t protein. our current work sheds some light on the invasion mechanism of the virus, and supports a molecular basis for prevention and control of ped. as s protein recognizing and interacting with the host receptor are crucial for pedv infection, further research needs to be carried out to identify the genuine receptor of pedv and elucidate the specific interaction between them. the authors declare that they have no conflicts of interest. papn ectodomain is colored as described for (a). the zinc ion is represented as red sphere and n-linked oligosaccharides are shown in stick representation. the n and the c termini are also labeled. (c) structural comparison of papn ectodomain in the current study with that determined by others (pdb code hom) in cartoon diagrams. the papn ectodomain in the current study is colored in green and the papn ectodomain previously determined is in cyan. their n and c termini are labeled. fig. . characterization of the interaction between papn ectodomain and pedv s or s t protein. (a): native-page analysis. lane : pedv s protein; lane : papn ectodomain and pedv s protein mixtures ( : ); lane : papn ectodomain; lane : papn ectodomain and pedv s t protein mixtures ( : ); lane : pedv s t protein. (b): spr analyses of the interaction between pedv s (top) or s t protein (bottom) and papn ectodomain. . gel filtration chromatography analyses of pedv s or s t protein (blue), papn ectodomain (green) and their mixtures (brown). the papn ectodomain, pedv s (a) or s t protein (b) and their mixtures were loaded on a calibrated superdex increase column ( / gl) individually. their chromatographs were superposed and shown in the figures. sds-page analyses of pooled samples (a: - and b: - ) were also presented. funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. coronavirus genomics and bioinformatics analysis a novel coronavirus associated with severe acute respiratory syndrome public health: broad reception for coronavirus coronavirus as a possible cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome severe respiratory illness caused by a novel coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission induction of transmissible gastroenteritis coronavirus specific immune responses using vectors with enteric tropism host cell proteases: critical determinants of coronavirus tropism and pathogenesis mechanisms of coronavirus cell entry mediated by the viral spike protein receptor recognition mechanisms of coronaviruses: a decade of structural studies structure, function, and evolution of coronavirus spike proteins angiotensin-converting enzyme is a functional receptor for the sars coronavirus structure of sars coronavirus spike receptorbinding domain complexed with receptor dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptorbinding domain complexed with human receptor dpp molecular basis of binding between novel human coronavirus mers-cov and its receptor cd human aminopeptidase n is a receptor for human coronavirus e receptor-binding loops in alphacoronavirus adaptation and evolution aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in central china based on the orf gene and the main neutralization epitopes complete genome sequence of porcine epidemic diarrhea virus strain aj isolated from a suckling piglet with acute diarrhea in china molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in gansu viral disease affects u.s. pigs: porcine epidemic diarrhea found in at least states distinct characteristics and complex evolution of pedv strains novel porcine epidemic diarrhea virus variant with large genomic deletion identification of a putative cellular receptor kda polypeptide for porcine epidemic diarrhea virus in porcine enterocytes porcine aminopeptidase n is a functional receptor for the pedv coronavirus receptor usage and cell entry of porcine epidemic diarrhea coronavirus development of transgenic mouse model expressing porcine aminopeptidase n and its susceptibility to porcine epidemic diarrhea virus contribution of the porcine aminopeptidase n (cd ) receptor density to porcine epidemic diarrhea virus infection identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains porcine aminopeptidase n is not a cellular receptor of porcine epidemic diarrhea virus, but promotes its infectivity via aminopeptidase activity aminopeptidase n is not required for porcine epidemic diarrhea virus cell entry the crystal structure of the fifth scavenger receptor cysteine-rich domain of porcine cd reveals an important residue involved in porcine reproductive and respiratory syndrome virus infection the cd longrange scavenger receptor cysteine-rich repeat: expression, purification and x-ray crystallographic characterization propagation of the virus of porcine epidemic diarrhea in cell culture a novel duplex taqman probe-based real-time rt-qpcr for detecting and differentiating classical and variant porcine epidemic diarrhea viruses structural basis for multifunctional roles of mammalian aminopeptidase n computational project, the ccp suite: programs for protein crystallography coot: model-building tools for molecular graphics the pymol molecular graphics system analysis of protein-dna interactions using surface plasmon resonance identification of n-linked carbohydrates from severe acute respiratory syndrome (sars) spike glycoprotein glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections the surfactant-induced conformational and activity alterations in rhizopus niveus lipase interplay of multiple interaction forces: binding of tyrosine kinase inhibitor nintedanib with human serum albumin synthesis, characterization and interaction studies of , , -oxadiazole derivatives of fatty acid with human serum albumin (hsa): a combined multi-spectroscopic and molecular docking study multispectroscopic and molecular modelling approach to investigate the interaction of riboflavin with human serum albumin binding of janus kinase inhibitor tofacitinib with human serum albumin: multi-technique approach biophysical and molecular docking insight into the interaction of cytosine β-d arabinofuranoside with human serum albumin cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding cryo-em structure of porcine delta coronavirus spike protein in the pre-fusion state the moonlighting enzyme cd : old and new functions to target the structure and main functions of aminopeptidase n the x-ray crystal structure of human aminopeptidase n reveals a novel dimer and the basis for peptide processing allosteric inhibition of aminopeptidase n functions related to tumor growth and virus infection isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene isolation and characterization of a korean porcine epidemic diarrhea virus strain knu- the dynamics of chinese variant porcine epidemic diarrhea virus production in vero cells and intestines of -day old piglets crystal structure of nl respiratory coronavirus receptor-binding domain complexed with its human receptor crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor we thank the staff of bl u beamline at national center for protein sciences shanghai (ncpss) and shanghai synchrotron radiation facility (ssrf), shanghai, china, for assistance during data collection. we also acknowledge qiang wei and hongfang ma from henan academy of agricultural sciences for their assistance on crystallization, and baolin xiao from henan university for his assistance on cd measurements. this work was supported by the key: cord- -ywwqnw authors: tomar, jasmine; biel, carin; de haan, cornelis a.m.; rottier, peter j.m.; petrovsky, nikolai; frijlink, henderik w.; huckriede, anke; hinrichs, wouter l.j.; peeters, ben title: passive inhalation of dry powder influenza vaccine formulations completely protects chickens against h n lethal viral challenge date: - - journal: eur j pharm biopharm doi: . /j.ejpb. . . sha: doc_id: cord_uid: ywwqnw bird to human transmission of high pathogenicity avian influenza virus (hpaiv) poses a significant risk of triggering a flu pandemic in the human population. therefore, vaccination of susceptible poultry during an hpaiv outbreak might be the best remedy to prevent such transmissions. to this end, suitable formulations and an effective mass vaccination method that can be translated to field settings needs to be developed. our previous study in chickens has shown that inhalation of a non-adjuvanted dry powder influenza vaccine formulation during normal breathing results in partial protection against lethal influenza challenge. the aim of the present study was to improve the effectiveness of pulmonary vaccination by increasing the vaccine dose deposited in the lungs and by the use of suitable adjuvants. two adjuvants, namely, bacterium-like particles (blp) and advax, were spray freeze dried with influenza vaccine into dry powder formulations. delivery of dry formulations directly at the syrinx revealed that blp and advax had the potential to boost either systemic or mucosal immune responses or both. upon passive inhalation of dry influenza vaccine formulations in an optimized set-up, blp and advax/blp adjuvanted formulations induced significantly higher systemic immune responses than the non-adjuvanted formulation. remarkably, all vaccinated animals not only survived a lethal influenza challenge, but also did not show any shedding of challenge virus except for two out of six animals in the advax group. overall, our results indicate that passive inhalation is feasible, effective and suitable for mass vaccination of chickens if it can be adapted to field settings. outbreaks of avian influenza in poultry are the major source of h n infection in humans [ ] [ ] [ ] . these outbreaks, caused by highly pathogenic strains of avian influenza virus, pose a significant risk of poultry-human transmission. currently, the only remedy to control these outbreaks is to cull the poultry. for safety reasons animals are not only culled on infected farmyards but also on non-infected neighboring farmyards [ , ] . the slaughter of millions of poultry not only leads to severe economic losses but also raises ethical questions and incomprehension within the society. to combat outbreaks of avian influenza virus, it could be useful to rapidly vaccinate the poultry in areas surrounding the outbreak, in a ring fencing strategy. however, this would require that very large numbers of poultry, potentially in the tens or hundreds of millions, should be able to be immunized within a very short time period. this would require a vaccine formulation and route of immunization that are suitable for such mass application. liquid or powder influenza vaccine formulations that can be aerosolized and administered via the respiratory tract could be appropriate formulations for mass vaccination. liquid formulations of inactivated influenza virus have been shown to be inadequate for aerosol vaccination by single administration [ ] . powder formulations, on the other hand, have shown an advantage over liquid formulations because of their long-term storage stability [ ] [ ] [ ] . this long term stability facilitates stockpiling and thus provides ease of administration during mass vaccination [ ] . in a previous study, we have shown that pulmonary immunization by dispersion of a dry powder influenza vaccine directly at the syrinx of chickens (active administration) was able to completely protect these animals against lethal viral challenge [ ] . however, in realistic situations, due to extreme time pressures, active administration of chickens is unsuitable for mass vaccination. a more realistic approach would be to let chickens inhale aerosolized dry powder influenza vaccine formulation during breathing (passive administration). indeed, passive inhalation of dry powder influenza vaccine formulation was feasible in chickens, but the animals were only partially protected indicated by a delay in time to death and reduced virus shedding [ ] . hence, the efficiency of influenza immunization by passive inhalation must be improved for pulmonary vaccination to become feasible. possible approaches could be to increase the concentration of aerosolized vaccine, to expose the animals to aerosolized powder for longer periods of time and to include an adjuvant in the vaccine formulation. an effective adjuvant should not only be cheap, readily available and potent, but also safe. an adjuvant that has been generally recognized as safe is blp. blp are produced by hot acid treatment (ph for min at °c) of lactococcus lactis, a non-pathogenic, food-grade gram-positive bacterium [ , ] . blp act as toll-like receptor- ligand and have shown potent immune boosting properties when administered together with vaccines against influenza, yersinia pestis, malaria, and pneumococcal disease [ ] [ ] [ ] [ ] . besides toll-like receptor ligands, naturally derived polysaccharides, for example advax™ adjuvant comprising an insoluble isoform of inulin, boosts vaccine responses through mechanism that are still not fully characterized [ ] . in (pre) clinical studies advax has been shown to enhance immune responses induced by a wide variety of vaccines including vaccines against hepatitis b, sars coronavirus, listeria and influenza [ ] [ ] [ ] [ ] [ ] [ ] . upon parenteral administration, advax has been shown to have a good safety and tolerability record both in animal studies and clinical trials [ ] [ ] [ ] . the aim of the current study was to investigate whether passive administration with dry non-adjuvanted or adjuvanted influenza formulations has the potential to completely protect chickens against lethal hpaiv challenge. for this, we initially tested whether (a) blp or advax could be co-formulated with influenza vaccine into dry powder formulations that are suitable for pulmonary immunization; (b) the adjuvants have the potential to boost systemic and mucosal immune responses to influenza; (c) passive administration with either non-adjuvanted or adjuvanted influenza formulations would protect chickens against a lethal hpaiv challenge. for immunization, a reassortant virus, nibrg- , prepared by reverse genetics from a/turkey/turkey/ / (h n ) virus and a/pr/ / (h n ) virus was used (nibsc code: / ). the virus was cultured in embryonated chicken eggs by allantoic inoculation of the seed virus. the virus was purified and inactivated as described previously to obtain whole inactivated virus vaccine (wiv) [ , ] . for challenge, a highly pathogenic avian influenza virus strain a/ turkey/turkey/ / (h n ) (clade . . ), obtained from the animal health and veterinary laboratories agency, weybridge, uk was used. for active administration, . , . , and . µg ha of nibrg- wiv was sfd either as such or admixed with adjuvants (blp, mucosis, groningen, the netherlands or advax, vaxine, adelaide, australia) in various ratios. for active administration, the dose of blp and advax in mg of sfd powder was µg and µg respectively. wiv was mixed with blp in weight ratios of ha:blp of : , : and : . for wiv-advax, the weight ratios of ha:advax were : , : and : . likewise, for passive administration, µg ha of nibrg- was sfd either as such or mixed with µg of blp or µg of advax (this dose corresponds to the amount of ha and adjuvants in mg of sfd powder). both non-adjuvanted and adjuvanted formulations were sfd using inulin ( kda, sensus, roosendaal, the netherlands) as the stabilizer. inulin used for stabilization of influenza vaccine is amorphous and water soluble with potentially no adjuvant effect whereas advax (delta inulin) is crystalline and insoluble in water with potent adjuvant activity. in brief, a % (w/v) solution of wiv and inulin with or without adjuvants was pumped at a flowrate of ml/min through a two-fluid nozzle of a büchi mini spraydryer (büchi, flawil, switzerland). an atomizing airflow of l/h was used to spray vaccine preparations in a vessel of liquid nitrogen. then, the frozen vaccine preparations were placed in a christ epsilon - freeze dryer precooled to a shelf temperature of − °c and at a pressure of . mbar. the shelf temperature was slowly increased from − °c to °c within the time period of h. during the next h, the temperature was further increased to °c and pressure was lowered to . mbar. the dry vaccine powder was collected in a climate box with a relative humidity of < % and stored under airtight conditions. a philips cm transmission electron microscope was used to make transmission electron microscopy images. sfd powders containing wiv ( µg ha formulation) with or without adjuvants i.e. blp ( µg in mg of sfd powder), advax ( µg in mg of sfd powder) were reconstituted in sterile filtered water. liquid and reconstituted sfd formulations were placed on a plain carbon grid, rinsed with water and then samples were stained twice with µl of wt-% uranyl acetate. a gatan type ultrascan sp ccd camera at a magnification was used to take images. the receptor binding activity of wiv after sfd with or without adjuvants was assessed by hemagglutination assay. sfd powders were reconstituted in phosphate buffer saline (pbs) to a concentration of µg/ml of ha. then, µl of the preparation was added to -well v bottom plates containing µl of pbs. the entire mixture was two-fold serially diluted, then µl of . % guinea pig red blood cell suspension was added to each well. the plate was allowed to stand undisturbed for two hours at room temperature. hemagglutination titers read after two hours were expressed as log of the highest dilution where red blood cell agglutination could be seen. the capacity of blp to activate nf-κb via toll-like receptor- was evaluated using raw-blue™ cells (invivogen, toulouse, france). raw-blue™ cells have a number of pattern recognition receptors which when bound to agonists leads to activation of nf-κb and thus the production of secreted alkaline phosphatase. a schematic illustration is shown in fig. s . cells were maintained in dmem with high glucose (gibco life technologies bv, bleiswijk, the netherlands), % fbs (lonza, basel, switzerland), µg/ml normocin™ (invivogen, toulouse, france), mm l-glutamine and passaged when - % confluency was reached. approximately × cells were added to -well flat bottom plates and were stimulated with . µg untreated blp or with liquid and reconstituted sfd powder formulations with ( µg ha + µg blp formulation) or without . µg blp. the incubation was maintained for h at °c with % co . to measure alkaline phosphatase levels, µl quanti-blue™ (invivogen, toulouse, france) was added to the cell supernatant and after h, absorbance was measured at nm using a spectrophotometer. sfd-wiv preparations were imaged using a jeol jsm -f microscope (jeol. ltd., tokyo, japan). powders were placed on doublesided sticky carbon tape on a metal disc and the particles were coated with approximately nm of gold using a balzer's b sputtering device (balzer union, liechtenstein). a magnification of × and × was used to capture the images. geometrical particle size distribution of the powders were measured using a helos compact model ka laser diffraction apparatus (sympatec gmbh, clausthal-zellerfeld, germany). to disperse the powders, the highly efficient rodos dispersing system (sympatec gmbh, clausthal-zellerfeld, germany) was used at a pressure of bar. aerodynamic particle size distribution was calculated using the equation described by bhide et al. [ ] . sixty -week old specific-pathogen free chickens (white leghorn) were randomly divided into groups of animals. animals were immunized twice i.e. on day and day . sfd-wiv formulations (either non-adjuvanted or adjuvanted with blp or advax) were administered to the animals using a dp- -c dry powder insufflator (penn-century inc., philadelphia, usa). a custom length delivery tube designed to deliver the powder directly at the syrinx was used. three puffs of ml air were used to aerosolize mg of powder filled in the insufflator. the animals were sacrificed on day . blood samples were taken on day (before the st immunization), day (two weeks after the st immunization) and day (two weeks after the nd immunization). on the day of sacrifice (day ), lung lavages were collected by flushing lungs with ml of pbs as described by holt et al. [ ] . the obtained sera and lung lavages were stored at − °c until further use. an overview of the immunization scheme and groups is shown in fig. a and b. for passive administration of non-adjuvanted and adjuvanted wiv formulations, -week old specific-pathogen free chickens were randomly divided into groups of chickens. one animal died of unknown causes before the start of the experiment. each group was placed in a customized box with a volume of . m . a ml eppendorf tube filled with mg of sfd powder containing . mg ha of nibrg- wiv with or without adjuvants was punctured at the bottom with a g hypodermic needle. the lid of the eppendorf tube was fitted to a pressurized air container while the bottom conical part of the tube was inserted airtight in a hole drilled in the customized box for the dispersion of powders. the sfd powders were aerosolized in the box by using several pulses of pressurized air, resulting in a theoretical concentration of mg ha/m (ha dose for dispersion: . mg, inhalation box volume: . m ). for the first vaccination, the animals were exposed to the aerosolized vaccine for min and after every min short pulses of medicinal oxygen ( s; flow rate . l/min) were supplied through another hole in the box. for the second vaccination ( weeks later i.e. on day ) the exposure time was min with short pulses of oxygen ( s; flow rate . l/min) every minute. based on the respiration rate for chickens of l/h/kg body weight [ , ] these exposure times would result in a maximum theoretical dose of µg ha per animal per application. the eid ( % embryo-infectious dose) titre of the challenge strain stock of a/turkey/turkey/ / ( . log eid /ml) was used to calculate the dilution for the challenge virus. the % chicken lethal dose of a/turkey/turkey/ / virus is . log eid [ ] . on day , animals were challenged with a lethal dose of . log eid of this virus by the combined intranasal/intratracheal route (liquid suspension; . ml each). for back titration, we determined tcid ( % tissue culture infectious dose). the titre ( log tcid /ml) after backtitration of the challenge virus that we received from the stables after the challenge inoculation was . whereas the titre of the original stock was . . thus, a titre of . eid /ml corresponds to . tcid /ml. the titre of . tcid /ml for the back-titration should therefore correspond to at least . eid /ml. since the % chicken lethal dose of the challenge virus is . log eid , a dose of . logeid ( . ml of . eid /ml) is more than sufficient (> × cld ) to kill nonvaccinated animals. in previous studies with the same virus batch and dilution, we found that this dose killed all non-vaccinated control group animals within - days after the challenge [ , ] . in this study, after challenge, animals were observed for clinical signs and on , , , and days post challenge choanal and cloacal swabs were collected for virus quantification. on day , the animals were sacrificed. an overview of the immunization-challenge scheme along with groups is shown in fig. c and d. passive inhalation of dry formulations could be seen in video file (supplementary data). virus titers in choanal and cloacal swabs were determined as per the method described by van der goot et al. [ ] . challenge virus was tenfold serially diluted, followed by rna extraction, and a standard curve consisting of the rna from these serial dilutions was prepared. as per the standard curve, pcr data was converted into equivalent virus titers (eqtcid /ml). serum samples and lung washes were used for the determination of igy and iga antibody titers. for the determination of igy titers, elisa was performed as previously described [ ] , except that the secondary antibody consisted of goat anti chicken igy-hrp (southern biotech, birmingham, usa). igy titers were determined as log of the reciprocal of the sample dilution corresponding to an absorbance of . at the wavelength of nm. iga titers were determined in lung washes using the commercially available chicken iga elisa kit as per manufacturer's instructions (abcam, cambridge, uk). hi assay was performed as described previously [ ] . briefly, hemagglutination units of inactivated virus a/turkey/turkey/ / (h n ) were added to two-fold diluted sera samples. hi titers were recorded as highest serum dilution capable of preventing hemagglutination. hi titers are presented on log scale. micro-neutralization titers were determined as previously described [ ] . briefly, two-fold serial dilutions of serum samples were prepared and tcid of nibrg- virus was added to each well of well plate. after h of incubation at °c, the mixture of serum and virus was transferred to mdck cells (atcc, germany) cultured in -well flat bottom plate. after h of incubation at °c, the supernatant was discarded and replaced with episerf-medium ( u/ml penicillin, mg/ml streptomycin, m hepes and . % sodium bicarbonate, all life technologies™ bv, bleiswijk, the netherlands) supplemented with µg/ml of tpck trypsine (sigma-adrich, the netherlands). after h of incubation, the supernatant was transferred to v bottom plate. micro-neutralization titers were calculated by finding the highest serum dilution capable of preventing hemagglutination. micro-neutralization titers are presented on log scale. mann-whitney one tailed test was used to compare whether the differences between non-adjuvanted and adjuvanted influenza vaccine formulations were significant. levels of significance are denoted as * p ≤ . , ** p ≤ . , *** p ≤ . respectively. in order to investigate the potential of blp and advax as mucosal adjuvants in chickens, it was essential to investigate whether the physical and biological properties of wiv and adjuvants remained unaltered during sfd. to evaluate whether admixing adjuvants with wiv and sfd had an influence on the physical appearance of wiv, blp or advax particles, transmission electron microscopy analysis was performed for these samples before and after sfd. the powder samples were reconstituted prior to use. transmission electron microscopy pictures showed that wiv, blp and advax particles had comparable morphological appearance before and after sfd ( fig. a-c) . thus, neither the stress encountered during sfd nor admixing adjuvants with wiv had any adverse effect on their physical appearance. the biological activity of wiv needs to be preserved both after the addition of adjuvants and after sfd. in order to investigate whether the receptor binding activity of ha was preserved, hemagglutination assay was performed. no differences in hemagglutination titers could be detected either by the addition of adjuvants or by sfd, thus indicating no detrimental effects of adjuvants or sfd on the biological activity of ha (fig. d) . furthermore, the effect of sfd on the biological activity of blp was evaluated. for this purpose, reconstituted blp-adjuvanted sfd wiv formulations were compared to unprocessed dispersion of liquid wiv and blp for their capacity to activate nfκb using raw-blue™ cells. nibrg- derived wiv alone was found to be a poor activator of nfκb. reconstituted blp-adjuvanted wiv formulations activated nfκb to a similar extent as native liquid wiv-blp dispersion. thus, stress encountered during sfd had no effect on the biological activity of blp to activate toll-like receptor (fig. e ). to assess whether the incorporation of blp or advax in the sfd formulation had an effect on the physical characteristics of powder particles, scanning electron microscopy was used to analyze physical appearance of sfd formulations. spherical shaped intact particles with an interconnected porous structure could be seen for both non-adjuvanted and adjuvanted wiv formulations. no major differences could be observed in the morphology of sfd particles by varying the dose of ha and by the addition of adjuvants. representative pictures of nonadjuvanted, blp and advax adjuvanted wiv formulation at an ha dose of . µg per mg of powder are shown in fig. a -c. further, upon determination of geometric particle size, average particle size (x ) was found to be between and µm both for non-adjuvanted and adjuvanted wiv formulations (fig. d) . however, for inhalation, an aerodynamic particle size of - µm is considered to be most suitable [ ] . the average aerodynamic particle size of non-adjuvanted as well as adjuvanted wiv formulations was calculated according to the formula described by bhide et al. [ ] . the average aerodynamic particle size was found to be ≤ . µm and the majority of the particles (x ) had size ≤ µm (fig. e) . particles with a size range between and . µm are considered to be suitable for deposition in the entire respiratory tract of chickens [ ] [ ] [ ] . overall, our results indicated that sfd can produce blp and advaxadjuvanted wiv particles with physical and biological characteristics that make them suitable for pulmonary vaccination. for active administration, the tip of the insufflator was placed directly at the syrinx of chickens and three puffs of ml air were used to disperse the powders. we next evaluated the systemic and mucosal immune responses induced in chickens after active administration. serum igy and hi titers were measured both at day and day whereas serum mn, lung igy and iga titers were measured only at day . on day , non-adjuvanted as well as adjuvanted wiv formulations had induced considerable serum igy titers. advax-adjuvanted wiv formulations induced significantly higher serum igy titers than the corresponding non-adjuvanted wiv formulations (fig. a ). however, no major differences were found among non-adjuvanted and blp-adjuvanted wiv formulations, except between corresponding . µg ha formulations (fig. a) . although, this difference was statistically significant, it was not major. however, on day , significantly higher titers were induced by blp-adjuvanted wiv formulations than by the non-adjuvanted wiv formulation at an ha dose of . and µg; the difference between non-adjuvanted and blp-adjuvanted formulations was not significant at ha dose of µg (fig. b) . for advax-adjuvanted wiv formulation, significantly higher titers than corresponding nonadjuvanted wiv formulation were only induced by the µg ha formulation (fig. b) . systemic immune responses were further assessed by determining serum hi titers. it was found that after the first vaccination i.e. at day , none of the vaccinated chickens had detectable hi titers (fig. c) . however, after the second vaccination, blp-adjuvanted wiv formulations, at a dose of and . µg ha, induced a trend towards higher hi titers (mean hi titers of . log and . log respectively) than corresponding non-adjuvanted wiv formulations (mean hi titers of . log and respectively) (fig. c ). though at an ha dose of µg, the difference was not statistically significant, but at an ha dose of . µg, significantly higher titers could be seen for blp-adjuvanted wiv formulation than corresponding non-adjuvanted formulation. also, compared to non-adjuvanted wiv formulation ( ), a small but non-significant increase in hi titers was seen for the advax-adjuvanted wiv formulation at the lowest ha dose of . µg ( . log ) (fig. c) . at day , micro-neutralization titers were found to be in line with hi titers. in comparison to non-adjuvanted wiv formulation, advax and blp-adjuvanted wiv formulations at a dose of . µg ha induced an increase in the micro-neutralization titers of about five and eight fold, respectively (fig. d) . no major differences were seen at µg and . µg ha dose. mucosal immune responses were assessed by determining iga and igy titers in the lung lavages obtained two weeks after the second immunization. lung igy titers were found to be consistent with serum igy titers after the second immunization. blp-adjuvanted wiv formulations induced significantly higher lung igy titers than respective non-adjuvanted wiv formulations at a dose of . and µg ha (fig. e ). in addition, advax-adjuvanted wiv formulations induced higher lung igy titers than non-adjuvanted wiv formulation at an ha dose of . µg, though the difference was not significant (fig. e) . though non-adjuvanted wiv formulations were found to induce considerable lung iga titers, the inclusion of blp and advax further boosted the immune responses (fig. f) . it was found that at a dose of µg ha, blp-adjuvanted wiv formulation boosted lung iga titers ( . log ) by approximately four fold in comparison to corresponding non-adjuvanted wiv formulation ( . log ) (fig. f) . for advax-adjuvanted wiv formulations, at a dose of . and µg ha, lung iga titers were augmented by three to five fold as compared to corresponding non-adjuvanted wiv formulations (fig. f) . passive administration might be a suitable method for mass vaccination of chickens. for passive administration, chickens placed in a custom made box were allowed to inhale aerosolized vaccine powders during breathing using two applications weeks apart. we next evaluated the systemic immune responses induced in chickens after passive administration of these aerosolized powders. serum igy titers were determined at day and day whereas micro-neutralization titers were determined at day . adjuvanting with blp or advax resulted in significantly higher serum igy titers at day ; an increase of about three to five fold could be seen by the co-administration of blp or advax with wiv (fig. a) . however, after the second vaccination, only blp and advax/blp adjuvanted wiv formulations had significantly higher serum igy titers ( . - . log ) than non-adjuvanted wiv formulation ( . log ) (fig. b) . the augmentation in serum igy titers was about six-fold for blp-adjuvanted wiv and three fold for advax/ blp-adjuvanted wiv (fig. b) . trends in hi titers were in agreement with serum igy titers both after the first and second immunization. after the first immunization, all three formulations i.e. blp, advax and advax/blp adjuvanted wiv formulations showed significantly higher hi titers (mean hi titers between . and . log ) than non-adjuvanted wiv formulation (mean hi titer . log ) (fig. c) . notably, after the second immunization, blp adjuvanted and advax/blp-adjuvanted wiv formulations induced significantly higher hi titers (mean hi titers between . and . log ) than non-adjuvanted wiv or advax adjuvanted wiv (mean hi titers of . log ). in addition, micro-neutralization titers determined after the second immunization were also in line with serum igy and hi titers. blp and advax/blp adjuvanted wiv formulations induced six-fold higher micro-neutralization titers ( . log ) than non-adjuvanted wiv formulation ( . log ) (fig. d ). to assess whether passive administration of non-adjuvanted or adjuvanted influenza vaccine formulations, has the potential to reduce/ diminish the shedding of challenge virus after lethal challenge, virus shedding was determined in choanal and cloacal swabs. in our previous study, we found out that non-vaccinated animals shed virus until they succumbed to infection ( days post challenge) [ ] . in this study, a similar dose of a/turkey/turkey/ / virus was used to challenge chickens by the combined intranasal/intratracheal route. no virus was found either in choanal or cloacal swabs of animals immunized with non-adjuvanted or blp adjuvanted or advax/blp adjuvanted wiv formulations (table ) . only two animals immunized with advax-adjuvanted wiv formulation showed virus in their choanal swabs ( table ) in this study, we demonstrated that blp and advax can be co-formulated with influenza vaccine into a dry formulation that is suitable for pulmonary immunization of chickens. upon active administration of chickens, the adjuvants blp and advax were shown to augment systemic and mucosal immune responses. remarkably, in an optimized setup, passive administration of chickens either with non-adjuvanted or blp or advax adjuvanted wiv formulations induced robust immune responses that were sufficiently high to protect chickens against lethal viral challenge. furthermore, blp and advax/blp adjuvantation significantly raised the level of antigen-specific serum antibodies. in real life situations, where passive inhalation would be performed in large rooms, a portion of the aerosolized vaccine formulation would also end up in food and water. however, previous studies have shown that ha of the influenza vaccine undergoes conformational changes at acidic ph and is thus susceptible to proteolytic cleavage in the acidic environment [ , ] . consequently, little-no neutralizing antibodies are induced by acidic ph treated influenza vaccine [ , ] . therefore, it is speculated that the acidic environment in the stomach of chickens would not really add up to the lung induced mucosal immunity. the crystalline nature of advax and peptidoglycans on the surface of blps might have detrimental effects on influenza vaccine during the production of adjuvanted dry powder formulations. however, the inclusion of blp or advax together with wiv in a formulation led to the formation of dry powder particles of comparable physical and biological characteristics as those of non-adjuvanted wiv formulation. moreover, the physical properties of adjuvants i.e. their size and shape were found to be unaltered after sfd. also, the biological activity of blp was found to be well preserved during sfd. previous studies have shown that administration of blp or advax adjuvanted influenza vaccine formulations via the respiratory tract results in substantial augmentation of systemic and mucosal immune responses in mice [ , [ ] [ ] [ ] [ ] . in this study we showed that after active administration, systemic immune responses (serum igy titers) after one immunization were predominantly enhanced by advax whereas after two immunizations they (serum igy, hi titers, micro-neutralization titers) were mainly enhanced by blp. after two immunizations, both non-adjuvanted and adjuvanted wiv formulations, at an ha dose of µg, induced average hi titers above log which was previously found to be required for protection against lethal dose of influenza virus [ ] . with respect to mucosal immune responses, lung igy titers were mainly enhanced by blp whereas lung iga titers were augmented by both blp and advax. though both blp and advax were found to enhance either systemic or mucosal immune responses or both, the observed discrepancies might be due to the dose of blp ( µg) and advax ( µg) used in this study. although doses of µg of blp and µg of advax were found to be sufficiently high to boost both systemic and mucosal immune responses in mice, however, these doses might be too low for chickens with a x higher body weight. this might have resulted in the adjuvant dose to be a limiting factor in the active administration study. the active administration study revealed a slight but significantly higher effect of both blp and advax adjuvants, in comparison to nonadjuvanted wiv formulations. hence, for the passive administration study, the relative dose of blp was increased to double, i.e µg/mg of sfd powder while keeping the dose of advax similar. since advax particles consisted of wt% of the sfd powder formulation, enhancing the dose of advax further would most likely have compromised the integrity of the powder particles. moreover, a pattern of advax being effective after one and blp after two immunizations could also be determined in the active administration study. therefore, one of the groups of passively administered animals inhaled advax-adjuvanted wiv for the first immunization and blp-adjuvanted wiv for the second immunization. in our previous study, passive inhalation led to in-efficient delivery of influenza vaccine powders to the lungs of chickens and thus provided only partial protection [ ] . hence, for this study, we aimed for complete protection against lethal influenza viral challenge. this was achieved by enhancing vaccine powder delivery to the lungs: by increasing the vaccine concentration (by reducing the size of inhalation box), by increasing the exposure time, and by the use of adjuvants. compared to our previous study, optimization of the vaccine concentration and exposure time resulted in an increment of ∼ fold in the theoretical vaccine dose (this study × µg ha/animal; previous study × µg ha/animal) [ ] . using this optimized set-up, passive inhalation of both non-adjuvanted and adjuvanted wiv formulations not only protected chickens against clinical signs after hpaiv challenge, but also almost completely prevented challenge virus shedding. these results are a significant improvement over our previous study in which chickens showed partial protection and challenge virus shedding even after three immunizations. though the immunological mechanism that governed complete protection in chickens by passive administration of non-adjuvanted and adjuvanted wiv formulations still needs to be investigated, the shedding data indicate that mucosal immune responses were high enough to prevent production of substantial amounts of virus. in addition to mucosal immune responses, an important class of immune responses are those elicited systemically. at both time points, blp and advax/blp adjuvanted wiv formulations, were found to induce higher systemic immune responses than non-adjuvanted or advax-adjuvanted wiv preparation. in addition, we found out that, either of the two adjuvants i.e. advax or blp would be appropriate for first immunization, however, for second immunization, blp was vital. although these adjuvanted wiv formulations elicited significantly higher immune responses than non-adjuvanted wiv formulation, the fact that even the non-adjuvanted wiv formulations provided complete protection, suggests that the use of an adjuvant was not critical for protection via passive inhalation, but it might add to dose-sparing of influenza vaccines for future passive inhalation studies. in conclusion, our results show that, vaccination by passive inhalation of dry influenza vaccine powders is suitable to induce protective immunity in chickens against highly pathogenic avian influenza virus. it not only has the potential to completely protect chickens from morbidity and mortality, and to prevent the virus from spreading, but also seems to be a feasible option for mass vaccination of chickens. the challenge now remains to translate passive inhalation vaccination from the box to real-world field settings. np is affiliated with vaxine pty ltd, which has commercial interests in advax adjuvant. the other authors declare no conflict of interest. this research was funded by the dutch ministry of economic affairs (wot- - - ; kb- - - ). probable limited person-to-person transmission of highly pathogenic avian influenza a (h n ) virus in china human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome limited human-to-human transmission of avian influenza a(h n ) virus avian influenza a virus (h n ) epidemic in the netherlands in : course of the epidemic and effectiveness of control measures influenza vaccines and vaccination strategies in birds a lack of antibody formation against inactivated influenza virus after aerosol vaccination in presence or absence of adjuvantia preservation of the immunogenicity of dry-powder influenza h n whole inactivated virus vaccine at elevated storage temperatures a comparison between spray drying and spray freeze drying to produce an influenza subunit vaccine powder for inhalation physical and immunogenic stability of spray freezedried influenza vaccine powder for pulmonary delivery: comparison of inulin, dextran, or a mixture of dextran and trehalose as protectants dry influenza vaccines: towards a stable, effective and convenient alternative to conventional parenteral influenza vaccination pulmonary immunization of chickens using nonadjuvanted spray-freeze dried whole inactivated virus vaccine completely protects against highly pathogenic h n avian influenza virus novel surface display system for proteins on non-genetically modi gram-positive bacteria mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria development of lactococcal gem-based pneumococcal vaccines immunogenicity of a malaria parasite antigen displayed by lactococcus lactis in oral immunisations intranasal delivery of influenza subunit vaccine formulated with gem particles as an adjuvant neonatal mucosal immunization with a non-living, nongenetically modified lactococcus lactis vaccine carrier induces systemic and local th -type immunity and protects against lethal bacterial infection advax™, a novel microcrystalline polysaccharide particle engineered from delta inulin, provides robust adjuvant potency together with tolerability and safety delta inulin: a novel, immunologically active, stable packing structure comprising β-d-[ → ] poly(fructo-furanosyl) αd-glucose polymers a novel hepatitis b vaccine containing advax™, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology advax, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broadbased enhancement of adaptive immune responses a gold glyco-nanoparticle carrying a listeriolysin o peptide and formulated with advax™ delta inulin adjuvant induces robust t-cell protection against listeria infection delta inulin polysaccharide adjuvant enhances the ability of split-virion h n vaccine to protect against lethal challenge in ferrets safety and immunogenicity of a delta inulin-adjuvanted inactivated japanese encephalitis virus vaccine in pregnant mares and foals randomized clinical trial of immunogenicity and safety of a recombinant h n / pandemic influenza vaccine containing advax™ polysaccharide adjuvant immunogenicity and safety of advax™, a novel polysaccharide adjuvant based on delta inulin, when formulated with hepatitis b surface antigen; a randomized controlled phase study development of a dried influenza whole inactivated virus vaccine for pulmonary immunization pulmonary delivery of influenza vaccine formulations in cotton rats: site of deposition plays a minor role in the protective efficacy against clinical isolate of h n pdm virus development of a lavage procedure to collect lung secretions from chickens for evaluating respiratory humoral immunity respiratory and heart rates of birds at rest lung volume of meat-type chickens the effect of inoculation dose of a highly pathogenic avian influenza virus strain h n on the infectiousness of chickens genetic versus antigenic differences among highly pathogenic h n avian influenza a viruses: consequences for vaccine strain selection transmission of highly pathogenic avian influenza h n virus in pekin ducks is significantly reduced by a genetically distant h n vaccine enhancement of the immunogenicity and protective efficacy of a mucosal influenza subunit vaccine by the saponin adjuvant gpi- induction of heterosubtypic cross-protection against influenza by a whole inactivated virus vaccine: the role of viral membrane fusion activity an aerosolized fluorescent microsphere technique for evaluating particle deposition in the avian respiratory tract deposition of differently sized airborne microspheres in the respiratory tract of chickens induction of respiratory immune responses in the chicken; implications for development of mucosal avian influenza virus vaccines immunogenicity of low-ph treated whole viral influenza vaccine an improved inactivated influenza vaccine with enhanced cross protection inactivated influenza vaccine adjuvanted with bacterium-like particles induce systemic and mucosal influenza a virus specific tcell and b-cell responses after nasal administration in a tlr dependent fashion enhanced pulmonary immunization with aerosolized inactivated influenza vaccine containing delta inulin adjuvant bacterium-like particles supplemented with inactivated influenza antigen induce cross-protective influenza-specific antibody responses through intranasal administration advax augments b and t cell responses upon influenza vaccination via the respiratory tract and enables complete protection of mice against lethal influenza virus challenge development of advax adjuvant was supported by the national institute of allergy and infectious diseases of the national institutes of health, under contracts hhsn c, hhsn c and u ai . we thank jacqueline de vries-idema for providing the inactivated vaccine, dr. marc stuart for transmission electron microscopy pictures and anko eissens for scanning electron microscopy pictures. also, we would like to thank the animal technicians from wbvr for the execution of the animal experiments, and diana van zoelen and cynthia baars for performing some laboratory tests. the content is solely the responsibility of the authors and the funders played no part in the writing of this paper. supplementary data to this article can be found online at https:// doi.org/ . /j.ejpb. . . . key: cord- -qnbgywgy authors: yilmaz, huseyin; faburay, bonto; turan, nuri; cotton-caballero, maira; cetinkaya, burhan; gurel, aydin; yilmaz, aysun; cizmecigil, utku y.; aydin, ozge; tarakci, eda altan; bayraktar, erhan; richt, juergen a. title: production of recombinant n protein of infectious bronchitis virus using the baculovirus expression system and its assessment as a diagnostic antigen date: - - journal: appl biochem biotechnol doi: . /s - - - sha: doc_id: cord_uid: qnbgywgy the avian coronavirus-infectious bronchitis virus (avcov-ibv) is recognized as an important avian pathogen, and new viral variants are a continuous threat to the poultry industry worldwide. sensitive diagnostics and efficacious vaccines are necessary to combat ibv infections in chickens. the aim of this study was to produce recombinant n protein of ibv in the baculovirus system to use in elisa diagnostic tests in order to enable the assessment of the sero-prevalence and risk of ibv infections in chickens in turkey. for this, the gene encoding the n protein of the beaudette strain of ibv was expressed using a recombinant baculovirus expression system. the recombinant n protein was purified using ni-nta affinity chromatography. an estimated -kda recombinant protein corresponding to the expected molecular weight of ibv n including the xhis tag was detected using an anti-his monoclonal antibody. specific immunoreactivity of the recombinant protein was confirmed by western blot using antiserum obtained from vaccinated and naturally infected chicken from turkey as well as using a monoclonal antibody raised against the n protein of the ibv massachusetts strain. the results obtained with the in-house elisa had high agreement with a commercial elisa. immunoreactivity analysis using antisera in western blotting and the in-house elisa suggests that the recombinant ibv n protein could be broadly cross-reactive with antisera produced against different ibv strains. we conclude that the recombinant baculovirus expressed ibv n protein could serve as a useful diagnostic antigen for detection of ibv infections in chickens by elisa. infectious bronchitis virus (ibv) is the causal agent of infectious bronchitis (ib), an acute and highly contagious disease, threatening the poultry industry worldwide [ , , , , ] . avian coronavirus-infectious bronchitis virus (avcov-ibv) is an enveloped, positive single-stranded rna virus, and belongs to the genus gamma-coronavirus within the family of coronaviridae in the order nidovirales [ ] . the virus is spread mainly by aerosol, consumption of contaminated feed and water, and contact with infected feces or equipment. ib is characterized by various clinical signs in broilers and layer hens: coughing, sneezing, and decreased weight gain [ , ] . specifically, in layers, egg production can drop up to % with eggs that have shells that are wrinkled, thin, and soft. in young chicks, ibv infection can lead to oviduct cysts and reduced laying potential [ , , ] . the to kb genome of ibv encodes nine genes, which includes spike (s), membrane (m), envelope (e), and nucleocapsid (n) [ ] . the spike protein, a viral surface glycoprotein, was shown to induce neutralizing antibody response, and the nucleocpasid protein was shown to elicit strong antibody responses [ , ] . despite the presence and application of ibv vaccines in poultry, there is a high rate of emergence of antigenic variants and recombinant strains, and the lack of cross-protection between different viral genotypes, making disease control difficult and vaccine development rather challenging [ , ] . therefore, genetic characterization of circulating strains of ibv, appropriate vaccination programs, and application of sensitive diagnostic tests to detect and assess disease risk are important regional, national, and international strategies to control ibv infections [ , , , , ] . several elisas have been developed for detection of antibodies to ibv in chickens. recombinant antigens used in these elisas were either based on the s protein [ ] , which is highly variable, or the n protein and often produced in an e. coli expression system [ ] . e. coli is a common flora or pathogen in chickens, thus creating the possibility of serological cross-reactivity and detection of false positives in these diagnostic assays. thus, it is necessary to assess the suitability of other expression platforms for production of diagnostic antigens for use in ibv serology. the objectives of this study were to clone and express ibv n protein using the recombinant baculovirus expression system and assess its use as diagnostic antigen for serological diagnosis of ibv infection in chickens in turkey. the complete coding sequence ( bp) of the ibv n gene of beaudette strain (genbank accession no. m . ) was initially amplified by pcr using primers, jar f: ′-cac cat ggc ttc cgg taa ggc tg- ′ and jar r: ′-cag ctc gtt ctc acc cag agc agc- ′. the pcr product was cloned into pfastbac vector (life technologies), to create a recombinant donor plasmid, pfastbac-n, which was transformed into one shot mach t chemically competent e. coli (life technologies). the donor plasmid was double digested with restriction enzymes bamhi and psti to determine the presence of the correct insert in the right orientation. the accuracy of the sequences was confirmed by dna sequencing. the donor plasmid was transformed into max efficiency dh bac competent e. coli to construct a recombinant bacmid via site-specific transpositioning. to rescue recombinant baculoviruses encoding the ibv n gene, recombinant bacmids were purified using highpure miniprep kit (life technologies) and used to transfect spodoptera frugiperda (sf ) cells grown in sf- ii serum-free medium (sfm) supplemented with % fetal bovine serum and % penicillin-streptomycin as manufacturer's instruction (life technologies). transfection was carried out using cellfectin ii reagent as previously described [ ] and according to the manufacturer's instructions (invitrogen-life technologies). recombinant ibv n protein was expressed using passage or higher passage recombinant baculovirus stocks (> pfu/ml). the protein was expressed with a carboxy-terminal xhis tag, and purification using ni-nta superflow resin (qiagen inc., valencia, ca) was performed as described previously [ ] . concentration of the purified protein was measured by the method of bicinchoninic acid (bca) assay (thermo scientific, rockford, il) at an absorbance of nm, using bovine serum albumin (sigma-aldrich, st. louis, mo) as the protein standard. aliquots of the protein were stored at − c until used. western blot analysis was performed to verify specific protein expression. for this, approximately μg of purified recombinant ibv n protein was resolved in % bis-tris polyacrylamide gel (life technologies) as described previously [ ] . following electroblotting of the proteins onto pvdf membrane, the blots were probed with mouse anti-his (c-terminal)-hrp monoclonal antibodies ( . mg/ml) (life technologies) diluted : to determine expression of the correct molecular size protein. further confirmation of expression was performed using a mouse anti-ibv n (massachusetts strain) monoclonal antibody ( mg/ml) diluted : (cat no. mbs ; mybiosource, san diego, ca) and antiserum (at : dilution) collected from chickens naturally infected with local turkish wild-type ibv strains. the membrane was then incubated with anti-species igg-hrp secondary antibody conjugate (santa cruz biotechnology, dallas, tx) at : dilution, and specific signals were detected using enhanced chemiluminescent (ecl) detection system. to assess the induction of specific ibv n-specific antibodies in the immunized chickens (described below), the antisera were tested in immunoblot assays. approximately, μl of sample containing approximately μg of recombinant ibv n protein was diluted in -μl laemmli sample dilution buffer (santa cruz biotechnology, dallas, tx) and resolved in % bis-tris polyacrylamide gel as described above. following transfer, the blot was probed with ibv n antisera obtained from the chicks immunized with the recombinant ibv n protein at a dilution of : and with antisera from wild-type ibv-infected chicken at : dilution [ ] . ibv negative chicken sera, at a dilution of : , and mouse anti-ibv n monoclonal antibody ( mg/ml) (thermo fisher, cat. no. ) at a dilution of : were used as negative and positive controls, respectively. thereafter, the blots were probed using goat anti-chicken hrp-conjugated secondary antibody ( μg/ . ml) (cat. no. sc ; santa cruz biotechnology, dallas, tx) at a dilution : , and goat anti-mouse hrp conjugated secondary antibody ( μg/ . ml) diluted : (cat. no. sc ; santa cruz biotechnology, dallas, tx). reactivity was detected using ′- ′-diamino benzidine peroxidase substrate system (sigma cat no: d - ) as recommended by the manufacturer. to assess immunogenicity of recombinant baculovirus-expressed ibv n protein, embryonated spf chicken eggs were obtained from the bornova veterinary control institute (izmir, turkey). the eggs were incubated in an incubator for hatching. after hatching, -day-old ibv antibody negative chicks (determined by commercial elisa-biochek, ibv elisa, cat no: ck ibv) were immunized with the recombinant ibv-n protein produced in this study. four chicks were immunized intramuscularly with μg each of the recombinant ibv-n protein mixed with isa vg adjuvant (seppic, france) at a ratio of : (adjuvant/ibv-n solution), and four chicks were given placebo as mock-immunized controls. on day , immunization was repeated using the same amount of adjuvant and antigen. chicks were bled on day (pre-immunization) and then on days , , and to test for presence of antibodies to recombinant ibv-n protein. optimization of elisa an indirect elisa protocol was first optimized by checkerboard titration following the methods as described previously [ , , ] . for this, two-fold dilutions of ibv-n protein were made starting from to ng in a carbonate bi-carbonate coating buffer (sigma, c- ). the negative control serum was from the spf chicks tested antibody negative by a commercial elisa (biochek). positive control was from chickens either vaccinated or had field infection tested ibv-positive by pcr. the ibv positive and negative sera were also diluted two-fold (from : to : ), and the conjugate (hrp-conjugated goat anti-chicken antibody, santa cruz, sc ) was diluted : , : , , : , , and : , , in blocking solution. per the results of the optimization, the optimal amount of the recombinant ibv protein for use in elisa ranged from to ng, whereas the optimal dilution of ibv positive chicken serum ranged from : to : , that of the conjugate at : dilution. following optimization of the elisa protocol, test sera were obtained from broiler chickens exposed to natural wild-type ibv infection, test sera from broiler chickens vaccinated with a live-attenuated commercial ibv vaccine, and sera obtained at different time-points from chicks immunized with recombinant ibv n protein (described above) were analyzed to detect ibv n-specific antibodies. for this, a -well plate (nunc maxisorb, thermo fisher scientific) was coated overnight at °c with ng ibv-n protein/per well in -μl carbonate-bicarbonate coating buffer (sigma, cat. no. c- ). the plate was then blocked for h at room temperature with a blocking buffer (pbs containing % skimmed milk and . % tween ) . a volume of μl of test sera, diluted : in blocking solution, was added and incubated for h at °c. each serum sample was tested in duplicate, and each test plate included duplicate positive and negative control sera. the negative control serum was from the spf chicks tested antibody negative by a commercial elisa (bochek). positive control serum was from chickens either vaccinated or had field infection and tested ibv positive by pcr. goat anti-chicken hrp-conjugated antibody (santa cruz biotechnology, cat. no. sc ), diluted : , was added to the wells and incubated at °c for h. between each step, the plate was washed four times by using the wash buffer (pbs containing . % tween ) . hundred microliters of tmb substrate solution was added to each well and incubated at room temperature for min. the reaction was stopped by adding μl of m h so and absorbance (optical density) was measured at nm using a microplate reader . (slt-spectra, slt lab instruments, germany). the elisa cut-off value was determined by addition of standard deviations to measurements of the mean od value of the negative control sera in each plate. od values above cut-off value were taken as positive. thus, a cut-off od value of . was determined for the in-house indirect elisa. to assess the reliability of the performance of our in-house indirect ibv n elisa, a panel of sera was obtained from chickens naturally infected with local wild-type ibv strains, chickens vaccinated with live-attenuated commercial ibv vaccine, and chickens immunized with recombinant ibv n protein (expressed in a baculovirus expression system). the serum samples were tested simultaneously in the in-house indirect elisa (using the procedure described above) and a commercial elisa (biocheck, san francisco, ca) per manufacturer's instruction. the cut-off value for the commercial elisa was defined according to the manufacturer and determined as samples with a sample to positive ratio (s/p ratio) of . or greater. a bp fragment representing the expected molecular size of the ibv n gene was successfully amplified by high fidelity pcr (fig. ) . the pcr amplicon was successfully cloned into pfastbac/ct plasmid resulting in the creation of a donor plasmid pfastbac-n ( bp), restriction enzyme analysis of the recombinant donor plasmid confirmed the presence of the correct insert in the correct orientation by release of two restriction fragments of the expected size ( and bp) (data not shown). the donor plasmid with the correct sequences was used to create recombinant bacmid for subsequent expression of the target protein. in order to express the recombinant n protein of ibv, sf cells were infected with a recombinant baculovirus encoding the n gene of ibv. at h post-infection, cells were harvested and the recombinant protein was purified via affinity chromatography. an estimated kda recombinant protein was overexpressed, which corresponded to the expected molecular size of ibv n protein as determined by coomassie staining (fig. a) and western blot analysis using anti-his (c-terminal)-hrp monoclonal antibody (fig. b) . to confirm specificity of the target protein, an immunoblot analysis was performed using monoclonal antibodies raised against the n protein of the massachusetts ibv strain (fig. a) and as well as antiserum obtained from local chickens naturally infected with turkish wild-type ibv strain (fig. b) . in both cases, specific reactivity was confirmed by detection of a single band ( kda) of the expected molecular size. to assess the ability of the recombinant ibv n protein to induce host specific antibody responses, four chicks ( -day old) were immunized with the recombinant protein and the sera examined for specific reactivity via immunoblot analysis. specific reactivity was detected with serum samples obtained from of the ibv n-immunized chickens at day post-immunization (example see fig. seen with the other two chicken sera. this result was supported by detection of specific ibv n-reactivity with the ibv-specific monoclonal antibody (fig. , lane ) and with antisera from a chicken exposed to a natural field infection (fig. , lane ) . no specific bands were detected in sera from mock-immunized chickens or sera taken from chicks before immunization (fig. , lanes , , , , , , ) . overall, sera from vaccinated and naturally infected (field-exposed) chickens that tested positive in the immunoblot assay were found to be correspondingly positive in the in-house indirect ibv n elisa (data not shown). to further assess the immunogenicity of the recombinant n protein and the reliability of our results, a panel of antisera from naturally infected ( samples plus negative and positive controls), from chickens vaccinated with live-attenuated ibv ( samples plus negative and positive controls) and from chickens immunized with recombinant ibv n protein ( samples), were tested using both the in-house indirect elisa and a commercial elisa. with the in-house elisa, cut-off value was set at od of . . there was relatively good agreement in performance between the two elisas in detecting ibv n-specific antibodies in the sera from naturally infected chickens (fig. a) and live-attenuated vaccinated chickens (fig. b) . of the serum samples obtained from the naturally infected chickens, % ( / ) tested positive in the in-house indirect elisa, whereas % ( / ) tested positive in the commercial elisa detected (fig. a) . all samples collected from vaccinated chickens tested positive in both the in-house and commercial indirect elisas (fig. b) . of the commercial elisa-positive field serum samples, tested positive in the in-house indirect elisa, indicating a sensitivity of % (using the commercial elisa as reference test). none of the sera obtained from preimmunized or naïve chicks tested positive in either elisas, indicating % specificity (n = ). of the four chicks immunized with the recombinant ibv n protein, two chicks seroconverted at day after immunization. all four chicks seroconverted at days post-immunization in both elisa tests, indicating a % agreement between the two assays. the od values of chicks immunized with recombinant ibv n protein were . , . , . , and . . production of immunogenic recombinant proteins derived from pathogenic organisms represents a good strategy for identification of antigenic proteins that may serve as targets for sero-diagnostic and vaccine development [ , ] . this represents the first study in turkey that expressed recombinant ibv n protein in baculovirus and examined its reactivity against antisera obtained from turkish chickens for potential use as antigen fig. detection of ibv n specific antibodies in sera obtained from naturally infected chicken (a) and vaccinated chickens (b) using an in-house ibv-n elisa and a commercial elisa. the cut-off value for inhouse indirect elisa is set at od . , and for the commercial elisa at s/p (sample to positive) ratio of . in serological investigation of ibv infection in domestic poultry. we have demonstrated that ibv n produced in a recombinant baculovirus expression system is immunogenic in local chickens and could detect ibv n-specific antibodies in sera obtained from chickens exposed to natural infection. the recombinant n protein was reactive in both immunoblot and indirect elisa assays. the performance of the in-house indirect elisa was comparable to the commercial elisa tested in this study, indicating the potential suitability of recombinant baculovirus ibv n as a diagnostic antigen that could be used for serological risk assessment of ibv in domestic chickens in turkey. indeed, the n protein of ibv, in contrast to the s protein, has been associated with high stability and immunogenicity and has been used by others as a diagnostic antigen [ , ] . the recombinant n protein used in the current study was based on nucleotide sequences of the beaudette ibv strain. the reactivity of the recombinant ibv n protein with a monoclonal antibody (b m) produced against the heterologous ibv massachusetts strain, and with sera from local turkish chicken indicate the conserved, cross-reacting nature of the n protein sequence and suggest potential broad cross-reactivity of the target protein among different ibv isolates and its suitability as a diagnostic antigen to detect ibv infection in local turkish poultry. other researchers have utilized recombinant ibv n expressed in various host systems in serological assays. for example, in a study performed by pradhan and others [ ] , ibv-n protein was produced using a prokaryotic expression system. the protein was used for diagnostic purposes and similar to this study, compared the performance with a commercial elisa kit (idexx). the authors reported a . % sensitivity and . % specificity for the in-house elisa based on the recombinant ibv n antigen and they concluded that an indirect elisa based on the recombinant antigen is suitable for use in development of serodiagnostic tools [ ] . in another study, the ibv-n protein was produced in both e. coli and baculovirus expression systems [ ] , and the recombinant antigens were used in elisas to analyze chicken sera collected from farms [ ] . their data from screening of sera for the presence of ibv antibodies indicated that using the recombinant n protein as coating antigen could achieve equivalent performance to an elisa kit based on extracts from infected material as coating antigen [ ] . in a study performed by ding and others [ ] utilizing a multi-fragment antigen composed of s (spike), m (matrix), and n proteins produced in e. coli [ ] , a good reactivity with an anti-ibv chicken serum was demonstrated. an in-house elisa using the multi-fragment protein exhibited a . % concordance with a commercial elisa [ ] . in the present study, recombinant ibv-n protein was produced using a baculovirus expression system and utilized as a diagnostic antigen in an indirect elisa. the chicken sera from farms and the sera from immunized chickens were reactive with the recombinant antigen with comparable performance to a commercial elisa test kit. this study describes the successful cloning and recombinant baculovirus expression of ibv nucleoprotein and its evaluation as a potential serodiagnostic antigen. the recombinant antigen was immunoreactive with hyperimmune sera from local turkish chickens and the performance of an in-house indirect elisa based on the antigen was comparable to a commercial elisa, suggesting the potential utility for serological detection of ibv infections in chickens in turkey. further studies to validate the assay using a larger sample size including comprehensive assessment of assay specificity will be performed. decreased neutralizing antigenicity in ibv s protein expressed from mammalian cells coronavirus avian infectious bronchitis virus evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus the long view: years of infectious bronchitis research infectious bronchitis virus variants: a review of the history, current situation and control measures development of an elisa based on a multi-fragment antigen of infectious bronchitis virus for antibodies detection rift valley fever virus structural and nonstructural proteins: recombinant protein expression and immunoreactivity against antisera from sheep. vector borne and zoonotic diseases a glycoprotein subunit vaccine elicits a strong rift valley fever virus neutralizing antibody response in sheep s gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the united states and israel avian infectious bronchitis virus review of infectious bronchitis virus around the world expression and purification of the kda periplasmic protein of brucella abortus: a reagent for the diagnosis of bovine brucellosis cleavage of structural proteins during the assembly of the head of bacteriophage t expression, purification, and improved antigenic specificity of a truncated recombinant bp protein of brucella melitensis m - : a potential antigen for differential serodiagnosis of brucellosis in sheep and goats detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay immune responses to mucosal vaccination by the recombinant a and n proteins of infectious bronchitis virus molecular epidemiology and evolution of avian infectious bronchitis virus recombinant nucleocapsid protein based single serum dilution elisa for the detection of antibodies to infectious bronchitis virus in poultry s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification an elisa for antibodies against infectious bronchitis virus using an s spike polypeptide a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from detection of antibodies to equine arteritis virus in horse sera using recombinant chimaeric n/g(l) protein. the veterinary record phylogeny and s gene variation of infectious bronchitis virus detected in broilers and layers in turkey compliance with ethical standards national and international ethical rules were followed during this study. the authors declare that they have no conflicts of interest. key: cord- -abs tc r authors: chong, ka chun; hu, pei; lau, steven; jia, katherine min; liang, wenjia; wang, maggie haitian; zee, benny chung ying; sun, riyang; zheng, huizhen title: monitoring the age-specificity of measles transmissions during - in southern china date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: abs tc r background: despite several immunization efforts, china saw a resurgence of measles in . monitoring of transmissions of individuals from different age groups could offer information that would be valuable for planning adequate disease control strategies. we compared the age-specific effective reproductive numbers (r) of measles during – in guangdong, china. methods: we estimated the age-specific r values for age groups: – months, – months, months to years, – years, – years, – years, and ≥ years adapting the contact matrix of china. the daily numbers of laboratory and clinically confirmed cases reported to the center for disease control and prevention of guangdong were used. results: the peak r values of the entire population were above unity from to , indicating the persistence of measles in the population. in general, children aged – years and adults aged – years had larger values of r when comparing with other age groups after . while the peaks of r values for children aged – years dropped steadily after , the peaks of r values for adults aged – years kept at a high range every year. conclusions: although the provincial supplementary immunization activities (sias) conducted in and were able to reduce the transmissions from to , larger values of r for children aged – years were observed after , indicating that the benefits of the sias were short-lived. in addition, the transmissions from adults aged between and years increased over time. disease control strategies should target children and adult groups that carry high potential for measles transmission. increased over time. disease control strategies should target children and adult groups that carry high potential for measles transmission. measles is a highly contagious acute viral disease. throughout the world, and most countries have set goals for its elimination. in , the national expanded program on immunization (epi) in china started to implement a standard schedule for the routine administration of one dose of measles-containing vaccine (mcv ) among children between and months of age. subsequently, the mean annual measles incidence decreased from per , in - to per , in - [ ] . in , a two-dose routine measles immunization program was implemented for children aged between months and years. the age schedule for the second dose of mcv (mcv ) was shifted to - months in . during - , the number of measles cases showed a remarkable decrease but remained around . per , on average [ ] . in , the government of china set a goal to eliminate measles by , for which purpose a series of programs was implemented, including strengthening routine immunization surveillance, supplementary immunization activities (sias), and casebased surveillance [ ] . an sia is defined as the administration of a supplementary dose of a vaccine to a specific age population in a certain area during a short period, regardless of the recipients' previous vaccination histories. sias enhance routine immunization programs, including catch-up campaigns, follow-up campaigns, and outbreak-response immunization. the estimated coverage rate of routine immunization with mcv increased from . % in to . % in , whereas the estimated coverage rate for mcv was < % before and . % in [ ] . in september , china conducted a synchronized, nationwide sia that targeted children aged months to years, covering million children with a reported coverage rate of . % [ ] [ ] [ ] . although the annual measles incidence had dropped to . per , in , it resurged to more than . per , in [ ] . despite the implementation of two-dose routine vaccines since , frequent outbreaks have occurred over the past years [ ] [ ] [ ] . guangdong, a highly populated province with million population in , is located in the southernmost part of mainland china (fig ) . the incidence of measles in guangdong dropped to a remarkable low in ( . / , ) after a series of sias targeting children aged months to years during and . however, guangdong had the highest number of reported cases in china in and , with incidences of . and . per , , respectively [ ] , even though the reported coverage of mcvs was kept above % every year after . to this end, the guangdong government implemented some mop-up vaccination programs after targeting children aged months to years (fig ) . monitoring the effectiveness of the measles control policy is done by surveillance. in china, measles is a category b infectious disease which indicates it is highly contagious and must be reported to the surveillance system within hours after confirming the laboratory samples [ ] . since , china has a direct network reporting system and automatic warning information system for infectious diseases. the system focusses on the number of reported cases, but does not evaluate the transmissibility of measles. the effective reproduction number, r, is a key epidemiologic variable that summarizes the transmissibility of infectious diseases. it is defined as the expected average number of secondary cases produced by an infectious individual in a population in which not all the individuals are susceptible [ ] . when r is larger than , an infectious individual is expected to infect more than one secondary case. when r is less than , an infectious individual tends to infect less than one secondary case, and the incidence will decrease. nevertheless, some infectious diseases have been shown to be strongly age-specific, for example, measles. age-specific r, defined as an average total number of secondary cases from all age groups generated by a single case with respect to his age group was recommended to study the differences in transmission potential taking account of social mixing [ ] [ ] [ ] [ ] [ ] [ ] . although a usual interpretation of age-specific r for gauging the control measures required to eliminate an infection is inappropriate [ , ] , age-specific rs provide valuable information on the underlying heterogeneous transmission between and within different groups of individuals. for example, glass et al. estimated the rs of pandemic influenza a(h n ) for children and adults and identified children had a higher transmission then adult cases. in china, the demography of measles infections has changed over time. while infants aged between months and months and young adults aged to years were the primary population of measles infections, children aged to months and adults aged - years became the primary sources after the national sia. apparently, the age specificity in measles transmissions could be affected by vaccination policies. chong et al. [ ] showed that even though substantial decreases in the numbers of cases were observed after mass vaccination campaign, measles could still persist in a population given a high value of r. the majority of the relevant epidemiological studies conducted in china have been based on reported cases and have aimed to describe the incidence and characteristics of population distribution [ , ] . however, the age specificity in measles transmissions had hardly been studied. in the present study, we compared the age-specific r of measles infections between different age groups by using laboratory and clinically confirmed data collected from to . daily notifications of measles cases from january , to december , were collected from the national infectious disease monitoring information system (nidmis), as complied by the center for disease control and prevention (cdc) of guangdong province. for some of the cases with typical clinical symptoms, case notifications were sent to the person in charge of reporting by outpatient or resident doctors, and the cases were then recorded as "clinically confirmed". blood samples from these cases were sent to cdc clinical laboratories for confirmation, if laboratory capacity allowed. other cases with atypical clinical symptoms were recorded as "suspected cases," and the blood samples of these cases were subsequently transferred to a diagnostic laboratory to obtain a confirmed diagnosis. the test results were returned to the patients' doctors. if the test results were positive, the cases were relabeled as "laboratory-confirmed cases," and the person in charge of reporting was notified. if the results were negative, the cases were relabeled according to the specific disease that had been detected before handing over to the reporting personnel. for the (clinically confirmed or suspected) cases without laboratory confirmation, epidemiological investigations were conducted to determine whether the patients' infections had any linkage to other confirmed cases within - days before the onset of any symptom. the epidemiological investigations were performed through direct contacts in the relevant village, community, or school, or through direct contacts for mass gathering events. the clinically confirmed and laboratory-confirmed cases were both regarded as cases, and reporting personnel were required to report such cases to the nidmis within hours. we divided the population into age groups according to the age of onset: - months (pre-vaccination age), - months (received the first dose of the measles containing vaccine, mcv- ), months to years (received the second dose of the measles containing vaccine, mcv- ), - years (primary and secondary school students), - years (high school and college students/young adults), - years (mature adults), and ! years (aged adults). this study was reviewed and approved by the medical ethics committee of the guangdong cdc. the application of the data in this study has been authorized by the guangdong cdc. all data were fully anonymized prior to access by any of the authors and does not involve patients' privacy prior collection. informed consents were exempt from the ethics committee in accordance to the cdc policy of continuing public health investigations of notifiable infectious diseases, in which the patient names, addresses, medical histories with infectious diseases, and their family information will not be disclosed to the public by guangdong cdc in any form. the data are available without restrictions (the link will be provided after the acceptance of the paper). the method for estimating the age-specific effective reproduction numbers suggested by white et al. [ ] , which is a modification of the wallinga and teunis approach [ ] , was adopted. let p d denote the probability of a serial interval of length d, (d = , ,. . .,d where d be the maximum serial interval length), n t;g i denote the frequency of symptom onset in age group g i (i = , ,. . .,g where g is the total number of age groups) on day t (t = , ,. . .,t where t is the length of the study period), r g i !g j denote the contact rate between two individuals from age group g i and age group g j (j = , ,. . .,g). the effective reproduction number of age group g i on day t, r t;g i , can be calculated by summing the expected number of individuals in each age group from t+ to t+d infected by an individual in age group g i whose symptom onset was on day t: denote the relative probability that an individual in group g j on day t+d was infected by an individual in group g i on day t. in this study, there were age groups (i.e., g = ) and the maximum serial interval length d was set at . p d was generated from a gamma distribution with a mean of days and standard deviation of days [ ] . r g i !g j was estimated by using the contact matrix of china, projected by the bayesian hierarchical model in prem et al [ ] . the estimation formulas were implemented in microsoft excel. we extended the probabilistic method described in white et al. to generate the statistical uncertainty [ ] . a parametric bootstrapping approach was employed to generate , realizations of fr t;g i g. in each iteration, we generated a new dataset by first simulating the total number of individuals in group g j (j = , , . . ., ) infected by those in group g i (i = , , . . ., ) with symptom onset of day t (t = , , . . ., t- ) from a poisson distribution with mean = n t;g i  x minðd;tÀ tÞ d¼ n tþd;g j  pði t;g i ! i tþd;g j Þ, which can be interpreted as the estimated total number of individuals in group g j infected by all the individuals in group g i with symptom onset on day t, where n t;g i and pði t;g i ! i tþd;g j Þ were directly obtained and calculated from the original dataset respectively. the simulated number was then distributed within the serial interval t + and t + according to a gamma distribution with a mean of days and standard deviation of days. the above procedure was repeated for all i,j, and t. the resulting data were used to calculate a realization of fr t;g i g and further averaged by months. the % credible intervals (ci) of the monthly estimates were obtained from the . th and . th percentiles over the , realizations. apart from china's contact matrix, contact matrixes from other countries were employed to test the sensitivity of our results [ ] . we also tried evaluating the estimates using days and days as the mean and standard deviation of the gamma distribution of the serial interval [ ] . the r values estimated for children aged - years were low across the study period in general, even though the values also increased since , indicating that primary and secondary school students had a limited contribution to measles transmissions. similarly, the results for the adults aged ! years were extremely low across the study period, indicating that these persons were unlikely to infect more than a case on average. the for adults aged between and years, a clear seasonal pattern of r values was observed after , which showed a similar trend to that observed in children. in and , the estimated annual peaks of r were . ( % ci: . to . ) and . ( % ci: . to . ) respectively. given that the r peak of the entire population was significantly above unity in , adults aged - years had the largest contribution to measles transmissions. s fig shows the sensitivity of the results to the use of contact matrices from other countries [ ] . in general, the major findings were robust with the variation of contact patterns, for example, children aged - years still had a large contribution in measles transmissions after . nevertheless, due to a difference in contact frequency, larger estimates were observed for children aged - months and - months when using the contact matrix of germany. moreover, while using poland's contact matrix drew a lower estimates for children aged - we also investigated using a different set of parameters for the distribution of the serial interval, and found that the results were generally consistent with the main analysis (s fig). the r values of children groups were slightly increased, whereas the r values of adult groups were slightly decreased. monitoring the age specificity of measles transmissions could provide information that would be valuable to officials who seek to develop adequate disease control strategies. for example, it could help to select appropriate age groups for supplementary vaccination. in this study, we compared the age-specific r of measles infections between different age groups, using laboratory and clinically confirmed data from to for guangdong province. according to the results, measles transmissions varied across most age groups before and after and the large values of r from the entire population indicated a persistence of measles in the population from to . in general, children aged - years and adults aged - years had higher contributions in measles transmissions when comparing with other age groups after . after , while the peaks of r values for children aged - years dropped steadily by years, the peaks of r values for adults aged - years remained unchanged and kept at a high range every year, demonstrating the highest contributions in measles transmissions. the findings suggest that disease control strategies should target children and adult groups that carry a high potential for measles transmission. as has been previously noted, we found that children aged - years had r values that increased after , even though sias targeted this population in and . the increasing r values could have resulted from low mcv coverage in this cohort, for which the official reported coverage was usually over-estimated [ , ] . an in-house survey of a similar cohort of children aged - months showed that mcv and mcv coverage rates were only % and %, respectively [ ] , results that were inconsistent with the generally reported figure of > % in china [ ] . the geographic heterogeneity of vaccine coverage in china could be another explanation [ , ] . a chinese study indicated that the measles antibody levels of children aged - years old were significantly lower for residents of rural areas than for residents of urban areas [ ] . the primary reasons why rural children had missed their mcvs were because they were living far from the clinics and because they were unable to access vaccination information [ ] . the incomplete immunization records of rural children also made it more difficult for public health officials to track them in order to administer the vaccine. we observed elevated transmissions in infants aged - months, which may primarily be attributed to the design of the immunization system, which regarded them as too young to be vaccinated by either routine immunization or sias. a longitudinal study of maternal measles antibody titers in infants in guangzhou (the provincial capital of guangdong province) showed that titers among infants decreased rapidly after months of age, and were generally undetectable at months of age [ ] . several other studies reported similar results [ , ] . hence, there was a remarkable immunity gap among children under months old. some studies showed that only around . % to . % of infants are seropositive for measles at months of age [ , ] . nevertheless, even though infants aged - months were identified as a high transmissibility group, reducing the minimum age for receiving mcv- to months is controversial. we also found that the transmissions from children aged - years were comparatively low, which was expected given that given that they were the main target of previous sias. moreover, many primary schools implemented screening of children's vaccination certificates and administered supplementary doses of the measles vaccine to fill immunity gaps before the annual entrance [ ] . the values of age-specific r for adults aged between and years kept at a high range from to and it could be attributed to the lower efficacy of measles vaccines, the low vaccination coverages during s and earlier, and the reduced chance of natural infections. persons aged - years at the time of the present study were thought to be the first recipients of the vaccination after the approval of routine immunization. liquid vaccines were used for immunization at that time. they had a lower effective dosage and may have resulted in the lower level or shorter protective duration of antibodies among the population. in addition, a functional cold chain, transportation, and communication system for the measles vaccine had not been established at that time; hence, the quality and efficacy of the vaccines could not be guaranteed. secondly, several parents knew nothing about the measles vaccine and underestimated the severity of measles, thereby resulting in a low vaccination rate and a high rate of unsure inoculation history. thirdly, secondary vaccine failure (i.e. measles onset after vaccination and successful seroconversion) due to waning immunity might have occurred among vaccinated adults. although our study could not identify secondary vaccine failure from other cases as serological evidence of previous successful vaccination were lacked, it has been concluded by who that waning immunity has not played a major role in the transmission of measles compared to the absence of initial immunity [ ] . the proportion of cases attributable to secondary vaccine failure varied greatly across outbreaks [ ] . in a cohort study (n = ) in zhuji county of zhejiang province, around - % of those given with single doses of domestic vaccines would become sero-negative (measured by haemagglutination-inhibition tests) after years, yet clinical measles cases rarely happened among them who had humoral immunity waned as they were still protected by secondary immune response [ , ] . finally, the subsequent sias did not cover these persons; thus, the immunity gaps among people aged to years increased. on the other hand, the transmissions from individuals aged ! years were the lowest among the age groups studied, even though there was almost no vaccination history in this group. we believe that the majority of these individuals acquired antibodies through natural infection, owing to the highly contagious nature of measles when they were young. moreover, many studies have shown that seropositivity after natural infection persists longer and generates a stronger response than the immunity acquired from vaccination [ ] [ ] [ ] . given the increasing values of r for the entire population observed after , some mopup vaccination campaigns in and appear to have had limited effectiveness, even though they aimed to control measles transmission. one reason for this is that rural families usually have a lower level of education and do not fully understand information regarding mop-up campaigns, which results in a lack of initiative to get the vaccine. moreover, some of the susceptibles were migrants, and officials reported difficulties tracking their vaccination histories. although door-to-door notifications, text messages, and telephone notifications have been used to inform migrant families to join mop-up campaigns [ ] , it is often difficult to contact these families because of changes to their addresses or phone numbers. from a policy-making perspective, these results imply that for a successful measles control campaign, the public health department should carry out control measures for appropriate age groups. for children between and months old, it is necessary to take measures to improve vaccination coverage, including providing more publicity to improve parents' awareness about vaccination against measles, creating integrated multichannel notifications to inform parents of the vaccination, and strengthening the supervision of kindergartens. for adjustments to the immunization strategy, adult-specific vaccination programs should be considered to fill the immunity gaps among adults, especially for those aged between and years. one of the major limitations in this study is the quality of notification data. indeed, a proportion (~ %) of the notification data in the early phase study ( ) ( ) ( ) was only clinically confirmed which may lead to some misdiagnosis as well as an underestimate of the age-specific rs. nevertheless, more than % of cases were laboratory confirmed after . particularly, the reliance on clinically confirmation was more in rural hospitals in which doctors may lack sufficient knowledge on measles diagnosis. the positive predictive value of a clinical definition would also be changed over time as measles has become rarer by time. to minimize the chance of misdiagnosis, the clinically confirmed cases were not only identified by clinical symptoms, but were also investigated with any potential epidemiological association with other laboratory-confirmed cases. moreover, the completeness of the data could be affected by underreporting as some of the parents might have regarded measles as a kind of skin disease or might have confused it with other diseases that involve skin rashes. apart from that, age-specific rs are common to be used as a metric to identify appropriate age groups most responsible for transmission as for a target of interventions [ , , ] . however, when determining the effort required to eliminate an infection, the interpretation of an age-specific r is different from that of an overall r in a heterogeneous population as using the original threshold of unity could lead to an underestimation of target population for interventions [ , ] . alternatively, roberts & heesterbeek [ ] suggested a type-reproduction number which can single out particular subgroup rather than averaging over all subgroups. further works such as generalizability and statistical inference [ ] on the alternative measures worth being investigated. in summary, we compared the age specificity in measles transmissions from to in guangdong province. although the provincial sias conducted in and were able to reduce the transmission rates from to , larger effective reproductive numbers for children aged - years were observed after , which indicates that the benefits of the sias were short-lived. in addition, the transmissions from adults aged between and years increased over time. based on the findings of the present study, we believe that disease control measures should strategically target those groups that carry a high potential for measles transmissions. monitoring progress towards the elimination of measles in china: an analysis of measles surveillance data beijing: world health organization china representative office world health organization. measles and rubella surveillance data investigation of a measles outbreak in china to identify gaps in vaccination coverage, routes of transmission, and interventions endemic and imported measles virus-associated outbreaks among adults new measles virus genotype associated with outbreak on the road to measles-free: where are we now? law of the people's republic of china on prevention and treatment of infectious diseases. database of laws and regulations definition and estimation of an actual reproduction number describing past infectious disease transmission: application to hiv epidemics among homosexual men in denmark determining the dynamics of influenza transmission by age estimating age-specific reproductive numbers-a comparison of methods. statistical methods in medical research estimating reproduction numbers for adults and children from case data pandemic influenza h n : reconciling serosurvey data with estimates of the reproduction number likelihood-based estimation of continuous-time epidemic models from time-series data: application to measles transmission in london estimation of the reproductive number and the serial interval in early phase of the influenza a/h n pandemic in the usa. influenza and other respiratory viruses the type-reproduction number t in models for infectious disease control. mathematical biosciences a new method for estimating the effort required to control an infectious disease interpreting the transmissibility of measles in two different post periods of supplementary immunization activities in hubei epidemiological analysis of measles in china between changes of epidemiological characteristics of measles in beijing before and after supplementary immunization campaigns of measles vaccine in different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures projecting social contact matrices in countries using contact surveys and demographic data social contacts and mixing patterns relevant to the spread of infectious diseases the correlation between infectivity and incubation period of measles, estimated from households with two cases measles vaccine coverage estimates in an outbreak three years after the nation-wide campaign in china: implications for measles elimination investigation of a case of measles outbreak in a kindergarten in hubei province immunization: measles, st dose (mcv ) immunization coverage estimates by country comparison of measles antibody levels and trends of children aged - years between and in jiujiang area, jiangxi province analysis on the immunization of measles and its influencing factors among freshmen in elementary school and kindergarten in shenzhen city analysis of the influencing factors of measles vaccine leakage dynamic maternal measles antibody level in infants: a longitudinal study fetal, and neonatal outcomes associated with measles during pregnancy: namibia early waning of maternal measles antibodies in era of measles elimination: longitudinal study pre-vaccination evolution of antibodies among infants , and months of age: a longitudinal analysis of measles, enterovirus and coxsackievirus . vaccine measles antibodies in mother-infant dyads in tianjin notice on the implementation plan for checking the vaccination certificate before children's kindergarten and primary school enrollment in guangdong province world health organization. measles vaccines: who position paper identification of primary and secondary measles vaccine failures by measurement of immunoglobulin g avidity in measles cases during the são paulo epidemic duration of immunity following immunization with live measles vaccine: years of observation in zhejiang province decreasing seroprevalence of measles antibodies after vaccination-possible gap in measles protection in adults in the czech republic immunoglobulin response in serum and secretions after immunization with live and inactivated poliovaccine and natural infection the influence of children's immunization leak replant measures on improving immunization vaccination rate we thank guangdong cdc for providing the data for analysis. we thank the two anonymous reviewers whose comments helped improve and clarify this manuscript. key: cord- -fxxceffl authors: razanajatovo, norosoa harline; guillebaud, julia; harimanana, aina; rajatonirina, soatiana; ratsima, elisoa hariniaina; andrianirina, zo zafitsara; rakotoariniaina, hervé; andriatahina, todisoa; orelle, arnaud; ratovoson, rila; irinantenaina, judickaelle; rakotonanahary, dina arinalina; ramparany, lovasoa; randrianirina, frédérique; richard, vincent; heraud, jean-michel title: epidemiology of severe acute respiratory infections from hospital-based surveillance in madagascar, november to july date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: fxxceffl background: few comprehensive data exist regarding the epidemiology of severe acute respiratory infections (sari) in low income countries. this study aimed at identifying etiologies and describing clinical features of sari-associated hospitalization in madagascar. methods: it is a prospective surveillance of sari in hospitals for years. nasopharyngeal swabs, sputum, and blood were collected from sari patients enrolled and tested for viruses and bacteria. epidemiological and clinical information were obtained from case report forms. results: overall, patients were enrolled in the study, of which . % ( / ) were tested positive for at least one pathogen. viral and bacterial infections occurred in . % ( / ) and . % ( / ) of tested samples, respectively. among all detected viruses, respiratory syncytial virus (rsv) was the most common ( . %; / ) followed by influenza virus a (flua, . %; / ), rhinovirus (rv, . %; / ), and adenovirus (adv, . %; / ). among bacteria, streptococcus pneumoniae (s. pneumoniae, . %, / ) was the most detected followed by haemophilus influenzae type b (hib, . %; / ), and klebsiella ( . %; / ). other streptococcus species were found in . % ( / ) of samples. compared to patients aged less than years, older age groups were significantly less infected with rsv. on the other hand, patients aged more than years (or = . ) were at higher risk to be infected with flua, while those aged – years (or = . ) and – years (or = . ) were more likely to be infected with flub (influenza virus b). conclusion: the frequency of influenza viruses detected among sari patients aged years and more highlights the need for health authorities to develop strategies to reduce morbidity amongst at-risk population through vaccine recommendation. amongst young children, the demonstrated burden of rsv should guide clinicians for a better case management of children. these findings reveal the need to develop point-of-care tests to avoid overuse of antibiotics and to promote vaccine that could reduce drastically the rsv hospitalizations. samples. compared to patients aged less than years, older age groups were significantly less infected with rsv. on the other hand, patients aged more than years (or = . ) were at higher risk to be infected with flua, while those aged - years (or = . ) and - years (or = . ) were more likely to be infected with flub (influenza virus b). the frequency of influenza viruses detected among sari patients aged years and more highlights the need for health authorities to develop strategies to reduce morbidity amongst at-risk population through vaccine recommendation. amongst young children, the demonstrated burden of rsv should guide clinicians for a better case management of children. these findings reveal the need to develop point-of-care tests to avoid overuse of antibiotics and to promote vaccine that could reduce drastically the rsv hospitalizations. severe acute respiratory infections (sari) are among the leading cause of hospitalization and deaths worldwide [ ] . it is estimated that about . million of deaths are attributed to sari annually [ ] . estimation in have shown that million of children aged less than years were admitted for sari in developing countries, with an estimated case fatality ratio of . %, compared to cases and a case fatality ratio of . % in developed countries [ ] . in low income countries, the annual incidence of pneumonia is estimated at million children of whom % are severe enough to warrant hospitalization [ , ] . nearly million children die from pneumonia every year, % of which occur in africa and southeast asia [ , ] . sari may be caused by various pathogens. while bacterial infections play a critical role in causing life-threatening pneumonia [ , [ ] [ ] [ ] , viral infections are associated with significant proportion that range from mild to severe infections [ , ] . however, due to the lack of gold standard methods to rapidly differentiate between viral and bacterial infections, most of the patients may be treated empirically with antibiotics [ ] . rapid etiologic diagnosis is therefore needed to select the most appropriate treatment protocol and to avoid the development of bacterial resistance. following the a/h n / influenza pandemic that was associated with a high morbidity and an increased risk of mortality among particular groups [ ] , a number of countries have strengthened vigilance for the surveillance of severe diseases and deaths in order to rapidly detect new viruses and to provide information in assessing the impact on the population and having operational preparedness plans. so far, only few sub-saharan african countries are collecting data on hospitalization associated to acute respiratory illness [ ] [ ] [ ] . in madagascar, the sentinel syndromic surveillance has been collecting sari data since . however, hospitals only collect few data that are not exploitable for analysis. thus, data are still scarce regarding the epidemiology of sari in this country. during the last influenza pandemic in , an excess of mortality was observed among inhabitant of the main capital city of antananarivo [ ] . following the pandemic, health authorities requested to develop an active surveillance of sari in order to better understand the epidemiology of this disease in the context of madagascar. the present study aimed to identify etiologies of sari and to assess clinical features of hospitalization linked to sari in hospitals during years of surveillance. a prospective hospital-based sari surveillance was conducted from november to july in selected sites: the soavinandriana hospital of antananarivo and the secondary level public hospital (chd ii) of moramanga. the cenhosoa is a national referral hospital and is among the largest hospitals that deserve antananarivo, the main capital city of madagascar which has around . million inhabitants. the chd ii in moramanga is the only local referral hospital for the health district of moramanga that has inhabitants. it is located km east from the capital city. antananarivo and moramanga present the same climatic profile: hot and rainy in summer and cold and dry in winter, but moramanga encompasses both semiurban and rural areas. all patients presenting sari symptoms at admission were enrolled. sari case definition was defined according to the who case definition as fever (t� ˚c) or history of fever and cough that required hospitalization. for children less than years, the eligibility criteria were suspected sepsis or sari diagnosed by physician including bronchiolitis, pneumonia, bronchitis, pleural effusion, and cough or difficult breathing as previously published [ ] . the onset of illness should be less than days before hospitalization. for each consented patient, demographic, socio-economic, clinical, and epidemiological data were recorded in case report forms (crfs). nasopharyngeal, blood, and sputum specimens were collected for each patient enrolled from monday to friday and shipped the same day to laboratories located at the pasteur institute of madagascar where they were immediately processed or stored at ˚c until testing (test were performed within hours post-sampling). all procedures for the biological analyses have been previously described [ , ] . briefly, nasopharyngeal swabs were screened for respiratory viruses using in-house multiplex real-time pcr [ ] . sputum was collected for cytobacteriologic testing while blood samples were used for cell blood count. single infection was defined as an infection caused by one pathogen (virus or bacteria) and multiple infection as an infection caused by at least pathogens (virus/virus, virus/bacteria or bacteria/bacteria) in a single sample. patients were divided into age groups: infants and young children (� years old); children ( - years old); adolescent and young adults ( - years old); adults , and elderly (� years old). demographic, clinical characteristics, and etiologies were compared by study sites and by age groups. univariate analysis and logistic regression were performed using r software. in univariate analysis, qualitative variables were compared with fisher's exact test and quantitative variables by non-parametric tests (wilcoxon test). furthermore, logistic regressions were performed to adjust odds-ratio (or) found via maximum-likelihood estimation according to age group for the different variables as dependent variables and to compare each of them according to age group by wald test. the age group less than years has been considered as reference group. statistical differences were considered significant for two-sided p-values < = . . although sari surveillance in madagascar is not considered as research, this prospective study requested extra sampling and the collection of personal information. for that reason, the present study was submitted and approved by the national ethics committee from the ministry of health in madagascar (authorization n˚ -msanp/ce). adult participants or parents were fully informed of the study's objectives and procedures. written informed consent was obtained from patients enrolled in the study. young participants aged from to years old were asked to sign an informed assent form if they were willing to take part in the study. for all patients aged less than years, consent was obtained from the parents or legal guardians. after obtaining the consent/assent form, the team survey proceeded to collect samples and fill out the crfs. refused consent and prior hospitalization within weeks before consultation were reasons for exclusion. from november to july , hospitalized patients completed the established sari case definition of whom cases ( . %) were from cenhosoa and cases ( . %) from chd ii moramanga. the mean age was . years ranging from day to years and . % of patients were male cases. the majority of recruited patients were children aged less than years and accounting for % of total inclusion. adult aged more than years represented % of cases ( table ) . differences of mean age ( . years vs. . years; p< . ; wilcoxon test) and age repartition (p< . ) of included patients were observed between the study sites with patients hospitalized in chd ii moramanga being younger than those hospitalized in cenhosoa (table ) . with exception of dyspnea and fever which are among the principal inclusion criteria, runny nose ( . %), intercostal recessions ( . %), productive cough ( . %), movement of nose wings ( . %), and anorexia ( . %) were the most recorded at the time of examination. bronchiolitis ( . %) was the most clinical diagnostic made by clinicians at admission followed by bronchopneumonia ( . %) and pneumonia ( . %). other low respiratory tract infections (bronchoalveolitis/exacerbation of chronic obstructive pulmonary disease (copd), pleuropneumonia, and acute lobar pneumonia) were observed in . % of inpatients (s table) . comparison of clinical symptoms between the sites showed that dry cough (p = . ), asthenia (p = . ), runny nose (p< . ), and anorexia (p = . ) were statistically frequently observed in patients hospitalized in chdii moramanga whereas productive cough (p = . ), sore throat (p< . ), headache (p = . ), thrill (p = . ), and cyanosis (p< . ) were significantly associated with patients hospitalized in cenhosoa (table ) . at least one pathogen was found in . % ( / ) of tested patients of which . % ( / ) were single infection and . % ( / ) were multiple infections. the highest rate of infection was obtained in patients aged less than years ( . %; / ). for this age group, single and multiple infections were reported in . % ( / ) and . % ( / ) of cases, respectively. the type of infection (mono-infection vs. multiple infection) differed significantly between age groups (p = . , fisher's exact test). indeed, patients aged - years were significantly less likely at risk for being co-infected (or = . ; p = . ) compared to those aged less than years (s table) . overall, viral and bacterial infections occurred in . % ( / ) and comparison of the prevalence of pathogens by study sites revealed that flua (p = . ), s. pneumoniae (p = . ), and hib (p = . ) were statistically relatively common in patients hospitalized in cenhosoa (s table) . in respect to multiple infections, flua (or = . ) . surprisingly, patients were co-infected with , , and different pathogens ( , , and , respectively) ( table ) . comparison of demographic and clinical characteristics of sari patients by age group. analysis of data by the age group showed that the frequency of clinical symptoms significantly varied across groups. indeed, by multivariate analysis adjusted by age, patients aged � years appeared to suffer chest pain compared to patients aged < years while dry cough and gpd were more often observed in the elderly. dyspnea and cyanosis were significantly more common in adults aged - years. headache, thrill, sweats, anorexia, weight loss, and asthenia were more often observed in the older age groups ( - years, - years and � years). sore throat was frequently reported in the age groups - years, - years and - years. runny nose and intercostal recession appeared to be relatively infrequent in the older age groups ( - years, - years and � years) ( table and s table) . regarding infection, prevalence of rsv (p< . ) and influenza viruses (p< . ) substantially varied depending the age group. as expected, rsv accounted for the highest proportion of sari in young children ( . %; / ). in fact, the older age groups were significantly less at risk to develop an rsv infection compared to the age group less than years (table ) ) and between - years (or = . ) were more likely to catch flub infection. by multivariate analysis, the age group between - years were less likely to suffer from s. pneumoniae infection (or = . ) than the age group less than years ( table ) . pattern of circulation of respiratory pathogens. sari cases were detected all year around during the years of surveillance with distinct peaks of detection observed each year: between january and march in and and between may and july in (fig ) . these peaks coincided with an active circulation of rsv and influenza viruses. nevertheless, peaks of sari appear to be more correlated to rsv circulation. unlike other respiratory pathogens that are detected all year along, rsv seems to circulate once a year and being responsible of an epidemic every year. to the best of our knowledge, this study is the first description of etiologies associated to hospitalized sari patients from both urban and peri-urban areas in madagascar. during the years of surveillance, consented patients meeting the established case definition of sari were enrolled. the observed different clinical spectrum across age groups may be helpful to avoid misclassification of patients presenting with respiratory illness at the triage level when no standardized protocol is available. the finding that about % of inpatients were tested positive for a pathogen is consistent with those reported elsewhere which showed etiologies ranging from % to % of hospitalized sari cases depending on case definition or methodology design [ , , ] . most of the included patients were children less than years old ( . %). this study confirms that young children are the most vulnerable group to suffer from sari [ , ] . nevertheless, these results could also be explained by a socio-economic behavior of the malagasy population. indeed, parents are keener to seek care for their children while older individuals try to cure themselves at home using self-medication or traditional medicine. in the present study, about % of inpatients presented multiple infections by at least pathogens. here, viral infections were commonly found in . % of tested patients which is similar to what was observed within the community during the influenza-like illness surveillance [ ] . taken together, the present study clearly demonstrates that respiratory viruses are the leading cause of severe acute respiratory illnesses in madagascar. overall, rsv was the most commonly detected followed by s. pneumoniae and influenza viruses. similar to other studies, substantial rate of rsv infection was observed in young population [ , , ] . a meta-analysis of data from africa reported that the incidence of rsv in lower acute respiratory infections that required hospitalization ranged from - per person year for infants and - per person year for children under years of age [ ] . the increased circulation of rsv that coincided with the peak of sari cases may be informative for clinicians to predict an epidemic due to rsv and to choose the most appropriate care for patients in order to avoid overuse of antibiotics. in addition, the epidemic pattern of this virus reveals the need to develop rapid diagnostic tests to rapidly manage patients and reduce the number of hospitalization, and to promote effective vaccine to prevent severe infections in high-risk populations. the role of influenza viruses in sari-associated hospitalization is increasingly defined because of available data from active influenza sentinel surveillance system initiated in several countries including sub-saharan africa [ ] [ ] [ ] [ ] . the proportion of influenza viruses in young population obtained here ( . %) is much higher than in other african countries were sari attributable to influenza are around % [ ] . as seen elsewhere, the highest rate of hospitalization is obtained with the subtype a/h n virus during its circulation [ , ] . in madagascar despite decades of reliable influenza surveillance, no national policy is implemented regarding vaccination. moreover, influenza vaccines are not widely accessible and affordable for the whole population. in the present study, about . % of sari cases were likely due to bacterial infection. nevertheless, it is not clear what this high proportion of bacteria means due to the notion of carriage. the substantial rate of s. pneumoniae detected might be partly explained by the very low pneumococcal vaccination rate among the younger population (estimated at . %) because most of the positive cases were detected before the introduction of pneumococcal vaccine into the expanded program on immunization (epi) in madagascar in [ ] . this vaccine is believed to reduce substantially the burden of pneumococcal disease. thus, it would be interesting to evaluate the proportion of sari associated to s. pneumoniae more than years after the introduction of the vaccine in madagascar. on the other hand, the role of atypical bacteria in hospitalized ari is poorly understood. one assumes that certain bacteria predispose to bacterial super-infections [ ] . while rv is considered as the common cold virus, the present study showed that this virus accounted for % of total sari cases. this finding highlights the potential role of rv in public health like other authors already postulated [ ] [ ] [ ] . our study presents some limitations. first, data were collected only from sites that are not representative of the whole population preventing us to real estimate the prevalence at the national level. indeed, prevalence of each pathogen may differ for regions having different bioclimatic or demographic patterns, access to healthcare, and connectivity. since children are more at risk to develop severe infection, our sampling was skewed toward the younger group and likely underestimate the prevalence in adult. indeed, few adult cases had been identified at the adult ward mainly because the duration of symptoms prior to admission exceeded the days limit of our sari case definition. it was also observed that severe cases were treated on external consultation or admitted at the emergency department for a few hours and then discharged. patients that did not recover, worsened at home, and returned to the hospital were then admitted resulting in prolonged duration of illness (usually more than days) at the time of admission. another limitation is to attribute the pathogen(s) that is (are) the main cause that lead to severe illness. indeed, we could not exclude that some pathogens may be present due to carriage. to conclude, the high prevalence of etiology associated to severe infections of relevant pathogens highlights the importance of sustaining national surveillance of sari to clearly estimate the role of associated pathogens and establish the burden of disease. further challenges in such program include efforts to implement strategies that capture adult cases as it is relevant to measure the impact of sari in this population. since vaccines play an important role in preventing sari, vaccination against important pathogens, including rsv, should be accessible at least for high-risk population in developing countries. indeed, although use of synthetic antibodies against rsv can be provided to certain at-risk infants, this treatment only provides a short-term protection, is not always effective, and is also expensive, putting it beyond the reach of developing countries like madagascar. supporting information s table. clinical diagnosis of patients hospitalized for sari, madagascar, november to july . � lrti: low respiratory tract infection including bronchoalveolitis/exacerbation of chronic obstructive pulmonary disease (copd), pleuropneumonia, and acute lobar pneumonia; �� other: neonatal infection, asthma, laryngitis, influenza-like illness etc. . . n = number of included patients. n = number of patients that responded by "yes" or "no" for a given symptom. only bronchiolitis, pneumonia, and bronchopneumonia were statistically analyzed. statistical analyses were performed using fisher's exact test. (docx) s table. univariate analysis of the distribution of monoinfection and multiple infection detected in sari adjusted by age groups, november to july . the age group less than years was considered as reference group. (docx) s epidemiology and etiology of childhood pneumonia the global burden of disease: update. geneva: world health organization global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in : a systematic analysis global estimate of the incidence of clinical pneumonia among children under five years of age global, regional, and national causes of child mortality in : a systematic analysis estimates of world-wide distribution of child deaths from acute respiratory infections burden of disease caused by haemophilus influenzae type b in children younger than years: global estimates burden of disease caused by streptococcus pneumoniae in children younger than years: global estimates community-acquired pneumonia in children childhood community-acquired pneumonia the association of newly identified respiratory viruses with lower respiratory tract infections in korean children study of community acquired pneumonia aetiology (scapa) in adults admitted to hospital: implications for management guidelines transmission characteristics of the h n influenza pandemic: comparison of southern hemisphere countries severe acute respiratory infection in children in a densely populated urban slum in kenya etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya severe acute respiratory illness deaths in sub-saharan africa and the role of influenza: a case series from excess mortality associated with the a(h n )v influenza pandemic in antananarivo outcome risk factors during respiratory infections in a paediatric ward in antananarivo viral etiology of influenza-like illnesses in antananarivo etiology of communityacquired pneumonia in hospitalized children respiratory viral coinfections identified by a -plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections viral and atypical bacterial etiology of acute respiratory infections in children under years old living in a rural tropical area of madagascar viral etiology of severe pneumonia among kenyan infants and children incidence of medically attended respiratory syncytial virus and influenza illnesses in children - months old during four seasons global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and metaanalysis viral etiology of severe acute respiratory infections in hospitalized children in cameroon sentinel surveillance system for early outbreak detection in madagascar unfinished business: severe acute respiratory infection in sub-saharan africa the national burden of influenza-associated severe acute respiratory illness hospitalization in rwanda the impact of influenza epidemics on mortality: introducing a severity index influenza-associated hospitalizations in the united states the burden of acute respiratory infections in crisis-affected populations: a systematic review epidemiology and clinical profile of pathogens responsible for the hospitalization of children in sousse area epidemiology of multiple respiratory viruses in childcare attendees we would like to express our gratitude to all clinicians and nurses involved in this study: emma rasoanirina, roland randriamirado, harimboahangy rakotoarison, lucien radozahana, rani razafindrakoto, domohina rakotovao, durandalle ny hanitrin'ny ala razana, dominique razafimandimby, françois de paul razanakoto, and saholiarisandy ndremihavana. we are deeply indebted to all the staff involved in the hospital surveillance program on a daily basis. we also would like to thank the laboratory technicians at the virology unit and the cbc for their tremendous work. the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the ministry of health or of the us centers for disease control and prevention. key: cord- -qrt zmz authors: miyakawa, kei; matsunaga, satoko; yamaoka, yutaro; dairaku, mina; fukano, kento; kimura, hirokazu; chimuro, tomoyuki; nishitsuji, hironori; watashi, koichi; shimotohno, kunitada; wakita, takaji; ryo, akihide title: development of a cell-based assay to identify hepatitis b virus entry inhibitors targeting the sodium taurocholate cotransporting polypeptide date: - - journal: oncotarget doi: . /oncotarget. sha: doc_id: cord_uid: qrt zmz sodium taurocholate cotransporting polypeptide (ntcp) is a major entry receptor of hepatitis b virus (hbv) and one of the most attractive targets for anti-hbv drugs. we developed a cell-mediated drug screening method to monitor ntcp expression on the cell surface by generating a hepg cell line with tetracycline-inducible expression of ntcp and a monoclonal antibody that specifically detects cell-surface ntcp. using this system, we screened a small molecule library for compounds that protected against hbv infection by targeting ntcp. we found that glabridin, a licorice-derived isoflavane, could suppress viral infection by inducing caveolar endocytosis of cell-surface ntcp with an ic( ) of ~ μm. we also found that glabridin could attenuate the inhibitory effect of taurocholate on type i interferon signaling by depleting the level of cell-surface ntcp. these results demonstrate that our screening system could be a powerful tool for discovering drugs targeting hbv entry. hepatitis b virus (hbv) is the causative agent of chronic hepatitis b (chb), which can lead to liver cirrhosis and hepatocellular carcinoma. the world health organization reported that over million people worldwide have chronic hbv [ ] . despite the effectiveness of the hbv vaccine, worldwide prevalence of the disease remains high, and chb is a major global health problem. current therapeutic regimens for chb include pegylated interferon (ifn) and nucleoside/ nucleotide analogues. both treatments aim to prevent progression of the disease to liver failure, cirrhosis, and hepatocellular carcinoma. however, these treatments have limited effectiveness for hbv clearance [ ] . in fact, pegylated ifn maintains viral suppression only in approximately % of patients [ ] . nucleoside and nucleotide analogues inhibit hbv replication by targeting viral dna polymerase, but long-term treatment is required to achieve clinical benefits. for example, a -month course of lamivudine achieves clearance of hepatitis b e antigen (hbeag) in approximately % of patients with chb [ ] . moreover, long-term treatment can be associated with a higher risk of side effects and emergence of drug resistant viruses, resulting in treatment failure and disease progression. therefore, it is vital to research paper www.oncotarget.com develop new types of antiviral drugs for hepatitis b treatment. in the hbv life cycle, the hepatitis b surface antigen (hbsag) initially attaches to heparan sulfate proteoglycans on the host cell surface [ ] . this attachment seems to be relatively low affinity and reversible, but is essential for the subsequent more specific interaction between hbs and the hepatocyte-specific bile acid transporter sodium taurocholate cotransporting polypeptide (ntcp) [ ] . although various membrane proteins have been reported to be hbv entry receptors, accumulating evidence now suggests that ntcp is an essential receptor for hbv infection [ ] . discovery of this receptor has dramatically increased our understanding of the molecular basis of hbv entry. viral entry is currently one of the most important targets in the search for new drugs to treat viral infections and identification of ntcp has kindled interest in exploring compounds that inhibit hbv entry. ntcp is exclusively expressed at the basolateral membrane of hepatocytes [ ] [ ] [ ] , and is involved in reuptake of conjugated bile acids from the bloodstream into hepatocytes [ ] . ntcp is one of the factors that highly restrict host tropism of hbv to hepatocytes. the specific interaction of the pres domain of hbsag with ntcp triggers hbv attachment and initiates entry into hepatocytes. the synthetic peptide drug myrcludex b exhibits significant anti-hbv activity by competitively inhibiting this interaction, both in vitro and in vivo, and is currently undergoing a phase ii clinical trial for chronically hbv-infected patients [ , ] . physiological substrates of ntcp such as taurocholic and glycocholic acids can also inhibit hbv infection, suggesting the competitive binding of bile acids and pres to ntcp [ ] . interestingly, pres -ntcp binding could trigger type i ifn signaling, whereas bile acid treatment does not [ ] . bile acid intake via ntcp was found to inhibit the ifn pathway in the physiologically relevant range of concentrations [ ] . these observations support the possibility that ntcp also takes part in the ifn-mediated innate antiviral response. because high expression often causes cell cycle inhibition, there are very few conventional hepatocellular carcinoma cell lines with ectopic expression of high levels of ntcp. moreover, there are no commercially available monoclonal antibodies (mabs) specifically recognizing cell-surface ntcp due to the difficulty of producing full-length ntcp protein with native structure. in the current study, we generated hbv-susceptible hepg cells with a tetracycline-inducible ntcp gene (intcp cells) without affecting cell growth. we also generated a high quality mab (clone a ) to efficiently detect cell-surface ntcp. using these tools, we identified the compound glabridin, which significantly inhibits hbv infection through the downregulation of ntcp from the cell surface. endogenous ntcp expression is rarely detectable in hepatoma cell lines such as hepg and huh , and these cells are not susceptible to hbv infection. therefore, we generated the intcp cell line, a hepg based cell line harboring a tetracycline-inducible human ntcp gene ( figure a) . treatment of the intcp cell line with the tetracycline analogue doxycycline (dox) caused expression of ntcp in a dox-dependent manner, and ntcp expression was -to -fold higher than endogenous expression in differentiated heparg cells and primary hepatocytes, as revealed by western blot and quantitative reverse transcription-pcr ( figure b- d) . to determine whether dox-induced ntcp protein localized to the plasma membrane, we performed a pres peptide binding assay. we found that the pres peptide detected dox-treated intcp cells but not untreated cells, indicating the presence of ntcp on the cell surface ( figure e ). consistent with a previous report [ ] , a western blot of ntcp showed major bands of - kd that were shifted to a single band of - kd after treatment with peptide n-glycosidase (pngase), implying that ntcp was modified by n-glycosylation (supplementary figure a) . although ntcp expression has been reported to affect cell proliferation [ ] , intcp cells showed no differences in cell cycle progression or cell expansion with or without dox treatment ( figure f, g) . we subsequently examined the susceptibility of intcp cells to hbv infection. at an moi of geq/cell, intcp cells showed high susceptibility to hbv infection (~ % infected) without dmso treatment ( figure h , supplementary figure b ). this infection was significantly inhibited by pres peptide treatment ( figure h ), which indicates that hbv infection was ntcp-mediated. taken together, these results show that dox-induced ntcp proteins are exposed on the cell surface and functionally interact with pres . although the above results suggest that ntcp proteins localize on the cell surface, this could not be directly demonstrated due to the lack of suitable antibodies for flow cytometry or immunofluorescence microscopy analysis. therefore, we generated a monoclonal antibody (mab) for this purpose. because recombinant ntcp protein tends to form insoluble aggregates, we utilized the wheat germ cell-free system, which has been shown to have advantages for the production of membrane proteins [ , ] . the synthesized ntcp proteins were purified and used to immunize mice, and more than hybridoma clones were established (figure a ). using a www.oncotarget.com flow cytometer-based screening assay with dox-treated and untreated intcp cells, we identified a hybridoma clone producing anti-ntcp mab, clone a ( figure b ). the a mab could recognize endogenous ntcp in differentiated heparg cells (supplementary figure ) . we performed immunofluorescence microscopy using the a mab in various cell lines and cell-surface ntcp was clearly observed ( figure c , d). because the three-dimensional organoid culture recapitulates cell-cell interactions and recent studies have indicated some advantages of hepatoma organoids in hepatitis virus infection [ ] , we investigated the localization of ntcp in hepatoma organoids. we embedded intcp cells in hydrogel before performing immunofluorescence microscopy with the a mab to visualize ntcp. ntcp localization was widespread in the membrane of internal cells as well as on the surface of the organoid ( figure e ). epitope mapping using recombinant ntcp mutants revealed that the a mab recognizes amino acids - of ntcp ( figure f ). to test whether the a antibody can inhibit hbv infection, we pretreated intcp cells and primary human hepatocytes with a mab and subsequently infected cells with wild type hbv and hbv encoding a luciferase reporter gene (hbv-nl) [ ] . the a mab failed to inhibit hbv infection ( figure g , h), suggesting that the interaction between a mab and ntcp neither blocks hbv-host cell interaction nor causes downregulation of ntcp from the cell surface. because the a mab binds ntcp but does not interfere with its localization and function, it can be used to screen for antiviral compounds that modulate the level of cell-surface ntcp. briefly, intcp cells were treated with different low-molecular-weight chemical compounds from a library derived from natural plant extracts for hours and we subsequently quantified cell viability and the amount of cell-surface ntcp using the a mab ( figure a ). we identified two compounds, geraldol and glabridin, that could decrease the amount of cell-surface ntcp without observable cytotoxicity ( figure b ). flow cytometry analysis using the a mab demonstrated that both geraldol and glabridin decrease the levels of cell-surface ntcp in a dose-dependent fashion ( figure c ), but further analysis revealed that geraldol negatively regulates the tetracycline-responsive promoter activity ( figure d ). microscale thermophoresis analysis of the biomolecular interaction [ ] revealed that glabridin could weakly but directly interact with ntcp ( figure e ). therefore, we focused on glabridin for further functional analyses. flow cytometry analysis showed that glabridin downregulated the amount of cell-surface ntcp in hepg -hntcp-c [ ] and heparg cells with ic values of µm and µm, respectively ( figure a ). notably, treatment with µm glabridin had no effect on the level of ntcp mrna (supplementary figure a) , suggesting that glabridin affects ntcp protein rather than ntcp gene expression. consistently, treatment with glabridin rapidly (within three hours) modulated the membrane localization of ntcp, but not that of the transferrin receptor ( figure b ). similar results were obtained with cell-surface biotinylation analysis in which only cell-surface proteins were purified and detected by western blotting (supplementary figure b ). immunofluorescence microscopy using the a mab showed that in glabridin-treated cells, ntcp mainly accumulated in the cytoplasm ( figure c ). to test the role of endocytosis in ntcp localization, we treated cells concurrently with glabridin and inhibitors of clathrinmediated or caveolar endocytosis. we found that genistein, a specific inhibitor of caveolar endocytosis, completely disrupted ntcp internalization ( figure d ), suggesting that glabridin causes the internalization of ntcp through caveolar endocytosis. because internalized membrane proteins are typically trafficked to degradation or recycling pathways, we next performed a cycloheximide chase assay. interestingly, ntcp protein levels were relatively stable in control cells, but the half-life of ntcp was significantly shorter in glabridin-treated cells ( figure e ). this suggests that glabridin reduces the amount of ntcp on the cell surface by promoting its endocytosis and subsequent intracellular degradation. previous reports have indicated that glabridin induces apoptosis in certain cancer cells [ ] , but we observed no negative effect on cell viability under our experimental conditions ( figure f ). we next assessed the impact of glabridin on hbv infection in hepatocytes. time-of-addition experiments demonstrated that µm glabridin inhibited hbv infection when added early in infection but became less effective when added later ( figure a ), suggesting that it acts at an early stage of the viral life cycle. indeed, when glabridin was added to hbv-producing cells, it did not affect the amounts of core-associated viral dna and/or rna (supplementary figure ) . consistent with these observations, intcp cells treated with glabridin three hours prior to infection showed a significant reduction in the percentage of infected cells and the amount of hbsag secretion ( figure b , c). we performed a parallel analysis with differentiated heparg cells and confirmed that glabridin blocked hbv infection and downregulated endogenous ntcp with an ic value of µm ( figure d ). these results suggest that glabridin inhibits hbv infection by removing ntcp from the cell surface. ntcp is a transporter for bile acid uptake. we investigated the effect of glabridin on ntcp-mediated www.oncotarget.com anti-ntcp mab generation. using a wheat germ cell-free protein production system, large amounts of ntcp were synthesized with high solubility. following mouse immunization, we established over hybridoma clones. (b) selection of a clones producing anti-ntcp mab. culture supernatants of hybridoma clones were used as primary antibodies for flow cytometry analysis of dox-treated (red line) or -untreated intcp cells (blue line). (c-e) immunofluorescence staining of cell-surface ntcp by a mab on intcp cells (c), hepg -hntcp-c cells (d), and intcp-derived spheroid (e). scale bars: µm. (f) epitope mapping of a mab. recombinant wild-type or partially truncated ntcp proteins tagged with his were generated using wheat germ extracts and subjected to western blotting using anti-his or a antibodies. the predicted epitope of a mab is shown in pink. (g, h) a mab fails to inhibit hbv infection. intcp cells (g) and primary human hepatocytes (h) were infected with hbv or its reporter virus (hbv-nl) respectively, in the presence of a mab. anti-hbs mab (clone a , which recognizes the pres domain) was used as a control. viral infectivity was determined by intracellular hbcag staining (g) or nanoluc activity (h) of infected cells. uptake of taurocholate (tca) in a sodium-containing condition and found that glabridin reduced tca uptake in a dose-and time-dependent manner ( figure a ). a previous study has shown that ntcp-mediated bile acid transport affects the expression of ifn stimulatory genes (isgs) in primary human hepatocytes [ ] , so we investigated whether glabridin counteracts this function of bile acid. primary human hepatocytes were treated with type i ifn and tca in the presence or absence of glabridin. treatment with ifn upregulated various isg proteins including mx and bst , which are known to inhibit hbv replication [ ] [ ] [ ] [ ] , while concurrent treatment with tca reduced this effect ( figure b ), in accordance with previous observations [ ] . interestingly, the addition of µm glabridin partially cancelled the effect of tca in isg expression ( figure b ). these findings suggest that glabridin enhances the innate immune response by suppressing bile acid uptake in hepatocytes through the downregulation of cell-surface ntcp. in this study, we generated intcp cells, which have high ntcp expression and high susceptibility to hbv infection, and also developed a monoclonal antibody (mab) that recognizes cell-surface ntcp. using these tools, we identified glabridin as a compound that inhibits hbv infection by downregulating levels of its entry receptor, ntcp. although primary hepatocytes express ntcp at low levels for the uptake of bile acids, endogenous ntcp in hepatocellular carcinoma cell lines is not sufficient to achieve successful infection with hbv in vitro. hepatocellular carcinoma cell lines stably expressing ntcp have been created, and exhibited increased susceptibility to hbv infection [ ] , but it has been shown that continuous ntcp expression can induce cell cycle arrest at the g /g phase [ ] , indicating that ectopic ntcp expression may be unfavorable for the long-term culture. to overcome these issues, we created the intcp cell line, featuring inducible ntcp expression, using the third-generation tetracyclineinducible gene expression system in hepg cells. upon transient treatment with tetracycline derivatives, this cell line exhibits high expression of cell-surface ntcp and becomes highly susceptible to hbv infection without any observable cell cycle arrest. this unique feature of our newly developed cell line makes it a useful tool for further biological analysis of ntcp in hepatic cells. we created a new mab recognizing cell-surface ntcp by using a wheat germ cell-free protein production system to synthesize full-length recombinant ntcp protein, which was then used to immunize mice. generally, the quality of an antibody is determined largely by the antigen used for immunization. preparation of immunogens derived from highly insoluble membrane proteins is challenging, and ntcp tends to be insoluble in conventional cell expression systems due to its multiple transmembrane domains. there are several methods for preparing antigens derived from insoluble proteins, including use of synthetic peptides containing the predicted immunogenic epitope of extracellular domains [ ] , but synthetic peptides are structurally linear and do not recapitulate the native structural features of membrane proteins. by contrast, the wheat germ cell-free protein production system utilizes a eukaryotic translation system to synthesize structurally intact and biologically active proteins similar to those expressed in mammalian cells [ , ] . this approach enabled us to create a mab capable of detecting a spatial antigen within the region of ntcp exposed on the cell surface. our work demonstrates the advantage of the cell-free system in the production of proteins with multiple transmembrane domains [ , ] . the three-dimensional structure of ntcp protein has not been well characterized and the number of transmembrane domains is controversial at present. several groups have predicted that ntcp has - transmembrane domains and that the c-terminus of ntcp is located in the cytoplasm [ , ] . we found that our a mab recognizes amino acids - of ntcp on intact cells without membrane permeabilization, implying that this epitope is possibly exposed to the extracellular space. more precise structural biological studies should be carried out to elucidate the topology of the ntcp protein. immunofluorescence microscopy experiments using our a antibody revealed that ntcp localized on the plasma membrane, frequently at cell-cell contact sites. this localization was more obvious in a hepatocyte organoid culture system, suggesting that the localization of ntcp may depend on cell polarity. because the threedimensional culture system recapitulates cell polarity, it is useful for analyzing the function of ntcp in sodium taurocholate transport as well as hbv infection. recent studies demonstrated that the three-dimensional culture system is suitable for analyzing polarized hbv transmission [ , ] . it is not well established whether ntcp plays a role in viral transmission in polarized cells, and our newly developed a mab may be useful for pursuing this intriguing question. several compounds have been shown to target ntcp. recent studies demonstrated that antagonists of retinoic acid receptor ro - [ ] or the cytokine interleukin- [ ] could reduce ntcp expression. a previous report also indicated that the green tea extract epigallocatechin- -gallate (egcg) also inhibited hbv entry into primary human hepatocytes [ ] . interestingly, egcg induced endocytosis of ntcp from the plasma membrane followed by protein degradation. in the current study, we found that glabridin, a compound from licorice extract, also causes ntcp receptor downregulation. although the precise mechanisms are unclear, our findings intcp cells were treated with glabridin ( , , and µm) for three or hours. after incubation with [ h]-taurocholate (tca) for minutes, cells were washed and intracellular radioactivity was quantified. cyclosporin a (csa, µm) was used as a positive control in this assay. * p < . , ** p < . , two-tailed unpaired t-test. (b) glabridin counteracts bile acids to promote innate immune signaling. primary human hepatocytes were sequentially treated with ifn-β ( u/ml), tca, and glabridin ( µm), as shown in left panel. the expression of representative isgs, including mx and bst , was quantified using qpcr. ns, not significant; ** p < . , two-tailed unpaired t-test. www.oncotarget.com suggest that glabridin directly binds ntcp and causes its internalization by caveolar endocytosis. by estimating protein turnover using a cycloheximide chase assay, we were able to infer that glabridin promotes the intracellular degradation of ntcp. interestingly, glabridin is orally bioavailable and is known to rapidly accumulate in the liver [ ] [ ] [ ] . since ic value of glabridin for inhibiting of hbv entry is relatively high (~ µm), this compound might have little translational benefit in hbv research. however, the data obtained from our study demonstrates the potential scope of our current methodology in drug discovery for hbv therapeutics. it is possible that the internalization of ntcp could inhibit its transporting activity thereby causing unfavorable effects on hepatocytes. however, people with i t or s f mutations in the ntcp gene show decreased surface expression and transporting activity of ntcp, but there have been no reports of serious diseases resulting from these mutations to date [ , ] . the frequent occurrence of physiological downregulation of ntcp is also notable. this downregulation is typically controlled by transcription factors, such as hnf α, under cholestasis conditions [ ] . furthermore, recent papers have shown that ntcp is the main transporter for conjugated bile acids into the liver, but other auxiliary transporters, such as oatp, may compensate when ntcp is absent [ , ] . although these reports hint that transient downregulation of ntcp might not induce immediate adverse effects on the liver, the effect should be further investigated in in vivo models to determine whether ntcp inhibition is a reasonable anti-hbv drug target. in conclusion, we identified as glabridin as a natural compound capable of inhibiting hbv infection by impairing viral entry into host cells, with an additional effect of enhancing the antiviral immune response. furthermore, our data demonstrate that our newly developed cell line and antibody will serve as powerful tools for drug discovery targeting hbv entry and for exploring molecular mechanisms underlying hbv spread. to generate intcp cells, a hepg tet-on advanced cell (clontech) parental cell line was transduced with a retroviral vector encoding the ntcp gene fused to a tetracycline-responsive element, then was selected with puromycin ( µg/ml), and cultured with dmem (wako) supplemented with % fbs. unless otherwise indicated, intcp cells were treated with doxycycline (sigma-aldrich) for hours before experiments. the intcp spheroid was made using the -d life dextran-cd hydrogel sg kit (cellendes) and cultured for seven days on a chamber slide (thermo fisher scientific). primary human hepatocytes (pxb-cells) were purchased from phoenixbio. heparg cells were purchased from biopredic international and differentiated according to the manufacturer's instructions. the chemical compound library from natural plant extracts was obtained from tokiwa phytochemical. nocodazole, cyclosporin a, genistein, and pitstop were purchased from sigma-aldrich. glabridin, staurosporine, and ifn-β were obtained from wako. a protemist xe robotic protein synthesizer (cellfree sciences) was used for the generation of full length ntcp and its truncated derivatives as previously described [ , ] . immunization of balb/c mice with recombinant ntcp and generation of hybridoma cells producing anti-ntcp antibody were performed as previously described [ ] . purification of antibodies in the culture supernatant of the hybridoma clones was performed by centrifugation at , rpm for minutes and elution with acrosep hyper df columns (pall). samples were then concentrated using amicon ultra filters (merck millipore). immunoglobulin characterization was carried out using the isostrip mouse monoclonal antibody isotyping kit (roche). cells were fixed with % paraformaldehyde, blocked with % normal goat serum (thermo fisher scientific), and stained with either anti-ntcp mab (clone a ) or anti-hbcag polyclonal antibody (dako) and alexa fluor-conjugated secondary antibodies (thermo fisher scientific). alexa fluor -conjugated phalloidin (thermo fisher scientific) was used for f-actin staining. for intracellular staining, cells were permeabilized with . % triton x- before blocking. for the pres -peptide binding assay, intcp cells pretreated with µg/ml dox for hours were incubated with nm fitc-conjugated pres peptide (the first amino acid residues of small hbs domain fused with n-terminal myristoyl group and c-terminal fitc) at ° c for two hours. cells were then washed with pbs and fixed with % paraformaldehyde. microscopic imaging was performed with an fv -d confocal laser scanning microscope (olympus) or bz- fluorescence microscope (keyence). cells were detached with mm edta in pbs and fixed with % formaldehyde before incubation with either anti-ntcp ( a ) or anti-transferrin receptor (genetex) antibodies at ° c. cells were then stained with pe-conjugated secondary antibody and analyzed using a facscanto ii instrument (bd biosciences). for the drug screen, cells were treated with candidate compounds www.oncotarget.com ( µm) hours before analysis. data were analyzed with flowjo software (treestar). wild-type hbv was derived from the supernatants of hepg . . cells, which were stably transfected with a complete hbv genome. hbv reporter viruses (hbv-nl) were produced by transient transfection of hepg cells with puc . -hbv/nl and puc-hbv-d, as previously described [ ] . the collected supernatants were filtered through a . -μm filter (merck millipore), and concentrated approximately times using a peg virus precipitation kit (biovision). cells were infected with wild-type hbv at a concentration of , genome equivalents per cell in the presence of % peg for hours. alternatively, cells in a -well plate were inoculated with µl of hbv-nl in the presence of % peg for hours. hbv-infected cells were cultured in fresh medium for an additional - days and their infectivity was determined by intracellular hbcag staining or extracellular hbsag quantification, as previously described [ ] . the infectivity of hbv-nl was quantified using the nano-glo luciferase system (promega), according to the manufacturer's instructions. samples in sds loading buffer were loaded onto % polyacrylamide gels, electrophoresed, and blotted onto pvdf membranes (merck millipore), as previously described [ ] . membranes were probed with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (ge healthcare). for protein degradation analysis, cycloheximide ( µg/ml) was added - hours before harvesting cells. the primary antibodies used were as follows: anti-ntcp, anti-vinculin, anti-α-tubulin (sigma-aldrich) and anti-his (genetex). detected proteins were visualized using a fluorchem digital imaging system (alpha innotech). band analysis was performed with imagej software (national institutes of health). messenger rna extraction and subsequent cdna synthesis was performed using trizol reagent (thermo fisher scientific) and revertra ace (toyobo), respectively, according to each manufacturer's instructions. gene expression was then analyzed by qpcr using sybr premix ex taq ii (takara) and a cfx real-time pcr detection system (bio-rad). the primer pairs used were ′-ggacttcgagcaagagatgg- ′ and ′-agcactgtgttggcgtacag- ′ for actb; ′-atggaggcccacaacg cgtctgccc- ′ and ′-cagaaggtggagcaggtggtcatcac- ′ for ntcp; ′-ggctgtttaccagactccgaca- ′ and ′-cacaaa gcctggcagctctcta- ′ for mx ; and ′-tctcctgcaaca agagctgacc- ′ and '-tctctgcatccagggaagccat- ' for bst . recombinant ntcp-gfp and gfp proteins were incubated with different concentrations of compounds in mm potassium phosphate buffer (ph . ) containing mm nacl, . % bsa, and . % ddm (n-dodecylβ-d-maltopyranoside) for one hour at room temperature. samples were loaded on monolith nt. standard treated capillaries (nano temper technologies) and analyzed with a monolith nt. blue red microscale thermophoresis instrument. cell cycle analysis was performed with a tali image-based cytometer (thermo fisher scientific). cell viability was measured with cell counting kit- (dojindo) or celltiter-glo assay (promega) according to the manufacturer's instructions. cells were treated with [ h]-taurocholic acid (tca) in a sodium-free or sodium-containing buffer at ºc for minutes to allow tca uptake into cells. cells were washed and intracellular radioactivity was measured using a liquid scintillation counter. all graphs represent means and standard deviations. the statistical significance of differences between two groups was tested using a two-tailed unpaired t test with prism software (graphpad). km designed and performed research, analyzed data, and wrote the manuscript; sm, yy, md, and kf performed research and analyzed data; hk and tc analyzed data; hn, kw, ks, and tw contributed reagents and analyzed data; and ar designed and supervised the research, analyzed the data, and wrote the manuscript. global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity virologic monitoring of hepatitis b virus therapy in clinical trials and practice: recommendations for a standardized approach pegylated interferon alfa- b alone or in combination with lamivudine for hbeag-positive chronic hepatitis b: a randomised trial nucleoside analogues for chronic hepatitis b: antiviral efficacy and viral resistance entry of hepatitis b and hepatitis d virus into hepatocytes: basic insights and clinical implications sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus hepatitis b and d viruses exploit sodium taurocholate co-transporting polypeptide for speciesspecific entry into hepatocytes characterization of cloned rat liver na(+)-bile acid cotransporter using peptide and fusion protein antibodies in situ localization of the hepatocytic na+/ taurocholate cotransporting polypeptide in rat liver chlorambucil-taurocholate is transported by bile acid carriers expressed in human hepatocellular carcinomas sinusoidal (basolateral) bile salt uptake systems of hepatocytes first-in-human application of the novel hepatitis b and hepatitis d virus entry inhibitor myrcludex b treatment of chronic hepatitis d with the entry inhibitor myrcludex b: first results of a phase ib/iia study kinetics of the bile acid transporter and hepatitis b virus receptor na+/taurocholate cotransporting polypeptide (ntcp) in hepatocytes solute carrier ntcp regulates innate antiviral immune responses targeting hepatitis c virus infection of hepatocytes bile acids modulate the interferon signalling pathway down-regulation of ntcp expression by cyclin d in hepatitis b virus-related hepatocellular www.oncotarget.com carcinoma has clinical significance production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system a cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters single particle imaging of polarized hepatoma organoids upon hepatitis c virus infection reveals an ordered and sequential entry process novel reporter system to monitor early stages of the hepatitis b virus life cycle molecular interaction studies using microscale thermophoresis evaluation and identification of hepatitis b virus entry inhibitors using hepg cells overexpressing a membrane transporter ntcp glabridin mediate caspases activation and induces apoptosis through jnk / and p mapk pathway in human promyelocytic leukemia cells mxa inhibits hepatitis b virus replication by interaction with hepatitis b core antigen the interferon-inducible protein tetherin inhibits hepatitis b virus virion secretion identification of bst- /tetherin-induced hepatitis b virus restriction and hepatocyte-specific bst- inactivation molecular dissection of hbv evasion from restriction factor tetherin: a new perspective for antiviral cell therapy cell culture models for the investigation of hepatitis b and d virus infection generation and identification of peptide-based monoclonal antibodies against vacuolar proton pyrophosphatase of toxoplasma gondii advances in genome-wide protein expression using the wheat germ cell-free system cell-free expression systems for eukaryotic protein production organization of the membrane domain of the human liver sodium/bile acid cotransporter cloning and functional characterization of human sodium-dependent organic anion transporter (slc a ) concise review: organoids are a powerful tool for the study of liver disease and personalized treatment design in humans and animals cultivation of hepg . . on cytodex- : higher yield of hepatitis b virus and less subviral particles compared to conventional culture methods dysregulation of retinoic acid receptor diminishes hepatocyte permissiveness to hepatitis b virus infection through modulation of sodium taurocholate cotransporting polypeptide (ntcp) expression interleukin inhibits hbv entry through ntcp down regulation epigallocatechin- -gallate inhibits entry of hepatitis b virus into hepatocytes determination of glabridin in human plasma by solidphase extraction and lc-ms/ms role of p-glycoprotein in limiting the brain penetration of glabridin, an active isoflavan from the root of glycyrrhiza glabra role of p-glycoprotein in the intestinal absorption of glabridin, an active flavonoid from the root of glycyrrhiza glabra ethnicity-dependent polymorphism in na+-taurocholate cotransporting polypeptide (slc a ) reveals a domain critical for bile acid substrate recognition genetic polymorphisms in na+-taurocholate co-transporting polypeptide (ntcp) and ileal apical sodium-dependent bile acid transporter (asbt) and ethnic comparisons of functional variants of ntcp among asian populations hepatocyte nuclear factor- alpha is a central transactivator of the mouse ntcp gene sodium taurocholate cotransporting polypeptide (slc a ) deficiency: conjugated hypercholanemia without a clear clinical phenotype hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice wheat germ cell-free system-based production of hemagglutininneuraminidase glycoprotein of human parainfluenza virus type for generation and characterization of monoclonal antibody development of monoclonal antibody and diagnostic test for middle east respiratory syndrome coronavirus using cell-free synthesized nucleocapsid antigen www.oncotarget.com we thank naohito nozaki for antibody production and haruka sato, kyoko ohnishi, and sho nonoyama for their technical assistance. the authors declare that they have no competing interests. this work was supported in part by an amed grant-in-aid for the program on the innovative development and the application of new drugs for hepatitis b (jp fk to ar and jp fk to km), the creation of innovation centers for advanced interdisciplinary research areas program (to ar), and by a gsk japan research grant (to km). key: cord- -y g yak authors: choi, eunjin; ha, kee-soo; song, dae jin; lee, jung hwa; lee, kwang chul title: clinical and laboratory profiles of hospitalized children with acute respiratory virus infection date: - - journal: korean j pediatr doi: . /kjp. . . . sha: doc_id: cord_uid: y g yak purpose: despite the availability of molecular methods, identification of the causative virus in children with acute respiratory infections (aris) has proven difficult as the same viruses are often detected in asymptomatic children. methods: multiplex reverse transcription polymerase chain reaction assays were performed to detect common respiratory viruses in children under years of age who were hospitalized with ari between january and december . viral epidemiology and clinical profiles of single virus infections were evaluated. results: of , patients, viruses were identified in , ( . %), with the assay revealing a single virus in , cases ( . %). while major pathogens in single virus-positive cases differed according to age, human rhinovirus (hrv) was common in patients of all ages. respiratory syncytial virus (rsv), influenza virus (if), and human metapneumovirus (hmpv) were found to be seasonal pathogens, appearing from fall through winter and spring, whereas hrv and adenovirus (adv) were detected in every season. patients with aris caused by rsv and hrv were frequently afebrile and more commonly had wheezing compared with patients with other viral aris. neutrophil-dominant inflammation was observed in aris caused by if, adv, and hrv, whereas lymphocyte-dominant inflammation was observed with rsv a, parainfluenza virus, and hmpv. monocytosis was common with rsv and adv, whereas eosinophilia was observed with hrv. conclusion: in combination with viral identification, recognition of virus-specific clinical and laboratory patterns will expand our understanding of the epidemiology of viral aris and help us to establish more efficient therapeutic and preventive strategies. acute respiratory infections (aris) are a main cause of morbidity and mortality in children, with viruses being responsible for more than % of aris worldwide. , ) clinical manifestations of ari can hardly differentiate bacterial from viral etiologies, which can lead to the unnecessary use of antibiotics. recent development of the multiplex reverse transcription polymerase chain reaction (rt-pcr) assay makes noninvasive identification of respiratory pathogens possible. however, a more accurate diagnosis of causative ari pathogens does not decrease hospital admissions or antibiotic use in children with ari. ) since viruses are often detected in asymptomatic children, ) identification of a virus by rt-pcr does not always imply that it is the culprit for a current ari. because these viruses are sometimes asymptomatic carriers and sometimes pathogens, a diagnosis should not be based solely on viral identification in respiratory samples. moreover, new respiratory viruses have been increasingly recognized, but their clinical significance remains unclear. , ) thus, in addition to multiplex rt-pcr assays, korean j pediatr ; ( ) : - a better understanding of these viruses is required to improve clinical management. in this study, we assessed the viral epidemiology of ari in hospitalized children under age of years, and we characterized the virusspecific clinical and laboratory profiles as well as clinical outcomes. together with rt-pcr results, this data will help us understand the clinical course of viral ari and to establish more effective preventive and therapeutic strategies. this retrospective cohort study was approved by the institutional review board of korea university guro hospital (kugh). hospitalized patients under years of age with a discharge diagnosis of ari from january to december were enrolled. ari was diagnosed based on one of symptoms or signs; fever of more than . °c, cough, rhinorrhea, sore throat, tonsillar injection, wheezing, crackle, chest wall retraction. nasopharyngeal aspirates from all patients were obtained within hours of admission for multiplex rt-pcr assay to detect the following common respiratory viruses: influenza virus a and b (ifa, ifb), respiratory syncytial virus a and b (rsv a, rsv b), parainfluenza virus - (piv , piv , piv , piv ), human coronavirus e and oc (hcv- e, hcv-oc ), human rhinovirus (hrv), human enterovirus (hev), adenovirus (adv), human bocavirus (hbv), and human metapneumovirus (hmpv). only a single sample was taken from the patients during the admission. rt-pcr results were used to evaluate the incidence of respiratory viruses. laboratory parameters on the first day of admission, as well as overall clinical profiles including baseline characteristics, presenting symptoms and signs, treatments and clinical outcomes, were analyzed in patients with single-virus infections. any possible bacterial coinfections were excluded by sputum and blood culture and clinical course. mycoplasma coinfections were excluded by sputum pcr or by a serial increase of antibody titer or a high initial igm titer without history of recent respiratory disease. respiratory samples from children were collected by nasopharyngeal aspiration. the presence of each virus was determined using the seeplex rv rt-pcr assay (seegene, inc., seoul, korea). two sets of primers were designed based on conserved regions of genetic sequences for the respiratory viruses mentioned above. amplified pcr products were analyzed by electrophoresis on % agarose gel and were compared with the reference band size provided by the manufacturer. ) statistical analysis was performed using ibm spss statistics ver. . (ibm co., armonk, ny, usa). data were expressed as mean± standard deviation or as percentages, as appropriate. a multipleway analysis of variance was run to test differences between viral groups. then, each viral group was compared with the other viral groups using chi-square tests for categorical data and unpaired student t tests for continuous data. a p-value < . was considered statistically significant. from respiratory samples of a total of , patients, viruses were detected in , patients ( . %). after excluding patients with bacterial coinfections (mycoplasma in cases, other bacteria in cases, and mycoplasma with other bacteria in cases), , patients ( . %) were found to have viral aris. in those cases, the most common were hrv ( of , , . %), adv ( of , , . %), and rsv a ( of , , . %) (fig. viruses were detected more frequently in younger cases (≤ year old vs. > years old was . % vs. . %, p= . ). cases with multiple viruses tended to be male-dominant when compared to single-virus cases ( . % vs. . %, p= . ) ( table ) . a total of , single-virus cases were analyzed for their distribution at different ages (fig. ) . seventy-five percent of single-virus cases were identified in patients younger than years old. while rsv a, rsv b, hbv, and piv were major pathogens in patients younger than one year of age, ifa, ifb, hev, and adv were common in patients older than three years of age. hrv was a predominant single virus that was seen across all ages. although not as common as hrv, hmpv was also prevalent across all ages. during the observation period, many viruses showed seasonal patterns. ifa and ifb were most common from winter to spring, while hmpv was predominant in the spring, and piv and hev common from spring to summer. rsv a and b peaked during fall and winter biennially. although not common, hbv was prevalent during the spring and hcv was seen during fall and winter. hrv and adv did not show a discrete seasonal pattern (fig. ). although each virus prevailed throughout preferred seasons during the observation period, there was annual deviations. there were no differences in comorbidities such as premature birth, congenital heart disease and chronic lung disease among viral ari cases. rsv a, rsv b, and hmpv were more associated with lower ari compared with other viruses. croup was most commonly associated with piv and hcv ( . %, . %), while pneumonia was associated with rsv a, rsv b, and hmpv ( . %, . %, and . %). additionally, bronchiolitis was associated with rsv a and b ( . % and . %), while asthma exacerbations were associated with hrv ( . %, p< . ) only. most cases of ari presented with cough and rhinorrhea (data not shown). while fever was associated with many viruses, rsv a, rsv b, and hrv were not significantly associated with fever ( . %, . %, and . %, p< . ). adv and piv were associated with fever lasting more than days ( . %, headache was significantly associated with hev and adv ( . % and . %, p< . ). antibiotics were used more frequently in cases with adv and hmpv ( . % and . %, p< . ), and steroids were used more frequently in cases with hrv ( . %, p< . ). rsv was associated with greater nebulizer use, o supplementation and intensive care unit care, and it was also associated with a longer hospital stay ( table ). cases of adv, hev, and hrv had a greater tendency to show leukocytosis over , /l when compared to other viruses ( . %, . %, and . %, p< . ). although leukopenia less than , /l was observed more with ifa, ifb ( . %, . %, p< . ), an absolute neutrophil count less than , /ml was more associated with piv ( . %, p< . ). while neutrophil-dominant inflammation, represented by a neutrophil-to-lymphocyte ratio ≥ , was more associated with ifa, ifb, adv, hrv, and hev, lymphocyte-dominant inflammation with a neutrophil-to-lymphocyte ratio ≤ was more associated with rsv a, rsv b, piv, and hmpv. monocytosis greater than , /l was observed more with rsv a, rsv b, and adv ( . %, . %, and . %), while eosinophilia greater than /l was observed more with hrv than with other viruses ( . %, p< . ). although not common, thrombocytopenia less than , /l was observed more with hcv-oc ( . %, p= . ). ele vated levels of aspartate aminotransferase and alanine aminotransferase were associated more with piv and rsv a. c-reactive protein levels less than mg/l were associated with ifb, rsv a, rsv b, and piv ( . %, . %, . %, and . %, p< . ), and an erythrocyte sedimentation rate less than mm/hr were associated with ifa, rsv a, and piv ( . %, . %, and . %, p< . ). both c-reactive protein levels greater than mg/l and erythrocyte sedimentation rates greater than mm/hr were associated with adv (p< . ) ( table ). in this study, viruses were detected in . % of all ari cases, but when cases of bacterial coinfection were excluded, viruses were detected in . % of cases. bacterial infection may be erroneously estimated because we counted all possible cases, even if not confirmed. additionally, we did not routinely test for bacterial infection at presentation. nevertheless, viral detection rates in this study were comparable to those of other studies. , ) although hrv, adv, and rsv are the most prevalent viruses, rsv was more present as a single pathogen, whereas hrv and adv were more seen with multiple pathogens. rsv, hrv, and piv were the most prevalent single pathogens in patients younger than years old. after the age of three, the prevalence of rsv decreased while the prevalence of if and adv increased, making hrv, if, and adv the most prevalent after three years of age. the younger age group shows higher virus positivity compared with the older age group, which may reflect prolonged shedding of respiratory viruses in young children. ) cases with multiple detected viruses are more likely to be males, while the sex ratio in single-virus cases is concordant with that of cases examined. the incidence of infectious disease is also reported higher among males. , ) it is possible that after viral infection, viral clearance may be delayed in males, resulting in a greater chance for viruses to accumulate. the significance of multiple viral coinfections has been controversial. while it is unlikely to be associated with more severe illness, , , ) specific pathogen pairs such as rsv with influenza virus may be associated with increased severity. ) studies report strong causal attribution of rsv, if, and hmpv, less strong evidence for hrv and no attribution of adv, hbv and hcv, [ ] [ ] [ ] which is in agreement with the ratio of single to multiple detections of each virus in our study. quantitative viral analysis may help to distinguish active infection from viral shedding in cases with multiple viral coinfection. ) because the clinical significance of multiple viral infections remains controversial and hard to interpret, we compared single-virus cases to understand characteristics of each virus. seasonality of some respiratory viruses is an important clue for early recognition. in addition to the strong seasonal patterns observed with if, rsv, and hmpv, it is interesting that rsv a and b have an apparent biennial seasonality. while hrv and adv are prevalent throughout the year, seasonal patterns of viruses are discrete. each respiratory viruses differ in seasonal onset as well as activity, but this can slightly vary from year to year. knowing the seasonality of each virus will help to plan effective control strategies and to make epidemiological diagnosis. analysis of virus-specific clinical profiles showed that cases of rsv a, rsv b, hbv, piv, and hcv affect patients who are signifi cantly younger than those with ifa, ifb, adv, and hev aris. premature birth history, and presence of congenital heart disease and chronic lung disease are not associated with any specific virus. while fever is a common symptom of ari, cases of rsv and hrv are significantly afebrile. infants younger than months with rsv infection tend to be afebrile, which may be due to a lack of a pyrogenic cytokine response. , ) while patients with afebrile rsv cases are significantly younger than febrile rsv cases ( . months vs. . months, p= . for rsv a, . months vs. . months, p= . for rsv b), there is no age difference between afebrile and febrile hrv cases ( . months vs. . months, p= . ) in this study, suggesting that pyrogenic immune responses might be different depending on the virus. some viral respiratory infections are associated with asthma and asthma exacerbation. ) cases of rsv and hrv ari present more commonly with wheezing, but eosi nophilia and a diagnosis of asthma exacerbation are associated only with hrv infection in this study. a study performed in children with acute wheezing illness reports that rsv is the predominant virus in patients with no previous wheezing, whereas hrv is predominant in patients with a history of wheezing. ) whether rsv infection causes asthma is still debatable, but it is possible that lower ari with pathogens such as hrv and rsv could precipitate the development of asthma, especially in children with atopic features. ) the total leukocyte, neutrophil, lymphocyte and monocyte counts, and specific associations between blood cells, especially the neutrophil-to-lymphocyte ratio have been used as markers of inflammation and infection. , ) viral aris show leukocytosis rather than leukopenia and furthermore, this study shows that if, hrv, hev, and adv are associated with neutrophil-dominant inflammation whereas rsv, piv, and hmpv are associated with lymphocyte-dominant inflammation. of note, monocytosis is observed with rsv a, rsv b, and adv aris. other inflammatory markers such as erythrocyte sedimentation rate and c-reactive protein levels are not significantly increased in most viral aris except for adv infection. thus, adv ari present with neutrophildominant leukocytosis, a high erythrocyte sedimentation rate and a high c-reactive protein level, which mimics the response to bacterial infections. the decision to use antibiotics is determined by clinical and laboratory evaluation. in this study, antibiotics are used more often in cases of hmpv and adv, and they are also used more when infection in young infants who are potentially immune-incompetent, manifests as a lower ari. concerns of bacterial superinfection often cause us to continue the use of antibiotics, even after identification of a viral pathogen. application of a clinical rule without consideration of virus-specific clinical and laboratory characteristics may lead us to make a wrong decision. ) this study has some limitations. not all patients were examined for a bacterial etiology at presentation, and the use of antibiotics might affect bacterial detection. thus, it is possible that this study overestimates the number of viral aris. also, the rt-pcr assay used in this study did not quantify the viral load. it is still possible that even the single virus detected in a patient with a symptomatic ari might not be the causative pathogen. in conclusion, the epidemiology of predominant viral pathogens associated with aris among children differs seasonally as well as regionally. using routinely available hospital laboratory data will be a great addition to current virus surveillance and will help to identify the viruses circulating in the community and to predict their timing, trends and impact. ) in addition to viral identification, recognition of the virus-specific clinical and laboratory patterns presented in this study will expand our understanding of the epidemiology of viral ari and will help us to improve management and prevention of viral infections. respiratory virus infections aetiology of acute respiratory tract infections in hospitalised children in cyprus clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial respiratory pathogens in children with and without respiratory symptoms new respiratory viral infections the role of infections and coinfections with newly identified and emerging respiratory viruses in children etiology and clinical outcomes of acute respiratory virus infection in hospitalized adults detection of viral and bacterial pathogens in hospitalized children with acute respiratory illnesses viral etiology and the impact of codetection in young children presenting with influenza-like illness dual respiratory virus infections the male predominance in the incidence of infectious diseases in children: a postulated explanation for disparities in the literature prevalence of respiratory viruses among children hospitalized from respiratory infections in shenzhen, china clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis etiology, seasonality, and clinical characterization of viral respiratory infections among hospitalized children in beirut, lebanon clinical utility of pcr for common viruses in acute respiratory illness aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia fever and the thermal regulation of immunity: the immune system feels the heat epidemiology, clinical characteristics, laboratory findings and severity of respiratory syncytial virus acute lower respiratory infection in malaysian children role of viral respiratory infections in asthma and asthma exacerbations a molecular epidemiological study of respiratory viruses detected in japanese children with acute wheezing illness rhinovirus and the initiation of asthma the neutrophil-lymphocyte count ratio in patients with community-acquired pneumonia are there standardized cutoff values for neutrophil-lymphocyte ratios in bacteremia or sepsis? development and internal validation of a clinical rule to improve antibiotic use in children presenting to primary care with acute respiratory tract infection and cough: a prognostic cohort study a new laboratory-based surveillance system (respiratory datamart system) for influenza and other respiratory viruses in england: results and experience from no potential conflict of interest relevant to this article was re ported. key: cord- - caevjvh authors: falanga, annarita; galdiero, massimiliano; morelli, giancarlo; galdiero, stefania title: membranotropic peptides mediating viral entry date: - - journal: pept sci (hoboken) doi: . /pep . sha: doc_id: cord_uid: caevjvh the means used by enveloped viruses to bypass cellular membranes are well characterized; however, the mechanisms used by non‐enveloped viruses to deliver their genome inside the cell remain unresolved and poorly defined. the discovery of short, membrane interacting, amphipathic or hydrophobic sequences (known as membranotropic peptides) in both enveloped and non‐enveloped viruses suggests that these small peptides are strongly involved in breaching the host membrane and in the delivery of the viral genome into the host cell. thus, in spite of noticeable differences in entry, this short stretches of membranotropic peptides are probably associated with similar entry‐related events. this review will uncover the intrinsic features of viral membranotropic peptides involved in viral entry of both naked viruses and the ones encircled with a biological membrane with the objective to better elucidate their different functional properties and possible applications in the biomedical field. significant improvements have been achieved in recent years in the understanding of the multiple alternative ways of virus entry into susceptible cells. [ , ] the strategies employed by viruses to enter cells are different according to the presence or absence of a lipid bilayer surrounding the virus. enveloped viruses present a membrane bilayer while non-enveloped viruses lack this membrane and present on their surface only capsid proteins. the mechanism of cell invasion by the two groups of viruses is rather diverse and the foremost difference is the direct consequence of their distinctive physicochemical state at the interface that occurs at the time of the encounter between the virus and the cell membrane; in particular, two lipid membranes confronting each other in the case of enveloped viruses as opposed to a layer consisting only of proteins that face a lipid layer in the entry of naked viruses. enveloped viruses entry exploits direct fusion with a cellular membrane through the involvement of specialized viral fusion proteins, present on the viral membrane and the consequent transfer of the nucleocapsid into the cytoplasm. [ ] on the other hand, the entry of non-enveloped viruses, which lacking the outer viral membrane are unable to take advantage of the cellular mechanism of membrane fusion, involves the activation of viral lytic factors that induce cell membrane rupture. [ ] the major features in membrane interaction by enveloped and non-enveloped viruses are reported in figure . notwithstanding the different mechanisms of penetration, the key step is the entry process and the modification of the cell membrane which allows the viral genome to penetrate into the host cell and start the replication cycle in the appropriate cellular compartment. [ , ] the most critical barriers for viral penetration and replication are the plasma membrane, the cytoplasm, and any other membrane that needs to be crossed in order to have access to the sites where viral replication or assembly take place. the overall picture of the mechanism of viral entry is becoming increasingly complete; in fact, depending on their dimension and structure, they have acquired different strategies to penetrate and take control of cell functions. [ , ] the molecular details of the interactions at the interface of virus and cell surfaces are quite complex and highly variable, but there is a common idea that only a limited number of pathways allowing viruses to reach the sites of penetration exist, with enveloped and non-enveloped viruses presenting different and unrelated processes, but with general principles driving all fusion events. the main difference between fusion peptides of enveloped viruses and lytic factors of non-enveloped viruses is the fact that fusion peptides promote membrane fusion while lytic factors promote membrane disruption. in this review, we summarize current knowledge on the mechanism of membrane fusion of both enveloped and non-enveloped viruses with a focus on common principles. fusion peptide derived from enveloped viruses and lytic peptides from non-enveloped viruses are described with an effort to find undisclosed differences and similarities. we conclude reporting ground breaking applications of membranotropic peptides (both fusion and lytic peptides) which open a new exiting field of research. enveloped viruses depend on membrane fusion for cell penetration. [ ] the two main routes used by enveloped viruses to enter the cell are the endocytic and non-endocytic pathways. [ ] most enveloped viruses undergo endocytosis while only few are able to fuse directly to the plasma membrane. [ ] viruses that use the endocytic route for cellular internalization are able to escape into the cytosol avoiding lysosomal degradation. therefore, penetration invariably involves membrane fusion mediated by specific viral glycoproteins (catalysts) with the main difference being that viruses using endocytic pathways fuse their envelope with the endosomal membrane from the luminal side. [ ] thus, membrane fusion constitutes the essential and ubiquitous mechanism of entry of enveloped viruses, irrespective of their route of entry. [ , , ] viral fusion proceeds through a hemifusion stalk with merging of proximal leaflets, which culminates in the opening and expansion of a pore connecting the two sides of the membrane. [ ] the overall process is supported by a catalyst responsible of the lowering of the transition barriers and fusion proteins constitute the catalytic agents fulfilling this function. [ ] the entry involves several other critical steps which include cellular receptor or co-receptor binding, internalization, uncoating, and release of viral nucleic acids at the proper site of replication. [ , , ] various factors can mediate an efficient interaction between cells and viruses; for example, cholesterol rich domains are platforms for the entry of many enveloped viruses such as the influenza virus [ , ] and many viral membranes contain much more cholesterol than the mammalian plasma membranes from which they are derived. [ , ] viral fusion proteins undergo significant rearrangements from the pre-fusion to the post-fusion conformations which are triggered by either receptor binding, proteolytic cleavage or low endosomal ph, and eventually determine the exposure of previously sequestered hydrophobic peptides, loops, or patches, able to interact with and destabilize one or both the opposing membranes. [ , ] crystallographic data on pre-and post-fusion structures of viral fusion proteins has resulted in the identification of at least three distinct classes based on their three-dimensional organization and mechanism of fusion ( figure ). [ , ] class i fusion proteins includes many of the most studied human pathogens such as influenza virus, human immunodeficiency virus (hiv), and ebola virus; the key features is the requirement of a proteolytic cleavage to initiate fusion, [ ] activating these proteins which form an extended intermediate on the surface of the virion with the fusion peptide at the n-terminus of the protein engaged in the interaction with the target membrane and forming a link between the two fusing bilayers. the pre-hairpin structure is a unique state in which the fusion protein is simultaneously connecting two distinct membranes, the target membrane through the fusion peptide or patches and its own viral membrane through its transmembrane domain (tm). further conformational changes produce trimers of hairpins with a central a-helical coiled-coil structure. [ , ] the -helix formation is related to the opening of the fusion pore and provides the major driving force for the process. thus, peptides able to prevent -helix formation, interfering with refolding of the fusion glycoproteins act as potent viral entry inhibitors. [ , ] class ii fusion proteins, which include flaviviruses, alphaviruses, and bunyaviruses, are consistently different from those of class i and are mainly composed of b structures. [ ] these fusion proteins are often heterodimers or homodimers lying almost flat on the virion surface. [ ] they are organized in three globular domains: domain i is char- class iii fusion proteins have a mixed secondary structure with the central a-helical trimeric core similar to class i and two fusion loops located at the tip of an elongated b-sheet similar to class ii fusion proteins; the main representatives of class iii fusion proteins are protein g of vesicular stomatitis virus (vsv), [ , ] gb protein of herpes simplex virus (hsv), [ ] and of epstein-barr virus (ebv), [ ] and gp from insect-cell baculovirus. [ ] the post fusion structure is characterized by the presence of an internal fusion peptide in domain i organized in two hydrophobic fusion loops flanked by a b-sheet domain with a pleckstrin-like fold (domain ii). domain ii is nested with the largely a-helical domain iii, which is composed of trimers that give rise to an elongated, rod-like shape molecule. the a-helical domain is inserted in domain iv which is made of b-sheets and a very long c-terminal extension (domain v). interestingly, the multicomponent herpesvirus fusion machinery requires the presence of gb, gh/gl, and gd and multiple cellular receptors are engaged in the entry pathway in a cascade of molecular interactions. [ ] gh/gl and gb are part of the core fusion machinery and need to cooperate in order to trigger the initial lipid destabilization which culminates in the fusion of the two bilayers. [ ] [ ] [ ] despite the fact that it is still debated whether gh is merely a fusion regulator or it plays a more direct role in the fusion process, studies leave little doubt that also the gh/gl complex undergoes dynamic rearrangements during the fusion process. [ ] the crystallographic post-fusion structure of gb shows that it is a canonical class iii fusion protein; [ ] and several synthetic peptides derived from gb induce the fusion of large unilamellar vesicles and inhibit herpes virus infection. [ , , ] irrespective of their structural differences, the three classes of fusion proteins seem to induce membrane fusion by essentially the same generic mechanism and a common refolding pathway is highly suggestive for the conservation of several phases of the process. the non-enveloped viruses exploit a different mechanism for entry because they lack a membrane which surrounds the protein capsid; as a consequence they require capsid-dependent mechanisms for penetrating the cell membrane or for exiting from the endosome. [ , ] the entry process is much less known and involves a series of triggers producing conformational and structural rearrangements which end in the exposure and/or release of lytic factors. low ph, receptor interactions, protease cleavage, chaperone-assisted morphological changes, divalent cation chelation, or any combination of these factors which take place at the appropriate site of membrane penetration are essential triggers to produce the activated viral intermediate. [ ] the mechanism of membrane disruption involves proteolytic cleavage of the coat protein and programmed exposure/release of small membrane-lytic peptides. in this scenario, viral penetration is mediated by short, membrane interacting, amphipathic and/or hydrophobic sequences present in proteins undergoing a conformational modification which allows the exposure of these domains and their interactions with membranes. [ ] similarly to enveloped viruses, capsid proteins seem to be trapped in a metastable state waiting for a trigger to expose their membrane active peptides and the membrane penetrating ability of these sequences is fundamental for entry but at the same time their premature exposure has to be avoided before host cells provide the triggers. flock house virus (fhv) is one of the simplest and most studied non-enveloped viruses. the infection starts with the binding to one or more host cell receptors leading to a receptor-mediated endocytosis mechanism. [ ] the receptor binding induces a conformational change that initiates uncoating or viral disassembly once the particle is exposed to low ph within the endocytic pathway blocking this event often inhibits infection. the immature provirion fhv is initially assembled from copies of the coat protein alpha and, subsequently, it undergoes autoproteolytic cleavage to generate the mature infectious virion which contains a large n-terminal fragment, b ( amino acids), and a small c-terminal fragment, g ( amino acids). [ ] the capsid shell is constituted by the central region of b forms, while the g peptides are the amphipathic helices non-covalently associated with the capsid interior. the g peptides are responsible of the interaction with the membrane and its local disruption which ends in viral entry. these peptides are composed of an n-terminal amphipathic helix separated by a proline-glycine-proline turn from a hydrophobic c-terminal region. the amphipathic g helices are located in the interior of the capsid and when fhv enters cells through receptor mediated endocytosis, the g peptides are exposed and determine the disruption of the membrane with consequent release of the viral genome in the cytosol. [ ] this is a dynamic process with g peptides continuously, transiently, and reversibly exposed to the exterior of the capsid; g peptides may not only be exposed but also released from the virus particle which may represent a common paradigm of non-enveloped virus entry. in this dynamic process g peptides may continuously "sample" the environment until they encounter the appropriate cellular trigger; at this point the virus undergoes an irreversible conformational change in which the g amphipathic helices insert into the target membrane, allowing the viral rna to enter the cytoplasm. [ ] | m em b ra n otrop i c p ep ti d es many studies are devoted to the comprehension of the mechanism of insertion of fusion peptides, loops, or patches into monolayers inducing nipple formation and curvature in the target cellular membrane through many synergic interactions. the insertion in one leaflet of a closed bilayer will cause the increase of the surface area of the leaflet and the formation of a spontaneous curvature, which is one of the driving forces to reduce the energetic barrier needed for the achievement of fusion. [ ] fusion and/or membranotropic domains in viral fusion proteins contain aromatic residues which together with alanines and glycines [ ] contribute to the interaction with just one bilayer leaflet. many biophysical and structural techniques using synthetic analogues and model membranes have been used to determine physiologically relevant states during membrane partitioning. [ , ] the two structural motif widely found in fusion proteins and able to produce membrane curvature are the amphipathic a-helix and tilted peptides. [ , ] the segregation of hydrophobic and polar residues on the two opposite sides of the amphipathic a-helix is responsible of the superficial insertion into the upper monolayer which modifies the pack- similarly, also tilted peptides are characterized by an asymmetric distribution of hydrophobic residues, which again produces a modification of the organization of the membrane into which they insert. [ ] the membrane bound conformation of the influenza virus fusion peptide is characterized by the presence of a hairpin of two tightly packed, antiparallel a-helices; [ , ] the asymmetric insertion is determined by the fact that hydrophobic residues insert into the proximal membrane leaflet and polar residues project outward. the precise angle of the kink between the two arms depends on the sequence, the ph, and the lipid environment and determines the functional features necessary for activity. as a matter of fact, the compact hairpin structure drives favorable insertion, while its expanded structures promote subsequent membrane destabilization. [ ] canonical class ii and class iii fusion peptides correspond to loops which do not undergo conformational changes upon insertion into the target membrane; the interaction clearly involves few hydrophobic residues, and the insertion into the outer leaflet of the membrane is superficial and probably inadequate to destabilize membranes. thus, a cooperative effect attained upon fusion activation is the only explanation for activity. [ ] the idea of a single fusion peptide being exclusively responsible for the membrane perturbing activity has been overwhelmed by evidences supporting the concerted action of different membranotropic peptides, [ ] which together with the canonical fusion peptide are involved in the modification of membrane curvature. [ ] [ ] [ ] [ ] [ ] [ ] many viral fusion proteins present an additional hydrophobic membrane proximal region at the intersection between the ectodomain and the transmembrane anchor (tm), the so-called mper (or pre-tm); [ ] the unusual clustering of aromatic amino acid in these regions prompted the idea of their strong involvement in the fusion process. indeed, peptides corresponding to the pre-tm region partition into membrane interfaces and likely cooperate with fusion peptides and tm domains during apposition of membranes, enhancing the overall hydrophobicity of the environment and contributing to the distortion of the lipid membranes required for fusion. [ ] the pre-tm of hsv- gh interacts strongly with membranes; [ ] the pre-tm of gp of foamy virus induces fusion of model membranes; [ ] the aromatic domain of the glycoprotein s of severe acute respiratory syndrome virus (sars) partitions into lipid membranes and perturbs their integrity; [ ] the pre-tm region of the gp protein from ebola promotes perturbations of membranes when in a helical structure. [ ] hsv fusion involves both gb and gh and several membranotropic sequences are present in both glycoproteins, [ , ] although the precise role played by each of these regions remains to be elucidated. the two fusion loops at the tip of the domain ii of gb constitute a structural subdomain with the hydrophobic amino acids forming a crest lined on both sides by charged residues; [ ] the concerted use of the two peptides produced a significant distortion of the target membrane bilayer, while when they were used separately they presented a lower membrane penetration. [ ] the two charged residues represent a novel feature of fusion peptides with the presence of hydrophilic residues on either side favoring insertion. [ ] fusion of inner and outer monolayers is clearly involved but not formation of pores, indicating that bilayer perturbation not complemented by leakage is a typical feature of viral fusion peptides which violate the host membrane without compromising its integrity. [ ] several gh peptides are able to interact with membranes and play a role in the process. among these, gh , represents a key achievement in grasping the role of hydrophobic viral peptides. [ ] [ ] [ ] [ ] the peptide contains residues critical for interaction such as aromatic residues (tryptophan and tyrosines) which are known for their preferred location at the membrane interface and for their ability to facilitate oligomerization [ ] together with numerous hydrophobic residues (glycines, leucines, alanines) which are critical for membrane insertion; [ ] at the c-terminus there is an arginine residue which is key for establishing peptide-lipid interactions. gh penetrates into membranes from its n-terminal side, and assumes an amphipathic helical conformation. [ ] the n-terminal histidine acts as a switch for triggering viral fusion and strongly enhances the fusion activity; [ ] the role of the histidine has been reported also for paramyxoviruses [ ] and togaviruses. [ ] yao et al. [ ] in order to investigate how the fusion peptide (fp) and lipids also play a key role in this process, generating membrane curvature thanks to their physico-chemical properties. cholesterol is key as it selectively intercalates into the leaflet of the bilayer favoring its distortion without producing unfavorable hydrophobic/hydrophilic interactions. [ ] the secondary structure of the fusion peptide of hiv changes according to the cholesterol content in the membrane, being a-helical in the absence of cholesterol, but shifting to a b conformation with the increase of cholesterol. [ ] both the fully a-helical and the fully b-structured peptides are able to insert deeply inside the membrane, while the mixed secondary structures, present at intermediate cholesterol concentrations, are more superficially inserted into lipid bilayers and less effective in inducing membrane fusion. [ ] it is likely that different secondary structures and domains with different content of cholesterol might be involved in different stages of fusion. [ ] probably, lipid phase discontinuities between liquid ordered and disordered domains containing cholesterol produce membrane defects with exposed hydrophobic surfaces favoring a deeper insertion of fusion peptides, and promoting membrane fusion. [ ] in conclusion, the clear view is that membrane fusion is a very complex process involving several domains of the fusion proteins which interact directly or indirectly with biological membranes, and contribute to the merging of the viral envelope and cell membrane. some peptides (as the lytic peptides of nodaviruses, picornaviruses, and reoviruses) can be generated by an autocatalytic cleavage step of a precursor, whereas others can be generated from the proteolytic activity of cellular enzymes. [ , ] essentially we can classify lytic peptides in amphipathic a-helices and myristoyl groups; or we can classify them according to their mechanism of membrane action in those causing transient modification of the cellular membrane, pore formation, and total disruption of the limiting membrane. [ ] although being clearly different among themselves and with fusion peptides of enveloped viruses, they present notable similarities. incubation of hela cells with various peptides corresponding to the c terminus of the l protein of papillomavirus, determines the entrance of propidium iodide into cells. [ ] the incubation of the adenovirus internal protein vi with liposomes loaded with a fluorophore, caused the release of the entrapped fluorophore. [ ] similarly, incubation of vp * of rotavirus with liposomes entrapping a fluorophore caused its release, suggesting that vp * is sufficient to perforate the lipid vesicles. [ , ] the membrane disrupting g peptide of fhv also triggers the release of the fluorophore. [ , ] as for enveloped virus fusion peptides, the amphipathic a-helix seems to be a key structural motif also for non-enveloped viruses. the n-terminal amino acids of the fhv g peptide form an amphipathic a-helix which is commonly referred to as g , [ ] while the g peptide comprises also a c-terminal region which is commonly considered to play a supporting role for the correct positioning of g . the g peptide assumes a random coil conformation in solution, while it adopts a kinked helical conformation in model membranes; similarly to other membranotropic peptides also viral lytic factors seem to be able to adopt a membrane-active conformation when interacting with the lipid bilayer. [ , ] the g peptide is able to spontaneously partition into lipid bilayers and increases the membrane permeability of liposomes; [ , [ ] [ ] [ ] ] in particular, it mediates liposome lysis through the insertion only into the outer leaflet of the lipid bilayer, and locating parallel to the membrane surface, with the hydrophobic face of the helix packed against the membrane surface. [ , ] a concentration dependent membrane leakage process [ ] similar to other non-enveloped viruses such as poliovirus vp [ ] and reovirus l n [ ] is observed. similarly to the influenza [ ] and hiv gp [ ] fusion peptides from enveloped viruses, the amphipathic region of g peptide presents a kinked helical structure in solution. the rigid, boomerang like structure assumed by the influenza fusion peptide in lipid environment is required to promote membrane fusion; [ ] in fact, abolishing the kink in the structure or making it flexible eliminates membrane fusion [ ] probably for the failure of the fusion peptide to insert deep into the lipid bilayer and pack against the hydrocarbon moieties. at neutral ph the g peptide is able to cause membrane disruption; while at low ph it is only able to alter its location relative to the capsid, but does not increase its membrane interacting ability. [ ] surprisingly, thanks to a kinked structure and a tight alignment of the hydrophobic residues on one side of the peptide at low ph similar to influenza virus, fhv g peptide shows localized perturbation of lipid arrangements with no proof of pore formation. [ , ] while membrane destabilization requires the simple insertion of the fusion peptide into the outer leaflet of the lipid bilayer, leakage needs both a deeper insertion and an interaction between peptides inside the membrane. the amphipathic region of g peptide oligomerizes in bilayers [ ] with a low content of cholesterol, which being more fluid would promote association between peptides. [ ] the presence of the g c-terminus is absolutely necessary for virus entry; [ ] as a matter of fact, truncations, or point mutations in the c-terminal region of g determine a disordering of the pentameric bundle formed by the n-terminal amphipathic helices of g in fhv particles and hamper in vitro and in vivo membrane lysis. the pentameric bundles constitute the viral "membrane attack module," and an essential function of the g c-terminal region is to maintain this module in its correct conformation [ ] (figure ). the g and g peptides cause a similar localized disorder of the target bilayer and the presence of the cterminal hydrophobic helical region in full-length g make the peptide effective at concentrations achievable in the context of viral infections. the kinked helical structure seems to be a common trait of enveloped and non-enveloped viruses and differences in length and angularity of the helices as well as differences in the amino acid content may cause variations in the mechanism of interaction with the bilayer; the presence of this motif in viral proteins may signal a membrane associated role for this component during a certain step of the viral life cycle which adds to eventual other roles. adenovirus requires acidic ph for exposure of the amphipathic helix contained in its protein vi and disrupts endosomal membranes to release its nucleocapsid. [ ] mutations in the amphipathic helix reduce infection and endosome escape, supporting the view that both the hydrophobic character and a-helical structure are key to allow maximal membrane disruption. the n-terminal amphipathic helix of protein vi, as fhv g peptides, lies parallel to model membranes with the hydrophilic face interacting with the phospholipid head groups, and probably causes disruption of the bilayer by introducing positive curvature in figure schematic representation of fhv capsid (a). an expanded view of the crystallographic structure (pdb: ftb) of one subunit (a protein) showing the location of the amphipathic region of g peptide in yellow (b). schematic representation of a protein, which undergoes auto cleavage during maturation producing b and g (c) with relative sequence of g peptide membranes. [ ] mutations in the amphipathic region of protein vi, which prevent insertion into the membranes, severely affects membrane penetration and cellular entry. [ ] proteins vp and vp play a fundamental role in membrane penetration by poliovirus [ ] with the exposure of amphipathic a-helical nterminal approximately amino acid region of vp being necessary for liposome binding; [ ] while the n-terminus of poliovirus vp contains a hydrophobic myristoyl (c acyl) group for insertion into membranes. [ ] following receptor binding, the amphipathic a-helix within the n-terminal portion of vp , is exposed and probably forms pores to transfer the genome to the cytoplasm. interestingly, vp also performs a role in membrane penetration and the hypothesis is that vp primarily function is to secure the particle to the limiting membrane, while vp participates directly in pore formation. mammalian orthoreovirus protein l contains a small hydrophobic peptide (l n) with a myristoyl group at its n-terminus. [ ] disruption of cellular membranes, requires the complete dissociation of l n peptides from the particle; auto-cleavage of l during reovirus entry generates l n peptides that are linked to an n-terminal myristoyl group. after lipid association, the l n peptide changes conformation from an extended to a b-strand rich secondary structure. [ ] l n is able to generate size-selective pores in erythrocyte or liposomal membranes. it is likely that the b-hairpins in l n associate with the membranes forming a b-barrel pore. [ ] probably, membrane disruption caused by l n is similar to that of b-barrel toxins. [ ] it is highly probable that a future more detailed knowledge of the mechanism used by lytic peptides of non-enveloped viruses will lead to the discovery of more common features between enveloped and nonenveloped membranotropic peptides. membranotropic peptides thanks to their adaptness to interact with the membrane, are opening the way for numerous applications. [ , ] their ability to bind lipid membranes is correlated to their simultaneous hydrophobic and amphipathic nature, while their insertion into the bilayer is due to their capability to change conformation according to the environment; moreover, they are able to penetrate deep into the hydrophobic core but do not span the bilayer in a pore-like manner; on the contrary, they tend to self-associate at the interface between the membrane and the aqueous compartments. below are reported main applications. one of the main applications of fusion peptides of enveloped viruses is as inhibitors of viral penetration. their ability to directly interact with the hydrophobic surfaces present on cell membranes and/or fusion proteins allows them to interfere with virus entry. [ , ] the inhibition mechanism is still unclear but it is likely that inhibition of infectivity is correlated to inactive aggregates formed between the fusogenic stretches present in the viral protein and in the peptides. in particular, the formation of aggregates is related to their ability to oligomerize or to mimic the mode of binding of their original domains in their partner protein; thus, stabilizing a pre-fusion intermediate and preventing merging of the bilayers. [ , [ ] [ ] [ ] self-oligomerization of fusion peptides has been proposed to be responsible of inhibition by several groups. [ , ] hiv fusion peptides form structurally defined oligomeric complexes which have been considered responsible of inhibition; [ , ] moreover, mutants of the native sequence with a lower helical content and tendency to self-associate into b-sheets are able to inhibit membrane fusion with different magnitude and at various stages. [ ] virip is a peptide designed to target gp fusion peptide and thus block hiv- infection; it has undergone clinical studies and was demonstrated to be as active as peptides targeting the coiled-coil. [ , ] its clinical evaluation represents the proof of concept that membranotropic sequences could inhibit viral replication in infected individuals and may have potential clinical effectiveness. moreover, the knowledge that several domains are implicated in the fusion mechanism and may interfere with the intramolecular interactions between the several domains, clearly demonstrates that they all represent potential targets for the design of entry inhibitors. [ ] membranotropic peptides are emerging as delivery vectors. [ ] [ ] [ ] until now, the most widely used delivery vectors are cationic cell penetrating peptides (cpps), which enter essentially by endocytosis causing the entrapment of the cargo into endosomes with only minor quantities of the cargo able to reach the target where to exert the biological function. on the contrary, membranotropic peptides are internalized by direct penetration of the membrane and thus determine immediate bioavailability of the delivered molecule. fusion membranotropic peptides are particularly noteworthy because they can physically interfere with the membrane hydrophobic interior forming bulges that protrude from the membrane and ease contacts between fusing bilayers; in particular, they are able to translocate molecules through the plasma membrane directly into the cell, promoting lipid-membrane reorganizing processes, and causing local and temporary membrane destabilization with subsequent reorganization, circumventing the endosomal entrapment by favoring the escape from the endosome. [ , ] the internalization mechanism is also related to the toxicity of the internalized drug and the development of resistance. the gh is able to directly translocate across the membrane bilayer and to transport several cargos such as quantum dots, [ ] liposomes, [ ] dendrimers, [ , ] nanoparticles; [ ] it is also able to cross the bbb in vivo. [ , ] mpg is an amphipathic peptide composed of the fusion peptide of hiv- associated to an hydrophilic domain with positively charged residues derived from the nuclear localization sequence (nls) of simian virus (sv ) large t antigen (pkkkrkv), through a spacer (wsq). [ , ] in vitro mpg is able to deliver both sirna and dna after just h. [ ] the principal internalization mechanism was shown to be independent of the endosomal pathway and to involve the study of the internalization of g peptide derived from fhv [ ] revealed that this is mediated by relatively high cell surface adsorption leading to enhanced macropinocytic uptake and cytosolic distribution and also revealed a higher efficiency of internalization compared with tat. [ ] influenza virus fusion peptide has been used for increasing transfection efficiency, its ph-dependent fusogenic and endosomolytic activities are able to enhance lysosomal degradation before the contents of the endosomes are delivered to lysosomes. [ ] the development of industrial applications in drug delivery is probably one of the most exciting and fastest growing fields, with the possibility of these peptides to pass through the bbb and become an important player in the fight against all pathologies correlated to neurosciences. scientists envisage also a possible use of viral membranotropic peptides as an alternative to classical antibiotics in order to combat the antibiotic resistance problem. [ ] antimicrobial peptides (amps) are widely exploited and represent attractive candidates for the development of anti-infective agents; [ ] [ ] [ ] recently, attention has been devoted also to the exploitation of membranotropic peptides derived from viral fusion proteins as antibacterial drugs. in fact, some amps with helical structure seem to share high sequence (preference for alanines and glycines) and structure (amphipathic a-helix) similarity with fusion peptides and suggest a convergent evolution correlated to their ability to disturb lipid bilayers. [ ] the fusion domain of influenza virus was evaluated for its antibacterial activity; analysis showed that the amidation of the c-terminus is a key factor to render the fusion peptide an antibacterial peptide and optimization of the amphiphilic balance can improve efficacy. [ , ] the antibacterial activity of viral membranotropic peptides is not yet widely evaluated and much work is still open in this field; in particular, their mechanism of perturbation of membrane bilayers may allow the design of novel sequences with the ability to denature the membrane bilayer of bacteria which will add to their many roles. development of effective vaccines against viruses is another worldwide concern. a potent vaccine needs to be able to induce both [ ] in vivo prime-boost immunization enhanced humoral and cellular immune responses, suggesting the promising application of membranotropic peptides as vaccine candidates in future (table ) . the membrane entry of enveloped and non-enveloped viruses employs fundamentally different mechanisms, although common themes have emerged in the entry process. this similarity is essentially represented by the presence/exposure of small membranotropic peptides which cause membrane disruption and/or promote membrane fusion. entry involves membrane fusion versus perforation, but cellular triggering factors and structural intermediates appear to share some similarities. interestingly there is also some similarity with the mechanism used by bacterial toxins to cross biological membranes in order to reach the cytosol; in fact, many toxins, undergo conformational changes which allow them to initiate the translocation process. [ , ] how exactly both enveloped and non-enveloped viruses overcome host cell membrane barriers to deliver their genomes remains an intriguing problem. comprehensive structural and biochemical studies on enveloped viruses have brought to the conclusion that a unifying mechanism for host cell entry exists; where a membranotropic fusion loop, peptide, or patches catalyze fusion of the two membranes. in contrast, interaction of non-enveloped viruses with host cells during entry is less defined; while membrane active peptides have been discovered as necessary elements for entry in several well-studied non-enveloped virus capsids. in conclusion, it is now evident that the success of membranotropic peptides further stimulates challenging research on the unraveling of the many roles and applications that could be developed for both enveloped virus fusion peptides and small lytic peptides in nonenveloped viruses; membranotropic peptides are attracting increasing attention from the scientific community and their future will be dictated by the progresses in their industrial applications. http://orcid.org/ - - - entry of enveloped viruses into host cells: membrane fusion biophysical modulation of peptide-membrane interactions author biographies annarita falanga received the degree in food science and technology at the university of naples he earned his phd in virology from the university of cambridge (uk) in . he was appointed lecturer in microbiology in at the department of animal health of the faculty of veterinary medicine of the university of naples 'federico ii'. he moved to the faculty of medicine of the second university of naples where he was appointed associate professor and then full professor of microbiology giancarlo morelli is a full professor of chemistry at the university of naples 'federico ii she carried out research activities at the columbia university of new york in - . in she earned the position of assistant professor of inorganic chemistry at the university of naples and since she is associate professor. since , she is adjunct professor at loyola university chicago. research activities focus on antimicrobial peptides and drug delivery mechanisms. how to cite this article membranotropic peptides mediating viral entry key: cord- -pd elo l authors: luo, wei; gao, peng; cassels, susan title: a large-scale location-based social network to understanding the impact of human geo-social interaction patterns on vaccination strategies in an urbanized area date: - - journal: computers, environment and urban systems doi: . /j.compenvurbsys. . . sha: doc_id: cord_uid: pd elo l abstract cities play an important role in fostering and amplifying the transmission of airborne diseases (e.g., influenza) because of dense human contacts. before an outbreak of airborne diseases within a city, how to determine an appropriate containment area for effective vaccination strategies is unknown. this research treats airborne disease spreads as geo-social interaction patterns, because viruses transmit among different groups of people over geographical locations through human interactions and population movement. previous research argued that an appropriate scale identified through human geo-social interaction patterns can provide great potential for effective vaccination. however, little work has been done to examine the effectiveness of such vaccination at large scales (e.g., city) that are characterized by spatially heterogeneous population distribution and movement. this article therefore aims to understand the impact of geo-social interaction patterns on effective vaccination in the urbanized area of portland, oregon. to achieve this goal, we simulate influenza transmission on a large-scale location-based social network to ) identify human geo-social interaction patterns for designing effective vaccination strategies, and ) and evaluate the efficacy of different vaccination strategies according to the identified geo-social patterns. the simulation results illustrate the effectiveness of vaccination strategies based on geo-social interaction patterns in containing the epidemic outbreak at the source. this research can provide evidence to inform public health approaches to determine effective scales in the design of disease control strategies. infectious diseases (e.g., influenza) have posed great challenges to society. dangerous infectious diseases, such as severe acute respiratory syndrome (sars), anthrax, h n flu, and ebola pandemics, have been exacerbated by increasing human density and population mobility seen in contemporary society (anderson et al. ; fraser et al. ; leung et al. ) . infectious disease outbreaks can spread worldwide via international transportation networks more rapidly than the distribution of vaccines (ferguson, fraser, donnelly, ghani, & anderson ; webby & webster ) . a key public health question is how to contain infectious diseases at the source or reduce transmission long enough to implement an effective response (longini jr et al. ) . network science has been used to study infectious disease transmission and to design effective control (germann, kadau, longini, & macken ; halloran et al. ; longini jr et al. ) . traditional network models are individual-centric approaches in which nodes represent individuals and links represent physical contacts (meyers ) . disease simulations based on network models primarily focus on health outcomes (e.g., the number of infected cases) (mao & bian ; ), but do not sufficiently consider the role of location in disease transmission and control (zhong & bian ) ; for example, what are the critical locations in the transmission? are location-based control strategies more effective than individual-based control strategies? how do the spatial flows of population mobility among locations influence epidemics and control strategy design? those are all critical public health questions in terms of spatially informed policies for infectious disease control and prevention (bian, , gao & bian, . to tackle the above questions, this research treats infectious disease spread as geo-social interaction patterns, in which locations interact with each other via viruses that transmit from one place to another because of human interaction and population movement (gushulak & macpherson ) . geo-social interaction patterns identified from human mobility data within cities have the potential to provide valuable insight for designing effective epidemic control measures (guo ) . luo ( ) empirically found that vaccination strategies considering geo-social interaction patterns have a high probability to contain pandemics at the source within a primary school environment. however, human interaction patterns at an urban scale, which are characterized by heterogeneous population interactions and movement in space, have not been used to inform prevention strategies. the spatial heterogeneity of population interaction and movement make the choice of appropriate containment areas for effective vaccination strategies challenging. this research, therefore, aims to understand the impact of geo-social patterns identified from human movement and interaction data on effective vaccination in the urbanized area of portland, oregon. we use a location-based network framework in which nodes represent locations and links represent population flows among locations. the framework explicitly represents spatial dynamics of disease transmission from one location to another as well as design control strategies according to population flows among locations. based on the location-based network, we simulate influenza transmission dynamics and evaluate the efficacy of different vaccination strategies according to the identified geo-social interaction patterns. our research questions follow: ( ) given geo-social interaction patterns, how does population distribution and movement impact the effectiveness of influenza control efforts? ( ) how does the size of the containment area impact the control effectiveness considering human geo-social interaction patterns? the remainder of this article is organized into the following sections. the second section introduces related work in agent-based epidemic models and prevention strategies. the third section introduces location-based network model and agent-based epidemic simulation model in the urbanized area of portland. the one section that follow present and discuss the simulation results. the last section concludes the article and discusses its implications. infectious diseases are transmitted from one individual to another through physical contacts. in many agent-based epidemic models, the concepts of networks are used to represent contacts among individuals (bian ; bian & liebner ) . each individual can have attributes, such as infection status and spatial locations, whereas each link can also have a set of attributes, such as the duration of the contact and the infection rate. based on human interaction networks, epidemiologists can use agent-based epidemic models to simulate disease transmission and design different control scenarios. recently, agent-based models for disease transmission at different spatial scales (e.g., city, nation) have been used to study spatio-temporal patterns of disease spread (bian et al. ; ferguson et al. ; ferguson et al. ; halloran et al. ; mao & bain ) . these models predict that interactions at homes and workplaces could cause local transmission followed by a long distance transmission to an areawide epidemic (eubank et al. ) . local transmission indicates that early disease transmission is concentrated around the infection sources. later, population movement and distribution determine the spatiotemporal spread of infection. prevention strategies for infectious disease can be classified into three broad categories: antiviral, vaccine, and non-pharmaceutical measures (ferguson et al. ) . antiviral measures require rapid identification of early infections for treatment in order to reduce their infectiousness. vaccination seeks to protect people from being infected by other infections via physical contacts. non-pharmaceutical measures (e.g., case isolation, household quarantine, school or workplace closure, restrictions on travel) aim to locally contain the spread of disease (ferguson et al. ; ferguson et al. ) . previous research has shown the effectiveness of the above prevention measures in reducing disease transmission with agent-based modeling (ferguson et al. ; ferguson et al. ; halloran et al. ), but those agent-based modelings are individual-centric without capturing explicitly spatial dynamics of the disease transmission for designing control strategy purpose. spatial locations, one attribute of each agent, are treated as spatial stamps of the transmission process in the individual-centric agent-based modeling (zhong & bian ) . such representations suffer from a number of drawbacks regarding transmission and control design. first, the importance of locations in understanding disease transmission and control has been missing. for example, antiviral "ring chemoprophylaxis" strategies aim to geographically targeted containment via applying a certain distance threshold (e.g., km) by means of prophylaxis to attempt to prevent spread to unaffected regions (lee et al. ) . in fact, infectious disease transmission is the mixing patterns of geographical mobility and social interactions that are much more complicated than a simple distance threshold. second, the design of control strategies based on individualbased networks (e.g., vaccinate the individual with a large number of contacts) is challenging because it is infeasible to keep track of all social contacts of infections (cohen, havlin, & ben-avraham ; gómez-gardenes, echenique, & moreno ; holme ), but the information to estimate population flows among locations based on intra-and inter-community travelers is widely available in the existing census data and travel survey reports (mao & bian ) . thus, location-based human interaction network models are required to easily represent explicitly spatial dynamics of the disease transmission and design control strategies to target certain critical locations first rather than prioritizing individuals. based on a location-based human interaction network model, luo ( ) proposed a new framework in terms of an effective disease control strategy that consists of identifying geo-social interaction patterns first, following by designing effective control measures according to those patterns, and ending with control measure evaluation. the work also showed that vaccination strategies considering the geo-social interaction patterns can reduce disease transmission within a primary school environment because they can prevent disease from spreading to other classes. though the above research has demonstrated the importance of locations considering human geo-social interaction patterns in designing effective control scenarios, little research has been conducted to evaluate the effectiveness of such approach in a larger scale (e.g., city). thus, this research develops an integrated two-layer location-based social network approach to investigate the impact of human geo-social interaction patterns on effective vaccination design in an urban environment. we use a previously built and parameterized network from a synthetic population for a normal day in the city of portland, oregon, usa (eubank et al. ) to construct the integrated two-layer locationbased social network framework. one layer is a location-based network for the vaccination scenario design and the other layer is an individualbased social network to simulate infectious disease transmission and control to evaluate the effectiveness of those scenarios. each is detailed next. the synthetic population data includes over . million individuals and more than . million locations. each synthetic individual has their demographic attributes such as income range and age, whereas each synthetic location has utm coordinates. based on the individual and location information, the simulated daily activity data also includes the purpose of the activities such as work, school, and shopping, in which the starting time and duration of activity are stored in seconds (fig. ) . the data also include the information representing dynamic personperson contact networks including contact hours, contact type, and location information. the total number of person-person contacts is approximately equal to millions. the simulated data are configured with social surveys, transportation simulations, and census data in the portland city. the data set is available at http://ndssl.vbi.vt.edu/ synthetic-data/. human interactions and population mobility show spatial and temporal co-occurrence that connect discrete events over the same or different locations to facilitate disease transmission (zhong & bian ) . spatial and temporal co-occurrence is the basis of the construction of location-based human interaction network, in which nodes represent locations and edges represent population flows between locations. the total number of travel activities by individuals between two locations (population flows) is recorded as the weight of the edge (fig. ).the total population flows among locations are more than millions. a spatially explicit agent-based epidemic model is built on the simulated daily activities of person-person contact network in the city of portland, oregon, usa (eubank et al. ) (fig. ) . the influenza transmission is simulated over the contact network with each individual as one agent changing their infection states over time. following the natural history of the influenza, each individual can take one of four statuses: susceptible, exposed, infectious, or recovered (seir) at a given time (anderson & may ) . all individuals in our model are initially considered as susceptible to influenza. after contact with an infection, susceptible individuals have a probability of infection. those newly infected change their statuses from susceptible to exposed. exposed individuals may progressively develop their infectious statuses, which further enable them to transmit the influenza viruses to susceptible contacts. finally, infectious individuals recover from influenza. first, the exposed and infectious periods are from the established literature (table ) . second, the infection probability is calculated based on the r (the basic reproductive number). r is defined as the average number of secondary cases produced by a single infection over the course of its infectious period in a completely susceptible population (diekmann, heesterbeek, & metz ) . the transmission probability is calculated in order to generate consistent values of r (the basic reproductive number) corresponding to the observed r of pandemic influenza ( . - . ) in previous studies (ferguson et al. ; mills, robins, & lipsitch ) . we use a monte-carlo method to randomize the infection probability of individuals to derive r . all parameters to describe the natural history of the influenza are in table . we implement three vaccination strategies: random-based, degreebased, and betweenness-based vaccination strategies over the influenza diffusion in the city of portland, oregon, usa. the three strategies are the most representative ones to compare the effectiveness of different vaccination scenarios in the established literature (mao & bian ; . the basic idea of vaccination strategies is first to rank the importance of individuals according to their network measures (e.g., degree, betweenness) and then target the individuals from the highest network measures to lowest. the randombased strategy treats all of individuals the same and randomly identifies a certain number of individuals for vaccination. the degree-based strategy prioritizes individuals who have the most contacts within the network for vaccination. the betweenness-based strategy ranks the importance of individuals according to their betweenness centrality that can capture the extent to which a particular node lies on those shortest paths that pass through the nodes (freeman ) . both degree-based and betweenness-based vaccination strategies have shown much more effective than random-based strategy. for example, betweenness-based vaccination strategy tend to prevent disease outbreak from one community to another via targeting individuals on the shortest paths, so it becomes more effective with stronger community structures methodology framework consists of the integrated two-layer location-based social network: location-based network to design control strategy considering geo-social interaction patterns and individual-based social network to evaluate effectiveness of the above control strategy with the implementation of agent-based epidemic model. the framework is implemented in a high performance computing environment. scale-free network (albert, jeong, & barabási ) . though identifying all targeted individuals with high degree or high betweenness may not be practical, information needed to identify these individuals is becoming available (e.g., gps, social media) . using the location-based human interaction network, the vaccination strategy is designed according to population flows among locations with infections and their neighboring locations. neighboring locations that have direct connections with infected locations are most likely to be infected, followed by locations that have indirect connections with those infected locations. thus, the vaccination strategy will target those direct neighboring locations first, followed by targeting indirect connected locations. in practice, it is much easier to target a small population size with direct neighboring locations instead of a large population size with indirect connected locations. thus, this study explores the impact of the size of containment areas on vaccination efficacy in order to provide evidence in public health practice. the strategy is implemented at the beginning of the influenza epidemic. the simulation will stop when there are no new infections for six consecutive days (length of exposed period + length of infectious period). we randomly picked one infectious individual as the infection source in the study area on the first day of the simulation. based on the strategy rationale, we considered the locations where the first infection visited as the original case cluster. the original case cluster and all of the rest locations which have the population flows to the original case clusters were used to determine the first level local containment scale. the second level geo-social local containment scale was determined by all of the locations in the first level local containment area and the locations which have the population flows to the locations in the first level local containment area, so were the third, the forth, and the fifth level local geo-social containment scales. though the location network was dynamic over time in a day, the strategy design was based on the total population flows among different locations within one day for a practice purpose. it is more practical to design vaccination strategy at a daily basis instead of at an hourly basis. in order to understand the impact of the spatially heterogeneous population distribution and mobility on the design of vaccination strategies, we randomly pick the first infection times with varying total number of individuals in the first level local containment area (i.e., , , and ). the larger number of individuals within the first level local containment scale is caused by the high local population density which potentially leads to more complicated population interaction and mobility to larger geographical regions. given that the vaccination results are sensitive to the number of available vaccines, we simulate results according to an increasing number of vaccines from , to , with an increment of , . there is one network, three different initial infection locations with times' randomness, three strategies (e.g., random-based, degree-based), four different local control regions (from the second to the fifth level local containment area), and twenty different possibilities for the number of available vaccines, yielding ( * * * * * ) combinations to simulate ( table ). the efficacy of vaccination strategies for each combination is estimated for , simulation runs, resulting in a total of , , epidemic simulation runs. because of the large number of individuals and locations in the study area and the massive number of simulations involved, the tasks in this research are extremely computation intensive. we tackle this issue by taking the advantage of a high performance platform of cpu clusters. parallel algorithms are developed to deal with the most computation intensive tasks: ) the calculation of the betweenness centrality and ) the simulation of influenza epidemics. for the former, it is widely understood that the definitions of betweenness centrality in very large networks have inherently high computational complexity. the parallel algorithm is adapted from the brande's algorithm (brandes ) , and the actual running time is about hours using computing nodes, each with cores and gb of ram memory. for the latter, the parallel algorithm is developed following the seir rules as discussed earlier, and the running time ranges from to hours for a given first infection and vaccination strategy using the same computing nodes. the parallel design does not only reduce the running time but also improve the scalability of this methodology framework, which are essential for analyzing extremely large network data. we use two metrics to measure the effectiveness of vaccination strategies in the city of portland: the number of infections and the success rate of local containment. for the first metric, a better vaccination strategy is expected to generate a lower number of infected cases. for the second metric, we measure how well we can contain the outbreak at the source. if the number of infections outside of the containment areas identified by geo-social interaction patterns is zero, we consider the local containment successful. otherwise we consider the local containment a failure. fig. show the containment effectiveness of three typical vaccination strategies in which the first initial infections occurring within the first level local containment scale with approximately , individuals in the city of portland according to the two metrics: the number of infections and the success rate of local containment. fig. illustrates that an increasing number of vaccines can produce a decreasing number of infections. the random-based vaccination strategy generates the largest number of infections, followed by the degreebased and betweenness-based vaccination strategies. the latter two vaccination strategies produce the similar control efficacy in which betweenness-based ones lead to a relatively faster decreasing number of infections. the explanation of such patterns is illustrated in the fig. . fig. shows that an increasing number of vaccines can produce a decreasing number of local containment failure rates. in other words, betweenness-based vaccination strategies can contain disease outbreak at the local geo-social scales more successfully than the other two vaccination strategies. one reason is that betweenness-based vaccination strategies target high risk individuals who lie on the bridge among different communities, so they can control epidemics more effective through preventing epidemic outbreak from the local geo-social containment areas to outside areas. from fig. (a-d) , there is an obvious trend that increasing geosocial scales of local containment areas result in an increasing number of infections. fig. suggests that increasing extents of local geo-social containment areas lead to difficulty in containing outbreaks at the local scale. for example, in order to successfully contain the disease locally larger than % with degree-based or betweenness-based approach, it cost , vaccines in fig. (a) , , vaccines in fig. (b) , , vaccines in fig. (c) and (d) . in addition to supporting the importance of reliable detection of early infections to contain outbreaks at the source, such patterns also illustrate that identifying an appropriate scale for the local containment is key for designing effective prevention strategies. fig. (c) and (d) show that when the local containment areas are larger than certain extent, there is no obvious difference in terms of containment efficacy. it is because that the number of available vaccines cannot effectively contain disease outbreak at the local scale in fig. (c) and (d). this study further explores the impact of spatially heterogeneous population distribution and mobility on the containment efficacy of local geo-social vaccination strategies. this section only displays the simulation results with the random-based vaccination strategies, because the other two strategies display the similar patterns. fig. and fig. show the results of the two metrics: the number of infections and the success rate of local containment, respectively. they both display consistent patterns: the initial infections occurring in the higher population density areas can lead to higher number of infections (fig. ) because they increase the local containment failure rates with the same number of vaccines (fig. ) . for example, the disease transmission caused by the first infection from locations with a medium population density can areas be confined locally at a very high percentage (i.e., %) with less than , infections using , vaccines ( fig. b and b ), whereas the same amount of vaccines can only successfully contain the disease outbreak from high population density locations less than percent with more than , infections. such patterns imply that we need more vaccine available in order to contain disease outbreak from critical locations with high human interaction density and large numbers of population flows from infected locations to others. thus, it is essential to place sensors in the hubs of the locations to allow highly efficient outbreak detection. fig. and show that increasing scales of local geo-social containment areas lead to decreasing efficacy of the same amount of vaccines. for example, the random-based control scenario with more than , vaccines can successfully contain the disease outbreak in a high population density area (fig. a) , but it generates less than , infections (fig. b) and more than , infections (fig. c and d) . from the both (c) and (d) of the fig. and , we can tell that there is no obvious difference in terms of containment efficacy when the local containment areas are larger than certain scales in both (c) and (d). one explanation is that a certain number of vaccines have its threshold of susceptible population pool. the number of local control failure increases after the susceptible population pool goes beyond the threshold with the increasing local geo-social containment areas. when the disease outbreak occurs outside of the containment areas in the most simulation runs, the number of total infections finally reach similar amounts. we also notice that it is unlikely to confine the disease outbreaks caused by the first infection from high population density areas, if the local geo-social containment areas are set too large (fig. c and d ). when the first infection is from low population density areas, the likelihood to contain disease outbreaks within a large local geosocial containment areas is still very high ( fig. c and d) . thus, we can reach the conclusion that an appropriate geo-social scale for the local containment and the population density where the first infection resides are two keys for prevention strategies to achieve their maximum control effectiveness. based on the comparison analysis above, broader scales of local geosocial containment areas lead to decreasing efficacy of the same amount of vaccines. we further examine and compare their spatial effectiveness through infection density maps (fig. ) . we choose degree-based vaccination strategies with the medium number of individuals (approximately equal to ) within the first level local containment scale. we pick the number of vaccines equal to , for the second level local containment scale (fig. a ) and the number of vaccines equal to , for the third, forth, and fifth level local containment scales (fig. b, c, and d) because they all have the local containment failure rates lower than % (fig. ) . fig. (a) induces an extremely low intensity of infections in the whole study area with a relatively higher intensity of infections in the central business district of the study area. it is caused by the densest residential population and business locations in the central business district. compared to the fig. (a), fig. (b) with a larger containment area greatly increases the infection intensities with an additional dense area: the city of vancouver on the north bank of the columbia river. vancouver is the largest suburb of portland, oregon with highly concentrated population density. the spatial effectiveness of fig. (c) and (d) lead to the wide spread of influenza over the study area with three of highest infection density areas. the similar spatial patterns and total infections in both fig. (c) and (d) indicate that a certain number of vaccinations have its upper limit of susceptible population pool. when the local containment scale is larger than the upper limit, there is no difference in terms of spatial effectiveness. the spatial effectiveness of fig. highlights the importance of early detection of infections with an appropriate local containment scale, which is capable of confining the wide spread of influenza over the study area. this research examined the impact of geo-social interaction patterns on effective vaccination strategies in the urbanized area of portland, oregon. to achieve this goal, we built a large-scale two-layer locationbased social network model including an innovative location-based network to design vaccination strategies and an individual-based spatially explicit disease model to evaluate the efficacy of the above vaccination strategies. we implemented the network model in parallel algorithms to take advantage of a high performance platform of cpu to tackle the challenge of extremely intensive computation with millions of nodes and edges. the simulation results suggested that geo-social interaction patterns can be used to design effective vaccination strategies to contain epidemic outbreaks at the source. increasing extents of local geo-social containment areas lead to decreasing control effectiveness because it becomes challenging to confine the spread of influenza within local containment areas. especially when the local containment scale is larger than the upper limit, there is little difference in terms of spatial effectiveness. vaccination designs should consider spatially heterogeneous population distribution and movement in an urban area. it is possible to confine disease outbreaks for early infections in the low population areas, even if local geo-social containment areas have been set too large. it is unlikely to confine disease outbreaks for early infections in the high population areas if local geo-social containment areas are set too large. this study highlighted the importance of identifying the population density for the early infections and an appropriate geo-social scale for local containment in order to achieve maximum control effectiveness with a limited number of vaccines. a successful vaccine strategy must meet a number of key criteria: ( ) early detection of the original case cluster, ( ) rapid delivery of treatment to targeted groups, and ( ) effective delivery of treatment to high risk individuals (eubank et al. ; ferguson et al. ) . the identified geo-social interaction patterns and their effectiveness in designing vaccination strategies provide valuable insights for helping meet the above challenges. they can help identify critical individuals, locations, and clusters of locations for disease control purposes. traditional prevention strategies, including ring vaccination and contact tracing, can only capture one aspect of infectious disease transmission (i.e., spatial, or social) and both of which have significant limitations. "contact tracing" strategies require the identification of people who may have had contact with infectious individuals (germann et al. ) , which is difficult to implement in real situations considering the limited resources and time before the disease outbreak. "ring vaccination" with a simple distance threshold (e.g., km) (lee et al. ) is easy to implement, but it may fail to capture the most likely and complicated disease transmission processes. many research have demonstrated that ring vaccination alone might not lead to containment of disease outbreak with a high r at the source (kucharski et al. ; wells et al. ) , but a combined intervention of contact tracing and ring vaccination might contribute increase the ability to contain outbreaks of emerging infectious disease threats (kretzschmar, van den hof, wallinga, & van wijngaarden ; longini jr et al. ; merler et al. ; diao and wang, ; . the proposed geo-social interaction patterns cannot only address the inherent challenges of contact tracing to identify contacts with infections, but also capture the mixed interactions of the social and spatial relationships among individuals that determine infectious disease transmission (luo & maceachren ) . in addition to taking advantages from both contact tracing and ring vaccination strategies, the proposed location-based network approach provides the potential solution to address the challenge that it is infeasible to identify individuals with high degree or high betweenness for the disease control purpose in critical and timely situations. the wide available data (e.g., sensor networks, twitter) can provide more reliable estimation on the critical locations than critical individuals with high degree or high betweenness. thus, public health workers should focus on those critical locations with their close neighbor locations first in a timely manner. the proposed location-based social network framework also suggests several directions for future research. first, the estimation of population flows among different locations is still based on the existing census data and travel survey reports. gao et al. ( ) found that large-scale social media data can provide reliable estimates of regional origin-destination trips on weekdays compared with the community survey data in greater los angeles area. it shows great promise to build location-based social networks with large-scale social media data to provide real-time estimation and prevention strategy design in a timely manner. second, the location-based social network framework is built on population interaction and movement patterns in a normal situation, but people may adjust their behaviors accordingly during an epidemic outbreak. thus, human preventive behaviors caused by inter-personal influence (mao & yang ) and mass media could be incorporated to refine the framework. third, this research focuses on the impact of vaccination strategies on geo-social interactions patterns, but has not evaluated such impact of other prevention strategies such as travel restrictions and case isolation. forth, this research also has implications for the control of other location-based spreading pheonmena, such as invasive species , diao & wang , and hiv transmission (luo et.al ) . to summarize, the proposed location-based social network framework can help geographically optimize the design of prevention strategies before epidemic outbreaks, especially for novel viruses with limited resources and time constraints. to understand advantages and disadvantages of the framework in a full picture, it is crucial to apply it for other regions with different population distributions and movements as well as epidemiological settings. the author declares that he has no competing interests. this material is supported in part from the nih/nichd r hd . error and attack tolerance of complex networks epidemiology, transmission dynamics and control of sars: the - epidemic infectious diseases of humans: dynamics and control spatial approaches to modeling dispersion of communicable diseases-a review a conceptual framework for an individual-based spatially explicit epidemiological model modeling individual vulnerability to communicable diseases: a framework and design a network model for dispersion of communicable diseases a faster algorithm for betweenness centrality efficient immunization strategies for computer networks and populations development of an invasive species distribution model with fine resolution remote sensing temporal partial unmixing of exotic saltcedar using landsat time series incorporating plant phenological trajectory in exotic saltcedar detection with monthly time series of landsat imagery. remote sensing of environment landsat time series-based multiyear spectral angle clustering (msac) model to monitor the inter-annual leaf senescence of exotic saltcedar on the definition and the computation of the basic reproduction ratio r in models for infectious diseases in heterogeneous populations modelling disease outbreaks in realistic urban social networks strategies for containing an emerging influenza pandemic in southeast asia strategies for mitigating an influenza pandemic public health risk from the avian h n influenza epidemic pandemic potential of a strain of influenza a (h n ): early findings centrality in social networks conceptual clarification detecting origin-destination mobility flows from geotagged tweets in greater los angeles area scale effects on spatially embedded contact networks mitigation strategies for pandemic influenza in the united states immunization of real complex communication networks visual analytics of spatial interaction patterns for pandemic decision support population mobility and infectious diseases: the diminishing impact of classical infectious diseases and new approaches for the st century modeling targeted layered containment of an influenza pandemic in the united states control of communicable diseases manual efficient local strategies for vaccination and network attack ring vaccination and smallpox control effectiveness of ring vaccination as control strategy for ebola virus disease oseltamivir ring prophylaxis for containment of h n influenza outbreaks the epidemiology of severe acute respiratory syndrome in the hong kong epidemic: an analysis of all patients containing pandemic influenza at the source visual analytics of geo-social interaction patterns for epidemic control development of an agent-based model to investigate the impact of hiv self-testing programs on men who have sex with men in atlanta and seattle journal of spatial information science a dynamic network with individual mobility for designing vaccination strategies coupling infectious diseases, human preventive behavior, and networks-a conceptual framework for epidemic modeling agent-based simulation for a dual-diffusion process of influenza and human preventive behavior containing ebola at the source with ring vaccination contact network epidemiology: bond percolation applied to infectious disease prediction and control transmissibility of pandemic influenza immunization of complex networks dynamics and control of diseases in networks with community structure a high-resolution human contact network for infectious disease transmission are we ready for pandemic influenza? harnessing case isolation and ring vaccination to control ebola effects of immunization in small-world epidemics a location-centric network approach to analyzing epidemic dynamics support provided by the center for computational research at the university at buffalo. the authors appreciate shaohua wang's contributions to data processing at the early stage. the authors are grateful to the anonymous reviewers and editors, whose valuable comments and suggestions helped improve the quality of the paper. key: cord- -qnijw y authors: morgene, m. fedy; botelho-nevers, elisabeth; grattard, florence; pillet, sylvie; berthelot, philippe; pozzetto, bruno; verhoeven, paul o. title: staphylococcus aureus colonization and non-influenza respiratory viruses: interactions and synergism mechanisms date: - - journal: virulence doi: . / . . sha: doc_id: cord_uid: qnijw y viral infections of the respiratory tract can be complicated by bacterial superinfection, resulting in a significantly longer duration of illness and even a fatal outcome. in this review, we focused on interactions between s. aureus and non-influenza viruses. clinical data evidenced that rhinovirus infection may increase the s. aureus carriage load in humans and its spread. in children, respiratory syncytial virus infection is associated with s. aureus carriage. the mechanisms by which some non-influenza respiratory viruses predispose host cells to s. aureus superinfection can be summarized in three categories: i) modifying expression levels of cellular patterns involved in s. aureus adhesion and/or internalization, ii) inducing s. aureus invasion of epithelial cells due to the disruption of tight junctions, and iii) decreasing s. aureus clearance by altering the immune response. the comprehension of pathways involved in s. aureus-respiratory virus interactions may help developing new strategies of preventive and curative therapy. the development of upper and lower respiratory tract infections is determined by the interaction between one micro-organism and the host immune response. more recently, the interaction between the resident microbiota and incoming pathogens may also participate in the development of respiratory tract infections. it has been postulated for a long time that viral infection of the respiratory tract indirectly predispose to bacterial superinfection by disruption of the respiratory mucosal epithelium [ ], or passively through anatomical and mechanical changes like eustachian tube dysfunction [ ], ostiomeatal obstruction and reduced mucocilliary clearance [ ] . over the last decades, there was an increasing interest to investigate the contribution of bacterial colonization in the outcome of viral respiratory tract infections. the human respiratory tract is known to be the reservoir of diverse commensals and potential pathogens including mainly staphylococcus aureus, streptococcus pneumoniae and haemophilus influenzae that compose a significant part of the respiratory tract microbiota [ ] . the role of interactions between viruses and bacteria in the pathogenesis of respiratory infections have been extensively studied in the literature and notably those between influenza viruses and s. aureus or s. pneumoniae [ ] [ ] [ ] [ ] [ ] [ ] . however, interactions between s. aureus and non-influenza respiratory viruses were not reviewed recently. the aim of this report is to describe the current knowledge on possible interactions between s. aureus and non-influenza viral pathogens in the respiratory tract, with a focus on the mechanisms by which these interactions are potentially mediated. impact of viral infections in the respiratory tract on staphylococcus aureus colonization s. aureus is a commensal bacterium of the skin and mucosa, colonizing to % of the whole population [ , ] . the main reservoir of s. aureus carriage in humans remains the nose [ ] , but other sites of carriage have been reported, such as the skin [ ] , pharynx [ , ] , vagina [ ] , and rectum [ ] . s. aureus is however a janus-faced bacterium and beyond its commensal status, it is also a life-threatening pathogen. it is in fact considered to be one of the leading causes of nosocomial and community-acquired bacterial infections [ ] . in addition to be the most common cause of bacteremia, with a % mortality rate despite appropriate treatment [ ] , it is also known as an etiological agent of other deep-seated infections including osteomyelitis, septic arthritis, endocarditis and device-related infections [ , ] . s. aureus is also an important pathogen in lung infection, mostly implicated in hospital-acquired pneumonia [ ] . s. aureus expresses a wide repertoire of surface proteins that recognize cellular adhesive molecules and it is therefore able to adhere to and internalize into lung epithelial cells, which protects the bacteria from the host immune system and facilitate chronic infection [ ] . in vitro experiments have demonstrated that the activation of nf-κb (nuclear factor kappa-light-chain-enhancer of activated b cells) signaling pathway in infected pulmonary epithelial cells results in inflammation enhancement via il- expression; furthermore, intracellular s. aureus can lead, after an initial lag period, to the apoptosis of these cells. [ , ] in necrotizing pneumonia, the key virulence factors of s. aureus associated with the apoptosis of lung cells were shown to be pore-forming toxins, namely panton-valentine leukocidin (pvl) and alpha-hemolysin [ , ] . besides, s. aureus is frequently involved in secondary bacterial pneumonia occurring during seasonal influenza outbreaks [ ] . during the a/h n influenza pandemic, bacterial co-infections complicated up to one-third of influenza cases in the united states in which s. aureus was the most common pathogen, accounting for % of the cases in both critically ill children and adults [ , ] . s. aureus co-infection was associated with significantly higher morbidity and mortality [ ] . to date, the clinical association between the healthy carriage of s. aureus and the secondary staphylococcal pneumonia is still unclear. however, recent evidence from in vitro and in vivo tests showed that host physiologic changes induced by influenza virus can lead to the transition from asymptomatic colonization to invasive disease [ ] . in addition to influenza viruses, s. aureus nasopharyngeal carriage has been found to be associated with some other respiratory viruses like rhinovirus and respiratory syncytial virus (rsv). rhinovirus is the second respiratory virus that has been most frequently reported to interact with s. aureus. several studies showed that natural or experimental rhinovirus infection in s. aureus nasal carriers leads to increased s. aureus airborne dispersal, especially when sneezing is a part of the syndrome [ ] [ ] [ ] [ ] [ ] [ ] . these studies emphasized that rhinovirus infection may facilitate the spreading of s. aureus from staphylococcal carriers to their environment and the transmission of the bacterium between humans. a recent study showed that rhinovirus infection is associated with changes in the upper respiratory tract microbiota [ ] . in this study, healthy adults who were experimentally infected by rhinovirus showed increase in the relative abundance of h. parainfluenzae, neisseria subflava and s. aureus, and returned to their baseline level after the infection was cleared [ ] . it has been also shown that experimental rhinovirus infection significantly increases s. aureus nasal load by % compared to baseline bacterial load [ ] . these findings suggest that changes in the composition of respiratory microbiota following rhinovirus infection may play a role in the development of bacterial superinfection. however, the role of rhinovirus infection on the onset or increase of staphylococcal carriage in human remains poorly studied. a prospective microbiological analysis showed that % of children with severe rsv bronchiolitis had a bacterial co-infection in their lower airways and were at increased risk for bacterial pneumonia [ ] . bacterial co-infection in children with rsv bronchiolitis seems to increase inflammatory markers, abnormal radiologic patterns and hospital stay [ , ] . in young infants with rsv bronchiolitis, s. aureus, h. influenzae, m. catarrhalis and s. pneumoniae are the most common pathogens colonizing the nasopharynx and the lower airways [ , ] . a recent prospective study investigated the differences in the nasopharyngeal microbiome during acute respiratory tract infections due to human rhinovirus or rsv in infants aged less than months [ ] . by contrast to previous studies, s. aureus was not found among the most abundant bacteria. however, the difference of s. aureus abundance was significantly higher in rsv than in rhinovirus-infected infants [ ] . another prospective study investigated the nasopharyngeal microbiota in young infants with rsv infections by s-rna sequencing; the authors found that rsv infection was positively associated with h. influenzae and s. pneumoniae, but negatively associated with s. aureus nasopharyngeal colonization [ ] . in another study concerning nasopharyngeal aspirates from children with rsv infection aged between months and years, s. aureus was shown to colonize % of rsvinfected patients with a positive association between rsv and s. aureus nasopharyngeal carriage [ ] . the conflicting data from the two latter studies may be explained by differences in target populations, sampling procedures and/or microbiological methods. to date, most of the studies dedicated to the impact of rsv infection on the bacterial colonization of the upper respiratory airways identified a synergistic interaction between rsv and s. pneumoniae [ , ] . nevertheless, nasopharyngeal colonization with s. aureus is clearly more than a passive phenomenon during rsv infection and further studies are needed to elucidate the interactions between these pathogens. there is no doubt that the impact of infection with respiratory non-influenza viruses on s. aureus colonization is important and should not be neglected as this may worsen the disease outcome and even be fatal in some cases [ ] . the recent technical breakthrough of molecular diagnostic will be of precious help to investigate the changes in nasopharyngeal microbiota composition and notably in staphylococcal carriage during non-influenza viral infections of the respiratory tract. the high incidence of staphylococcal superinfection during pandemic and seasonal influenza and the important mortality risk associated to this condition promoted the in-depth investigation of the molecular and immunologic mechanisms that are involved in the bidirectional synergism between both pathogens. influenza virus is known to promote staphylococcal superinfection by alteration of the host immune system via increased production of pro-inflammatory cytokines and interferons (ifns), impairment of phagocytic cell functions and suppression of type immunity [ , ] . in addition, influenza virus has been shown to promote s. aureus adhesion and internalization within non-professional phagocytic cells through at least two distinct mechanisms: binding of bacteria to the membrane-associated hemagglutinin of influenza-infected cells, and binding of bacteria to free virions, followed by internalization of virus-coated bacteria into non-infected cells [ ] . besides, s. aureus coinfection promotes influenza virus replication and pathogenicity; indeed, extracellular bacteria secrete staphylokinase that facilitates the binding of influenza virus to the host cells, whereas intracellular s. aureus inhibits influenza virus-induced type i ifn signaling through impaired signal transducers and activators of transcription (stat and stat ) dimerization [ , ] . while the mechanisms of interaction between s. aureus and influenza virus seem to be deeply understood, the interactions between s. aureus and other respiratory viruses were less investigated. table summarizes the main mechanisms and pathways potentially involved in the interactions between s. aureus and non-influenza respiratory viruses. rhinovirus, the most common cause of upper respiratory tract infections (urti), primarily targets the nasal and nasopharyngeal epithelial cells [ ] . the innate immune system recognizes rhinovirus by different pattern recognition receptors (prrs) including membrane and endosomal toll-like receptors (tlrs) and cytoplasmic inducible rna helicases like retinoic acid-inducible gene- (rig-i) or melanoma differentiation-associated protein (mda ) [ ] . rhinovirus infection promotes proinflammatory cytokines and ifn production mainly through the activation of nfκb [ , ] . several potential mechanisms through which rhinovirus increases susceptibility to bacterial infection have been demonstrated in vitro in epithelial cells of the upper and lower airways. in , selinger, reed and mclaren developed an in vitro model for studying bacterial adherence to virus-infected epithelial cells [ ] . they reported that the adherence of s. aureus was significantly higher in rhinovirusinfected cells compared to uninfected cells. only recently, various in vitro studies have shown that inflammation due to rhinovirus infection increased cellular patterns that facilitate the adhesion and internalization of s. aureus within host cells [ ] [ ] [ ] [ ] [ ] . in primary human nasal epithelial cells, it has been demonstrated that rhinovirus infection up-regulates the expression of cellular fibronectin [ , ] . the upregulation of fibronectin was observed at both transcriptional and translational levels and seems to be related to the rhinovirus-induced nfκb activation the intercellular adhesion molecule (icam- ), which is the receptor of the major group of rhinoviruses [ ], have been found to be overexpressed during rhinovirus infection [ , ] . adhesion and internalization of s. aureus to epithelial cells is also mediated by ] . both immortalized pneumocytes (a cell line) and primary human nasal epithelial cells have been found to release higher levels of il- and il- when infected by rhinovirus and subsequently overexpressed icam- . the pro-inflammatory effect of il- and il- also induces an overexpression of icam- in the surrounding uninfected cells, which increases s. aureus uptake by the epithelial cells [ ] . the up-regulation of icam- and pro-inflammatory cytokines in rhinovirus-infected epithelial cells seems to follow the nfκb pathway [ ] [ ] [ ] . interestingly, the extracellular adherence protein (eap), an adhesin naturally secreted by s. aureus, was found to bind icam- [ ]. eap belongs to secreted expanded repertoire adhesive molecules (serams) and is involved in bacterial aggregation [ ], adhesion and invasion of epithelial and fibroblastic cells [ ] [ ] [ ] , and in preventing neutrophil recruitment [ , [ ] [ ] [ ] [ ] . in addition to icam- , eap can also bind a large variety of extracellular matrix and plasma proteins including collagen, fibrinogen, fibronectin, vitronectin, laminin, thrombospondin and prothrombin [ , , [ ] [ ] [ ] . taken together, it can be hypothesized that the ligation of s. aureus to icam- , whose expression is enhanced by rhinovirus infection, is mediated by eap. the mucosal barrier of the nasal cavity is the first site of exposure to inhaled respiratory pathogens and plays an important role in host defenses in terms of innate immunity. its integrity is regulated in large part by tight junctions of epithelial cells [ ] , which is a complex of several internal and membrane proteins including occludin and zona occludens (zo) proteins [ ] . rhinovirus infection disrupts the barrier function of the airway epithelium through the dissociation of zo- and occludin from the tight junction complex [ ] . rhinovirus infection has been shown to induce oxidative stress in respiratory epithelial cells by generating reactive oxygen species (ros) [ ] . ros generation is induced by double-strands of viral rna (dsrna) that appear transiently during rhinovirus replication, and is produced by nadph oxidase [ ] . in nonphagocytic cells, ros act as a molecular switch to stimulate pro-inflammatory responses [ ] . nevertheless, ros generation disrupts the barrier function of rhinovirusinfected cells [ ] . thus, a perturbation of the tight junction barrier function increases paracellular permeability, facilitates translocation of pathogens and their soluble products, and exposes basolateral receptors. it has been found that the infection of primary human airway epithelial cells with both major and minor groups of rhinovirus promoted the paracellular migration of s. aureus and its epithelium invasion [ ] . the rhinovirus infection facilitates also the transmigration of other bacterial species like h. influenzae and pseudomonas aeruginosa [ ] . this provides insights into another mechanism by which rhinovirus can predispose the host to secondary bacterial infections. interactions between rhinovirus and s. aureus have been shown to be bi-directional: whereas rhinovirus promotes s. aureus adhesion and internalization within host cells, s. aureus enhances the rhinovirus replication [ ]. s. aureus secretes several toxins including pore-forming toxins, toxic shock syndrome toxin and enterotoxins [ ] . in vitro, staphylococcal enterotoxins a and b (sea and seb) were shown to enhance the rhinovirus replication in a epithelial cells in a dose-dependent manner; however, they were found able to enhance neither icam- expression nor il- β, il- and il- secretion [ ] . in contrast, studies using other cellular models (normal human keratinocytes, coculture of human peripheral blood mononuclear cells and a cells, or primary nasal epithelial cell cultures) showed that these enterotoxins induced icam- expression or the secretion of pro-inflammatory cytokines [ ] [ ] [ ] . therefore, the mechanisms involved in the promotion of rhinovirus replication via staphylococcal enterotoxins need to be studied more in-depth. all these experimental data provide evidence of bidirectional synergism between rhinovirus and s. aureus. the different mechanisms described above are summarized in figure . nevertheless, the molecular pathways of these mechanisms remain poorly understood and deserve further investigation. rsv is a worldwide seasonal virus that affects mainly young children. rsv disease manifestations can vary from mild urti to severe pneumonia or bronchiolitis, which can lead to hospitalization and serious complications like respiratory failure [ ] . despite the high incidence of bacterial and rsv co-infection in both children and adults, only few studies aimed to investigate the possible interactions between s. aureus and this virus [ ] [ ] [ ] [ ] . rsv is supposed to exacerbate s. aureus co-infection via at least two mechanisms: enhancing the adhesion of s. aureus to the rsv-infected cells [ , ] , and decreasing the bacterial clearance by altering the immune response to s. aureus [ , ] . in vitro studies had shown that rsv-infected epithelial cells bind more s. aureus than uninfected cells [ , ] . study of nasal washings in patients with respiratory virus diseases has shown that secretion of lewis blood group antigens is associated with rsv infection [ ] . in addition, epithelial cells (hep ) expressing high concentrations of antigens of the lewis blood group bound significantly more s. aureus than cells expressing low concentrations of these antigens [ , ] . while rsv infects about % of infants by the first year of life, lewis antigen is expressed in secretions of nearly % of month-aged infants [ , ] . this may explain the high susceptibility of rsv-infected infants to secondary staphylococcal infection, sometimes resulting in lethal issue [ ] . nevertheless, the molecular patterns involved in rsv-related increase of s. aureus adhesion to host cells remain unknown. pro-inflammatory cytokines and type i ifns are known to be secreted by rsv-infected epithelial cells after viral sensing by different prrs, mainly through nfκb and interferon regulation factors (irfs) pathways [ ] . in addition, ifn-γ (type ii ifn) seems to play an important role in the immune response to rsv as it activates the t cell response [ ] . it has been demonstrated that rsv infection of human adult or neonatal mononuclear leukocytes results in significant inhibition of the lymphoproliferative response to heat-killed s. aureus [ ] . this inhibitory effect on the development of cell-mediated immune response could be due in part to the increased secretion of ifn-γ [ ]. ifn-γ was shown to reduce rsv replication in epithelial cells and also to inhibit b cell responses, which may alter the humoral response to s. aureus infection and decrease the bacterial clearance [ , ] . murine models had been used to demonstrate that, despite the increased number of inflammatory cells, rsv decreases the clearance of s. aureus and other pathogenic bacteria like s. pneumoniae and p. aeruginosa from the lungs of mice following secondary bacterial infection [ ] . it was suggested that rsv alters neutrophil function via changes in inflammatory response and cytokine secretion in the lung. further studies are needed to delineate the different immunological pathways that sustain the decrease of bacterial clearance and the increase of susceptibility to secondary infections by s. aureus following rsv infection. s. aureus interactions with rhinovirus or rsv have been actively, yet insufficiently, investigated. however, bacterial interactions with other respiratory viruses are poorly studied and only partial studies are available. for example, parainfluenza virus has been shown to enhance the ability of non-typeable h. influenzae and s. pneumoniae to adhere to human respiratory epithelial cells [ ] . however, there is no information available about the interaction of these agents with s. aureus in humans. only one in vitro study conducted on bovine embryonic lung cells investigated the effect of bovine parainfluenza virus infection on adherence of several bacterial agents, including s. aureus, and found no virus-specific effect on any of the tested bacteria [ ] . besides, normal human bronchial epithelial cells that were pre-incubated with s. pneumoniae resulted in an increased susceptibility to infection with human metapneumovirus. nevertheless, this was not the case for cells pre-incubated with h. influenzae, m. catarrhalis or s. aureus [ ] . on the contrary, synergistic effect between s. aureus and coronavirus has been demonstrated in vivo in a swine model. lipoteichoic acid from s. aureus increased the susceptibility to coronavirus infection in pigs via increased secretion of pro-inflammatory cytokines il- , il- , . to date, the lack of clinical and experimental data about the relationship between s. aureus colonization and other respiratory viruses complicates the understanding of potential interactions between these pathogens. while clinical and mechanistic aspects of the synergism between s. aureus and influenza virus have been deeply studied, the interactions between this bacterium and other common respiratory viruses were only partially investigated [ ] . literature still lacks more data about the relations between s. aureus carriage and non-influenza respiratory virus infections, as well as deeper insights into mechanisms of interactions between these different pathogens. the evaluation of the concrete risk of switch from commensal s. aureus colonization to invasive disease during viral respiratory tract infections may help us to propose efficient strategies of decolonization when needed. furthermore, the comprehension of the involved molecular pathways may help to develop new strategies of preventive and curative treatments. factor kappa-light-chain-enhancer of activated b cells; nkg d: natural killer group d receptor; nod : nucleotide-binding oligomerization domain-containing protein ; nox: membrane-bound nadph oxidase; rac : ras-related c botulinum toxin substrate ; rantes: regulated on activation, normal t cell expressed and secreted chemokine; rig-i: retinoic acid inducible gene- ; ros: reactive oxygen species; s. aureus: staphylococcus aureus; sea: staphylococcal enterotoxin a; seb: staphylococcal enterotoxin b; ssrna: single-stranded rna; tlr: toll-like receptor; zo- : zona occludens . no potential conflict of interest was reported by the authors. viral potentiation of bacterial superinfection of the respiratory tract alterations of the eustachian tube, middle ear, and nose in rhinovirus infection acute community-acquired sinusitis colonization and infection of the respiratory tract: what do we know? paediatr child health insights into the interaction between influenza virus and pneumococcus axis of coinfection evil immune dysfunction and bacterial coinfections following influenza severe influenza and s.aureus pneumonia the immunology of influenza virus-associated bacterial pneumonia influenza and bacterial superinfection: illuminating the immunologic mechanisms of disease the role of nasal carriage in staphylococcus aureus infections detection and clinical relevance of staphylococcus aureus nasal carriage: an update exclusive staphylococcus aureus throat carriage: at-risk populations does pharyngeal sampling improve the detection of nasopharyngeal persistent carriers of staphylococcus aureus? maternal vaginal colonisation by staphylococcus aureus and newborn acquisition at delivery epidemiology and clinical relevance of staphylococcus aureus intestinal carriage: a systematic review and meta-analysis staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management thirty-day mortality in uk patients with community-onset and hospital-acquired meticillin-susceptible staphylococcus aureus bacteraemia management of staphylococcus aureus bacteremia and endocarditis: progresses and challenges staphylococcal adhesion and host cell invasion: fibronectin-binding and other mechanisms staphylococcus aureus rn replicates and induces apoptosis in a pulmonary epithelial cell line cystic fibrosis pathogens activate ca +-dependent mitogen-activated protein kinase signaling pathways in airway epithelial cells staphylococcus aureus panton-valentine leukocidin causes necrotizing pneumonia poring over pores: alpha-hemolysin and panton-valentine leukocidin in staphylococcus aureus pneumonia critically ill children during the - influenza pandemic in the united states critical illness from pandemic influenza a (h n ) virus and bacterial coinfection in the united states host physiologic changes induced by influenza a virus lead to staphylococcus aureus biofilm dispersion and transition from asymptomatic colonization to invasive disease a cloud adult: the staphylococcus aureus-virus interaction revisited airborne dispersal as a novel transmission route of coagulase-negative staphylococci: interaction between coagulase-negative staphylococci and rhinovirus infection dispersal of staphylococcus aureus into the air associated with a rhinovirus infection gesundheit!" sneezing, common colds, allergies, and staphylococcus aureus dispersion preventing the airborne spread of staphylococcus aureus by persons with the common cold: effect of surgical scrubs, gowns, and masks coinfection can trigger multiple pandemic waves changes in microbiota during experimental human rhinovirus infection high incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (rsv) bronchiolitis nasopharyngeal bacterial burden and antibiotics: influence on inflammatory markers and disease severity in infants with respiratory syncytial virus bronchiolitis impact of bacteria in nasal aspirates on disease severity of bronchiolitis differences in the nasopharyngeal microbiome during acute respiratory tract infection with human rhinovirus and respiratory syncytial virus in infancy nasopharyngeal microbiota, host transcriptome, and disease severity in children with respiratory syncytial virus infection pathogen transcriptional profile in nasopharyngeal aspirates of children with acute respiratory tract infection hospitalization for respiratory syncytial virus infection and invasive pneumococcal disease in danish children aged < years: a population-based cohort study association between respiratory syncytial virus activity and pneumococcal disease in infants: a time series analysis of us hospitalization data epidemiology, microbiology, and treatment considerations for bacterial pneumonia complicating influenza viral hemagglutinin is involved in promoting the internalisation of staphylococcus aureus into human pneumocytes during influenza a h n virus infection interactions between bacteria and influenza a virus in the development of influenza pneumonia super-infection with staphylococcus aureus inhibits influenza virusinduced type i ifn signalling through impaired stat -stat dimerization viruses and bacteria in the etiology of the common cold rhinoviruses, allergic inflammation, and asthma: rhinovirus infections, allergy, and asthma human rhinovirus recognition in non-immune cells is mediated by toll -like receptors and mda- , which trigger a synergetic pro-inflammatory immune response co-ordinated role of tlr , rig-i and mda in the innate response to rhinovirus in bronchial epithelium model for studying bacterial adherence to epithelial cells infected with viruses rhinoviruses promote internalisation of staphylococcus aureus into non-fully permissive cultured pneumocytes staphylococcal enterotoxins a and b enhance rhinovirus replication in a cells rhinovirus enhances various bacterial adhesions to nasal epithelial cells simultaneously clarithromycin inhibits rhinovirus-induced bacterial adhesions to nasal epithelial cells levocetirizine inhibits rhinovirus-induced bacterial adhesion to nasal epithelial cells through down-regulation of cell adhesion molecules sticky connections: extracellular matrix protein recognition and integrin-mediated cellular invasion by staphylococcus aureus pathogenic bacteria attach to human fibronectin through a tandem beta-zipper fibronectin-binding protein acts as staphylococcus aureus invasin via fibronectin bridging to integrin α β an update on the pathophysiology of rhinovirus upper respiratory tract infections levocetirizine inhibits rhinovirus-induced icam- and cytokine expression and viral replication in airway epithelial cells rhinovirus infection induces expression of its own receptor intercellular adhesion molecule (icam- ) via increased nf-κb-mediated transcription role of nf-kappa b in cytokine production induced from human airway epithelial cells by rhinovirus infection staphylococcus aureus extracellular adherence protein serves as antiinflammatory factor by inhibiting the recruitment of host leukocytes adherence of staphylococcus aureus is enhanced by an endogenous secreted protein with broad binding activity insertional inactivation of eap in staphylococcus aureus strain newman confers reduced staphylococcal binding to fibroblasts extracellular adherence protein from staphylococcus aureus enhances internalization into eukaryotic cells the staphylococcus aureus extracellular adherence protein promotes bacterial internalization by keratinocytes independent of fibronectinbinding proteins the staphylococcus aureus map protein is an immunomodulator that interferes with t cell-mediated responses the adhesive and immunomodulating properties of the multifunctional staphylococcus aureus protein staphylococcus aureus interactions with the endothelium: the role of bacterial "secretable expanded repertoire adhesive molecules" (seram) in disturbing host defense systems the extracellular adherence protein (eap) of staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms extracellular matrix proteins (fibronectin, laminin, and type iv collagen) bind and aggregate bacteria evidence for three different fibrinogenbinding proteins with unique properties from staphylococcus aureus strain newman identification of a staphylococcus aureus extracellular matrix-binding protein with broad specificity regulation of tight junctions in upper airway epithelium the tight junction: a multifunctional complex rhinovirusinduced barrier dysfunction in polarized airway epithelial cells is mediated by nadph oxidase the role of oxidative stress in rhinovirus induced elaboration of il- by respiratory epithelial cells free radicals in the physiological control of cell function rhinovirus disrupts the barrier function of polarized airway epithelial cells staphylococcus aureus toxins possible influences of staphylococcus aureus on atopic dermatitis-the colonizing features and the effects of staphylococcal enterotoxins stimulant-dependent modulation of cytokines and chemokines by airway epithelial cells: cross talk between pulmonary epithelial and peripheral blood mononuclear cells proinflammatory effects of staphylococcus aureus exotoxin b on nasal epithelial cells respiratory syncytial virus hospitalization and mortality: systematic review and metaanalysis: incidence of rsv hospitalization and mortality respiratory syncytial virus modulation of adult and neonatal lymphocyte mitogenic responses and the role of interferon-gamma susceptibility to infection in relation to sids factors enhancing adherence of toxigenic staphylococcus aureus to epithelial cells and their possible role in sudden infant death syndrome decreased bacterial clearance from the lungs of mice following primary respiratory syncytial virus infection association between secretor status and respiratory viral illness an infectious aetiology of sudden infant death syndrome innate immune recognition of respiratory syncytial virus infection immunity to rsv in early-life respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species-and cell type-dependent manner lack of virus-specific bacterial adherence to bovine embryonic lung cells infected with bovine parainfluenza virus type streptococcus pneumoniae exposure is associated with human metapneumovirus seroconversion and increased susceptibility to in vitro hmpv infection lipoteichoic acid from staphylococcus aureus exacerbates respiratory disease in porcine respiratory coronavirus-infected pigs natural killer cells are involved in acute lung immune injury caused by respiratory syncytial virus infection key: cord- -zlah u s authors: günther, sonja; felten, sandra; wess, gerhard; hartmann, katrin; weber, karin title: detection of feline coronavirus in effusions of cats with and without feline infectious peritonitis using loop-mediated isothermal amplification date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: zlah u s feline infectious peritonitis (fip) is a fatal disease in cats worldwide. the aim of this study was to test two commercially available reaction mixtures in a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay to detect feline coronavirus (fcov) in body cavity effusions of cats with and without fip, in order to minimize the time from sampling to obtaining results. rna was extracted from body cavity effusion samples of cats, including samples from cats with a definitive diagnosis of fip, and samples of control cats with similar clinical signs but other confirmed diseases. two reaction mixtures (isothermal mastermix, optigene ltd.and pcrun™ molecular detection mix, biogal) were tested using the same primers, which were designed to bind to a conserved region of the fcov membrane protein gene. both assays were conducted under isothermal conditions ( °c– °c). using the isothermal mastermix of optigene ltd., amplification times ranged from and min with a sensitivity of . % and a specificity of . % for the reported sample group. using the pcrun™ molecular detection mix of biogal, amplification times ranged from to min with a sensitivity of . % and a specificity of . %. although the rt-lamp assay is less sensitive than real time reverse transcription pcr (rt-pcr), it can be performed without the need of expensive equipment and with less hands-on time. further modifications of primers might lead to a suitable in-house test and accelerate the diagnosis of fip. feline coronavirus (fcov), a member of the genus alphacoronavirus of the subfamily coronavirinae, family coronaviridae within the order nidovirales (de groot et al., ) , belongs to a group of enveloped, positive-sense rna viruses that cause diseases in several species, such as severe acute respiratory syndrome (sars) in humans or transmissible gastroenteritis (tge) in pigs. despite the high prevalence of fcov infections in the cat population worldwide, only - % of fcov-infected cats develop the fatal disease feline infectious peritonitis (fip) (addie and jarrett, ) . this change of virulence of a harmless fcov biotype that usually causes no clinical signs into the pathogenic variant is thought to be caused by mutations in the fcov spike protein gene (chang et al., ; vennema et al., ) . these mutations cause a change in tropism from enterocytes to macrophages, giving fcov the ability to infect and effectively replicate within cells of the macrophage lineage and cause a lethal systemic disease with multi-organ involvement (pedersen, ) . the median survival time of cats with effusive fip is only a few days (ritz et al., ) , and the diagnosis of fip commonly leads to euthanasia, since to date, no treatment has been proven to be effective. cats with fip show nonspecific clinical signs such as fever, weight loss and anorexia, often accompanied by body cavity effusions and/or ocular and neurological signs. a definitive diagnosis of fip ante mortem remains challenging, especially when no body cavity effusions can be detected (hartmann et al., ) . presently, the gold standard for the diagnosis of fip is considered to be immunostaining of fcov antigen in macrophages within tissue lesions, a technique that requires invasive tissue collection (kipar and meli, ) . in cats with fip, fcov can be detected by rt-pcr in cell-free body cavity effusions in more than % of the cases, while serum or blood samples often are negative (doenges et al., ) . for both immunostaining and rt-pcr, samples have to be sent to specialized laboratories, resulting in the delay of diagnostic results. this leads to further unnecessary testing for other diseases, to withholding necessary therapy of other treatable diseases, or to delayed euthanasia in cats suffering from severe signs of fip. therefore, a fast and simple point of care test would be very beneficial in the diagnostic process. loop-mediated isothermal amplification (lamp) is a simple, rapid, and cost-effective nucleic acid amplification method (notomi et al., ) and is already used for the detection of coronaviruses in humans and several animal species (hong et al., ; nemoto et al., ) . a set of four to six primers is used, that form products with self-hybridizing loop structures. by using a dna polymerase with strand displacement activity, no melting or annealing steps are required, and amplification products of different lengths are formed at a constant temperature of - °c (nagamine et al., ) . since lamp reactions only require a simple heat block with constant temperature, and dna amplification can be detected by fluorescence or color change, the method can be applied for point-of-care diagnostics (surabattula et al., ) . the aim of this study was to test specificity and sensitivity of two commercially available reaction mixtures in a reverse transcription lamp (rt-lamp) to detect fcov in body cavity effusions of cats with and without fip, and to minimize the time from sampling to obtaining results. this study included cats that were presented to the clinic of small animal internal medicine, lmu munich, germany. all cats included had body cavity effusions. in every cat presenting with body cavity effusions, fip is a potential differential diagnosis. an earlier study showed that fip is responsible for about % of effusions, while most of the remaining cases were caused by malignomas, cardiac insufficiency or purulent serositis (hirschberger et al., ) . the fip group (n = ) included cats with a definitive diagnosis of fip by one or more methods: all effusions of cats with fip tested positive for fcov by rt-pcr by a commercial laboratory, and in / samples putative disease-causing mutations could be detected. the rt-pcr detection method has been described previously (felten et al., ) . in / cats fip diagnosis was achieved by post-mortem examination, including full body necropsy with histopathological examination. diagnosis of fip was confirmed when typical histologic lesions where detected (surfacebound multi-systemic pyogranulomatous and fibrinonecrotic disease with venulitis with or without high-protein exudate). in / cats with full body necropsy immunohistological staining for fcov-antibody was done on tissue sections and returned a positive result. immunofluorescent staining of fcov antigen in macrophages of thoracic or abdominal effusion was done in / cats, and all samples returned a positive result. a summary of the cases in the fip group can be found in the supplementary table . cats were included in the control group (n = ) if they were definitively diagnosed with a disease other than fip that explained the effusion. cats of the control group suffered from neoplasia (n = ), decompensated cardiac diseases (n = ), inflammatory diseases (n = ), such as bacterial peritonitis and pleurisy, or other diseases (n = ). one cat had chronic thoracic chylous effusion of unknown origin and secondary fibroplastic pleurisy. in another cat, an end stage kidney disease caused effusion, and one cat had thoracic effusion after subcutaneous urethral bypass placement, which resolved after treatment. the diseases of the cats of the control group (n = ) were definitively confirmed ante-mortem (n = ) or at necropsy with histopathological examination (n = ). ante-mortem diagnosis was established by echocardiography for cardiac diseases (n = ), and by cytology for neoplasia (n = ). immunofluorescent staining of fcov antigen in macrophages of thoracic or abdominal effusion was done in / cats, with three positive and eight negative results. all effusions of the cats in the control group were tested for fcov by rt-pcr, and all results were negative. the rt-pcr detection method has been described previously (felten et al., ) . a summary of the cases in the control group can be found in supplementary table . body cavity effusion samples of all cats were obtained ante mortem with ultrasound guidance for diagnostic purposes. the use of samples for this study was approved by the institutional animal care and use committee ('ethikkommission des zentrums für klinische tiermedizin'), permission number - - - . samples were stored at - °c in a . ml eppendorf safe-lock microcentrifuge tube until assayed. all samples were centrifuged for s at , × g. the supernatant of centrifuged thoracic and abdominal fluids was used for rna extraction. when using fresh fluid samples, omission of the centrifugation step should be considered to include intact cells with a high viral burden (pedersen et al., ) . in thawed samples, cell integrity is lost and cell debris can be removed. viral rna was isolated using the commercial zr viral rna kit™ (zymo research corp.) following the manufacturer's instructions. briefly, μl aliquots of samples were mixed with a buffer that facilitates viral particle lysis and allows for rna adsorption onto the matrix of the zymo-spin™ column. then the rna was washed and eluted with μl of rnase free water. extracted rna aliquots were stored at − °c in an eppendorf . ml safe-lock microcentrifuge tube until further processing. the rt-lamp primer design was assisted by the software primerexplorer (https://primerexplorer.jp/e/). based on sequence analysis, the gene for the membrane protein (m) was selected as a target because it is highly conserved among fcov strains. the dna sequence from position , to , of the fcov strain black (genbank accession number: eu . ) was used to design the rt-lamp primers used in this study. a set of six primers, including two outer primers (forward primer f and backward primer b ), two inner primers (forward inner primer fip and backward inner primer bip), and two loop primers (forward loop primer loopf and backward loop primer loopb) were selected as the target sequence ( fig. and table ). detection of fcov was performed using rt-lamp. two different commercial reaction mixtures (isothermal mastermix by optigene ltd., uk, pcrun™ molecular detection mix by biogal, israel,) were compared using the same set of primers. for the amplification following the isothermal mastermix protocol, the total volume of μl per reaction tube included μl isothermal master mix, μl template, μl primer mix and . μl superscript® iii reverse transcriptase (thermo scientific). the primer mix consisted of pmol each of f and b primers, pmol each of fip and bip primers and pmol each of loopf and loopb primers. for negative control, μl water were added instead of μl template. the reaction mix was incubated at °c for min in a real-time pcr system (applied biosystems). during rt-lamp, fluorescence of dna products was measured once every minute (fam detection channel, λ max nm), and the time to threshold crossing was analyzed. a positive sample (positive in rt-pcr and sequenced for mutations) was included in every run. all samples run in the realtime pcr system were subjected to a melt curve analysis after the run. a single sharp peak in the melt curve analysis demonstrates amplification of a single pcr product. the positive samples all showed a single peak and the same melting temperature as the positive control sample. following the pcrun™ molecular detection protocol the reaction mix contained . μl of luminescent reagent, μl of template, μl of primer mix and additional . μl superscript iii reverse transcriptase (thermo scientific). the primer mix consisted of . pmol each of f and b primers, pmol each of fip and bip primers and pmol each of loopf and loopb primers. for negative control, μl water were added instead of μl template. the reaction mix was incubated at °c for min in a pcrun™ reader (biogal). amplification was detected by measuring bioluminescence twice every minute (fig. ) . pcr products of both methods were verified on an agarose gel showing a typical pattern of multiple bands of different molecular weights (fig. ) . the results of the samples in the fcov rt-lamp assays using two different commercial reaction mixtures are shown in table . two samples tested false positive using the isothermal mastermix and one sample tested false positive with the pcrun™ molecular detection mix. sensitivity and specificity for the reported sample groups of both rt-lamp assays are shown in table . the pcrun™ molecular detection mix performed better both in sensitivity and specifity than the isothermal mastermix. the amplification times for the isothermal mastermix positives ranged between and min and for the pcrun™ molecular detection mix positives between - min. in the present study, rt-lamp assays were evaluated as a diagnostic tool for detection of fcov, in order to distinguish cats with and without fip that are presented with body cavity effusions. time from sample to result was kept to a minimum by isolating rna in about to min using a simple rna extraction kit. both commercially available reaction mixtures allowed an easy and fast preparation of the amplification reaction. with lamp assays, direct detection methods built into the amplification device are preferred, since opening lamp reaction tubes after amplification is not advisable to decrease the risk of carry-over contamination (parida et al., ; zanoli and spoto, ) . in the present study, a portable device results was used for the pcrun™ molecular detection mix. positive and negative amplification reactions were indicated by the device with '+' and '-', making it compatible as a point-of-care instrument. the isothermal mastermix is intended for use with a portable device, which was not part of this study. the machine used instead replicates the reaction conditions with a constant block temperature and uses a comparable fluorescence detection system. both assays tested have similar demands concerning handling skills and preparation time. detection of positive samples took about half the time with isothermal mastermix compared to the pcrun™ molecular detection mix, yet both tests require less than min. the specificity of both methods for the reported sample group was comparable, with . % and . %, respectively. however, false positive results in a test that diagnoses a fatal disease are very critical and should not occur. a cross-reaction of the lamp-primers or carry-over contamination might be the cause of these false positive results. however, the negative controls without sample material did not show any indication for carryover contamination and stayed negative in the lamp assays. another possibility for false positive results is the detection of fcov in cats without fip, since systemic spread of fcov does occur, but does not inadvertently result in fip (porter et al., ) . this explanation is not very likely for the three samples of cats without fip that were positive by rt-lamp, since all three were negative by rt-pcr. the sensitivity of the pcrun™ molecular detection mix was superior for the reported sample group with . % compared to . % of the isothermal mastermix, using the same primers and pcr conditions. since the samples for the rt-pcr and for the rt-lamp had the same preanalytical treatment (frozen, thawed, and centrifuged), the results can be directly compared and showed that the rt-pcr for fcov performed much better than the rt-lamp in our sample group. this is in agreement with a study on other coronaviruses, where rt-lamp also exhibited a lower analytical sensitivity compared to rt-pcr (bhadra et al., ) . false negative results can occur in samples with a lower viral burden. the fcov viral load determined by rt-pcr in effusions has been found to be quite low in some samples of fip-suspected cats (lorusso et al., ) . in our study, the results for rt-pcr were only returned as 'positive' or 'negative' without quantification, leaving open the question whether only effusions with a high viral load resulted in a positive rt-lamp detection. another possible reason for the lack of sensitivity might be that sequences of current fcov strains show sequence variations compared to the sequences deposited in the genbank database, which were used to design the rt-lamp primers. although the primers were chosen to bind in highly conserved regions, variations can occur, which might impair binding and eventually lead to low or no amplification, resulting in poor sensitivity. reliable primers for rt-pcr target the ′ utr of the fcov sequence, but the rt-lamp primers that were suggested by the primerexplorer software in this region included more sequence differences than the m region that we selected. modifications of the primers might enhance binding and improve sensitivity. two studies on lamp-based identification of fcov have been published to date. a study from thailand tested samples of body cavity effusions from cats that were suspected to have fip both by rt-pcr and by rt-lamp. more samples tested positive with the rt-lamp than with the rt-pcr ( % vs. %) (techangamsuwan et al., ) . however, the inclusion criteria for cats to be suspected of having fip were not described in that study. their control samples consisted of plasma and fecal samples from healthy cats without any contact to other cats. the samples from this healthy control group also had more positive results by rt-lamp than by rt-pcr ( % vs %). the authors mention high rates of false positives in the negative controls (no template controls) when using rt-lamp, so it remains unclear whether their rt-pcr was less sensitive or their rt-lamp was prone to unspecific amplification. in the second study, different sample types of cats with a clinical suspicion of fip and fecal samples for screening for fcov-shedding cats were tested (stranieri et al., ) . in most sample types, including effusions, their rt-pcr had about twice as many positive results as their rt-lamp, and none of the rt-pcr-negative samples was positive in the rt-lamp method. in agreement with our findings, the sensitivity of the rt-lamp appears to be inferior to the rt-pcr. for detection of amplification, both studies used gel electrophoresis and one study (stranieri et al., ) additionally used detection of color change from violet to blue with hydroxynaphtol blue. while gel electrophoresis is quite time-consuming, the color change can be difficult to detect in samples with low amplification. amplification detection with fluorescence or luminescence as used in the present study is preferable, since the results can be read immediately and could be easily converted to a quantitative format with a standard curve. as a perspective for the future, rt-lamp reactions for virus detection could be run on new devices that integrate nucleic acid extraction, amplification and detection in a miniature format to achieve a true point-ofcare diagnosis (stumpf et al., ) . in conclusion, the rt-lamp in the present study was relatively specific but not very sensitive. the sample type was restricted to effusions of cats unequivocally diagnosed with fip and of cats without fip but with clinical signs indicative of fip. this is a realistic setting in which a veterinarian would use a test for detection of fcov in the effusion sample. the rt-lamp assay with the pcrun™ molecular detection mix can be used in a clinical setting to a certain extent. however, before it can replace conventional rt-pcr methods, sensitivity and specificity have to be enhanced by optimizing primers and amplification conditions. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the pcrun™ molecular detection mix and the pcrun™ reader were provided by biogal free of charge. biogal was not involved in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. the fcov rt-pcr and subsequent mutation detection was done by idexx free of charge. idexx was not involved in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. a study of naturally occurring feline coronavirus infections in kittens real-time sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (mers-cov) spike protein fusion peptide and feline coronavirus virulence virus taxonomy: ninth report of the international committee on taxonomy of viruses comparison of realtime reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis comparison of different tests to diagnose feline infectious peritonitis clinical symptoms and diagnosis of feline infectious peritonitis development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus feline infectious peritonitis: still an enigma? discrepancies between feline coronavirus antibody and nucleic acid detection in effusions of cats with suspected feline infectious peritonitis accelerated reaction by loop-mediated isothermal amplification using loop primers rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplification loop-mediated isothermal amplification of dna loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases a review of feline infectious peritonitis virus infection: - levels of feline infectious peritonitis virus in blood, effusions, and various tissues and the role of lymphopenia in disease outcome following experimental infection amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis reverse transcriptase loopmediated isothermal amplification for the detection of feline coronavirus labdisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza a h n virus simple, rapid, inexpensive platform for the diagnosis of malaria by loop mediated isothermal amplification (lamp) development and application of reverse transcription loop-mediated isothermal amplification (rt-lamp) for feline coronavirus detection feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses isothermal amplification methods for the detection of nucleic acids in microfluidic devices we thank biogal for providing the pcrun™ molecular detection mix and the pcrun™ reader for this study. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jviromet. . . . key: cord- - hf qw authors: romette, j. l.; prat, c. m.; gould, e. a.; de lamballerie, x.; charrel, r.; coutard, b.; fooks, a. r.; bardsley, m.; carroll, m.; drosten, c.; drexler, j. f.; günther, s.; klempa, b.; pinschewer, d.; klimkait, t.; avsic-zupanc, t.; capobianchi, m. r.; dicaro, a.; ippolito, g.; nitsche, a.; koopmans, m.; reusken, c.; gorbalenya, a.; raoul, h.; bourhy, h.; mettenleiter, t.; reiche, s.; batten, c.; sabeta, c.; paweska, j. t.; eropkin, m.; zverev, v.; hu, z.; mac cullough, s.; mirazimi, a.; pradel, f.; lieutaud, p. title: the european virus archive goes global: a growing resource for research date: - - journal: antiviral research doi: . /j.antiviral. . . sha: doc_id: cord_uid: hf qw abstract the european virus archive (eva) was created in with funding from the fp -eu infrastructure programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. within three years, it developed from a consortium of nine european laboratories to encompass associated partners in africa, russia, china, turkey, germany and italy. in , the h research and innovation framework programme (infras projects) provided support for the transformation of the eva from a european to a global organization (evag). the evag now operates as a non-profit consortium, with partners and associated partners from eu and non-eu countries. in this paper, we outline the structure, management and goals of the evag, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. organisations or individuals who would like to be considered as contributors are invited to contact the evag coordinator, jean-louis romette, at jean-louis.romette@univmed.fr. the european virus archive (eva) was created in with funding from the fp -eu infrastructure programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry (gould et al., ) . within three years, it developed from a consortium of nine european laboratories to encompass associated partners in africa, russia, china, turkey, germany and italy. following the h , call for research and innovation framework programme (infras projects) the concept of developing the eva into a global organization was a natural progression and the project was funded for a four-year period. in april , the evag started to operate as a non-profit consortium including partners and associated partners from eu and non-eu countries. based on excellent progress during the first two operational years, we have every reason to believe that the consortium will continue to expand and will ultimately constitute the world's largest virus collection. in this paper, we outline the structure, management and goals of the evag, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. laboratory isolation and characterisation of viral pathogens, such as vaccinia virus, poliovirus, foot and mouth disease virus, yellow fever virus and others, began at the turn of the twentieth century. this represented a pioneering era in the development of medical and veterinary virology. over the years, diagnostic and research centres were established worldwide to investigate viruses causing a wide range of diseases including those that had hitherto escaped all attempts to be identified and cultured under laboratory conditions. in the st century, ever-growing numbers of mammalian viruses continue to be isolated. they may represent only the tip of the iceberg, as more viruses are being discovered and reported using rapidly developing molecular technologies. as a result of these advances, the isolation, identification, characterisation and preservation of growing virus collections has inevitably generated new challenges. many of the known viruses are in collections that may be lost or discarded when the virologist in charge retires or when a laboratory closes or is reorganized. other collections may not be available to the wider virology community, because scientists are not always willing or in a position to share their viral isolates. they may lack the infrastructure, time (manpower), facilities and experience to characterize and present their collection in an accessible catalogue and further be able to ship their collected strains to foreign countries due to the restraints imposed by increasingly complex transportation regulations. furthermore, many governments have introduced additional security measures that specifically regulate the transfer and/or exchange, between countries, of high-risk-group pathogens that may attract the interest of terrorist organisations. additionally, laboratories in russia, china, india, southeast asia and south america hold their own virus collections, many of which, to date, have been relatively inaccessible to european laboratories. viruses from many taxonomic families have also been identified on the african continent and some well-developed laboratories in africa have made their collections readily accessible to researchers in europe and the americas. currently, however, except for a few countries, including senegal, south africa and kenya, authenticated virus collections from other regions of africa are virtually nonexistent. one objective of the evag therefore has been to establish trust among these widely disparate institutes and to associate the scientists in these countries with partners in the consortium under carefully monitored conditions that protect these developing laboratories, to the mutual benefit of everyone. the current project aims at the development of virus collections in low-income countries where, in some cases, there has been an understandable reluctance to exchange viruses for fear of losing their resource to institutes from more highly developed countries. the evag infrastructure provides assured access to modern capacities, thereby assisting these institutes to train their staff in developing their own resources and expanding and maintaining these capacities locally. end-user requests for viruses and reagents from these developing laboratories will be entirely managed from within, but supported by the quality management and distribution systems of the consortium. in common with all other members of the consortium, these developing laboratories will automatically retain full ownership of their collections. elsewhere, research laboratories often accumulate collections of viruses that are primarily dependent on the speciality of the laboratory scientists. for example, the american type culture collection (atcc) and the us centres for disease control and prevention (cdc) maintain large collections of mammalian viral pathogens, including risk group (rg ) agents. laboratories in russia, china, india, southeast asia, south africa, australia and new zealand hold collections that have been isolated specifically in their countries. relatively large collections of arboviruses are currently held in the usa (galveston national laboratory, texas), france, the czech republic, slovakia and also in some countries of scandinavia. european collections of specified animal pathogens, particularly lyssaviruses, pestiviruses, arboviruses and influenza viruses are maintained at the animal and plant health agency and at the pirbright institute in the united kingdom; in italy at the istituto zooprofilattico sperimentale delle venezie (izsve) hosting the world organization for animal health/food and agriculture organization (oie/fao) and eu reference laboratory for avian influenza; and in france at the institut pasteur. rg viruses such as ebola and nipah are maintained in specialised facilities in france, germany russia, the usa, china, south africa and the uk. diverse viruses of medical or veterinary importance (coronaviruses, herpesviruses, retroviruses, adenoviruses, enteroviruses, etc.) are held in laboratories around the world. as the evag continues to develop and to expand its boundaries, access to a greater proportion of these viruses will be enabled where possible. recently, in meeting the challenges of emerging viruses, the consortium has voluntarily oriented a part of its activities to the support of the world health organization (who) during emerging health crises by dedicating a new work-package entitled "response to emergence". moreover, the evag is currently an associated partner of the global outbreak alert and response network (goarn). current processes of coordinating virus standardisation, characterisation, preservation and distribution are inevitably relatively arbitrary, and are largely dependent on the speciality of each laboratory. in fact, besides the evag, we are unaware of any non-profit organization that is concerned with facilitating reliable access globally to viruses and associated reagents from individual virus collections for research and/or diagnostic laboratories, teaching centres or industries involved in the production of diagnostic reagents, pharmaceuticals and vaccines solely for the benefit of science, in a safe and carefully regulated manner. it would be virtually impossible to establish a single laboratory to maintain supplies of all recognised pathogenic mammalian viruses. based on the knowledge and experience gained from the former eva project, we have addressed these problems by using web-based tools to centralise access to quality-controlled, standardised preparations of viruses held in laboratories worldwide. the underlying concept is simple, but because virologists are naturally protective of their collections, its success has required a paradigm shift in the mind-set of the partners or associated partners in the individual laboratories. the h -infraia- - framework programme of the european union (eu) placed a call for funding applications under the heading "research and innovation action," with sub-headings, one of which was "research infrastructures." accordingly, the concept of the evag was created and a funding proposal was submitted. in preparing the proposal, it was emphasised that it would be virtually impossible to generate a wide-ranging virus archive in a single laboratory complex. thus, although many high-calibre virology laboratories operate in europe, it was decided to retain the "virology club tradition" by developing the principle of an integrated international infrastructure via consortia, operating through validated networks. as far as we are aware, this concept had not previously been encompassed by virologists in the context of producing and providing access to viruses. the evag was therefore conceived to fill the gaps in accessing viruses through identification of qualified international laboratories that could become partners of the consortium. it was agreed that by integrating their collections or resources and devising appropriate validated protocols and effective dissemination procedures, the partner laboratories would be able to achieve common high standards in producing and supplying authenticated viruses to the scientific community, both within and outside europe. the evag is unique. it was conceived to move beyond the current state of the art and provide access to the largest collection of mammalian viruses and associated reagents in the world to the global scientific community, to government health and environmental departments, to higher education institutes and to industry, through appropriate information systems. the new approach was, in the first instance, to integrate non-eu partners as beneficiaries of the project. this was possible because the rule of reciprocal benefit was agreed: the non-eu partner provision activity of material to an eu entity (research institute, health organization, animal health-care structure, etc.) was eligible for financial compensation from the consortium eu budget. consequently, highcalibre european and non-european partner laboratories agreed to become the primary members of the evag consortium. these are listed in table partner laboratories that form the core of the evag, listed in chronological order of their integration into the project. both medical and veterinarian institutes are included to cover viral zoonotic diseases. it also illustrates the wide geographic coverage already attained and demonstrates that bsl laboratories are major contributors to this consortium. the choice of institutes also reflects the specific available skills and know-how of scientists in institutes closely associated with the most well-known contemporary emerging viruses. we have also begun to integrate network organisations such as the global approach to biological research, infectious diseases and epidemics in low-income countries (gabriel) network, of the fondation merieux and the international pasteur institutes. future plans include expansion of partnerships to countries not currently represented, such as malaysia, vietnam, cambodia, indonesia, south america, and additional african countries. in addition, associate partners who share the same interest in the dissemination of viruses and reagents, but are not beneficiaries of the eu grant, have now been included (see table ). this association was formalized by the signature of a memorandum of understanding. the archive is founded on the principle that members and associate members of the consortium retain ownership of the viruses that they disseminate via the centralised distribution centre. the virologist or institution that supplies the viruses to the customer retains the ownership and enters into agreements with the customer via material transfer agreements (mtas). all orders for viruses and associated reagents (cell cultures, recombinant proteins, rna, dna, recombinant viruses, plasmids, monoclonal antibodies), are placed via the website https://www. european-virus-archive.com/which is managed centrally in marseille by the management team (partner# , aix-marseille university). during the early stages of the existence of the original eva, emphasis was placed on establishing the collections of viruses and associated reagents and ensuring that quality control was managed centrally. this ensured that all laboratories complied with identical standards. this principle has been further developed in the project. a manager was appointed to head up a management quality committee with the responsibility of establishing and maintaining appropriate standards through close monitoring of procedures, protocols and record maintenance. to ensure uniformity throughout the consortium, the quality managers representing each partner form the body of the committee. previously, non-eu participants were given the status of associate partners, i.e. they were associated with the consortium, but were not full members. the objective was to evaluate the feasibility of this approach concerning both the collaborative exchanges related to development of a common infrastructure and the operational effectiveness of resource distribution. from the point of view of scientific collaboration, the process was established rapidly and functioned efficiently. however, standardisation of the procedure for the distribution of materials to end-users proved to be challenging, and it is still being developed and improved within the current consortium. the reason for this slow progress is almost entirely due to the complexity of national regulations. whilst the european regulations retain the principle of being national and are enforced primarily to control the circulation of pathogens, they have been developed to maintain uniformity throughout the eu countries. thus, the administrative pathway to obtaining authorisation for an exchange of resources between, for example, france and germany or the netherlands and italy are very similar and are principally based on agreements provided by the national biosafety agencies. in contrast, the process of exchanging resources between the eu and russia or between the eu and the usa involves many more constraints: the intervention of different administrations in processing authorisation for delivery of hazardous goods including, biosafety agencies, customs administration, commerce and trade administration, ministry of health, etc. (also, the law in russia does not permit the import or export of pathogens.) nevertheless, our expertise in overcoming these hurdles is exemplified by the report of studies on the rg virus crimean-congo haemorrhagic fever virus in which the virus was supplied by a european partner to an end-user in the usa (haddock et al., ) . not surprisingly, when zika virus emerged in south america and was found to be responsible for microcephaly, we received numerous requests for zika virus, and many papers have been published with recognition of the evag as the source of virus. five examples encompassing, i) simultaneous transmission with chikungunya virus, ii) diagnosis, iii) signal processing pathways, iv) pathogenesis in relation to foetal infections and v) virus structure, are cited here (goertz et al., ; chan et al., ; zhang et al., ; miner et al., ; sirohi et al., ) . these anticipated problems of transporting pathogenic viruses between countries, particularly outside europe, were considered during the preparation of the project. each potential barrier to efficient exchanges between different countries was extensively discussed and product dissemination objectives for each non-eu partner were defined depending on their specific status. once established and operating satisfactorily, the management team continued the process of contacting high-calibre laboratories outside europe, organising meetings and conferences with these laboratories and subsequently inviting them to become associate partners. currently associate partnerships have been negotiated and agreed with laboratories in africa, russia, china, turkey, australia, japan, germany, italy, hungary, south korea, south east asia and south america ( table ). the initiation and early progress of the evag are already tremendous achievements, and subject to its continued success and expansion, it is anticipated that it will become the largest global network of laboratories contributing to the sharing of resources and promoting advances in virology. currently agreed associate partners in the evag. the integration of institutes that share the same interest in research and in the provision of high quality products to the scientific community is made through an association formalized by the signature of a memorandum of understanding. the choice of the associate partners has been established to fill gaps or to extend the list of viruses currently offered in the catalogue, and to have evag present in geographical areas concerned by emerging virus outbreaks. dissemination of resources including know-how and material to the scientific community is one of the major deliverable of the project. it has been addressed by the creation of web-based catalogue accessible on the evag portal. quality of the offer is a key factor to differentiate evag from the other infrastructures proposing similar products to the end-user. the gold standard products listed on the catalogue have been clearly at the origin of the fast growing dissemination activity of the consortium and of its robust reputation as reliable provider of key components for the detection of emerging viruses acting under the umbrella of international health organisations. the evag infrastructure provides wide-ranging and efficient access to virus collections held in laboratories worldwide, with the potential for access to recently isolated viruses from clinical, veterinary and field samples. the collection will continually expand, as the number of contributing laboratories increases and will provide participants with access to materials and capacities, including: • freeze-dried viruses for long-term storage; • viruses validated by sequencing, including the "rescue" and characterisation of archived material; • reagents and kits, including primer sequences and appropriate protocols for virus identification; • standardised diagnostic kits and recombinant proteins representing genes of the viruses, or hyperimmune antisera with defined specificities; • custom-prepared kits, proteins and antibodies; • high-quality associated research laboratories accessible to perform experiments and theoretical and practical training. relevant information can be obtained by placing an enquiry on the website (www.european-virus-archive.com/evag-portal). at the time of writing the catalogue shows more than products consisting of viruses and associated reagents. the list which is constantly increasing includes human, animal, insect, fish and plant viruses of medical, veterinary, academic, industrial and economic importance in addition, a range of arthropod tissue culture cell lines, monoclonal antibodies, recombinant protein expression systems, purified viral rna, specialised kits for diagnosis and custom-prepared proteins, can also be obtained through the website. the approach to quality management is directed by the project's own quality standard, based upon organization for economic cooperation and development (oecd) guidelines, and critically focusses on the acquisition, characterisation and storage of virus products. each partner institute seeks to comply with this way of working to harmonise laboratory processes across the consortium. the quality manager conducts "best practice" audits to quantify improvement progress. all facilities are inspected and regulated by their own national bio safety authorities, and employ staff trained in procedures compliant with the requirements of the international air transport association (iata) for the safe dispatch of potentially hazardous materials. trans-national access (tna) ensures free of charge access to european research infrastructures and the costs of the research, products and associated activities. this opportunity is open to all european researchers and to some extent to researchers from non-eu countries. tna dispatches include regulatory documentation such as import/export permits, and a certificate of analysis (coa) provided by the supplying partner. the evag project has established a grading system that defines virus product quality ranked from to , in which grade is the basic level, described as partially sequenced to confirm identity; not checked for mycoplasma contamination; unknown infectivity; and stored frozen. the "gold standard" is a fully sequenced virus, mycoplasma-free, with defined infectivity and lyophilised for long-term storage. derived products such as proteins, nucleic acids or antigens are also prepared using standardised protocols and checked for quality before storage or dispatch. security at the consortium institutes is maintained using cctv, pincoded passes and at remote sites, -h manned security/vehicle identification. entry into bio containment laboratories is strictly controlled. supply of products to the applicants operates via the tna approach defined above, whereby research groups apply through the web portal by submission of a completed request form. a successful application must be justified by an explanation of scientific objectives, techniques to be utilised/developed, list of named researchers, list of relevant publications and confirmation that appropriate expertise and facilities to handle the virus are in place. a review of these criteria is carried out by the project quality manager and a selection panel that includes external experts. a team of scientists and administrators is dedicated to the management of the evag, to ensure continuity and to maximize efficiency. whilst the individual laboratories operate through the web-based catalogue, they supply to the end-user only the viruses and products that are held in their collections. the website is the main communication tool with which to inform the end-user and to disseminate the products through the dedicated portal. the portal is conceived as a user-friendly marketplace that lists the viruses and associated reagents of each partner. this web-based catalogue constitutes a single entry point to the archived viruses and reagents. it provides access for end-users to place any enquiry about products of interest through a commonly used e-shop format. any end-user could be entitled to free access to the virus or associated reagent(s). the provider of the requested item(s) receives financial compensation from the eu budget. eu support for free-ofcharge access provided to the end-user has as its main objective to facilitate access of the scientific community to high quality resources. however, this is conditional on the excellence of the project which the end-user aims to develop using the resource. an international panel, consisting of independent experts, reviews the request for free access and subject to approval the customer will receive the item without charge, other than covering the cost of transportation. in the case of a charge being required to supply the virus or reagent, the evag only recovers the cost of production and transportation to the customer. free-of-charge access to end-users employed outside eu-member or euassociate states is limited to % of the total accesses provided in the grant. in addition to core partners, the project also integrates associate partners as an efficient means of enlarging the collection of viruses and reagents in the web-catalogue. easy and efficient access to the viruses and reagents and to the service offered to the scientific community is attracting increasing numbers of requests for the products in the catalogue. in addition, the quality of the products distributed and the assistance provided by the management team to answer specific questions from scientists are establishing the evag as a key biological resource centre worldwide. it is now a recognised entity acting under the umbrella of medical and veterinarian international health organisations which include the who, goarn, oie and the major centres for disease control and prevention around the world which underpin the control of emerging viral diseases. to place an enquiry and receive a quotation for a product, open a web browser and type in "evag." then click on login/register to create an account. the online catalogue can then be accessed via the portal link on the home page. products in the catalogue can be found by typing in a keyword/and/or the appropriate taxonomic name. the filters on the right hand side of the portal can also be used to browse through the products by category. the product(s) of interest should then simply be added to an enquiry cart, and the enquiry form should be completed. a material transfer agreement (mta) and a quotation for the products and shipping costs will then be sent to the end-user. the enquiry then becomes an order when the quote is accepted and the mta is signed. all enquiries are evaluated in terms of biosafety, and an official (signed and stamped) end-user qualification form of the laboratory to receive and handle the products of interest is requested. the emergence of sars at the end of has clearly demonstrated the lack of preparedness of countries to face emerging virus outbreaks. when the concept of eva was discussed in , this aspect of the consortium activity was clearly stated in the project submitted to the european commission. this was reinforced in the second project: evag, submitted in , with a contingency fund dedicated to support this activity. the emergence of the novel mers-cov in humans in saudi arabia in provided an opportunity for consortium partners and members of a european epidemic response network to develop a precise and rapid mers cov diagnostic tool, suitable for field diagnosis under emergency conditions (corman et al., ) . during december , the who recommended pcr assays as the method to detect the virus in blood samples, and identified the evag as a reliable source for the delivery of positive control reagents (who, ) . to date more than kits have been distributed worldwide, to laboratories in countries. this achievement demonstrated the capacity of the consortium to mobilize high-calibre scientists capable of rapidly developing and supplying a technical solution to a high-level emergency situation involving human pathogenic agents. another key feature of this achievement was the efficiency of our logistics platform to distribute the material worldwide under the demanding conditions prevailing at the time. zika virus emerged in french polynesia and spread rapidly to south america and the caribbean, causing at least , clinical cases, within the first year according to paho/who estimates. on the st of february , who declared a public health emergency of international concern (pheic) (see ref ). on the th of february, the us centre for disease control (uscdc) elevated its response to "level activation" i.e. the highest level. prior to may , there were two strains of zika virus in the evag online catalogue. by the first trimester of , at the peak of international concern, partners had added eight more products of direct relevance to the emergency situation. since november, , the evag has received enquiries for more than products to be distributed in countries. a total of products were provided in february, alone, with the highest demands in the usa, singapore, netherlands, germany, france and china. in its interim guidance for zika virus laboratory testing (march , ), who identified the evag as the provider for pcr quality-control material (corman et al., ) . this emergency situation and the resulting need for a coordinated distribution logistic was very challenging. consequently, the management team introduced procedures to alleviate the burden on partners and to improve product access. this was achieved by establishing additional distribution hubs for zika virus products obtainable from: the centre for molecular diagnostics and therapy (crie), moscow, russia; wuhan institute of virology (wiv), wuhan, southern china; national institute of infectious diseases (niid), tokyo, japan. the management team also negotiated a common material transfer agreement (mta), with industry on behalf of all consortium members and a centralised exchange logistics platform was established. this experience underlines the reality that anticipation and preparedness are key components for an effective response to public health emergencies. from december, through may, brazil reported its largest yellow fever (yf) outbreak in decades, with a total of suspected cases and deaths (fischer et al., ) , prompting widespread yellow fever virus (yfv) vaccination campaigns and the need to distinguish between vaccine-and wild-type yfv-associated disease. novel multiplex real-time reverse transcription pcrs that differentiate between vaccine and american wild-type yfv were developed by consortium partners in response to this outbreak and validated under field conditions (fischer et al., ) . centres for disease control (cdc) in countries play an important role for the control of virus outbreaks by the collecting data and samples during the identification phase of the pathogens. however the academic research combined with material collections appears to be an unavoidable actor for the preparedness and the control of an emerging virus disease, each time a variant form or a new form of a pathogen is responsible of it. combining, in the same organization, cdc and high calibre research laboratories was foreseen as the best solution to reach both an efficient control of the virus propagation and a preparedness for the next outbreak. at the same time, to cover the all range of pathogens from rg to rg was only made possible by the constitution of a group of high containment facilities within the organization. sixteen registered biosafety level- (bsl- ) laboratories, with the resources to handle the highest risk hg pathogens and five cdcs are partners or associate partners in the consortium. each cdc contributes directly to the management and control of emerging virus diseases, under the umbrella of the who. the evag is also associated with the global outbreak alert and response network (goarn) and is recognised by goarn as a supplier of high quality viruses and virusrelated reagents and resources. during the - ebola virus epidemics in west africa, all of the bsl- institutes were directly involved in helping local authorities to contain the epidemic. they all gained important experience in the management of such health crises, collecting field samples and isolated relevant virus strains many of which are now characterised and stored in the facilities of the evag bsl- partner at the bernard nocht institute in hamburg, germany. in addition, they addressed specific issues involving management of the exchange of high risk pathogens and activation of platforms dedicated to offering services to appropriate endusers. the consortium is now in its third year of existence under the h framework programme. its ultimate objective is to become a permanent archive that provides access to a very wide range of viruses and reagents globally. this will be achieved firstly, through extension of the funding arrangements, secondly through further increasing the range of contributors to the collection, and thirdly by bridging large european or international infrastructures having activities related to those of evag, including: it would be naive to suggest that the long-term survival of the consortium will be straightforward. for example, its perpetuation will require a continuous and increasingly large funding stream to meet the almost restrictive costs of research, laboratory development and upkeep in the face of newly emerging highly pathogenic viruses. sustainability is a key issue for such an infrastructure and the consortium plan will be to remain a non-profit organization. the obvious consequence of this fundamental choice is to have access to diversified sources of funding. the nations to which partners belong will have to support the collection activities including infrastructure management, as well as research concerning the development of new tools necessary to keep the archiving process up to date. the financial support for all the actions involved in preparedness for emerging viral diseases e.g., distribution of bio-resources to supply reagents to detect infected patients at local level will have to come from government health departments but at the international level, from the european commission, charitable international health funding agencies such as the wellcome trust foundation, the bill and melinda gates foundation, the who, the oie. future financial planning also includes the creation of a specific interface between the industrial sector and the consortium. in the first instance, this interface will explore bi-directional opportunities to organize scientific exchanges, priority access to bio-resources including reference material development of diagnostic services, validation of diagnostic assays and commercialization by industrial partners to the benefit of both parties. signed contracts of collaboration will be facilitated, and procedures harmonized. as noted above, the concept of the evag is unique. as far as we are aware, there is no equivalent viral archive, nor does any other collection provide the accessibility, reagent backup, sequence data, provenance, quality control, and capacity to advertise, inform, negotiate and conduct the entire transaction to the end-user via the web and internationally registered secure transportation companies the reputations, high quality, experience and knowledge of the consortium partners, combined with the integration of the european commission infrastructure, will provide endusers with opportunities to approach new fields of research in structural viral genomics, evolutionary biology, control of infectious diseases, antiviral drug design, fundamental research, public and environmental health, pathogenesis, immunology and a wide variety of associated disciplines. subsequently, subject to continuation of funding and the appropriate justification, the evag aims to extend the diversity of its disciplines to encompass fish, plant, bacterial, fungal, protist and other currently unrecognised viruses. this will be achieved first through expansion within the european community, followed by integration of specialist laboratories in the americas, russia, asia, southeast asia, australia and africa. the recently created african cdc will provide a unique opportunity to establish strong links with public health institutes in africa. the substance of this relationship will be based on trust and mutual benefit. for instance, training workshops and staff exchanges will be organized with the objective of developing appropriate infrastructures that can be directly integrated into the evag format and the levels of scientific expertise and quality and range of products that will justify their integration into evag as full partners within the consortium. the evag provides a range of viruses and relevant reagents to endusers engaged in a wide variety of established infrastructures. for instance, the newly created eu infrastructure, infravec (european infrastructures for insect disease vector research and control -https:// infravec .eu/) supplies infectious vectors (e.g. mosquitoes and ticks) to their end-users. the evag is closely associated with this activity, via the provision of viruses that can infect the vectors. erinha (http://www. erinha.eu/), a european strategy forum on research infrastructures (esfri www.esfri.eu/), offering access to high containment facilities to industrial end-users for their preclinical trials including bsl pathogens, recommends evag as the provider of the viruses necessary for their trials. bbmri, another esfri, plans to create a service unit dedicated to the provision of viruses and derived material from viruses. evag is identified as the recommended supplier. the evag contributes to the functioning of other eu infrastructures involved in related research topics including zoonotic diseases, drug discovery, vaccine development, viral epidemiology and emerging virus diseases. these eu-funded projects include prepare (platform for european preparedness against (re-) emerging epidemics -https://cordis. europa.eu/project/rcn/ _en.html), compare (collaborative management platform for detection and analyses of (re-) emerging and foodborne outbreaks in europe -http://www.compare-europe.eu/about), emerge (efficient response to highly dangerous and emerging pathogens at eu level -https://www.emerge.rki.eu/), zikalliance (a multinational and multidisciplinary research consortium coordinated by insermhttps://zikalliance.tghn.org/about/). as a future objective, one can imagine the generation of links among all of these organisations to constitute a "life science infrastructure cooperative" dedicated to virology-related topics, in which the evag could play a central role. for the long-term future, we are planning to establish closer collaborative links with other compatible scientific infrastructures by initiating teaching and training programmes amongst younger scientists to ensure that people of the right calibre will be able to undertake the roles currently occupied by experienced but "maturing" scientists. indeed, one could envisage e-learning programmes being built into diploma courses in universities. as we continue our ambitious mission to build a scientific infrastructure that can benefit scientists operating in all areas of the viral world, we recognise that other agencies and research teams can provide valuable datasets, and analytical expertise from which disease intervention strategies might be developed. indeed, other compatible agencies are approaching the consortium to share their resources and to promote their activities through the consortium website. this is yet another avenue that is being explored, based on the complementarities of these parties with the existing group of partners. at the time of writing, we are witnessing the massive impact of nextgeneration sequencing technology that will undoubtedly continue to evolve and provide even more discoveries, any one of which might then be exploitable by a future larger and more comprehensive form of agency. since the first recognition of viruses and our obsession with trying to understand and control them, it has been an exciting time for virologists. long may the evag and its successors ensure that this is always the case! this publication was supported by the european virus archive global (evag) project that has received funding from the european union's horizon research and innovation programme under grant agreement no . improved detection of zika virus rna in human and animal specimens by a novel, highly sensitive and specific real-time rt-pcr assay targeting the '-untranslated region of zika virus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assay optimization for molecular detection of zika virus lineage-specific real-time rt-pcr for yellow fever virus outbreak surveillance mosquito co-infection with zika and chikungunya virus allows simultaneous transmission without affecting vector competence of aedes aegypti the european virus archive: a new resource for virology research a cynomolgus macaque model for crimean-congo haemorrhagic fever zika virus infection during pregnancy in mice causes placental damage and fetal demise the . a resolution cryo-em structure of zika virus laboratory testing for middle east respiratory syncytial coronavirus: interim guidance a crispr screen defines a signal peptide processing pathway required by flaviviruses we wish to thank the following scientists for their expertise and contributions during the successful establishment and development of supplementary data related to this article can be found at https:// doi.org/ . /j.antiviral. . . . key: cord- -lq yr authors: cunningham, steve title: bronchiolitis date: - - journal: kendig's disorders of the respiratory tract in children doi: . /b - - - - . - sha: doc_id: cord_uid: lq yr acute viral bronchiolitis is a common viral lower respiratory tract infection in young children. most typically caused by respiratory syncytial virus in % of cases, the condition lasts for to days, with a prolonged cough in many. children with comorbidity, particularly those born prematurely or with significant congenital heart disease, are at risk of more severe disease. nasal obstruction progresses over to days to difficulty with feeding and increased work of breathing with hypoxemia. crackles and/or wheeze may be auscultated. apnoea may be a presenting sign in those less than months of age. viral load is highest at peak of symptoms and in those with more severe disease. approximately % to % of all children are admitted to hospital with bronchiolitis. the differential diagnosis may include bacterial pneumonia, congenital lesions of the lung or heart, or an interstitial lung disease. there are no effective treatments, and admission is for feeding support (by nasogastric or intravenous fluids) or treatment of hypoxemia. critical care support is required for some infants experiencing respiratory failure, though mortality rates remain unchanged. practice within and between countries varies significantly and alignment of practice is a common goal of guidelines. vaccines for rsv are in advanced development, as are several antiviral therapies for rsv. in most children, acute symptoms improve within to days and cough by weeks. recurrent wheeze is common following acute bronchiolitis and a good association with a diagnosis of asthma in childhood. rsv infects % to % of infants in the first year of life. , the rapid development of vaccines and treatment therapies for rsv has added impetus to the need to better define the burden of rsv disease. globally there are an estimated . million cases of rsv lower respiratory tract infection each year in children under years of age, resulting in . million admissions to the hospital and to thousand deaths (with the majority in low-and middle-income countries). in the united states an estimated % of children will attend primary care each year with rsv bronchiolitis, and up to % attend an emergency department (ed). admission to hospital with rsv bronchiolitis is typically around . % of all infants, , though in previously healthy term infants, the admission rate to hospital with rsv bronchiolitis can be as low as . %. in infants who are born preterm at to weeks' gestation, % will develop bronchiolitis and % require admission to the hospital. the risk of bronchiolitis is increased in a range of conditions compared with term infants, including preterm birth (respiratory rate [rr] . ) , cystic fibrosis (rr . ), congenital heart disease (rr . ), chronic lung disease (rr . ), immunodeficiency (rr . ), down syndrome (rr . ), and cerebral palsy (rr . ). , epidemiology bronchiolitis is the most common lower respiratory tract infection in children. the condition forms part of the spectrum of viral lower respiratory tract infection that includes bronchiolitis, viral pneumonia, and viral-induced wheeze. in polar hemispheres (north and south), bronchiolitis is a seasonal disease, dominating winter months, with a peak over to weeks around the winter solstice. in tropical climates, the disease is associated with rainy months and is seasonally more dispersed. climate and environment appear to influence both season and severity. , bronchiolitis is diagnosed clinically by integrating characteristic but variable signs and symptoms across a broad age range, though the majority of cases occur in children under year of age. the condition can be caused by any respiratory virus and has a wide spectrum of disease severity. a "classic" case would be an infant aged to months of age who develops coryza and over the subsequent to days has increased difficulty with breathing, and consequent inability to maintain adequate oral feeding. wheeze or crackles can be heard on auscultation. improvement occurs by days to , though a characteristic harsh cough may persist for days or more. , while the diagnosis often appears straightforward, the wide range of disease severity across a skewed but broad age range and the need for clinical diagnosis (with associated inconsistency) creates difficulty in establishing precise data. in addition, while bronchiolitis is a clinical diagnosis applied to any infecting agent, the majority of data available relate to bronchiolitis caused by respiratory syncytial virus (rsv) infection; and within rsv bronchiolitis is a focus on those at high risk, in particular, those born prematurely. reference to these groups synonymously with bronchiolitis can make interpretation of epidemiological data difficult and may reduce the understanding of bronchiolitis caused by non-rsv and in lower risk patients (particularly children born at term). there are only limited estimates of population risk for bronchiolitis associated with all respiratory virus infections, but approximately % of infants are affected by bronchiolitis in the first year of life. in the united kingdom, using primary care databases, the year incidence of children given a specific diagnosis of bronchiolitis is to per children, , rising to per when a broader definition of bronchiolitis was used to capture potential cases. this study highlights that in children with typical lower respiratory tract signs and symptoms, clinicians may not ascribe the discrete diagnosis of bronchiolitis; a finding also found keywords bronchiolitis viral lower respiratory tract infection wheeze respiratory syncytial virus abstract acute viral bronchiolitis is a common viral lower respiratory tract infection in young children. most typically caused by respiratory syncytial virus in % of cases, the condition lasts for to days, with a prolonged cough in many. children with comorbidity, particularly those born prematurely or with significant congenital heart disease, are at risk of more severe disease. nasal obstruction progresses over to days to difficulty with feeding and increased work of breathing with hypoxemia. crackles and/or wheeze may be auscultated. apnoea may be a presenting sign in those less than months of age. viral load is highest at peak of symptoms and in those with more severe disease. approximately % to % of all children are admitted to hospital with bronchiolitis. the differential diagnosis may include bacterial pneumonia, congenital lesions of the lung or heart, or an interstitial lung disease. there are no effective treatments, and admission is for feeding support (by nasogastric or intravenous fluids) or treatment of hypoxemia. critical care support is required for some infants experiencing respiratory failure, though mortality rates remain unchanged. practice within and between countries varies significantly and alignment of practice is a common goal of guidelines. vaccines for rsv are in advanced development, as are several antiviral therapies for rsv. in most children, acute symptoms improve within to days and cough by weeks. recurrent wheeze is common following acute bronchiolitis and a good association with a diagnosis of asthma in childhood. for infants born ≤ weeks gestation, % of infants will have a lower respiratory tract infection in the first year of life, with % rsv positive and % rsv negative; of these infants, % of rsv positive will be admitted to the hospital versus % of rsv negative. recent studies suggest that hospitalization rates for high-risk infants due to rsv are reducing over time and are now similar to those for rsv negative, possibly as a result of improvements in neonatal care or immunoprophylaxis in high-risk groups ( fig. . ). , risk of death is much higher amongst high-risk groups who are rsv positive, including preterm ( . %), congenital heart disease ( . %), and bronchopulmonary dysplasia ( . %). bronchiolitis has a viral etiology, with rsv the most common cause, reported in % to % , of cases. other viruses associated with bronchiolitis are human rhinovirus ( %), influenza, coronavirus, human metapneumovirus, adenovirus, parainfluenza virus and human boca virus , ; that is, "any respiratory virus." rsv has two strains, a and b, with rsv a associated with more severe disease. , reinfection in the same season with the same or different strain is possible. as a sole infecting agent, rsv is associated with more severe bronchiolitis than other single respiratory virus infections. coinfection of rsv with rhinovirus can produce even more severe disease. rsv is the most common infectious agent in children admitted to the hospital with radiological features consistent with pneumonia (occurring in % of children-most commonly those under years of age). in young children who are well immunized, rsv represents the most common cause of lower respiratory tract infection. what commences as an upper respiratory tract infection becomes a lower respiratory tract infection over the course of to days. infants are particularly susceptible as they have small bronchi that are more likely to become blocked by secretions and edema, and a less well-developed ability to respond to and clear viral infection. histopathology is naturally limited to the most severe cases who have died, where the bronchioles are edematous and blocked by necrotic epithelium and neutrophils, with some mucus binding this debris together. airway obstruction is intensified by poor airway clearance associated with loss of cilia function occurring within hours and persisting for up to months after the illness. destruction of cilia is considered to be caused by virus replication and not mediated by inflammation. rsv is associated with more severe airway pathology than that found in children dying from other respiratory viruses, even in those not mechanically ventilated. viral shedding is higher and more prolonged in younger infants and those with more severe disease. increased disease severity, longer hospital stay and use of intensive care is associated with higher viral load for rsv in nasopharyngeal secretions. , severity of disease is associated with both infant risk factors (including lack of adaptive t cell response), , but also rsv virus specific factors (viral antigen load and direct cytotoxic effects). determining the relative contribution of both of these to disease severity will be important; if the latter is dominant, antiviral agents provided early in the course of the disease may reduce severity, whereas dominance of the former might need additional immunomodulators. biomarkers are now sought to better characterize those at risk of greater disease severity and to indicate recovery. infants hospitalized with rsv bronchiolitis have increased interleukin (il)- and il- in secretions. polymorphisms of surfactant protein a are associated with increased risk of intensive care admission. cysteinyl leukotrienes are increased in infants with rsv bronchiolitis and are still increased month following infection. more severe disease is also associated with increased serum cathelicidin, lactate dehydrogenase, caspase and il- . there is some evidence that more severe disease may be associated with an insufficient inflammatory response. the interrelationship of the microbiome in bronchiolitis is also being actively explored. bronchiolitis is diagnosed clinically. variance in the clinical interpretation of symptoms and physical findings lead to inconsistency in diagnosis, particularly in milder cases and children over year of age. typical symptoms are rhinorrhea, proceeding over to days to a characteristic harsh moist cough with pyrexia that is typically below °c, although fever above . °c is seen in % of infants. ability to achieve adequate oral feeding declines as nasal obstruction with secretions develops and work of breathing increases. the time to peak symptoms of days is associated with the peak in viral load, , varying from infant to infant. in younger children (particularly < weeks of age), apnea may be a presenting sign, sometimes in the absence of other features of bronchiolitis. apnea may be temporarily improved by nasal suctioning, but it is most likely a direct viral effect in young infants. apnea is a "red flag" sign in bronchiolitis that warrants a period of review in a supervised clinical setting to ensure that it has resolved. patients more likely to require intensive care include preterm infants and those with apnea, low birth weight, or a respiratory rate greater than /min. , children tend not to relapse during the improving phase of the illness, which should give confidence to clinicians when considering discharge from ed or hospital. , physical findings include an increased respiratory rate, chest recession, use of accessory muscles, hyperinflation, wheezing, crackles, and reduced arterial oxygen saturations. physical findings vary depending on sleep state (and associated changes in tidal volume). respiratory rate is a key marker of disease severity, with ≥ /min considered severe and ≥ / min critical. , oxygen saturation may be improved (at least temporarily) by removal of nasal secretions. bronchiolitis is a highly variable disease that requires assessment of disease severity by clinicians for decision making, some of which is subjective. clinical scoring systems have been developed in an attempt to standardize care and minimize variance. many early scores derived from asthma scores. the most commonly applied scores for bronchiolitis are outlined in ) and to improve the ability to identify those at risk of deterioration. the ability of clinical scores to retain precision and reliability, when scoring is performed by larger numbers of health care professionals in the context of multicenter phase iii trials, is of current interest. symptoms in bronchiolitis vary across a wide but skewed continuum from mildly increased work of breathing with cough to respiratory failure and death. often divided into mild, moderate, and severe disease, the perspective on these gradations varies across health care systems. a world health organization (who) workshop has provided candidate definitions differentiating a diagnosis of rsv lower respiratory tract infection (spo < %) from severe (< %) and very severe rsv disease (spo < %, inability to feed orally, or reduced level of consciousness). infants can display variance in spo within this range ( % to %) over short periods of observation without significant change in clinical status, , , which may limit the discriminatory reliability of these definitions. from a secondary care perspective, moderate severity is often considered a need for admission to hospital and severe by need for critical care (positive pressure support). clinical scores are often designed to identify transition points in the level of care required. the currently available evidence concerning transition points in level of care is poor. treatment guidance, particularly benefit from use of interventions at the ed/ward (i.e., spo ) and ward/critical care floor interface (i.e., highflow nasal cannula [hfnc] oxygen and continuous positive airway pressure [cpap]) is much needed. guidelines have provided signs and symptoms that should alert clinicians to a child at risk of deterioration and suggested criteria for admission to the hospital. in hospitals, those most likely to deteriorate to the extent of being provided with critical care support are of lower birth weight (< lbs, . kg) and/or have a respiratory rate ≥ /min on day of admission. chest radiography is not required to confirm a diagnosis of bronchiolitis. a chest radiograph often leads to increased diagnostic uncertainty as the features may be similar to those of pneumonia (atelectasis, mucous plugging, and loss of volume) and consequently lead to greater inappropriate use of antibiotics. chest radiography should be reserved for a child who is atypical, for example, showing persistently focal crackles, a temperature remaining above °c despite antipyretics, or respiratory failure requiring critical care support. , laboratory tests do not aid in the clinical diagnosis of bronchiolitis. serious bacterial infection is unusual and complete blood counts and blood cultures are unhelpful (though recent evidence suggests that although still uncommon, it may be more frequent than previously considered). dehydration is usually mild and best assessed clinically without electrolyte measurement. approximately % of infants with bronchiolitis can have concurrent urinary tract infection, so urine culture may be of value in persistently febrile infants, particularly those under months of age. measurement of arterial/capillary carbon dioxide is commonly performed, but can be restricted to those children with increased respiratory rate and work of breathing despite oxygen supplementation. the clinical interpretation of signs and symptoms is difficult in a condition where age boundaries are loose (and skew to older ages in those with comorbidity) and symptoms vary from patient to patient and time to time. this naturally leads to variation in diagnosis and differential diagnosis. there is a common understanding that a clearer diagnosis is possible in those under year of age and most guidelines reflect this. however, constraining a diagnosis of bronchiolitis to those less than year of age may reduce the ability to identify the whole population of children with bronchiolitis who could benefit from potential interventions. in general, a broader bronchiolitis) is often performed to aid cohorting of patients within hospitals. the increasing recognition that multiple viruses may be identified in those with acute bronchiolitis has called into question the benefit of cohorting based on rsv status. pcr diagnostics may sometimes be considered oversensitive to the detection of virus fragments postinfection, and multiplex pcr results should be interpreted with this understanding. differential diagnosis includes bacterial pneumonia or an alternative cause of crackles, wheeze, and increased work of breathing in a young child. persisting crackles (crepitations) in one lung zone, fixed focal wheeze, persistent pyrexia (> °c) or persistently increased work of breathing in a child who appears otherwise recovered warrant further evaluation. prescient in the mind of most clinicians is that a bacterial pneumonia may be missed. chest radiographs have similar appearances and are poor discriminators. we can assume that bacterial coinfection risk is low, as the use of antibiotics in bronchiolitis is not associated with faster recovery. further investigation could be limited to those with the persisting clinical features noted above. in children with more severe disease, there may be a role for antibiotics as bacteria are isolated in % to % of lavage samples in children with severe bronchiolitis who are intubated and ventilated. [ ] [ ] [ ] definition of bronchiolitis is used in north america and asia that captures a higher percentage of older children with wheezing, where rhinovirus is the dominant infecting agent. such children may be given a diagnosis of viral induced wheeze in other countries. there is most likely a continuum of viral lower respiratory tract infection across age ranges that moves from current diagnoses of viral bronchiolitis to viral pneumonia and viral-induced wheeze/wheezy bronchitis. , the clinical features consistent with a diagnosis of bronchiolitis across different guidelines are presented in table . . diagnosis has a typical onset of a viral respiratory tract prodrome proceeding to lower respiratory symptoms over to days. the south african guideline ( ) considers hyperinflation the most reliable clinical sign in bronchiolitis. the uk guideline ( ) provides a more proscriptive definition. testing of nasal secretions for virus may help consolidate the clinical diagnosis of bronchiolitis and inform health care logistics. most commonly used, and with highest precision, are polymerase chain reaction (pcr) diagnostics for a range of respiratory viruses, but point of care (poc) testing for a more limited range of viruses (most often rsv) is increasingly precise and cost effective. testing for rsv (as the most common infecting agent in therapies in addition to supplemental oxygen and hydration are poorly supported by current evidence. there is some evidence that infants handled less get better quicker, and the use of additional therapies should be considered with that in mind. there is widespread variation across hospitals and countries in the management and treatment of bronchiolitis reflecting local custom and individual clinician practice. reducing variation and associated health care costs is a key aim of bronchiolitis management presented through guidelines. guidelines for the care of infants with bronchiolitis based on systematic review and published in english are available from the united kingdom ( ), and south africa ( ), which has been updated as a critical review . no therapies receive support across all guidelines for use with the exception of supplemental oxygen. chest physiotherapy does not speed recovery. antibiotics, though still widely used, are of no benefit in bronchiolitis. in addition, bronchodilators are less likely to be recommended in more recent guidelines, and the theory that they may be of greater benefit in infants more likely to develop asthma has been refuted. nebulized hypertonic saline has been of benefit in cystic fibrosis and in early trials in bronchiolitis, but larger well-designed trials have not demonstrated a persuasive benefit. [ ] [ ] [ ] [ ] [ ] recent years have seen the increasing use of hfnc oxygen in acute bronchiolitis. though clinical trials have not yet demonstrated important clinical or physiological benefits, large well-designed trials are in progress and are beginning to report. a, cpap has some benefit in bronchiolitis, and may prevent deterioration when used early. as with all management in bronchiolitis, the use of hfnc oxygen, cpap, and intubation varies across sites irrespective of disease severity, and better understanding of the risks and benefits of these interventions is required. there are no current effective pharmacological treatments for rsv. while ribavirin was previously used as an antiviral treatment for rsv, it is now considered ineffective. novel treatments for acute infection are in development: antivirals and nebulized immunoglobulin. in this rapidly moving field, it seems probable that a treatment for rsv will become available in the next years. reduction in viral load has been demonstrated in adult challenge models of rsv treated with the antivirals als- and gs- prevention of spread of rsv depends on good hygiene, in particular, hand washing, as rsv may survive for up to hours on surfaces contaminated by droplets. similar precautions are appropriate for other respiratory virus infections associated with bronchiolitis. many hospitals use poc testing for rsv to determine cohorting of infants as inpatients. while this is still common, the practice is called into question by the range of coinfection with other respiratory viruses revealed by pcr panel testing; up to % of children with viral respiratory tract infection have more than one virus detected. prevention of rsv (as the most common cause of bronchiolitis) has been a long-term goal. early formalin inactivated there are no trials of outcome for antibiotic use in children with bronchiolitis receiving intensive care. though uncommon, congenital lesions may masquerade as bronchiolitis, and this should be borne in mind for children with atypical clinical features or those slow to recover. congenital heart disease may present as bronchiolitis when pulmonary vascular resistance falls increasing left to right shunt. more difficult to differentiate are children with congenital (or less commonly acquired) pulmonary malformations. fixed focal wheeze may be a sign of tracheomalacia or bronchomalacia, stenosis, or compression from lobar emphysema or a bronchogenic cyst and would warrant a chest radiograph. a slow recovering course with persistent chest signs could be an infected congenital pulmonary malformation (such as a congenital cystic adenomatoid malformation [ccam] or sequestration). children with persistent fine crackles, tachypnea, and low (often borderline) oxygen saturation may have an interstitial lung disease, particularly neuroendocrine cell hyperplasia (nehi) presenting as recurrent "bronchiolitis." young children with persistent, sometimes focal, crackles postadenovirus (though may also be other respiratory viruses and mycoplasma pneumonia) should be evaluated for postinfectious bronchiolitis obliterans (pibo); see section of the book. management of bronchiolitis is supportive, assisting hydration and hypoxemia until improvement. with increased respiratory rate and nasal secretions, oral feeding is challenged, and those with severe disease require assistance with feeding by enteral or parenteral means. the threshold for supporting hydration is typically when an infant's intake is reduced to % to % of usual volume. the chosen percentage of intake depends on the child's status: an expreterm -week-old infant on day of illness may be supported at % understanding that they most likely will deteriorate, whereas a robust -month-old term infant may be able to tolerate % feed volume for a couple of days until disease resolution. nasogastric feeding is easier to administer than intravenous fluids but has no advantage in recovery from acute disease. oxygen may be used to treat hypoxemia. the threshold oxygen saturation at which to use supplemental oxygen varies across guidelines and is typically set between % and % at sea level. in children admitted to hospital with bronchiolitis, management at a threshold of % spo is safe and as clinically effective as a % target. the threshold oxygen saturation for admission to hospital is often %, as some data suggest that infants have a higher risk of desaturating further at this oxygen saturation. oxygen desaturation may however have a disproportionate influence on decisions to admit children to the hospital, and much like hydration status (previously mentioned), the context of the measurement should be considered. many infants discharged home from ed with bronchiolitis experience desaturation events subsequently that are not associated with clinical deterioration. in hospitals, the use of intermittent oxygen saturation monitoring is much discussed, and though the benefit below % spo is not established, once stable above % spo , oxygen saturation monitoring should be stopped. use of bronchiolitis occurs in % of those who are rsv positive and % of those who are rsv negative. recurrent postinfectious wheeze is not reduced by montelukast or inhaled corticosteroids, , but there is good evidence of benefit from palivizumab, and potentially azithromycin, though the latter requires further study. in the longer term, there is good evidence that children who have had an admission to the hospital for rsv bronchiolitis are times more likely to have a diagnosis of asthma and lower lung function at age years and a higher incidence of asthma at age and years. the question remains whether such children are predisposed to bronchiolitis because of premorbid anatomy and the consequent interrelationship between host and virus specific effects on the development of asthma. vaccines were associated with more severe enhanced rsv disease and deaths, possibly resulting from inadequate t cell priming. subsequent vaccine development has been cautious in view of this experience. in the s, rsv intravenous immunoglobulin was developed , but was rapidly superseded by palivizumab, a monoclonal antibody delivered by monthly intramuscular injection. when administered over the rsv season, it reduces hospital admission in high-risk infants. palivizumab's monthly injections and limited efficacy have prompted the development of extended life monoclonal antibodies that are undergoing licensing trials in preterm infants. they will hopefully be evaluated in the future for high-risk infants born at term. rsv vaccine development has gained significant impetus over the last years with a wide range of candidate vaccines in development both for pediatric and maternal use; maternal immunization could provide passive transplacental protection to infants in the first to months of life (http:// www.path.org). a phase iii trial of maternal immunization by novovax is expected to conclude in . for most children, bronchiolitis is a self-limiting disease, with cough as the most persistent symptom resolving at a median of to days. , many children, however, develop recurrent respiratory symptoms. in the first few months following illness, this is considered in part to result from loss of cilia from the airway epithelial surfaces during the acute illness. for those who experience chronic symptoms, the debate continues on whether children with more severe bronchiolitis and recurrent postinfectious wheezing have premorbid susceptibility, with some evidence suggesting poorer preexisting lung function. recurrent wheeze in the year following oxygen saturation targets in infants with bronchiolitis (bids): a double-blind, randomised, equivalence trial sabre: a multicentre randomised control trial of nebulised hypertonic saline in infants hospitalised with acute bronchiolitis nebulized hypertonic saline for bronchiolitis in the emergency department: a randomized clinical trial % hypertonic saline in acute bronchiolitis: a randomized controlled trial % hypertonic saline versus normal saline in inpatient bronchiolitis: a randomized controlled trial the effect of % and % hypertonic saline in viral bronchiolitis: a randomised controlled trial nebulized hypertonic saline for bronchiolitis: a randomized clinical trial risk factors for bronchiolitis, recurrent wheezing, and related hospitalization in preterm infants during the first year of life chronic diseases, chromosomal abnormalities, and congenital malformations as risk factors for respiratory syncytial virus hospitalization: a population-based cohort study bronchiolitis: diagnosis and management in children prospective study of healthcare utilisation and respiratory morbidity due to rsv infection in prematurely born infants trends in respiratory syncytial virus and bronchiolitis hospitalization rates in high-risk infants in a united states nationally representative database respiratory syncytial virus hospitalization trends in infants with chronic lung disease of infancy viral etiologies of infant bronchiolitis, croup and upper respiratory illness during consecutive years viral etiology of bronchiolitis among pediatric inpatients in northern taiwan with emphasis on newly identified respiratory viruses occurrence of groups a and b of respiratory syncytial virus over years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children severity of respiratory syncytial virus infection is related to virus strain immunity to and frequency of reinfection with respiratory syncytial virus association of rhinovirus infection with increased disease severity in acute bronchiolitis community-acquired pneumonia requiring hospitalization among u.s. adults aetiology of childhood pneumonia in a well vaccinated south african birth cohort: a nested casecontrol study of the drakenstein child health study respiratory syncytial virus (rsv) and its propensity for causing bronchiolitis pecularities of lesions in viral and mycoplasma infections of the respiratory tract recovery of the ciliated epithelium following acute bronchiolitis in infancy respiratory syncytial virus infections in infants: quantitation and duration of shedding respiratory syncytial virus genomic load and disease severity among children hospitalized with bronchiolitis: multicenter cohort studies in the united states and finland respiratory syncytial virus load, viral dynamics, and disease severity in previously healthy naturally infected children type and type cytokine imbalance in acute respiratory syncytial virus bronchiolitis viral specific factors contribute to clinical respiratory syncytial virus disease severity differences in infants respiratory syncytial virus disease is mediated by age-variable il- surfactant protein a polymorphisms and disease severity in a respiratory syncytial virus-infected population latitudinal variations in seasonal activity of influenza and respiratory syncytial virus (rsv): a global comparative review altitude and environmental climate effects on bronchiolitis severity among children presenting to the emergency department environmental drivers of the spatiotemporal dynamics of respiratory syncytial virus in the united states viral bronchiolitis in children epidemiology of respiratory syncytial virus bronchiolitis in hospitalized infants in greece duration of illness in ambulatory children diagnosed with bronchiolitis duration of illness in infants with bronchiolitis evaluated in the emergency department the clinical burden of respiratory syncytial virus (rsv) bronchiolitis among infants in the united kingdom (uk) [phd]: imperial college epidemiology of respiratory syncytial virus infection in washington, dc. ii. infection and disease with respect to age, immunologic status, race and sex a comparison of the recording of common childhood conditions in the doctor's independent network and general practice research databases viral respiratory tract infections in the first six months of life evaluation of hospitalized infants and young children with bronchiolitis-a multi centre study trends in bronchiolitis hospitalizations in the united states admission to hospital for bronchiolitis in england: trends over five decades, geographical variation and association with perinatal characteristics and subsequent asthma risk factors for hospital admission with rsv bronchiolitis in england: a population-based birth cohort study international variation in the management of infants hospitalized with respiratory syncytial virus. international rsv study group the risk of mortality among young children hospitalized for severe respiratory syncytial virus infection risk of primary infection and reinfection with respiratory syncytial virus respiratory-syncytialvirus infections, reinfections and immunity. a prospective, longitudinal study in young children global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis the burden of respiratory syncytial virus infection in young children hospitalizations associated with influenza and respiratory syncytial virus in the united states increased risk of wheeze and decreased lung function after respiratory syncytial virus infection g blood gas analysis in acute bronchiolitis-who and when? a clustering approach to identify severe bronchiolitis profiles in children deciphering clinical phenotypes in acute viral lower respiratory tract infection: bronchiolitis is not an island variability in the diagnostic labeling of nonbacterial lower respiratory tract infections: a multicenter study of children who presented to the emergency department south african guideline for the diagnosis, management and prevention of acute viral bronchiolitis in children rapid point of care diagnostic tests for viral and bacterial respiratory tract infections-needs, advances, and future prospects co-infections in children hospitalised for bronchiolitis: role of roomsharing antibiotics for bronchiolitis in children under two years of age high incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (rsv) bronchiolitis pulmonary and systemic bacterial co-infections in severe rsv bronchiolitis concurrent bacterial infection and prolonged mechanical ventilation in infants with respiratory syncytial virus lower respiratory tract disease nasogastric hydration versus intravenous hydration for infants with bronchiolitis: a randomised trial oxygen saturation targets in infants with bronchiolitis (bids): a double-blind, randomised, equivalence trial predictors of major intervention in infants with bronchiolitis intermittent monitoring of oxygen saturation in infants and children with acute bronchiolitis: peekaboo pediatrics or good clinical care? racemic adrenaline and inhalation strategies in acute bronchiolitis variability in inpatient management of children hospitalized with bronchiolitis bronchiolitis in children: summary of nice guidance clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis clinical practice guideline on acute bronchiolitis. clinical practice guidelines in the spanish national healthcare system finnish guidelines for the treatment of laryngitis, wheezing bronchitis and bronchiolitis in children acute viral bronchiolitis in south africa: strategies for management and prevention allergic diseases and the effect of inhaled epinephrine in children with acute bronchiolitis: follow-up from the randomised, controlled, double-blind, bronchiolitis all trial nebulized hypertonic saline for acute bronchiolitis: a systematic review % hypertonic saline versus normal saline in inpatient bronchiolitis: a randomized controlled trial nasal lavage leukotrienes in infants with rsv bronchiolitis serum cathelicidin level is associated with viral etiology and severity of bronchiolitis lactate dehydrogenase and caspase activity in nasopharyngeal secretions are predictors of bronchiolitis severity. influenza other respir viruses interleukin- is associated with disease severity in viral bronchiolitis a robust cytokine and chemokine response in nasopharyngeal secretions is associated with decreased severity in children with physician diagnosed bronchiolitis nasopharyngeal bacterial burden and antibiotics: influence on inflammatory markers and disease severity in infants with respiratory syncytial virus bronchiolitis north ryde: nsw ministry of health virus type and genomic load in acute bronchiolitis: severity and treatment response with inhaled adrenaline the g glycoprotein of respiratory syncytial virus depresses respiratory rates through the cx c motif and substance p risk factors for requiring intensive care among children admitted to ward with bronchiolitis effect of oxygen supplementation on length of stay for infants hospitalized with acute viral bronchiolitis hospital course and discharge criteria for children hospitalized with bronchiolitis inter-observer agreement between physicians, nurses, and respiratory therapists for respiratory clinical evaluation in bronchiolitis bronchiolitis: recommendations for diagnosis, monitoring and management of children one to months of age is nasal suctioning warranted before measuring o saturation in infants with bronchiolitis? wheezing in infants: the response to epinephrine measuring clinical severity in infants with bronchiolitis bacteremia in children hospitalized with respiratory syncytial virus infection who consultation on respiratory syncytial virus vaccine development report from a world health organization meeting held on - effect of oxygen desaturations on subsequent medical visits in infants discharged from the emergency department with bronchiolitis effect of oximetry on hospitalization in bronchiolitis: a randomized clinical trial variation in inpatient diagnostic testing and management of bronchiolitis predictors of airspace disease on chest x-ray in emergency department patients with clinical bronchiolitis: a systematic review and meta-analysis assessing the utility of urine testing in febrile infants aged to months with bronchiolitis respiratory syncytial virus (rsv) immune globulin intravenous therapy for rsv lower respiratory tract infection in infants and young children at high risk for severe rsv infections: respiratory syncytial virus immune globulin study group reduction of respiratory syncytial virus hospitalization among premature infants and infants with bronchopulmonary dysplasia using respiratory syncytial virus immune globulin prophylaxis palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics epinephrine and dexamethasone in children with bronchiolitis decreased lung function precedes severe respiratory syncytial virus infection and post-respiratory syncytial virus wheeze in term infants clinical prediction rule for rsv bronchiolitis in healthy newborns: prognostic birth cohort study study of montelukast for the treatment of respiratory symptoms of post-respiratory syncytial virus bronchiolitis in children randomised placebo controlled trial of nebulised corticosteroids in acute respiratory syncytial viral bronchiolitis on behalf of the rsvcsg. lack of long term effects of high dose inhaled beclomethasone for rsv bronchiolitis-a randomized placebo-controlled trial respiratory syncytial virus and recurrent wheeze randomized trial to evaluate azithromycin's effects on serum and upper airway il- levels and recurrent wheezing in infants with respiratory syncytial virus bronchiolitis severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age asthma and allergy patterns over years after severe rsv bronchiolitis in the first year of life nebulized hypertonic saline for bronchiolitis: a randomized clinical trial the effect of % and % hypertonic saline in viral bronchiolitis: a randomised controlled trial % hypertonic saline in acute bronchiolitis: a randomized controlled trial sabre: a multicentre randomised control trial of nebulised hypertonic saline in infants hospitalised with acute bronchiolitis high-flow nasal cannula oxygen for bronchiolitis in a pediatric ward: a pilot study cpap and high-flow nasal cannula oxygen in bronchiolitis high-flow warm humidified oxygen versus low-flow nasal cannula oxygen for moderate bronchiolitis (hfwho rct): an open, phase , randomised controlled trial early high flow nasal cannula therapy in bronchiolitis, a prospective randomised control trial (protocol): a paediatric acute respiratory intervention study (paris) improved clinical and economic outcomes in severe bronchiolitis with pre-emptive ncpap ventilatory strategy variability of intensive care management for children with bronchiolitis mechanical ventilation drives inflammation in severe viral bronchiolitis bronchiolitis in children (sign ). nhs quality improvement: scotland respiratory syncytial virus, an ongoing medical dilemma: an expert commentary on respiratory syncytial virus prophylactic and therapeutic pharmaceuticals currently in clinical trials. influenza other respir viruses activity of oral als- in a respiratory syncytial virus challenge study oral gs- activity in a respiratory syncytial virus challenge study possible transmission by fomites of respiratory syncytial virus viral coinfection in childhood respiratory tract infections respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine key: cord- -ur g vp authors: miłek, justyna; blicharz-domańska, katarzyna title: coronaviruses in avian species – review with focus on epidemiology and diagnosis in wild birds date: - - journal: j vet res doi: . /jvetres- - sha: doc_id: cord_uid: ur g vp coronaviruses (covs) are a large group of enveloped viruses with a single-strand rna genome, which continuously circulate in mammals and birds and pose a threat to livestock, companion animals, and humans. covs harboured by avian species are classified to the genera gamma- and deltacoronaviruses. within the gamma-covs the main representative is avian coronavirus, a taxonomic name which includes the highly contagious infectious bronchitis viruses (ibvs) in chickens and similar viruses infecting other domestic birds such as turkeys, guinea fowls, or quails. additionally, ibvs have been detected in healthy wild birds, demonstrating that they may act as the vector between domestic and free-living birds. moreover, covs other than ibvs, are identified in wild birds, which suggests that wild birds play a key role in the epidemiology of other gammacovs and deltacovs. development of molecular techniques has significantly improved knowledge of the prevalence of covs in avian species. the methods adopted in monitoring studies of covs in different avian species are mainly based on detection of conservative regions within the viral replicase, nucleocapsid genes, and ’utr or ’utr. the purpose of this review is to summarise recent discoveries in the areas of epidemiology and diagnosis of covs in avian species and to understand the role of wild birds in the virus distribution. among the most abundant viruses infecting a wide variety of animals, including birds and humans are representatives of the large coronaviridae family. their virions contain the largest single-stranded positive sense rna (ssrna) genome, the feature which distinguishes them from other known viral rna genomes ( ) . similarly to other rna viruses, coronaviruses (covs) are characterised by high genetic diversity driven by mutation and recombination, which can lead to the emergence of new viruses. such new pathogens can have new features which even enable them to switch to new hosts ( ) . these newly created viruses can acquire zoonotic potential, as witnessed by the severe acute respiratory syndrome (sars), the epidemic from southern china in caused by sars-covs. this disease, termed "atypical pneumonia", was diagnosed in humans in countries and had a nearly % mortality rate. in , there emerged a subsequent disease caused by a novel coronavirus, the so-called middle east respiratory syndrome (mers) with even higher mortality rates. both sars-and mers-covs crossed the species barrier from bats to humans through civet cats and camels as intermediate organisms ( ) . wild bird species serve as a natural reservoir of many emerging zoonotic pathogens and thus have a significant impact on public health. they are also the source of pathogens dangerous to domestic animals, and such infections could have socio-economic consequences. this is the reason why over the years wild birds have been under epidemiological surveillance. among the viruses transmitted by wild birds, the most well-known are influenza a viruses ( ) . wild birds are also implicated in the spread of the west nile virus, borrelia burgdorferi, and other bacterial infections such as salmonella or campylobacter, and with them also resistance genes to antibiotics ( ) . studies from the last years have also shown the presence of covs in wild birds ( , , , ) . among factors which make birds an excellent reservoir of various pathogens and also a bioreactor contributing to their variability, there are the high biodiversity of bird species, their ecological traits such gathering/grouping during feeding and roosting, but most importantly their capability to fly long distances ( ) . in this paper, we will focus more on different aspects related to covs identified in wild birds. however, acknowledging the gaps in understanding of the biology of these viruses, we will also refer to the most attention-worthy representatives of avian coronavirus species, i.e. infectious bronchitis viruses (ibv). taxonomic classification. covs belong to the family coronaviridae, subfamily coronavirinae, and order nidovirales. initially, classification of members of this subfamily was based on their serological relationships as opposed to the new taxonomic revision based on a threshold level of sequence identity of a few replicase regions (the pp ab polyprotein and the orf ab gene). according to these criteria, coronavirinae are divided into four genera: alpha-, beta-, gamma-, and deltacoronavirus, replacing the traditional division into antigenic groups , , and ( ) . the final list of species proposed by the international committee on taxonomy of viruses (ictv) including the genus identified in birds, is presented in table . generally, alpha-and betacovs infect humans and domestic animals, while gamma-and deltacovs are largely associated with avian hosts although they were also detected in marine mammal species as well as in some asian carnivores ( , ) . the main representative of the gammacoronavirus genus is avian coronavirus. this taxonomic name includes ibv which causes a highly contagious disease of chickens, and genetically similar viruses isolated from other domestic galliformes: turkey coronavirus (tcov), responsible for turkey enteritis, and the more recently detected guinea fowl coronavirus (gfcov), the aetiological factor of fulminating disease in this species ( , , ) . analogous viruses were also detected in pheasants, peafowl, and quails, but also in nongalliformes, namely columbiformes, pelecaniformes, ciconiiformes, psittaciformes, and anseriformes ( , , , , ) . there are plenty of ibv variants with divergent molecular and biological properties. due to the lack of a clear method for ibv classification, new rules based on the spike gene fragment (s ) sequence were recently proposed. it distinguished and named lineages, categorised into six genotypes (gi to gvi) ( ) . the difference between other members of the avian coronavirus family, namely tcov and gfcov, is also in the s gene structure. interestingly, all other regions of the genome were found to be similar to ibv, suggesting that they come from the same ancestor ( , ) . in , surprising information about a new gammacov species from the white beluga whale appeared, which challenged the prevailing opinion of the specificity of gammacovs only to birds ( ) . further studies identified another coronavirus in ducks in whose genome fulfilled the official ictv criteria required to distinguish a new species in the gammacoronavirus genus. ictv approval is still awaited for this new species designation ( ) . coronaviruses identified in in birds of the passeriformes order, namely munia, bulbul, and thrush, appeared to be similar to each, but distinct from known coronaviruses, and they formed a unique cluster in the phylogenetic tree which was the basis for generation of a novel genus: deltacoronavirus ( ). interestingly, deltacovs were also identified in pigs in hong kong and the united states ( ) . additionally, these viruses clustered with previously unclassified coronaviruses detected in the asian carnivores, the asian leopard cat, and ferret badger ( ) . currently, the deltacoronavirus genus comprises eight species, including seven avian and one swine coronaviruses. it is suspected that covs appeared over million years ago, corresponding in time to the coevolution and codivergence of bat and avian species ( ) . their subsequent diversification was a product of differences in alimentation, reproduction, and roosting ecology. genomic organisation of avian coronaviruses. the coronavirinae subfamily is characterised by an exceptionally large rna genome ( ) . the genome size among viruses, included under the taxonomic species name avian coronavirus, is about , nucleotides (nt), the smallest being the , nt of / ibv strain (genbank no kx ), and the largest the , nt of ck/ch/lgd/ (kc ). duck cov (genbank no. km ), a candidate for a separate species within the gammacoronavirus genus, has a similar size of , nt. the complete genomic sequences of deltacov strains, identified in wild birds available in genbank, are about , nucleotides shorter, their sizes ranging from , nt in white-eye cov hku (nc ) to , nt in magpie-robin cov hku (nc ) strains. the genomes of all coronaviruses have similar structures and organisation, but also display unique groups or even strain-specific genomic structures, including accessory genes. generally, the genome is contained between the 'capped end and poly(a) tail at the '-end, comprising short untranslated regions (utrs). about two-thirds of the genome is occupied by two overlapping large open reading frames (orfs), encoding replicase (rna-dependent rna polymerase (rdrp)) polyproteins a and ab. these two large polyproteins are formed by a ribosomal frameshift mechanism and subsequently cleaved by viral proteases into the nonstructural proteins (nsp) ( ) . the number of nsps of viral replicase in the most complex of the family of positive-strand rna viruses and in alphaand betacovs is , whereas gamma-and deltacovs have nsps because they lack nsp . part of these replicase domains, the seven most conserved, which are nsp , nsp , and nsp - , serve as species demarcation among coronaviruses ( ) . the other third of the genome includes four structural protein genes organised in the following, canonical way: s, e, m, and n ( ) . among these four genes, there are a set of lowmolecular accessory proteins the presence of which could be strain-dependent, and these are a, b, b, c, a, b, and b, this being the most recently identified in some ibv and tcov strains and only orf downstream of n protein, - b ( , ) . it is believed that accessory proteins are not essential for virus replication, but could play a role in virus virulence as b protein does. in addition to these four canonical structural proteins s, e, m, and n, deltacovs seem to have a smaller number of nonstructural accessory proteins. they contain nonstructural protein ns and a number of proteins located downstream of the n protein, designated ns a, b, and sometimes also c and d ( ) . the functions of nonstructural proteins of gamma-and deltacovs are largely unknown. viral proteins. the name "coronavirus" aptly represents their appearance of solar corona in negatively stained electron micrographs, which displays characteristic club-shaped spike (s) proteins on the envelope of the virion. in addition to protruding s proteins, the envelope is formed by the most abundant membrane (m) proteins and non-glycosylated envelope proteins (e) present in lower quantities. such an envelope surrounds the viral genomic rna which forms the complex with a few copies of the nucleocapsid (n) protein ( ). information about the role of virus proteins in the course of infection comes from studies on ibv and tcov. the crucial stage in the virus life cycle relies on interaction of a viral attachment protein with a particular host cell receptor and then release of the genome inside the cell through the fusion with this cell membrane. the key player in both stages is the s protein, which is therefore recognised as a determinant of tissue and cell tropism and pathogenesis. this protein consists of two subunits: the n-terminal s subunit which forms a globular head structure and the c-terminal s subunit which is a transmembrane stalk. the s subunit is responsible for recognising and binding the receptor cell to the host, and the s domain specialises in the fusion process ( ) . coronaviruses identify their various specific receptors and co-receptors, which may be proteins and sugars. the ibv has a primary affinity for the respiratory systems of chickens, but its variants could have tropism also to other organs such as kidneys, oviduct, testes, bursa of fabricius, caecal tonsils, or the alimentary system ( ). the main attachment factor for respiratory ibv is α , -linked sialic acid glycan, widely distributed on host tissue, and this explains why such strains can also have affinity to other organs. additionally, the diversity of the s domain sequence as high as %- % among different ibv variants could further contribute to the binding capacity of these viruses. it is suspected that nephropathogenic ibv strains could use a different or additional receptor for tissue binding. studies on enteric coronaviruses (tcov and gfcov) also revealed their different receptor specificity, as binding of their s protein to the host glycan receptor is independent of the presence of sialic acid residues and recognises poly-lacnacon complextype n-glycans. the difference in receptor binding observed between respiratory ibvs and enteric gammacovs could be concluded from the difference between their s coding regions which is as high as % ( ) . the function of the s protein in avian deltacov seems to be analogous to ibv or tcov, but nothing is known about their receptor specificity. diagnosis. information about the range of avian species infected with covs and the prevalence of these infections has been accumulated from the increasing number of studies based on the use of virus-specific molecular tests. however, such detection methods depend on the specificity of the primers used. the method which was applied for the first time in a systemic monitoring of coronaviruses in wild birds amplified bp of the replicase gene of all coronaviruses known at that time ( ) . this allowed the identification of novel coronaviruses infecting graylag geese, feral pigeons, and mallards ( ) . next, the methods targeting the conserved regions of ibv viruses, i.e. 'utr, 'utr, or even s gene fragments ( , , ) were used for monitoring purposes. additionally, in a molecular survey of avcov in ducks the viral nucleocapsid (n) gene was amplified in a reverse-transcription polymerase chain reaction (rt-pcr) ( ) . the viruses identified in such way were designated ibv-like. the summary of monitoring studies of coronaviruses in wild birds is given in table . however, this approach changed after a few findings; firstly, it turned out that other gammacovs such as tcov or gfcov have genomes closely related to ibv, and the only diverged gene between them is the s gene; secondly, recombination events were discerned to be a quite common phenomenon in these groups of viruses; and the most important discovery was that of deltacov and determination of the criteria which enable discrimination between gamma-and deltacov ( , , ) . more recently, primers specific to the appropriate conserved site of the replicase gene have commonly been applied ( , , ) . all these primers are within a region of about nt occupied by the nsp of orf ab which is instrumental in coronavirus species differentiation and contains different numbers of degenerative nucleotides to detect the broader spectrum of coronavirus strains. the position of the used primers, aimed at the replicase gene, is shown in table . epidemiology. it is well known that ibv is ubiquitous in most parts of the world in regions with intensive poultry production, where it causes huge economic losses. ibv strains are responsible for diseases of the respiratory, urogenital, and digestive tracts of domestic fowl (gallus gallus). however, there are many reports of ibv presence in other bird species, which indicate that the virus can cross the species barrier. recently, ibvs homologous to vaccine h and field ibv strains were found in healthy domestic teal (anas) and peafowl (pavo cristatus), respectively. this indicates that the virus can replicate in these bird species without clinical signs ( ) . moreover, a virus similar to field ibv inoculated into specific-pathogen-free chickens caused nephritis and high mortality, in contrast to h -like ibv which revealed itself to be apathogenic. these findings indicate the possible role of these birds as asymptomatic carriers of ibv and the possibility of virus transmission to susceptible chicken populations. similar conclusions were drawn from chinese and brazilian studies which showed the presence of mass-like ibv strains in wild peafowl and pigeons ( , ) . the detection of virus closely related to the h ibv vaccine strain in faeces of free living ducks and whooper swans also suggested cross-species infection from a poultry population to synanthropic birds ( ) . the presence of gammacov was also found in wild birds sampled in poland. ibv-like strains were identified in anseriformes, charadriiformes, and galliformes, and the detected gene fragments were highly similar to the most frequently detected lineages of ibv in this geographical region, i.e. mass, /b and qx ( ) . similarly, ibv strains were identified in studied samples from wild birds of the corvidae, ardeidae and anatidae families in egypt, and some of them had s gene fragments highly homologous to the ma vaccine strain. such findings suggest the possibility for vaccine strains to spillover to wildlife which may serve as the asymptomatic host, enabling these strains to undergo some genetic changes. such modified virus could then spillover in the reverse direction back to a poultry population, and the possibility of its higher pathogenicity could not be ruled out ( ) . recently, interesting results for the presence of both gamma-and deltacoronaviruses in quails were reported ( ) ( ) ( ) . the identified gammacovs revealed a similarity to ibv strains unique to south america ( ) . furthermore, in some cases, both genera of the viruses were identified simultaneously ( ) . the summary of monitoring studies on the occurrence of coronaviruses in wild birds is presented in table . the rate of positives of gamma-and deltacovs differs greatly from . % to %, depending on temporal/seasonal and spatial features and depending on applied detection methods. however, even using the same method, the rate of positives varies from . % to %. among factors influencing the prevalence of cov in wild birds, there could also be the age of the birds sampled, bird order/species, and their behaviour (migratory versus resident, water birds or land-dwelling birds). very interesting is the identification of betacovs in south american wild birds, these being viruses which were previously detected only in mammals ( ) . it seems, however, that these studies may suffer from methodological deficiencies and require a thorough re-analysis. there is much evidence that cov could be transmitted from poultry to wild birds and from wild birds to poultry. in this way, the virus could spread over long distances. wild birds are suspected of spreading different ibv variants into new geographical regions such as variant qx (gi- lineage) from china to europe or var (gi- lineage) from the middle east to poland ( , ) . if the hosted wild bird acquires infections of various coronaviruses, it could be an excellent environment for recombination events, which may lead to the emergence of a new disease dangerous to humans as evidenced by sars-or mers-covs. that is the reason why wild birds have to be continuously studied for the presence of various coronaviruses. the authors declare that there is no conflict of interests regarding the publication of this article. financial disclosure statement: this publication was possible due to the polish ministry of science and higher education (grant no. know /cb/pro / within the scientific consortium "healthy animal -safe food"). animal rights statement: none required. phylogenetic analysis of partial s and n gene sequences of infectious bronchitis virus isolates from italy revealed genetic diversity and recombination first complete genome sequence of european turkey coronavirus suggests complex recombination history related with us turkey and guinea fowl coronaviruses development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens ratification vote on taxonomic proposals to the international committee on taxonomy of viruses detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens coronaviruses in poultry and other birds coronavirus avian infectious bronchitis virus interspecies transmission and emergence of novel viruses: lessons from bats and birds cross-species transmission and emergence of novel viruses from birds identification and survey of a novel avian coronavirus in ducks avian coronavirus in wild aquatic birds coronavirus associated with an enteric syndrome on a quail farm detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations detection of a novel and highly divergent coronavirus from asian leopard cats and chinese ferret badgers in southern china coronaviruses detected in brazilian wild birds reveal close evolutionary relationships with beta-and deltacoronaviruses isolated from mammals genetic diversity of avian infectious bronchitis virus isolated from domestic chicken flocks and coronaviruses from feral pigeons in brazil between emerging animal coronaviruses: first sars and now mers think globally, act locally: phylodynamic reconstruction of infectious bronchitis virus (ibv) qx genotype (gi- lineage) reveals different population dynamics and spreading patterns when evaluated on different epidemiological scales nidovirales: evolving the largest rna virus genome infectious bronchitis viruses with naturally occurring genomic rearrangement and gene deletion novel coronavirus and astrovirus in delaware bay shorebirds genetically diverse coronaviruses in wild bird populations of northern england. emerg infect dis emergence of a group coronavirus through recombination molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards (anas platyrhynchos) identification of avian coronavirus in wild aquatic birds of the central and eastern usa surveillance of avian coronaviruses in wild bird populations of korea novel avian coronavirus and fulminating disease in guinea fowl diverse gammacoronaviruses detected in wild birds from madagascar first characterization of a middle-east gi- lineage (var -like) of infectious bronchitis virus in europe isolation of avian infectious bronchitis coronavirus from domestic peafowl (pavo cristatus) and teal (anas) retrospective testing and case series study of porcine delta coronavirus in us swine herds identification of a novel coronavirus from a beluga whale by using a panviral microarray broadly targeted multiprobe qpcr for detection of coronaviruses: coronavirus is common among mallard ducks (anas platyrhynchos) prevalence and phylogeny of coronaviruses in wild birds from the bering strait area (beringia) birds, migration and emerging zoonoses: west nile virus, lyme disease, influenza a, and enteropathogens reverse spillover of avian viral vaccine strains from domesticated poultry to wild birds phylogenetic analysis of a highly conserved region of the polymerase gene from coronaviruses and development of a consensus polymerase chain reaction assay a massachusetts prototype like coronavirus isolated from wild peafowls is pathogenic to chickens an avian coronavirus in quail withr respiratory and reproductive signs gammacoronavirus and deltacoronavirus in quail gamma and deltacoronaviruses in quail and pheasants from northern italy s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification a case for the ancient origin of coronaviruses the avian coronavirus spike protein novel receptor specificity of avian gammacoronaviruses that cause enteritis temporal dynamics, diversity, and interplay in three components of the virodiversity of a mallard population: influenza a virus, avian paramyxovirus and avian coronavirus high prevalence and putative lineage maintenance of avian coronaviruses in scandinavian waterfowl urbanization and the dynamics of rna viruses in mallards (anas platyrhynchos) coronavirus diversity, phylogeny and interspecies jumping comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group c coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group c 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 genomic analysis and surveillance of the coronavirus dominant in ducks in china key: cord- -jso mbx authors: lee, sunhee; lee, changhee title: genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from south korea, date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: jso mbx porcine epidemic diarrhoea virus (pedv) is a globally emerging and re‐emerging enteric coronavirus in pigs causing serious economic threats to the world swine industry. since the re‐emergence of massive pedv outbreaks in south korea in − , domestic pig farms have continued to experience ped epidemics or endemics. this study represents the molecular characterization of pedv isolates identified in diarrhoeic animals collected across the country in . initial sequencing analysis of the full‐length s genes revealed that % of the isolates ( / ) belong to the g b subgroup, while the remaining isolates were classified as g b. the data indicated that both variant g b and global epidemic g b strains were responsible for current ped outbreaks in south korea. the g b and g b isolates shared . %– . % and . %– . % amino acid sequence identity at the s gene level and . % and . %– . % nucleotide sequence homology at the genome level compared to the corresponding korean prototype g b and g b strains, respectively. in an interesting manner, one g b‐like knu‐ strain was found to possess a large ‐nucleotide deletion in the orf a region theoretically encoding nonstructural protein . phylogenetic analysis based on the entire genome and spike protein sequences indicated that the isolates were most closely related to other global g b or g b strains but formed different branches within the same genogroup. these results indicate that pedvs undergo continuous evolution in the field. in addition, one pedv strain, kor/knu‐ / , was successfully isolated and propagated in vero cells. the antisera raised against the korean prototype g b strain efficiently neutralized knu‐ virus infection, suggesting antigenic homology between the and pedv strains. our data advance the understanding of the molecular epidemiology and antigenicity of pedv circulating in south korea. polyadenylated tail and consists of seven canonical coronaviral genes, including open reading frame (orf) , in the following conserved order: untranslated region (utr)-orf a-orf b-s-orf -e-m-n- utr (kocherhans, bridgen, ackermann, & tobler, ) . the first two large orfs, orf a and b, encompass the -proximal two-thirds of the genome and code for nonstructural proteins (nsps). orf a translation yields a replicase polyprotein (pp) a, whereas orf b is expressed by a À ribosomal frame shift that c-terminally extends pp a into pp ab. these pp a and pp ab are proteolytically matured by internal viral proteases to generate processing end-products, named as nsp - . the remaining orfs in the -proximal region of the genome encode four canonical structural proteins, spike (s), envelope (e), membrane (m) and nucleocapsid (n), as well as one accessory gene, orf (duarte et al., ; kocherhans et al., ; lai, perlman, & anderson, ; lee, ; saif et al., ) . porcine epidemic diarrhoea was first observed in feeder and fattening pigs in england in (oldham, ) and caused widespread epidemics in multiple swine-producing countries in europe during the s (opriessnig, ) . a marked decrease in acute ped epizootics occurred in europe in the s and s, and only sporadic outbreaks have occurred in recent years (opriessnig, ) . in asia, ped was first reported in , and unlike in europe, it has since posed a huge economic threat to the asian pork industry (chen et al., ; kweon et al., ; li et al., ; puranaveja et al., ; takahashi, okada, & ohshima, ) . in may , ped outbreaks suddenly appeared in the united states and swiftly spread across the nation, as well as to adjacent countries. this outbreak caused the death of more than million newborn piglets in the united states alone during a -year-epidemic period, leading to annual losses in the range of $ million to $ . billion (langel, paim, lager, vlasova, & saif, ; mole, ; ojkic et al., ; stevenson et al., ; vlasova et al., ) . the us emergent strain-like viruses further reached east asian countries, resulting in nationwide ped disasters (lee, ; lin et al., ; maff, ) . during the - pandemics, ped rapidly swept across mainland south korea and jeju island, killing hundreds of thousands of piglets in domestic herds , . since then, ped epizootics or enzootics have regionally occurred through provinces in south korea with intensive swine industries. to investigate the diversity of pedvs responsible for the ongoing outbreaks in south korea, in this study, we determined the full-length sequences of the s proteins of field isolates and complete genome sequences of representative strains identified throughout . in addition, we isolated and serially propagated a kor/knu- / strain and assessed the antigenic cross-reactivity between and pedv field isolates. the small intestine or stool specimens were collected from piglets showing acute watery diarrhoea at various swine farms located in eight different provinces from march through december . intestinal homogenates were prepared as % (wt/vol) suspensions in phosphate-buffered saline (pbs) using a magna lyser (roche diagnostics, mannheim, germany) by three repetitions of s at a speed of , g. faecal samples were also diluted with pbs to % (wt/vol) suspensions. the suspensions were then vortexed and centrifuged for min at , g (hanil centrifuge fleta , incheon, south korea). the clarified supernatants were initially subjected to rt-pcr using a tge/ped detection kit (intron biotechnology, seongnam, south korea) according to the manufacturer's instructions. pedv-positive samples were filtered through a . -lm-poresize syringe filter (millipore, billerica, ma) and stored at À °c until subsequent sequencing analysis and virus isolation. the s glycoprotein gene sequences of the virus isolates were determined by the traditional sanger method. two overlapping cdna fragments spanning the entire s gene of each isolate were amplified by rt-pcr as described previously (lee, park, kim, & lee, ) . the individual cdna amplicons were gel-purified, cloned into a pgem-t easy vector system (promega, madison, wi) and sequenced in both directions using two commercial vector-specific t and sp primers and gene-specific primers. the full-length s sequences of pedv, designated knu- to - , were deposited in the gen-bank database under the accession numbers shown in figure a . in addition, the complete genomes of representative pedv field strains were sequenced by the traditional sanger method. ten overlapping cdna fragments spanning the entire genome of each virus strain were rt-pcr-amplified as described previously lee et al., , and each pcr product was sequenced as described above. the and ends of the genomes of individual isolates were determined by rapid amplification of cdna ends (race) as described previously . general procedures for dna manipulation and cloning were performed according to standard procedures (sambrook & russell, ) . the complete genomic sequences of the viruses were deposited in the gen-bank database under the accession numbers shown in figure b. the sequences of the fully sequenced s genes and complete genomes of global pedv isolates were independently used in sequence alignments and phylogenetic analyses. multiple sequence alignments were generated with the clustalx . program (thompson, gibson, plewniak, jeanmougin, & higgins, ) , and the percentages of nucleotide sequence divergences were further assessed using the same software program. phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences using the neighbour-joining method and subsequently subjected to bootstrap analysis with , replicates to determine the percentage reliability values of each internal node of the tree (saitou & nei, pedv isolation was conducted from faecal suspensions on vero cells in the presence of trypsin (usb, cleveland, oh) as described previously . virus isolation was confirmed by cytopathic effect (cpe) observation, immunofluorescence assay (ifa) and nucleotide sequencing as described previously . the isolated pedv strain was propagated for serial passages in vero cells, and virus titres were determined as described previously . the cross-reactivity of antisera collected from sows inoculated with a korean pandemic g b strain knu- isolated in (baek et al., ) was evaluated by a serum neutralization (sn) test in -well microtiter plates against the past and present isolates as previously described oh, lee, choi, & lee, ) . the neutralization titre was calculated as the reciprocal of the highest dilution of serum that inhibited virus-specific cpe in all duplicate wells. the pedv s glycoprotein is a suitable viral gene for investigating genetic relatedness among isolates and the molecular epidemiology of pedv (chen et al., ; gerber et al., ; lee, ; lee et al., ; oh et al., ) . based on the s gene sequences, therefore, pedv can be genetically separated into two genogroup clusters, genogroup (g , classical and recombinant: low-pathogenic) and genogroup (g , field epizootic or panzootic: high-pathogenic), which are further divided into subgroups a and f i g u r e phylogenetic analysis based on nucleotide sequences of the spike genes (a) and full-length genomes (b) of porcine epidemic diarrhoea virus strains. a region of the spike protein and complete sequence of tgev were included as an outgroup in each tree. multiple sequence alignments were performed using the clustalx program, and the phylogenetic tree was constructed from the aligned nucleotide sequences using the neighbour-joining method. numbers at each branch represent bootstrap values greater than % of , replicates. names of the strains, countries, years of isolation, genbank accession numbers, and genogroups and subgroups proposed in this study are shown. the pedv isolates identified in this study are indicated by solid circles. scale bars indicate nucleotide substitutions per site lee and lee | b as well as a and b (lee, ; figure s ). in an interesting manner, the g b virus s genes were well-conserved, sharing . %- . % aa identity with each other, whereas the g b s genes were relatively variable, exhibiting . %- . % aa homology with each other (table ) (table s ). the number of nt/aa differences and percent identity shared between the isolates and genogroup representative strains is summarized in table s . to establish the genetic relationships involved, phylogenetic analyses were carried out using the nucleotide sequences of the s gene and full-length genome of the isolates, which were determined in this study and are available from genbank (figure ) . consistent with previous studies (lee, ; lee et al., ) , phylogenetic analysis based on the pedv s genes revealed clear separation among the g a, g b, g a and g b subgroups. all g b strains identified in were grouped within the g b clade; however, they were in different branches from the emergent us strains and past re-emergent korean field isolates (figure a) . the g b isolates were most closely clustered together, forming an independent branch within the g b subgroup. furthermore, a phylogenetic tree subsequently reconstructed from the complete genome showed the same grouping structure as the s gene-based tree (figure b) . as shown previously (lee, ; lee et al., ) , the entire genome-based phylogenetic tree revealed that the g b strains including knu- were grouped within the g clade because of the similarity between the g b and g b genomes, except for the n- the percent nucleotide identity was shown in the upper right and the percent amino acid identity was presented in the lower left. lee and lee | which were less than -log lower but not significantly different compared to those against knu- . taken together, our data indicate that the antisera cross-reacted well between the homologous g b field isolates, suggesting antigenic similarity between the and pedv strains. pedv has emerged or re-emerged as one of the deadliest and most contagious viral pathogens in swine, leading to large financial losses in the global swine industry. along with strict biosecurity, vaccination is a fundamental tool for managing and eradicating pedv during epidemic or endemic outbreaks. although g a-based vaccines against pedv were developed and used to combat this disease in south korea over the past decade, their efficacy in the field, as well as the advantages and disadvantages of their use, is continuously debated. furthermore, a growing body of evidence suggests that their incomplete effectiveness may result from antigenic, genetic (> % aa variation between respective s proteins) and phylogenetic (g versus g ) differences between vaccine and field epidemic strains (lee et al., ; oh et al., ; kim et al., ; lee et al., ; lee, ) . the advent of the - pedv pandemic led to a breakthrough in the development of g bbased vaccines phenotypically and genotypically homologous to field strains responsible for global ped epidemics, and these g b vaccines are currently applied to prevent pedv in south korea. another important policy for controlling pedv is to operate a monitoring and surveillance system (moss) to monitor genetic diversity among field isolates and surveil the emergence of novel variants in the field, which will contribute to preventing future outbreaks. to provide insight into the understanding of the current epidemiological status of pedv in south korea, the present study aimed to investigate the genetic, phylogenetic and antigenic characteristics of pedvs responsible for regional outbreaks in south korea in . nucleotide sequencing analysis revealed that two different pedv genotypes, low-pathogenic g b and high-pathogenic g b, caused regional outbreaks in south korea, with the latter genotype more % nucleotide sequence variations at the genome level with the - pandemic strains. however, field g b isolates with nearly % amino acid sequence divergence compared to previous g b strains at the s gene level were identified in the present study. furthermore, mutations within the s protein were randomly and extensively distributed in the s and s regions among the isolates ( figure s ). replication. however, this unique del is in the glu-rich acidic region, which does not affect the authentic roles of nsp and thus is nonessential for coronavirus replication (lei, kusov, & hilgenfeld, ) . although the virus can tolerate the large nsp -del which is dispensable for pedv replication as shown in figure conditions. therefore, the timeline of this situation is unclear and it is unknown whether antigenic differences among pedv epidemic strains will contribute to the failure of current g b vaccines. to counteract the prospective scenario, further studies are critical for securing culturable pedv epidemic strains that are genetically, phenotypically and antigenically characterized in the laboratory. in summary, genetic and phylogenetic analyses indicated that the epidemic-related isolates are closely related with corresponding global g b or g b strains identified in previous outbreaks and that the virus continues to evolve in its host environment. despite their genetic diversity, antigenicity currently seems to remain unchanged among g b strains, indirectly confirming the efficacy of g b-based vaccines against homologous g b pedvs responsible for current epidemics. because the virus is assumed to undergo an evolutionary process to accumulate mutations to ensure viral fitness in the field, new genotypes or variants of pedv, against which the current g b vaccine may provide partial protection, will eventually emerge. furthermore, this circumstance may advent earlier than expected if pedv outbreaks fade from our attention following sporadic or endemic outbreaks without serious economic problems. therefore, it is important to execute mandatory notification of ped-like outbreaks essentially followed by activating an moss, including early diagnosis, to survey forthcoming pedv strains that may emerge locally or globally through genetic drift (e.g., nonsilent point mutations) or genetic shift (e.g., recombination events) and obtain and characterize epidemic field isolates to predict and prepare for future epizootics or panzootics. we would like to acknowledge the swine veterinarians and choon- the authors declare that they have no conflict of interest. lee http://orcid.org/ - - - efficacy of an inactivated genotype b porcine epidemic diarrhea virus vaccine in neonatal piglets nidovirales: a new order comprising coronaviridae and arteriviridae isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa nidovirales: evolving the largest rna virus genome genetic characterization of porcine epidemic diarrhea virus in korea from completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence isolation of porcine epidemic diarrhea virus (pedv) in korea coronaviridae lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus isolation and characterization of a korean porcine epidemic diarrhea virus strain knu- reemergence of porcine epidemic diarrhea virus on jeju island complete genome sequence of a novel porcine parainfluenza virus isolate in korea outbreak-related porcine epidemic diarrhea virus strains similar to us strains complete genome sequence of a novel s-insertion variant of porcine epidemic diarrhea virus from south korea heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea full-genome sequence analysis of a variant strain of porcine epidemic diarrhea virus in south korea genetic characteristics, pathogenicity, and immunogenicity associated with cell adaptation of a virulent genotype b porcine epidemic diarrhea virus nsp of coronaviruses: structures and functions of a large multi-domain protein new variants of porcine epidemic diarrhea virus, china us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan ministry of agriculture, forestry, and fisheries deadly pig virus slips through us borders immunogenicity and protective efficacy of recombinant s domain of the porcine epidemic diarrhea virus spike protein the first case of porcine epidemic diarrhea in canada letter to the editor porcine epidemic diarrhea (ped) in europe and strategies to control outbreaks a new coronavirus-like particle associated with diarrhea in swine chinese-like strain of porcine epidemic diarrhea virus diseases of swine the neighbor-joining method: a new method for reconstructing phylogenetic trees molecular cloning: a laboratory manual emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan mega : molecular evolutionary genetics analysis (mega) software version . the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools distinct characteristics and complex evolution of pedv strains additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article: lee s, lee c. genomic and antigenic characterization of porcine epidemic diarrhoea virus strains isolated from south korea key: cord- -n ncamqh authors: donaldson, braeden; lateef, zabeen; walker, greg f.; young, sarah l.; ward, vernon k. title: virus-like particle vaccines: immunology and formulation for clinical translation date: - - journal: expert rev vaccines doi: . / . . sha: doc_id: cord_uid: n ncamqh introduction: virus-like particle (vlp) vaccines face significant challenges in their translation from laboratory models, to routine clinical administration. while some vlp vaccines thrive and are readily adopted into the vaccination schedule, others are restrained by regulatory obstacles, proprietary limitations, or finding their niche amongst the crowded vaccine market. often the necessity to supplant an existing vaccination regimen possesses an immediate obstacle for the development of a vlp vaccine, despite any preclinical advantages identified over the competition. novelty, adaptability and formulation compatibility may prove invaluable in helping place vlp vaccines at the forefront of vaccination technology. areas covered: the purpose of this review is to outline the diversity of vlp vaccines, vlp-specific immune responses, and to explore how modern formulation and delivery techniques can enhance the clinical relevance and overall success of vlp vaccines. expert commentary: the role of formation science, with an emphasis on the diversity of immune responses induced by vlp, is underrepresented amongst clinical trials for vlp vaccines. harnessing such diversity, particularly through the use of combinations of select excipients and adjuvants, will be paramount in the development of vlp vaccines. virus-like particles (vlp) are a type of subunit vaccine based on virus-derived proteins, assembled to form a particle. vlp hold several advantages over other particulate subunit vaccines. these include a morphological resemblance to their parent virus, a highly repetitive immunogenic surface structure, and the retention of cell uptake and immune processing pathways associated with their parent virus [ ] . vlp themselves are nonpathogenic, devoid of an intact virus genome, and are incapable of infection or replication. the noninfectious nature of vlp significantly improves their safety profile over live-attenuated vaccines, while also possessing advantages when compared to other forms of subunit, killed, or particulate vaccines. commercially successful vlp vaccines include hepatitis b virus (hbv) vlp, such as recombivax hb (merck), engerix-b (glaxosmithkline), elovac b (human biologicals institute), genevac b (serum institute) and shanvac b (shantha biotechnics), hepatitis e virus (hev) vlp, such as hecolin (innovax), and human papillomavirus (hpv) vlp, including gardasil (merck) and cervarix (glaxosmithkline). vlp vaccines currently under investigation in clinical trials include influenza a virus (iav) vlp (nct , nct ) [ , ] , chikungunya virus vlp (nct ) [ ] , human cytomegalovirus (hcmv) vlp (nct ) [ ] , and human norovirus (hunv) vlp (nct , nct , nct ) [ ] [ ] [ ] . recent and current vlp clinical trials registered in the united states of america and the european union are summarized in table . vlp can be bulk expressed in bioreactor cultures, utilizing advanced in vitro protein expression systems for producing vaccine-grade vlp suitable for clinical administration. the manufacture of some vlp also includes additional processing steps, such as disassembly and reassembly of vlp. the manufacture of the hpv vlp utilizes disassembly and reassembly to improve vlp morphology and stability [ ] . similarly, the manufacture of qβ vlp includes this method [ ] . different vlp expression systems and the applications of vlp disassembly and reassembly was explored in a recent review [ ] . compatibility with commercial upscaling technology, gmp production, and with minimal postproduction manipulation or modification supports large-scale use of vlp vaccines; however, some vlp vaccines struggle in their translation from laboratory research and development, to clinical trials and routine public access [ ] [ ] [ ] . open accessibility to the molecular or genetic components of derivative constituents may place some limitations on the commercialization of specific vlp vaccines. this may be circumvented through tactical use of proprietary modification, or by utilizing the vaccine as a constituent within a composite formulation [ ] . the development of some vlp vaccines can be challenged by issues with stability and longevity, which may be alleviated with formulation excipients, or other vaccine additives that facilitate vaccine distribution and storage [ , ] . the purpose of this review is to explore some of the challenges in the translation [ ] , and negative-sense rna viruses such as human parainfluenza virus type [ ] . variety can be observed in vlp size, ranging from ms bacteriophage vlp at around . nm in diameter [ ] , to hpv vlp at around nm [ ] , and influenza vlp at around nm [ , ] . vlp also vary in structural complexity, as illustrated in figure , including mono-layer vlp such as hbv vlp formed from hbv surface antigen (hbsag) or hbv core antigen (hbcag) [ ] [ ] [ ] , and multi-layer vlp such as rotavirus vlp [ , ] . vlp can be encapsulated within a phospholipid bilayer envelope to resemble their parent virus, such as hiv [ ] or sendai virus vlp [ ] . the envelope itself can also form the primary particle structure of some vlp, with recombinantly expressed virus envelope-stabilizing proteins embedded within the membrane, such as with iav virosomes [ ] . some vlp are compatible with the formation of polyvalent or mosaic vlp, derived from multiple virus strains [ ] . while this increases the diversity of vlp vaccines beyond the variety of parent viruses, the increased complexity of polyvalent or mosaic vlp may require post-production manipulation to facilitate stable particle formation [ , ] . in addition to facilitating the development of complex vlp vaccine constructs, introduction of postproduction manipulation has also been demonstrated to improve the consistency and stability of some standard structure vlp vaccines [ , ] . the diversity of vlp can also be characterized based on the variety of diseases these vaccines have been developed to prevent or treat. these include vlp vaccines developed for both human and veterinary pathologies, with some examples including an hbv hbcag core particle-based vaccine for her + cancer [ ] , an adenovirus vlp-based vaccine for placental malaria [ ] , and a qβ vlp-based vaccine for leishmania infection [ ] . a selection of recently developed and investigated novel vlp vaccines are provided in table . vlp can be produced using a variety of expression systems, usually involving vlp assembly by spontaneous polymerization upon the expression of virus protein constituents. vlp can be expressed in cells derived from bacteria, yeast, insects and mammals, in cell-free expression systems, and in live organisms such as silkworm pupae and various plants [ ] [ ] [ ] . selection of an appropriate expression system is important, as each expression system can differ in their efficacy for expressing specific vlp, their post-translational modification (e.g. phosphorylation, glycosylation), and their potential for contamination with biologically compatible zoonotic viruses. protein expression for the production of vlp is highly amenable to modification, such as substituting strain-specific amino acid sequences [ ] , or inserting foreign peptide sequences or proteins [ , ] . manipulation of this process can facilitate the engineering of recombinant chimeric vlp containing xenogeneic antigens, inducing an immune response against targets other than the parent virus [ ] . vlp can also present self-antigens in an immunogenic context, which can be particularly useful in immunotherapeutic vaccination for cancer [ ] . a diverse range of applications for this type of modification have been investigated prophylactically and therapeutically for various conditions, including protection against infection from other organisms [ ] , autoimmune inflammatory disease [ ] , and as an antitumor immunotherapy [ , , ] . chimeric vlp have been investigated as a potential therapy for conditions not usually associated with vaccination, such as atherosclerosis [ ] , type ii diabetes mellitus [ ] , and nicotine addiction [ ] . some of these vlp vaccines utilize chemical conjugation for incorporation of antigenic sequences as opposed to recombinant insertion. vlp can serve as an effective immunogenic scaffold for chemical conjugation, compatible with a broad range of conjugation chemistries. the applications of chemical conjugation with vlp has been comprehensively reviewed in recent articles [ , [ ] [ ] [ ] ; however, it is worth noting that introducing unique forms of chemical conjugation may provide some proprietary claim over methods involving vlp vaccines already in the public domain. vlp are a form of subunit vaccine, inducing a characteristic immune response shared amongst exogenous antigens. while vlp are taken up by cells through non-specific pathways such as phagocytosis and macropinocytosis, some vlp can also utilize specialized uptake pathways inherited from their parent virus. in general, protein-based subunit vaccines like vlp are internalized into antigen-presenting cells (apcs), digested within the phagolysosome, and the resulting antigen peptides are presented loaded on major histocompatibility complex (mhc) ii molecules to cd + t helper cells (t h cells). t h cells recognize epitopes presented on mhc-ii through their specific t cell receptor (tcr), and activate dependent on the strength of this interaction, in addition to the degree of signaling through costimulatory receptors and cytokines. these additional signals are usually supplied by the apc, which is activated to increase the presentation of costimulatory receptors and the release of cytokines in response to a variety of stimulatory signals. these stimulatory signals correspond with the activation of pattern recognition receptors (prrs), such as tolllike receptors (tlrs), nod-like receptors (nlrs), and rig -like receptors (rlrs), which each recognize specific pathogenassociated molecular patterns (pamps) and damage-associated molecular patterns (damps). as vlp vaccines usually contain minimal or negligible amounts of these stimulatory molecules, these signals are instead induced by vaccine adjuvants. manipulation and selective targeting of specific apcs or other target cell populations may be an effective means of developing a novel proprietary vlp vaccine. chemical conjugation of receptor ligands onto the surface of compatible vlp should promote binding and uptake into cells expressing the corresponding receptor, while primary vlp uptake and processing pathways should continue unperturbed. this type of modification may be particular effective when targeting a specific apc population is desired for the induction of a specific type of immune response. for example, chemical conjugation of mannoside-based saccharides on the surface of rabbit hemorrhagic disease virus (rhdv) vlp selectively targets the mannose receptor expressed on the surface of apcs, inducing increased uptake and alteration of antigen cross-presentation in murine dendritic cells [ ] . chemical conjugation of unmethylated cpg oligonucleotides on the surface of rhdv vlp has also been investigated for targeting uptake through the dec receptor [ ] . similarly, coating of hiv vlp with phosphatidylserine-laced liposomes enhanced uptake into macrophages by mimicking apoptotic bodies [ ] . although the immune response to vlp may be generalized due to similarities shared with other subunit vaccines, there are some unique components of a vlp-induced immune response. the pathways for uptake of vlp into apcs vary depending on the size and morphology of each vlp, including the potential retention of receptor binding motifs. particles smaller than nm, such as norovirus p particles [ ] , can drain directly from the vaccination site into the lymph through pores in the fenestrated lymphatic vessels [ ] . trafficking of molecules within the lymphatics is likewise size dependent, with soluble proteins smaller than kda or nm transported by specialized small antigen conduits [ , ] . vlp are larger than nm, and many are instead transported passively to the lymph nodes bound on the surface of myeloid immune cells [ ] [ ] [ ] . vlp larger than nm cannot drain directly through the fenestration pores into the lymph, and instead require active transport following internalization by apcs at the vaccination site, such as in dendritic cells (dcs), macrophages and b cells [ ] . uptake of virus antigens into antigen presenting cells can occur through multiple pathways, including phagocytosis, macropinocytosis, caveolae-mediated uptake, clathrinmediated uptake and clathrin noncaveolae-mediated uptake [ ] . vlp have been identified to utilize a similar repertoire of uptake pathways, including phagocytosis [ ] , size-dependent macropinocytosis [ ] , and clathrin-dependent or independent forms of receptor-mediated endocytosis [ ] [ ] [ ] . the retention of receptor-mediated endocytosis indicates that some vlp can inherit functional receptor binding domains, and are compatible with the corresponding uptake pathways derived from their parent virus. these mechanisms of receptor-mediated uptake can also be introduced to vlp through post-expression modification, such as the chemical conjugation of superantigen on hbv vlp for internalization through mhc-ii molecules [ ] , or the chemical conjugation of transferrin on qβ vlp for internalization through the transferrin receptor, which is upregulated in some cancer cells [ ] . non-specific uptake into apcs subjects vlp to standard components of exogenous antigen processing pathways, including lysosomal fusion, enzymatic digestion, mhc-ii loading and antigen presentation (figure (a) ). parent viruses of vlp internalized through these pathways engage additional mechanisms to progress toward virus replication, including capsid disassembly or uncoating, exportation of the virus genomic material, and disruption of vesicular fusion. while the retention of intracellular processing mechanisms resembling those utilized by parent viruses has been investigated for some vlp [ , ] , it remains unclear whether vlp universally mimic the intracellular processing of their parent virus. vlp can retain access to cross-presentation pathways, facilitating presentation of antigens on mhc class i (mhc-i) for induction of a cd + t cell-mediated cytotoxic immune response. there are several known mechanisms of cross-presentation in apcs, including antigen escape from the early endosome, fusion of the endosome with the endoplastic vlp can be internalized and processed through a variety of intracellular pathways. (a) vlp internalized through non-specific pathways such as phagocytosis and macropinocytosis can be processed, with peptides presented on mhc-ii as exogenous antigen. (b) some vlp can also utilize cross-presentation pathways, facilitating the presentation of peptides on mhc-i [ ] . (c) influenza vlp, in this example an influenza virosome, is internalized through receptor-mediated endocytosis prior to fusion of its envelope with the endosomal membrane [ , , ] . (d) jcv vlp is thought to utilize the processing pathway of its parent virus to facilitate delivery of exogenous nucleic acids, including clathrin-dependent endocytosis, nuclear trafficking, and uncoating of the capsid within the nucleus [ , ] . reticulum, and recycling of mhc-i receptors from the cell surface. rhdv vlp are known to utilize the mhc-i receptor recycling pathway of cross-presentation [ ] (figure (b) ). additional examples of vlp known to induce cross-presentation of antigens include vlp derived from hbv [ , ] , hepatitis c virus (hcv) [ ] , hpv [ ] , papaya mosaic virus [ ] , and parvovirus [ ] . the ability to induce cross-presentation is important when a cellmediated cytotoxic immune response is the primary desired outcome of a vlp vaccine. the retention of receptor-mediated internalization in vlp illustrates that these mechanisms of uptake in their parent virus are autonomous, independent of the integrity of the complete virion. vlp derived from influenza a or b viruses are prime examples of this process. influenza vlp can be produced in several forms, including enveloped vlp formed by expressing the influenza matrix (m ) protein [ ] , retroviral gag proteins [ ] , or as independent virosome particles without a traditional protein core [ , ] . each of these constructs includes the expression of influenza hemagglutinin (ha) with or without neuraminidase (na). these influenza proteins are crucial for the induction of an influenza-specific immune response, and they also play essential roles the budding of influenza virus-like particles [ , , ] , and in receptor-mediated endocytosis. influenza vlp can bind to sialylated glycoproteins and glycolipids on the surface of cells from the inherent activity of ha, and are internalized through clathrin-mediated endocytosis [ , ] (figure (c)); however, alternative endocytic pathways have also been identified [ ] [ ] [ ] [ ] . as the early endosomal ph lowers, the envelope fuses with the endosome membrane. ha and na proteins distributed along the internal surface of the endosome membrane are enzymatically degraded, with peptides presented as exogenous antigens on mhc-ii (figure (c) ). specialized vlp vaccines designed to deliver a genetic payload add further complications to vlp uptake and processing. retention of the ability to deliver dna/rna requires conservation of intracellular processing pathways utilized by the parent virus for replication, or the induction of an alternative method of nucleic acid translocation. john cunningham virus (jcv) vlp is a polyomavirus vlp that has received significant attention over its ability to facilitate gene delivery and genetic manipulation [ , , ] . jcv is internalized by clathrin-dependent endocytosis upon binding to an undefined plethora of receptors and molecules known to include the serotonin receptor and sialic acid [ , ] . the virus then colocalizes to endosomes along with transferin, prior to cytosolic translocation, and subsequent trafficking through nuclear pores, mediated by the n-terminus of the vp capsid protein [ ] . the virus then uncoats, exposing the genomic material within the capsid. jcv vlp are thought to mimic this process due to their ability to deliver dna plasmids or other nucleic acids to the nucleus [ , ] (figure (d) ). vlp that demonstrate this ability to deliver nucleic acids likely utilize similar processing pathways to their parent virus [ , ] , but vlp can also be modified to utilize alternative processing pathways [ , ] . the primary desired outcome of most commercial vlp vaccines is the production of antibodies specific for the vlp parent virus. anti-vlp antibodies produced by these vaccines correspondingly neutralize the parent virus, protecting against infection. the production of anti-vlp antibodies involves the activation of a humoral immune response. b cells that specifically recognize vlp surface domains through their b cell receptor (bcr) bind and internalize vlp. binding of vlp to the bcr provides a stimulatory signal that primes the b cell for activation. most vlp consist of structurally identical capsid proteins arranged in a repetitive quasicrystalline pattern, which can crosslink multiple bcrs to provide a stimulatory advantage over other types of subunit vaccines. crosslinking of bcrs is important for inducing a robust humoral response, as b cells can ignore some monomeric soluble antigens [ ] . potent stimulation through bcr crosslinking can override inherent tolerogenic mechanisms, and can activate unresponsive or anergic b cells [ ] . highly repetitive structures also promote binding to low-affinity bcrs through multivalent, or highavidity interactions [ ] . antigen primed b cells receive additional activation signaling induced by pamps delivered with the vlp vaccine. these signals are usually provided by vaccine adjuvants, but may also be induced by other virus-associated or expressionderived endogenous molecules associated with the vlp. vlpderived antigens are presented to t h cells loaded on mhc-ii, inducing the cytokine and costimulatory receptor signaling from the t h cell that continues b cell activation. activated b cells initially become plasmablasts, a transient extrafollicular activation state that can provide some immediate antibody production [ ] . plasmablasts migrate to the follicular region of lymph nodes to form germinal centers, a specialized region where activated b cells proliferate. plasmablasts within germinal centers under affinity maturation and immunoglobulin class switching, guided by signaling from t follicular helper (t fh ) cells [ , ] . this specialized t fh cell guidance results in the development of high-affinity antibody-producing plasma cells, and long-lived memory b cells. rapid t cellindependent activation can also be induced in marginal zone b cells and b cells, providing an alternative pathway for activation and antibody production over the predominant follicular b cell population [ ] . some vlp have been identified utilizing this alternative form of b cell activation [ ] . the production of anti-vlp antibodies has been investigated and characterized amongst a broad range of vlp. this process can be influenced by various vaccine components, constituents, and even unexpected molecules, such as those derived from the vlp expression system. for example, the presence of bacterial rna packed inside qβ vlp plays a role in the induction of igg a/c antibodies in mice, and igg antibodies in humans [ , ] . qβ vlp can also induce the production of iga class antibodies in immunocompetent mice when immunized through the intranasal route [ ] , while t h cell-deficient mice required subcutaneous rather than intranasal vaccination to induce robust iga production through t-cell independent b cell activation [ ] . tlr- , simulated by singlestranded rna in endosomes, was also identified as crucial for the induction of both igg c and iga antibodies against qβ vlp in mice. this further indicates the importance of endogenous bacterial rna present in these vlp. the induction of anti-hpv vlp antibodies has also been extensively studied. although the production of secretory iga antibodies is desired for protection against viral infection at mucosal surfaces, the predominant antibody class found in secretions from the female genital tract following vaccination with hpv vlp is igg [ ] [ ] [ ] . while the presence of igg in these secretions may be due to transudation from the serum, some active transportation or local production in the mucosa may be possible [ , ] . vaccination with hpv vlp induces the production of both igg and iga antibodies in the serum and cervical secretions of humans [ ] . while serum titers of anti-hpv antibodies may initially decline post-vaccination, these levels plateau and remain stable to provide prolonged immunity [ ] . while the induction of a potent humoral immune response and the subsequent production of anti-vlp antibodies is the primary desired outcome of most commercial vlp vaccines, these is increasing appreciation for the role of vaccine-induced cell-mediated immunity [ ] [ ] [ ] . measurements of anti-vlp titers can provide an important indication of vaccine efficacy with respect to the neutralization of a virus challenge, a cellmediated immune response also plays an important role in antivirus immune defense [ , ] . activation of a cellmediated immune response can also be the primary desired outcome of vlp vaccines, particularly for chimeric or other modified vlp vaccines that target non-virus pathologies. a potent cell-mediated response to vlp vaccines is dependent upon cross-presentation of vlp-derived antigens on mhc-i. cd + t cytotoxic (t c ) cells can recognize through mhc-i antigen complexes through their tcr, and become primed for activation. these antigen-primed t c cells require additional signaling for activation, including interaction with costimulatory receptors, and cytokines from t h cells. while many vlp may be capable of cross-presentation, some may be more effective at inducing cross-presentation due to uptake and processing pathways inherited from their parent virus [ , ] . the induction of a potent cell-mediated immune response is particularly important for immunotherapeutic cancer vaccines. tumor cell-specific antibodies can enhance the inherent cytotoxic activity of natural killer cells through mechanisms such as antibody-dependent cell-mediated cytotoxicity, or directly through complement-dependent cytotoxicity; however these mechanisms tend to be restricted to passive vaccination with monoclonal antibodies, such as the her -specific monoclonal trastuzumab [ ] . a cell-mediated immune response combines target specificity with the in vivo expansion of effector cell populations, with the capacity to establish prolonged memory. rhdv vlp are particularly effective as a vector for cancer vaccines, capable of inducing the cross-presentation of vlp-derived antigens [ ] , and compatible with recombinant insertion and chemical conjugation [ ] . rhdv vlp vaccines have been investigated in models of hpvinfected cervical cancer [ ] , melanoma [ ] , lewis' lung carcinoma [ ] , and colorectal cancer [ ] . additional examples of vlp that can induce cross-presentation and a potent cellmediated immune response include qβ vlp [ ] , ms vlp [ ] , and alphavirus vlp [ ] . preexisting immunity in the form of preformed anti-vlp antibodies can be an important consideration for some vlp vaccines [ , ] . binding of anti-vlp antibodies may interfere with the normal uptake and presentation pathways of vlp vaccines. anti-vlp antibodies may be present in unvaccinated individuals due to environmental exposure to prevalent strains of the parent virus, or induced following vlp vaccination. while the presence of preexisting antibodies may decrease the efficacy or alter the immune recognition of some vlp vaccines [ , ] , they can also be associated with measures of enhanced vaccine efficacy [ ] . this may be due to accessing fc receptor-mediated uptake pathways, the formation of antibody-vlp complexes, or antigenic boosting of specific b cells. the presence of anti-vlp antibodies have also been found to have no observable effect on the intended vaccine outcome for several vlp vaccines, such as polyomavirus [ ] , and rhdv vlp [ ] . when anti-vlp antibodies present deleterious interference, alternating between different vlp vaccine vectors may be a suitable solution. alternating between rotavirus and adenovirus vlp has been found to enhance both the humoral and cell-mediated immune responses against target antigens in comparison to repeat delivery with the same vlp vector [ ] . similarly, alternating between closely related vlp such as rhdv and human norovirus (hunv) vlp can be sufficient to enhance vaccine immunogenicity [ ] . further complication can arise when vlp vaccines induce a phenomenon referred to as carrier-induced epitopic suppression (cies), in which the intended immune response is outcompeted by a strong anti-vlp response [ ] . cies may be prevented by masking vlp surface antigens, or by avoiding recognition through recombinant modification. for example, recombinant insertion of the p domain of hiv into hev vlp was identified to avoid recognition by anti-hev antibodies [ ] . the formulation of a vaccine refers to the constituents that make up the final administrable solution, including the vaccine vector, adjuvants, and excipients. for vlp vaccines, these excipients can include a variety of salts and compounds prepared as an aqueous solution or emulsion, which maintains the physical stability of vlp for enhanced shelf life of the vaccine. appropriate use of buffers can limit fluctuations in ph, while protection from fluctuations in temperature and desiccation may be provided by thermoprotectants and lyoprotectants, respectively. formulation science involves investigating the various components of a vaccine under different environmental conditions, with the intention of formulating a stable vaccine product suitable for the route of administration, and with maximized immunogenicity. the combination of formulation components, vlp vaccine preparation states and routes of administration is outlined in figure . the majority of vlp vaccines currently on the market and under clinical evaluation are liquid suspensions, ready for administration. this places strict limitations on vlp vaccine storage and distribution for safe and compliant administration. for example, the commercial hpv vaccine gardasil (merck) must be refrigerated at - °c, and protected from light. gardasil cannot be frozen, and guidelines for the use of gardasil advise that the vaccine must be used within h when removed from refrigeration at temperatures below °c, or when stored at - °c. these guidelines mirror those of many other commercial vlp vaccines, and outlines the primary stability, storage and distribution challenges for formulation science to investigate. maintaining the integrity and stability of vlp in solution is largely dependent on the combination of salts, and the buffering chemicals and compounds used. optimization of buffer ph, ionic strength, and other stabilizing components is imperative to developing a marketable, stable, liquid vlp vaccine [ ] . an investigation into the stability of ev vlp identified that sodium phosphate-based buffers were superior to citrate or tris buffered solutions, with vlp stored in sodium phosphate buffer remaining stable for month at both and °c [ ] . such prolonged stability at room and core body temperature suggests that this vlp may be suitable for importation into countries where maintaining cold-chain storage and distribution can be difficult. other important considerations for the selection of an appropriate buffer include compatibility with downstream applications, such as chemical conjugation, and the availability of scavengable nutrients that may promote the growth of potentially harmful organisms in an improperly stored vaccine. various additive molecules, particularly carbohydrates such as trehalose, sucrose and glycerol, have been investigated extensively in vlp vaccine formulations. the addition of these molecules to vaccine formulations demonstrated enhancement in vlp stability the role of formulation science in vlp vaccine manufacture includes the chemical composition of buffers, preservatives, additives and other stabilizing compounds for maintaining intact vlp. this includes protecting vlp from chemical or physical instability, and enzymatic degradation. formulations can also include targeted delivery compounds, such as muco-adhesives, and immunogenic components such as adjuvants. storage and distribution of vlp vaccines, and the subsequent route of administration are also important considerations in formulation science, critical in determining the efficacy and immunogenicity of the vaccine. within a liquid suspension of norwalk virus vlp [ ] and rotavirus vlp [ ] . other than sugar-based formulation additives, polyanionic solutions can also stabilize vlp that would otherwise be unstable at neutral ph, such as chikungunya virus vlp [ ] . the majority of commercial vlp vaccines are distributed as a liquid suspension, requiring a cold chain maintaining - °c throughout distribution and storage. even under stable cold chain conditions, the longevity of vlp can be limited. this can be prolonged by storage at temperatures at or below − °c, stabilized with the addition of a cryopreservative such as glycerol or trehalose [ ] ; however, this only further exacerbates the issue of delivering these vaccines intact where they are needed, such as in developing countries without reliable cold-chain delivery infrastructure. alternative storage methods such as freeze-drying, or lyophilization, can have variable effects on the stability of vlp. mechanical damage induced by ice crystallization, or exposure to varying salt concentrations and ph changes during the freezing process may adversely affect the integrity of vlp [ ] . exposure to these factors can be limited through the addition of specific cryoprotectants and lyoprotectants. for example, red-spotted grouper nervous necrosis virus (rgnnv) vlp [ ] reportedly retain stable particles and remain immunogenic when freezedried in the presence of sorbitol, but were adversely affected in the presence of mannitol. qβ vlp [ ] , murine polyomavirus (mupyv) vlp [ , ] , and hbv vlp [ ] have likewise demonstrated some capacity to survive varying freeze-drying methodologies. spray-drying has also gained traction as an alternative vaccine storage mechanism, avoiding the potential mechanical damage induced by freeze-drying by instead forming a dry powder formulation through a combination of nebulization and dehumidification [ ] ; however, spray-drying involves exposure to elevated temperatures, which can be similarly damaging to thermolabile vlp. hpv vlp suspended in a formulation containing mannitol, trehalose, dextran, l-leucine and inositol are capable of surviving this procedure [ ] [ ] [ ] . spray-dried ms - l vlp stored at room temperature for months were found to induce high titer anti-hpv l igg antibodies, and significantly protected vaccinated mice from challenge with hpv [ ] . air or vacuum drying methods have also been reported for coating microneedles with influenza vlp suspended in a formulation containing carboxymethylcellulose (cmc). microneedle-based delivery of dried h n (a/pr ) [ ] and h n (a/aichi) [ ] were found to induce immune responses comparable to intramuscular injection, but with the advantage of prolonged longevity in storage. investigation of similar formulation and preparation strategies with different types of vlp may uncover some universality in their application, with the potential for these methods to eradicate the cold-chain limitation imposed on many vlp vaccines. as has been previously described, many vlp possess structural or molecular features that can confer some auto-immunostimulatory properties. these properties facilitate the induction of immune responses by vlp without the need for adjuvants; however, the use of adjuvants with vlp vaccines may enhance vaccine immunogenicity, and promote the activation of a specific type of immune response. currently licensed adjuvants for use with vaccines include aluminum sulfate salts (alum), proprietary combination adjuvants such as as (glaxosmithkline), as (glaxosmithkline) and mf (novartis), thermo-reversible oil-in-water immersions, and montanide isa [ ] . while many standard vaccine adjuvants may be suitable for vlp vaccines where the generation of anti-vlp antibodies is the desired outcome, these adjuvants may fail to overcome immunotolerance and induce antitumor immunity in cancer vaccine formulations [ ] ; however, where traditional adjuvants may fail, novel tlr agonist adjuvants have had success. the tlr agonist adjuvant imiquimod was recently approved for use with vaccines for melanoma, and another clinical trial is assessing its candidacy for treatment of bladder cancer [ ] . tlr / stimulate anti-viral interferon pathways in apcs, promoting the activation of cd + t cells and nk cells [ , ] . the use of the adjuvant gardiquimod in combination with vaccination has been found to induce tumor regression in murine models of melanoma [ ] , human hepatocellular carcinoma [ ] and pancreatic cancer [ ] . tlr agonist adjuvants such as unmethylated cpg oligonucleotides have drawn considerable interest due to their inherent ability to associate with some vlp [ , ] . similarly, the presence of other nucleic acids can have downstream ramifications on the immune response induced by vlp. for example, the presence of rna inside qβ vlp can skew the humoral response induced in mice to produce igg a antibodies, while the removal of this rna results in the production of igg antibodies [ ] . qβ vlp can also contain additional adjuvant molecules derived from their expression system, encapsidated during particle formation. a recent study in mice investigated the use of various adjuvants in conjunction with a filovirus vlp vaccine, including the tlr agonist adjuvant poly-iclc (hiltonol), tlr agonist adjuvant monophospholipid a (mpla), cpg odn and alhydrogel [ ] . poly-iclc was found to induce a predominantly t h response, with an igg c subtype antibody production that correlated with protection from virus challenge. in comparison, administration of the vlp vaccine with alhydrogel elicited a strong t h response with high antibody titers, but conferred no protection from virus challenge. this study highlighted the importance of carefully considering what kind of adjuvant is suitable to accompany a vlp vaccine, as the most immunogenic adjuvant may not necessarily provide the desired vaccine outcome. vlp vaccines approved for clinical use have utilized various administration modalities, including vaccination subcutaneously, intradermally, intramuscularly or at mucosal surfaces [ ] . live attenuated vaccines are traditionally injected subcutaneously, while inactivated or subunit vaccines are often administered intramuscularly. in infants, intramuscular injection of vaccines tends to confer enhanced immunogenicity over subcutaneous administration [ ] . modern vaccine delivery technologies, such as intradermal microneedle patches, have also been investigated for their potential applications with vlp vaccines [ , , ] . while the majority of recent clinical trials involving the administration of vlp favor intramuscular injection, alternative routes of administration are being investigated based on differential immunogenicity at different delivery sites. for example, an investigation into the routes of administration of hunv vlp found that intranasal inoculation of mice induced the production of mucosal anti-hunv igg and iga antibodies, while intramuscular injection only induced igg production [ ] . the study identified that only mucosal iga was suitable for neutralization of infectious hunv virions, rendering intramuscular injection unsuitable for administration of hunv vlp. a similar finding was identified regarding the administration of respiratory syncytial virus (rsv) vlp, with the desired t h response, neutralizing mucosal antibodies and cd + t cell responses identified following intranasal inoculation, rather than intramuscular injection [ ] . vaccination with vlp at mucosal surfaces also requires higher doses of vlp in comparison to parental administration. oral delivery of virus-like particles is also being investigated as an alternative, convenient route of administration. vlp expressed in plant-based systems represents a particularly efficient means of both production and delivery, with expression in an edible plant species potentially forgoing the need for vlp purification and vaccine formulation. examples of this concept include the expression of hbv and norwalk virus vlp in solanum tuberosum potato and lycopersicon esculentum tomato plants [ , ] . nicotiana benthamiana tobacco are also used as an efficient plant expression system for production of vlp. hbv vlp purified from n. benthamiana are capable of withstanding a simulation of the acidic environment within the stomach [ ] ; however, the hbcag polypeptide of hbv vlp was digested when exposed to porcine pepsin. the protection of protein-based vlp from enzymatic digestion is a significant hurdle for oral delivery, despite the promise of this vaccination route. the translation of vlp vaccines from preclinical research to routine clinical administration is a multifaceted task combining studies into stability, formulation science, immunogenicity, and clinical vaccine efficacy. these issues are not necessarily unique to the development of vlp vaccines, encompassing the breadth of research and development necessary for bringing any vaccine candidate to market. the expression of stable vlp is only the first step toward the development of a novel vlp vaccine, which may eventually include various excipients, adjuvants and other compounds in the administrable form. advancements in virology and vaccinology that have facilitated the development of novel vlp vaccines seemingly parallel regulatory and proprietary restrictions placed upon new vaccines; however, vlp vaccines also possess unparalleled potential due to their balance between clinical vaccine efficacy and safety, and their versatility as a vaccine vector. the field of vlp vaccines has continued to experience significant growth over the past decade, both in the diversity of vaccines and in the translation of vaccines toward routine clinical administration. our research focuses upon the development of rhdv vlp as a versatile vaccine scaffold, particularly for immunotherapeutic vaccination as an alternative treatment option for cancer. the translation of vlp vaccine constructs from proof of concept models toward clinical administration is no menial feat. we have observed a corresponding shift toward focusing on clinical translation amongst our colleagues in the field. focal points of discussion highlighted by the process include the relevance and translatability of research animal models, adaptation to the established dogma within the field of human vaccination, and the establishment of a developmental and commercial niche conducive to the advancement of novel vaccines throughout the rigors of clinical trials and regulatory approval. the immune response to vlp vaccines tends to strike a desirable balance between outcomes indicative of optimal vaccine efficacy, such as the induction of high-titer mucosal iga antibodies, while also maintaining an almost unparalleled vaccine safety profile. the inherent inability of vlp to infect or replicate alleviates potential vaccine risks, such as spontaneous reversion to pathogenesis, or incomplete inactivation. retention of the ability to facilitate cross-presentation of associated antigens, and the induction of a potent cell-mediated immune response can also have significant implications for vlp vaccines, diversifying the field to cover a broader range of disorders and diseases. the intracellular processing pathways remain largely unexplored for many vlp, and elucidation of the underlying mechanisms involved in some of the more advanced applications of vlp vaccines, such as gene delivery and immunomodulation. the role of formulation science appears underrepresented amongst clinical trials and clinical reports for vlp vaccines, with at times minimal elucidation of the vaccine administration route, the excipients included, and even exclusion of adjuvant delivered with the vaccine. the importance of formulation science and the relevance of excipients and adjuvants may be expectedly underappreciated during earlier phases of research and development, where establishment of vaccine efficacy may be paramount; however, early adoption of these vital constituents may ease clinical translation, potentially expanding the repertoire of marketable vlp vaccines. novelty, proprietary design, and versatility are almost as important in the development of vlp vaccines as the actual vaccine efficacy itself. maintaining compatibility with the upscaling of production and gmp manufacture pipelines is another important component in the development of a vlp vaccine for routine clinical administration. the benefit of such foresight is clear, and the promotion of clinical translation will be advantageous across the field of vlp vaccines. upon review across the field including current trends in new and emerging research, the progression of established vaccine research, and clinical trial schedules, vlp vaccine research is experiencing a period of rapid growth. this includes both the repertoire of organisms and conditions being targeted with vlp vaccines, and the advancement of established vaccines through clinical trials, translation, and clinical applications. over the next five years, we predict that there will be a significant increase in the diversity of vlp vaccines in active circulation. we expect that vlp will be used in more complex applications than as a vaccine against the cognate virus from which they were derived. we also predict the development of novel vlp vaccine constructs for human and zoonotic pathogenic organisms and conditions with or without current vaccination options, and the incorporation of advanced vaccination methodologies and techniques into routine vlp vaccine production, distribution and administration. in particular, we predict increased incorporation of alternative vaccine-storage techniques such as freeze-dried or spray-dried methods. the development of vlp vaccines is often focused on facilitating distribution amongst populations and communities where these vaccines are most needed. we predict that over the next five years, the capability to support vlp vaccine distribution will increase by limiting the current reliance on cold-chain delivery. these predictions outline an exciting future over the next five years for vlp vaccine research and development. • virus-like particles consist of virus-derived proteins and associated molecules that spontaneously form a particulate structure. • vlp vaccines have the safety profile of a subunit vaccine, but with efficacy and vaccination outcomes that can be comparable to killed or live attenuated vaccines, depending on the particular vlp vaccine. • while the predominant desired immune response amongst the majority of vlp vaccines is a potent humoral response producing high titer antibodies, some vlp vaccines can induce a potent cytotoxic immune response for selective elimination of cells currently infected by the target virus, or target tumor cells. • formulation science plays a major role in the development of vlp vaccines, facilitating the selection of appropriate combinations of excipients and adjuvants. excipients are used to increase vlp particle stability in the vaccine formulation, while adjuvants enhance vaccine efficacy, and help to select for a specific desired immune response and outcome. this research was funded by the university of otago. interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. braeden donaldson http://orcid.org/ - - - vernon k. ward http://orcid.org/ - - - papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers antigen delivery by virus-like particles for immunotherapeutic vaccination this review covers the structural diversity of vlp, post-production modification, and the immune response to vlp vaccines with a specific focus on immunotherapeutic vaccine development immunogenicity of a quadrivalent virus-like particles (vlp) influenza vaccine in healthy adults. nct immunogenicity, safety and tolerability of a plant-derived seasonal virus-like-particle quadrivalent influenza vaccine in adults. nct national institute of allergy and infectious diseases (niaid) study to evaluate safety, tolerability, and immunogenicity of candidate human cytomegalovirus vaccine in healthy adults. nct efficacy and immunogenicity of norovirus gi. /gii. bivalent virus-like particle vaccine in adults. nct . takeda long-term immunogenicity of the norovirus gi.i/gii. bivalent virus-like particle (vlp) vaccine in adults. nct . takeda safety and immunogenicity of norovirus gi. /gii. bivalent virus-like particle vaccine in an elderly population. nct . takeda disassembly and reassembly improves morphology and thermal stability of human papillomavirus type virus-like particles this research article investigates the effects of disassembly and reassembly on the morphological consistency and thermal stability of human papillomavirus vlp. disassembly and reassembly is including as a post-production modification in the manufacture of select commercial vlp vaccines inventors; cytos biotechnology ag, assignee. vlp-antigen conjugates and their uses as vaccines gmp issues for recombinant plant-derived pharmaceutical proteins methodological issues for trials of vaccine efficacy against hpv types and raising expectations for subunit vaccine intellectual property policies in early-phase research in public-private partnerships vaccine instability in the cold chain: mechanisms, analysis and formulation strategies improved storage stability and immunogenicity of hepatitis b vaccine after spray-freeze drying in presence of sugars expression of animal virus genomes novel epstein-barr virus-like particles incorporating gh/gl-ebna or gb-lmp induce high neutralizing antibody titers and ebv-specific t-cell responses in immunized mice expression and characterization of yeast derived chikungunya virus like particles (chik-vlps) and its evaluation as a potential vaccine candidate viral protein requirements for assembly and release of human parainfluenza virus type viruslike particles cell-specific delivery of diverse cargos by bacteriophage ms virus-like particles structure of small virus-like particles assembled from the l protein of human papillomavirus influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles influenza virus-like particles comprised of the ha, na, and m proteins of h n influenza virus induce protective immune responses in balb/c mice. vaccine dynamic of immune response induced in hepatitis b surface antigen-transgenic mice immunized with a novel therapeutic formulation recombinant hepatitis b vaccine (engerix-b): a review of its immunogenicity and protective efficacy against hepatitis b [review] immunological characterization of two hepatitis b core antigen variants and their immunoenhancing effect on co-delivered hepatitis b surface antigen characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells assembly of double-shelled rotaviruslike particles by simultaneous expression of recombinant vp and vp proteins expression of human immunodeficiency virus type gag protein precursor and envelope proteins from a vesicular stomatitis virus recombinant: highlevel production of virus-like particles containing hiv envelope paramyxovirus sendai virus-like particle formation by expression of multiple viral proteins and acceleration of its release by c protein a virosomal formulated her- /neu multi-peptide vaccine induces her- /neu-specific immune responses in patients with metastatic breast cancer: a phase i study influenza virus-like particle can accommodate multiple subtypes of hemagglutinin and protect from multiple influenza types and subtypes. vaccine generating enveloped virus-like particles with in vitro assembled cores core-like particles of an enveloped animal virus can self-assemble efficiently on artificial templates human parvovirus b virus-like particles: in vitro assembly and stability engineering hepatitis b virus core particles for targeting her receptors in vitro and in vivo novel adenovirus encoded virus-like particles displaying the placental malaria associated var csa antigen virus-like particle display of the α-gal carbohydrate for vaccination against leishmania infection molecular pharming-vlps made in plants the large-scale production of an artificial influenza virus-like particle vaccine in silkworm pupae virus-like particles: the future of microbial factories and cell-free systems as platforms for vaccine development chimeric gii. norovirus virus-like-particle-based vaccines induce broadly blocking immune responses chimeric bivalent virus-like particle vaccine for h n hpai and nd confers protection against a lethal challenge in chickens and allows a strategy of differentiating infected from vaccinated animals (diva) novel recombinant chimeric viruslike particle is immunogenic and protective against both enterovirus and coxsackievirus a in mice multi-target chimaeric vlp as a therapeutic vaccine in a model of colorectal cancer an enhanced heterologous virus-like particle for human papillomavirus type tumour immunotherapy protective effect of a germline, il- -neutralizing antibody in murine models of autoimmune inflammatory disease virus-like particles from rabbit hemorrhagic disease virus can induce an anti-tumor response memory and effector cd t-cell responses after nanoparticle vaccination of melanoma patients a vlp-based vaccine against interleukin- α protects mice from atherosclerosis development of an interleukin- β vaccine in patients with type diabetes a vaccine against nicotine for smoking cessation: a randomized controlled trial virus-like particles, a versatile subunit vaccine platform bioengineering virus-like particles as vaccines construction and characterization of virus-like particles: a review this review covers the breadth of expression systems available for vlp vaccine production, while also discussing vlp structural diversity and characterization mannosylation of virus-like particles enhances internalization by antigen presenting cells coupling the adjuvant cpg oligonucleotides to rhdv vlp. dunedin: university of otago αenv-decorated phosphatidylserine liposomes trigger phagocytosis of hiv-virus-like particles in macrophages norovirus p particle: a subviral nanoparticle for vaccine development against norovirus, rotavirus and influenza virus nanoparticles target distinct dendritic cell populations according to their size conduits mediate transport of low-molecular-weight antigen to lymph node follicles the humoral immune response is initiated in lymph nodes by b cells that acquire soluble antigen directly in the follicles virus-like particle (vlp) lymphatic trafficking and immune response generation after immunization by different routes follicular shuttling of marginal zone b cells facilitates antigen transport b cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node a molecular assembly system that renders antigens of choice highly repetitive for induction of protective b cell responses. vaccine dissecting virus entry via endocytosis despite differences between dendritic cells and langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime t cells. virology cross-presentation of epitopes on virus-like particles via the mhc i receptor recycling pathway the length of vesicular stomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes differential uptake and crosspresentation of human papillomavirus virus-like particles by dendritic cells and langerhans cells an engineered non-toxic superantigen increases cross presentation of hepatitis b virus nucleocapsids by human dendritic cells multivalent display and receptormediated endocytosis of transferrin on virus-like particles low ph-dependent endosomal processing of the incoming parvovirus minute virus of mice virion leads to externalization of the vp n-terminal sequence (n-vp ), n-vp cleavage, and uncoating of the full-length genome efficient gene transfer using the human jc virus-like particle that inhibits human colon adenocarcinoma growth in a nude mouse model cross-presentation of virus-like particles by skin-derived cd -dendritic cells: a dispensable role for tap hepatitis b virus-like particles access major histocompatibility class i and ii antigen presentation pathways in primary dendritic cells uptake and presentation of hepatitis c virus-like particles by human dendritic cells proteasome-independent major histocompatibility complex class i cross-presentation mediated by papaya mosaic virus-like particles leads to expansion of specific human t cells in vivo, dendritic cells can crosspresent virus-like particles using an endosome-to-cytosol pathway influenza-pseudotyped gag virus-like particle vaccines provide broad protection against highly pathogenic avian influenza challenge eleven years of inflexal ® v-a virosomal adjuvanted influenza vaccine inflexal ® v a trivalent virosome subunit influenza vaccine: production influenza virus assembly and budding in raftderived microdomains: a quantitative analysis of the surface distribution of ha, na and m proteins influenza virus m ion channel protein is necessary for filamentous virion formation infectious entry pathway of influenza virus in a canine kidney cell line studies on the mechanism of influenza virus entry into cells epsin is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway endocytosis of influenza viruses influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis gene therapy for human lung adenocarcinoma using a suicide gene driven by a lung-specific promoter delivered by jc virus-like particles in vivo sirna delivery using jc virus-like particles decreases the expression of rankl in rats the human polyomavirus, jcv, uses serotonin receptors to infect cells jc virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis contrasting roles of endosomal ph and the cytoskeleton in infection of human glial cells by jc virus and simian virus use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles human jc virus-like particles as a gene delivery vector encapsulation and delivery of plasmid dna by virus-like nanoparticles engineered from macrobrachium rosenbergii nodavirus a novel polyethyleneimine-coated adeno-associated virus-like particle formulation for efficient sirna delivery in breast cancer therapy: preparation and in vitro analysis gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified dna encapsidation capacity by addition of packaging sequences from the l or l protein of human papillomavirus type novel mir- delivery system based on ms virus like particle surface displaying cell-penetrating peptide tat for hepatocellular carcinoma neutralizing antiviral b cell responses virus-like display of a neo-self antigen reverses b cell anergy in a b cell receptor transgenic mouse model low-affinity b cells transport viral particles from the lung to the spleen to initiate antibody responses this research article investigates the transportation of vlp following intranasal delivery, mediated by low-affinity b cells. intranasal vaccination and the induction of mucosal iga anti-vlp production is an important vaccination outcome for vlp vaccines against viruses that infect through mucosal surfaces extrafollicular antibody responses germinal center b and follicular helper t cells: siblings, cousins or just good friends competence and competition: the challenge of becoming a long-lived plasma cell plasma cell development: from b-cell subsets to long-term survival niches b lymphocyte activation by human papillomavirus-like particles directly induces ig class switch recombination via tlr -myd t cell-dependent and-independent iga responses: role of tlr signalling alveolar macrophages and lung dendritic cells sense rna and drive mucosal iga responses efficient induction of mucosal and systemic immune responses by virus-like particles administered intranasally: implications for vaccine design mucosal immunity in the female genital tract mucosal immunity in the female genital tract: relevance to vaccination efforts against the human immunodeficiency virus a comparison of antibody titres in mouse uterine fluid after immunization by several routes, and the effect of the uterus on antibody titres in vaginal fluid systemic and secretory humoral immunity in the normal human vaginal tract comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women specific antibody levels at the cervix during the menstrual cycle of women vaccinated with human papillomavirus virus-like particles induction of immune memory following administration of a prophylactic quadrivalent human papillomavirus (hpv) types / / / l virus-like particle (vlp) vaccine. vaccine induction of virusspecific cytotoxic t lymphocytes as a basis for the development of broadly protective influenza vaccines influenza vaccines: t-cell responses deserve more attention this review article discusses the importance of inducing a potent cytotoxic immune response for vaccination against select virus infections t-cell-mediated and humoral approaches to universal influenza vaccines better influenza vaccines for older people: what will it take? influenza hemagglutinationinhibition antibody titer as a correlate of vaccine-induced protection efficient homologous prime-boost strategies for t cell vaccination based on virus-like particles use of chemotherapy plus a monoclonal antibody against her for metastatic breast cancer that overexpresses her antigen incorporated in virus-like particles is delivered to specific dendritic cell subsets that induce an effective antitumor immune response in vivo messenger rna vaccine based on recombinant ms virus-like particles against prostate cancer anti-tumor effect of the alphavirus-based virus-like particle vector expressing prostate-specific antigen in a hla-dr transgenic mouse model of prostate cancer universal influenza a m e-hbc vaccine protects against disease even in the presence of preexisting anti-hbc antibodies segments of puumala hantavirus nucleocapsid protein inserted into chimeric polyomavirus-derived virus-like particles induce a strong immune response in mice carrier induced epitopic suppression of antibody responses induced by virus-like particles is a dynamic phenomenon caused by carrier-specific antibodies. vaccine innate immunity mediates follicular transport of particulate but not soluble protein antigen this research article investigates the transportation and delivery of qβ vlp to follicular dendritic cells within the lymph node, and the impairment of this delivery in the presence of anti-qβ vlp antibodies effects of preexisting anti-carrier immunity and antigenic element multiplicity on efficacy of a modular virus-like particle vaccine immunomodulation and vaccination with rhdv vlp: a thesis submitted for the degree of doctor of philosophy prime immunization with rotavirus vlp / followed by boosting with an adenovirus expressing vp induces protective immunization against rotavirus in mice can different virus-like particles be used for primeboost vaccination? dunedin: university of otago chimeric hepatitis e virus-like particle as a carrier for oral-delivery. vaccine evaluation of the stability of enterovirus virus-like particle physical stabilization of norwalk virus-like particles downstream processing of triple layered rotavirus like particles development of a stable viruslike particle vaccine formulation against chikungunya virus and investigation of the effects of polyanions stability studies of hiv- pr gag virus-like particles made in insect cells after storage in various formulation media rational design of a stable, freezedried virus-like particle-based vaccine formulation stability of virus-like particles of redspotted grouper nervous necrosis virus in the aqueous state, and the vaccine potential of lyophilized particles modular virus-like particles for sublingual vaccination against group a streptococcus. vaccine virus-like particle formulation optimization by miniaturized high-throughput screening freeze-drying of plant tissue containing hbv surface antigen for the oral vaccine against hepatitis b developments in the formulation and delivery of spray dried vaccines preclinical refinements of a broadly protective vlp-based hpv vaccine targeting the minor capsid protein, l . vaccine optimized formulation of a thermostable spray-dried virus-like particle vaccine against human papillomavirus characterization of a spraydried candidate hpv l -vlp vaccine stored for multiple years at room temperature. papillomavirus res this research article demonstrates the compatibility of a recombinant ms - l vlp vaccine with spray-drying, and the retention of vaccine immunogenicity after prolonged storage at room temperature immunization by vaccine-coated microneedle arrays protects against lethal influenza virus challenge transdermal influenza immunization with vaccine-coated microneedle arrays vaccine adjuvants: from to and beyond vaccine adjuvants as potential cancer immunotherapeutics distinct indirect pathways govern human nk-cell activation by tlr- and tlr- agonists tlr / -mediated activation of human nk cells results in accessory cell-dependent ifn-gamma production the tlr agonists imiquimod and gardiquimod improve dc-based immunotherapy for melanoma in mice tlr / agonists promote nk-dc crosstalk to enhance nk cell anti-tumor effects in hepatocellular carcinoma activation of toll-like receptor inhibits the proliferation and migration, and induces the apoptosis of pancreatic cancer cells immunotherapy of type- allergies with virus-like particles and cpg-motifs a universal virus-like particle-based vaccine for human papillomavirus: longevity of protection and role of endogenous and exogenous adjuvants. vaccine adjuvant-enhanced cd t cell responses are critical to durable vaccine immunity influence of parenteral administration routes and additional factors on vaccine safety and immunogenicity: a review of recent literature immunogenicity and safety of measles-mumps-rubella-varicella (mmrv) vaccine followed by one dose of varicella vaccine in children aged months- years or - years primed with measles-mumps-rubella (mmr) vaccine long-term protective immunity from an influenza virus-like particle vaccine administered with a microneedle patch mucosal antibodies induced by intranasal but not intramuscular immunization block norovirus gii. virus-like particle receptor binding a single intranasal administration of virus-like particle vaccine induces an efficient protection for mice against human respiratory syncytial virus virus-like particle expression and assembly in plants: hepatitis b and norwalk viruses expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice plant-expressed hepatitis b core antigen virus-like particles: characterization and investigation of their stability in simulated and pig gastro-intestinal fluids key: cord- -c v jz authors: chae, sangwon; kwon, sungjun; lee, donghyun title: predicting infectious disease using deep learning and big data date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: c v jz infectious disease occurs when a person is infected by a pathogen from another person or an animal. it is a problem that causes harm at both individual and macro scales. the korea center for disease control (kcdc) operates a surveillance system to minimize infectious disease contagions. however, in this system, it is difficult to immediately act against infectious disease because of missing and delayed reports. moreover, infectious disease trends are not known, which means prediction is not easy. this study predicts infectious diseases by optimizing the parameters of deep learning algorithms while considering big data including social media data. the performance of the deep neural network (dnn) and long-short term memory (lstm) learning models were compared with the autoregressive integrated moving average (arima) when predicting three infectious diseases one week into the future. the results show that the dnn and lstm models perform better than arima. when predicting chickenpox, the top- dnn and lstm models improved average performance by % and %, respectively. the dnn model performed stably and the lstm model was more accurate when infectious disease was spreading. we believe that this study’s models can help eliminate reporting delays in existing surveillance systems and, therefore, minimize costs to society. infectious disease occurs when a person is infected by a pathogen from another person or an animal. it not only harms individuals, but also causes harm on a macro scale and, therefore, is regarded as a social problem [ ] . at the korea center for disease control (kcdc), infectious disease surveillance is a comprehensive process in which information on infectious disease outbreaks and vectors are continuously and systematically collected, analyzed, and interpreted. moreover, the results are distributed quickly to people who need them to prevent and control infectious disease. the kcdc operates a mandatory surveillance system in which mandatory reports are made without delay to the relevant health center when an infectious disease occurs and it operates a sentinel surveillance system in which the medical organization that has been designated as the sentinel reports to the relevant health center within seven days. the targets of mandatory surveillance consist of a total of infectious diseases from groups to by the kcdc. the targets of sentinel surveillance include influenza from group along with infectious diseases from group . overall, a total of infectious diseases in six groups are monitored. in the current korean infectious disease reporting system, if there is a legally defined infectious disease patient at a medical organization, a report is made to the managing health center through the infectious disease web reporting system. the managing health center reports to the city and province health offices through another system and the city and province health offices report to the kcdc. in the conventional reporting system, some medical organizations' infectious disease reports are incomplete and delays can occur in the reporting system. for instance, in the traditional influenza surveillance system, around two weeks elapses between when a report is made and when it is disseminated [ ] . the kcdc has been running an automated infectious disease reporting system as a pilot project since . however, by , only . % of all medical organizations were participating in the pilot project. in medical organizations using the conventional infectious disease reporting system, a large number of missing and delayed reports can occur, which hinders a prompt response to infectious disease. as such, it is necessary to create a data-based infectious disease prediction model to handle situations in real time. furthermore, if this model can understand the extent of infectious disease trends, the costs to society from infectious disease can be minimized. an increasing number of researchers recognize these facts and are performing data-based infectious disease surveillance studies to supplement existing systems and design new models [ ] [ ] [ ] [ ] [ ] [ ] [ ] . among these, studies are currently being performed on detecting infectious disease using big data such as internet search queries [ ] [ ] [ ] [ ] [ ] [ ] . the internet search data can be gathered and processed at a speed that is close to real time. according to towers et al., internet search data can create surveillance data faster than conventional surveillance systems [ ] . for example, when huang et al. predicted hand, foot, and mouth disease using the generalized additive model (gam), the model that included search query data obtained the best results. as such, it has been reported that new big data surveillance tools have the advantage of being easy to access and can identify infectious disease trends before official organizations [ ] . in addition to internet search data, social media big data is also being considered. tenkanen et al. report that social media big data is relatively easy to collect and can be used freely, which means accessibility is satisfactory and the data is created continuously in real time with rich content [ ] . as such, studies have used twitter data to predict the occurrences of mental illness [ ] and infectious disease [ ] [ ] [ ] [ ] in addition to predictions in a variety of other scientific fields [ ] [ ] [ ] [ ] . in particular, a study by shin et al. reported that infectious diseases and twitter data are highly correlated. there is the possibility of using digital surveillance systems to monitor infectious disease in the future [ ] . when these points are considered, using search query data and social media big data should have a positive effect on infectious disease predictions. in addition to these studies, there are also studies that have used techniques from the field of deep learning to predict infectious disease [ , , , ] . deep learning is an analysis method and, like big data, it is being actively used in a variety of fields [ ] . deep learning yields satisfactory results when it is used to perform tasks that are difficult for conventional analysis methods [ ] [ ] [ ] . in a study by xu et al., a model that used deep learning yielded better prediction performance than the generalized linear model (glm), the least absolute shrinkage and selection operator (lasso) model, and the autoregressive integrated moving average (arima) model [ ] . as such, methods of predicting infectious disease that use deep learning are helpful for designing effective models. there are also examples of infectious disease prediction based on environmental factors such as weather [ ] [ ] [ ] [ ] . previous studies have confirmed that weather data comprises a factor that has a great influence on the occurrence of infectious diseases [ ] [ ] [ ] . liang et al. showed that rainfall and humidity are risk factors for a hemorrhagic fever with a renal syndrome [ ] . in addition, a study by huang et al. reported that trends in dengue fever show a strong correlation with temperature and humidity [ ] . previous studies indicate that infectious disease can be predicted more effectively if weather variables, internet big data, and deep learning are used. most previous research has attempted to predict infectious disease using internet search query data alone. however, as discussed above, it is necessary to also consider various big data and environmental factors such as weather when predicting infectious disease. in addition, in the case of models that use deep learning, it is possible to improve prediction performance by optimizing the deep learning model by optimizing its parameters. therefore, the aim of this study is to design a model that uses the infectious disease occurrence data provided by the kcdc, search query data from search engines that are specialized for south korea, twitter social media big data, and weather data such as temperature and humidity. according to a study by kwon et al., a model that considers the time difference between clinical and non-clinical data can detect infectious disease outbreaks one to two weeks before current surveillance systems [ ] . therefore, this study adds lag to the collected dataset to take temporal characteristics into account. in addition, in the design process, a thorough testing of all the input variable combinations is performed to examine the effects of each resulting dataset on infectious disease outbreaks and select the optimal model with the most explanatory power. the model's prediction performance is verified by comparing it with an infectious disease prediction model that uses a deep learning method and an infectious disease prediction model that uses time series analysis. ultimately, using the results obtained by this study, it should be possible to create a model that can predict trends about the occurrence of infectious disease in real time. such a model can not only eliminate the reporting time differences in conventional surveillance systems but also minimize the societal costs and economic losses caused by infectious disease. the remainder of this paper is organized as follows. section describes the data sources and standards used in this study and introduces the analysis methodology used to design the prediction model. in section , the analysis results are described and their implications are discussed. section discusses the results. section concludes the paper. as mentioned above, this study uses four kinds of data to predict infectious disease, which includes search query data, social media big data, temperature, and humidity. the standards for the non-clinical data are as follows. data from days between january, and july, was used. the infectious diseases selected for this study are subject to mandatory reporting. unlike those diseases subject to mandatory reporting, diseases subject to sentinel reporting aggregate data on a weekly basis. since prediction is also performed on a weekly basis, it is difficult to cope with infectious diseases in real time. therefore, diseases that are subject to sentinel reporting were excluded from the study. moreover, the study excluded infectious diseases with an annual occurrence rate of less than as well as infectious diseases that have a statistically insignificant model with an adjusted r-squared value of less than . when regression analysis is performed using all variables. three infectious diseases satisfied all conditions, which include malaria, chickenpox, and scarlet fever. the search data was collected from the naver data lab (https://datalab.naver.com/keyword/trendsearch.naver). the usage share data provided by internettrend (http://internettrend.co.kr/trendforward.tsp) on search engines in the health/medicine field in the first half of shows that the naver search engine had the highest usage share ( . %) in south korea. therefore, it was chosen as the search engine for extracting search data. note that the collected search data consists of only korean terms because the search engine is specific to south korea. the search queries used in this study consisted of the infectious disease's proper name and symptoms (e.g., "chickenpox" and "chickenpox symptoms" in korea). the frequency of inquiries using these search queries were used as the search data. the number of searches were normalized with respect to the largest number of searches within the study period. weather data (temperature and humidity) were collected from the korea meteorological administration's weather information open portal (https://data.kma.go.kr). hourly data collected from weather stations nationwide was converted into daily average data for each station. in gyeonggi-do province, where around half of south korea's population lives, there are many weather stations crowded together. there was a concern that simply finding the averages of the daily data for each station would cause errors to occur, so the following process was performed. first, the averages of the data from each station were collected for the eight provinces in south korea (gyeonggi-do, gangwon-do, chungcheongnam-do, chungcheongbuk-do, jeollanam-do, jeollabuk-do, gyeongsangbuk-do, and gyeongsangnam-do). next, the averages of the data for each of the eight provinces were found to obtain south korea's national average weather data. average temperature (degrees celsius) and average humidity (percentage) were recorded. social media big data was collected for each infectious disease from twitter through a web crawler that used the python selenium library. for the twitter data, the daily number of tweets mentioning infectious disease was recorded. lastly, infectious disease data was collected from the infectious disease web statistics system (https://is.cdc.go.kr/dstat/index.jsp). this data consists of the daily number of people who were infected throughout south korea. table shows the sources and descriptions of the data. table shows the statistics for each of the infectious disease variables used in this study. in the case of temperature and humidity, the same conditions were used, which means they were put in a shared category. the data in table shows that an average of . people are infected with chickenpox daily with a standard deviation of . and the daily naver frequency average is . with a standard deviation of . . we observed that all the statistics for chickenpox are higher than those for other infectious diseases. figure shows the overall framework of the model used in this study including the data collection process and the comparison of models designed using the deep neural network (dnn) method, the long-short term memory (lstm) method, the autoregressive integrated moving average (arima) method, and the ordinary least squares (ols) method. this study constructed an infectious disease surveillance model that uses non-clinical search data, twitter data, and weather data. to design the optimal prediction model, the ols models that use all possible combinations of variables in the dataset were created. the adjusted r-squared values of each model were compared. in addition, lags of - days were added to each infectious disease and their adjusted r-squared values were compared in a preliminary analysis. a lag of seven days, which had high explanatory power for all infectious diseases, was selected as the optimal lag parameter. the optimal parameters were used to create the ols, arima, dnn, and lstm models. before analysis, this study applied a lag of seven days between the input variables (optimal variable combination) and their associated output variable (disease occurrence). the ols dataset was divided into a training data subset and a test data subset using a ratio of : . this means all rows of collected data were divided such that there were rows for the training data subset and rows for the test data subset. the training data subset was only used for model training. the test data subset was only used for prediction and performance evaluation in the model after training. the arima dataset was also divided into a training data subset and test data subset using a ratio of : , but only the disease occurrences were required for arima. similarly to the data above, the rows of disease occurrence data were divided into rows for the training data subset and rows for the test data subset. in the dnn and lstm models, the whole dataset was divided into training, validation, and test data subsets at a ratio of : : and training was performed. this means all rows of collected data were divided into rows for the training data subset, rows for the validation data subset, and rows for the test data subset. the training data subset was used for model training. the validation data subset was only used for performance evaluation during training. the final model after training was the model that yielded the best performance when the validation data subset was used in training. the test data subset was only used for the prediction and performance evaluation. to compare the models, the root mean squared error (rmse) was used to evaluate the prediction rates. rmse is a common measurement for the difference between predicted and actual values. it is usually used in the other fields as well as in the prediction of infectious diseases [ , , ] . rmse is calculated using the equation below. ( ) this study constructed an infectious disease surveillance model that uses non-clinical search data, twitter data, and weather data. to design the optimal prediction model, the ols models that use all possible combinations of variables in the dataset were created. the adjusted r-squared values of each model were compared. in addition, lags of - days were added to each infectious disease and their adjusted r-squared values were compared in a preliminary analysis. a lag of seven days, which had high explanatory power for all infectious diseases, was selected as the optimal lag parameter. the optimal parameters were used to create the ols, arima, dnn, and lstm models. before analysis, this study applied a lag of seven days between the input variables (optimal variable combination) and their associated output variable (disease occurrence). the ols dataset was divided into a training data subset and a test data subset using a ratio of : . this means all rows of collected data were divided such that there were rows for the training data subset and rows for the test data subset. the training data subset was only used for model training. the test data subset was only used for prediction and performance evaluation in the model after training. the arima dataset was also divided into a training data subset and test data subset using a ratio of : , but only the disease occurrences were required for arima. similarly to the data above, the rows of disease occurrence data were divided into rows for the training data subset and rows for the test data subset. in the dnn and lstm models, the whole dataset was divided into training, validation, and test data subsets at a ratio of : : and training was performed. this means all rows of collected data were divided into rows for the training data subset, rows for the validation data subset, and rows for the test data subset. the training data subset was used for model training. the validation data subset was only used for performance evaluation during training. the final model after training was the model that yielded the best performance when the validation data subset was used in training. the test data subset was only used for the prediction and performance evaluation. to compare the models, the root mean squared error (rmse) was used to evaluate the prediction rates. rmse is a common measurement for the difference between predicted and actual values. it is usually used in the other fields as well as in the prediction of infectious diseases [ , , ] . rmse is calculated using the equation below. the optimal variable combinations for the model were selected by considering all possible models in the regression analysis. the models are combinations of the four types of data in the dataset (naver searches (n), twitter searches (tw), temperature (t), and humidity (h)). figure shows the adjusted r-squared values of regression models for each infectious disease. among the observed regression models, the models that are combinations of all variables had the best explanatory power. therefore, this combination was chosen as the optimal variable combination. the optimal variable combinations for the model were selected by considering all possible models in the regression analysis. the models are combinations of the four types of data in the dataset (naver searches (n), twitter searches (tw), temperature (t), and humidity (h)). figure shows the adjusted r-squared values of regression models for each infectious disease. among the observed regression models, the models that are combinations of all variables had the best explanatory power. therefore, this combination was chosen as the optimal variable combination. previous results [ ] have shown that it is possible to predict infectious disease at an early stage if a model is designed to consider the time difference between clinical data and non-clinical data. based on this observation, our model was designed to consider the time difference in each data set. in this situation, "lag" refers to the time delay between the date the data is collected and the date at which the effects actually occur. this means analysis was performed by establishing the time difference between the four input variables used in this study and the output variable that is actually affected. for example, a lag of means that the output variable of january is calculated using the input variables of january . figure shows the adjusted r-squared values of regression models when - days of lag were tested for each of the infectious diseases in order to select the optimal lag. in the case of chickenpox, it was found that lags of , , and days yielded the highest explanatory power. for scarlet fever, it was found that lags of , , and days yielded the highest explanatory power. in the case of malaria, it was found that lags of , , and days yielded the highest explanatory power. for chickenpox and malaria, the lag with the highest explanatory power was one day. however, it was decided that this lag was not suitable for the ultimate goal of reducing the length of delay from reporting to dissemination. in the observed regression models, the explanatory power of a lag of seven days was high for all infectious diseases. therefore, it was decided that this lag was the most suitable and was used for later predictions. previous results [ ] have shown that it is possible to predict infectious disease at an early stage if a model is designed to consider the time difference between clinical data and non-clinical data. based on this observation, our model was designed to consider the time difference in each data set. in this situation, "lag" refers to the time delay between the date the data is collected and the date at which the effects actually occur. this means analysis was performed by establishing the time difference between the four input variables used in this study and the output variable that is actually affected. for example, a lag of means that the output variable of january is calculated using the input variables of january . figure shows the adjusted r-squared values of regression models when - days of lag were tested for each of the infectious diseases in order to select the optimal lag. in the case of chickenpox, it was found that lags of , , and days yielded the highest explanatory power. for scarlet fever, it was found that lags of , , and days yielded the highest explanatory power. in the case of malaria, it was found that lags of , , and days yielded the highest explanatory power. for chickenpox and malaria, the lag with the highest explanatory power was one day. however, it was decided that this lag was not suitable for the ultimate goal of reducing the length of delay from reporting to dissemination. in the observed regression models, the explanatory power of a lag of seven days was high for all infectious diseases. therefore, it was decided that this lag was the most suitable and was used for later predictions. in this study, the ols model was used to select the optimal parameter values. it was also used as a comparison model to evaluate the prediction performance of the deep learning models. linear regression is a regression analysis technique that models the linear correlation between the output variable y and one or more input variables x in the collected data. the model has the following form. ols is the most simple and commonly used form of linear regression. it is a technique that minimizes the sum of squared errors and can solve the mathematical expression for ß, which is the parameter to be predicted, by using the equation below. ols analyses were performed by r version . . (https://www.r-project.org/). because ols is the simplest form of linear regression analysis, it is not sufficient for comparison with deep learning models. therefore, we also compare the arima model, which is often used for the prediction of infectious diseases [ ] [ ] [ ] . this will more clearly compare traditional analysis methods (ols and arima) with deep learning (dnn and lstm). the arima model is a method for analyzing non-stationary time series data. one characteristic of arima analysis is that it can be applied to any time series. in particular, it shows the detailed changes when the data fluctuates rapidly over time. in this study, we used seasonal arima because the collected data is seasonal. the seasonal arima model is denoted as arima(p, d, q)(p, d, q)s. where p is the order of the autoregressive part, d is the order of the differencing, q is the order of the moving-average process, and s is the length of the seasonal cycle. (p, d, q) is the seasonal part of the model. the seasonal arima model is written below. in this study, the ols model was used to select the optimal parameter values. it was also used as a comparison model to evaluate the prediction performance of the deep learning models. linear regression is a regression analysis technique that models the linear correlation between the output variable y and one or more input variables x in the collected data. the model has the following form. ols is the most simple and commonly used form of linear regression. it is a technique that minimizes the sum of squared errors and can solve the mathematical expression for ß, which is the parameter to be predicted, by using the equation below. ols analyses were performed by r version . . (https://www.r-project.org/). because ols is the simplest form of linear regression analysis, it is not sufficient for comparison with deep learning models. therefore, we also compare the arima model, which is often used for the prediction of infectious diseases [ ] [ ] [ ] . this will more clearly compare traditional analysis methods (ols and arima) with deep learning (dnn and lstm). the arima model is a method for analyzing non-stationary time series data. one characteristic of arima analysis is that it can be applied to any time series. in particular, it shows the detailed changes when the data fluctuates rapidly over time. in this study, we used seasonal arima because the collected data is seasonal. the seasonal arima model is denoted as arima(p, d, q)(p, d, q) s . where p is the order of the autoregressive part, d is the order of the differencing, q is the order of the moving-average process, and s is the length of the seasonal cycle. (p, d, q) is the seasonal part of the model. the seasonal arima model is written below. where y t refers to the value of the time series at time t, µ is the mean term, a t is the independent disturbance, b is the backshift operator, φ(b) is the autoregressive operator, and θ(b) is the moving average operator. φ s b s and θ s b s are the seasonal operators of the model. the arima analyses were carried out using the r version . . . the dnn model is a feedforward analysis method that is a basic model for deep learning. dnn is composed of a minimum of three node layers and, with the exception of the input node, each node uses a nonlinear activation function. dnn uses a supervised learning technique called backpropagation. in this study, an infectious disease prediction model that uses dnn was designed and the basic dnn model was compared with this more advanced deep learning model. the variables used in dnn are bias b, input x, output y, weight w, calculation function σ and activation function f (σ). each neuron in dnn uses the following equation. figure shows the structure of a neuron in the dnn model. the dnn analyses were carried out using the "dense layer" option of the keras package in the python version . . (https://keras.io/). there are parameters available in the dense layer. we only modified the units, activation function, and dropout. the rest of the parameters used the default values (e.g., use_bias = true and kernel_regularizer = none). where refers to the value of the time series at time , is the mean term, is the independent disturbance, is the backshift operator, ( ) is the autoregressive operator, and ( ) is the moving average operator. ( ) and ( ) are the seasonal operators of the model. the arima analyses were carried out using the r version . . . the dnn model is a feedforward analysis method that is a basic model for deep learning. dnn is composed of a minimum of three node layers and, with the exception of the input node, each node uses a nonlinear activation function. dnn uses a supervised learning technique called backpropagation. in this study, an infectious disease prediction model that uses dnn was designed and the basic dnn model was compared with this more advanced deep learning model. the variables used in dnn are bias , input , output , weight , calculation function and activation function ( ). each neuron in dnn uses the following equation. figure shows the structure of a neuron in the dnn model. the dnn analyses were carried out using the "dense layer" option of the keras package in the python version . . (https://keras.io/). there are parameters available in the dense layer. we only modified the units, activation function, and dropout. the rest of the parameters used the default values (e.g., use_bias = true and kernel_regularizer = none). the lstm model is suitable for predicting time series data when there is a time step with a random size [ ] . it was thought that prediction performance could be improved by creating an infectious disease prediction model using lstm and the time series data collected in this study. an important advantage of recurrent neural networks (rnns) is that contextual information is available when mapping io sequences. however, there is a gradient problem in that the effect of a given input on the hidden layer can be increased or decreased significantly during the circular connection. as new inputs are overwritten, the sensitivity of the first input decreases over time. therefore, the network is "forgotten". the input gate, output gate, and forget gate are non-linear summation units that control the activation of the cell. the forget gate multiplies the previous state of the cell while the input and output gates multiply the io of the cell. the activation function f of the gate is a logistic sigmoid. the io activation functions g and h of the cell usually use hyperbolic tangents or logistic sigmoids. however, in some cases, h uses the identity function. as long as the forget gate is open and the input gate is closed, the memory cell continues to remember the first input. in this way, lstm is an algorithm that resolves a problem in traditional rnns [ ]. the lstm model is suitable for predicting time series data when there is a time step with a random size [ ] . it was thought that prediction performance could be improved by creating an infectious disease prediction model using lstm and the time series data collected in this study. an important advantage of recurrent neural networks (rnns) is that contextual information is available when mapping io sequences. however, there is a gradient problem in that the effect of a given input on the hidden layer can be increased or decreased significantly during the circular connection. as new inputs are overwritten, the sensitivity of the first input decreases over time. therefore, the network is "forgotten". the input gate, output gate, and forget gate are non-linear summation units that control the activation of the cell. the forget gate multiplies the previous state of the cell while the input and output gates multiply the io of the cell. the activation function f of the gate is a logistic sigmoid. the io activation functions g and h of the cell usually use hyperbolic tangents or logistic sigmoids. however, in some cases, h uses the identity function. as long as the forget gate is open and the input gate is closed, the memory cell continues to remember the first input. in this way, lstm is an algorithm that resolves a problem in traditional rnns [ ] . the equations for forgetting, storing, renewing, and outputting information in the cell are shown below, respectively. when data (x t ) is input to the lstm cell in equation ( ), function f t determines the information to be forgotten in the cell layer. in equations ( ) and ( ), information that will be newly saved in the cell layer is created in i t and c t in equation ( ), the cell layer c t is renewed using f t , i t , and c t in equation ( ), the cell layer's information is used and h t is the output. in equation ( ), the cell state gets a value between − and through the tanh function. the values of c t and h t are kept for the next iteration of lstm. lstm analyses were carried out using the "lstm layer" of the keras package in the python version . . . there are parameters available in the lstm layer. we only set the units, activation function, return sequence, and dropout. the rest of the parameters used the default values (e.g., use_bias = true, recurrent regularizer = none, recurrent_constraint = none, and unit_forget_bias = none). figure shows the parameter selection method for the deep learning approach used in this study. the adadelta, adagrad, adam, adamax, nadam, rmsprop, and stochastic gradient descent (sgd) optimizers were compared. all parameters of each optimizer used the default values of the keras package. for instance, in sgd, the learning late is . , the momentum is , the decay is , and the nesterov momentum is false. in addition, the following activation functions were evaluated: exponential linear unit (elu), rectified linear unit (relu), scaled elu (selu), and softplus. lastly, various numbers of epochs ( , , , and ) were evaluated. the other parameters were fixed as follows: number of hidden layers = , number of units in each hidden layer = , batch size = , and drop out = . prediction models with variable and fixed parameters were trained on the data and the resulting models were compared to determine the optimal prediction model. to ensure the amount of dnn model data was the same as that of the lstm model, previous data from the same time period as the lstm was inserted. all deep learning models were implemented using the keras package in the python version . . . and drop out = . prediction models with variable and fixed parameters were trained on the data and the resulting models were compared to determine the optimal prediction model. to ensure the amount of dnn model data was the same as that of the lstm model, previous data from the same time period as the lstm was inserted. all deep learning models were implemented using the keras package in the python version . . . the regression model was formed based on days of data in which a lag of seven days was applied to each infectious disease dataset. the dataset was divided up in an : ratio and each part was used for constructing the regression model and prediction. table presents the ols results. each regression model had results that were below the level of significance (p < . ). the adjusted r-squared value was greater than . for all three infectious diseases, which means the models can be said to have significant explanatory power. of the infectious disease regression models, the chickenpox model yielded significant results for the naver search queries, temperature, and humidity. the scarlet fever model yielded significant results for the naver search queries and humidity. additionally, the malaria model yielded significant results for the naver search queries and temperature (p < . ). looking at these results together, the naver search query data was significant for all three infectious diseases and the twitter data was not significant for any of the three. it can be seen that the internet search query data can be used to design an infectious disease prediction model, which was reported by previous studies. however, the results for the twitter data differ from the results of previous studies. this is believed to be because naver accounted for the largest share ( . %) of korean search engine use in the health/medicine field for the first half of while twitter accounted for the smallest share ( . %) of social media use in the health/medicine field for the same time period (http://internettrend.co.kr/trendforward.tsp). however, the twitter data had an effect on the process of finding the model with the highest adjusted r-squared value. therefore, it is expected to have an effect on future analysis as well. the temperature had a significant relationship with all infectious diseases except for scarlet fever and humidity had a significant relationship with all infectious diseases except for malaria. the values of the coefficients show that the most significant variables for chickenpox and scarlet fever was the naver search query data ( . and . , respectively) and, for malaria, it was the temperature values ( . ). the effect of naver search query data in particular was significant for all three infectious diseases, which confirms that it can be suitable for predicting infectious disease. the seasonal arima model was evaluated using the same data used for ols. the autocorrelation function and the partial autocorrelation function were checked for the seasonality of infectious diseases and seasonality was observed. it was considered inappropriate to select the parameters (e.g., p, d, q) because cuts off and tails off are unclear. therefore, the optimal model for each infectious disease was selected based on the akaike information criterion (aic) and rmse. the aic and rmse were used to compare the arima models. table shows the aic and rmse of the seasonal arima model for each infectious disease, which allows us to identify the top three arima model. in addition, the choice of parameter values did not substantially affect the aic and rmse of the model for a single infectious disease. to compare the performance of each model, figure shows the models with the lowest rmse of the test data subset. the numbers inside the parentheses in each model's name represent the optimizer, the activation function, and the number of epochs used in the models (e.g., dnn ( , , ) indicates that the optimizer, the activation function, and the number of epoch are adadelta, relu, and , respectively). the metric used to compare the models is rmse, which shows the difference between the actual and predicted values. a smaller rmse value indicates a smaller difference between the actual and predicted values and indicates a higher prediction performance. table s shows the rmse and prediction graphs of the dnn and lstm models with the lowest rmse for chickenpox. it can be seen that the prediction graphs for each analysis method have similar shapes overall. the dnn models for chickenpox had a mean rmse of . and a standard deviation of . , which shows stable model performance. when the prediction performances of each model were compared based on rmse, the top dnn models showed a . % performance improvement compared to the arima model. the mean rmse of the lstm models was . , which is higher than the dnn models. the standard deviation was . , which shows that the difference among lstm models was more marked than the difference among dnn models. despite this, the top lstm models achieved an . % performance improvement over the arima model on average. there was a difference between the dnn and lstm models' average figures and standard deviations. however, in the models with the lowest rmse for each analysis method, there was not a big difference, which indicates that there was not a large difference in performance when the optimal parameters for each analysis method were used. table s shows the rmse and the prediction graphs of the dnn and lstm models with the lowest rmse for scarlet fever. the shapes of the graphs for the dnn models are similar. for the lstm models, the shapes of the graphs are similar except for the model with the lowest rmse. unlike the graphs of the other lstm models, the graph lstm model with the lowest rmse showed a strong tendency to follow the actual trend. this result infers that a prediction model that is better than existing prediction models can be designed by changing the deep learning parameters to achieve optimization. as is the case with chickenpox, the mean rmse of the dnn model ( . ) for scarlet fever was lower than that of the lstm model ( . ) . the standard deviation of the dnn model ( . ) was also lower than that of the lstm model ( . ) . when comparing each model based on rmse, the top dnn models showed a . % performance improvement over the arima model and the lstm models showed a . % performance improvement over the arima model. table s shows the rmse and the prediction graphs of the dnn and lstm models with the lowest rmse for malaria. like other infectious diseases, the dnn model prediction graphs have a similar shape. however, the shapes of the lstm model prediction graphs have a tendency to not follow the trend. the rmses of each prediction model, excluding the arima model, showed little difference. this is believed to be because the number of malaria occurrences is fewer than those of the other infectious diseases. therefore, adequate predictions could not be formed. diseases, the dnn model prediction graphs have a similar shape. however, the shapes of the lstm model prediction graphs have a tendency to not follow the trend. the rmses of each prediction model, excluding the arima model, showed little difference. this is believed to be because the number of malaria occurrences is fewer than those of the other infectious diseases. therefore, adequate predictions could not be formed. it is difficult to understand the special characteristics of each analysis method by simply comparing rmse figures alone. therefore, a detailed comparison was performed on the basic comparison models (ols and arima) and the analysis methods that use deep learning (dnn and lstm) . the deep learning models used for comparison were the models with the optimal performance and lowest rmse so that they could best represent each analysis method. with the best performance figure shows the chickenpox predictions of the model with the lowest rmse out of the models with the lowest rmse for each analysis method. the dnn model with the best performance it is difficult to understand the special characteristics of each analysis method by simply comparing rmse figures alone. therefore, a detailed comparison was performed on the basic comparison models (ols and arima) and the analysis methods that use deep learning (dnn and lstm). the deep learning models used for comparison were the models with the optimal performance and lowest rmse so that they could best represent each analysis method. with the best performance. figure shows the chickenpox predictions of the model with the lowest rmse out of the models with the lowest rmse for each analysis method. the dnn model with the best performance had the following specifications, which include optimizer = adadelta, activation function = relu, and number of epochs = (dnn ( , , ) ). the lstm model with the best performance had the following specifications, which include optimizer = nadam, activation function = softplus, epochs = (lstm ( , , ) ). the ols model's predictions had a smaller range of fluctuation than the deep learning models. from day , it seems to follow the trend, but it does not follow the small changes. after day , it cannot predict the downward shape even within a stable graph model. in short, the ols model is not suitable as a prediction model. the arima model's prediction graph has a very simple shape. this model is cyclic and there is a slight increasing trend in which the predicted value per cycle increases by a factor of about . each time. this model cannot predict the trend at all. it can only predict a stable cyclic behavior. in contrast, the dnn ( , , ) predictions followed the actual occurrence trend well. moreover, it had a large range of fluctuation, which means it made accurate predictions overall. however, when the number of occurrences rose rapidly in days - , it was unable to follow these values. the lstm ( , , ) predictions had a smaller range of fluctuation than the dnn ( , , ) model. its range of variance was small, which means it had a stable shape and it performed better than dnn ( , , ) when the number of occurrences rose rapidly. predictions overall. however, when the number of occurrences rose rapidly in days - , it was unable to follow these values. the lstm ( , , ) predictions had a smaller range of fluctuation than the dnn ( , , ) model. its range of variance was small, which means it had a stable shape and it performed better than dnn ( , , ) when the number of occurrences rose rapidly. figure shows the scarlet fever predictions of the models with the best performance for each analysis method. the dnn model with the best performance had the following specifications: optimizer = adadelta, activation function = elu, and number of epochs = (dnn ( , , ) ). the lstm model with the best performance had the following specifications: optimizer = adamax, activation function = elu, number of epochs = (lstm ( , , ) ). the ols model's predictions were completely unable to follow the trend, which was similar with the chickenpox case. the arima model's prediction has no particular merit because it also predicts a simple cycle. much like its figure shows the scarlet fever predictions of the models with the best performance for each analysis method. the dnn model with the best performance had the following specifications: optimizer = adadelta, activation function = elu, and number of epochs = (dnn ( , , ) ). the lstm model with the best performance had the following specifications: optimizer = adamax, activation function = elu, number of epochs = (lstm ( , , ) ). the ols model's predictions were completely unable to follow the trend, which was similar with the chickenpox case. the arima model's prediction has no particular merit because it also predicts a simple cycle. much like its chickenpox prediction, it can only predict a stable cyclic behavior. the dnn ( , , ) predictions were relatively close when the number of occurrences was low, but they were too low when the number of occurrences was high. lstm ( , , ) had a larger range of variance than dnn ( , , ) and its predictions were close when the number of occurrences was high. in the prediction graphs for all of the top performing scarlet fever models, none of the models were able to follow the trend on days - when there was a severe variance in the number of actual occurrences. looking at the mean of each model's predicted value for the number of occurrences, the lstm model ( . ) was larger than the mean of the dnn model ( . ). these same results were also seen in the case of chickenpox (dnn model mean = . , lstm model mean = . ). this showed that more suitable results can be obtained if the lstm model is used to predict the maximum value for the number of occurrences and the dnn model is used to predict the minimum value. figure shows the malaria predictions of the models with the best performance for each analysis method. the dnn model with the best performance had the following specifications: optimizer = adamax, activation function = softplus, number of epochs = (dnn ( , , ) ). the lstm model with the best performance had the following specifications: optimizer = adadelta, activation function = softplus, number of epochs = (lstm ( , , ) ). the predictions of the analysis methods were not satisfactory, but the dnn ( , , ) model's predictions seemed to follow the trend relatively well. the arima model predicts values close to . it is believed that the occurrences in the malaria data are less than those of other diseases and, therefore, not suited to time series analysis because occurrences are concentrated in the summer seasons. as seen in section . , the lowest rmses of each prediction model excluding the arima model showed little difference. lstm ( , , ) had a lower range of variance than ols and it seemed completely unable to make predictions. as mentioned before, the reason that predictions were inadequate for all of the models in addition to lstm ( , , ) was that figure shows the malaria predictions of the models with the best performance for each analysis method. the dnn model with the best performance had the following specifications: optimizer = adamax, activation function = softplus, number of epochs = (dnn ( , , ) ). the lstm model with the best performance had the following specifications: optimizer = adadelta, activation function = softplus, number of epochs = (lstm ( , , ) ). the predictions of the analysis methods were not satisfactory, but the dnn ( , , ) model's predictions seemed to follow the trend relatively well. the arima model predicts values close to . it is believed that the occurrences in the malaria data are less than those of other diseases and, therefore, not suited to time series analysis because occurrences are concentrated in the summer seasons. as seen in section . , the lowest rmses of each prediction model excluding the arima model showed little difference. lstm ( , , ) had a lower range of variance than ols and it seemed completely unable to make predictions. as mentioned before, the reason that predictions were inadequate for all of the models in addition to lstm ( , , ) was that the number of malaria occurrences was small and proper results could not be produced. the deep learning model showed outstanding performance compared to the traditional arima method. of all the dnn and lstm prediction models for chickenpox, the optimal models with the lowest rmse yielded . % and . % better performance than the arima model, respectively. the top dnn models for chickenpox improved performance by an average of . % and the lstm models improved performance by an average of . %. the lowest rmses of the dnn and lstm prediction models for scarlet fever showed . % and . % improved performances compared to arima models. the top dnn models for scarlet fever improved performance by an average of . %. the lstm models improved performance by an average of . %. as noted in the previous sections, it was difficult to predict infectious diseases when the number of infections was small and concentrated in one season. in effect, we observed that the incidence of malaria was high over days - and after day . this period corresponds to the summer season in korea. predicting infectious diseases with this particular data set was difficult and it was not suitable for the arima analysis. even using this particular data set, when dnn was used, the trend of infectious diseases was followed comparatively (figure ). moreover, there is a possibility that the performance would be improved in the dnn model if more diverse parameters were adjusted. this means using deep learning has the advantage of scalability and this can be further investigated in future studies. the arima model that was used in this study was observed to be effective if the number of incidences of infectious diseases was regular and had no increasing or decreasing trends. however, when the predictions were compared according to each analysis method, the dnn and lstm deep learning models performed better than the ols and arima models by assuming that there was a sufficiently large number of occurrences. when comparing the dnn and lstm models, the best models had similar performance, but the dnn models were better in terms of average performance. however, when the number of occurrences was large, the lstm model made close predictions. it seems to be an analysis method that is suitable for circumstances when the number of occurrences is rapidly increasing and infectious disease is believed to be spreading. the deep learning model showed outstanding performance compared to the traditional arima method. of all the dnn and lstm prediction models for chickenpox, the optimal models with the lowest rmse yielded . % and . % better performance than the arima model, respectively. the top dnn models for chickenpox improved performance by an average of . % and the lstm models improved performance by an average of . %. the lowest rmses of the dnn and lstm prediction models for scarlet fever showed . % and . % improved performances compared to arima models. the top dnn models for scarlet fever improved performance by an average of . %. the lstm models improved performance by an average of . %. as noted in the previous sections, it was difficult to predict infectious diseases when the number of infections was small and concentrated in one season. in effect, we observed that the incidence of malaria was high over days - and after day . this period corresponds to the summer season in korea. predicting infectious diseases with this particular data set was difficult and it was not suitable for the arima analysis. even using this particular data set, when dnn was used, the trend of infectious diseases was followed comparatively (figure ). moreover, there is a possibility that the performance would be improved in the dnn model if more diverse parameters were adjusted. this means using deep learning has the advantage of scalability and this can be further investigated in future studies. the arima model that was used in this study was observed to be effective if the number of incidences of infectious diseases was regular and had no increasing or decreasing trends. however, actual data can have trends and be irregular. therefore, deep learning can be an excellent analytical method when analyzing such data and predicting future situations. according to the results of the previous analyses, the deep learning model follows increasing and decreasing trends sufficiently well. moreover, the dnn and lstm models were observed to be sensitive to decreasing trends and increasing trends, respectively. infectious disease is a social problem in that it can cause not only personal damage but also widespread harm. for this reason, research is being conducted to minimize social losses by predicting the spread of infectious diseases. the aim of this study was to design an infectious disease prediction model that is more suitable than existing models by using various input variables and deep learning techniques. therefore, in this study, the optimal parameters were set using a variable selection method based on ols. the relationship between actual instances of disease occurrence and the internet search query data tends to have a time lag, which means a lag was added to each infectious disease's dataset to find the future trend. next, an analysis of arima, dnn, and lstm was performed with optimal parameters. the results of ols analysis using optimal parameters showed that the regression models for each infectious disease had significant results. of the four input variables, the naver search frequency had a significant relationship with all three infectious diseases. the performance of the ols and arima analysis was used to evaluate the deep learning models. looking at the results for dnn and lstm, both the deep learning models made much better predictions than the ols and arima models for all infectious diseases. moreover, the dnn models had the best performance on average, but the lstm models made more accurate predictions when infectious diseases were spreading. however, in the case of malaria, there were few occurrences of the disease compared to other infectious diseases, which means the predictions were not comparatively accurate. this study was also able to reveal special characteristics of the dnn and lstm models. the dnn model produced smaller values than the lstm model on average when predicting infectious diseases. suitable predictions can be made using the dnn model when predicting the minimum value for disease occurrence and using the lstm model when predicting the maximum value. in previous studies, deep learning algorithms were not used [ ] [ ] [ ] [ ] [ ] [ ] ] or the amount of data considered was small [ , , , ] . this study used social media big data and weather data, which have not been sufficiently considered in existing studies. it also used deep learning analysis, which yields high prediction performance to increase the performance of infectious disease predictions. the results showed that, when selecting the optimal parameters, adding all input variables had the highest explanatory power. this means that, by adding various data, it was possible to design a model with higher explanatory power. moreover, the lstm model results for scarlet fever indicate that it is possible to optimize a deep learning model by changing its parameters in various ways and, therefore, design a prediction model that is better than existing prediction models. this study has reviewed the factors involved with infectious disease occurrence using search query data and social media big data, which exist because of the development of the internet as well as temperature and humidity weather data. it also constructed traditional prediction models such as ols, arima, and deep learning prediction models such as dnn and lstm and compared their prediction performance to confirm that the models that use deep learning are the most suitable for infectious disease prediction. it is believed that infectious disease prediction models that employ deep learning can be used to supplement current infectious disease surveillance systems and, at the same time, predict trends in infectious disease. if this can reduce the time differences in reporting systems so that infectious disease trends can be known immediately, it is expected that immediate responses to infectious disease will become possible and costs to society can be minimized. according to a study by shin et al., an emerging infectious disease known as the middle east respiratory syndrome (mers) has a deep correlation with internet search data [ ] and it will become possible to expand these methods to the real-time surveillance and prediction of emerging infectious diseases as well. however, this study has three limitations, which include a relatively short data collection period, regionally combined predictions, and a consideration of a narrow range of parameters in the deep learning model. the search query data collection time period used in this research was relatively short extending from january to july . the particular spatial ranges of data were averaged across the whole of south korea. it is believed that, if the data is expanded and the spatial ranges are subdivided, the model's performance will improve. in addition, an effort was made to change the dnn and lstm model parameters and create a variety of prediction models, but the deep learning prediction models used in this study did not cover all the prediction models that could be implemented. parameters such as hidden layers and batch size were not considered. therefore, it is difficult to conclude that the most effective model was created. if more parameters are considered and more prediction models are made in future research, it is believed that prediction performance can be increased somewhat. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : the root mean squared error (rmse) and prediction graphs of top deep neural network (dnn) and long-short term memory (lstm) models for chickenpox. the seasonal autoregressive integrated moving average (arima) model is denoted as arima(p, d, q)(p, d, q) s . where p is the order of the autoregressive part, d is the order of the differencing, q is the order of the moving-average process, and s is the length of the seasonal cycle. (p, d, q) is the seasonal part of the model. the numbers in parentheses indicate each deep learning model's optimizer, activation, and number of epochs, respectively. (optimizer) : adadelta, : adagrad, : adam, : adamax, : nadam, : rmsprop, and : sgd, (activation function) : elu, : relu, : selu, and : softplus, (number of epochs) : , : , : , and : , table s : the rmse and prediction graphs of the top dnn and lstm models for scarlet fever, table s : the rmse and prediction graphs of top dnn and lstm models for malaria. infectious disease, safety, state: history of infectious disease prevention and mers situation a profile of the online dissemination of national influenza surveillance data multiscale mobility networks and the spatial spreading of infectious diseases modeling the worldwide spread of pandemic influenza: baseline case and containment interventions seasonal transmission potential and activity peaks of the new influenza a(h n ): a monte carlo likelihood analysis based on human mobility modelling disease outbreaks in realistic urban social networks strategies for mitigating an influenza pandemic controlling pandemic flu: the value of international air travel restrictions mitigation measures for pandemic influenza in italy: an individual based model considering different scenarios monitoring pertussis infections using internet search queries disease surveillance based on internet-based linear models: an australian case study of previously unmodeled infection diseases advances in nowcasting influenza-like illness rates using search query logs correlation between national influenza surveillance data and google trends in south korea dynamic forecasting of zika epidemics using google trends influenza forecasting with google flu trends mass media and the contagion of fear: the case of ebola in america monitoring hand, foot and mouth disease by combining search engine query data and meteorological factors assessing the usability of social media data for visitor monitoring in protected areas forecasting the onset and course of mental illness with twitter data high correlation of middle east respiratory syndrome spread with google search and twitter trends in korea defender: detecting and forecasting epidemics using novel data-analytics for enhanced response applying gis and machine learning methods to twitter data for multiscale surveillance of influenza forecasting influenza-like illness dynamics for military populations using neural networks and social media twitter in the cross fire-the use of social media in the westgate mall terror attack in kenya real-time diffusion of information on twitter and the financial markets bibliographic analysis of nature based on twitter and facebook altmetrics data frequent discussion of insomnia and weight gain with glucocorticoid therapy: an analysis of twitter posts forecasting influenza in hong kong with google search queries and statistical model fusion construction and evaluation of two computational models for predicting the incidence of influenza in nagasaki prefecture deep learning applications and challenges in big data analytics deep learning for digital pathology image analysis: a comprehensive tutorial with selected use cases dermatologist-level classification of skin cancer with deep neural networks deep learning based tissue analysis predicts outcome in colorectal cancer time series analyses of hand, foot and mouth disease integrating weather variables short term effects of weather on hand, foot and mouth disease weather and virological factors drive norovirus epidemiology: time-series analysis of laboratory surveillance data in england and wales imported dengue cases, weather variation and autochthonous dengue incidence in cairns a large temperature fluctuation may trigger an epidemic erythromelalgia outbreak in china implications of temperature variation for malaria parasite development across the impact of variations in temperature on early plasmodium falciparum development in anopheles stephensi mapping the epidemic changes and risks of hemorrhagic fever with renal syndrome in shaanxi province a threshold analysis of dengue transmission in terms of weather variables and imported dengue cases in australia monitoring seasonal influenza epidemics in korea through query search forecast model analysis for the morbidity of tuberculosis in xinjiang, china time series analysis of dengue incidence in guadeloupe, french west indies: forecasting models using climate variables as predictors the authors declare no conflict of interest. key: cord- -piust s authors: oh, hyang soon title: knowledge, perceptions, and self-reported performance of hand hygiene among registered nurses at community-based hospitals in the republic of korea: a cross-sectional multi-center study date: - - journal: j prev med public health doi: . /jpmph. . sha: doc_id: cord_uid: piust s objectives: to assess the nurses’ hand hygiene (hh) knowledge, perception, attitude, and self-reported performance in small- and medium-sized hospitals after middle east respiratory syndrome outbreak. methods: the structured questionnaire was adapted from the world health organization’s survey. data were collected between june and july , . results: nurses showed scores on knowledge ( . ± . ), perception ( . ± . ), self-reported hh performance of non-self ( . ± . ), self-reported performance of self ( . ± . ), and attitude ( . ± . ). hh performance rate of non-self was y( )= . + . x (hh performance rate of self) (adjusted r( )= . , p< . ). the regression model for performance was y( )= . + . x( ) (peception)+ . x( ) (attitude)+ . x( ) (role model); coefficients were significant statistically except attitude, and this model significant statistically (adjusted r( )= . , p< . ). conclusions: advanced hh education program would be developed and operated continuously. perception, attitude, role model was found to be a significant predictors of hh performance of self. so these findings could be used in future hh promotion strategies for nurses. health care-associated infections (hais) critically impact patient outcomes, increase hospital costs, and extend hospital pissn - eissn - stays [ ] . at any given time, about in inpatients has an hai. this leads to the loss of tens of thousands of lives and costs the us health care system billions of dollars annually [ ] . the primary transmission route of pathogens between patients in hais is via health care workers (hcws)' hands [ ] . thus, hand hygiene (hh) is the single most important factor for preventing hais [ ] . proper hh among hcws is one of the foremost techniques for reducing hais [ , ] . however, the reported rates of hh performance among hcws are low, ranging from . to . %, which is insufficient to satisfactorily prevent hais [ , ] . during the middle east respiratory syndrome (mers) outbreak from may to july , in the republic of korea (hereafter korea), there were confirmed mers cases nationwide [ , ] . we learned several important lessons from the mers outbreak, including that hh is the key to infection control and prevention. thereafter, various types of educational initiatives, campaigns, and training about hh have been implemented nationally, ranging from large cities to small-tomedium cities. in korea, infection control programs have been developed and implemented at university-affiliated hospitals in large cities, and these programs have spread to hospitals in small-tomedium cities [ ] . studies of hh performance and knowledge among hcws have been conducted in larger hospitals with good resources for hh [ ] [ ] [ ] . however, few studies have investigated knowledge and perceptions of hh among hcws in small-to-medium hospitals with relatively limited resources. because of their frequent contact with patients, nurses' proper execution of hh plays an especially important role in the prevention of hais, and nurses should therefore be provided with essential and up-to-date hh information. it has also been established that nurses' knowledge, perceptions, and attitudes about hh influence their hh performance [ ] [ ] [ ] . as such, this study was conducted to assess the status of knowledge, perceptions, and performance of hh among nurses in community-based hospitals in a small-to-medium-sized city in an urban region in korea after the mers outbreak, to identify the factors that influenced their knowledge, perceptions, and performance, and to identify relationships among their knowledge, perceptions, and performance. a cross-sectional design was used to administer a self-reported questionnaire, which took approximately minutes to complete. before the start of the study, the author contacted the hospital directors to explain the purpose of the study and to obtain permission for recruitment. the enrolment criteria were hospitals that were affiliated with teaching hospitals and permitted nurses to participate voluntarily in this study. finally, community-based hospitals affiliated with teaching hospitals located in a small-to-medium-sized city in the south jeolla province of korea were enrolled in this study. we performed a power analysis using g*power version . . . (franz faul, universitat kiel, germany) to determine that a sample size of would be required to achieve a power of . with an effect size of . (a medium effect size for multiple correlations) with an alpha of . . a convenience sample of registered nurses (rns) was recruited from the hospital study sites. the hospitals fully understood the purposes of the study and permitted voluntary recruitment; subjects' participation was voluntary and anonymous. questionnaires were delivered directly to, and later collected from, each hospital. data were collected from june to july , . a total of questionnaires were distributed and were returned (response rate, . %). after excluding incomplete questionnaires, questionnaires were used for the analysis. the questionnaire included domains: (a) hh knowledge, (b) hh perceptions, (c) hh attitudes and role models, and (d) participant demographics and hospital characteristics. the knowledge domain (a) was adapted from the revision of the world health organization (who) hand hygiene knowledge questionnaires for health-care workers, which are composed of questions about the main route of transmission of germs, sources of hais, the timing of hh to prevent transmission of germs to patients or to other healthcare workers, knowledge about handwashing and alcohol-based hand rubs, knowledge about hh methods for clinical situations, and practices for increasing the prevalence of hh [ ] . the items included multiple-choice, true/false, and yes/no (coded as right answer= , wrong answer= ) items, with a total score range of - points (table s ). the perceptions domain (b) was also adapted from the who questionnaire to identify perceptions and performance [ ] . three questions (b , b , and b in table s ) were excluded from the total score because they reduced the reliability of the questionnaire (cronbach alpha, . with all questions; . after exclusion). eleven of the items were on a -to -point scale from 'not effective' to 'very effective', or 'very low' to 'very high', with a total score range of - points. question b was used to assess self-reported hai rates. two other questions (b and b ) were analysed separately as indicators of the self-reported hh performance of non-self and self. the attitudes and role models domain (c) was adapted from hand hygiene knowledge and performance a previous study [ ] . it was a self-report questionnaire consisting of items on a -to -point scale from 'not effective' to 'very effective' [ ] . the total scores ranged from to . hh role models were assessed with items on a -to -point scale from 'do not agree' to 'strongly agree', with a total score range of - points. a higher score in each domain indicated greater knowledge, more positive perceptions, more frequent performance, better attitudes, and higher scores for role models, respectively. the who-based questions were translated into korean and a pilot study was conducted from june to , , during which the translated items were reviewed by nursing professors and rns to assess the content validity and to refine a checklist. pilot study participants were asked to comment on whether the questionnaire items adequately sampled each domain; the questions were accurate, clear, and easy to understand; the instructions were clear and complete; and any of the questions or statements might lead to discord. the suitability of the questionnaire for use was confirmed by the pilot study. participants' time to complete the questionnaire was recorded and within-domain reliability was calculated. demographic variables included participants' age, sex, religion, marital status, education level, clinical work experience (years), department, and position. hospital characteristics included the type of hospital, number of beds, hh guidelines, presence of an infection control department (icd), presence of an infection control nurse (icn), number of hh sinks, number of alcohol-based hand rubs, experiences of hh education within the last year, hh campaigns, hh monitoring and feedback, and mass media information. yes/no answers were coded as 'yes'= , 'no'= . data were analysed using spss version . (ibm corp., armonk, ny, usa), and alpha values < . were considered to indicate statistical significance. the cronbach alpha was calculated to determine reliability. descriptive statistics were calculated. descriptive data for knowledge, perceptions, attitudes, role models, and self-reported hh performance are presented as mean±standard deviation (sd), minimum, maximum, and median. the per-centage of correct answers to each question in the knowledge domain was scaled as high ( % and over), medium ( - %), and low ( % and below). this scale was established after the proportion of correct answers of each question was analysed (table s ) ; the mean and sd were calculated as . and . %, respectively. thus, % was assigned as a medium score, and a high score would be recommended to correspond to the mean or sd; however in this study, that figure would be over %, so % was assigned as the cutoff for a high score. the data were found not to be distributed normally based on the kolmogorov-smirnov test (p< . ). non-parametric univariate statistical analyses were conducted using the mann-whitney u and kruskal-wallis tests. simple linear regression analysis was conducted to confirm the relationship between the self-reported hh performance of self and that of non-self. pearson correlation analysis was conducted to identify associations among knowledge, perceptions, attitudes, and self-reported hh performance of self. multivariate analysis with multiple linear regression with stepwise variable selection was conducted using variables that were confirmed to be statistically significant in the univariate analysis and correlation analysis. the cronbach alpha values were . (domain a, knowledge), . (domain b, perceptions), . (domain c, attitudes) and . (domain c, role models) in this study. these values were . , . , . , and . , respectively, in the pilot study. the study was approved by the institutional review board of sunchon national university ( - -hr- - , - -hr- - ). prior to participating, written informed consent was obtained from each participant; participants were also informed that their consent could be withdrawn at any time during the study. in the hospitals included in this study, there were . ± . beds (mean±sd) and . ± . (mean±sd) hcws, had an icd and had an icn ( had a part-time icn, had full-time icns), and were general hospitals. the demographics and general characteristics of the participants are presented in table . participants' mean±sd score of knowledge was . ± . ; the proportion of high and medium levels of correct answers for knowledge was . % (table ) . some questions showed a low proportion of correct answers, as follows (table s ): "a : what is the most frequent source of germs responsible for health care-associated infections?" ( . %); "a - : hand rubbing causes skin dryness more than hand washing" ( . %); "a - : after exposure to the immediate surroundings of a patient" ( . %); "a - : after exposure to the immediate surroundings of a patient" ( . %); "a - : regular use of a hand cream" ( . %). participants' mean±sd perception score was . ± . (table s ). the following questions about perceptions showed low mean±sd scores: "b . : hh posters are displayed at points of care as reminders" ( . ± . ), "b : what importance does the head of your department attach to the fact that you perform optimal hh?" ( . ± . ), "b : what importance do your colleagues attach to the fact that you perform optimal hh?" ( . ± . ), and "b : what importance do patients attach to the fact that you perform optimal hh?" ( . ± . ). the self-reported hai rate (%) was identified as . ± . (mean±sd). the self-reported hh performance of non-self (other hcws) was . ± . (mean±sd). the self-reported hh performance of self was . ± . (mean±sd); this was highest after body fluid exposure/risk ( . ± . ) and lowest before touching a patient ( . ± . ). participants' mean±sd scores in the attitudes and role models of c domains were . ± . and . ± . (table s ) . some questions on attitudes showed extraordinarily low mean±sd scores. in particular, the scores of "c : hh is convenient." and "c : hh is protective" were . ± . , and . ± . , respectively. some questions on role models showed low mean±sd scores, as follows (table s ) : "cr : i think that the charge nurse is performing according to the hospital's regulations" and "cr : i think that my colleague nurses are performing hh according to the hospital's regulations" both received a score of . ± . . the mean knowledge scores were significantly higher among participants who had received hh education within the past year; those who worked at a hospital with an icd, icn, or hh campaign, or where hh performance was monitored; those who worked in general hospitals; and those whose hospitals employed a full-time icn (compared with those with no icn or a part-time icn) ( table ) . the mean perceptions scores were significantly higher among participants whose hh performance was monitored; those who monitored their colleagues' hh performance; those who had experienced hh campaigns; those who were married; those who had higher education levels; and those who had higher positions ( table ). the mean scores for self-reported hh performance of self were significantly higher among hcws who had received hh education within the past year; those whose hh performance was monitored; those who monitored their colleagues' hh performance; and those who had higher positions (table ) . no independent variables were associated with significant differences in the scores for attitudes or role models in the univariate analyses. the correlation analysis among knowledge, perceptions, attitudes, and self-reported hh performance of self identified significant positive correlations among all categories except knowledge (table ). the model for the self-reported hh performance rates of self and non-self was follows. the hh performance rate of non-self was calculated as y = . + . x (hh performance rate of self), and a significant linear relationship was found (adjusted r = . , p< . ) ( table ). the regression model for knowledge was calculated as y = . + . x (receiving education within the past year)+ . x (icd)+ . x (icn) - . x (type of hospital). the coefficient of each predictor was not statistically significant, but the model as a whole did show statistical significance (adjusted r = . , p< . ) ( table ). the regression model for perceptions was calculated as y = . + . x (hh performance was monitored)+ . x (monitoring colleagues' hh performance)+ . x (hh campaign)+ . x (marital status)+ . x (education level)+ . x (position). the coefficients were not statistically significant, but the model as a whole did show statistical significance (adjusted r = . , p< . ) ( table ). the regression model for self-reported hh performance of self was not calculated by multiple linear regression using the variables found to be significant in the univariate analysis. the regression model for performance was calculated as y = . + . x (perceptions)+ . x (attitudes)+ . x (role model); the coefficients were statistically signifi- in terms of infection control infrastructure [ ] , icds and icns were not fully allocated across the hospitals analysed in this study. the values for numbers of sinks and the placement of alcohol-based hand rub stations in this study were no worse than has been reported in previous studies (in ) of large korean hospitals [ ] . however, as resources for hh, the placement of sinks in every room and alcohol-based hand rub stations by every bed, as well as supplying alcohol-based hand rub to every hcw should be improved continuously. in terms of hh activities, most participants ( . %) had received hh education; however, the scores for other activities such as hh campaign experience and hh monitoring activities were low. these issues can be easily resolved with icd and icn placement [ , ] . such improvements should be made continuously until the korean health care quality standards are satisfied [ ] . the mean score of knowledge among our participants ( . ± . ) was higher than was reported in previous studies ( . ± . [ ] , . ± . [ ] , and . ± . [ ] ) conducted by the same method (who questionnaire). however, the proportion of medium and high levels of correct answers was . %. moreover, serious weaknesses in knowledge were found in response to the following questions: "what is the most frequent source of germs responsible for health care-associated infections?", "hand rubbing causes skin dryness more than hand washing", "a - : after exposure to the immediate surroundings of a patient", "a - : after exposure to the immediate surroundings of a patient", and "regular use of a hand cream". therefore, hh education programs should be promptly reviewed, and systemic and advanced hh education and training programs must be developed and implemented to enhance hh knowledge broadly, not just focusing on these specific knowledge questions. the perceptions in this study ( . ± . ; total score, ) were somewhat higher than observed in a previous study ( . ± . ; total score, ) [ ] . however, some perceptions-related items that received low scores should be improved, because perceptions have been shown to be significant predictors of nurses' hh intentions and adherence [ ] , and an important predictor of hh performance [ , ] . self-reported hh performance of self was highest after body fluid exposure/risk and lowest before touching a patient. this finding corresponds to those of previous observational studies [ , ] and is consistent with a previous self-reported performance study [ ] . interestingly, the relationship between the hh performance of self and that of non-self was positive and linear. this finding is also consistent with a previous report [ ] . participants evaluated the hh performance of non-self at . times the hh performance of self. attitudes about hh were relatively poor in responses to both "hh is convenient" and "hh is protective". these findings may represent barriers to maintaining good hh, and also demonstrate the need for strategies to promote the perceived convenience and protectiveness of hh [ ] . in the role model domain, "it is important for my colleagues to perform hh according to the hospital's regulations" showed high endorsement, and nursing colleagues were identified as the most important hh role models. these results are consistent with previous studies [ ] [ ] [ ] , ] . moreover, the perception of being a role model for one's colleagues can be used to improve hh compliance [ , ] . according to the multiple linear regression analysis, receiving education within the past year (yes), having an icd (yes), and having an icn (yes) positively affected knowledge, while type of hospitals negatively affected knowledge. hh performance being monitored (yes), monitoring colleagues' hh performance (yes), and the presence of an hh campaign (yes) positively affected perceptions. therefore, these findings can be used to improve knowledge and perceptions. as this study was not an observational study of hh performance, this study has some limitations in terms of the self-reporting of hh performance. the regression model of self-reported hh performance of self showed increases with . (perceptions)+ . (attitudes)+ . (role models). knowledge was excluded from this model. consistently with previous studies [ , , , ] , our participants' self-reported hh performance rate of self was positively correlated with their scores for perceptions, attitudes, and role models. an explanation for this is that general perceptions of hh [ , , ] and the perception of being a role model for one's colleagues [ , ] are very important for improving hh compliance among hcws [ ] . as such, these findings could be used in future hh promotion strategies for nurses. in this study, the hh knowledge, perceptions, attitudes, and role models of rns in community-based hospitals in a small-to-medium urban area were characterized. the presence of some items with relatively low scores revealed some room for improvements in knowledge. receiving education within the past year, having an icd, and having an icn were found to be related to knowledge. in addition, hh campaigns and monitoring were associated with perceptions. the self-reported hh performance rate of self was associated with perceptions, attitudes, and role models. world health organization. who guidelines on hand hygiene in health care: first global patient safety challenge clean care is safer care office of disease prevention and health promotion evidence-based model for hand transmission during patient care and the role of improved practices point of care hand hygiene-where's the rub? a survey of us and canadian health care workers' knowledge, attitudes, and practices mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, : epidemiology, characteristics and public health implications national survey of the status of infection surveillance and control programs in acute care hospitals with more than beds in the republic of korea knowledge and beliefs about hand hygiene among hospital nurses hand hygiene compliance of healthcare workers in a children's hospital p : knowledge and perception toward hand hygiene among health-care workers in teaching hospital perceptions, attitudes, and behavior towards patient hand hygiene key beliefs of hospital nurses' hand-hygiene behaviour: protecting your peers and needing effective reminders improved hand hygiene compliance is associated with the change of perception toward hand hygiene among medical personnel world health organization. hand hygiene knowledge questionnaire for health-care workers world health organization. perception survey for health-care workers study on the efficacy of nosocomial infection control (senic project): results and implications for the future the status of healthcare-associated infection control among healthcare facilities in korea development and application of evaluation indices for hospital infection surveillance and control programs in the republic of korea reference material knowledge, attitudes, and practices study on hand hygiene among imam hossein hospital's residents in understanding the determinants of australian hospital nurses' hand hygiene decisions following the implementation of a national hand hygiene initiative nurses and physicians' perceptions of the importance and impact of healthcare-associated infections and hand hygiene: a multi-center exploratory study in hong kong analysis of hand hygiene practices of health care personnels understanding healthcare workers self-reported practices, knowledge and attitude about hand hygiene in a medical setting in rural india importance of structured training programs and good role models in hand hygiene in developing countries nurses' knowledge regarding hand hygiene and its individual and organizational predictors this research was supported by sunchon national university funds ( ). the author has no conflicts of interest associated with the material presented in this paper. key: cord- -bdpp qmf authors: lanzavecchia, antonio title: dissecting human antibody responses: useful, basic and surprising findings date: - - journal: embo mol med doi: . /emmm. sha: doc_id: cord_uid: bdpp qmf human memory b cells and plasma cells represent a rich source of antibodies that have been selected in response to human pathogens. in the last decade, different methods have been developed to interrogate the human memory repertoire and isolate monoclonal antibodies. i will discuss how a target‐agnostic approach based on high‐throughput screening of antibodies produced by cultured b cells and plasma cells has not only provided potent and broadly neutralizing antibodies against a range of pathogens, but has also advanced our understanding of basic aspects of the immune response, from host–pathogen interaction to the role of somatic mutations in affinity maturation and in the diversification of the antibody response. most surprisingly, this approach has also revealed a new mechanism of diversification based on templated insertion of non‐ig dna into antibody genes that we discovered in the context of the immune response to malaria infection. human memory b cells and plasma cells represent a rich source of antibodies that have been selected in response to human pathogens. in the last decade, different methods have been developed to interrogate the human memory repertoire and isolate monoclonal antibodies. i will discuss how a target-agnostic approach based on highthroughput screening of antibodies produced by cultured b cells and plasma cells has not only provided potent and broadly neutralizing antibodies against a range of pathogens, but has also advanced our understanding of basic aspects of the immune response, from host-pathogen interaction to the role of somatic mutations in affinity maturation and in the diversification of the antibody response. most surprisingly, this approach has also revealed a new mechanism of diversification based on templated insertion of non-ig dna into antibody genes that we discovered in the context of the immune response to malaria infection. a trove of antibodies m emory b cells represent the repository of the immune experience of an individual. they are selected in the germinal centres, where they undergo a process of somatic mutations and selection by antigen and t helper cells. memory b cells persist for a life time and can rapidly respond to a booster immunization by generating large numbers of plasma cells that are transiently present in the blood and localize to surviving niches in the bone marrow as long-lived plasma cells that are the main source of serum antibodies. the interrogation of memory b cell and plasma cell repertoires of humans offers unique opportunities. as compared to mice, humans are naturally exposed to their own pathogens, often through recurrent infections, and are therefore a better source of antibodies that target the most relevant antigens and can reveal mechanisms of host-pathogen interaction. furthermore, given the genetic variability of the human population and the idiosyncrasies of the immune response, some individuals may generate antibodies with unusual potency or breadth. finally, studies of the antibody response in humans can shed light on basic aspects of the immune response, such as the role of somatic mutations and the mechanisms that underpin the generation of broadly neutralizing antibodies or autoantibodies, and possibly uncover examples of such mechanisms that may be uniquely developed or better identified in humans. a target-agnostic approach to identify functional antibodies in the last decade, several methods have been developed to isolate human monoclonal antibodies. in principle, they come down to two basic approaches. the first approach consists of the isolation of single antigen-specific b cells using a fluorescently tagged antigen as a bait, followed by cloning of immunoglobulin genes and expression of the recombinant antibodies in a cell line. this method is very effective, but requires that the target antigen is known and available in a purified form. in my laboratory, we developed an alternative approach based on high-throughput screening of the antibodies produced by cultured b cells or plasma cells (fig a) . memory b cells from selected donors are immortalized with high efficiency (> %) by combining epstein-barr virus (that delivers signal and signal ) with a tlr or tlr agonist (that delivers signal required for potent activation of memory b cells) (traggiai et al, ) . the immortalized b cells proliferate and secrete large amounts of antibodies in the culture supernatant. antigen-specific plasma cells recovered following a booster immunization have lost proliferative capacity, but can be kept alive in cultures supplemented with il- and stromal cells. in these cultures that mimic the survival niches of the bone marrow, a single plasma cell secretes antibodies at a constant rate of pg/day (corti et al, ) . to facilitate the isolation of specific antibodies and the cloning of the vh/vl genes, both immortalized b cells and plasma cells are seeded in clonal conditions and cultures are handled with an automated liquid handling system. there are distinctive advantages in screening the repertoire of secreted antibodies. the first is that the approach is target-agnostic, since screening can be done using functional assays, such as virus neutralization, without a priori knowledge of the target antigens, using even whole bacteria or parasites. second, it is well suited to identify antibodies that cross-react with different antigens, since parallel screenings can be performed against multiple targets. third, it bypasses the need to sequence and express large numbers of antibody sequences since cells of interest are selected based on the initial screenings. a relevant example of the utility of the target-agnostic approach comes from a study of the antibody response to human cytomegalovirus (hcmv), a complex herpesvirus expressing different surface glycoproteins that causes serious pathology in the foetus and in immunocompromised patients. by screening for the capacity to neutralize the wild-type virus, we isolated antibodies that were , -fold more potent than antibodies to the fusion protein gb and identified their viral target as a pentameric complex formed by gh, gl, pul , pul and pul a. we then produced a soluble form of this pentamer and found that it can elicit, in mice, neutralizing antibody titres that exceeded by more than -fold those induced in humans by natural infection (kabanova et al, ) . these studies illustrate a general approach of "analytic vaccinology" where the neutralizing antibodies are used for the identification of the target antigen and for the quality control of the recombinant vaccine. the possibility of performing parallel screening against multiple targets was instrumental for the identification of broadly reactive antibodies capable of neutralizing multiple viruses. these include the antibodies fi and fy , which bind to conserved epitopes in the stem region of influenza hemagglutinin (ha) and neutralize all influenza a viruses (corti et al, ; kallewaard et al, ) , and mpe , an antibody that binds to a unique conserved site on the pre-fusion form of the f protein and neutralizes four different paramyxoviruses, including human rsv and mpv (corti et al, ) . these antibodies are promising candidates for prophylaxis and therapy of infections since they trigger effector functions and target conserved structures, thus limiting the possibility of selecting escape mutants. in addition, they represent useful tools for the design of stabilized pre-fusion glycoprotein vaccines. a straightforward application of the method lies in the rapid isolation of neutralizing antibodies against emerging pathogens, such as ebola virus, sars (traggiai et al, ) and mers coronaviruses. in the case of mers, it was possible, in only months, to go from a sample of memory b cells to preclinical validation of a neutralizing antibody and the generation of a cell line producing the antibody in grams/l amounts. similar studies on dengue and zika viruses defined different classes of antibodies endowed with neutralizing or infection-enhancing activity. the interrogation of the memory b-cell repertoire offers the opportunity to investigate, in biologically and medically relevant settings, fundamental aspects of the human immune response, such as the relative contribution of germ line v-gene polymorphism, vdj junctional diversity and somatic mutations to antigenic specificity. an interesting example comes from the study of antibodies that neutralize group influenza viruses and that are produced by most individuals following infection or vaccination. as reported by several laboratories, these "public" antibodies use vh - and bind, exclusively through the vh, to a conserved region in the ha stem. by isolating a large number of sister clones and by performing a genealogical analysis of different clonal families, we found that the unmutated common ancestors (uca) have low affinity for group ha and do not neutralize infection (fig b and c) . remarkably, the first branch points acquire through a single p aa mutation in hcdr , high affinity and neutralizing activity, comparable to that of the antibodies isolated from memory b cells that carried up to - amino acid substitution (pappas et al, ) . these findings demonstrate that affinity maturation can be achieved rapidly, through a single mutation, and that numerous and redundant somatic mutations continue to accumulate, providing an extensive diversification within the proliferating clone. interestingly, in these antibodies, a critical contact residue, f , is germline-encoded, but this position is dimorphic, with different alleles carrying either f or l. consequently, individuals who lack a f -encoding allele cannot produce the public vh - antibody response, while individuals that have a f allele have a high frequency of precursors, with the only additional requirement being a -amino acid hcdr with a y that makes a critical contact site with the antigen. these findings underline the impact that vh-gene polymorphism can have on the antibody response and suggest that certain alleles may play the role of immune response (ir) genes and become positively selected in the population exposed to a given pathogen. the genealogical analysis of the two paninfluenza neutralizing antibodies, fi and fy , showed that the corresponding ucas have already high binding affinity and neutralizing activity, but only on group viruses and acquire the capacity to neutralize group viruses through somatic mutations (kallewaard et al, ) . these findings are consistent with a model where pan-influenza neutralizing antibodies arise from the priming of high-affinity precursors by a group virus (in this case h n ), followed by somatic mutations and generation of a variant with broader reactivity to group viruses that can be boosted by a group virus such as h n (fig d) . importantly, the isolation of cells producing the identical fi antibody for consecutive years in response to vaccination or infection indicates that secondary antibody responses are to a large extent independent from germinal centres, a factor that allows the antibody to retain the original specificity. the genealogical analysis of the neutralizing antibody mpe revealed a similar mechanism, with the uca possessing high affinity for rsv only and mpe acquiring cross-reactivity to mpv through somatic mutations (corti et al, ) . the extensive antibody diversification observed in humans can increase antibody affinity and promote breadth, but comes at the price of generating many new specificities that may cross-react with self-antigens. the notion that autoantibodies can be generated by somatic mutations is supported in humans by the finding that the ucas of autoantibodies specific for desmoglein or gm-csf found in patients with severe autoimmune diseases do not bind to the respective self-antigens (di zenzo et al, ; piccoli et al, ) . thus, it is tempting to speculate that these autoantibodies may have been generated by somatic mutations in b-cell clones responsive to a foreign antigen in the absence of structural mimicry. importantly, the mechanisms of deletion or anergy that restrain naïve autoreactive b cells may not apply to activated/memory b cells, which have different triggering requirement and can traffic to peripheral tissues. as mentioned above, the target-agnostic approach is especially suited to dissect the antibody response to complex pathogens in the absence of a detailed knowledge of the target antigens. we were interested in identifying antibodies that could broadly recognize the variant surface antigens (vsas) of plasmodium falciparum, the parasite causing malaria, which are expressed on the surface of infected erythrocytes (ies). vsas mediate adhesion of ies to endothelia and are targets of antibodies that control disease, but their high number (> genes), their extensive polymorphism and their clonal expression provide the pathogen with a powerful chameleon-like escape strategy. out of a large kenyan cohort, we initially selected two individuals with serum antibodies that cross-agglutinated erythrocytes infected by different p. falciparum strains. from their memory b cells, we isolated a panel of broadly reactive monoclonal antibodies using staining of infected erythrocytes as a screening strategy. surprisingly, all the broadly reactive antibodies had a unique structure, since they carried a large insert between the v and the dj segments. the inserts, of approximately bp, comprised the exon encoding the extracellular domain of the collagen-binding inhibitory receptor lair /cd , encoded in the leucocyte receptor cluster on chr. (tan et al, ) . given the insertion between v and dj, the lair domain is positioned at the tip of the hcdr (fig a and b) . the target antigens of lair -containing antibodies were identified in some members of the rifins, a family of polymorphic vsas expressed on the surface of ies (fig c) . importantly, the inserted lair domain was both necessary and sufficient for binding to rifins, as demonstrated by its insertion into an irrelevant antibody or by the production of lair -ig fusion proteins. to investigate how frequently lair containing antibodies are produced, we screened two additional cohorts from mali and tanzania and found that up to % of the individuals have such antibodies in the serum (pieper et al, ) . this study led not only to the isolation of more lair containing monoclonal antibodies, but also to the finding of a new insertion modality where the lair exon with and intronic sequences is inserted into the immunoglobulin switch region. this insertion leads, by exon shuffling, to the production of the original antibody and of a bispecific antibody containing the lair domain precisely placed at the elbow between the vh and ch domains (fig a and b) . in one case, the lair insertion in the switch region was accompanied by the genomic deletion of vh and ch , generating a "camel-like" lair containing antibody. in each individual analysed so far, the lair -containing antibodies dominate the response to ies and are produced by a single b-cell clone that contains a unique insert and undergoes extensive expansion and somatic mutations, thus demonstrating an extraordinary fitness. somatic mutations in lair were more frequent when the insertion was at the v-dj junction, which is continually targeted by aid in germinal centres, and less frequent when the insertion was in the switch region, which is only transiently targeted by aid during switch recombination. interestingly, somatic mutations in the lair domain eliminate collagen binding and modulate affinity and cross-reactivity with different rifins. the finding that collagen binding was always lost, even when the somatic mutation mechanism was less effective, suggests that loss of self-reactivity is a prerequisite for expansion of b cells making lair -containing antibodies. the insertion of non-vdj sequences encoding structured domain into ig genes is a new and potentially general concept. our finding was facilitated by the huge expansion of lair -containing b cells and by the unique properties of the antibodies made. in an initial attempt to determine the generality of this phenomenon, we found frequent insertions in the switch region of memory b cells from european blood donors. interestingly, the inserts largely originate from genes that are transcribed in b cells and are encoded in virtually all chromosomes and, in rare cases, comprised exons with frames compatible with expression ( fig d) . malaria infection can cause chromosomal instability and translocations involving the switch region, suggesting that it may also play a causative role in the generation of templated insertions. however, the finding of frequent templated insertions in european blood donors is more consistent with a general mechanism that remains to be molecularly defined. the sites of insertion suggest a mechanism of patch-repair of dna double-strand breaks induced by either rag or aid during v-dj recombination or class switch. the integrity of the genomic lair loci in b-cell clones producing lair containing antibodies suggests a copy-andpaste mechanism. in view of the finding that the inserts contain intronic sequences and are derived from expressed genes, we favour the hypothesis that the templated dna may originate from the resolution of stalled replication forks caused by a clash between transcription and dna replication. our findings suggest that the insertion of templated dna represents a novel and general mechanism of antibody diversification that can provide a broad range of protein domains to be further diversified by somatic mutations. as exemplified by lair , the insertion of a domain encoding a pathogen receptor can generate public antibodies that are effective against the pathogen. distinctive advantages of these receptor-based antibodies lie in the broad recognition of all pathogen variants, leaving no room for the selection of escape mutants, and in the possibility of diversifying the receptor domain through somatic mutations to increase binding to pathogen and decrease self-reactivity. the new antibodies are reminiscent of ehrlich's "side-chain" theory of antibody production, where the antibody was essentially a secreted form of the receptor for the pathogen. we predict that other examples of receptor-based antibodies will be found in response to a variety of human pathogens. besides lair , other candidates for the generation of receptor-based antibodies to p. falciparum are icam , an adhesion molecule that binds to ies, and lilrb , an inhibitory receptor that, like lair , has been shown to bind to rifins. similarly, insertions of icam or slam domains may generate receptor-based antibodies that neutralize rhinoviruses or measles virus. as a final remark, we realize that, by uncovering the ingenuity of nature, we have learned new ways to engineer antibodies and, possibly, to edit antibodies in primary b cells using the endogenous aid activity. we also discovered how strong antibody responses can be generated by a single b-cell clone, raising a case for adoptive b-cell therapy. a neutralizing antibody selected from plasma cells that binds to group and group influenza a hemagglutinins cross-neutralization of four paramyxoviruses by a human monoclonal antibody pemphigus autoantibodies generated through somatic mutations target the desmoglein- cis-interface antibody-driven design of a human cytomegalovirus ghglpul l subunit vaccine that selectively elicits potent neutralizing antibodies structure and function analysis of an antibody recognizing all influenza a subtypes al is scientific founder of humabs biomed, has substantial interest in antibodies developed by humabs and is a shareholder of vir bio. key: cord- - lzjhn j authors: tian, bin; zhou, ming; yang, yu; yu, lan; luo, zhaochen; tian, dayong; wang, ke; cui, min; chen, huanchun; fu, zhen f.; zhao, ling title: lab-attenuated rabies virus causes abortive infection and induces cytokine expression in astrocytes by activating mitochondrial antiviral-signaling protein signaling pathway date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: lzjhn j rabies is an ancient disease but remains endemic in most parts of the world and causes approximately , deaths annually. the mechanism through which the causative agent, rabies virus (rabv), evades the host immune response and infects the host central nervous system (cns) has not been completely elucidated thus far. our previous studies have shown that lab-attenuated, but not wild-type (wt), rabv activates the innate immune response in the mouse and dog models. in this present study, we demonstrate that lab-attenuated rabv causes abortive infection in astrocytes, the most abundant glial cells in the cns. furthermore, we found that lab-attenuated rabv produces more double-stranded rna (dsrna) than wt rabv, which is recognized by retinoic acid-inducible gene i (rig-i) or melanoma differentiation-associated protein (mda ). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor molecule for rig-i and mda , results in the production of type i interferon (ifn) and the expression of hundreds of ifn-stimulated genes, which suppress rabv replication and spread in astrocytes. notably, lab-attenuated rabv replicates in a manner identical to that of wt rabv in mavs−/− astrocytes. it was also found that lab-attenuated, but not wt, rabv induces the expression of inflammatory cytokines via the mavs- p /nf-κb signaling pathway. these inflammatory cytokines increase the blood–brain barrier permeability and thus enable immune cells and antibodies infiltrate the cns parenchyma, resulting in rabv control and elimination. in contrast, wt rabv restricts dsrna production and thus evades innate recognition by rig-i/mda in astrocytes, which could be one of the mechanisms by which wt rabv evades the host immune response in resident cns cells. our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated rabv in the cns. rabies is an ancient disease but remains endemic in most parts of the world and causes approximately , deaths annually. the mechanism through which the causative agent, rabies virus (rabv), evades the host immune response and infects the host central nervous system (cns) has not been completely elucidated thus far. our previous studies have shown that lab-attenuated, but not wild-type (wt), rabv activates the innate immune response in the mouse and dog models. in this present study, we demonstrate that lab-attenuated rabv causes abortive infection in astrocytes, the most abundant glial cells in the cns. furthermore, we found that lab-attenuated rabv produces more double-stranded rna (dsrna) than wt rabv, which is recognized by retinoic acidinducible gene i (rig-i) or melanoma differentiation-associated protein (mda ). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor molecule for rig-i and mda , results in the production of type i interferon (ifn) and the expression of hundreds of ifn-stimulated genes, which suppress rabv replication and spread in astrocytes. notably, lab-attenuated rabv replicates in a manner identical to that of wt rabv in mavs−/− astrocytes. it was also found that lab-attenuated, but not wt, rabv induces the expression of inflammatory cytokines via the mavs-p /nf-κb signaling pathway. these inflammatory cytokines increase the blood-brain barrier permeability and thus enable immune cells and antibodies infiltrate the cns parenchyma, resulting in rabv control and elimination. in contrast, wt rabv restricts dsrna production and thus evades innate recognition by rig-i/mda in astrocytes, which could be one of the mechanisms by which wt rabv evades the host immune response in resident cns cells. our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated rabv in the cns. introduction rabies is an acute encephalomyelitis. the hallmark of rabies is that the disease is almost always fatal once clinical signs develop ( , ) . the causative agent, rabies virus (rabv), is a negative-strand rna virus belonging to the genus lyssavirus in the family rhabdoviridae ( ) . rabv enters neurons from the neuromuscular junction closest to the site of infection. after a short incubation, rabv travels to the central nervous system (cns) through sensory or motor neurons. only slight tissue damage and neuroinflammation can be observed in the brains of rabid patients ( ) . in contrast, lab-attenuated rabv induces extensive inflammation and apoptosis, as well as increases in the expression levels of innate immunity-related genes in the cns of infected mice ( ) ( ) ( ) ( ) ( ) ( ) . these findings suggest that wt, but not lab-attenuated, rabv evades the host immune responses. the innate immune system is the first line of defense against viral invasion. viruses are usually confronted by various pattern recognition receptors (prrs), including toll-like receptors (tlrs) and retinoic acid-inducible gene i (rig-i) like helicases (rlrs) ( ) . tlr family members, such as tlr and tlr , are generally involved in recognizing negative-strand rna viruses ( ) . tlr binds double-stranded rna (dsrna), whereas tlr recognizes single-strand rna. the main sources of dsrna in infections with single-strand rna viruses are the replicative intermediates generated by viral rna-dependent rna polymerase ( ) . tlr and tlr initiate signaling though the adaptor molecules trif and myd , respectively. both signaling cascades triggered by these proteins lead to irf phosphorylation in the c terminal region at serine , which is critical for irf activation by two iκb kinases (tbk- and ikkε) ( , ) . activated irf homo-or hetero-dimerizes with irf and then translocates into the nucleus, to interacts with the creb binding protein cbp/p and stimulates the transcription of interferon (ifn)-β, as well as some ifn-stimulated genes (isgs) ( , ) . alternatively, rna viruses can be recognized by two rlrs, rig-i, and melanoma differentiation-associated protein (mda ), located in the cytoplasm ( , ) . rig-i solely senses short and blunt dsrna of negative-strand rna viruses containing ′ triphosphate rna in the panhandle region of their single-stranded genome ( ) . unlike rig-i, mda preferentially binds blunt-ended dsrna, with or without ′ triphosphate ( ) . rig-i and mda signaling is mediated through mitochondrial antiviral-signaling protein (mavs), which is also known as ips , visa or cardif ( ) . similar to tlr signaling, rlr signaling results in irf activation and nuclear translocation ( ) . several groups have attempted to identify the prrs that recognize rabv. prehaud et al. found that tlr mrna expression is upregulated following rabv infection in postmitotic human neurons ( ) . furthermore, enhanced tlr expression has also been observed in the cerebellar cortical tissues of rabies patients ( ) . the observation that tlr is upregulated following infection suggests that tlr plays a role in the innate recognition of rabv. faul et al. found that both rig-i and mda were responsible for inducing dendritic cell (dc) activation and type i ifn production upon rabv infection ( ) . mavs, the adaptor protein for both rig-i and mda , is essential for inducing innate immune responses in dcs. in another study, li et al. found that mice lacking tlr exhibited a phenotype associated with intermediate mortality rates between those of myd −/− and control mice, indicating that tlr may play an important role in controlling rabv infections. however, the role of tlr in rabv infection is not entirely understood ( ) . astrocytes are the most abundant glial cells in the cns and constitute the blood-brain barrier (bbb) along with endothelial cells and pericytes ( ) . astrocytes are generally involved in regulating the cns microenvironment and also play roles in neuronal metabolic support, synaptic transmission, and neurotropism. moreover, astrocytes participate in developing and maintaining the bbb and guiding neuronal migration during development. the roles of astrocytes in innate immunity and inflammation have been reported recently ( ) . astrocytes are the reservoir for many neuroinvasive viruses, such as human immunodeficiency virus, theiler's murine encephalomyelitis virus (tmev), john cunningham virus, and herpes simplex virus (hsv) ( ) ( ) ( ) ( ) . astrocytes have also been shown to play multiple roles in viral infections. specifically, they can increase bbb permeability ( ) by producing cytokines and degrading tight junction proteins ( ) . moreover, astrocytes have been shown to induce the innate immune response to produce ifn ( ) . an early report showed that both wt and lab-attenuated rabv could successfully infect primary astrocytes in the early stages of rabv infection ( ) . a recent study demonstrated that a rabv vaccine strain sad-l caused an abortive infection in astrocytes, but the detailed mechanism was not revealed ( ) . in this study, it was found that lab-attenuated rabv produces higher level of dsrna than wt rabv, which is recognized by rig-i/mda and results in the activation of mavs signaling pathway. following mavs activation, the production of isg and inflammatory cytokines helps to clear the lab-attenuated rabv from the cns. in contrast, wt rabv can maintain a persistent infection in astrocytes by evading the innate recognition. mouse bend. cells and vero cells were obtained from the american type culture collection (atcc; manassas, va, usa) and were maintained in dulbecco's modified eagle medium (dmem) supplemented with fetal bovine serum (fbs; gibco, carlsbad, ca, usa). mouse neuroblastoma (na) cells were maintained in rpmi- medium (thermo-fisher, usa) supplemented with % fbs. the drv-ah (drv) was isolated from a rabid dog in anhui province, china ( , ) . cvs-b c (b c), originated from cvs- virus by passage in bhk- cells ( ), has been used as a lab-attenuated rabv ( ) ( ) ( ) . both drv and cvs-b c were propagated in suckling icr mouse brains. all the viruses were manipulated under the standard biosecurity procedures made by the ministry of agriculture of china. a rabbit anti-ubiquitin protein c (ubc) polyconal antibody was purchased from abclonal technology (woburn, ma, usa). a rabbit anti-rig-i monoclonal antibody was purchased from enzo life technology (farmingdale, ny, usa), and rabbit polyclonal antibodies against irf , stat and occludin were obtained from santa cruz biotechnology (santa cruz, ca, usa). a rabbit antiphospho-irf monoclonal antibody was purchased from cell signaling technology (washington, dc, usa), and rabbit anti-ifit and irf monoclonal antibodies were purchased from abcam (cambridge, ma, usa). a rabbit polyclonal anti-claudin- antibody, a biotinylated goat anti-rabbit or mouse antibody, and an alexa fluor -conjugated goat antimouse or rabbit antibody were purchased from invitrogen (grand island, ny, usa). mouse monoclonal anti-zoluna occludens- (anti-zo- ) antibodies were obtained from sigma (st. louis, mo, usa), and mouse monoclonal anti-gapdh antibody was purchased from proteintech (wuhan, china). the mouse anti-rabv n and p monoclonal antibodies were prepared in our laboratory, and the fluorescein isothiocyanate (fitc)-conjugated anti-rabv nucleoprotein antibody used herein was obtained from female wt or mavs-knockout (mavs−/−) c bl/ mice ( - weeks old) were infected intracerebally (i.c.) with µl of drv-ah ( ffu), b c ( ffu), or mock infected with the same volume of dmem. at days postinfection (d.p.i.), the mice were euthanized with co when moribund, and their brains were collected for immunohistochemistry analysis. blood-brain barrier permeability was determined by measuring sodium fluorescein (naf) uptake as described previously with minor modifications ( ) . briefly, µl of naf ( mg/ml) was injected intraperitoneally (i.p.) into each mouse. after anesthetization, peripheral blood and brains of each mouse were collected. the fluorescence in serum and brain homogenate samples was analyzed by a spectrophotometer (biotek instruments, vt, usa) with excitation at nm and emission at nm. standards ( - , g/ml) were prepared to calculate the naf content. naf uptake into tissue is calculated as (μg of fluorescence spinal cord/mg of tissue)/(μg of fluorescence sera/ml of blood) to normalize values for blood levels of the dye at the time of tissue collection. to detect cd + cell in the brain, infected mice were anesthetized with ketamine-xylazine ( . ml/ g body weight), perfused with ml pbs, and then the brains were transferred into % neutral buffered paraformaldehyde (pfa) for at least h ( ) . briefly, the brain sections were antigenic recovered and blocked with donkey serum, incubated with primary antibodies overnight at °c, and then secondary antibodies was applied. pbs was treated as a negative control by replacing primary antibodies. sections were photographed and analyzed using an olympus bx microscope (tokyo, japan). primary mixed glial cell cultures were established as described previously ( ) . briefly, the brain cells of -to -day-old neonatal c bl/ mice were dissociated by repeated pipetting and then passed through a -nm nylon mesh (corning, ny, usa). the cells were subsequently washed once in cold pbs and cultured in dmem (with high glucose) supplemented with % fbs and % penicillin-streptomycin. the medium was changed on days , , and for the astrocytes and on day only for the microglia. on day , the flasks were shaken at rpm for h to remove any non-adherent cells (mainly microglia). the remaining adherent astrocytes were detached with trypsin-edta and then plated again for further experiments. the purity of the astrocyte cultures was greater than %. mouse neurons were obtained from embryonic mouse brains as previously described ( ) and then dissociated by repeated pipetting (approximately times) before being passed through a -nm nylon mesh. the cells were then washed once in cold pbs and cultured in dmem (with high glucose) supplemented with % fbs and % penicillin-streptomycin for h, after which the medium was replaced with serum-free neural-basal medium (invitrogen, carlsbad, ca, usa) supplemented with % b- (invitrogen, carlsbad, ca, usa). viral titers were determined by direct fluorescent antibody assay ( ) . na cells cultured in -well plates were inoculated with viruses diluted serial -fold and then incubated at °c for h. then the culture supernatant was subsequently removed, and the cells were fixed and stained with fitc-conjugated anti-rabv n antibodies. the antigen-positive foci were counted under a fluorescence microscope (zeiss, germany), and the viral titers were calculated as ffu per milliliter. all titrations were conducted in quadruplicate. confocal microscopy table | primers used for quantification of viral mrna, ifn-stimulated genes, chemokines, and cytokines. retinoic acid-inducible gene i (rig-i) f gcgtctcagtgcagcacatcatt qrt-pcr antibodies against dsrna, rabv n, rabv p, claudin- , occludin, zo- , or dapi. infected wt or mavs−/− mice were anesthetized with ketamine-xylazine ( . ml/ g body weight) and then perfused with pbs followed by % neutral buffered formalin, as described previously ( ) . three independent mouse brain samples were collected from each group and embedded in paraffin for coronal sectioning. the sections were subsequently stained with antibodies against gfap, map , rabv p, rabv n, or dapi. after being washed, the cells or sections were incubated with an alexa fluor -conjugated goat antirabbit or mouse secondary antibodies or an alexa fluor -conjugated goat antirabbit or mouse secondary antibodies for h at room temperature. staining was visualized with a nikon a confocal laser microscope system equipped with nis-elements imaging software (nikon, melville, ny, usa) and was quantified using fiji, an imagej distribution package manufactured by nih (http://imagej.net/introduction). mean fluorescence intensity (mfi) was quantified using the region of interest, which encompassed the entire cell to include the membrane, and background staining was quantified using three negatively stained regions per cell. these regions were subtracted from the total mfi. for the protein synthesis inhibition tests, primary astrocytes were pretreated with cycloheximide (chx) (invivogen, san diego, ca, usa) for h at a dose of µg/ml, infected with drv or b c at an moi of . and then continued to be incubated with chx for another h. for the tbk activation blockage assays, bx (invivogen, san diego, ca, usa) at a dose of µm was used to treat primary astrocytes. for the inflammatory pathway blockage assays, the primary astrocytes were pretreated with a p inhibitor (skepinone-l; sellek, houston, tx, usa), a jnk inhibitor (jnk inhibitor ix; sellek, houston, tx, usa), an nf-κb inhibitor (sc ; sellek, houston, tx, usa), or dmso as control at a dose of µg/ml for h. then the cells were infected with drv or b c at moi and incubated with the above inhibitors for h. the concentrations of tnf-α and il- in the supernatant of astrocytes were measured by the commercial elisa kits according to the manufacturer's instruction (abclonal technology, woburn, ma, usa). rna was isolated with trizol ® reagent (invitrogen), according to the manufacturer's instructions, and qrt-pcr was performed as described previously ( ) . briefly, ng of total rna (from either cells or tissue) was transcribed into cdna in a reaction mixture with a total of volume of µl using a superscript iii reverse transcription kit (toyobo). the reaction mixture comprised μl of cdna combined with µl of iq sybr green mix (biorad, hercules, ca, usa), µl of diethyl pyrocarbonatetreated water, and . µl of primer mix (the concentration of each primer was mm). the cdna was amplified using an iq icycler (bio-rad), and the cycle threshold (ct) values were recorded. the ct value was inversely correlated with the mrna concentration, and each ct unit represented a twofold change in the mrna concentration. basal mrna expression levels were expressed as Δ ct values and were normalized to β-actin mrna expression levels [ Δ ct ct (isg)/ Δ ct (β-actin)]. induced mrna expression levels were expressed as fold changes relative to mock-infection levels using the ΔΔct method. all the primer sequences are listed in (table ) . to quantify cellular rabv n rna levels, we transcribed the total rna using avian myeloblastosis virus reverse transcriptase xl (takara, kusatsu, japan) and a primer specific for the rabv n genomic sequence. a standard curve was generated from serially diluted plasmids carrying a rabv n gene and the copy numbers of n mrna were normalized to mg of total rna. the cells were lysed with ripa buffer containing protease inhibitors (roche), and the protein concentrations were measured using a dc protein assay kit (bio-rad). equal quantities of protein were resolved by or % sds-page and then transferred to polyvinylidene difluoride membranes (bio-rad), which were blocked with % nonfat milk before being incubated with primary antibodies against rig-i, phosphorylated irf (p-irf ), stat , ifit , rabv n, claudin- , occludin, zo- , or gapdh and then probed with the appropriate secondary antibodies. the blots were then visualized using ecl reagent (ge, pittsburgh, pa, usa) and detected under an intelligent dark box ii (ge, pittsburgh, pa, usa). the -gain pmt scan generated the optimal standard curve, and the results of this scan were analyzed using q-analyzer software for qam-cyt- (raybiotech). transendothelial permeability assay was performed according to the methods described previously ( ), with some modifications. b.end cells were grown on -μm-pore transwell filter inserts until they reached % confluency. the medium was then treated with uv-inactivated cell culture supernatants collected from rabv-infected astrocytes. after the cells had incubated for h, they were treated apically with dextran-fitc at a dose of . µg/ml for min. the samples were then removed from the lower chamber and subjected to fluorescence measurement, which were performed using a fluorimeter (biotek, winooski, vt, usa; the excitation wavelength was nm, and the emission wavelength was nm). the fluorescence values for the experimental cells were subsequently compared to corresponding values for a control cell monolayer. data are expressed as the mean and standard error of the mean (sem), and the significance of the differences between groups was evaluated by student's t test or one-way analysis of variance followed by tukey's post hoc test. the survival ratios were analyzed by log-rank (mantel-cox) test. the asterisks indicated statistical significance (*, p < . ; **, p < . ; ***, p < . ). graphs were plotted and analyzed using graphpad prism software, version . (graphpad software, la jolla, ca, usa). results comparison of the pathogenicity of drv-ah and cvs-b c in mice first, the pathogenicity of the wt rabv strain drv-ah (drv) and lab-attenuated strain cvs-b c (b c) used in this study were compared in a mouse model. c /bl mice were i.c. inoculated with ffu b c or drv and the development of rabies was observed. as expected, drv-infected mice displayed development of the diseases at d.p.i. and all moribund at d.p.i., earlier than b c-infected mice which all succumb to rabies at d.p.i. (figure a) . we found that b c replicated faster than drv at the early stage of the infection in the cns. to ensure that the viral load in the brains is similar, c /bl mice were i.c. inoculated with ffu b c or ffu drv. then the viral load in the mice brain were determined at , , , , , and d.p.i. the results showed that the genomic rna of drv was lower than that of b c at and d.p.i., but reached the same level as that of b c at and d.p.i. (figure b) . previous studies have shown that the lab-attenuated rabv infection enhances the bbb permeability and induces inflammation in the brain ( , ) . thus, the na-fluorescence (na-f) uptake of both strains at d.p.i. was measured, and the data showed that na-f uptake in b c-infected brain was significantly higher than that of drv or mock-infected brains ( figure c) . consistently, there were more cd + lymphocytes in b c-infected brains than drv-infected mouse brains (figures d,e) . all these results are consistent with the previous findings related to the cns inflammation and bbb permeability change during comparison of the pathogenicity between wt and lab-attenuated rabv ( , , ) . astrocytes play an important role in the induction of innate immunity in the cns. to investigate the role of astrocytes in the pathogenesis of rabv, we isolated primary astrocytes and neurons from suckling mice and infected them with drv or b c. the growth kinetics of both viruses in astrocytes was assessed by virus titration and immunofluorescence assay (ifa). the viral loads in the cell culture supernatants infected with b c quickly reached peak at d.p.i., and then gradually decreased until d.p.i. in contrast, the virus titer of drv was initially relatively low but subsequently increased steadily until the endpoint (figure a) . to be noted, the viral titer of drv in the cell supernatant maintained at a relatively low level than that of b c in astrocyte at and d.p.i., but n mrna transcription levels of drv was significantly higher than that of b c at and d.p.i. (figure c) . the ifa results showed that drv persistently replicated in astrocytes and large immunofluorescence foci could be observed at d.p.i., while no obvious immunofluorescence foci could be found in b c-infected cells ( figure d) . as a control, virus replication kinetics in primary neurons was also compared. it was found that viral titers of b c were always higher than those of wt rabv at the indicated time points (figure b) . the ifa results also showed that both drv and b c could efficiently replicate in neuron ( figure d) . to confirm these observations in vivo, c bl/ mice were i.c. infected with ffu b c or ffu drv. infected mice were euthanized when moribund and the brains were harvested for fluorescence ihc analysis. gfap was a well-known surface marker for astrocytes ( ) , and the gfap staining demonstrated drv could effectively infect astrocytes in the brains ( figure e) . however, few infected astrocytes could be observed in b cinfected mouse brains (figure e ). in contrast, neuron was intensively infected by both drv and b c ( figure f) . together, these results show that the b c causes abortive infection in astrocytes both in vitro and vivo. previous studies have demonstrated that rabv can be recognized by rig-i and mda , which share a common adaptor mavs in dcs ( ) . to assess innate immune responses in astrocytes, cells were infected with drv or b c at an moi of . and the expression of several proteins involved in the mavs signaling pathway, namely, rig-i, p-irf , stat and ifit (isg ), was measured by western blot. the ubiquitin ligase trim mediates lysine -linked ubiquitination of rig-i's n-terminal card domains is indispensable to induce type i ifn production and antiviral immunity ( ) . thus, the immunoprecipitation of rig-i was carried out and then resolved by western blot by using an anti-ubiquitin antibody. as expected, rig-i was much more robustly ubiquitinated in astrocytes infected with b c than that in astrocytes infected with drv. consistently, the expression levels of rig-i, p-irf , stat , and ifit in b c-infected astrocytes were higher than those in drv-infected cells ( figure a) . next, we attempted to determine which viral product (rna or protein) activates mavs signaling pathway in astrocytes. primary astrocytes were treated with chx, a protein synthesis inhibitor, to inhibit viral protein synthesis. it was found that b c and drv n transcription levels were similar between chxtreated and mock-treated astrocytes at h p.i. (figure b) . however, the expression levels of the genes involved in the mavs signaling pathway, namely, rig-i, mda , mavs, irf , ifn-β, stat , and ifit , were significantly upregulated by b c compared with drv in both chx-treated and mock-treated astrocytes, indicating that viral rna rather than proteins activates ifn pathway depending on mavs (figures c-i) . taken together, these data demonstrate that the lab-attenuated rabv, but not wt rabv, activates the mavs signaling pathway by viral rna, resulting in the production of ifn as well as isgs in astrocytes. retinoic acid-inducible gene i and mda activation is induced mostly by dsrna, which is produced during viral replication ( ) . to compare the amount of dsrna produced by wt and labattenuated rabv, primary astrocytes were infected with drv or b c at an moi of . . at and d.p.i., dsrna was stained with specific antibody and observed by a confocal fluorescence microscope. the results demonstrate that more dsrna is synthesized in b c-infected astrocytes than those in drv-infected cells ( figure a) . the dsrna intensity per cell in b c-infected cell was significantly higher than that in drv-infected cells (figure b) , considering the similar rabv intensity per cell (figure c) . the ratio of dsrna intensity to rabv intensity in b c-infected astrocyte was significantly higher than that in drv-infected cells ( figure d) . these results suggest that lab-attenuated rabv produces more viral dsrna than wt rabv during viral replication, ( ffu) . (e) at d.p.i., mice were euthanized, perfused with pbs and then fixed with % pfa. the brains were subsequently coated with paraffin, and the brain sections were stained with antibodies against gfap (red), rabv p protein (green), or dapi (blue). the white arrows indicate rabv-infected astrocytes. the scale bars represent µm. (f) the same brain sections as (e) were stained with antibodies against map (green), rabv p protein (red), or dapi (blue), then visualized under a confocal microscope. the scale bars represent µm. notably, b c titers were significantly increased in mavs−/− astrocytes compared with wt astrocytes (figures a,b) . moreover, the cell numbers of immunofluorescence plaques in mavs−/− astrocytes caused by b c infection were significantly more than those in wt astrocytes (figures e,f) . in contrast, mavs deficiency had no significant influence on drv replication and spread in astrocytes (figures a,e) . tbk is an iκb kinase downstream of the mavs signaling pathway and is critical for irf phosphorylation. treatment with the tbk specific inhibitor bx significantly increased b c titers in astrocytes (figures c,d) . to verify these observations in vivo, mavs−/− mice were i.c. infected with drv or b c, and virus infection of astrocytes was determined by ifa. we found that both drv and b c could efficiently infected mavs−/− astrocytes (figures g,h) . taken together, these findings suggest that mavs signaling significantly restricts the replication and spread of lab-attenuated but not wt rabv in astrocytes. recent studies have shown that tlr may be another innate immune molecule that recognizes rabv ( ) . thus, the role of tlr in rabv replication was investigated in astrocytes. astrocytes from tlr −/− and control mice were isolated and then infected with drv or b c at an moi of . . at different time points p.i., viral titers (figures a,b ) and the formation of viral immunofluorescence foci was analyzed (figures e,f) . the results demonstrated that tlr deficiency did not significantly affect the replication and spread of either drv or b c in astrocytes, suggesting that tlr does not play a role in containing rabv replication and spread in astrocytes. astrocytes are one of the major sources of inflammatory cytokines in the cns post viral infection, and these cytokines play an important role in regulating bbb permeability. to investigate rabv-induced cytokine production, primary astrocytes from wt and mavs−/− mice were prepared and infected with drv or b c at an moi of . . at indicated time points, the cell culture supernatants were harvested and analyzed with a cytokine array kit. the results showed that b c induced significantly higher levels of cytokine expression, namely, tnf-α, il- , il- β, ifn-γ, il- , and vegf expression, than drv in wt and mavs−/− astrocytes. the levels of cytokine expression in mavs−/− astrocytes were significantly lower than those in wt astrocytes, indicating that rabv-induced inflammatory cytokine production in astrocytes is dependent on the mavs signaling pathway (figures a-f) . the specific pathway through which rabv induces cytokine production was subsequently identified in astrocytes. a previous study demonstrated that rabv induces cytokine production in macrophages mainly through p , jnk, and nf-κb pathways ( , ) . thus, primary astrocytes were treated with p , jnk, and nf-κb pathway inhibitors skepinone-l, jnk ix, and sc , respectively, and the concentrations of tnf-α and il- in the cell supernatant were measured by elisa. the results showed that skepinone-l and sc caused greater reductions in tnf-α ( figure g ) and il- ( figure h ) protein levels than jnk ix in b c-infected astrocytes. none of these inhibitors significantly altered the expression levels of tnf-α and il- in drv-infected astrocytes. these findings suggest that lab-attenuated rabv induces cytokine expression in astrocytes mainly through the p and nf-κb pathways and that wt rabv suppresses cytokine production in astrocytes. our previous studies demonstrated that the chemokines/ cytokines induced by rabv infection are responsible for reducing tj protein expression and enhancing bbb permeability ( ) . to investigate the effect of these cytokines on bbb permeability, a mouse brain microvascular endothelial cell line b.end , was cocultured with uv-inactivated supernatants infected with drv or b c and collected them at different time points after infection. treatment with the supernatants from b c-infected astrocytes for h (figure a ) induced significant increase in dextran-fitc infiltration from to h p.i. notably, treatment with the supernatants from b c-infected mavs−/− astrocytes elicited significant increases in dextran-fitc permeability at h p.i. (figure b) . no significant increases in dextran-fitc permeability were observed after treatment with the supernatants from wt or mavs−/− astrocytes infected with drv. to gain an insight into the mechanisms by which labattenuated rabv enhances bbb permeability, the expression levels of the indicated tj proteins (claudin- , occludin, and zo- ) were assessed in astrocytes by western blot. it was found that the expression levels of claudin- and occludin were unchanged, while zo- was significantly reduced after the cells were treated with the supernatants from b c-infected wt astrocyte ( figure c) . however, this reduction was attenuated in cells treated with the supernatants from mavs−/− astrocytes infected with b c. no significant changes in zo- expression were observed in b.end cells treated with supernatants from wt or mavs−/− astrocyte infected with drv ( figure d) . similarly, ifa results showed that zo- expression was significantly decreased in b.end cells cocultured with the supernatants from b c-infected astrocytes collected at h p.i. (figure e) . zo- fluorescence intensity was only slightly decreased in b.end cells cocultured with the supernatants from b c-infected mavs−/− astrocytes collected at h p.i., indicating that cell-to-cell contact was not significantly decreased in these cells. moreover, no zo- degradation was observed in b.end cells cocultured with the supernatants from wt or mavs−/− astrocytes infected with drv. to be noted, no significant changes in claudin- and occludin expression were detected in either wt or mavs−/− astrocytes upon rabv infection (figures c,e) . together, these results illustrate that lab-attenuated, but not wt, rabv upregulates the production of inflammatory cytokines in astrocytes, resulting in zo- degradation in bmecs and subsequent increases in bbb permeability. furthermore, these results indicate that cytokine production is dependent on mavs signaling pathway. astrocytes play critical roles in host defense during viral infections of the cns ( ) . prr activation in astrocytes results in the expression of many immune mediators, including type i ifns and inflammatory cytokines ( , ) . during infections caused by pathogens for which glia are not susceptible targets, activation of the innate immune system caused by pathogen recognition in astrocytes may promote antiviral immune responses in susceptible neurons, as well as cns leukocyte trafficking ( , , ) . in an early report, primary murine, feline, and human astrocytes were infected with wt (srv) and lab-attenuated rabv (era) ( ), after which viral loads and replication were assessed by infectivity assay and immunofluorescence. the results showed that astrocytes can be infected by rabv, suggesting that astrocytes may play a role in viral spread and persistence and/or neuronal dysfunction ( ) . however, in that study, viral loads and replication were evaluated only at the early time point after infection ( d.p.i.) . in this present study, the growth of drv with that of b c, which were used as a pair of wt and lab-attenuated rabv in previous studies ( ) ( ) ( ) , was compared in a long-term experiment. surprisingly, we found that lab-attenuated rabv, but not wt rabv, caused abortive replication in astrocytes, a feature that may be associated with the ability of the virus to evade the innate immune response. productive infection of the astrocyte is critical for neurotropic pathogens to induce encephalitis. astrocytes sensed viral entry into the cns and mounted a type i ifn response, which rapidly restricts the virus after neuronal transport into the cns. previous studies demonstrated that tlr −/− astrocytes were more permissive to hsv infection and caused severe symptom of encephalitis and tissue damage, which was due to impaired type i ifn production in the absence of tlr ( ) . a recently work found that abortively infected astrocytes are the major producers of ifn-β after infection of the brain with diverse neurotropic viruses, including tmev, rabv, and vesicular stomatitis virus (vsv) ( ) . consistent with these studies, we also found that the abortive infection of lab-attenuated rabv in astrocytes was related to its ability to activate ifn signaling pathway. basal isg expression levels are an important determinant of susceptibility to viral infection. we have found that astrocytes have higher basal expression levels of the mrnas encoding isg proteins, such mda and stat , and other molecules crucial for recognizing viral invasion and creating an more antiviral environment than neurons ( ) , which may explain why lab-attenuated rabv activates the innate immune response to a degree sufficient to restrict viral replication in astrocytes but not in neurons. double-stranded rna is a viral product that plays an essential role in inducing innate immunity, which leads to the production of type i ifns and the activation of hundreds of isgs. early biochemical studies of viral replication suggested that most viruses produce dsrnas ( ) . however, in , weber et al. reported that dsrna could be detected by ifa in cells infected with positive-stranded rna viruses, but not with negative-stranded rna viruses ( ) . this notion was challenged by two other studies on dsrna production in cells infected with negative-stranded rna viruses ( , ) . moreover, a recent study demonstrated the dsrna formation in cells infected with several negative-stranded rna viruses, such as vsv, measles virus (mev), and influenza a virus, although the intensity of the staining of dsrna tended to be weaker in cells infected with negative-stranded rna viruses when compared with those infected with positive-stranded rna viruses ( ) . consistent with this finding, the production of dsrna was detected in both lab-attenuated and wt rabv-infected astrocytes, although lab-attenuated rabv produced significantly more dsrna in the cytoplasm than wt rabv. we attempted to investigate why lab-attenuated rabv produces more dsrna than wt rabv. pfaller et al. found that dsrna expression was much lower in cells infected with wt mev than in cells infected with a mutant mev whose c protein was knocked out which is known to control genome replication and transcription ( ) . similarly, takeuchi et al. observed dsrna formation in cells infected with sendai virus ( ) with c protein knocked out but not in cells infected with wt sev, indicating that the c protein limits or masks dsrna production ( ) . both mev and sev are within the family paramyxoviridae, thus it is possible that their c protein possess the similar function to subvert the production of dsrna. ebola virus protein vp adopts a unique strategy to mask key cellular recognition sites on dsrna ( ) . a recently study showed that the coronavirus endonuclease (endou) activity is the key to prevent early induction of dsrna. replication of endou-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of virus infection ( ) . in this present study, by exchanging viral genes between wt and lab-attenuated rabv, we found that single n, p, and g of wt rabv could not suppress dsrna formation (data not shown). however, multiple viral proteins of rabv may work together to limit the production of dsrna. further studies are needed to elucidate the mechanism through which wt rabv restricts dsrna formation and thus evades recognition by the innate immune system in infected cells. the bbb, which is composed of specialized bmecs joined by tjs and ensheathed by astrocytes and pericytes, plays an important role in protecting the cns. our previous studies have shown that rabv does not infect bmecs, nor does it modulate tj protein expression in bmecs ( ) . however, brain extracts prepared from mice infected with lab-attenuated rabv but not wt rabv reduced tj protein expression in bmecs, indicating that the above enhancements of bbb permeability and reductions in tj protein expression are not caused by rabv infection. rather, they are caused by virus-induced inflammatory chemokines/ cytokines. the innate immune mechanisms that regulate bbb function in the setting of infectious diseases have been appreciated only recently. multiple inflammatory cytokines, including tnf-α, il- , il- β, ifn-γ, il- , and vegf, disrupt bbb and tj integrity in bmecs ( , , ( ) ( ) ( ) , and inflammatory cytokine signaling at the bbb during infection facilitates leukocyte trafficking into the cns, which is essential for the clearance of many pathogens ( , ) . our present study demonstrates that lab-attenuated rabv induces production of several inflammatory cytokines in astrocytes, especially tnf-α, il- , il- β, ifn-γ, il- , and vegf, which cause disruption of the bbb and tj integrity. our findings suggest that astrocytes play a critical role in regulating bbb permeability as a major source of cytokines during viral infection. furthermore, we found that the production of inflammatory cytokines in astrocytes by lab-attenuated rabv was dependent on mavs signaling pathway, underscoring the critical role of mavs signaling in defensing against rabv infection in cns. | the proposed model for the mechanism through which wild-type (wt) and lab-attenuated rabies virus (rabv) infect astrocytes. during infection, lab-attenuated rabv produces double-stranded rna (dsrna), which is recognized by retinoic acid-inducible gene i (rig-i)/melanoma differentiation-associated protein (mda ). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor protein for rig-i and mda , leads to enhanced production of interferon, as well as some ifn-stimulated genes, which limit rabv replication and spread. mavs activation stimulates the p and nf-κb signaling pathways and induces cytokine production in astrocytes. inflammatory cytokines promote blood-brain barrier (bbb) permeability, enabling peripheral inflammatory cells and antibodies to infiltrate into the central nervous system, thereby facilitating rabv clearance. in contrast, wt rabv prevents activation of the mavs signaling pathway by restricting dsrna production. conclusion based on the results, we propose the following model: labattenuated rabv produces dsrna recognized by rig-i, mda , or both, resulting in the activation of the mavs signaling pathway in astrocytes. ifn expression induces the transcription of hundreds of isgs to inhibit rabv replication in astrocytes and causes the abortive infection by lab-attenuated rabv. the inflammatory cytokines induced by lab-attenuated rabv enhance bbb permeability, enabling immune cells and antibodies to infiltrate the cns and facilitate rabv clearance. conversely, wt rabv restricts dsrna production and then evades recognition by the innate immune system, resulting in persistent viral replication in astrocytes (figure ). current status of rabies and prospects for elimination the cell biology of rabies virus: using stealth to reach the brain rhabdoviruses: rabies virus role of apoptosis in rabies viral encephalitis: a comparative study in mice, canine, and human brain with a review of literature silver-haired bat rabies virus variant does not induce apoptosis in the brain of experimentally infected mice attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system rabies virus-induced apoptosis involves caspase-dependent and caspase-independent pathways failure to open the bloodbrain barrier and deliver immune effectors to central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection the roles of chemokines in rabies virus infection: overexpression may not always be beneficial neuronal apoptosis does not play an important role in human rabies encephalitis toll-like receptors in innate immunity toll-like receptor function and signaling double-strand rna is detected by immunofluorescence analysis in rna and dna virus infections including those by negative-strand rna viruses myd functions as a negative regulator of tlr /trif-induced corneal inflammation by inhibiting activation of c-jun n-terminal kinase regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp involvement of the ubiquitin-like domain of tbk /ikk-i kinases in regulation of ifn-inducible genes recognition of ' triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus structural basis of double-stranded rna recognition by the rig-i like receptor mda mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response induction of irf- and irf- phosphorylation following activation of the rig-i pathway virus infection switches tlr- -positive human neurons to become strong producers of beta interferon toll-like receptor (tlr ) plays a major role in the formation of rabies virus negri bodies rabies virus infection induces type i interferon production in an ips- dependent manner while dendritic cell activation relies on ifnar signaling the role of toll-like receptors in the induction of immune responses during rabies virus infection blood-brain barrier biology and methodology immune players in the cns: the astrocyte susceptibility to theiler's virus-induced demyelinating disease correlates with astrocyte class ii induction and antigen presentation longlasting astrocyte reaction to persistent junin virus infection of rat cortical neurons astrocyte infection by hiv- : mechanisms of restricted virus replication, and role in the pathogenesis of hiv- -associated dementia tlr deficiency renders astrocytes permissive to herpes simplex virus infection and facilitates establishment of cns infection in mice potential mechanisms for astrocyte-timp- downregulation in chronic inflammatory diseases viral disruption of the blood-brain barrier astrocyte response to junin virus infection rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes abortively infected astrocytes appear to represent the main source of interferon beta in the virus-infected brain complete genome sequence of a street rabies virus isolated from a rabid dog in china rabies viruses leader rna interacts with host hsc and inhibits virus replication role of chemokines in the enhancement of bbb permeability and inflammatory infiltration after rabies virus infection enhancement of blood-brain barrier permeability and reduction of tight junction protein expression are modulated by chemokines/cytokines induced by rabies virus infection the inability of wild-type rabies virus to activate dendritic cells is dependent on the glycoprotein and correlates with its low level of the de novo-synthesized leader rna expression of neuronal cxcl induced by rabies virus infection initiates infiltration of inflammatory cells, production of chemokines and cytokines, and enhancement of blood-brain barrier permeability cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus infection of pericytes in vitro by japanese encephalitis virus disrupts the integrity of the endothelial barrier regional astrocyte ifn signaling restricts pathogenesis during neurotropic viral infection influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i overexpression of tumor necrosis factor alpha by a recombinant rabies virus attenuates replication in neurons and prevents lethal infection in mice rabies virus-induced activation of mitogen-activated protein kinase and nf-kappab signaling pathways regulates expression of cxc and cc chemokine ligands in microglia doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses measles virus c protein impairs production of defective copyback double-stranded viral rna and activation of protein kinase r production of il- , il- , ifn-gamma and ip- in human astrocytes correlates with alphavirus attenuation sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna ebolavirus vp coats the backbone of double-stranded rna for interferon antagonism early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication astrocytes produce and release interleukin- , interleukin- , tumor necrosis factor alpha and interferon-gamma following traumatic and metabolic injury cellular mechanisms of il- -induced blood-brain barrier disruption expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type i interferon the authors wish to thank the staff members of the animal facility at huazhong agricultural university for caring for the mice. this work was partially supported by the national natural science foundation of china ( and to zf and and to lz); the national program on key research project of china ( yfd to lz). key: cord- -wels wt authors: gottlieb, jens title: community-acquired respiratory viruses date: - - journal: semin respir crit care med doi: . /s- - sha: doc_id: cord_uid: wels wt the incidence of community-acquired respiratory viruses (carvs) is ∼ cases per patient-years after lung transplantation (ltx). paramyxoviruses account for almost % of the cases of carv infection in ltx. most patients will be symptomatic with a mean decline of to % in forced expiratory volume in second. the attributable death rate is low in recent years to % carv infected ltx patients will develop chronic lung allograft dysfunction within a year after carv infection. this risk seems to be increased in comparison to the noninfected ltx recipient. detection rate of carv dependent on clinical awareness, sampling, and diagnostic method with nucleic acid testing by polymerase chain reaction in bronchoalveolar lavage is the gold standard after ltx. there is no approved treatment for paramyxoviruses, most centers use ribavirin by various routes. toxicity of systemic ribavirin is of concern and some patients will have contraindication to this treatment modality. treatment may reduce the risk to develop chronic lung allograft dysfunction and respiratory failure. agents under development are inhibiting viral attachment and use silencing mechanisms of viral replication. the respiratory tract is a major entry through which viruses initiate infection. the respiratory tract can be divided anatomically into the upper respiratory tract and the lower respiratory tract divided by the lymphoid tissue of waldeyer's ring. community-acquired respiratory viruses (carvs) are highly developed and ubiquitous pathogens which can cause infection of both the upper and lower respiratory tract. infection with these viruses usually results in a mild, self-limited disease in the nonimmunocompromised adult. some carv may result in respiratory failure even with fatal outcome (e.g., middle east respiratory syndrome coronavirus, corona virus associated severe acute respiratory syndrome). carvs have been increasingly recognized as common pathogens after lung transplantation (ltx). carv are a diverse group of viruses including the paramyxoviridae: respiratory syncytial virus (rsv), parainfluenza virus (pv), human metapneumovirus (hmpv); the orthomyxoviridae: influenza a and b (flu); the picornaviridae: rhinovirus (rv) and enterovirus; the coronaviridae: coronavirus (cov); and the adenoviridae: adenovirus (av) (see ►table ). most viruses will cause local infection in the respiratory tract first with secondary dissemination to other sites in the body, while other viruses typically remain limited to the respiratory tract and induce tissue injury locally. various defense mechanisms have evolved in the respiratory tract to prevent and control infection. advances in the pathomechanisms involved in carv infection has been made recently. excessive inflammation associated with severe infection can be controlled by blocking costimulatory signals, without altering immune-mediated virus clearance. recent studies suggest that the activation of effector t cells in virus-infected lungs is generated by inflammatory cells locally. this modification of the immune response in the infected epithelium may lead to virus clearance and will regulate acute inflammation, and possibly immunological memory. resolution of respiratory virus infection requires not only the elimination of the keywords ► lung transplantation ► community-acquired respiratory viruses ► ribavirin ► bronchiolitis obliterns syndrome the incidence of community-acquired respiratory viruses (carvs) is $ cases per patient-years after lung transplantation (ltx). paramyxoviruses account for almost % of the cases of carv infection in ltx. most patients will be symptomatic with a mean decline of to % in forced expiratory volume in second. the attributable death rate is low in recent years to % carv infected ltx patients will develop chronic lung allograft dysfunction within a year after carv infection. this risk seems to be increased in comparison to the noninfected ltx recipient. detection rate of carv dependent on clinical awareness, sampling, and diagnostic method with nucleic acid testing by polymerase chain reaction in bronchoalveolar lavage is the gold standard after ltx. there is no approved treatment for paramyxoviruses, most centers use ribavirin by various routes. toxicity of systemic ribavirin is of concern and some patients will have contraindication to this treatment modality. treatment may reduce the risk to develop chronic lung allograft dysfunction and respiratory failure. agents under development are inhibiting viral attachment and use silencing mechanisms of viral replication. virus but also the repair and regeneration of normal lung structures and restoration of normal pulmonary function. cells and molecules regulate processes such as epithelial cell to mesenchymal cell transitions, as well as the transformation of fibroblasts and fibrocytes into myofibroblasts. dysregulation of these processes in response to lung inflammation is associated with progressive pulmonary injury and lung fibrosis. in ltx recipients, carv infection may cause inflammatory processes mediated by both innate and adaptive immune responses that result in injury to airway epithelial cells and subepithelial structures leading to obliteration of small airways. apart from direct sequelae, carv may promote immunologically mediated lung injury resulting in the development of acute rejection (ar). a clinical term for chronic lung allograft dysfunction (clad) is bronchiolitis obliterans syndrome (bos). the clinical picture is an irreversible decline in forced expiratory volume in second (fev ). histopathologically obliteration of terminal bronchioli by fibromyxomatous tissue is recognized. bos is the most important prognosis limiting factor following ltx, and remains the major impediment to long-term graft and patient survival after ltx. on average, it affects every second recipient after years. the annual incidence of bos after ltx is $ %. bos is a progressive disease and % of bos patients die from respiratory failure with a -year survival in affected patients of %. in ltx recipients, symptoms of carv infections may resemble those of other clinical problems like other causes of infection (bacterial or fungal), acute rejection, and drug toxicities. bacteria (mycoplasma pneumoniae, chlamydia pneumoniae, and legionella pneumophila) also cause community outbreaks, share similar clinical features, and may present diagnostic difficulties. in contrast to the nonimmunosuppressed host, carv infection usually leads to more severe illness in the lung transplanted recipient with a higher incidence of respiratory failure. in a recent clinical trial with rsv-infected ltx patients, median decline in fev compared with the preinfection level was %. seventy-four percent of these patients had symptoms of a lower respiratory tract infection and % a significant infiltrate on chest x-ray. in a retrospective singlecenter study over a -year time span, % of ltx patients presenting with carv infection ( % coxsackie and rv, % pv, % cov, % rsv, % hmpv, % flu) demonstrated a pulmonary infiltrate and % were asymptomatic. acute consequences of rsv infection include bronchiolitis, pneumonia, and respiratory failure. permanent long-term effects following ltx include the development of new or progressive chronic allograft dysfunction that manifest clinically as bos. , rhinoviral infection can be persistent in lung transplant recipients with graft dysfunction. no temporal association was observed between carv infection and ar. , cumulative infection rates of carv in lung transplant recipients in earlier retrospective single-center studies of to years ranged from to %. [ ] [ ] [ ] [ ] retrospective studies did not test for viruses newly recognized as important pathogens for bos, especially hmpv. therefore, apart from methodological issues, bos incidence may not be exactly estimated from these studies. there are several published prospective studies using polymerase chain reaction (pcr) techniques of carv in ltx involving between and patients during surveillance periods of to months. , - symptoms of respiratory tract infection (rti) were frequent to % in screened patients but incidence of carv was variable between . and %. the different incidences can be explained by different methodology and inclusion criteria. in the swiss study, only patients who underwent bronchoscopy were studied, making it difficult to calculate the true incidence of respiratory viral infections and comparing it with uninfected patients. in a canadian study, only stable patients were included and screened for carv and matched with uninfected patients, which excluded an estimation of the true incidence of carv during the observation period. in a u.s. study, ltx recipients were contacted weekly by telephone and screened for symptoms. symptoms of rti were, therefore, more frequent. serology identified most of the carv in this study and hmpv was not investigated. pvs were the dominant pathogens in a german surveillance study using pcr testing in upper respiratory samples and antigen testing in sample from the lower respiratory tract followed by rsv ( - % in other prospective studies). in the german surveillance study, an annual incidence of % in carv-positive recipients was described, which is higher than to % in other prospective studies using pcr techniques. , in summary, prospective studies have described an incidence of to cases per patientyears (►table ). infected ltx recipients in early studies presented with respiratory failure. interestingly, in retrospective studies , , the bos incidence in carv-positive lung transplant recipients was much more common after year than in the prospective studies ( - % versus - %). , , several surveillance and retrospective studies confirmed an association of carv infection (as well other carv) with the onset of bos. these studies are heterogeneous and have limitations in design, case selection, and diagnostic procedures. this association was identified mainly for paramyxoviruses, and the association with influenza and av is less well documented. according to the published literature, the incidence of bos was to % in ltx recipients infected with carv during the following months. , , , , patients may present with onset of clad immediately with carv infection or later after an initial recovery of fev after the viral infection. there is a lack of information on the potential role of viruses that are difficult to grow, such as rv, human cov, enteroviruses, and hmpv. some of them have recently been recognized as important pathogens in ltx. , , virus isolation is still the gold standard for the laboratory detection of respiratory viruses. however, virus isolation in cell cultures is slow and not always technically successful. therefore, this method is no longer used in the transplant setting where rapid workup is crucial. rapid antigen detection tests or antibodies for immunofluorescence testing (ift) are not available for all carv. the detection of viral antigens is reported to be less sensitive and less specific than cell cultures but allows rapid detection. nucleic acid amplification tests for carv by pcr are rapid and sensitive. they are commercially available in single or multiplex format. the significance of virus detection by pcrs in asymptomatic patients is unknown and virus detection by pcr may not reflect active infection. serology plays no role in detecting carv infection after ltx. upper respiratory sampling by naso-oropharyngeal swab (nos) are useful in combination with pcr techniques avoiding the need for bronchoscopy. special swabs samples should be used. mouth rinses are an acceptable alternative. in ltx recipients, bronchoalveolar lavage (bal) is a widespread diagnostic tool. given the broad spectrum of other possible diagnosis in a ltx patient with symptoms of upper respiratory tract infection (urti) or lower respiratory tract infection (lrti), bronchoscopy with bal is advised in most circumstances. bal was most sensitive do detect carv in surveillance studies. in a recent randomized controlled trial (rct), % of all rsv infections were detected by pcr. the potential incidence of bos after untreated carv infections and the threat of respiratory failure in infected ltx recipients illustrate the need for effective treatment of carv infections. unfortunately, respiratory viruses are a hard target for various reasons. most of the time the virus spends in the host and will be inside the host cell. the virus is protected from the host immune system as well as from available circulating enzymes. there are a limited number of potential drug targets since viruses use the host biochemical mechanisms to multiply. viruses multiply very quickly, so antiviral drugs will often have little effect by the time symptoms appear. in addition, resistance to commonly used antivirals develops early. theoretically, viruses can be targeted by inhibiting the stage of viral attachment, internalization, fusion and uncoating, replication, assembly, and release. unfortunately, except the neuraminidase inhibitors for influenza, no registered drugs are available for most the treatment of carv infections in adults. treatment strategy may be symptomatic in most cases. it usually consists of steroids, oxygen, and antibiotics in case of concomitant bacterial infection. ribavirin is known since and is registered for use in chronic hepatitis c virus infection. ribavirin, a purine community-acquired respiratory viruses gottlieb nucleoside analogue with efficacy against many rna viruses, including rsv, hmpv, and pv, represents a viable treatment option for pv infection. ribavirin has been shown to have in vitro activity against rsv and the aerosolized form has been approved for the treatment of lower respiratory tract disease due to rsv in certain at-risk populations. data for intravenous use of ribavirin remain limited in ltx recipients. drawbacks with inhaled ribavirin include difficulties in administration, requiring continuous inhalation, along with associated risks of bronchoconstriction and respiratory distress, which may necessitate discontinuation. in addition, aerosolized ribavirin is considered to be potentially hazardous and teratogenic to the environment including health care workers as well as being excessively expensive. considering these limitations, widespread acceptance of the nebulized administration is missing. several centers use oral ribavirin as an off-label alternative treatment in paramyxovirus infections in ltx. a retrospective study demon-strates no significant differences in -month outcomes between oral and inhaled ribavirin therapy for rsv infection after ltx. in a recent multicenter trial involving rsv-infected patients from sites in australia, austria, germany, france, canada, and the united states, inhaled ribavirin was used in % infected individuals, oral ribavirin in %, intravenous ribavirin in %, intravenous immunoglobulin (ivig) in %, pulsed steroids in %, and palivizumab in %. the optimal duration of ribavirin therapy is unknown, although treatment orally for days led to virus elimination in % of patients in a retrospective study. of particular note, ribavirin was contraindicated in % of pv-infected recipients in this study and was terminated early in another quarter of cases due to adverse effects. twice-weekly monitoring of blood cell counts (risk of hemolytic anemia) and renal function is mandatory while under ribavirin therapy. compared with other patient populations treated with ribavirin, more frequent discontinuation due to toxicity was necessary in the ltx population. the most likely explanation for this remains combined toxicity of ribavirin and that inherent to the maintenance medication required after ltx. distinction must be made between the short-and longterm effects of treatment with ribavirin. prior studies have focused on short-term effects of ribavirin treatment, such as survival of the acute phase or shorter hospital length of stay, and found no beneficial effect. retrospective single-center studies suggest that ltx patients treated with ribavirin have a better pulmonary function months after paramyxovirus infection and have found that oral ribavirin reduces the number of complicated courses of paramyxovirus infection and reduces the long-term risk of bos %. recovery of pulmonary function postinfection was significantly better in ribavirin-treated patients (n ¼ ) than in patients treated with supportive care (n ¼ ) in a retrospective study. patients treated with oral ribavirin had a lower incidence of bos ( % of the ribavirin group versus % of the nonribavirin group [p ¼ . ]) within months. the latter subgroup was treated with supportive care in this study and had contraindications for ribavirin (e.g., advanced kidney disease, anemia). all ribavirin studies in ltx lack a randomized placebocontrolled design, which makes them unsuitable to make evidence-based recommendations. there is clearly the need for a rct to determine the efficacy of oral ribavirin. since unnecessary treatment with drugs should always be avoided and ribavirin has some unfavorable side effects, a firm conclusion is needed about the clinical benefits of this treatment. oral ribavirin seems to be a promising and inexpensive therapy which may have a significant impact on long-term morbidity and mortality of lung transplant recipients. anecdotal cases have reported the off-label use of pavilizumab and ivig in rsv-infected ltx recipients. several agents have been studied against influenza including das (fludase) and nitazoxanide, but no data are published for ltx patients. rna interference is a natural biological process whereby small interfering rnas (sirnas) can direct sequence-specific degradation of mrna, leading to reduced expression of the corresponding protein. aln-rsv is a sirna targeting the rsv nucleocapsid messenger rna, preventing the formation of the nucleocapsid protein and thereby reducing viral replication. intranasal aln-rsv administration significantly inhibited the rate of rsv infection in a phase experimental infection study in healthy adults. in a pivotal study in rsv-infected ltx patients, nebulized aln-rsv treatment proved to be safe and well-tolerated and incidence of new or progressive bos was decreased at day in patients treated with aln-rsv compared with placebo. the efficacy of aln-rsv administration in addition to standard of care on preventing new or progressive bos in rsvinfected ltx patients was evaluated in this large randomized, double-blind, placebo-controlled trial. in this phase b, trial subjects were randomized to receive aerosolized aln-rsv or placebo daily for days. aln-rsv was found to be safe and well tolerated. at day in aln-rsv -treated patients (n ¼ ) compared with placebo (n ¼ ), there was a trend toward a decrease in new or progressive bos ( . % vs. . %, p ¼ . ), which was significant in the per-protocol cohort (p ¼ . ). treatment effect was enhanced when aln-rsv was started < days from symptom onset, and the effect was independent from ribavirin treatment. presatovir (gs- ) is an oral rsv fusion inhibitor with potent and selective anti-rsv activity in vitro. a phase a, randomized, double-blind, placebo-controlled study was conducted to evaluate the safety, tolerability, and efficacy of presatovir in healthy adult volunteers infected with an rsv challenge virus. treatment with presatovir resulted in lower mean area under the curve (auc) viral load from initial dose through end of quarantine. viral load was assessed twice daily using nasal washes and in total symptom score during the entire quarantine period. results from a phase b, rct evaluating the effect of gs- in ltx recipients with rsv infection (nct ) are expected soon. there are several unmet needs in the development of effective therapies for carv. one is a wider treatment window. most agents are reported to be less effective if the patient presents more than hours of symptom onset. therapies should be cost-effective and reduce the threat of resistance by continuous antigenic drifting and antigenic shifting of viruses. alternative formulations (e.g., intravenous drugs) for hospitalized patients should be available as well as drugs for severely ill, hospitalized patients, and for pediatric patients. prevention of carv infection is of utmost importance in ltx. annual influenza vaccination is strongly recommended for all lung transplant recipients including all their household members. wearing face masks, avoiding contact with infected persons, and skin disinfection are usually recommended as preventive measures in ltx recipients, although they were not systematically evaluated in this high-risk cohort. viral pneumonia community-acquired respiratory viral infections in lung transplant recipients regulating the adaptive immune response to respiratory virus infection an international ishlt/ats/ers clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome post-transplant bronchiolitis obliterans international society for heart and lung transplantation. the registry of the international society for heart and lung transplantation: thirty-third adult lung and heart-lung transplant report- ; focus theme: primary diagnostic indications for transplant survival after bronchiolitis obliterans syndrome among seminars in respiratory community-acquired respiratory viruses gottlieb unauthorized distribution is strictly prohibited. bilateral lung transplant recipients chlamydia pneumoniae infection after lung transplantation aln-rsv for prevention of bronchiolitis obliterans syndrome after respiratory syncytial virus infection in lung transplant recipients graft loss and cladonset is hastened by viral pneumonia after lung transplantation chronic rhinoviral infection in lung transplant recipients incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients impact of human metapneumovirus and human cytomegalovirus versus other respiratory viruses on the lower respiratory tract infections of lung transplant recipients respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant community respiratory viral infection in adult lung transplant recipients a prospective molecular surveillance study evaluating the clinical impact of communityacquired respiratory viruses in lung transplant recipients a single-season prospective study of respiratory viral infections in lung transplant recipients the value of polymerase chain reaction for the diagnosis of viral respiratory tract infections in lung transplant recipients community-acquired respiratory viral infections in lung transplant recipients: a single season cohort study respiratory viruses and severe lower respiratory tract complications in hospitalized patients incidence and morbidity of human metapneumovirus and other communityacquired respiratory viruses in lung transplant recipients human metapneumovirus in lung transplant recipients and comparison to respiratory syncytial virus intravenous ribavirin is a safe and cost-effective treatment for respiratory syncytial virus infection after lung transplantation oral ribavirin for the treatment of noninfluenza respiratory viral infections: a systematic review oral versus inhaled ribavirin therapy for respiratory syncytial virus infection after lung transplantation single-centre experience with oral ribavirin in lung transplant recipients with paramyxovirus infections rna interference therapy in lung transplant patients infected with respiratory syncytial virus oral gs- activity in a respiratory syncytial virus challenge study lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact the impact of viral respiratory tract infections on long-term morbidity and mortality following lung transplantation: a retrospective cohort study using a multiplex pcr panel key: cord- - n f wrz authors: węglarz-tomczak, ewelina; talma, michał; giurg, mirosław; westerhoff, hans v.; janowski, robert; mucha, artur title: neutral metalloaminopeptidases apn and metap as newly discovered anticancer molecular targets of actinomycin d and its simple analogs date: - - journal: oncotarget doi: . /oncotarget. sha: doc_id: cord_uid: n f wrz the potent transcription inhibitor actinomycin d is used with several cancers. here, we report the discovery that this naturally occurring antibiotic inhibits two human neutral aminopeptidases, the cell-surface alanine aminopeptidase and intracellular methionine aminopeptidase type . these metallo-containing exopeptidases participate in tumor cell expansion and motility and are targets for anticancer therapies. we show that the peptide portions of actinomycin d and actinomycin x( ) are not required for effective inhibition, but the loss of these regions changes the mechanism of interaction. two structurally less complex actinomycin d analogs containing the phenoxazone chromophores, questiomycin a and actinocin, appear to be competitive inhibitors of both aminopeptidases, with potencies similar to the non-competitive macrocyclic parent compound (k(i) in the micromolar range). the mode of action for all four compounds and both enzymes was demonstrated by molecular modeling and docking in the corresponding active sites. this knowledge gives new perspectives to actinomycin d's action on tumors and suggests new avenues and molecules for medical applications. natural products are excellent leads for drug development. particularly in the areas of antibiotic and anticancer therapies, natural products and their derivatives comprise a significant percentage of clinically used drugs [ ] . actinomycin d (actd), also known as dactinomycin, a small molecule naturally produced by streptomyces bacteria [ ] , was the first antibiotic shown to have anticancer activity. approved for medical use in [ ] , dactinomycin has been mostly used as a remedy for a variety of mainly pediatric tumors, such as wilms' tumor, rhabdomyosarcoma and ewing's sarcoma [ ] [ ] [ ] [ ] [ ] [ ] . actd is a neutral molecule consisting of a planar phenoxazone ring substituted with two carboxylatelinked cyclic pentapeptides ( figure ). its anticancer role has been associated with dna functionality, leading to rna, and consequently, protein synthesis inhibition [ ] . actd binds to double and single-stranded dna (but not to rna) with its phenoxazone ring intercalating with high specificity between gpc base pairs. moreover, it stabilizes cleavable topoisomerase i and ii complexes with dna, in which the polypeptide lactone rings occupy a position in the minor groove of the dna helix or the drug penetrates the topoisomerase-dna binding area [ , ] . after years of actinomycin d usage, its action was also correlated with apoptosis; at low doses, the drug is a potent inducer of apoptosis [ , ] , triggering this process in normal and tumor cells, as well as in cells that rarely undergo apoptosis [ ] . it was shown that the molecular mechanism of actinomycin d-induced apoptosis was associated with activation of the apoptosis fas surface protein, which is also known as cd (cluster of differentiation ) [ ] , the non-mitochondrial stress-activated protein/junminoterminal kinases apoptotic pathway, and with increased expression of the apoptosis regulator bax [ ] . cells inducing apoptosis in the immune response pathway via actinomycin d may also be associated with the expression of the cd surface antigen [ ] . additionally, the presence of intracellular atp is necessary for the induction of apoptosis by actinomycin d [ , ] . in recent years, intensive research in laboratories and clinics has focused on the correlation between actinomycin d activity and cellular tumor antigen p functionality and expression [ , ] . actd induces p expression [ , ] . apoptosis in patients with mutated or deleted p suggests a p -independent cell death mechanism [ ] . however, the function of p was restored after low doses of actinomycin d in various tp wildtype tumor cell lines [ , ] . -amino- h-phenoxazin- -ones missing the cyclic pentapeptide fragments, such as actinocin and questiomycin a (figure ), are also naturally produced by streptomyces [ ] . they occur in nature as pigments, fungal metabolites and allelo-chemicals [ ] [ ] [ ] . compounds composing the -aminophenoxazin- -one skeleton exhibit antitumor, antimicrobial, and antiviral activities in vitro and in vivo [ ] [ ] [ ] [ ] [ ] . soon after the discovery of actinomycin d and its properties, the anticancer actions of questiomycin a were also described [ ] , but the success of actinomycin d turned attention away from its simpler analogues. human alanine aminopeptidase (hsapn) and methionine aminopeptidase type (hsmetap ) are representative metalloproteases that have been targeted as a therapeutic intervention against pathological disorders. among others, they participate in proteolytic pathways associated with angiogenesis, apoptosis and proliferation, which makes them interesting objects of oncological research [ ] [ ] [ ] . metap is the best recognized member of the three known intracellular methionine aminopeptidases that catalyze the removal of the n-terminal methionine residue from a number of nascent proteins. this step is required before folding into their functional forms [ , ] . the enzyme binds two cobalt [ ] or manganese ions [ ] in its active site. as higher metap expression was observed in tumor (mesothelioma, [ ] neuroblastoma, [ ] and colorectal carcinoma [ ] ) cells compared with normal cells, the enzyme serves as a target for anti-angiogenic compounds of natural origin, such as fumagillin and ovalicin [ ] . selective inhibition of metap stops vascularization and tumor growth in animal models [ , ] . aminopeptidase n is a membrane-bound, zincdependent metalloproteinase also known as cd (cluster of differentiation ) [ ] . apn specificity is directed towards the cleavage of a neutral or basic amino acid from the n-terminal position of peptides and proteins [ ] . in contrast to metap , its active site is accessible to the exterior environment of the cell and the cell membrane anchoring its n-terminus [ ] . aminopeptidase n is described as a moonlighting protease with multiple functions, including antigen presentation. it is also a receptor for some human viruses, e.g., coronaviruses. these functions are connected not only to peptide cleavage but also to endocytosis, signaling and ligation or the inhibition of its enzymatic activity, which might result in complex and systemic effects [ ] . apn overexpression has been observed in tumor cells from melanoma [ ] , renal [ ] , colon [ ] , gastric [ , ] , pancreatic [ ] , and thyroid [ ] cancers and on leukemic blasts in acute myeloid leukemia [ ] . compelling studies have implicated apn protease activity in tumor-associated processes, particularly angiogenesis, apoptosis and metastasis [ ] [ ] [ ] . here, we show that actinomycin d and its analogues inhibit human metap and apn. developments in synthetic ligands of metalloaminopeptidases, substrates and inhibitors, indicated that not only amino acid and peptide mimetics but also heterocyclic compounds may effectively interact with these enzymes [ , [ ] [ ] [ ] [ ] . inspired by these accounts we have suggested the -amino- h-phenoxazin- -one scaffold as capable of binding to the aminopeptidase active sites. indeed, the well-known antibiotic and anticancer drug actinomycin d inhibits the two aminopeptidases hsapn and hsmetap with inhibition constants approximately μm (table and figure a ). kinetic studies allowed us to determine the mechanism www.oncotarget.com of inhibition of actinomycin d as non-competitive, with steady-state binding being achieved immediately ( figure b ). comparable results (within the experimental error) were obtained for actinomycin x , a structure closely related to actd. a minor variation in the amino acid composition of the α peptide side chain ( figure ) did not influence their binding affinities. despite structural similarity to actd medicinal properties of actinomycin x have been scarcely investigated. for example, actinomycin x was reported to show higher cytotoxicity of the human leukemia cell line hl- (lc = nm, lc is the lethal concentration for a half of population) than actinomycin d (lc = nm) [ ] . further studies proposed actinomycin x as an inducer of apoptosis of human prostate cancer cells through the mtor pathway compounded by mirna [ ] . the analogs that do not possess the macrocyclic polypeptide fragments, actinocin ( figure c and d) and questiomycin a, exhibited competitive inhibition mechanisms for each aminopeptidase. regarding their potency, they differed significantly from one another. actinocin was twice as effective as actinomycin d/x in the case of hsapn and four times as effective in the case of hsmetap . in contrast, questiomycin was times less effective; the unsubstituted -aminophenoxazone ring gave rise to a five-fold decrease in activity for hsapn and hsmetap compared to the lead compound. cytotoxicity of actinomycin d and questiomycin a reported in the literature is visibly different to each other and to some extend corresponds with these results. -aminophenoxazine- -ones induced apoptosis in many types of cancer cells [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , however, in comparable cases of human acute b-and t-lymphoblastic leukemia, cervix carcinoma and larynx carcinoma, the cytotoxicity of questiomycin a is up to three orders of magnitude lower (ic = - μm) than actinomycin d (ic = - nm) [ ] . the mechanisms of binding for each inhibitor must be significantly different at the molecular level due to the the inhibitors were screened in tris buffer. the release of the fluorophore was monitored continuously. the linear portion of the progress curve was used to calculate the velocity. each experiment was repeated at least three times and the results are presented as the average with standard deviation. for more details, please see the materials and methods section. * calculated based on the cheng-prusoff equations for non-competitive and competitive inhibitors [ ] . fundamental variations in their structures. this issue was studied in additional detail by molecular modeling. the modeled binding of actd/actinomycin x with hsapn ( figure ) and hsmetap ( figure ) showed complex sets of contacts based on typical peptide-protein interactions at different conformations. in the case of apn [ ] , both drugs most typically penetrated the interior of the enzyme and filled the characteristic central cavity, which was apt to accept large peptide substrates ( figure a and b). the modeled assembly was generally stabilized by a hydrogen bonding network ( figure c and d), particularly welldefined for actinomycin x . arg of hsapn has been identified as a residue highly involved in binding. for actinomycin d its terminal guanidine nitrogen atom forms a convenient hydrogen bond bridge between both c=o groups of the heteroaromatic ring-substituting carboxyamides ( . Å and . Å, respectively, figure c ). in contrast, for the most favorable actinomycin x conformation ( figure d) , the corresponding n atom of arg is engaged in interactions with the α cyclic peptide via two tight contacts with c=o of d-val ( . Å) and thr ( . Å). the extra oxygen atom of oxopro is pointed towards the α-nitrogen of the same enzyme residue ( . Å). the β peptidyl ring of the inhibitor is also favorably bound by the guanidine residue of arg , with distances . Å and . Å to c=o of thr and n-meval, respectively. the thr residue is in additional contacts with the hydroxyl group of ser ( . Å, via c=o) and the carboxylate of asp ( . Å, via n-h). finally, the amino group of phenoxazinone interacts with -oh of tyr , a group which is involved in the transition state stabilization during the natural substrate cleavage. for metap [ ] , protein-actinomycin d/x interactions mainly involved the surface of the enzyme ( figure a and b). the inhibitors are favorably located at the entrance of the active site, thereby blocking substrate inclusion and processing. in the lowest energy conformation, moieties of the cyclic peptide α of actinomycin d are partially inserted into the active site cavity and surrounded by the hydrophobic enzyme residues: his , leu , his , ile , pro and leu ( figure c ). the heterocyclic system is exposed to the outside and hardly contributes to this lipophilic binding. nevertheless, the functional groups of -amino- hphenoxazin- -one form a set of hydrogen bond contacts. the -amino group is bridged between the carboxylate of asp ( . Å) and the hydroxyl o atom of tyr ( . Å). the latter is also in a favorable position to n-h of d-val of the cyclic peptide α ( . Å). in addition, the -oxo group of the inhibitor forms a contact with the n-h of phe ( . Å). the β peptidyl ring of actinomycin d interacts less tightly with the enzyme and only two specific hydrogen bonds are recognized. they involve the thr residue, the c=o group binds with the side chain -nh of asn , and n-h with the hydroxyl o atom of thr . for actinomycin x the overall ligand-enzyme architecture is somewhat different as the β ring and the heterocyclic system are mostly involved in hydrophobic interactions with phe , his , ile , his , phe , his , met , ala and leu of the protein surface ( figure d ). this fit is emphasized by hydrogen bond contacts of the hydroxyl group of tyr bridged between n-h and c=o of thr ( . Å and . Å, respectively), and the guanidino group of arg with c=o of d-val ( . Å). the α peptidyl ring of the ligand forms two specific interactions which involve asn and asn (the side chain -nh ) of the protein, and oxopro and n-meval (oxo/c=o, . Å and . Å, respectively) of actinomycin x . elimination of the cyclic peptide fragments from the structure of actinomycin d/x allowed the resulting actinocin to penetrate much further into the active sites of the studied metallopeptidases and to act as a classical competitive ligand by interacting with the metal ions (figures and ). for human apn, the heteroaromatic ring is conveniently positioned in the hydrophobic environment while a functional group oxygen atom is directed towards the zn ion. interestingly, flipping the molecule does not seem to disturb the preferential binding. the highest rating docking score was shown for the conformation where the carbonyl oxygen atom is involved in an interaction with the metal cation ( . Å, figure a ). the oxygen was also in a tight hydrogen bond proximity to the tyr -oh group ( . Å). these two contacts and the stabilization of the neighboring amino group by glu ( . Å) and gln ( . Å), similar to the peptide n-termini, closely resembles the substrate-enzyme complex, including the carbonyl positioning for the nucleophilic water attack. the zinc bidentate complexation with the -carboxylate group ( . Å and . Å) was an alternative thermodynamically favored conformation ( figure b ). in this case, the -oxo group was in close hydrogen bond proximity to the arg guanidine nitrogen atoms ( . Å and . Å, respectively). the -carboxylate also formed a potential o-h … o=c contact with the glu side chain group ( . Å). similarly, the main driving force for the binding of actinocin to hsmetap was a tight bidentate complexation between one of the cobalt ions and -carboxylate ( . Å and . Å, figure ). this interaction was further strengthened by the close three-dimensional fit of the molecule in the active site provided by hydrophobic contacts with phe , ala , ile and his and hydrogen bonds. in particular, the latter involved interactions between the -amino group and the ala ( . Å) and lys ( . Å) oxygen atoms, as well as the -oxo group and the ser hydroxyl ( . Å). this set of favorable interactions gave rise to the highest theoretical docking score for actinocin to metap and was consistent with the lowest obtained inhibition constant values. lacking both carboxylate groups, questiomycin a loses the affinity of actinocin to both apn and metap. the decrease in inhibitory activity is not dramatic, approximately one order of magnitude, but demonstrates that the metal-binding moieties are important but not critical for the structure and potency of a good ligand. indeed, according to the molecular modeling results, the general fit of the heteroaromatic system and specific hydrogen bonds are reproduced for questiomycin a in the active sites of the human metallopeptidases ( figure a and b). for hsapn ( figure a ) the -oxo group of questiomycin a is firmly bridged between arg guanidine nitrogen atoms ( . Å and . Å, respectively) while the heteroaromatic ligand core is nested in the hydrophobic environment of ala , ala , val and his . similarly, the structurally simplest inhibitor reproduces interactions of the functional groups with metap. -amino group is hydrogen-bonded with c=o of ala ( . Å), lys ( . Å) and gln ( . Å), while the -oxo group with the ser hydroxyl ( . Å). the lipophilic contacts involve ala , his and ile . taking in account these close similarities of questiomycin a versus actinocin binding, it can concluded that the difference in their activities are predominantly caused by the absence of metal coordination by carboxylates. actinomycin d is a long-known drug that was developed as an anticancer agent years before apoptosis and other cell death mechanisms and cancer progression were elucidated. it is still present in the world health organization's list of essential medicines [ ] , which contains the most effective and safest medicines required by any proper health system. while actd is widely used in the treatment of cancer, intensive research on its mechanism of action, as well as on clinical trials with actinomycin d in combination with other drugs, are progressing. at low concentrations, actinomycin d inhibits proliferation and induces cell death in neuroblastoma cell lines, as well as tumor regression in xenograft tumor models. cortes et al. proved experimentally that apoptosis is the major cell death mechanism caused by actinomycin d treatment in p wt cells, though less so in p -deficient cell lines [ ] . the combination of actinomycin with drugs as nutlin- [ ] and the immunotoxin rg [ ] have improved the anticancer activities of the latter by activating apoptosis and p . questiomycin a was demonstrated as costive agent of apoptotic cell death in the gastric and colon cancer cell line by dysregulating the function of mitochondria, and activating the caspase signaling [ , , ] . questinomycin a also induced cellular apoptosis by causing rapid intracellular acidification in several cancer cells [ , , , ] , generating reactive oxygen species in human lung adenocarcinoma cells and human glioblastoma cell line [ , ] and by dna laddering and upregulated fas expression in mouse melanoma b cells [ ] . direct connection of questiomycin with p still remains an issue to be elucidated. there are deep premises that it exists. the cell-surface receptor fas, a member of the tnf-r family of receptors, is a key component of the extrinsic death pathway [ ] . it has been reported that p can activate the extrinsic apoptotic pathway through the induction of genes encoding transmembrane protein fas [ ] that could be unregulated by questiomycin a [ ] . in contrast, aminopeptidases have also been subject to intensive research in oncological studies. accordingly, in recent years, methionine aminopeptidase type , as well as alanyl aminopeptidase, have become extremely specialized targets for the development of new inhibitors [ , - , , , , ] . most known metap inhibitors are irreversible binding synthetic analogues of fumagillin [ , ] , two of which, tnp- and beloranib, have advanced to clinical trials. tnp- has been shown to inhibit angiogenesis in vitro and in vivo. it entered clinical development for cancer as an anti-angiogenic agent and has achieved phase i/ii trials for kaposi's sarcoma, renal cell carcinoma, brain cancer, breast cancer, cervical cancer and prostate cancer. however, the trials have not led to its approval for clinical applications. beloranib was originally designed as an angiogenesis inhibitor similar to tnp- for the treatment of cancer. once the potential anti-obesity effects of metap inhibition became apparent during trials, clinical development began to focus on these effects, and beloranib has shown positive results inhibitor and enzyme amino acid residues are shown as sticks. the catalytic zinc ion is shown as a dark blue sphere. hydrogen bonds and hydrophobic contacts are shown as green lines. www.oncotarget.com in preliminary clinical trials for this indication [ ] . moreover, there are tens of reversible inhibitors of both natural and synthetic origin. they include anthranilic acid sulfonamides [ ] , bengamides [ ] , benzoselnazalones [ ] and compounds based on bestatin [ ] , , , -triazole [ ] , and pyrazolo [ , -b] indole [ ] . research on new, reversible agents that are safe for organisms is ongoing. the most known and widely studied reversible apn inhibitors, bestatin and chr- , also known under the commercial names ubenimex and tosedostat, respectively, have been investigated clinically for their anticancer effects. ubenimex is approved for the treatment of lung cancer and nasopharyngeal cancer, and tosedostat showed significant anti-leukemic activities in phase i/ii clinical trials. currently, phase i/ii studies of tosedostat in combination with other chemotherapeutic agents are in progress for aml and metastatic pancreatic adenocarcinoma [ ] . compounds with apn inhibitory activities improved the anti-metastasis and anti-angiogenesis effects in vivo [ , ] . additionally, intensive studies on enzyme structure specificity led to the development of highly active competitive inhibitors of apn, with inhibition constants in the nanomolar range. phosphonic and phosphinic analogues of amioacid and dipeptides [ , ] , benzosuberone [ ] , naturally occurring curcumin [ ] and even more recently, a cyclic peptide inhibitor [ ] can be counted in this group of compounds. the recent research on actinomycin d highlights its huge therapeutic potential and suggests that low doses of this drug could be used in combination with other agents to take advantage of its unique activity and avoid non-specific effects. nevertheless, there is no correlation between these properties and the inhibition of the neutral aminopeptidases metap and apn that is highly important in cancer progression. blocking the activity of metap and apn with actinomycin d or its analogs seems to be promising for the development of new generations of potent anticancer agents that would be implicated in different mechanisms of action and directed against multiple molecular targets. in view of the network nature of cancer [ ] , the use of combinations of inhibitors appears to be indicated, as well as alternative drugs with multiple specificities. actinomycin d may prove to be such a drug for targeting a network rather than a single molecule. the discovery reported here may contribute to the development of better therapies. actinomycin d was purchased as a lyophilized powder from sigma-aldrich (poznan, poland), actinomycin x was purchased form adipogen (liestal, switzerland). actinocine and questiomycin a were synthesized according to a previously described procedure [ ] . recombinant human apn and metap were purchased as a lyophilized powder from r&d systems (minneapolis, usa). fluorogenic substrates ala-amc (l-alanine- -methylcoumaryl- -amide) and met-amc (l-methionine- -methylcoumaryl- -amide) were purchased from peptanova (sandhausen, germany). spectrofluorometry was performed on a spectra max gemini em fluorimeter (molecular devices, sunnyvale, usa) in a -well plate format (excitation = nm, emission = nm). calculations of the kinetic parameters were performed using the softmax pro, graphpad prism and microsoft excel programs. recombinant human apn enzyme was dissolved in mm tris-hcl buffer (ph = . ) and used directly in the kinetic experiments. recombinant human metap was obtained as a lyophilized powder from r&d systems. the enzyme was dissolved in mm tris-maleate buffer containing . mm cocl , mm nacl and . % v/v albumin at ph = . and incubated for minutes. after this activation, the enzyme was used directly in the kinetic experiments. the inhibitors were screened against human apn and human metap at °c in the assay buffer as described above. for the steady-state measurements, the enzymes were preincubated for - min at °c with an inhibitor before adding the substrate (ala-amc, μm for human apn or met-amc and μm for metap ) to the wells. for the initial state measurements, the enzymes were preincubated for - min at °c before adding the substrate and inhibitor to the wells. the following conditions were used: ( ) total volume μl, ( ) eight different inhibitor concentrations, and ( ) enzyme concentration of . nm for human apn and nm for metap . the release of the amc fluorophore was monitored. the linear portion of the progress curve was used to calculate the velocity. hphepch phe [ , ] for apn and ebselen [ ] for metap were used as a positive control. the inhibition mechanism was determined from the lineweaver-burk and dixon plot ( figure b and d) . the ic values were calculated from the reaction velocity to inhibitor concentration plot fitted to the equation log[i] vs. response. the k i values were equal to the ic for non-competitive inhibitors [ ] and were calculated for competitive inhibitors using the following equation: k i = (ic -[e]/ )/ ( + ([s]/km)) [ ] , where the ic is the concentration of the inhibitor that effected % inhibition, [s] and k m are the substrate concentration and michaelis constant in the absence of inhibitor, respectively, and [e] is the concentration of the enzyme. www.oncotarget.com molecular modeling studies were performed using the biovia discovery studio program package (san diego, ca, usa). the crystal structures of human aminopeptidase n (cd ) in complex with amastatin (pdb id. fyt) [ ] and methionine aminopeptidase type (pdb id. d e) [ ] were used as the starting points for the calculations of the enzymes complexes with actinomycin d, actinomycin x , actinocin and questiomycin a. the enzyme models were protonated at ph . and minimized with the charmm force field. the water molecules and former ligands were removed. new ligands were protonated at the same ph and the partial charges of all the atoms were computed using the momany-rone algorithm. minimizations used the charmm force field with the smart minimizer algorithm up to an rms gradient of . Å. actinomycin d, actinomycin x , actinocin and questiomycin a were docked into the enzyme active center using the libdock docking algorithm. the number of hotspots was changed from to (standard method). the 'best' conformation method was selected and the minimization of the results was achieved using the charmm force field. natural products: a continuing source of novel drug leads bacteriostatic and bacteriocidal substances produced by soil actinomycetes dactinomycin basis of actinomycin action. i. dna binding and inhibition of rna-polymerase synthetic reactions by actinomycin the treatment of wilms' tumor: results of the second national wilms' tumor study results with ema/co (etoposide, methotrexate, actinomycin d, cyclophosphamide, vincristine) chemotherapy in gestational trophoblastic neoplasia and national wilms tumor study group. treatment of wilms tumor relapsing after initial treatment with vincristine, actinomycin d, and doxorubicin. a report from the national wilms tumor study group immune-mediated thrombocytopenia following dactinomycin therapy in a child with alveolar rhabdomyosarcoma: the unresolved issues improved outlook for ewing's sarcoma with combination chemotherapy (vincristine, actinomycin d and cyclophosphamide) and radiation therapy inhibition of rna synthesis by actinomycin d: characteristic dose-response of different rna species the structure of a pseudo intercalated complex between actinomycin and the dna binding sequence d(gpc) crystal structure of the : complex between d(gaagcttc) and the anticancer drug actinomycin d actinomycin d induces apoptosis and inhibits growth of pancreatic cancer cells actinomycin d and staurosporine, potent apoptosis inducers in vitro, are potentially effective chemotherapeutic agents against www.oncotarget.com glioblastoma multiforme inhibitory effect of thyrotropic hormone on apoptosis induced by actinomycin d in a functioning rat thyroid cell line molecular mechanism(s) of actinomycin-d induced sensitization of pancreatic cancer cells to cd mediated apoptosis induction of surface antigen cd expression in t-lymphocytes following exposure to actinomycin d intracellular atp is required for actinomycin d-induced apoptotic cell death in hela cells actinomycin d and its mechanisms of action akt mediates actinomycin d-induced p expression methylated actinomycin d, a novel actinomycin d analog induces apoptosis in hepg cells through fasand mitochondria-mediated pathways specific activation of the p pathway by low dose actinomycin d: a new route to p based cyclotherapy actinomycin d induces p -independent cell death and prolongs survival in high-risk chronic lymphocytic leukemia activation of p in cervical carcinoma cells by small molecules phenoxazinone synthase: mechanism for the formation of the phenoxazinone chromophore of actinomycin contributions of phenoxazone-based pigments to the structure and function of nanostructured granules in squid chromatophores plectosphaeroic acids a, b, and c, indoleamine , -dioxygenase inhibitors produced in culture by a marine isolate of the fungus plectosphaerella cucumerina isolation and synthesis of allelochemicals from gramineae: benzoxazinones and related compounds the new antibiotics, questiomycins a and b antiviral activity of -amino- , α-dihydro- α- -dimethyl- h-phenoxazine- -one on poliovirus antitumor effects of a novel phenoxazine derivative on human leukemia cell lines in vitro and in vivo glucosylquestiomycin, a novel antibiotic from microbispora sp. tp-a : fermentation, isolation, structure determination, synthesis and biological activities phenoxazine compounds produced by the reactions with bovine hemoglobin show antimicrobial activity against non-tuberculosis mycobacteria methionine aminopeptidase and cancer the moonlighting enzyme cd : old and new functions to target role of aminopeptidase in angiogenesis steady-state kinetic characterization of substrates and metal-ion specificities of the full-length and n-terminally truncated recombinant human methionine aminopeptidases (type ) physiologically relevant metal cofactor for methionine aminopeptidase- is manganese methionine aminopeptidase- regulates human mesothelioma cell survival: role of bcl- expression and telomerase activity prospects for therapeutic inhibition of neuroblastoma angiogenesis high expression of methionine aminopeptidase in human colorectal adenocarcinomas methionine aminopeptidase (type ) is the common target for angiogenesis inhibitors agm- and ovalicin high expression of methionine aminopeptidase type in germinal center b cells and their neoplastic counterparts. mori lab. invest cloning and characterization of complementary dna encoding the eukaryotic initiation factor -associated -kda protein (p ) ectopeptidases in pathophysiology aminopeptidase fingerprints, an integrated approach for identification of good substrates and optimal inhibitors the structure and main functions of aminopeptidase n m aminopeptidases as drug targets: broad applications or therapeutic niche? human melanoma invasion and metastasis enhancement by high expression of aminopeptidase n/cd urinary excretion of glycine.prolile dipeptidile aminopeptidase, n-acetyl-beta-d-glucosaminidase, alanine aminopeptidase and low molecular protein in patients with renal cell carcinoma aminopeptidase n is involved in cell motility and angiogenesis: its clinical significance in human colon cancer aminopeptidase n (apn/cd ) as a target molecule for scirrhous gastric cancer serum apn/cd as a novel diagnostic and prognostic biomarker of pancreatic cancer biological significance of aminopeptidase n/cd in thyroid carcinomas hematopoietic stem cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia role of aminopeptidase n (cd ) in tumor-cell invasion and extracellular matrix degradation aminopeptidase-n/ cd is a potential proapoptotic target in human myeloid tumor cells aminopeptidase n (cd ) as a target for cancer chemotherapy s pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors exploring s plasticity and probing s ′ subsite of mammalian aminopeptidase n/ cd with highly potent and selective aminobenzosuberone inhibitors metalloaminopeptidase inhibitors identification of methionine aminopeptidase as a molecular target of the organoselenium drug ebselen and its derivatives/analogues: synthesis, inhibitory activity and www.oncotarget.com molecular modeling study characterization of streptomyces mitkk- apoptosis of human prostate cancer cells induced by marine actinomycin x through the mtor pathway compounded by mirna ic -to-ki: a web-based tool for converting ic to ki values for inhibitors of enzyme activity and ligand binding synthesis, biological evaluation, and molecular modeling of h-pyrido[ , -a]phenoxazin- -one, a compound with potent antiproliferative activity anticancer activity of phenoxazines produced by bovine erythrocytes on colon cancer cells prevention of carcinogenesis and development of gastric and colon cancers by -aminophenoxazine- -one (phx- ): direct and indirect anti-cancer activity of phx- suppression of the proliferation of cancer cell lines, kb- - and k cells preceded by a decrease in intracellular ph caused by phenoxazine derivatives -aminophenoxazine- -one-induced apoptosis via generation of reactive oxygen species followed by c-jun n-terminal kinase activation in the human glioblastoma cell line ln -aminophenoxazine- -one and -amino- , α-dihydro- α, -dimethyl- h-phenoxazine- -one cause cellular apoptosis by reducing higher intracellular ph in cancer cells apoptosis induction preceded by mitochondrial depolarization in multiple myeloma cell line u by -aminophenoxazine- -one rapid decrease of intracellular ph associated with inhibition of na+/h+ exchanger precedes apoptotic events in the mnk and mnk gastric cancer cell lines treated with -aminophenoxazine- -one -aminophenoxazine- -one prevents pulmonary metastasis of mouse b melanoma cells in mice the x-ray crystal structure of human aminopeptidase n reveals a novel dimer and the basis for peptide processing spiroepoxytriazoles are fumagillin-like irreversible inhibitors of metap with potent cellular activity effect of low doses of actinomycin d on neuroblastoma cell lines low-dose actinomycin-d treatment re-establishes the tumoursuppressive function of p in rela-positive ependymoma actinomycin d enhances killing of cancer cells by immunotoxin rg through activation of the extrinsic pathway of apoptosis the fas death factor apoptosis -the p network the rational design of therapeutic peptides for aminopeptidase n using a substrate-based approach allosteric inhibition of aminopeptidase n functions related to tumor growth and virus infection phase i trial of the angiogenesis www.oncotarget.com inhibitor tnp- for progressive androgen-independent prostate cancer methionine aminopeptidase type- inhibitors targeting angiogenesis development of sulfonamide compounds as potent methionine aminopeptidase type ii inhibitors with antiproliferative properties the bengamides: a mini-review of natural sources, analogues, biological properties, biosynthetic origins, and future prospects -amino- -hydroxyamides and related compounds as inhibitors of methionine aminopeptidase- novel reversible methionine aminopeptidase- (metap- ) inhibitors based on purine and related bicyclic templates discovery of potent, reversible metap inhibitors via fragment based drug discovery and structure based drug design-part phase i/ii clinical study of tosedostat, an inhibitor of aminopeptidases, in patients with acute myeloid leukemia and myelodysplasia design, synthesis and biological evaluation of novel amino acid ureido derivatives as aminopeptidase n/cd inhibitors structureguided, single-point modifications in the phosphinic dipeptide structure yield highly potent and selective inhibitors of neutral aminopeptidases a structural insight into the p s binding mode of diaminoethylphosphonic and phosphinic acids, selective inhibitors of alanine aminopeptidases irreversible inhibition of cd /aminopeptidase n by the antiangiogenic agent curcumin cancer: a systems biology disease catalytic oxidative cyclocondensation of o-aminophenols to -amino- h-phenoxazin- -ones the authors declare no conflicts of interest. key: cord- -y mxcj authors: hoppe, ingrid bortolin affonso lux; medeiros, andréa souza ramos de; arns, clarice weis; samara, samir issa title: bovine respiratory syncytial virus seroprevalence and risk factors in non-vaccinated dairy cattle herds in brazil date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: y mxcj background: the cattle industry is one of the most important brazilian agribusiness sectors and is a strong contributor to the national economy. annually about . million calves are bred, which makes the optimal management of these animals extremely important. several diseases can affect the initial stages of the bovine production chain, being the bovine respiratory syncytial virus (brsv) one of the most relevant pathogens. this study aimed to characterize the epidemiology of brsv infection in dairy cattle herds of são paulo state, brazil, using serological and risk factors analyses. for that, blood samples were collected of animals from farms and a questionnaire about possible risk factors for brsv prevalence was performed. the obtained blood sera were analyzed using virus neutralization test (vnt). results: vnt results showed high brsv prevalence in dairy cattle herds, reaching . % of seropositivity. the brsv seroprevalence among studied farms ranged from to %. the analysis of risk factors indicated that the age group and the occurrence of coinfection with bovine herpesvirus (bohv- ) and bovine viral diarrhea virus (bvdv- ) should be associated with a higher prevalence of brsv, while natural suckling was considered a protective factor. conclusions: the study showed that adult animals over year old are an important risk factor for the high seroprevalence of brsv in herds. the high brsv prevalence associated with bohv- and bvdv- suggests that biosecurity measures should be applied in order to reduce viral dissemination. additionally, the natural suckling may be an important management to protect calves from high brsv seroprevalence. bovine respiratory syncytial virus (brsv) is an economically significant pathogen in cattle production [ ] , as it is one of the most important causes of lower respiratory tract infections in calves [ ] . in dairy cattle, brsv infection usually occurs in young calves aged between weeks and months [ ] . adult animals with subclinical infection are the main source of infection, since reinfections are common in the herds [ , , ] . brsv, bovine herpesvirus (bohv- ), bovine viral diarrhea virus (bvdv) and bovine parainfluenza type- (pi- ) are considered primary agents involved in the bovine respiratory complex. additionally, secondary infection by pasteurella multocida, histophilus somni and mycoplasmas contribute to the aggravation of the disease [ ] . clinical signs are characterized by respiratory symptoms, initially with moderated intensity, such as nasal and ocular discharges which can be aggravated leading to pneumonia. however, mainly in calves, an acute and severe onset is also observed, due to maternal antibodies not effectively protect against brsv infection [ ] . considering the high prevalence of the disease, several studies determined risk factors involved in the epidemiology of brsv. in europe, risk factors were mainly attributed to herd size, herd density, purchasing of new animals, geographic location of the farms, herd type and concomitant bvdv infection [ ] [ ] [ ] [ ] [ ] . similar studies have also been performed in some latin american countries and they showed that most of the animals probably have already been exposed to the virus with consequent high brsv prevalence in cattle herds. in these countries, herd size, age group, presence of bordering farms, herd type and geographic location of the farms were the main risk factors associated with brsv infection [ ] [ ] [ ] [ ] [ ] . in brazil, brsv was first diagnosed in calves in the state of rio grande do sul [ ] and some studies have shown that brsv infection is widespread in southern and southeastern brazil, with high serological prevalence rates [ ] [ ] [ ] . nevertheless, research has not been conducted in order to verify possible risk factors involved in brsv epidemiology. due to this, the current study aimed to determine antibody prevalence against brsv and investigate some risk factors associated with brsv seroprevalence in herds of an important milk producing region in são paulo state, brazil. the study was performed on dairy cattle herds in municipalities in the northern region of the são paulo state, southeastern brazil. this region produces about million liters of milk annually [ ] . the evaluated herds had between and animals and no animals were vaccinated against the pathogens associated with respiratory diseases. the farms were located in a region classified as aw by the climatic classification of koeppen, characterized by dry winter with average temperature higher than °c in the coldest month and precipitation below mm in the driest month. the altitude of these areas was to m above sea level [ ] . sampling of each farm was calculated [ ] with an expected brsv prevalence of % [ ] with acceptable error of % and confidence level of %. after setting the number of samples, bovines of all categories and age group were randomly selected. according to the management adopted in the herds, the age group was defined as ≤ months old "calf" and > months old "adult". in some farms, the number of samples collected was higher than that suggested by the mathematical calculation, once it was also used for serodiagnosis of bohv- and bvdv- . on farms with a small herd size, up to animals, all of them were sampled. the samples from animals were collected from april to june as shown in table . blood samples were collected by jugular or coccygeal vein puncture using disposable needles and vacuum tubes. samples remained at room temperature for h for coagulation and after being transported refrigerated to the laboratory, they were centrifuged at ×g for min to obtain the sera. the sera were aliquoted in . ml identified microtubes that were stored in freezer at − °c until analyses. the presence of antibodies to brsv was tested by virus neutralization test (vnt). serum samples were thawed, inactivated in water bath at °c for min, and diluted in duplicates from : to : in -well microplates with μl of tcid brsv suspended in eagle's minimum essential medium (difco e-mem®). the viral strain was previously titrated [ ] . following during the sample collection, a questionnaire was administered to the owner or manager of each farm with the purpose of identifying potential risk factors related to epidemiology of brsv. thus, the questionnaire was designed according to data of risk factors described in the literature [ , , , ] in addition to the information of the management adopted in dairy farms of são paulo state. the variables explored were: herd size (≤ , > animals), herd density (≤ , > animals/hectare), age group (≤ months old "calf", > months old "adult"), breed (holstein, mixed), type of reproduction (natural mating, artificial insemination), purchase of animals (last months: yes, no), cleaning of facilities (often, rarely), historical of respiratory disease (last months: yes, no), quarantine (yes, no), abortions (yes, no), bohv- infection (serodiagnosis: yes, no), bvdv- infection (serodiagnosis: yes, no), type of calves housing (individual, collective), type of calves feeding (natural suckling, artificial), presence of others domestic animals (yes, no), presence of wild ruminant animals (yes, no). the variables "herd size" and "herd density" were based on the average data; "age group" was defined from the management adopted on the farms; "quarantine" refers to the isolation of purchased animals before adding them to the herd. the serodiagnosis of bohv- and bvdv- was performed at the same time as brsv (unpublished data). the variables "disinfection of the umbilical cord" and "colostrum feeding" were not analyzed because these practices were applied in all dairy farms visited. additionally, the data related to climatic classification and altitude were not included in the analysis because they were similar in all farms. the chi-square tests were employed to compare the brsv, bohv- and bvdv- seropositivity and also with "age group". fisher's exact test was used to compare brsv status according to the expected prevalence of % (≥ %, "high"; < %, "low") and the other variables. only the variables with p < , (two-tailed fisher) were analyzed by a logistic regression model. the analyses were performed using the epi info™ program v. . . serum samples from animals belonging to dairy cattle herds were taken and tested by vnt, in which ( . %) were seropositive to brsv. regarding the age group, % ( / ) of the serum samples were positives for adult animals while the prevalence rate in calves was . % ( / ). antibodies to brsv were detected in all cattle herds, with prevalence rates ranging from to %. the mean antibody titers for adult animals was to , and for calves, to . therefore, prevalence of brsv both in herds and animals was considered high. the chi-square test showed association of brsv seropositivity with "age group" and animals tested seropositives to bohv- and bvdv- (table ). nevertheless, the fisher's exact test only detected statistical difference with the variable "type of calves feeding" (table ). in this case, the relative risk (rr) value was less than one, i.e., the factor "natural suckling" was considered protective. logistic regression (values of p < . , two-tailed fisher) did not show significant results, suggesting that the variables analyzed were not risk factors for the high seroprevalence of brsv in the studied population. this is the first epidemiological study to assess risk factors for brsv seroprevalence carried out in brazil. even though brsv prevalence of . % in the animals sampled was similar to that estimated, the prevalence in adult animals was higher than that expected, reaching % of samples. in calves, the seroprevalence was lower than that found in adult animals ( . %) and could be even lower once vnt does not allow the distinction between antibodies from colostrum and natural infection. thus, this study demonstrated that the prevalence of brsv antibodies was higher in adult animals, as previously reported in other countries [ , ] . adult animals are associated with high seroprevalence of brsv as consequence of a repeated exposure to the virus infection throughout their life and possibility of reinfections. similarly, the highest antibody titers were associated with non-vaccinated adult cattle, probably due to the exposure to successive viral reinfections, which results in a booster effect on antibody titers [ ] . other factor related to high antibody titers is recent brsv infections, which can be confirmed only by paired serology, antibody screening in calves after the period of colostral antibody detection or viral detection by direct methods. as respiratory disease was not reported in half of the herds studied, it is indicative that brsv infection can be subclinical. this is consistent with previous reports [ ] . herds can remain free of clinical brsv infection for many years even in areas of high prevalence of the virus [ ] . the presence of other pathogens is also associated with the prevalence of brsv [ , , , ] . this information explains the association of brsv serological prevalence with the prevalences of bohv- and bvdv- . the infection by these viral agents is also reported in brazilian herds, with high prevalences [ , ] . bvdv infection can cause impairment of the animal's immune function and thereby decrease resistance to other infections [ ] . the synergistic effects of bvdv with other respiratory pathogens have been observed [ , ] . thus, health status of the herds may also be affected indirectly by bvdv control measures [ ] . dairy cattle herds in são paulo state usually have poor biosecurity measures, such as the lack of quarantine of newly purchased animals, lack of diagnosis of respiratory diseases (particularly for brsv) and vaccination is rarely performed against these viruses. therefore, we hypothesized that risk factors for the seroprevalence of bohv- , bvdv- and brsv in the studied population likely to overlap. despite the logistic regression not confirming "type of calves feeding" variable as a risk factor for high prevalence of brsv, the fisher's exact test detected "natural suckling" as a protective factor. "natural suckling" would be important as it may be able to reduce the risk of calves becoming infected by brsv. weaning can be stressful and results in impaired immune function, which may further exacerbate a brsv exposure. suckling reduces the occurrence of diarrhea, prevents the abnormal behavior of cross-suckling of other calves and improves animal health [ , ] . prior to the current study there have been no report about "natural suckling" and its relationship with brsv seroprevalence or its role as a protective factor, therefore, based on the results presented, it has the potential to decrease seroprevalence to brsv. similarities were observed among the results found at the present study and those previously obtained by others conducted in brazil [ ] [ ] [ ] . in latin america countries, equivalents prevalences of brsv have also been reported [ , [ ] [ ] [ ] , as well as difficulties in detecting the risk factors involved in the dissemination of the agent, even using different forms of sampling and analyzing a considerable number of variables. thus, the dynamics of infection may differ even in a particular country or geographic area [ ] . the high serological prevalence of brsv found in this study shows the importance to know more about this infection since it is not considered important in the country, mainly due to the lack of diagnosis. the awareness of the risk factors involved in the brsv dissemination can allow understanding its mechanisms, even though, as in other studies, these factors were not very clear. thereby, further studies as a complement to the current one should be performed until concrete information has been found. adult animals over year old can present high seroprevalences of brsv and are an important risk factor for the virus maintenance in herds. additionally, the concomitant seroprevalence of bohv- and bvdv- leads us to suggest that biosecurity measures to reduce viral dissemination could be applied. non-stressful management, like keeping calves with their mothers promoting natural suckling, may constitute a practical management strategy to reduce the seroprevalence of brsv in brazilian dairy herds. bovine respiratory syncytial virus bovine respiratory syncytial virus (brsv): a review bovine respiratory syncytial virus) infection: its pathogenesis, diagnosis, prevention and treatment seroepizootiologic study of bovine respiratory syncytial virus in a dairy herd respiratory syncytial virus: brief review identificación de agentes virales por inmunohistoquímica en enfermedades respiratórias de bovinos en corral de engorda severe respiratory disease in dairy cows caused by infection with bovine respiratory syncytial virus bovine respiratory syncytial virus seroprevalence and risk factors in endemic dairy cattle herds risk factors for epidemic respiratory disease in norwegian cattle herds risk factors for seropositivity to bovine coronavirus and bovine respiratory syncytial virus in dairy herds seroprevalence of bovine respiratory viruses in north-western turkey bovine respiratory syncytial virus: first serological evidence in uruguay detection on antibodies and risk factors for infection with bovine respiratory syncytial virus and parainfluenza virus in dual purpose farms in colima seroprevalence to bovine virus diarrhoea virus and other viruses of the bovine respiratory complex in venezuela (apure state) prevalence of and risk factors for bovine respiratory syncytial virus (brsv) infection in non-vaccinated dairy and dual-purpose cattle herds in ecuador detection of antibodies and risk factors for infections with bovine respiratory syncytial vírus and parainfluenza virus- in beef cattle of yucatan detection of bovine respiratory syncytial virus in calves of rio grande do sul detection of antibodies against bovine respiratory syncytial virus (brsv) in dairy cattle with different prevalences of bovine herpesvirus type (bhv- ) in são paulo state vírus respiratório sincicial dos bovinos (brsv): situação no brasil serological evidence of bovine respiratory syncytial virus in brazil produção da pecuária municipal campinas: unicamp encuestas por muestreo para estudios epidemiologicos en poblaciones animales simple method of estimating per cent end point dynamics of bovine respiratory syncytial virus infections: a longitudinal epidemiological study in dairy herds a longitudinal study of the dynamics of bovine corona virus and respiratory syncytial virus infections in dairy herds herpesvirus bovino tipo (hvb- ): revisão e situação atual no brasil a infecção pelo vírus da diarreia viral bovina (bvdv) no brasil -histórico, situação atual e perspectivas synergistic effects of bovine respiratory syncytial virus and noncytopathic bovine viral diarrhea virus infection on selected bovine alveolar macrophage functions prevalence, outcome and health consequences associated with persistent infection with bovine viral diarrhoea virus in feedlot cattle the effects of early separation on the dairy cow and calf practical implications of increasing natural living through suckling systems in organic dairy calf rearing the authors thank coordenadoria de assistência técnica integral (cati/ saasp) for providing the dairy cattle herds for the study, dr. estevam g. lux hoppe and andressa de souza pollo for assistance with manuscript revision. this work was supported by fundação de amparo à pesquisa do estado de são paulo, fapesp (financial grant / - and doctoral scholarship / - ). the datasets analyzed within the current study are available from the corresponding author upon request.authors' contributions ibalh and asrm performed the experiment. ibalh analyzed the data and prepared the manuscript. ibalh, cwa and sis designed the study and revised the manuscript. all authors read and approved the final manuscript. the work is in accordance with ethical principles in animal experimentation, adopted by the brazilian code of experimentation (cobea) and approved by the ethics committee on animal use (ceua). protocol / . not applicable. the authors declare that they have no competing interests. key: cord- -upogtny authors: viboud, cécile; lessler, justin title: the influenza pandemic: looking back, looking forward date: - - journal: am j epidemiol doi: . /aje/kwy sha: doc_id: cord_uid: upogtny in commemoration of the centennial of the influenza pandemic, the american journal of epidemiology has convened a collection of articles that further illuminate the epidemiology of that pandemic and consider whether we would be more prepared if an equally deadly influenza virus were to emerge again. in the present commentary, we place these articles in the context of a growing body of work on the archeo-epidemiology of past pandemics, the socioeconomic and geographic drivers of influenza mortality and natality impact, and renewed interest in immune imprinting mechanisms and the development of novel influenza vaccines. we also highlight persisting mysteries in the origins and severity of the pandemic and the need to preserve rapidly decaying information that may provide treasure troves for future generations. initially submitted august , ; accepted for publication september , . in commemoration of the centennial of the influenza pandemic, the american journal of epidemiology has convened a collection of articles that further illuminate the epidemiology of that pandemic and consider whether we would be more prepared if an equally deadly influenza virus were to emerge again. in the present commentary, we place these articles in the context of a growing body of work on the archeo-epidemiology of past pandemics, the socioeconomic and geographic drivers of influenza mortality and natality impact, and renewed interest in immune imprinting mechanisms and the development of novel influenza vaccines. we also highlight persisting mysteries in the origins and severity of the pandemic and the need to preserve rapidly decaying information that may provide treasure troves for future generations. age patterns; history of epidemiology; influenza; mortality; pandemic; prior immunity one hundred years after the fact, the influenza pandemic remains one of the most important epidemics of the modern medical era; it was significant for its impact on both human health and the development of epidemiology and other medical sciences. still, as we mark its centennial, it is sobering to realize how little we understand about the origins and lethality of this unusual outbreak despite decades of intense multidisciplinary research. although it would be years before it was possible to fully characterize the virus responsible for the pandemic ( ), contemporaneous medical authorities put commendable effort into reporting detailed epidemiologic data on the progression of the pandemic that ranged from individual-level clinical records to aggregated city-level vital statistics ( , ) . in addition to quantitative epidemiologic data, there exist many anecdotal reports from clinicians, particularly those who served military populations, that have been mined to provide modern audiences a comprehensive account of the pandemic ( ). yet, many important questions remain about the evolutionary origins of the pandemic virus; the contribution of world war i and troop displacements to pandemic emergence and progression; the unique age profile of pandemic deaths, with its signature of high mortality rate among healthy young adults; the consequences of such a large mortality event on natality; and the heterogeneity of the pandemic experience around the world. such mysteries have captured the attention of the lay public and scientific community alike. in commemoration of the centennial of the pandemic, the american journal of epidemiology has convened a collection of articles that further illuminate the epidemiology of that pandemic and consider whether we would be more prepared if an equally deadly influenza virus were to emerge today. five of the articles touch on the origins of the pandemic virus, addressing the role of swine as mixing vessels in this and other pandemic events ( ), the age-specific mortality patterns of the pandemic ( ) ( ) ( ) , and prior population immunity ( ) . others include reports on geographic and social heterogeneities in the pandemic experience in which the authors describe the spatial diffusion of the pandemic in india and portugal ( , ) , the socioeconomic predictors of high mortality risk in sweden and globally ( , ) , and the consequences of the pandemic on us natality rates ( , ) . finally, commentaries address preparedness for future influenza pandemics ( , ) . the influenza virus is remarkable for its ability to infect a variety of animal species, from bats to birds to mammals. although successful cross-species transmission events may be rare, they play a key role in the genesis of new pandemic strains. nelson and worobey ( ) discussed different lines of evidence informing the origins of the virus, including the genetic make-up of the virus and other pandemic strains, the characteristics of influenza receptors across different influenza hosts, and the frequency of cross-species transmission events. they concluded that the pandemic virus must have emerged in mammals just before , most likely from the avian reservoir, with onward transmission from humans to swine. more broadly, a re-analysis of virologic data from the and pandemics, together with a modern understanding of the swinehuman interface, suggested a twist on the long-standing concept of swine as a "mixing vessel" for influenza virus. the authors proposed that swine should be viewed as a repository of historic human viruses rather than a conduit for reassortment of genetic material between avian and human viruses. van wijhe et al. ( ) returned to the question of the origins of the virus by exploring the epidemiologic imprint of the virus on danish mortality records, echoing recent work on immune imprinting ( ) ( ) ( ) . they identified several age breakpoints in pandemic mortality that were suggestive of the cycling of different influenza strains between the mid- th century and the pandemic. most notably, they argued for co-circulation of subtypes of influenza virus (carrying type i and ii hemagglutinin surface antigens) between and . as a result, persons born between and (aged - years during the pandemic) may have been primed by either hemagglutinin type, potentially explaining the intriguing age profile of pandemic mortality in adults. cilek et al. ( ) used a similar epidemiologic approach to explore the pandemic mortality patterns in madrid, spain. madrid is particularly interesting because a lethal pandemic wave was reported in the city in june , the earliest such event recorded. similar to other regions of the world, madrid experienced a signature pandemic pattern of higher mortality rates among young adults. however, seniors in madrid suffered equally high rates of excess influenza mortality. this is unlike the experience of the rest of europe and north america, where seniors were reportedly spared, presumably because of antigenic recycling (i.e., exposure to a related strain in childhood that conferred partial protection) ( , ) . this is an intriguing finding, and it will be important for future work to reconcile the well-accepted idea that a -like virus may have circulated in europe and north america in the second half of the th century, with the notion that madrid would have escaped this virus. to understand the unique epidemiology of the virus, it can be useful to document the experience of remote populations, in which prior immunity to influenza would be expected to be low because of less frequent circulation of the virus. rice ( ) built on a rich literature in this area to document mortality patterns in new zealand between and . he found that the s were a decade associated with high rate of influenza mortality in new zealand, despite the low global connectivity of this island in the era before air travel. he also noted that influenza mortality in was highest among young adults, with a more pronounced intensity in males than in females. these patterns are broadly consistent with the those among young adults in europe and the americas, pointing to the near universality of increased influenza mortality risk in this age group in . chuah et al. ( ) used seroepidemiology and structural equation modeling to answer the inverse question: how did early-life exposure to the pandemic virus impact how people responded to the pandemic, which was caused by an antigenically similar virus? they found evidence for immunologic priming from the virus in the oldest people they studied (individuals years of age or older) that impacted both baseline titers and vaccine response in . this work adds to a growing body of evidence that early-life exposures can have profound effects on immune response and mortality patterns decades after they occur ( ) . information about global connectivity in the th century is tenuous, and influenza records before are scarce. epidemiologic reconstructions of "modern" pandemics of the type presented here ( - ) provide indirect information on the exposures of populations that are now long gone, generating valuable hypotheses about influenza circulation patterns and disease dynamics well into the th century. such reconstructions offer precious insights into what influenza may have looked like years ago in a very different world and how long-term changes in human demography and mobility may affect disease dynamics ( ) . active research topics in the field of archeo-epidemiology include the search for predictors of influenza mortality, such as socioeconomic indicators or geography, and the drivers of influenza spatial diffusion. in articles in the present issue, the authors concentrated on the spatial diffusion of influenza, focusing on british india and portugal, countries that have been poorly studied in the context of the pandemic ( , ) . both studies revealed a highly heterogeneous spread of the pandemic and geographic variation in pandemic mortality impact, albeit at different spatial scales. although portugal as a whole was severely hit by the pandemic compared with other european countries, some provinces nearly fully escaped ( ). analysis of district-level mortality records in india revealed a northeastward wave of infection from september to november that was associated with climate and population density ( ) . diffusion was driven by long-distance jumps via the railroad network, superimposed on local diffusion between neighboring provinces. further, the authors found moderate heterogeneity in the mortality experiences of different indian provinces. spreeuwenberg et al. ( ) also made use of recently unearthed data from india to revisit the global mortality impact of the pandemic. india is a particularly important country for global burden estimation because it was the one most severely hit by the pandemic, with annual pandemic excess mortality rates that were -fold higher than those in denmark for instance ( ) . in the new study, the authors placed the burden of the pandemic at a much lower number than did previous work ( ) , in part by using more detailed data to better adjust for high background mortality unrelated to flu. the results of the portuguese study by nunes et al. ( ) echoed these conclusions-that careful analyses of more detailed data tend to decrease estimates of pandemic burden. the risk factors responsible for increased mortality and morbidity from influenza remain elusive, whether at the population level (e.g., effect of population density or weather on transmission) or the individual level (socioeconomic status, comorbid conditions, etc.). this is still an active area of contemporary influenza research, with direct applications to design targeted intervention strategies. in the present issue, bengtsson et al. ( ) explored the role of social class on pandemic mortality by linking individual death records with historical census data on occupation in a powerful study that captured the entire swedish population. the authors found that low-skilled or unskilled adults had higher death rates than did more skilled workers during the pandemic period relative to prepandemic years, whereas farmers (especially men) fared particularly well. social differences tended to be smaller in women, and there was no clear gradient between social class and mortality. the authors hypothesized that these social differences were linked to differential crowding in the workplace (hence an effect on transmission) rather than differences in income or nutrition. this is a topical issue because the effects of socioeconomic status and baseline health on influenza mortality are still debated today ( ) . researchers have long thought that the pandemic could have affected birth rates ( ) because of the large impact of this event on young adult mortality, the increased risk of severe flu outcomes during pregnancy, and a possible association between influenza infection and miscarriage. two papers in this issue address the topic of natality ( , ) . key questions here include the trimester of pregnancy during which the risk of death is highest for the mother and/or the unborn child and the impact of influenza on (increased) stillbirths and (decreased) live births. the duration of the pandemic effect on natality is also important because it informs the biological mechanism at play. if influenza impacts the probability of conception or fetal deaths, one would expect a temporary natality drop in the aftermath of the pandemic, followed by a rebound in births a few months later. in contrast, a high mortality rate among young women of childbearing age due to influenza infection would result in a long-lasting natality trough. dahal et al. ( ) explored these questions using individual birth and death certificates from arizona, where there was a drop in natality - months after pandemic mortality peaked. this was a temporary depletion, consistent with a detrimental effect of influenza early in pregnancy. in a larger study of populationlevel vital statistics in us states, chandra et al. ( ) found a % drop - months after peak influenza mortality, which they ascribe to a drop in conception during the period of intense pandemic activity. they also found a natality drop in the months after peak mortality, which they linked to excess preterm births and stillbirths due to influenza infections in the last trimester. interestingly, these patterns were also found in the aftermath of the influenza pandemic wave, albeit with a less pronounced effect. one reason we still look back at the pandemic years later is because doing so will make us better able to prepare for the future. the last articles of this collection are focused on preparedness for future pandemic threats ( , ) , building on the lessons learned in and later pandemics, and on new tools to protect populations, including the very active (but still elusive) topic of universal influenza vaccines. jester et al. painted an optimistic picture of progress made in influenza surveillance domestically and internationally, antiviral treatments, and robustness in the infrastructure for vaccine production ( ) . epstein reviewed the progress of the development of a broadly cross-protective flu vaccine, focused on conserved parts of the influenza virus, such as the matrix protein, nucleoprotein, the hemagglutinin stem, and various cocktail combinations ( ) . these vaccines offer promising broad protective effects against new influenza antigenic variants and could potentially be used in pandemic situations. however, some of the candidate vaccine formulations permit limited viral replication and may foster the emergence of escape mutants fit enough to cause disease. these features could have adverse epidemiologic consequences, and these risks need to be projected and monitored carefully. the pandemic is traditionally considered a worst-case scenario for pandemic preparedness, but there were many other pandemics before about which we know very little regarding mortality impact, circulating strains, or prior immunity. in fact, the pandemic has only recently drawn attention among epidemiologists ( , ) . most european and north american countries began formal collection of vital statistics in the mid-to-late th century, so that any pandemic predating can only be explored using church or cemetery records (or indirectly through reconstruction of modern pandemics). digitization of historic records is time consuming, data lack standardization, and information is generally limited to small populations. even with regard to the pandemic, crucial questions may never be answered, including which specific virus (or even subtype) circulated before and further back into the th century and what the population immunity profile was before the pandemic. the search for archival influenza specimens predating has remained elusive, and to our knowledge no archived sera exist from this period. in the absence of further virologic evidence, our understanding of the origins of the pandemic is limited to a handful of influenza virus sequences collected during may to november and to the epidemiologic signature of the virus in different populations. as nelson and worobey noted ( ), more work can be done in this area, particularly to explore the uracil content of post- viruses in different hosts, reconstruct their evolutionary trajectories, and better characterize host receptors and barriers to cross-species jumps. further, as rice ( ) and dahal et al. ( ) noted, a systematic analysis of the age mortality profiles of the pandemic in a sample of remote and wellconnected locations would be most useful, together with modeling of plausible biological hypotheses and immune histories most consistent with the data. databases, which are often crowdsourced or maintained by state health departments ( ) . further analyses of such data could shed light on the mortality profile of the pandemic in understudied locations and would also allow identification of family linkage and host genetic risk factors, which could be tested among descendants. many other library archives exist, although paper-based records rapidly decay and need to be digitized as quickly as possible. the detailed, sometimes freeform, notes typically kept by the scientists at the time mean that careful examination of these archives can sometimes yield surprising fruit. one such resource is the work of wade hampton frost, who was the first chair of the department of epidemiology at johns hopkins university and a critical figure in the fight against the pandemic. modern reanalysis of dr. frost's detailed work ( ) has already yielded abundant insights, and we included digital copies of his papers on the pandemic from the chesney archive in the web appendix (available at https://academic. oup.com/aje). the pandemic is remarkable for the large amount of extremely detailed epidemiologic data collected by public health officials ( , ), in part because it was an era that valued epidemiology, at a time when analytical approaches and knowledge of infectious agents were limited. these exquisitely detailed records have been particularly useful in the attempt to understand the pandemic retrospectively. as a thought experiment, we can imagine ourselves in : we may ask how scientists would look back at the large amount of data we archive on a daily basis in . on the one hand, much if not all of the modern data are digital, meaning that they do not run the risk of being destroyed by fire or floods and they can be more accessible to a wide audience, spurred by the open access movement. however, digital data can also be corrupted (intentionally or unintentionally) and disappear. much from the floppy-disk era has already been permanently lost, and it is unclear if modern cloud-based archives would survive a major disruption (whether technological or civil). further, even today, there is a systematic dearth of epidemiologic and molecular data from low-and middle-income settings (including data on the pandemic ( )). we are just beginning to scratch the surface of the intricate relationship between the influenza virus and the complex immune history of a host who has had repeated influenza exposures ( , ( ) ( ) ( ) ) . it is unclear whether we will be able to fully understand these interactions in the foreseeable future; in the meantime, population birth cohorts carrying important influenza immune histories disappear. we echo earlier calls for a time-stamped global repository of human sera and pathogen specimens, ideally together with epidemiologic information (biobanks) for current and future use ( ) . we also applaud the push by the us national institute of allergy and infectious diseases to fund international influenza birth cohort studies and help untangle the complex mechanisms of influenza immunity ( ) . if again confronted with a deadly flu pandemic, we would be in a better place than we were in because of the availability of drugs, vaccines, and antibiotics and the general improvements in health and nutrition. there are high hopes for the development of universal vaccines, but we need to keep in mind that influenza is a rapidly evolving virus that has a large and diverse animal reservoir and presumably many tricks in store. we can only anticipate another hundred years of very active, and always surprising, influenza research. structure of the uncleaved human h hemagglutinin from the extinct influenza virus preliminary statistics of the influenza epidemic influenza transmission in households during the pandemic the great influenza: the story of the deadliest pandemic in history origins of the pandemic: revisiting the swine "mixing vessel" hypothesis loose ends in the epidemiology of the pandemic: explaining the extreme mortality risk in young adults age-specific excess mortality patterns during the - influenza pandemic in madrid, spain influenza in new zealand before : a preliminary report investigating the legacy of pandemic on age-related seroepidemiology and immune responses to subsequent influenza a(h n ) viruses through a structural equation model the - influenza pandemic in portugal: a regional analysis of death impact spatiotemporal patterns and diffusion of the influenza pandemic in british india reassessing the global mortality burden of the influenza pandemic social class and excess mortality in sweden during the influenza pandemic natality decline and spatial variation in excess death rates during the - influenza pandemic in arizona, united states short-term birth sequelae of the - influenza pandemic in the united states: state-level analysis readiness for responding to a severe pandemic years after universal influenza vaccines: progress in achieving broad cross-protection in vivo potent protection against h n and h n influenza via childhood hemagglutinin imprinting re-examining the evidence regarding circulation of a human h influenza virus immediately prior to the spanish flu age-and sex-specific mortality associated with the - influenza pandemic in kentucky epidemiological evidence of an early wave of the influenza pandemic in new york city influenza epidemics in iceland over decades: changes in timing and synchrony with the united states and europe estimation of potential global pandemic influenza mortality on the basis of vital registry data from the - pandemic: a quantitative analysis global mortality estimates for the influenza pandemic from the glamor project: a modeling study natality decline and miscarriages associated with the influenza pandemic: the scandinavian and united states experiences transmissibility and geographic spread of the influenza pandemic age-specific excess mortality patterns and transmissibility during the - influenza pandemic in madrid, spain evidence for antigenic seniority in influenza a (h n ) antibody responses in southern china use of serological surveys to generate key insights into the changing global landscape of infectious disease the ghost of influenza past and the hunt for a universal vaccine this article does not necessarily represent the views of the national institutes of health or the us government.conflict of interest: none declared. key: cord- -z ekgtv authors: babu, tara m; perera, ranawaka a p m; wu, joseph t; fitzgerald, theresa; nolan, carolyn; cowling, benjamin j; krauss, scott; treanor, john j; peiris, malik title: population serologic immunity to human and avian h n viruses in the united states and hong kong for pandemic risk assessment date: - - journal: j infect dis doi: . /infdis/jiy sha: doc_id: cord_uid: z ekgtv background: influenza a pandemics cause significant mortality and morbidity. h n viruses have caused a prior pandemic, and are circulating in avian reservoirs. the age-related frequency of current population immunity to h viruses was evaluated. methods: hemagglutinin inhibition (hai) assays against historical human and recent avian influenza a(h n ) viruses were performed across age groups in rochester, new york, and hong kong, china. the impact of existing cross-reactive hai immunity on the effective reproduction number was modeled. results: one hundred fifty individual sera from rochester and from hong kong were included. eighty-five percent of patients born in rochester and hong kong before had hai titers ≥ : against a/singapore/ / , and > % had titers ≥ : against a/berkeley/ / . the frequency of titers ≥ : to avian h n a/mallard/england/ / and a/mallard/netherlands/ / in subjects born before was % and %, respectively. there were no h hai titers > : in individuals born after . these levels of seroprevalence reduce the initial reproduction number of a/singapore/ / or a/berkeley/ / by %– %. a basic reproduction number (r( )) of the emerging transmissible virus < . predicts a preventable pandemic. conclusions: population immunity to h viruses is insufficient to block epidemic spread of h virus. an h n pandemic would have lower impact in those born before . influenza pandemics may occur when influenza a viruses of animal origin with a novel hemagglutinin (ha, or h) with or without neuraminidase (na, or n) subtypes to which the human population has little or no immunity infect humans and transmit efficiently from person to person. there were influenza pandemics in the th century. in , an influenza a virus of the h n subtype emerged and caused widespread disease; subsequently, h n viruses caused seasonal epidemics until . in a new influenza a virus of the h n subtype, sometimes referred to as the "asian" influenza, emerged to cause a second pandemic, and subsequently h n viruses replaced h n viruses as the cause of seasonal influenza from to . in a third pandemic was caused by an h n virus, which replaced h n viruses and continues to circulate in humans to the present day. influenza a pandemics are associated with significant mortality, morbidity, and financial burden. for example, the pandemic of - resulted in at least million influenza-related deaths [ ] , while the pandemics of (h n ) and (h n ) combined had estimated economic losses around us $ billion (estimated in dollars) [ ] . although not designated a pandemic, the reemergence of h n in also shared some characteristics with the other pandemics. the h n pandemic was caused by a virus subtype that was then circulating in humans and was unexpected because it was generally assumed that that population immunity would prevent emergence of a pandemic virus of a subtype currently endemic in humans. there continues to be concern regarding the potential for new influenza a viruses to be transmitted to humans, with documented severe zoonotic disease caused by influenza a h and h infections. however, because h n virus has already caused a pandemic, and h subtype viruses are currently circulating in wild and domestic birds [ ] , reemergence of an h n virus is one of the most likely scenarios for a new pandemic. anti-ha antibody, anti-na antibody, and cell-mediated immunity have all been correlated with protective immunity in both experimental animals and in humans [ ] [ ] [ ] [ ] . anti-ha antibody in peripheral blood sera, as assessed by the hemagglutination inhibition (hai) assay, has a strong correlation with protection against influenza infection and disease. although complicated by significant interlaboratory variation, an hai titer of : is generally accepted as a marker of reduced susceptibility [ ] . therefore, analysis of the population level of hai antibody can be used to estimate the population susceptibility to infection [ ] . for example, the seroprevalence of hai antibody to ph n in individuals older than years correlated with a significantly decreased influenza-associated mortality for these individuals during [ ] . it has been suggested that exposure to antigenically related h n influenza virus - years earlier provided older adults with some degree of immunity against the h n pdm virus [ ] . similarly, individuals who were exposed to h n viruses during the period from to may have persistent antibody to these viruses and be relatively protected from an emerging h n virus. however, more than two-thirds of the world population in was born after [ ] , suggesting that there may be substantial susceptibility to these viruses. because pandemics spread across the world within weeks after emergence [ ] , much faster than the process of developing and rolling out a vaccine to the newly emerged pandemic virus, which takes > months [ ] , attention has recently focused on developing systematic risk assessment algorithms for animal viruses of possible pandemic threat so that preemptive preparations including the development of vaccine seed strains can be initiated in advance. examples of these include the influenza risk assessment tool and the tool for influenza pandemic risk assessment [ ] . an integral aspect of this risk assessment process is assessment of population immunity to the relevant virus. in this study, we evaluated population immunity using hai assays against human and avian h n influenza strains in different age groups in the united states and hong kong. we then estimated the impact of existing cross-reactive hai immunity on reducing the effective reproduction number (r) of a potentially pandemic h subtype virus and characterized the minimum basic reproduction number (r ) that such a virus must possess to cause a pandemic. samples in rochester were collected between january and march from nonimmunosuppressed individuals who were healthy or had stable medical conditions and were from months to years of age. in the united states, influenza activity typically peaks in january or february. according to the centers for disease control and prevention, in - influenza activity peaked in early february and in - it remained low through february and did not peak until mid-march. in - , - , and - , influenza activity peaked in late december with some variability [ ] . sera from children and adults in hong kong were collected as part of a previous serological study between august and december , prior to the winter influenza season, which typically commences around february-march in hong kong [ ] . the preceding influenza season peaked in february-march and the dominant virus subtype was pandemic h n . two hundred ninety-five serum samples from this study were selected for testing in age strata. selection of viruses for hai testing was based on phylogenetic lineages of the h influenza virus subtypes: human and avian eurasian lineages. viruses were selected from each lineage to represent temporal and geographic diversity. a/singapore/ / (h n ) and a/berkeley/ / (h n ) represented the human lineage, whereas a/mallard/ england/ / (h n ) and recent h n virus isolate a/ mallard/netherland/ / (h n ) were of the eurasian avian lineage [ ] . the a/mallard/england/ / virus was generated by plasmid-based reverse genetics with ha and na of a/ mallard/england/ / and other internal virus genes of a/ puerto rico/ / origin. antigenic relatedness of the selected test viruses was determined by reciprocal hai assays shown in supplementary table . viruses with pandemic potential were handled in a us department of agriculture-approved animal biosafety level (absl )-enhanced facility. β-propiolactone (bpl)-inactivated virus using standard procedures for use as antigen in the hai test was prepared. the bpl-treated virus preparation was inoculated into -day-old hen's eggs following standard virus culture procedures to confirm complete inactivation of the virus. the antigen was then removed from the absl -enhanced laboratory for hai analysis. in rochester and hong kong, hai studies were performed in biosafety level conditions. serology hai tests were performed using turkey red blood cells in rochester, and chicken red blood cells in hong kong, otherwise the laboratories used the same procedure for the test. sera were pretreated with receptor-destroying enzyme (denka seiken co ltd) and tested at a starting dilution of : . hai was performed using "v" bottom microtiter plates as previously described [ ] . the positive control sera used were ferret hyperimmune sera against a/kruitt/ , a/mallard/netherlands/ / , a/swine/ missouri/ / , a/mallard/netherlands/ / , and a/bakker/ viruses, and goat hyperimmune sera against a/ japan/ / and a/singapore/ / . negative controls consisted of antigen alone wells and the reagent control contained phosphate-buffered saline with red blood cells. statistical significance was analyzed using graphpad prism software (graphpad, san diego, california) using -way analysis of variance, followed by bonferroni post hoc analysis. p values <. were considered statistically significant. the reproduction number of each virus in each population was calculated as follows. we partitioned the population into n = age groups ( - , - , - , - , - , - , - , ≥ ) and m = hai titer levels (< : , : , : , ≥ : ). let p i be the proportion of population in age group and s ij be the proportion of age group i with the jth hai titer. the age distribution p i was based on the most recent census data from the united states ( ) and hong kong ( ). to estimate s s for each age group, we used bayesian inference with dirichlet y i was the number of individuals in age group i in our serologic study and x ij was the number of subjects in age group i with the jth hai titer level [ ] . we assumed noninformative priors, that is, all priors were dirichlet distributions with parameters a j = for j m = ¼ , , , and hence the joint posterior distributions of s s we assumed that an hai titer of < : , : , : , and ≥ : reduced susceptibility by %, %, %, and %. as such, the proportion of the population that were immune was ij j , where z j was the reduction in susceptibility conferred by the jth hai titer level (ie, , and z = . ). the basic reproduction number r was the largest eigenvalue of the matrix q ij { } , where q ij was the average number of secondary cases in age group i generated by an infector in age group j when everyone in the population was susceptible [ ] . we constructed the matrix q ij { } using the united kingdom social contact matrix [ ] because analogous data are not available from the united states and hong kong. because the immune proportion of age group i was table . the demographics of the study population matched the demographics of the source population (data not shown). the results of hai testing of the sera from rochester and hong kong gave very similar results, despite the populations and different laboratories. the geometric mean titers (gmts) of antibody in the populations against the test viruses by decade of birth are shown in table . as expected, there were substantial levels of anti h hai antibody in the sera of persons old enough to have been infected with h n viruses between and , and essentially no detectable antibody in persons born after . among persons born before , the gmt of antibody against the early human a/singapore/ / was significantly higher compared with titers against the later human a/berkeley/ / virus, whereas in persons born from to there was a trend toward relatively higher titers against the a/berkeley/ / virus. titers against the avian h n viruses were lower. there were no significant differences in the gmt against a/singapore/ / , a/berkeley/ / , or a/ mallard/england/ / in sera tested in hong kong and rochester, but sera tested in rochester had significantly higher titers against a/mallard/netherlands/ / than the sera tested in hong kong. the prevalence of titers ≥ : against the test viruses is shown for sera from rochester and hong kong in persons born before the h n pandemic, during the years that h n circulated ( - ), or after is shown in figure . ninetyeight percent of individuals from rochester and hong kong born before had titers ≥ : against the a/singapore/ / virus, whereas > % of subjects born between and had titers ≥ : . in contrast, none of those born after had titers > : to a/singapore/ / . generally, persons born prior to had a lower prevalence of titers ≥ : to a/ berkley/ / , while the prevalence of titers ≥ in persons born during circulation of these viruses was similar against a/ ( ) ( ) - ( ) ( ) - ( ) ( ) - ( ) ( ) - ( ) ( ) - ( ) ( ) or later ( ) ( ) data are presented as no. (%). singapore/ and a/berkley/ . the prevalence of titers ≥ : against the avian h n viruses tested were significantly lower in these groups. no person born after had titer > : against any of the h viruses. after combining the results from both populations, the cumulative distribution of antibody titers against the test viruses in these age groups is shown in figure . eightyfive percent of individuals born in both the united states and hong kong before had hai titers ≥ : against a/ singapore/ / ( figure ). more than % of subjects had hai titers ≥ : to a/berkeley/ / if born before . the frequency of titers to avian h n viruses was %, and % of subjects born before had titers of ≥ : to a/mallard/ england/ / and a/mallard/netherlands/ / , respectively, with such titers seen in % and % of individuals born from to . successful pandemic emergence and spread of a virus depends on the proportion of the population that is immune and the initial r of the potentially pandemic strain. the median r for the a/h n pandemic was . (interquartile range [iqr], . - . ) [ ] . to assess the impact of these age-dependent population immunity profiles on the pandemic potential of each of these viruses if they were to emerge in the human population, we computed the impact of population immunity in reducing r (figure ). the current population immunity in the united states and hong kong would reduce the initial r of a/singapore/ / by around % ( %- %) and that of a/ berkeley/ / by around % ( %- %). as such, a pandemic of a/singapore/ / and a/berkeley/ / would be prevented if initial r of the emerging virus was below . ( . - . ) and . ( . - . ), respectively. the threshold r below which a pandemic with the avian subtype h viruses a/mallard/england/ / and a/mallard/netherlands/ / would be prevented was slightly lower. the comparability of data from geographically separated areas of the world, rochester and hong kong, argues for the representativeness and generalizability of such studies that aim to assess population immunity to viruses of pandemic concern. our study suggests that those individuals born prior to or during the period of h n virus circulation were more likely to have higher hai titers against the human h n viruses than those born after when h infection in humans had been displaced by the h n virus. in our study, we also confirmed evidence of cross-reactive hai antibodies to unrelated avian h viruses, albeit at lower prevalence and titer. the prevalence of such cross-reactive antibodies was higher in those born prior and the gmt of these cross-reactive antibodies was higher in those born prior to than in the birth cohort of - . this is possibly because those who were infected in the early pandemic waves of the h n virus in - were reinfected some years later by antigenically drifted h n viruses, possibly broadening cross-reactive immunity. as reported by others, we also observed low hai titers < : spanning across the age groups and this could be due to nonspecific data are presented as geometric mean titer ( % confidence interval). a samples in hong kong and rochester were tested at a starting dilution of < : and negative tests are given an imputed value of . inhibition by the sera. however, we checked the sera for nonspecific agglutinins where about % of the samples contained nonspecific inhibitors. since the assays were run on sera which were confirmed not to contain nonspecific inhibitors, these low titers may be due to a certain type of antigen exposure that needs further investigation. sera from those born after had higher hai titers to a/berkeley/ / than to a/singapore/ / whereas the converse was true in those born before . the older group of individuals who were first infected by a/singapore/ / like viruses likely had a boost of these titers when they were subsequently infected by later drift variants (ie, a/ berkley/ / -like viruses), the phenomenon known as "original antigenic sin" [ ] . the broader cross-reactivity may also occur due to targeting different antigenic sites, as a recent study points out that antibody response against h is mainly to the receptor binding domain resulting in a greater degree of cross-reactivity whereas for h or h viruses it is the hypervariable regions of ha resulting in a lesser cross-reactivity [ ] . soon after the h n pandemic of , studies done on sera collected prior to this pandemic were carried out and it was reported that people born prior to had detectable hai antibody to h viruses. it was therefore suggested that the historical pandemic that was believed to have occurred in - was likely caused by an h subtype virus [ ] . this prior exposure has also shown to elicit cross-reactive antibody for h strains that are currently circulating in animals, potential candidate pandemic stains [ ] . sera tested in rochester yielded higher titers against a/mallard/ netherlands/ / than the sera tested in hong kong. the differences between laboratories are not unexpected given the known laboratory-to-laboratory variation in the hai test, and what remains remarkable is the closeness of results. one technical difference between the laboratories was the source of erythrocytes for the hai test; rochester used turkey erythrocytes whereas hong kong used chicken erythrocytes. it is not known if this may contribute to this difference in hai test results. an alternative reason for the discrepancy in titers between countries may be attributed to variation in vaccination rates. vaccine uptake in hong kong is lower than in the united states. potentially, repeated vaccination may increase cross-reactive antibody to this avian h strain. assuming that the h seroprevalence in rochester and hong kong reflects global population seroprevalence, we modeled the impact of such immunity on possible emergence of an h n pandemic. this model was based on the candidate pandemic h strain originating from escape of the human adapted h n virus or from the avian gene pool that acquires transmissibility in humans. we estimate that the current levels of population immunity would reduce r of a/singapore / / - a recent risk assessment for viruses currently known to be circulating in wild birds has been carried out and the risk of these viruses acquiring transmissibility in humans was assessed to be low; most isolates replicated in human bronchial epithelial cells and ferrets. several did transmit between ferrets by direct contact, but all has retained a preference for avian-like α , -linked sialic acid receptors [ ] . these viruses still remain primarily in wild birds and have not been established in mammalian species including swine. our study provides the systematic assessment of the impact of human population immunity that goes toward such an overall assessment of pandemic risk. a limitation of the study is that other potential contributors to population immunity, such as cross-reactive na-inhibiting antibody, stalk-specific antibody, cell-mediated immunity, and heterosubtype reactive hai or neutralizing antibody [ ] [ ] [ ] [ ] ] , were not assessed. furthermore, in many parts of the world (especially in developing countries), the proportion of individuals born in or later might be larger than that in rochester and hong kong. the level of immunity against a(h n ) in these populations would be lower than that estimated here and the pandemic potential of a(h n ) in these populations would thus be higher than estimated here. in summary, we find that levels of population immunity to h subtype viruses are not substantial enough to block epidemic spread of an h virus that had acquired efficient transmissibility between humans. furthermore, the existing levels of population immunity to h viruses will continue to decline with new birth cohorts being added to the human population. given that human-adapted h n viruses that arose subsequent to the pandemic are present in many laboratories worldwide, these findings support the need to have preparedness for h viruses as a credible pandemic threat. the approach used in the case study of h viruses is more broadly applicable in defining the impact of population immunity to viruses to which there is some level of cross-reactive hai antibodiesfor example, swine h and h viruses and avian h n viruses [ ] . reliable methods for assessing population immunity are a key to reliable risk assessment of viruses for pandemic threat. it was the lack of such risk assessment of population immunity to the h n triple reassortant swine viruses that led to these viruses not being recognized as potentially pandemic viruses prior to ; indeed, it was the triple reassortment swine ha that was the key protective antigen for the h n pandemic virus. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. figure . estimations of overall population-level immunity against h viruses and the potential impact of population immunity on reproduction number, using potential titer cutoffs for % immunity. bars updating the accounts: global mortality of the - "spanish" influenza pandemic origin of the pandemic h influenza a virus and the persistence of its possible progenitors in the avian reservoir correlates of protection to influenza virus: where do we go from here? antibody to influenza virus neuraminidase: an independent correlate of protection preexisting influenza-specific cd + t cells correlate with disease protection against influenza challenge in humans cellular immune correlates of protection against symptomatic pandemic influenza seroprevalence to influenza a(h n ) virus-where are we? complex patterns of human antisera reactivity to novel h n and historical h n influenza strains cross-reactive antibody responses to the pandemic h n influenza virus vaccinate for the next h n pandemic now the infection attack rate and severity of pandemic h n influenza in hong kong pandemic preparedness and the influenza risk assessment tool (irat) relative incidence and individual-level severity of seasonal influenza a h n compared with pandemic h n adaptation of pandemic h n influenza a viruses in humans world health organization. manual for the laboratory diagnosis and virological surveillance of influenza the bugs book: a practical introduction to bayesian analysis inferring influenza infection attack rate from seroprevalence data social contacts and mixing patterns relevant to the spread of infectious diseases estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature original antigenic sin responses to influenza viruses host versus flu: antibodies win a round? pre-epidemic antibody against , strain of asiatic influenza in serum of older people living in the netherlands studies on the content of antibodies for equine influenza viruses in human sera risk assessment of h n influenza viruses from the avian reservoir safety and antigenicity of whole virus and subunit influenza a/hong kong/ / (h n ) vaccine in healthy adults: phase i randomised trial key: cord- -ol ofj authors: abdul-cader, mohamed sarjoon; ehiremen, godsent; nagy, eva; abdul-careem, mohamed faizal title: low pathogenic avian influenza virus infection increases the staining intensity of kul + cells including macrophages yet decrease of the staining intensity of kul + cells using clodronate liposomes did not affect the viral genome loads in chickens date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: ol ofj the effect of depletion of macrophages using clodronate liposomes as well as macrophage response following viral infections have been studied in various mouse-virus infection models, but they have not been extensively studied in chickens relevant to virus infections. when we infected day chickens with h n low pathogenic avian influenza virus (lpaiv), we observed that h n lpaiv infection increased the staining intensity of kul + cells in trachea, lungs and duodenum of chickens at days post-infection. then, we used clodronate liposomes intra-abdominally in day-old chickens and found significant reduction of staining intensity of kul + cells in trachea and duodenum but not in lungs at days post-treatment. when we infected the clodronate liposome and pbs liposome treated chickens with h n lpaiv intra-nasally at day , we found no effect on h n lpaiv genome loads in trachea, lungs and duodenum of chickens. this study indicates that although kul + cell intensity are increased in respiratory and gastrointestinal tissues in chickens following h n lpaiv infection, the decrease of kul + cell intensity using clodronate liposomes did not change the h n lpaiv genome loads in any of the examined tissues suggesting that kul + cells may not be critical during h n lpaiv infection in chicken. the innate immune system, which mounts potent, nonspecific and broadly effective host responses, is equipped with an array of immune cells. one of the key immune cells indispensable in this regard is the macrophages. in addition to phagocytic activities of macrophages against various microbes and harmful substances, they also act as antigen presenting cells and a source of cytokines and chemokines facilitating the induction of antigen specific adaptive immune responses (arango duque and descoteaux, ) . in avian species, mobilization of macrophages into the site of infection contribute more than resident macrophages in phagocytic activity (maina, ) . liposome encapsulated clodronate (dichloromethylene diphosphonate or cl -mdp) has been widely used to deplete macrophages (benoit et al., ; kameka et al., a; leemans et al., ) . once phagocytosed by macrophages, clodronate liposomes get accumulated in the cytosol, resulting in apoptosis and depletion of macrophages (van rooijen et al., ) . it has been shown that the use of clodronate liposomes significantly depletes macrophages in chickens (jeurissen et al., ) . the effect of depletion of macrophages using clodronate liposomes has been studied against various virus infections in mouse models, such as measles (roscic-mrkic et al., ) and influenza (tate et al., ) . in chickens, increased marek's disease virus genome load in the blood and spleen following treatment with clodronate liposomes has been shown (rivas et al., ) . however, macrophage depletion using clodronate liposomes has not been extensively studied during other viral infections in chickens. macrophage recruitments following viral infections in chickens such as marek's disease virus, infectious bursal disease virus, adenovirus and infectious bronchitis virus infections have been shown previously (abdul-careem et al., ; abdul-careem et al., ; fulton et al., ; kameka et al., b; nakamura et al., ) . however, studies that investigated macrophage response in chickens following avian influenza virus infection are scarce (cornelissen et al., ; rebel et al., ) . avian macrophages have been shown to be targeted for viral replication during infectious bronchitis corona virus , marek's disease virus (barrow et al., ; chakraborty et al., ) , avian influenza virus (barjesteh et al., ) , infectious laryngotrcaheitis virus (calnek et al., ) and reovirus (swanson et al., ) infections. avian macrophages are also have been shown to elicit antiviral response depending on nitric oxide (no) production against avian influenza virus , infectious laryngotracheitis virus (haddadi et al., ) and marek's disease virus (xing and schat, ) . following avian influenza virus infection, macrophages secrete pro-inflammatory cytokines and chemokines, facilitating the development of immune response, which further reduce the replication and spread of avian influenza virus in the host (herold et al., ; peschke et al., ; seo et al., ) . the effect of macrophage depletion on avian influenza virus replication in chickens has not been studied. kim et al. reported that the depletion of macrophages using clodronate liposomes in pigs, results % mortality after infection with h n influenza virus while zero mortality in infected control pigs (kim et al., ) . similarly, tate et al. have shown that the depletion of macrophages in a mouse model of influenza virus infection leads to severe viral pneumonia in h n influenza virus infected mice (tate et al., ) . we hypothesized that lpaiv infection will increase the staining intensity of kul + cells, which include macrophages, monocytes and interdigitating cells (mast et al., ) in respiratory and gastrointestinal tracts. we also hypothesized that the decrease of the staining intensity of kul marker + cells following intra-abdominal administration of clodronate liposomes will augment replication of low pathogenic avian influenza virus (lpaiv) in respiratory and intestinal tracts of chickens. the veterinary science animal care committee (vsacc) and health science animal care committee (hsacc) have approved the use of specific pathogen free (spf) eggs, embryos, and chickens used in all our experimental procedures. the eggs were purchased from the canadian food inspection agency (cfia), ottawa, canada and incubated ( - % relative humidity and . - . °c temperature depending on the stage of the incubation) at health research innovation center (hric), university of calgary. a lpaiv, a/duck/czech/ (h n ), propagated in the embryonated chicken eggs, was used in the studies. the titer of h n was determined by plaque assay using madin-darby canine kidney (mdck) cells. clodronate liposomes (foundation clodronate liposomes, amsterdam, netherland) were used for decreasing the staining intensity of kul + cells in chickens as described earlier (kameka et al., a) and phosphate buffered saline (pbs) liposomes were used as a control. six day-old chickens (n = ) were infected with . × pfu of h n lpaiv per chicken intra-nasally with uninfected controls (n = ). at days post-infection, the chickens were euthanized and the trachea, lungs and duodenum were collected. the collected samples were preserved in optimum cutting temperature (oct, vwr international, mississauga on, canada) and immunofluorescent assay was performed to quantify the staining intensity of kul + cells as described earlier (abdul-cader et al., ). we delivered . ml ( mg/ml) of clodronate liposomes (n = ) or pbs liposomes (n = ) intra-abdominally to each day-old chicken. at day , subsets of chickens (clodronate liposome, n = and pbs liposome, n = treated groups) of both groups were infected with . × pfu of h n lpaiv per chicken intra-nasally with uninfected controls (n = per group). at days post-infection, the chickens were euthanized and trachea, lung and duodenum were collected. previously, it has been shown that lower gastrointestinal tract is also a site of lpaiv replication (slemons and swayne, ) as has been the duodenum (wang et al., ) . from the tissue samples collected, rna extraction was done to quantify the genome loads of h n lpaiv. a portion of samples were preserved in oct and immunofluorescent assay was performed to quantify the staining intensity of kul + cells as described earlier (abdul-cader et al., ). for the quantification of staining intensity of kul + cells in the tissues, five areas with highest dylight ® fluorescent signals and corresponding nuclear stained , -diamidino- -phenylindole (dapi) areas were captured under x magnification from each tissue section. then, these images were subjected to fluorescent intensity quantification using image-j software (national institute of health, bethesda, maryland, usa). the resultant fluorescent intensities for dylight ® positive signals were expressed relative to the total nuclear stained areas as a percentage. to identify group differences, we analyzed all the data (graphpad prism software , la jolla, ca, usa) using non-parametric test, mann-whitney u test due to low number of animals per group. before being analyzed each set of data, the outlier test was conducted using the grubbs' test (graphpad software inc., la jolla, ca, usa). the differences between groups were considered significant at p ≤ . . first, we questioned whether h n lpaiv infection contributes to the increase of staining intensity of kul + cells including macrophage populations in trachea, lungs and duodenum. when we infected day-old chickens with h n lpaiv intra-nasally, we found that, at days post-infection, h n lpaiv infection significantly increased the staining intensity of kul + cells in trachea (fig. a , p < . ), lungs (fig. b , p < . ) and duodenum (fig. c , p < . ) compared to the uninfected control chickens. in trachea, the staining intensity of kul + cells distributed mainly mucosal and submucosal areas with some distribution between cartilages and serosal surface. in the lungs and duodenum, the kul + cells mainly distributed throughout the lung parenchyma and mucosa respectively (fig. a-c) . increased recruitment of kul + cells following viral infections other than avian influenza virus in chickens are shown (abdul-careem et al., ; abdul-careem et al., ; fulton et al., ; kameka et al., b; nakamura et al., ) . similarly, it has been shown that avian influenza virus infection also recruit kul + cells in chickens (cornelissen et al., ; rebel et al., ) . the recruitment of kul + cells following viral infections may potentially be due to the availability of pathogen associated molecular patterns (pamps) of h n lpaiv during the infection, increasing the recruitment of these cells. it has been previously shown that toll-like receptor (tlr) of the innate immune system recognizes pamps of influenza virus and fig. . h n lpaiv infection increases the staining intensity of kul + cells in trachea, lungs and duodenum of chicken. at days of age, a group of chickens (n = ) were infected with h n lpaiv intra-nasally while another group was left as uninfected controls (n = ). at days post-infection, trachea, lung and duodenum were collected for immunofluorescent assay to quantify the staining intensity of kul + cells. representative images from trachea (a), lung (b) and duodenum (b) are shown. the mann-whitney u test was performed to identify group differences and the differences were considered significant at p < . . fig. . clodronate liposomes decrease the staining intensity of kul + cells in trachea and duodenum of chicken. at days of age, the chickens were treated with clodronate liposomes (n = ) or pbs liposomes (n = ) intra-abdominally. at days post-treatment, trachea, lung and duodenum were collected. the immunofluorescent assay was performed for the quantification of the staining intensity of kul + cells. representative images obtained following the immunofluorescent assay for the quantification of the staining intensity of kul + cells in trachea (a), lung (b) and duodenum (c) are shown along with quantitative data. the mann-whitney u test was performed to identify group differences and the differences were considered significant at p < . . activate mouse mononuclear cells (lund et al., ) . furthermore, lee et al. have shown that activation of tlr induces differentiation of myeloid-derived suppressor cells into one of the kul + cells, macrophages (lee et al., ) . we observed that intra-abdominally delivered clodronate liposomes in day-old chickens significantly reduced the staining intensity of kul + cell populations in trachea and duodenum but not in lung at days post-treatment when compared to the controls that received pbs liposomes ( fig. a and c, p < . ; fig. b , p > . ). in previous studies, it has been found that treatment of clodronate liposomes significantly decrease staining intensity of cv -chnl- . antibody specific cells populations in chickens (jeurissen et al., ) . cv -chnl- . antibody specific cells include macrophages, monocytes and interdigitating cells which are essentially similar to kul + cells. in agreement with this finding, we observed decreased staining intensity of kul + cells in this study in trachea and duodenum following intra-abdominal clodronate liposome treatment. since we observed that the clodronate liposomes significantly decreased the staining intensity of kul + cells in trachea and duodenum, then we investigated to see whether this reduction in the staining intensity of kul + cells influences the h n lpaiv infection. we found that the clodronate liposome-mediated reduction of the staining intensity of kul + cells was associated with no change of h n lpaiv infection at days post-infection ( days post-treatment) in trachea (fig. a , p > . ), lung (fig. b , p > . ) and duodenum (fig. c , p > . ). kul + cells including macrophages are important immune cells that can be beneficial in innate antiviral response against h n lpaiv infection via several mechanisms. first, it is known that macrophages can lead to the production of a number of antiviral cytokines including ifnγ (barjesteh et al., ; dimier et al., ) , il- β (lavric et al., ) and release of reactive nitrogen species such as no (setta et al., ) . previously, it has been shown that no originated from avian macrophages are inhibiting infectious laryngotracheitis virus (haddadi et al., ) , reo virus (pertile et al., ) and marek's disease virus (xing and schat, ) infections in vitro. we have previously observed that inhibition of h n lpaiv replication is attributable to no produced from kul + cells . second, tracheal fig. . reduction of the staining intensity of kul + cells with clodronate liposomes did not change h n infection in trachea, lungs and duodenum. at days of age, the chickens were treated with clodronate liposomes (n = ) or pbs liposomes (n = ) intra-abdominally. after h, all chickens were infected with h n lpaiv intra-nasally. at days post-infection, trachea (a), lung (b) and duodenum (c) were collected. real-time pcr assay was performed for h n lpaiv genome quantification. the mann-whitney u test was performed to identify group differences and the differences were considered significant at p < . . macrophages can play a role in clearing h n lpaiv infected cells as has been shown in mouse model of influenza infection (hashimoto et al., ) . however, the lack of significant differences in h n lpaiv genome loads between clodronate liposome treated and controls could be two-fold. first, immune cell functions are redundant (hamada et al., ) and it is possible that even the kul + cell staining was decreased following clodronate liposome treatment, the functions of these cells may have been compensated by other intact immune cells in chicken. second, it is possible that the number of animals used per group (n = ) may not have provided adequate power in our experiments to show differences in h n lpaiv genome loads in all the examined tissues in chickens although number of animals used are adequate for differentiating of kul + cells in trachea and duodenum. although our investigations led to generation of significant new knowledge, there were some limitations. first, the monoclonal antibody used for the quantification of macrophages detects kul mannose receptor and these receptors are also expressed by monocytes and dendritic cells of chicken (mast et al., ) as such it is possible that the quantified macrophages may also include monocytes and dendritic cells. however, only macrophages and their precursor, blood monocyte will uptake the liposomes; since it has been shown that liposomes of the size > nm will not be internalized by non-phagocytic cells and other immune cells such as granulocytes (claassen et al., ) . dendritic cells are also unaffected upon administration of clodronate encapsulated-liposomes (van rooijen and hendrikx, ). however, we believe that kul cell staining alone is not adequate for characterizing cells reduced by clodronate liposomes and further immune markers should have been included in the experiments. second, we did not observe that intra-abdominal clodronate liposome treatment completely decrease the staining intensity of kul + cells. third, in our experiments we used a lpaiv strain h n which does not lead to clinical signs due to the fact that it establishes low grade infection. consequently, we resorted to real-time pcr technique to quantify this low grade h n lpaiv infection in the observed tissues, which is more sensitive than the immunofluorescent assay (landry et al., ; perosa et al., ) , although immunofluorescent assay would have given more meaningful data relevant to h n lpaiv infection in the observed respiratory and gastrointestinal tissues. in conclusion, we found that h n lpaiv infection contributes to increase of the staining intensity of kul + cell populations in trachea, lungs and duodenum of chickens. although, we observed that intra-abdominal treatment of clodronate liposomes reduced the staining intensity of kul + cells in trachea and duodenum of chickens, the decrease of the staining intensity of kul + cells did not alter the h n lpaiv genome load in examined tissues. further investigations are warranted to investigate other roles of kul + cells in the host responses elicited against h n lpaiv infection. the authors declare that there is no conflict of interest. toll-like receptor (tlr) signalling-mediated antiviral response against avian influenza virus infection correlates with macrophage recruitment and nitric oxide production host responses in the bursa of fabricius of chickens infected with virulent marek's disease virus induction of innate host responses in the lungs of chickens following infection with a very virulent strain of marek's disease virus infectious bronchitis corona virus establishes productive infection in avian macrophages interfering with selected antimicrobial functions macrophage cytokines: involvement in immunity and infectious diseases tlr ligands induce antiviral responses in chicken macrophages infection of macrophages by a lymphotropic herpesvirus: a new tropism for marek's disease virus effects of alveolar macrophage depletion on liposomal vaccine protection against respiratory syncytial virus (rsv) in vitro infection studies with infectious laryngotracheitis virus marek's disease virus infection of phagocytes: a de novo in vitro infection model a new method for removal of mononuclear phagocytes from heterogeneous cell populations in vitro, using the liposomemediated macrophage 'suicide' technique differences in highly pathogenic avian influenza viral pathogenesis and associated early inflammatory response in chickens and ducks inhibition of eimeria tenella development in vitro mediated by chicken macrophages and fibroblasts treated with chicken cell supernatants with ifn-gamma activity cellular response of the respiratory tract of chickens to infection with massachusetts and australian t infectious bronchitis viruses induction of toll-like receptor signaling in avian macrophages inhibits infectious laryngotracheitis virus replication in a nitric oxide dependent way multiple redundant effector mechanisms of cd + t cells protect against influenza infection evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules inadequate anti-polysaccharide antibody responses in the chicken clodronate treatment significantly depletes macrophages in chickens induction of innate immune response following infectious bronchitis corona virus infection in the respiratory tract of chickens alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs real-time pcr compared to binax now and cytospin-immunofluorescence for detection of influenza in hospitalized patients mycoplasma synoviae lipoprotein mspb, the n-terminal part of vlha haemagglutinin, induces secretion of nitric oxide, il- and il- beta in chicken macrophages resiquimod, a tlr / agonist, promotes differentiation of myeloid-derived suppressor cells into macrophages and dendritic cells depletion of alveolar macrophages exerts protective effects in pulmonary tuberculosis in mice recognition of single-stranded rna viruses by toll-like receptor some recent advances on the study and understanding of the functional design of the avian lung: morphological and morphometric perspectives characterisation of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody kul proliferation of lung macrophages in acute fatal viral infections in chickens comparison of the direct fluorescence assay and real-time polymerase chain reaction for the detection of influenza virus a and b in immunocompromised patients an antiviral effect of nitric oxide: inhibition of reovirus replication role of macrophage cytokines in influenza a virus infections highly pathogenic or low pathogenic avian influenza virus subtype h n infection in chicken lungs: small differences in general acute responses intravenous treatment with liposome-encapsulated dichloromethylene bisphosphonate (cl mbp) suppresses nitric oxide production and reduces genetic resistance to marek's disease roles of macrophages in measles virus infection of genetically modified mice no apoptotic deaths and different levels of inductions of inflammatory cytokines in alveolar macrophages infected with influenza viruses immune dynamics following infection of avian macrophages and epithelial cells with typhoidal and non-typhoidal salmonella enterica serovars; bacterial invasion and persistence, nitric oxide and oxygen production, differential host gene expression, nf-kappab signalling and cell cytotoxicity replication of a waterfowl-origin influenza virus in the kidney and intestine of chickens restricted growth of avirulent avian reovirus strain in macrophage derived hd cells critical role of airway macrophages in modulating disease severity during influenza virus infection of mice liposomes for specific depletion of macrophages from organs and tissues apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine cytokine expression in three chicken host systems infected with h n influenza viruses with different pathogenicities inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of marek's disease virus we acknowledge funding from margaret gunn endowment for animal research (grant #, , university of calgary). we acknowledge the routine animal management by the staff at prion-virology animal facility of the university of calgary. key: cord- - lyw gh authors: guzman, efrain; montoya, maria title: contributions of farm animals to immunology date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: lyw gh by their very nature, great advances in immunology are usually underpinned by experiments carried out in animal models and inbred lines of mice. also, their corresponding knock-out or knock-in derivatives have been the most commonly used animal systems in immunological studies. with much credit to their usefulness, laboratory mice will never provide all the answers to fully understand immunological processes. large animal models offer unique biological and experimental advantages that have been and continue to be of great value to the understanding of biological and immunological processes. from the identification of b cells to the realization that γδ t cells can function as professional antigen presenting cells, farm animals have contributed significantly to a better understanding of immunity. great advances in immunology are usually supported by experiments carried out in animal models and by far, inbred lines of mice and their corresponding knock-out or knock-in derivatives, are the most commonly used animal systems in immunological studies. though with much credit to their usefulness, laboratory mice will never provide all the answers to fully understand immunological processes. also, some answers provided in mouse models are not applicable to other species of animals or humans. large animal models offer unique biological and experimental advantages that have been and continue to be of great value to the understanding of biological and immunological processes. the humble cow, the underestimated pig and the unassuming chicken have greatly influenced our current understanding of human immunology. for most immunologists dedicated to fundamental and applied research, it is easy to forget that b cells were first identified in chickens and vaccination first occurred because of a cow. although there are far too many important events to discuss in this paper, we have chosen to highlight a few of the most important contributions of farm animals to the current understanding of immunology ( table ) . edward jenner published in a booklet entitled "an inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the western counties of england, particularly gloucestershire and known by the name of cow pox" ( , ) and although strictly speaking jenner did not discover vaccination, he was the first person to use scientific rigor to prove protection from disease through targeted intervention. the english dairy farmer benjamin jesty ( - ) was the first person known to vaccinate against smallpox ( ) protecting his family against the virus even after numerous exposures ( ) . however, the idea and indeed the term vaccination, only came into the light spot years later thanks to louis pasteur. this time the chicken takes a privileged position and the story was beautifully explained by pasteur himself ( , ) and has been romanticized in paul de kruif 's book "microbe hunters" ( ) . in pasteur inoculated chickens with "stale" cultures of pasteurella multocida. the chickens became sick but recovered so he decided to re-inoculate them with a fresh culture. the chickens that had received the "stale" culture recovered whereas chickens that had not been pre-exposed to the stale cultures died. pasteur recognized the similarities between his studies in chickens and what jenner had published with smallpox. he coined the term "vaccine" ( , , ) in honor of jenner. by the early s, william smith greenfield in the uk ( , ) and pasteur working with henri thullier, charles chamberland and pierre paul Émile roux in france ( , ) had begun developing and testing vaccines against anthrax in sheep and cattle. a decade later, the german scientists friedrich loffler and paul frosch identified the first ever filterable infectious agent in mammals: foot and mouth disease virus (fmdv) and developed a fully protective heat-inactivated vaccine against it ( , ) ; however an effective long-lasting and broadly protective vaccine against fmdv remains elusive. pigs also played an important role in early vaccinology studies. by the late s swine plague or hog cholera (later discovered to be caused by a virus now called classical swine fever virus, csfv ( ) was killing hundreds of thousands of pigs across the word and was particularly of concern to the us pig producing industry, causing an impressive us$ million a year in losses in ( ) and us$ million by . once again, pasteur and thullier developed a vaccine to what is now thought to be the first ever vaccine against a viral infectious disease ( ) and the first mass-vaccination campaign in history. in addition, it is rarely recognized that csfv was the first animal virus ever to be cultured in vitro ( ) and the techniques developed by carl tenbroek continue to be used today. horses have also contributed to the understanding of fundamental immunological mechanisms. in a series of experiments, emile roux working with alexandre yersin and followed by emil von behring and shibasaburo kitasato immunized horses to produce an "antidote" or immune sera against the diphtheria toxin that was eventually used to treat humans, an important step in understanding antibodies and humoral immunity ( ) . behring won the nobel prize for medicine in for this work. another milestone in vaccine development was the generation in the 's of vaccines to control marek's disease (md), a naturally occurring neoplastic disease in chickens caused by an oncogenic herpesvirus ( ) . md vaccines are the first examples of the use of vaccination to protect against cancer ( , ) . with the discovery of molecular biology techniques in the 's and 's, the race was on to develop recombinant vaccines against numerous infectious diseases. the first report of a biosynthesized polypeptide vaccine was published in ( ) . the structural protein vp of fmdv was cloned ( ). swine plague. science , and expressed in e. coli and the purified protein used to vaccinate six cattle and two swine, which developed neutralizing antibodies and were protected against challenge with fmdv ( ) . and new technologies have only helped to highlight the importance of farm animals in vaccine development: using a computational approach to assess protein-protein stability, kotecha and colleagues ( ) used molecular dynamic ranking to predict fmdv capsid stabilities and produced stabilized fmdv capsids based on these predictions, assessed their stability using x-ray crystallography and demonstrated their improved immunogenicity in vivo by vaccinating cattle. this demonstrates the potential value of structure-based design of vaccines to develop stabilized vaccine antigens for animals and humans alike. although the innate immune system of animals is largely conserved, there are significant variations in the pattern-recognition-receptor (prr) structures of various species ( ) . it has been suggested that laboratory mice have not been subjected to the selective pressures that other animals have and so innate immune studies carried out in laboratory animals do not accurately inform human biology ( ) . it has been demonstrated that human and farm animal prr responses to their ligands ( , ) are more similar to each other than human-mouse prr responses ( ) ( ) ( ) . because prr recognition is associated with adaptive immunity, a better understanding of these molecules in farm animals is likely to better inform on their effect in these animals as well as humans. a major contribution of the chicken to fundamental innate immunity was the description in of the first interferon. chicken embryos were exposed to influenza virus by alick issacs and jean lindenmann ( ) and they identified an immune soluble element responsible for regulating virus infection. this discovery was certainly one of the scientific landmarks in cell biology in the twentieth century and one which opened the doors of what we now know as innate immunity. perhaps the most recognizable contribution of the chicken to science, and immunology in particular, was in the definition of the two elements of adaptive immunity: the b-dependent and the t-dependent immunity. the avian bursa of fabricius, named after hieronymus fabricius of aquapendente ( ) is a sac-like structure located in the cloacal passage of the bird and its function remained elusive until well into the twentieth century. bruce glick and timothy chang working at the poultry science department at ohio state university ( , ) described how following the surgical removal of the bursa, chickens injected with salmonella typhimurium "o" antigen failed to develop bacteria-specific antibodies. glick and chang wrote a paper entitled: "the role of the bursa of fabricius in antibody production" and was rejected by leading scientific journals ( ) and eventually published in poultry science ( ) . several years later, the bone marrow in mammals was shown to be the equivalent of the bursa in birds ( ) , and so the term "b-lymphocyte" originated from "bursa-derived lymphocyte". several years later, cooper et al. published a fundamental paper on the demarcation of the thymic and bursal and systems in birds and proposed the existence of equivalent systems in mammals ( ) . the cannulation of lymphatic vessels was developed in the early twentieth century in rats to study the lymphatic system but due to the complexity of the surgical procedure, sheep and then cattle were used extensively in the s and s in lymphatic cannulation studies ( ) . in a series of adoptive transfers of lymph-migrating cells and fluid, hall and colleagues first identified in sheep that antibody-secreting cells (acs) encounter antigens and are activated in the lymph nodes ( ), then migrate via the efferent lymphatics to the circulatory system, and that the immune response depends on an intact lymphatic system. cattle have also contributed to fundamental b cell immunology and the generation of a highly diverse antibody repertoire. most vertebrates encode a large number of variable (v), diversity (d), and joining (j) gene segments and antibody diversity is achieved by recombination of these segments. in contrast, cattle only express a limited number of v genes and so it is thought that antibody diversity is achieved recombination events and endogenous mutation mechanisms in the cdr region ( ) . another unusual feature of bovine antibodies is their exceptionally long cdr regions ( ) . these long cdr and unusual mutation mechanisms result in "microfolds" within the cdr region that allow bovine antibodies to bind antigens that would normally be inaccessible ( ) . a recent report demonstrated that cows can be immunized with a single hiv env trimer and this results in potent hivspecific nabs which are dependent on the length of the cdr loops of bovine ig ( ) . it has been suggested that this could be an efficient way of producing super-antibodies against other human pathogens. transchromosomal cows have been engineered to produce large amounts of full human igg molecules with pathogen specificity: mers-cov ( ) , hanta virus ( ), veev ( ) , and ebola virus ( ) . this technology has the potential to generate prophylactic antibodies against emerging viral diseases. on the other hand, chickens have serum igm and iga both of which are homologs of their mammalian counterparts; in addition, they express igy, not found in mammals but thought to be an evolutionary ancestor for mammalian igg and ige. chickens however do not have either ige nor igd but instead use a distinct process for generating antibody diversity that is distinctly different to mammals ( ) . engineers frequently look to nature for inspiration. antibody engineers are no exception, modeling new therapeutics on molecules found in animals such as camels and cows. indeed, % of bovine antibodies have unusually long heavy-chain cdr s as part of their antigen-recognition sites. stanfield et al. ( ) have solved crystal structures of three new bovine fab fragments and analyzed the five known structures to show that their ultra-long cdr h s all adopt similar architectures composed of a knob domain containing a small conserved β-sheet connected by diverse disulfide-bonded loops that is separated from the antibody surface by a long conserved stalk. they propose that varying the length of the stalk and the positions and number of disulphide links in the knob may help drive antibody diversity. these structural insights could be leveraged to tailor antibody-based therapeutics. in contrast to all other mammals, camelids (dromedaries, camels, llamas, etc) also have an unique antibody type similar igg but with identical heavy chains lacking the ch domain and which do not pair with their corresponding light chains. these "heavy-chain antibodies" (hcabs) display antigenspecific variable domains or "vhh" which are structurally and functionally similar to an igg fv but have only three cdr loops defining the antigen biding sites. camel vhh domains, also called "nanobodies, " have been of great interest because of their stability and small size and strong affinity to their corresponding antigens. in fact, several camel vhh domain antibodies are in early preclinical development in oncology, infectious, inflammatory, and neurodegenerative diseases ( ), the most recent example being the generation of broadly neutralizing antibodies to influenza in llamas ( ) . cytotoxic and helper t cells are generally considered to be phenotypically different due to the mutually exclusive expression of the co-receptors cd αβ and cd and differences in mhcrestriction (class i vs. class ii). however, between (in normal individuals) and % (in certain pathologies) of human peripheral blood lymphocytes have been shown to be cd /cd double positive (dp) t cells ( ) . thymic and extra-thymic development of t cells has been studied mainly in mice and because the expression of cd and cd in mouse t cells for the most part mutually exclusive, cd /cd dp lymphocytes have generally been ignored. nevertheless, the presence of cd /cd dp t cells in many animals makes it impossible to ignore these cells. studies in pigs have shown that cd /cd dp are a distinct subset of activated and/or memory t helper cells ( ) and in humans the increase in circulating cd /cd dp t cell frequency has been identified in autoimmune and chronic inflammatory diseases ( ) ( ) ( ) ( ) ( ) suggesting the importance of this particular t cell population in human health. most circulating t cells in humans and mice are conventional t cells expressing the αβ t cell receptor (tcr) and either cd or cd . unlike mice, other species like cattle, pigs and chickens possess a substantial proportion of t cells expressing the γδ tcr cells in the circulation suggesting that circulating γδ tcr t cells have a more important role immunity than previously thought ( ) . the fact that the phenotype and frequency of circulating and tissue resident t cells is so vastly inconsistent in different species suggests that immune responses to (vaccine) antigens are also distinct. it is assumed that that all animal species have a similar immune response to a particular antigen, but this is a statement to be reviewed in light of each host particularities. in addition, the th /th polarization of t cells observed in response to particular antigens is a phenomenon of certain strains of laboratory mice and not of outbred mammals including farm animals and humans ( , ) . in fact, it has been shown that cytokine profiles defining th /th responses to antigens in cattle are more similar to human responses than those observed in mice ( ). dendritic cells (dc) as such, and their role in immunity were first described in the s and in ralph steinman published a series of papers describing that a cellular receptor called "dec- " (now cd ) was expressed on mouse dc, was involved in antigen processing ( , ) and was detected by the monoclonal antibody nldc- . in fact, it was years earlier in , that chris howard, a bovine immunologist working at the then called "institute for animal heath" in the uk published a series of papers identifying an important and until then uncharacterized marker expressed on pseudoafferent lymph veiled cells (also called aldc) detected by the monoclonal antibody wc (now cc ) ( - ). although steinman's identification of mouse cd helped characterize the binding of cc to bovine cd ( ), the importance of cd in identifying dc was first evident in cattle. as mentioned above, steinman's seminal work in characterizing dc using the mouse system has been one of the most important developments in cellular immunology of the twentieth century, and one which lead to his nobel laureate. however, the idea that a component of the immune system was involved in antigen processing and presentation had been proposed many times before. as mentioned above, cannulation of the lymphatic vessels is more practical in large than small animals, and this technique has been used to investigate dc biology. afferent or peripheral lymph dc were first described in sheep in ( ) as "very phagocytic dendritic macrophages that are involved in long term immunological reactions" that are very potent antigen presenting non-lymphoid cells ( ) and that their phagocytic and antigen presentation capacities differed from "classical" peritoneal macrophages ( ) , therefore indicating that dc and macrophages were different cell types ( ) several years before steinman's observations ( ) . in addition, lymphatic cannulation of sheep has revealed important ontologic, phenotypic and functional characteristics of dc subsets that are relevant in other mammals, particularly humans ( , ) . similitudes and differences between swine and human dc/macrophage populations have recently been described ( ) . in one striking example and in contrast to studies performed in mice, swine and human cdc , which are associated with th responses, both express fcεriα and are localized in or next to the tracheal and bronchial epithelia. these observations have been proposed to imply that swine and humans have similar allergen responses as opposed to mice. this theory is supported by the fact that localization of cdc helps them access antigens such as airborne allergens, and fcεriα expression on these cells might help proliferation observed in allergic responses. as mentioned before and unlike mice, horses and humans, most other animals have a large γδ t cell compartment. for example, up to % of all blood lymphocytes in young calves are t cell expressing the γδ t cell receptor (tcr). although the reasons for the enlarged t cell compartment in cattle, pigs and chickens is still unknown, their large numbers and ease of collection has resulted in great advances in γδ t cell biology knowledge not only for farm animals, but also for humans. for example, apcs were shown to influence γδ t cell proliferation ( , ) . cynthia baldwin's lab has defined antigen-specific bovine γδ t cell responses in various systems ( ) ( ) ( ) and adrian smith's lab has done similar observations in chickens ( , ) . it has also been shown that bovine γδ t are potent regulatory t cells ( ) , an observation that is also true for a subset of human γδ t cells ( , ) . these results in farm animals have and continue to enhance our understanding of human γδ t biology ( ) . perhaps the most important one was the realization that a subset of bovine γδ t cells expressed mhc class ii and co-stimulatory molecules on their surface, a characteristic normally attributed to macrophages, b cells and dc but not t cells ( ) . bovine γδ t cells were also shown to phagocyte antigens and of mhc iirestricted presentation to cd + t cells ( ) . this function of bovine γδ t cells was subsequently reported in pigs ( ) and much later in mouse ( ) and human ( ) ( ) ( ) γδ t cells. perhaps the best known contribution of any farm animal to scientific progress was the somatic cell nuclear transfer that gave origin to dolly, the sheep ( ) . although nuclear transfer itself is not a direct contribution to immunology, nuclear transfer technology has directly influenced many immunological concepts underpinned by technologies such as induced pluripotent stem (ips) cells and crispr-cas systems. clustered regularly interspaced short palindromic repeats (crispr) is a rna-guided endonuclease used both in vivo and in vitro. genetically modified animals becoming more common and their availability can be exploited in many applications such as comparative immunology, physiology and disease, to generate in vivo bioreactors to produce complex proteins, or to produce genetically modified organs for transplantation in humans ( ) . although the majority of pharmaceutical research is performed in laboratory mice models, it is clear that humans are not "large mice." by a large extent, studies in laboratory mice have been the victim of over interpretation; for example, by extrapolating successful pre-treatment in mice to therapeutic treatment in men. the weakness of the mouse model in pharmaceutical research was recently highlighted in a study showing that inflammatory responses in mouse models do not correlate with human inflammatory disease ( ) . an additional study showed a close similarity in expression profiles of immune-related genes between humans and pigs ( ) . cattle, pigs, and chickens, are useful, valid, and valuable models to study human infectious diseases and important clinical targets in their own right. both humans and cattle are the natural hosts for tuberculosis (being infected with the geneticallyrelated mycobacterium bovis and mycobacterium tuberculosis, respectively) and the bovine and human diseases share many similarities in terms of immunity and pathology ( ) , whereas the mouse model of tuberculosis does not provide a faithful representation of the disease in humans ( ) . similarly, bovine respiratory syncytial virus (brsv) is closely related to human (h) rsv and the pulmonary pathology, immune responses and epidemiology seen in young calves and children are very similar ( ) . swine have been shown to be a more faithful model for human influenza infection and immunity studies and the same strains of influenza infect both humans and pigs because the distribution of influenza virus receptors and physiopathology are similar in both species. the transfer of maternal-derived antibodies (mda) to new born pigs enables fancy vaccine study design to elucidate the role of mda in immunity ( , ) , vaccine efficacy and in enhancement of respiratory disease ( ) . gnotobiotic piglets have been used to study various human gastrointestinal pathogens. for example, human noroviruses are antigenically and genetically related to swine noroviruses and unlike mice, humans and pigs show genetic susceptibilities to noroviruses depending on their histoblood group antigen phenotypes and the virus strain. similarly, gnotobiotic pigs have been used in rotavirus research to study disease pathogenesis and identify virus-specific iga and asc as correlates for protection and vaccine efficacy in children ( ) . pigs have also been proposed to be better models than mice for many other infectious diseases including female genital infection with chlamydia trachomatis, helycobacter pylori, neisseria meningitides, and nipah virus among others because of the natural susceptibility of pigs to these pathogens ( ) . endogenous retroviruses were first discovered in pig kidney cell lines in ( ) and are now known to be present in most, if not all, mammalians. the presence and potential reactivation of endogenous retroviruses has very important consequences in both allo-and xeno-transplantation. immunotherapy is becoming more popular in clinical trials and vaccine efficacy studies. the success of immune cell therapies partially depends on the effective delivery of cells to target organs, a process that invariably involves the lymphatic system. dc migration in mice has not proven to be very informative, however, dc migration in pigs may be able to answer several question on dc migration that cannot be addressed otherwise. these studies demonstrate that using large animals to investigate immune cell trafficking will help improve immunotherapies in humans ( ) . in the french surgeon mathieu jaboulay ( - ) implanted a pig's kidney into one woman and a goat's liver into another thus starting the idea of xenotransplantation; unsurprisingly, both women died ( ) . the acceptance or rejection of a donor's organ or cells is fundamentally an immunological event. cellular rejection involves nk and t cells that recognize foreign antigens on the grafted tissue. using xenotransplantation models (pig-to-rat, pig-to-primate, and pigto-human), the main mechanism for organ and tissue rejection has been proposed to involve arteriosclerosis, or thickening of the arterial walls. this process if thought to be caused by activated and allo-reactive lymphocytes that migrate over time to the transplanted organ ( ) . arteriosclerosis is a major cause of chronic organ rejection ( ) . studies in laboratory mice have underpinned many concepts of immune tolerance and the generation of immune responses in the neonate. however, the peripheral immune system in mice remains unpopulated during pregnancy and it is only after birth that b and t cells begin to emmigrate to the periphery. in contrast, lymphoid cells circulate through the fetus in humans and large animals well-before birth and specialized lymphoid tissues are also well-developed and populated by the time of birth and are able to respond to a number of antigens ( , ) . certainly the immune system in neonate humans and large animals is not matured, but calves, lambs and piglets can be more useful than mice in understanding immune responses during pregnancy and in new borns and these studies can be used to better inform human developmental immunology. this advantage over mice has recently been used to develop extracorporeal support technologies using neonatal lambs with the ultimate objective to use these technologies in premature children ( ) . perhaps one of the most common uses of large animal models is in the development of vaccines with several advantages over mice. the serial collection of peripheral blood from animals such as pigs, cattle, chickens, and horses allows for immunokinetic studies to be possible in response to vaccination or infection at the level of the individual. these immunokinetic studies can be used to correlate immune responses generated with protection after challenge with the relevant pathogen. in vaccinology studies using mice, the typical approach would be to sacrifice groups of mice sequentially and harvest spleen and blood, so the immune response to vaccination at the individual level is not normally achieved. large animals also provide several advantages over mice when investigating mucosal immunity. when mice are vaccinated or inoculated intranasally, it is common for the inoculum to be digested because anesthetized mice can both swallow and inhale the material placed on their nose. in addition, the structure of the mucosal associated lymphoid tissue (malt) differs significantly in mice from that of large animals and humans; for example mice do not have tonsils but instead have undefined networks of malt, whereas cattle, pigs and sheep have well-defined tonsils ( ) ( ) ( ) . in the case of vaccine delivery through the skin, cattle, and pigs appear to be better suited than mice for these studies. skin thickness, structure of the epidermis and the presence and distribution of langerhan's cells are among many characteristics that humans and cattle and pigs have in common and which are practically relevant in transcutaneous immunizations ( ) . the process of selecting a relevant and appropriate animal model is a balanced and complicated exercise due to the diversity in vertebrate physiology, adaptive and innate immunity. studies in mice, for example, have shown the efficacy of vaccines against fmdv, however these efficacy studies have failed to be translated to the target species (cattle and pigs), presumably due to fundamental differences in the immune systems of model organisms and target species and the ability of the virus to mutate in these animals ( ) . it has recently been shown that because immunoglobulin subclass diversification occurred after speciation ( , ) a particular immunoglobulin subclass in one species bears no functional homology to one of the same name of another species ( ) . thus, our knowledge of the functions of igg in mice cannot be extrapolated to other mammals. characterizing generating reagents for each animal model hinders the development and usefulness of any of these models and therefore limiting the usefulness of cows, cattle or chickens as models for human immunology. mice and rats are and will probably continue to be the chosen model organisms over farm animals. mice can be readily mutated (knock in or knock out) to study immunological pathways; so far this has been proven to be very difficult-and expensive-in large animals. as mentioned above, the availability of reagents to study immune cells and processes in mice far out competes the availability of these reagents for large animals. pharmacokinetic and toxicology studies would be prohibitively expensive in pigs, horses or cattle, so small rodents and rabbits are the best organisms to use in these studies. in addition, studies in mice have been fundamental in the discovery of antibiotics, chemotherapy agents and more recently car-t cell therapies that can be directly applied to humans. genetic homogeneity, low cost, the availability of biologically-relevant mutants and reagents make the mouse the optimal animal model for many academic and industrial researchers. farm animals have historically contributed and continue to contribute to fundamental and applied immunology. the use of these animals in research is not difficult as long as the appropriate facilities and reagents are available. dedicated housing, cost, biosecurity, and genetic variability are some of the many disadvantages confronted when using farm animals in research. however, selecting an appropriate animal model should be more than just a matter of accessibility and common practice ( ) but should be based on the scientific question to be addressed and its relevance. not just a country doctor: edward jenner, scientist edward jenner and the eradication of smallpox new light in the dawn of vaccination on the germ theory on chicken cholera: study of the conditions of non-recidivation and of some other characteristics of this disease microbe hunters the life and works of louis pasteur lectures on some recent investigations into the pathology of infective and contagious diseases professor superintendent, the brown animal sanatory institution concerning the priority due to him for the production of the first vaccine against anthrax remarks on anthracic vaccination as a prophylactic of splenic fever summary report of the experiments conducted at pouilly-le-fort, near melun, on the anthrax vaccination, summarischer bericht uber die ergebnisse der untersuchungen der kommission zur erforschung der maul-und klauenseuche bei dem institut fur infektionskrankheiten in berlin. centralblatt fur bakteriologie berichte der kommission zur erforschung der maulund klauenseuche bei dem institut fur infektionskrankheiten in berlin. centralblatt fuxr bakteriologie the survival of the hog-cholera virus in laboratory animals, particularly the rat swine plague, or hog cholera correspondence of pasteur and thuillier concerning anthrax and swine fever vaccinations cultivation of the hog cholera virus remembering emil von behring: from tetanus treatment to antibody cooperation with phagocytes studies on the epidemiology of marek's disease herpesvirus in broiler flocks effect of vaccination with herpesvirus of turkeys (hvt) on horizontal spread of marek's disease herpesvirus protective efficacy of marek's disease vaccines cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design toll-like receptors in domestic animals variation matters: tlr structure and species-specific pathogen recognition human but not murine toll-like receptor discriminates between tripalmitoylated and tri-lauroylated peptides human toll-like receptor recognizes host-specific lps modifications identification of full length bovine tlr and functional characterization of lipopeptide recognition by bovine tlr / heterodimer virus interference. i the interferon a landmark contribution to poultry science-immunological function of the bursa of fabricius growth of the bursa of fabricius and its relationship to the adrenal gland in the white pekin duck, white leghorn, outbred new-hampshire, and inbred new-hampshire the bursa of fabricius and antibody production cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells restoration of gamma globulin production in agammaglobulinemic chickens surgical techniques for the collection of lymph from unanaesthetized sheep the ultrastructure and function of the cells in lymph following antigenic stimulation maintenance of antibody to pathogen epitopes generated by segmental gene conversion is highly dynamic during long-term persistent infection reshaping antibody diversity rapid elicitation of broadly neutralizing antibodies to hiv by immunization in cows human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo dna vaccine-derived human igg produced in transchromosomal bovines protect in lethal models of hantavirus pulmonary syndrome antibody preparations from human transchromosomic cows exhibit prophylactic and therapeutic efficacy against venezuelan equine encephalitis virus production of potent fully human polyclonal antibodies against ebola zaire virus in transchromosomal cattle structural and genetic diversity in antibody repertoires from diverse species conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin coexpression of t and t on peripheral blood t cells demonstrated by two-color fluorescence flow cytometry phenotypic maturation of porcine nk-and t-cell subsets evaluation of t cell subsets in myasthenia gravis using anti-t cell monoclonal antibodies circulating cd + cd + cd + t lymphocytes in multiple sclerosis circulating cd +cd + t lymphocytes in patients with kawasaki disease cd + cd + double positive (dp) t cells in health and disease peripheral cd cd double positive t cells with a distinct helper cytokine profile are increased in rheumatoid arthritis porcine gammadelta t cells: possible roles on the innate and adaptive immune responses following virus infection the relative magnitude of transgene-specific adaptive immune responses induced by human and chimpanzee adenovirus vectors differs between laboratory animals and a target species differences in immune responses against leishmania induced by infection and by immunization with killed parasite antigen: implications for vaccine discovery type and type responses in regulation of ig isotype expression in cattle the receptor dec- expressed by dendritic cells and thymic epithelial cells is involved in antigen processing dec- , a -kda protein abundant on mouse dendritic cells and thymic epithelium that is detected by the monoclonal antibody nldc- : purification, characterization, and n-terminal amino acid sequence summary of workshop findings for cattle (tables and ) leukocyte antigens of cattle and sheep. monoclonal antibodies submitted to the second workshop studies of monoclonal antibodies identifying two novel bovine lymphocyte antigen differentiation clusters: workshop clusters (wc) and phenotypic variation and functional differences within dendritic cells isolated from afferent lymph dec- expression on migrating dendritic cells in afferent lymph the cells of lymph and their role in immunological reactions. handbuch der allgemeinen pathologie the properties and functional activity of non-lymphoid cells from bovine afferent (peripheral) lymph some properties of dendritic macrophages from peripheral lymph identification of a novel cell type in peripheral lymphoid organs of mice. morphology, i., quantitation, tissue distribution plasmacytoid dendritic cells migrate in afferent skin lymph existence of cd alpha-like dendritic cells with a conserved functional specialization and a common molecular signature in distant mammalian species the respiratory dc/macrophage network at steady-state and upon influenza infection in the swine biomedical model monocytes control gamma/delta t-cell responses by a secreted product bovine gamma/delta t-cell proliferation is associated with self-derived molecules constitutively expressed in vivo on mononuclear phagocytes protective killed leptospira borgpetersenii vaccine induces potent th immunity comprising responses by cd and gammadelta t lymphocytes the role of bovine γδ t cells and their wc co-receptor in response to bacterial pathogens and promoting vaccine efficacy: a model for cattle and humans the bovine model for elucidating the role of γδ t cells in controlling infectious diseases of importance to cattle and humans an alphabeta t-cell-independent immunoprotective response towards gut coccidia is supported by gammadelta cells γδ t cells play a protective role during infection with nippostrongylus brasiliensis by promoting goblet cell function in the small intestine bovine γδ t cells are a major regulatory t cell subset antigen-specific regulation of ige antibodies by non-antigen-specific γδ t cells a new effect of il- on human γδ t cells: promoting regulatory vδ t cells via il- production and inhibiting function of vδ t cells bovine γδ t cells: cells with multiple functions and important roles in immunity gammadelta t cells present antigen to cd + alphabeta t cells a sub-population of circulating porcine gammadelta t cells can act as professional antigen presenting cells mouse gammadelta t cells are capable of expressing mhc class ii molecules, and of functioning as antigen-presenting cells professional antigen-presentation function by human gammadelta t cells cross-presenting human gammadelta t cells induce robust cd + alphabeta t cell responses prolonged antigen survival and cytosolic export in cross-presenting human gammadelta t cells sheep cloned by nuclear transfer from a cultured cell line crispr is knocking on barn door genomic responses in mouse models poorly mimic human inflammatory diseases structural and functional annotation of the porcine immunome tuberculosis immunity: opportunities from studies with cattle animal models of tuberculosis for vaccine development animal models of respiratory syncytial virus infection influence of antibody-mediated immune suppression on clinical, viral, and immune responses to swine influenza infection live attenuated influenza virus vaccine reduces virus shedding of newborn piglets in the presence of maternal antibody. influenza other respir viruses universal vaccines and vaccine platforms to protect against influenza viruses in humans and agriculture the gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses the pig: a model for human infectious diseases de madrid at. c-type virus particles in pig kidney cell lines in vivo tracking and immunological properties of pulsed porcine monocyte-derived dendritic cells xeno's paradox: why pig cells are better for tissue transplants than human cells xenoislet rejection following pig-to-rat, pig-to-primate, and pig-to-man transplantation expanding the role of peyer's patches in b-cell ontogeny induction of immune responses in newborn lambs following enteric immunization with a human adenovirus vaccine vector an extra-uterine system to physiologically support the extreme premature lamb of mice and not men: differences between mouse and human immunology revisiting the b-cell compartment in mouse and humans: more than one b-cell subset exists in the marginal zone and beyond the lymphoid system: a review of species differences transcutaneous immunization of domestic animals: opportunities and challenges laboratory animal models to study foot-and-mouth disease: a review with emphasis on natural and vaccine-induced immunity antibody repertoire development in fetal and neonatal piglets. xiii hybrid vh genes and the preimmune repertoire revisited genomic organization of the immunoglobulin light chain gene loci in xenopus tropicalis: evolutionary implications therapeutic administration of broadly neutralizing fi antibody reveals lack of interaction between human igg and pig fc receptors model organisms: there's more to life than rats and flies all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors were funded by the uk's bbsrc grants bbs/e/i/ and bbs/e/i/ . 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 © guzman and montoya. 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- - dk authors: gulla, krishna mohan; balaji, arvind; mukherjee, aparna; jat, kana ram; sankar, jhuma; lodha, rakesh; kabra, sushil k title: course of illness after viral infection in indian children with cystic fibrosis date: - - journal: j trop pediatr doi: . /tropej/fmy sha: doc_id: cord_uid: dk objective: to study the clinical impact of respiratory viral infection in children with cystic fibrosis (cf). design: retrospective cohort study. setting: tertiary care referral centre for cf in india. participants/patients: children with cf attending a pediatric chest clinic. methods: case records of the children with cf who had a pulmonary exacerbation with documented acute respiratory viral infection between october and december (group i) and an equal number of controls (group ii) with pulmonary exacerbation in absence of acute respiratory viral infection were reviewed. outcome measures: the two groups were compared for the following outcomes over a period of – months: bacterial colonization, antibiotics usage, pulmonary exacerbations, numbers of outpatient visits, hospitalization and oxygen therapy and spirometric parameters. results: in total, children [ each with viral infection (group i) and without viral infection (group ii)] of age – months were enrolled; baseline clinical status and pulmonary function tests were comparable. mean (sd) follow-up duration in those who had viral infection and who had no viral infection was . ( . ) and . ( . ) months, respectively. on follow-up, children with viral infection (group i) had adverse outcome in form of greater worsening of shwachman clinical scores, number of pulmonary exacerbations requiring antibiotic usage [ ( . %)] and [ . ( . %)], need for intravenous antibiotics . % vs. . %, hospitalization rates . % vs. . % and mortality . % vs. . %, respectively. conclusion: acute viral infection in children with cf affected course of illness on follow-up, including frequent and severe pulmonary exacerbations requiring hospitalization, intravenous antibiotics, decline in cf scores and increased mortality over next – months. respiratory failure is the major cause of morbidity and mortality in patients with cystic fibrosis (cf); bacterial infections are generally thought to be the major cause of clinical deterioration [ ] . however, literature suggests that respiratory viruses do play an important role in pulmonary exacerbations, disease progression and an increase in bacterial adherence to the cf airways [ ] . even with appropriate use of antibiotic therapy, chronic obstructive airway disease continues to develop in patients with cf and is the major cause of morbidity and mortality. in children with cf, approximately half of the pulmonary exacerbations are associated with viral infections and are associated with worsening of spirometric parameters over time, especially in young children [ ] [ ] [ ] [ ] [ ] . if respiratory virus infections do lead to secondary bacterial infection in cf, there may be implications for the management of cf. hence, we conducted a retrospective cohort study to compare the effects of viral infection on the clinical course of children with cf over a period of months. in this retrospective cohort study, data were collected from the case records of cf patients attending the pediatric chest clinic of an indian tertiary care centre. cf was diagnosed based on typical clinical characteristics and two elevated sweat chloride levels on different occasions. nasopharyngeal aspirates/ nasal swabs of children with cf who presented with pulmonary exacerbations during october to december had been tested for viral pathogen using real-time polymerase chain reaction, pcr (fast-track diagnostics, luxembourg) as a part of another study done at the same institute [b. arvind, unpublished dissertation]. this pcr test could detect rhino, human corona, influenza a and b, parainfluenza, adeno, respiratory syncytial, human metapneumo and echo viruses. children with cf were routinely followed up in the pediatric chest clinic every months or earlier if indicated. the clinical information is recorded using a predesigned performa. children with cf whose nasopharyngeal aspirates/nasal swabs were tested and found positive for viral infection during an exacerbation were included as group i. children with cf and pulmonary exacerbation, whose throat swabs tested negative for viral pathogens were enrolled as controls after matching for age and sex (group ii). the children had been followed up every months or earlier, if needed. details of the follow-up visits during the months after throat swab collection were collected in a predesigned performa, which included the following: age; gender; weight; height at time of throat swab collection and during follow-up; bacterial colonization; intravenous and oral antibiotics usage; episodes of outpatient visits; hospitalization; percentage of predicted fev (forced expiratory volume in s), fvc (forced vital capacity), mmef (maximum mid-expiratory flow) and fev /fvc for the age and height; and clinical cf score. clinical cf score designed by shwachman and kulczycki [ ] was used to assess the clinical status of the children in follow-up. assessment of pancreatic insufficiency was done with history of frequent, foul-smelling and oily stools. written informed consent had been taken from parent/guardian before collecting respiratory specimen for viral detection in the earlier study [b. arvind, unpublished dissertation]. ethical clearance had been obtained from the institutional ethics committee for the current study. as we collected data from case record forms of children with cf who are routinely followed up in the pediatric chest clinic of the institute, consent for this retrospective study was not obtained from parent/guardian. data were analysed using stata ver. (college station, tx, us) and significance of difference of various parameters between groups i and ii were examined using student's t-test and mann-whitney test, according to the distribution of the parameter. weight for age and height for age z-score were calculated using the anthroplus software (who, geneva). the visit where throat swab for viral detection was collected was considered as the baseline visit for this study. multivariate analysis was performed using performing logistic regression model. twenty-three children with cf were included in each group. the baseline characteristics of the study children were comparable in both the groups and are presented in table . the distribution of viruses detected in the respiratory specimen in the enrolled children is shown in intravenous antibiotic usage and episodes of hospitalizations were significantly more in children who had a viral infection as compared with the others. mortality was also significantly higher in children in whom virus had been detected ( ; . %) as compared with those in whom no virus could be detected in respiratory specimen ( ; . %), p ¼ . (table ). in total, children in group and children in group could perform spirometry and parameters were shown in table . of the seven children who died in group i, rhinovirus had been detected in five, adenovirus in one and coronavirus in one during the pulmonary exacerbation evaluated for viral infections. the course of illness of children infected with rhinovirus was compared with those who were infected with other viruses (table ). in group i, three patients were newly colonized with pseudomonas aeruginosa and one patient was colonized with beta-haemolytic streptococcus. whereas in group ii, three patients were colonized with pseudomonas and two patients were colonized with staphylococcus aureus after virus isolation. allergic bronchopulmonary aspergillosis (abpa) was present in three children in group i and five children in group ii. one patient was home oxygen-dependent this retrospective cohort study demonstrated that cf children who had suffered a viral respiratory infection had worse outcome in follow-up in the form of lower cf scores, higher use of intravenous antibiotics, higher rate of hospitalization and higher mortality as compared with matched controls who did not have viral respiratory infection. respiratory viruses associated with the respiratory exacerbations of cf include influenza a and b, respiratory syncytial virus (rsv), parainfluenza virus (piv) types - , rhinovirus, human metapneumovirus, coronavirus and adenovirus. viral infections predispose the respiratory epithelium to secondary bacterial infections by multiple mechanisms resulting in frequent pulmonary exacerbations [ , ] . rhinovirus has been reported as major viral pathogen in cf with detection rates reaching up to % from samples collected both during exacerbations and routine visits [ ] . in our cohort too, rhinovirus was the most commonly detected ( . %) virus. in a prospective multi-birth cohort study by armstrong et al. [ ] on viral infection in infants with cf during infancy, children of ( %) were hospitalized for respiratory distress and of them had viral infection, and they concluded that viral infections are important cause of hospitalization in children with cf. abman et al. [ ] followed infants with cf over a period of . months (range - months); ( %) infants had been hospitalized times for respiratory distress. among these children, rsv was detected in infants. reports from earlier research on cf children have documented that patients with virus-associated infections had higher use of antibiotics at the longterm follow-up, compared with those without virusassociated respiratory tract infections [ , ] . in our cohort, usage of oral antibiotic was similar in both groups. but the requirement of intravenous antibiotics was significantly higher during follow-up of cf patients with viral respiratory infection compared with those who did not have any virus detected in the respiratory specimen ( . % vs. . %, p ¼ . ). in our cf patients with viral infection, of ( %) children required hospitalization for pulmonary exacerbation during their follow-up period, whereas only patient of ( . %) from the other group required hospital admission. hospitalization was higher in cf children in whom rhinovirus was detected as compared with other virus. over few decades, it has been presumed that viruses may be the harbinger of infection with typical bacterial cf pathogens. a temporal link was proposed between viral infections and new pseudomonas infection [ ] [ ] [ ] . there are currently limited data available to determine the effects of viral infection on the cf lung microbiota. in vitro studies have shown that rsv and influenza virus increase the adherence of pseudomonas to human airway [ , ] . it was also shown that rsv promotes growth of pseudomonas biofilms through modulation of the immune response and pathogen iron metabolism [ ] , while rhinovirus promotes the free movement of pseudomonas from biofilms [ ] . in contrast to what was believed, a recent study on dynamic interaction between respiratory viruses and the lung microbiota in children recently reported that viral infection had no impact on bacterial diversity [ ] . in our cohort, three children in each group were found to have new pseudomonas infection in follow-up, which suggests that viral infection may not be the only factor involved in colonization of pseudomonas in airway epithelium of cf children. viral infections in cf patients are associated with decline in pulmonary function. the majority of studies had shown declining trend of fev and fvc in long-term follow-up in those cf patients who had virus-associated lower respiratory tract infection [ , , ] . some studies like that by ramsey et al. [ ] showed no effect on pulmonary function tests (vital capacity (vc) or residual volume (rv)/total lung capacity (tlc)) in the follow-up of children with cf after viral infection. a recent review on clinical impact of respiratory viruses in cf suggests that high burden of viral respiratory infection in children with cf is likely to be linked with progression of cf lung disease [ ] . in our cohort, both the groups had shown decrement of lung function tests over the follow-up period with more decrement in group , though statistically not significant. it suggests that lung disease progresses over the period of time in these patients irrespective of viral infection. disease progression in cf is assessed through clinical data, chest radiography and tomography and pulmonary function tests. shwachman-kulczycki (sk) [ ] score is the most commonly used score to follow the progression of disease activity in cf children. we reviewed clinical data on general activity, physical examination and nutrition in our cohort. at the baseline, both the groups were comparable in above said three domains. however, at the last visit, children in group i had poorer scores in the domains of general activity, physical examination and nutrition as compared with group ii suggesting that viral infection may play a role in worsening of clinical status in children with cf. nevertheless, the subjectivity in the score needs to be kept in mind. apart from morbidity of cf caused by pulmonary exacerbations, the annual number of exacerbations has a considerable impact on survival also. in our cohort, . % of mortality was constituted by group i who had viral infection. this can be attributed to the severe pulmonary exacerbations requiring hospitalization in this group of children. in total, of ( . %) children who required hospitalization in group i (cf with viral respiratory infection) died by the end of follow-up period. in a predictive model, cf patients who required hospitalization more than twice a year were . times more likely to die within years [ ] . in a similar survival prediction model comprising data from cf patients selected from cystic fibrosis foundation patient registry, each acute pulmonary exacerbation within the first year of follow-up increased the risk of death by . times in the following years [ ] . in a prospective cohort study conducted on adult cf patients, more than two exacerbations per year had a greater risk of lung transplant or death during the study than those with less than one exacerbation per year (adjusted hazard ratio, . ) [ ] . all of our patients had at least moderate pancreatic insufficiency as suggested by presence of history of frequent, foul-smelling and oily stools. we also note that our patients were malnourished at baseline in both the groups along with low fev , which might have contributed to the high mortality in part. supportive care apart from antibiotics during pulmonary exacerbation plays an important role in the care of cf patients. in our clinic, we do advise and demonstrate regarding chest physiotherapy, inhalation therapy such as hypertonic saline ( % saline) and daily enzyme replacement therapy. however, we do not routinely use dnase, potentiators or correctors because of cost constraints. we also want to highlight the fact that all of our patients were pancreatic insufficient because patient with milder diseases were not suspected to have cf because of lack of awareness and belief that cf does not occur in indian children. strengths of the study include the use of highly sensitive assay for detection of virus and long-term follow-up of months in indian children with cf. the major limitation of the study is its retrospective design. the determination of viral aetiology of pulmonary exacerbation was done only at enrolment; subsequent exacerbations were not investigated for viral etiology. c o n c l u s i o n following the viral respiratory infections in children with cf who were already nutritionally compromised, there were frequent and severe pulmonary exacerbations requiring hospitalization, intravenous antibiotics, decline in cf scores and increased mortality over next - months. the seattle virus watch. ii. objectives, study population and its observation, data processing and summary of illnesses community study of role of viral infections in exacerbations of asthma in - year old children detection of viral, chlamydia pneumoniae and mycoplasma pneumoniae infections in exacerbations of asthma in children the role of respiratory viruses in cystic fibrosis the art and science of the use of antibiotics in cystic fibrosis severe viral respiratory infections in infantswith cystic fibrosis effects of upper respiratory tract infections in patients with cystic fibrosis the effect of respiratory viral infections on patients with cystic fibrosis respiratory viral infection predisposing for bacterial disease: a concise review rsv mediates pseudomonas aeruginosa binding to cystic fibrosis and normal epithelial cells long-term study of one hundred five patients with cystic fibrosis; studies made over a five-to fourteen-year period viral and atypical bacterial infections in the outpatient pediatric cystic fibrosis clinic role of respiratory syncytial virus in early hospitalizations for respiratory distress of young infants with cystic fibrosis prevalence and impact of respiratory viral infections in young children with cystic fibrosis: prospective cohort study respiratory infections in cystic fibrosis patients caused by virus, chlamydia and mycoplasma-possible synergism with pseudomonas aeruginosa seasonal onset of initial colonisation and chronic infection with pseudomonas aeruginosa in patients with cystic fibrosis in denmark adherence of pseudomonas aeruginosa to tracheal cells injured by influenza infection or by endotracheal intubation respiratory syncytial virus infection enhances pseudomonas aeruginosa biofilm growth through dysregulation of nutritional immunity rhinovirus infection liberates planktonic bacteria from biofilm and increases chemokine responses in cystic fibrosis airway epithelial cells insights into the respiratory tract microbiota of patients with cystic fibrosis during early pseudomonas aeruginosa colonization infective respiratory exacerbations in young adults with cystic fibrosis: role of viruses and atypical microorganisms association of respiratory viral infections with pulmonary deterioration in patients with cystic fibrosis the diagnosis and management of respiratory viral infections in cystic fibrosis developing cystic fibrosis lung transplant referral criteria using predictors of -year mortality predictive -year survivorship model of cystic fibrosis exacerbation frequency and clinical outcomes in adult patients with cystic fibrosis key: cord- -ax i d f authors: karampourian, arezou; ghomian, zohreh; khorasani-zavareh, davoud title: exploring challenges of health system preparedness for communicable diseases in arbaeen mass gathering: a qualitative study date: - - journal: f res doi: . /f research. . sha: doc_id: cord_uid: ax i d f background: infectious diseases are common problems in mass gatherings, especially when there is a lack of health system preparedness. since iran is one of the most important countries on the walking path of arbaeen and has a vital role in providing health services to pilgrims, the experiences of health challenges by participants is of key importance. the aim of this study is to explore stakeholders’ experiences on the health system's preparedness and challenges, and to provide suggestions for preventing infectious diseases during the arbaeen mass gathering. methods: a qualitative research method was used with a conventional content analysis approach. the number of participants was , including executive managers and health policymakers who entered the study among participants. semi-structured interviews were used to generate the data. interviews were analyzed by means of content analysis after face-to-face interviews. results: data analysis resulted in the extraction of four main themes and sub-themes. health infrastructure defects in iraq has three sub-themes (health abandonment in iraq, the weaknesses in health culture and problems related to the health system); poor control of the causative factors of infectious diseases has three sub-themes (the underlying factors of the prevalence of contagious diseases, health system response to communicable diseases and ignoring the risks of the arbaeen ceremony); the low perception of risk in pilgrims has three sub-themes (lack of awareness in pilgrims, fatalism in pilgrims and unhygienic belief in pilgrims); and the ineffectiveness of health education has two sub-themes (training shortage in the targeted group and educational content problems) that shows participant’s experiences of the health system's challenges for coping with infectious diseases during the arbaeen ceremony. conclusion: pilgrim-based training, planning and controlling other challenges may change these threats to opportunities and improve the health of participants of the mass gathering of arbaeen in the region. according to the definition of the world health organization (who), any structured or spontaneous event leading to a certain number of people gathering in a particular site, for a specific aim in a determined period, putting pressure on the response resources and social programs, is called a mass gathering. mass gatherings are divided into different types based on their purpose. the expansion of interconnectivity between societies and increases in the number of national and international events in communities has led to an increase in the number of mass gatherings, which, despite the benefits like cultural exchange, have health challenges such as infectious disease transmission and, should therefore be considered by health planners . one of the health challenges of mass gatherings is the prevalence of infectious diseases and the outbreak of diseases, which, along with, complicated health needs of participants increases the health burden on the host country. the public health system can be under severe pressure, even with advanced equipment and the proper resources for prevention and control of infectious diseases - . various factors, such as the type and location of gathering, the number of participants and the lack of access to health facilities, can affect the incidence of infectious diseases in mass gatherings. planners must therefore pay attention to these factors in preparation , [ ] [ ] [ ] . since a mass gathering is a collection of many people together in one particular site, the possibility of infectious disease transmission due to the high population density always exists. studies on mass gatherings such as hajj, ashura day in karbala, and kumbh mela and sabarimala in india show the prevalence of infectious diseases in these ceremonies [ ] [ ] [ ] [ ] [ ] [ ] . the dates of religious ceremonies like hajj and arbaeen are decided using the lunar calendar (the islamic or hijri calendar) which is not only shorter than the gregorian calendar, meaning that these events occur days earlier each year and can synchronize with different seasons and season-associated diseases. accordingly, planners and policy makers of public health are faced with changing goals, requiring health system preparedness , . one of the world's largest religious gatherings is the arbaeen ceremony, which happens on the th day after the anniversary of imam hussein's martyrdom, the third shiite imam. in the ashura event, pilgrims walk to karbala, south of baghdad. based on the statistics of , the number of iranian pilgrims taking part in the arbaeen ceremony was , , . iran is a neighboring country of iraq and shares a common land border with it. pilgrims from other neighboring countries of iran, such as afghanistan and pakistan, cross iran to reach iraq and the arbaeen ceremony. on the basis of the mutual memorandum of cooperation between the two countries of iran and iraq, iran is committed to providing health services to other pilgrims in addition to iranian pilgrims . therefore, it is necessary to have a plan for preparedness in dealing with infectious diseases during arbaeen ceremony. if a mass gathering is not carefully managed, it can lead to the distribution of infectious diseases. in mass gatherings, infectious diseases are threats to global health security and even the political security of countries. therefore, planning, communication and public health supervision are important in these religious ceremonies , . mass gatherings are different from structured disasters so that in case of occurrence, many people will be affected . since the arbaeen ceremony is held with the presence of many pilgrims from many different countries and, like the hajj pilgrimage, is based on the lunar calendar, and it is held in iraq, there is the possibility of the occurrence and transmission of infectious diseases. it is therefore essential to be prepared to control and prevent these diseases. dealing with infectious diseases in arbaeen is considered a challenge for policy makers . according to arbaeen's social and cultural context, it seems essential to take a deeper look in this field. on the other hand, there is relatively little knowledge about the arbaeen ceremony; therefore, a qualitative method for clarifying the concept and challenges of health system preparedness in arbaeen ceremony is necessary. the aim of this study was to explore challenges of health system preparedness for communicable diseases in arbaeen ceremony. we collected data from june to march . since the study attempted to explore preparedness challenges of health systems, a qualitative research method with the approach of conventional content analysis was used . the health system's challenge in arbaeen is multidimensional, owing to the cultural difference between iran and iraq, the challenges faced by the health system during the arbaeen pilgrimage should be investigated in both countries. indeed, the cultural practices of the participants, especially those surrounding health, differs during the arbaeen ceremony. therefore, a qualitative research method, with the aim of describing phenomena, providing new knowledge, insight and a practical health guide, is the method used in this study , . the study was conducted using in-depth interviews, based on stakeholder's experiences in iraq-iran land terminals (mehran, shalamche, and chazaba) and also in health care posts in iraq. the interviews were conducted with health care providers and policy makers, as well as pilgrims in the arbaeen ceremony. participants were chosen among executive managers and policymakers of the ministry of health and medical education, medical training and treatment and other related organizations, including the red crescent organization, mobilizing the medical society, the hajj and pilgrimage organization, medical universities in the border cities and the social security organization. in total, participants, consisting of executive managers and health policymakers, were selected in this study through purposeful sampling with the aim of exploring challenges of health system preparedness for communicable diseases in the arbaeen ceremony. the existence of practical experience in planning or participate in arbaeen ceremony and the ability to communicate and willingness to participate were inclusion criteria in the research. we recruited participants either by phone call or by approaching them in person. "maximum variety sampling" was used to explore the experiences of the participants in those selected so that they were chosen from the ministry of health and medical education, medical training and treatment and other related organizations, including the red crescent organization, mobilizing the medical society, the hajj and pilgrimage organization, medical universities in the border cities and the social security organization, with different experiences of work, education and gender. inclusion criteria included the existence of practical experience in planning or participate in arbaeen ceremony, the ability to communicate and willingness to participate in the research (table ). the study was done through face-to-face interviews followed by telephone interviews for concept saturation. the data was collected using audio recorders with permission from the participants. ak conducted the interviews, ak and dkz transcribed the data. ak and dkz and zgh coded the data. dkz and zgh performed rigor. initially the first two interviews were conducted in a non-structured format, with the following interviews being semi-structured. open questions used to generate the data were developed by experienced and/or knowledgeable policy makers and health care providers. the individual's experiences and beliefs were used without considering their specialty . interviews were continued until data and concept saturation were reached . the interview duration was between and minutes, based on the tolerance, amount of information and desire of the participants. interviews were performed individually and based on participants' willingness in terms of time and site. firstly, interview questions began with the following general questions based on the participants' level and the main questions of the research: "how was your organization preparedness plan to deal with infectious diseases in arbaeen ceremony?"; "please express your experiences of related challenges in infectious diseases in arbaeen ceremony"; "what problems were in your preparedness plan?"; "what problems did you face in the vaccination program of the health team and pilgrims in arbaeen ceremony?"; and "what is your offer to pilgrims for a safe pilgrimage?" following this, exploratory questions were gradually used to clarify the concept and deepen the interview process: e.g. "please explain further what you mean?" and "why?" at first, the interviews were transcribed verbatim and then typed up using microsoft word office. the data were analyzed through a conventional content analysis method . first, the main researcher converted the interviews to written texts. the digital files were listened to several times and the texts were read repeatedly. next, the meaning units were determined based on the aim and the question of the research. meaning units were a collection of words and sentences that were related to each other in terms of content and were grouped together. meaning units reached the level of abstraction and conceptualization and were coded considering the research question. the key points and subjects were extracted as open codes. these codes have been put under the broader headings based on the present similarities and differences; in other words, the data were reduced in order to describe the phenomenon and gain a better understanding, and this abstraction process continued until concept extraction . data first emerged as meaning units, then condensed meaning units, codes, sub-themes and finally themes. the study was approved by ethics committee of shahid beheshti university of medical sciences on / / , no. ir.sbmu. retech.rec. . . the interviews were conducted and recorded with participants' consent. written or verbal consent was taken from participants to participate in the study. verbal consent was only taken for telephone interviews; this was due to the distance between interviewer and interviewee. anonymity, confidentiality and the right of resignation were informed to participants and considered during the study. the interview time was set according to participants' willingness. the researchers used the trustworthiness criteria recommended by guba and lincoln to establish rigor . all authors were engaged in the environment and field of research. in addition, the principle investigator always had suitable involvement with the participants for in-depth interviews. credibility was established by the prolonged engagement of researchers with participants. researcher triangulation was also used to verify the accuracy of the coding process. the research team also retained raw data, codes, and themes for control of the reliability. at the same time, sampling was carried out with maximum diversity in order to provide triangulation by means of credibility and confirmability. a detailed description of the method was used to establish transferability. engaging participants in the research increases the interaction between researchers and participants and then the credibility . the research supervisor monitored the data collection and data analysis process. it is necessary to mention that the research team participated in the arbaeen ceremony as pilgrim and conducted field notes. also, memberand peer-checking were used to ensure credibility. therefore, many interviews with related topics were sent to some external expert reviewers and participants (policymakers of the ministry of health and medical education, medical training and treatment and the red crescent organization) to be checked, and they were requested to assess the degree of relevance between the findings and raw data. moreover, transferability was established by sampling with maximum variation from various centers, including the ministry of health and medical education and other peer organizations including the red cross organization, the medical community mobilization, the hajj and pilgrimage organization, the country's medical universities in the border cities and the social security organization with different experiences of work, education and gender. a completed srqr checklist is available in supplementary file . themes and sub-themes the mean age of participants was and the mean time of work experience was years (table ) . overall, original codes were extracted and after integration using conventional concept analysis, four original themes consisting of sub-themes were identified. the theme of "health infrastructure defection in iraq" had three sub-themes: "health abandonment in iraq", "the weakness of health culture" and "problems related to health system". the theme of "poor control of factors effective in infectious diseases" had three sub-themes: the "underlying factors in prevalence of contagious diseases", "health system response to communicable diseases" and "ignoring the risks of the arbaeen ceremony". the theme of "low perception of risk in pilgrims" had three sub-themes: "lack of awareness in pilgrims", "fatalism in pilgrims" and "unhygienic belief in pilgrims". the theme of "ineffectiveness of health education" had two sub-themes: "training shortage in the targeted group" and "educational content problems" (table ) . according to the findings of this study, the main theme is pilgrim-based education. it seems that the biggest issue with the prevention of communicable diseases in the arbaeen ceremony is pilgrim-based education. educating pilgrims can directly help people to create or reform health infrastructures in iraq. education can also help to identify health risks and respond to them by identifying effective factors in combating infectious diseases. the training of health instructors and guidelines from people who are trusted and accepted by pilgrims, such as missionaries and religious leaders, can have a positive impact on their beliefs. the main point of training is to determine the targeted group both at the level of pilgrims and health directors and consider the training needs of each group in preparing training guidelines. health abandonment in iraq. most of the participants believed that the iraqi health system has been abandoned because foodstuff distribution is not under the supervision of a special organization and does not have a special trustee in the execution and monitoring of health rules or, if there is one, he is inconspicuous. the existence of a trustee or supervisor in the health system can prevent the delivery and distribution of unsafe food and reduce the prevalence of infectious diseases. unhealthy foodstuff preparation, production and distribution, and a lack of food evaluation and supervision system can lead to gastroenteric disease. based on the participants' experiences, the lack of a health system trustee means system weakness. the following quotation is an example of the above: "sometimes donations are prepared in an unhealthy manner and so lead to acute digestive problems… the health system is weak in iraq because health rules are not enforced and there is no supervision for these centers…" (executive manager, male, - years, - years job experience). the weakness of health culture. according to the participants' point of views, observation of unhealthy behaviors, such as neglect of the individual and public health standards, and the existence of cultural differences between iran and iraq, are considered by pilgrims to bring about an unsafe culture. policy makers and executives should be familiar with the kind of health culture in iraq so that they can develop a program to prevent contagious diseases. in iranian culture, the non-use of spoons and forks is considered as lack of health belief and neglect of health, whereas in iraq, eating food with the hands is part of the food culture. iran's health system can help train the iraqi people in sanitary practices alongside iranian pilgrims. the following quotation is an example of the above: "some food providers don't meet health … the culture of using spoons and forks for food serving is different in iran and iraq… one of the cultural works which we can do in iraq is health education…" (executive manager, male, - years, - years job experience). problems related to health system. most participants acknowledged that despite annual health care improvements in iraq, there are also some shortages in this field due to the lack of a long history of a health service system. health system weakness, incomplete health service implementation and insufficient supervision of environment health are indicative of the weakness of the health infrastructure; this has made it impossible to provide environmental health, sanitation and waste disposal. the following quotation is an example of the above: "health background in iraq was poor… there were no waste bins and waste sanitation there… failure to implement health services has led to failure in meeting health conditions … there was no health the probability of an outbreak of endemic and non-native diseases the ineffectiveness of the health system in screening the impossibility of requirement flu vaccination the inability of the system to provide services in an epidemic the underlying factors in prevalence of contagious diseases. most participants have identified underlying factors of infectious diseases as one of the challenges affecting the preparedness of the health system in dealing with infectious diseases. various factors, such as population density and diversity, not paying attention to the principles of personal and general health, for example through a lack of health facilities, weather conditions during the trip and changes in nutrition can cause the spread of infectious diseases. identifying these factors helps pilgrims and planners to prevent infectious diseases. the following quotation is an example of the above: "there were few toilets or no healthy facilities. somehow, pilgrims would have to sleep in the desert or in a limited space with a lot of people… congestion of pilgrims from different countries increases the risk of spreading infectious diseases…" (executive manager, male, - years, over year job experience). health system response to communicable diseases. on the basis of the view of participants, mass population movements from different countries and their gathering with different population diversity can transfer and spread endemic diseases and also emerging diseases such as plague and anthrax. there is also the possibility of bioterrorism events occurring during the arbaeen ceremony. the health system needs facilities such as equipped laboratories to diagnose and treat infectious diseases in a timely manner. the inaccessibility or lack of experimental equipment for disease identification leads to the failure of timely diagnosis of diseases, a lack of disease control and finally the incidence of epidemics. syndromic surveillance system can be used to diagnose infectious diseases. there is a need to correctly pass the treatment period of infectious diseases in order to prevent epidemics, although drug shortages can affect treatment completion and is one of the causes of epidemics. vaccination also helps to prevent infectious diseases, but the requirement of pilgrims to vaccinate is always up to the host country and since vaccination is not one of iraq's priorities, the ministry of health and medical education can only advise pilgrims to vaccinate. the following quotation is an example of the above: "there is the probability of spread of local and new-appeared diseases and even bioterrorism due to the gathering of pilgrims from different countries… some diseases can't be diagnosed due to lack of facilities but syndromic surveillance system can be used. …the large number of pilgrims and lack of medication have led to not having complete course of antibiotic treatment… the need for vaccination is one of the requirements of the host country". (policy makers, male, - years, - years job experience). the arbaeen pilgrimage has special features that distinguish it from other mass gatherings. participation of different peoples with a diverse range of socio-demographic statuses, cultures and nationalities makes this distinction. the arbaeen pilgrimage has some specific hazards like other trips that are sometimes neglected. the population of arbaeen pilgrims and its time of year are changeable. financial management of travel expenses at the ceremony is carried out by volunteers. considering these features is essential for the readiness program. in the opinion of the majority of participants, one of the challenges of the health system is the lack of attention to the risks of arbaeen ceremony and the lack of planning based on these features. the following quotation is an example of the above: "arbaeen is a new phenomenon that children and adults, men and women with different cultures and ethnicities take part in the ceremony … even with the knowledge of the dangers of the route, people attend arbaeen ceremony… arbaeen is a spontaneous and popular event and doesn't cost much … the population is moving and the time of the ceremony changes every year …" (executive manager, male, - years, - years job experience). lack of awareness in pilgrims. one of the challenges in the view of the participants, especially executives, is the pilgrims' low awareness of health hazards, such as not using personal hygiene products and non-compliance with health standards. unhealthy and dangerous practices, such as unsanitary food consumption, are not understood by pilgrims, and this low awareness and inadequate knowledge of the risks are the causes of infectious diseases in the pilgrims. respect for personal and public health, such as hand washing, providing food from health food centers and avoiding overeating, is effective in preventing digestive diseases. the following quotation is an example of the above: "some pilgrims do not wash their hands or do not use personal hygiene products …or they do not get foods from healthy centers … overeating and then digestive problems are one of the pilgrims' problems … the other problem is not being familiar with arabic language …" (executive manager, female, - years, less than years job experience). participants believed that one of the health challenges is belief in destiny and fatalism; so that most pilgrims who participate in the arbaeen walking ceremony, based on their belief in destiny, have a relatively low understanding of the dangers and diseases, and begin their trip merely confident in allah and without any plan for dealing with infectious diseases, such as vaccination, and continue the way using food and drinks that are often unhealthy. the following quotation is an example of the above: "it's enough to decide to travel and you do not need to have a special plan … if you think openly, you won't get sick, and nothing bad will happen…" (policy makers, male, - years, - year job experience). based on the participants' experiences, low perception of danger in pilgrims is sometimes seen as insanitary beliefs in preventing medication consumption and disregarding hygiene recommendations. believing in the use of medication during illness, preventing self-curing and trying to abide by hygiene recommendations prevents infectious diseases. the following quotation is an example of the above: "sometimes we see pilgrims using traditional treatments instead of antibiotics consumption… they don't pay attention to hygiene recommendations such as masking and not using suspicious food…" (executive manager, male, - years, - year job experience). training shortage in the targeted group. based on stakeholders' views, one of the present challenges is the quantitative and qualitative shortage in the stakeholder's training level. this means that providing a personal and public health plan should cover all stakeholders, including executives, policymakers and volunteers in the arbaeen ceremony, and be considered with respect to each participant. furthermore, the timing of training is also important and should be before the days of arbaeen ceremony in order to have a greater effect on the individuals' knowledge. indeed, training courses should be held separately for each of the groups, pilgrims, executive managers, and policymakers, and at a proper time before arbaeen. the following quotation is an example of the above: "training should not exactly be in the days of arbaeen. personal and public health training must be held several months before arbaeen… the training is not only for pilgrims, but also anyone involved in arbaeen ceremony. everyone should be trained from pilgrims to policymakers…" (policy maker, male, - years, - year job experience). another problem was the provision of educational content. most of the participants believed that training should be fitted with pilgrims' needs and respond to the problems of pilgrims. pilgrims should be divided into different groups based on the level of education, their problems and illnesses, and individual and general education should be planned accordingly. participants also believed that training in the area of personal, general and nutritional health should be provided more comprehensively. the following quotation is an example of the above: "we should divide pilgrims to diverse groups and send targeted training messages to each group, not the same training for everyone… the benefactors should be trained…pilgrims should have more training…" (policy maker, male, - years, - year job experience). the aim of this study was to explore the challenges of health system preparedness for infectious diseases in the arbaeen ceremony as the first qualitative study in iran. the most important findings of the study are the ineffectiveness of health training, the low perception of risk in pilgrims, poor control of the causative factors of infectious diseases, and deficient and defective health infrastructure in iraq. based on the views of the majority of participants, pilgrim-based training is the most effective factor in health system readiness in dealing with infectious diseases in the arbaeen ceremony. ineffectiveness of health training is one of the challenges of health system preparedness. one of the plans which should be considered to ensure arbaeen ceremony preparedness is health training. educational planning must be done before holding the arbaeen ceremony, with consideration of the training content and targeted groups. indeed, according to the needs of the targeted group, training must be given to pilgrims, executives and volunteer treatment teams, and the training content for pilgrims should be different to that of executives and policy makers. past conducted studies of religious gatherings such as the hajj in saudi arabia and ashura day in iraq, as well as other mass gatherings, such as the tamworth country music festival, australia, indicate the reality that a crowd of people from diverse nations and cultures is a source of infectious disease; disease transmission is one of the most important challenges of public health in these kinds of events, so using training strategies in relation to hand washing, masking and vaccination is an important factor in preventing the diseases and health improvement , , , - . since religious ceremonies are rooted in people's beliefs and have great popularity among people, people-centered education therefore has great potential to reduce the gap between knowledge and practice in pilgrims, empowering individuals and enhancing their ability to deal with health threats. this goal is achieved by identifying accurate and targeted needs, developing relevant content and through educational planning . in this study, like other studies on mass gatherings, a personal health training plan, such as the importance of hand washing, using healthy food and masking, should be included in the pilgrims' training plan, and public health training such as vaccination, environmental health, monitoring donations, cooking and distribution, controlling bioterrorism and establishing mobile toilets should be considered in executives and policymakers' preparedness plan. regarding the aim of holding the arbaeen ceremony, which is a popular religious-ideological ceremony, training can be performed in mosques before the ceremony by clergy. a low understanding of the risks of infectious diseases in pilgrims is one of the other challenges mentioned by the majority of participants, especially policymakers. a lack of risk understanding refers to the inability to identify and respond to dangerous situations. top documents such as sendai framework have identified that understanding risks is the first priority to decrease the incidence of disasters, and that it needs people-based and vast preventive approaches. besides this, the hyogo framework and sustainable development goals emphasize the role of training in increasing risk understanding and decreasing the vulnerability of individuals to hazards - . in this study, one of the health issues was fatalism and a low understanding of risk. a study concerning the beliefs and methods of infection control in hajj pilgrims residing in australia also showed that the majority of participants had low understanding of the occurrence of respiratory infections and the need for an influenza vaccine in hajj, and refused the vaccine, using trust in allah as an excuse and belief in destiny when dealing with the risk of disease . another problem is also the prevalence of self-treatment among pilgrims. in a study aimed at assessing the knowledge, attitude and the performance of australian pilgrims on using antibiotics in hajj showed that they did not have proper understanding of using these drugs and used medications arbitrarily, meaning that more training on proper use is needed . a lack of understanding of health instructions and disregard for public and personal health in any situation can endanger human health. it seems that the fatalism of pilgrims with regards to diseases increases their vulnerability if the understanding of danger is reduced. islamic instructions state that the person is obliged to preserve his health and life in any location and position, even in holy lands, and to avoid risks . given that arbaeen is a religious gathering, religious leaders have an important position in ceremony implementation and can affect the pilgrims' beliefs and understanding of risks during pilgrimage. the influence of religious leaders on the people's beliefs can provide the opportunity to promote health. we should consider that religious scholars must be trained first and then transfer health instructions and methods of infectious disease control to the people in cultural and religious gatherings such as mosques. another problem in this field is the poor control of the effective factors on infectious diseases. one of the most common causes of infectious disease is the epidemiology triangle, which has three components-pathogen, host and environment. the interaction of these agents causes infectious diseases, and considering these factors can help control infectious diseases . considering the three factors in arbaeen is very important. the 'host' factor includes different pilgrims with diverse cultures and nationalities. the 'environment' factor includes holding the ceremony in iraq, which, due to the many years of internal and external conflicts, has had little attention paid to its health infrastructure, and also the overall environmental factors effective in the occurrence of infectious diseases. different studies , , have shown that factors such as crowd size, equipment, climate, the event duration and location, the type of ceremony, and features and behavior of participants affect the occurrence of diseases in mass gatherings, and planners must consider it during preparations. to prevent the occurrence of contagious diseases and their consequences, comprehensive planning, rapid diagnosis and effective management is required , , . one of the factors that affects the occurrence of disease is the time of year that the arbaeen ceremony is held. arbaeen ceremony is held based on the lunar calendar and so its needs and challenges differ and are dependent on the season that the ceremony is held, so that if the ceremony is held in cold seasons, respiratory diseases are more common and if is held in hot seasons, the majority of infectious diseases are of the digestive system. additionally, population movement among different countries leads to the transfer of local diseases, so health considerations and cooperation between states are needed.. policymakers should consider three components-pathogen, host and environment-before the beginning of arbaeen mass gathering. it is also necessary that the health system is aware of all possible scenarios and the methods for dealing with them. the preparedness plan at the local level includes assessing the risk, resources capacity, equipment, surveillance system and an expert team for providing services to pilgrims. health infrastructure defects in iraq was another challenge to the health system from the participants' perspective. the term 'health infrastructures' refers to health facilities and their related factors. the infrastructure includes staff instructions, processes and the development of systematic approaches related to personnel resources and medical support plans . various studies indicate that readiness for structured mass gatherings depends on investing in health infrastructures and the size of gatherings, and strengthening infrastructures and post-event coordination of mass gatherings must be continued. the inappropriate location of gatherings, the weakness of facilities and the lack of infrastructure increase the vulnerability of communities , . the remoteness of health facilities and a lack of needed road infrastructure can make medical services and emergency assistance ineffective. limitation of infrastructure and medical care system, increase the incidence of injuries , . arbaeen is held in a country that has long been involved with interior and exterior wars; therefore, it seems that due to economic difficulties, it does not have the capacity to support the necessary health infrastructure required by pilgrims. although according to the participants, the health system in iraq seems somewhat weak, given that arbaeen ceremony is of particular popularity among shia muslims and is held annually, therefore, some of the activities serving pilgrims and its management during the arbaeen ceremony is voluntarily conducted. in recent years, numerous health facilities have been constructed on religious places, as well as along the path of pilgrimage using pilgrims' donations. management of pilgrims' donations can help to build and maintain health infrastructure in iraq. with this policy, the iranian pilgrims will benefit from the arbaeen ceremony, and the level of health in the region will be improved. this study is the first qualitative study on the experiences of arbaeen, so it provides rich information in this regard; however, since the results have been collected from semi-structured interviews, it is considered subjective. it is recommended that in future studies, by creating a quantitative instrument for examining the challenges and measuring, the subjective concepts can be objectively transformed and analyzed. the present study can be used as a basis for this purpose. the ineffectiveness of health training, low perception of risk in pilgrims, poor control of the effective factors on infectious diseases and deficient health infrastructure in iraq are important challenges of the health system in dealing with contagious diseases in the arbaeen ceremony from the stakeholder's perspective. therefore, pilgrim-based educational planning, along with the control of other challenges, represents an opportunity to improve the health of pilgrims taking part in the arbaeen ceremony. the full data for this study are not provided because the transcripts of the interviews contain identifiable and sensitive information. researchers can apply to access limited deidentified transcripts of interviews from the first author, arezou karampourian (a.karampourian@sbmu.ac.ir) under no strict conditions. please note that transcripts are only available in persian. the study was supported by the shahid beheshti university of medical sciences, tehran, iran. . . the paper is interesting and adds to the medical literature of arbaeen ceremony which has been poorly addressed from a public health research perspective. we hope this paper will serve as a key reference on health aspect of arbaeen ceremony in near future. a few minor issues should be addressed before the manuscript is published. the reference list needs to be revised, we note some misplacement error. for example, in the third paragraph of the introduction (p ), "based on the statistics of , the number of iranian pilgrims taking part in the arbaeen ceremony was , , ", the reference given for this is number . ineffectiveness of health education. emerging of all four themes has been verified by the participants' comments which made results acceptable. the method of analysis is appropriately explained which can facilitate the reproducibility. the authors conclude that the main factor in preventing communicable disease is pilgrim-based training long before the ceremony. this conclusion is adequately supported by the results. is the study design appropriate and is the work technically sound? yes are all the source data underlying the results available to ensure full reproducibility? yes no competing interests were disclosed. i have read this submission. i believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. the benefits of publishing with f research: your article is published within days, with no editorial bias you can publish traditional articles, null/negative results, case reports, data notes and more the peer review process is transparent and collaborative your article is indexed in pubmed after passing peer review dedicated customer support at every stage for pre-submission enquiries, contact research@f .com establishment of public health security in saudi arabia for the hajj in response to pandemic influenza a h n knowledge, attitude and practice (kap) survey concerning antimicrobial use among australian hajj pilgrims fatalism at the arbaeen ceremony methodology of applied research in medical sciences letter to editor: mortality trends of pilgrims in hajj: an implication for establishment of surveillance system. health in emergencies and disasters qurterly should cities hosting mass gatherings invest in public health surveillance and planning? reflections from a decade of mass gatherings in pubmed abstract | publisher full text | free full text human stampedes during religious festivals: a comparative review of mass gathering emergencies in india pattern of morbidity and mortality in karbala hospitals during ashura mass this study is part of a phd thesis. the authors thank shahid beheshti university of medical sciences for approving the study, as well as all participants in this study who participated in the study, despite being busy. click here to access the data. competing interests: we have read this submission. we believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. it is better to discuss the results respectively as the results mentioned. so, the reader can insure . it is better to discuss the results respectively as the results mentioned. so, the reader can insure that all results have considered in the discussion part. it is better to explain what pilgrim-based education is and how it controls cd in discussion part and conclusion as well. are all the source data underlying the results available to ensure full reproducibility?no source data required no competing interests were disclosed. this is an interesting and well-written piece of work. infectious disease prevention strategies in mass gatherings, especially religious or cultural gathering, need to start with such the qualitative study. having a predetermined program, implementation, evaluation, and upgrade of the strategies in preventing and responding to communicable diseases requires the proper recognition of the contextual factors and challenges, experiences and perception of healthcare providers and decision-makers of the key agencies involved. the authors carried out a qualitative study with conventional content analysis approach about the arbaeen ceremony, a religious mass gathering in iran and iraq and found four main themes: -health infrastructure defects in iraq, poor control of the causative factors of infectious diseases, -the low perception of risk in pilgrims, and ineffectiveness of health education. emerging of all four themes has been verified by the participants' key: cord- -vu b a authors: panahi, heidar ali; bolhassani, azam; javadi, gholamreza; noormohammadi, zahra title: a comprehensive in silico analysis for identification of therapeutic epitopes in hpv , , and oncoproteins date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: vu b a human papillomaviruses (hpvs) are a group of circular double-stranded dna viruses, showing severe tropism to mucosal tissues. a subset of hpvs, especially hpv and , are the primary etiological cause for several epithelial cell malignancies, causing about . % of all cancers worldwide. due to the high prevalence and mortality, hpv-associated cancers have remained as a significant health problem in human society, making an urgent need to develop an effective therapeutic vaccine against them. achieving this goal is primarily dependent on the identification of efficient tumor-associated epitopes, inducing a robust cell-mediated immune response. previous information has shown that e , e , and e early proteins are responsible for the induction and maintenance of hpv-associated cancers. therefore, the prediction of major histocompatibility complex (mhc) class i t cell epitopes of hpv , , and oncoproteins was targeted in this study. for this purpose, a two-step plan was designed to identify the most probable cd + t cell epitopes. in the first step, mhc-i and ii binding, mhc-i processing, mhc-i population coverage and mhc-i immunogenicity prediction analyses, and in the second step, mhc-i and ii protein-peptide docking, epitope conservation, and cross-reactivity with host antigens’ analyses were carried out successively by different tools. finally, we introduced five probable cd + t cell epitopes for each oncoprotein of the hpv genotypes ( epitopes in total), which obtained better scores by an integrated approach. these predicted epitopes are valuable candidates for in vitro or in vivo therapeutic vaccine studies against the hpv-associated cancers. additionally, this two-step plan that each step includes several analyses to find appropriate epitopes provides a rational basis for dna- or peptide-based vaccine development. hpvs are a large branch of the papillomaviridae family, grouped in different genera (alpha-, nu-/mu-, beta-and gamma-papillomaviruses), with more than genotypes [ ] [ ] [ ] [ ] . the classification of papillomaviruses (pvs) has been based on l gene sequence. they are clinically divided into two groups: low-risk hpvs, like hpv and , which cause benign lesions (warts and benign papillomas), and high-risk hpvs (hrhpvs), like hpv and , which are a a a a a response. however, the addition of cd + t cell epitopes can significantly augment its strength and duration [ , , ] . cd + ctls commonly recognize intracellular-originated peptides presented by mhc-i molecules. they accommodate peptides with - residues; the ideal length is residues. while cd + helper t lymphocytes (htls) commonly recognize extracellular-originated peptides presented by mhc-ii molecules. they accommodate peptides with - residues or even more; the ideal length is [ ] [ ] [ ] [ ] [ ] . the strength of the interaction between a t cell receptor and a peptide-mhc complex (pmhc), depends on the presented peptide and the mhc structure [ , ] . the binding of a peptide to mhc-i molecule is the most selective stage in the way of peptide presentation [ ] . bioinformatics tools can predict the potential immunogenic epitopes from thousands of epitopes in a short time [ ] . generally, the algorithms of these tools range from ones programmed to determine peptide-mhc molecule binding data to those based on structural similarity, molecular modeling, and molecular docking [ ] . peptides that bind to a specific mhc molecule have sequence similarity. therefore, peptide sequence patterns have been used to predict their binding to mhc molecules [ ] . in recent years, the accuracy of these methods has increased strikingly, and more than % of natural epitopes have been recognized at a high specificity of % [ ] . this improvement in performance was achieved by the expanding experimental binding data, available in the immune epitope database (iedb) and analysis resource (http://www.iedb.org/), and by the improvement of machine-learning algorithms [ ] . regarding the fundamental importance of epitope prediction in vaccine development, we investigated the best potential cd + t cell epitopes from the e , e , and e oncoproteins of four prevalent hrhpv genotypes ( , , and ) in the world and iran [ ] , as shown in a two-step plan was designed to identify the most probable cd + t cell epitopes (fig ) . for the first step, mhc-i and ii binding, mhc-i processing, mhc-i population coverage and mhc-i immunogenicity prediction analyses, and for the second step, mhc-i and ii protein-peptide docking, epitope conservation, and cross-reactivity with host antigens analyses were considered. the second step analyses were performed only for the selected peptides in the first step. in jan , in order of priority, the refseq, reviewed or unreviewed sequences of hrhpv oncoproteins (e , e , and e ) were retrieved from the national center for biotechnology information database (ncbi) (http://www.ncbi.nlm.nih.gov/) and uniprotkb/swiss-prot database (http://www.uniprot.org/). the isoform sequences of hpv , , , and oncoproteins were retrieved from hpv t cell antigen database (http://cvc.dfci.harvard.edu/hpv/ html/search.php). all the sequences are accessible in supporting information (s file). binding of epitopes to mhc-i molecules is an essential step for antigen presentation to ctls. herein, it was predicted by four online servers, as illustrated in table . the hla supertypes and frequently occurring hla-i alleles provided by the servers were included in the analysis. however, when an allele (e.g., hla-b � : ) was not provided, but its allele group (i.e., hla-b � ) was available, we used the allele group instead of the allele. the used human and mouse alleles, or allele groups are provided in supporting information (s table) . currently, eight prediction methods are available in the iedb mhc-i binding prediction tool, i.e., iedb recommended [ ] , consensus [ ] , netmhcpan [ , ] , artificial neural network (ann) [ , ] , smm with a peptide-mhc binding energy covariance matrix (smmpmbec) [ ] , stabilized matrix method (smm) [ ] , comblib_sidney [ ] , pickpocket [ ] , netmhccons [ ] and netmhcstabpan [ ] . the iedb-recommended and consensus are not independent methods; they use ann, smm and comblib_sidney methods to generate a representative index for each predicted pmhc; the median of percentile ranks (prs) or binding scores obtained from the used methods is reported as a representative pr or consensus score in the iedb-recommended or consensus method respectively. the pr is calculated by comparing the half maximal inhibitory concentration (ic ) of subjected peptide against a group of random peptides from swiss-prot database. the ic value, expressed as nanomolar, shows binding affinity. the lower ic or pr means higher binding affinity. as a rough guideline, peptides with ic values < nm are considered as high affinity, - nm intermediate affinity and more than - nm low affinity. no known t cell epitope has got an ic value > nm to date [ ] . in this study, iedb recommended method was used. the outputs for each pmhc in this method consisted of a median pr, a method-specific ic , and a method-specific pr. predictions were made against frequently occurring human mhc-i alleles (including hla supertypes) and mhc-i mouse alleles. epitope length was set on , , , and mer. peptides with median pr < . are applied for the analysis. netmhcpan mhc-i binding prediction. netmhcpan server predicts binding of peptides to the known mhc molecules using anns method. it is trained on a combination of naturally eluted ligands ( human and mouse mhc-i alleles) and binding affinity data ( mhc molecules from human, mouse, cattle, primates, and swine). besides, the user can perform a prediction against any custom mhc-i molecule by uploading its full-length sequence [ ] . in this study, predictions were performed for , , , and mer peptides against frequently occurring human mhc-i alleles and mhc-i mouse alleles. pr thresholds for strong and weak binders were set on . and . , respectively. peptides with pr < . were applied for the analysis. rankpep mhc-i binding prediction. rankpep predicts binder peptides of a given protein sequence or sequence alignments to mhc-i and ii molecules. the algorithm of rankpep based on the comparison of sequence similarities, using position-specific scoring matrices (pssms) method. it employs profiles of a group of aligned peptides recognized to bind to a specific mhc molecule and creates a consensus sequence by determining the most common residue for each position. then, it allocates an optimal score to the consensus sequence, compares the score of the subjected peptide with the optimal score, and gives the peptide a percentile optimal value for comparison. finally, it highlights strong binders in red [ , ] . herein, the prediction was made against frequently occurring hla-i and h -i alleles. the server did not provide all common lengths of epitopes for all the mhc alleles. thus, the used alleles and their provided epitope lengths are shown together, as given in supporting information (s table) . syfpeithi mhc-i binding prediction. syfpeithi (http://www.syfpeithi.de/ -home. htm/) is a database of over published and verified peptide sequences of human, mouse, and other organisms, known as natural binders of mhc-i and ii molecules. when syf-peithi analyzes a peptide for binding prediction against a specific mhc-i allele, its scoring system evaluates every residue of the query and gives it an arbitrary value between and , according to whether it is an anchor, auxiliary anchor, or preferred residue. it allocates the value to those residues which slightly preferred in that particular position, to the ideal anchor residues, and - to - to those residues which exhibit an adverse effect on the binding ability. the sum of these values is the score of the peptide. the maximal score could vary between different mhc alleles [ , ] . herein, the prediction was made against frequently occurring hla-i alleles and h -i alleles. epitope length was set on , , , and mer. every predicted pmhc which got a score less than % of the reference sequence score was excluded from the analysis. the allele-specific reference sequence was selected from rankpep's consensus sequence [ ] , or our syfpeithi predicted epitopes, whichever got the highest score in syfpeihi server. the reference sequences, their sources, and their scores are given in supporting information (s table) . recognition of high immunogenic cd + t cell epitopes was the primary aim of this study. therefore, all predictions were primarily made against epitopes with - residue length. however, it was valuable to determine that which mer mhc-i epitope is the core peptide of the mhc-ii epitope(s) too. the core peptide lies on the mhc-ii molecule grooves, and play the central role in constructing pmhc. with this strategy, the short minimal predicted epitopes could be used in designing of synthetic long peptides (slps), resulting in peptide loading to both mhc-i and ii molecules. iedb mhc-ii binding prediction. in this study, the mhc-ii binding prediction was made by iedb mhc-ii binding predictor (http://tools.iedb.org/mhcii/) [ , , ] . iedb possess seven prediction methods for mhc-ii binding prediction: iedb-recommended, consensus [ ] , netmhciipan [ ] , nn-align [ ] , smm-align [ ] , combinatorial libraries [ ] and sturniolo's method [ ] . herein, the iedb-recommended method was used, and all peptides with pr< . were selected for the analysis. the prediction was made against human alleles (iedb reference set) and three mouse alleles, given in supporting information (s table) . the server has fundamentally set the epitope length on mer. each iedb-recommended method participated in the prediction process offered a core peptide ( mer) for each predicted epitope ( mer). we associated the mer mhc-ii core peptides with the mer mhc-i predicted epitopes to determine that which mhc-i epitope is the core peptide of the mhc-ii epitope(s) too. mhc-i t cell epitope processing predictions of e , e , and e oncoproteins are made by the iedb combined predictor (http://tools.iedb.org/processing/). this tool combines predictors of three main steps of mhc-i antigen presentation pathway (proteasomal processing, transporter associated with antigen processing (tap) transport, and mhc-i binding) and calculates a total processing score for each predicted epitope. it allows the user to choose a method from ann, smm, smmpmbec, comblib_sidney , netmhcpan, netmhccons and pickpocket methods for the binding prediction. in the current update ( ), the iedb team has changed the choice of the recommended prediction method for the processing tool to be netmhcpan . rather than a consensus, since the processing tools requiring an ic value, which the consensus method does not provide. furthermore, netmhcpan . has provided all mhc alleles and has performed the predictions very well in recent comparisons [ ] . there are two types of proteasomes, the housekeeping types which are expressed instinctively, and immuno types which are provoked by ifn-γ secretion. the immunoproteasomes are believed to improve the efficiency of antigen presentation [ , ] . in this study, the immunoproteasome option was selected. the program outputs for every predicted epitope consisted of proteasome score, tap score, mhc score, processing score (proteasome + tap score), total score (proteasome + tap + mhc score), and mhc-i ic . the tap scoring system calculates a-log (ic ) value for the binding of a peptide (or n-terminal of its precursors) to the tap molecules. the higher tap score, the higher transport rate. [ , , ] . herein, the analysis was made against the human and mouse mhc-i alleles used later in the iedb binding prediction, with the iedb-recommended method and other default settings of the program. epitopes with ic < nm for hla-i alleles and < nm for h -i alleles were included in the analysis. several factors could clarify the difference between epitope and non-epitope peptides; an essential factor is epitope immunogenicity, i.e., it could be recognized by t cells. some amino acids, particularly those with large and aromatic side chains (especially tryptophan, phenylalanine, and isoleucine), are associated with immunogenicity. moreover, the positions p - of a peptide are more critical for immunogenicity [ ] . in this study, the mhc-i immunogenicity of all predicted epitopes was determined by the iedb web server (http://tools.iedb.org/immunogenicity/) [ ] . this tool uses the properties of amino acids and their locations to predict the immunogenicity of a pmhc. the default option was selected to specify which positions of the query peptide to be masked from the analysis, because it masked positions which are also suggested for the most frequent human mhc-i allele, hla-a � : . iedb population coverage prediction tool (http://tools.iedb.org/population/) [ ] is used to predict the hla-i population coverage of all - mer predicted epitopes in the first step. this tool can accept a target population by two query levels: ) area-country-ethnicity and ) ethnicity alone. it can integrate allele frequency information retrieved from the allele frequency net database (afnd) (http://www.allelefrequencies.net/default.asp) [ ] . iedb also accepts custom populations with allele frequencies defined by users. since, hla-i and hla-ii t cell epitopes elicit immune responses from two different t cell populations (ctl and htl, respectively), the server provided three different population coverage modes: ) hla-i lonely, ) hla-ii lonely, and ) hla-i and hla-ii together. herein, the mhc-i promiscuous predicted epitopes and their binding hla-i alleles (ic < nm or pr< . ) were entered as inputs for the analysis against the world population. the primary aim of molecular docking is the prediction of the binding site of a ligand at a protein receptor surface, and then docking and modeling the ligand into the recognized site. in this study, the binding ability of the first step selected peptides to human and mouse mhc molecules, was analyzed by cabs-dock (http://biocomp.chem.uw.edu.pl/cabsdock/) server. the server uses a multistage procedure that involves multiple programs, with the cα-cβ-side chain (cabs) model at its heart. the detailed information about these stages is given in supporting information (s file) [ , ] . also, fig shows the pipeline of cabs-dock protocol [ ] . cabs-dock gets the d structure of the receptor and the sequence of the peptide as obligatory inputs. furthermore, there are some non-obligatory inputs as recommendations which could improve outputs. in this study, duplicate dockings for each peptide ( dockings in total) were done against the most significant human/mouse mhc-i and ii molecules which had at least one well-structured protein data bank (pdb) file in the rcsb protein data bank (https://www.rcsb.org/), as shown in table . these pdb files are in the complex with their peptidic ligand and some x-ray crystallography solution molecules (heteroatoms). thus, these excess molecules, as well as redundant mhc molecules were removed before executing docking process. since, the binding site of epitopes on the mhc molecules was well-known previously, the unlikely regions to bind masked before the analysis. cabs-dock returns ten representative models (medoids) as the best-simulated models and ranks them by cluster density (cd). cluster density is equal to the number of elements in a cluster divided by their average ligand root mean square deviation (rmsd). the higher cd value implies greater accuracy. ligand rmsd value shows the differentiation measure between cluster elements. as a guideline; rmsd < . Å means high accuracy; rmsd � . and � . Å means medium accuracy and rmsd > . Å means low accuracy [ ] . herein, the rmsd and cd of the best-simulated models were selected for the analysis. the best model, which has the highest cd value, is not necessarily the top-ranked model, because, in some cases, peptides were not attached to their binding site properly. thus, these malformed models were excluded from the analysis. it is important to note that, due to the different frequency of mhc alleles in human populations, the equal cd value of different mhc alleles, don't have equal value regarding population coverage. thus, to involve the effect of population coverage, the cd value of every model was multiplied by its allele population coverage (divided by hundreds for more facility) to obtain a weighted index. then, the sum of all hla-i or ii weighted indexes of each peptide was calculated to get a total docking score (tds), used as a score to compare the candidate peptides. it is the first time that the tds has been formulated and used for this purpose. this formula is also applicable to the similar docking scores obtained from other servers. the use of highly conserved epitopes in a vaccine formulation reduces the risk of tumor immune escape and provides broader protection against different virus strains or genotypes. thus, the conserved areas are preferred to use in therapeutic vaccines, if they are appropriate epitopes. herein, the epitope conservancy analyses for the first step selected peptides were done in three levels: . inter-isoform conservancy: the percent of conservancy between all isoforms of each e , e , or e oncoprotein. . inter-type conservancy: the percent of conservancy between hpv and (alpha-papillomavirus ), as well as between hpv and (alpha-papillomavirus ). . inter-hrhpv conservancy: complete ( %) conservancy between all hrhpvs (hpv , , , , , , , , , , , and ). the selected peptides in the first step were analyzed to find inter-strains and inter-types conservancy percentages by iedb tool, conservation across antigens, (http://tools.iedb.org/ conservancy/). the inter-hrhpvs conservancy analysis was done by the iedb and expasy clustalw servers (https://embnet.vital-it.ch/software/clustalw.html). cross-reactivity with host antigens can cause adverse immune responses. therefore, the selected peptides in the first step were checked for similarities with the mouse and human proteomes by the ncbi blastp tool (https://blast.ncbi.nlm.nih.gov/blast.cgi). regarding the studies, different peptides usually get different scores/ranks in different analyses. this inconsistency indicates that these results needed to be analyzed with an integrated approach. indeed, integrated approach is more practical and efficient in such conditions in comparison with analysis by analysis filtering approach, in which those epitopes are chosen for the next analysis that have gotten an acceptable score in the previous analysis. herein, the integrated approach was applied in both steps of epitope selection. since the ultimate goal of the discovery of therapeutic epitopes is to use them in human vaccines, only the scores/ranks of human alleles were used to rank epitopes in some studies. however, investigators usually test therapeutic vaccines on mouse species in preclinical trials, thus in the current study, the binding status of the predicted epitopes to mouse mhc-i alleles was also studied by several binding predictors and molecular docking, as well. as stated above, ctl-mediated responses play a crucial role in killing the malignant cells. besides, the binding of epitopes to mhc-i molecules is the most selective step for antigen presentation to ctls. therefore, in the first step, the selection was made primarily by the comparison of obtained mhc-i binding, processing and immunogenicity scores/ranks, and population coverage percentages. however, the mhc-ii binding ranks were actually of secondary importance to the selection process as an added advantage. additionally, the population coverage has a dual application. first, it determines the coverage of a given peptide in the target population. second, it is the best index for summarizing and evaluating of the hla-i binding predictions too, since it is calculated from the results of hla-i binding prediction analyses. in the first step, ten peptides (tables - ) from each hpv genotype oncoprotein ( peptides in total), which got better results in the first step analyses were selected for the second step analyses, including protein-peptide molecular docking, epitope conservation, and crossreactivity with host antigens. the individual detailed results of the mhc-i and ii binding (s file), mhc-i immunogenicity (s file) and mhc-i population coverage (s file) predictions, as well as, mhc-i and ii molecular docking (s file) and epitope conservation (s file) analyses are given in supporting information, as excel files. indeed, cabs-dock returns ten representative medoids as the best-simulated models and ranks them by cluster density (cd). cluster density is derived from two factors (the number of elements in a cluster and their average ligand rmsd) that is an advantage for this server. in the second step, five peptides out of ten selected peptides in the first step (tables - ) , which got better results in all analyses of both steps, were selected as the final-predicted epitopes. none of the final predicted epitopes showed more than % sequence similarity with mouse and human proteomes. high prevalence and mortality of oncogenic infectious pathogens such as hpv and helicobacter pylori have caused serious problems for humans. currently, people who are infected with hrhpvs but show normal cytology or precancerous lesions do not have any treatment option, causing the disease progress toward invasive carcinoma in some cases. unfortunately, no fda-approved immunotherapy exists for pre-existing hpv infections or their related cancers to date. immunotherapy of hpv-associated cancers by dna or peptide-based vaccines, depends on the recognition of highly immunogenic epitopes, inducing robust and specific immune responses, particularly cell-mediated responses against the malignant cells. the primary aim of this study was the prediction of cd + t cell epitope from the e , e and e oncoproteins, using a comprehensive two-step selection plan. these proteins chose because they play a pivotal role in the cell transformation, immune evasion, and maintenance of malignancy, as well as, their permanent expression (e and e ) by the malignant cells [ ] [ ] [ ] . expression of e oncoprotein occurs in the early phase of hpv infection. evidence indicates that e play a prominent role in the genesis of hpv-associated cancers, but is not essential for cancer progression [ ] , since when hpv genome integrates into the host genome, it usually results in the disruption of e , e , and e genes. therefore, targeting e protein provides an opportunity for treatment of hpv infections and preventing the precancerous lesions from the progression to established carcinomas [ , ] . some genotypes of hrhpvs are more involved in the genesis of epithelial tissue malignancies [ ] . thus, in this study, hrhpv , , and were targeted due to their high prevalence in the hpv-associated cancers, especially cervical carcinoma. there are several limitations for epitope prediction: ) the major drawback of peptidebased vaccines is low immunogenicity [ , ] . many studies have focused on enhancing immunogenicity using immune stimulating agents or adjuvants to avoid this problem. another solution is the use of agonist epitopes [ ] . epitope immunogenicity is a crucial factor in vaccine development. however, many of known natural epitopes when are analyzed in silico by iedb mhc-i immunogenicity predictor, do not obtain a high score. therefore, in this study, epitope selection was based on the integrated approach, in which one analysis does not play an important role alone. ) there are certain drawbacks associated with the function of each method invented for the mhc-peptide binding prediction [ ] . for this reason, several predictors and a molecular docking program were used to augment the prediction accuracy. ) some web tools have been developed for mhc-ii epitope prediction. since mhc-ii groove can bind to peptides with variable lengths, and different peptides have the different number of residues between their n-terminus and first anchor [ ], the exact assignment of mhc-ii core peptide would be a difficult problem which reduces the success rate of these prediction tools. therefore, most mhc-ii prediction tools did not usually make epitope predictions as accurately noted for mhc-i molecules [ , ] . in cancer immunotherapy, the ctl-mediated responses play the central role in eradication of malignant cells, and the binding of epitopes to mhc-i molecules is an essential step for antigen presentation to ctls. thus, in this study, predicted epitopes were primarily selected by their mhc-i binding and processing scores. however, the mhc-ii binding scores were actually of secondary importance to the epitope selection process as an extra advantage. additionally, there are several other essential determinants which significantly affect the outcomes, such as antigen processing, immunogenicity, population coverage, conservancy and cross-reactivity with host antigens. vaccine development requires a comprehensive approach to cover all these effectual elements, covered in this study. the primary aim of molecular docking is the recognition of binding site of a ligand at a protein receptor surface, and docking and modeling the ligand into this recognized site. in this study, cabs-dock server was used for molecular docking analyses. cabs-dock has several main advantages: ) the method does not require any data about the peptide structure and its binding site. ) during docking process, peptide conformation is entirely flexible. ) it is possible to apply dynamic conformational changes in the receptor structure and ) to exclude some receptor regions from the docking search, leading to the more efficient search in the vicinity of the binding site at a sensible time. [ , ] . in comparison with protein-ligand (small molecules) docking, protein-peptide docking analysis is more problematic, since significant conformational changes occur during the process. as a general rule, how much the length of the query peptide to be longer, there are more torsions and conformational flexibilities. additionally, in comparison to protein-protein interactions, protein-peptide dockings are more transient, and their binding affinities are notably weaker [ ] . these factors make structural predictions of long peptides very challenging. therefore, in this study, mer peptides were preferred for selection compared to other possible lengths. they are also preferred by all mhc-i molecules as epitope and by mhc-ii molecules as the core peptide of epitopes. moreover, expansion of or mer ctl epitopes to longer peptides may create a practical alternative, containing both cd + htl and cd + ctl epitopes; especially, when cd + htl epitopes, covering ctl epitopes, are not recognized [ ] . [ , , [ ] [ ] [ ] [ ] [ ] [ ] . however, the prediction of t cell epitopes inducing strong responses has remained a big challenge. for therapeutic hpv vaccines, many candidates have been designed to trigger the activation of ctls or htls, mostly by targeting two major hpv oncoproteins, e or e [ ] , and in a few studies, e oncoprotein [ , ] . as well as, several clinical trials have been launched for immunotherapy of hpv-associated cancers [ ], although, they have not been so immunogenic, to induce a sufficient cellular immunity and eradicate malignant cells completely. some studies have suggested that the use of e and e slps, containing both cd + htl and cd + ctl epitopes, led to more potency and durability of cd + t cell reactivity in vivo, in comparison with the minimal ctl epitopes [ , ] . in , as pioneers in hpv epitope studies, feltkamp et al. recognized the hpv -e sequence rahynivtf as an mhc-i epitope that can provoke ctl-mediated responses and eradicates established hpv l -induced tumor cells in mice [ , ] . this sequence is the first hpv -e predicted epitope in our study as well. in , kumar et al. studied hpv -e oncoprotein to predict the candidate t-cell and bcell epitopes [ ] . they have screened potent epitopes for mhc-i molecules according to pr and the immunogenicity score, using iedb mhc-i binding and immunogenicity predictors. they found a mer potent epitope, safrcfivyiifvy, having the lowest pr and the highest immunogenicity score, i.e., . and . , respectively. notably, our second hpv -e predicted epitope, safrcfivy, is the n-terminal part of safrcfivyiifvy, and our first predicted epitope, flihtharf, is the c-terminal part of the third epitope of their study, vyiplflihtharf. in , tsang et al. scanned the hpv -e and e oncoproteins for the match peptides with the consensus motif of hla-a binding peptides [ ] . the bimas algorithm [ ] was employed to rank probable binding peptides according to the predicted one-half-time dissociation of pmhcs. three potential ctl predicted epitopes of the e protein (klpqlctel, kiseyr-hyc, and qqynkplcdl) and three of the e protein (ymldlqpet, tlheymldl, and rtledllmgt) were selected. they showed the immunogenicity of these peptides was enhanced when their agonist epitopes were used. the klpqlctel and tlheymldl sequences are the seventh and the fifth predicted epitopes of hpv -e and hpv -e in our study, respectively. experimental evidences about hrhpv-derived epitopes in literatures are mostly limited to e and e oncoproteins of hpv and . among our first-step predicted epitopes: fllcfcvll and yiifvyipl from the e -derived epitopes [ ] , fafrdlcivy [ ] , cyslygttl [ ] , vydfafrdl [ , ] , kfyskisey [ ] , klpqlctel [ ] [ ] [ ] , iseyrhycy [ ] , eyrhycysl [ ] , klpdlctel [ , [ ] [ ] [ ] , fafkdlfvv [ , ] and klpdlctel [ , [ ] [ ] [ ] from the e -derived epitopes, rahynivtf [ ] , ledllmgtl [ ] , tlheymldl [ , [ ] [ ] [ ] , llmgtlgiv [ , , , ] , qaepdrahy [ ] , gtlgivcpi [ , ] , fqqlflntl [ ] and tlqdivlhl [ ] from the e -drived epitopes were reported as t-cell epitopes experimentally. besides, ivyrdgnpy, cyslygttl, klpqlctel and iseyrhycy from the e -derived epitopes, and rahynivtf and gtlgivcpi from the e -derived epitopes were also reported as hla ligands [ ] . others are novel epitopes that they also require experimental studies for validation. as far as we know, this is the first time that in a laborious in silico study for epitope prediction, e , e and e oncoproteins of hrhpv , , and have been investigated altogether. moreover, in previous studies, usually only one predictor tool was used for making epitope prediction, or if several tools were used, no integrated approach was employed to make the conclusion. we believed that our predicted epitopes are valuable candidates for further in vitro and in vivo therapeutic vaccine studies. additionally, the introduction of the ten epitopes for each hpv genotype oncoprotein in the first step of the study shows which region of each oncoprotein is rich of the epitope, and thus, is more suitable for use in the design of slps. notably, the previous in vivo studies have been conducted using slps of hrhpv-e and/or-e oncoproteins, in particular hpv oncoproteins [ , [ ] [ ] [ ] [ ] [ ] . furthermore, the two-step plan of this in silico study, which each step includes several analyses to find proper epitopes by an integrated approach, would provide a basis for rational epitope prediction. however, it could be more efficient by adding other useful analyses. further studies are recommended on the peptide binding assays, the design of polyepitope constructions including e , e and e epitopes, the expansion of the minimal ctl epitopes to longer peptides (slps), the use of various adjuvants, involvement of delivery routes, mouse immunization with the designed constructs, evaluation of immune responses such as cytokines, antibodies, ctls and tumor growth for finding the best construct for clinical trials. it is important that improper vaccine design and immunosuppressive microenvironment were known as the main reasons of the failure in cancer immunotherapy by therapeutic cancer vaccines [ ] . cross-roads in the classification of papillomaviruses hpv vaccine: current status and future directions the natural history of human papillomavirus infection. best practice & research clinical obstetrics & gynaecology classification of papillomaviruses (pvs) based on pv types and proposal of taxonomic amendments global burden of human papillomavirus and related diseases carcinogenic human papillomavirus infection human papillomavirus molecular biology and disease association tumour virus vaccines: hepatitis b virus and human papillomavirus global burden of cancers attributable to infections in : a review and synthetic analysis. the lancet oncology worldwide burden of cancer attributable to hpv by site, country and hpv type comprehensive control of human papillomavirus infections and related diseases human papillomavirus genome variants human papillomavirus types in , hpv-positive women: a meta-analysis from cervical infection to cancer human papillomaviruses. iarc monographs on the evaluation of carcinogenic risks to humans e protein of human papillomavirus downregulates hla class i and interacts with the heavy chain via its first hydrophobic domain the bovine papillomavirus oncoprotein e retains mhc class i molecules in the golgi apparatus and prevents their transport to the cell surface the e oncoprotein encoded by human papillomavirus types and promotes the degradation of p degradation of the retinoblastoma tumor suppressor by the human papillomavirus type e oncoprotein is important for functional inactivation and is separable from proteasomal degradation of e human papillomavirus and related diseases in the world modeling the mhc class i pathway by combining predictions of proteasomal cleavage, tap transport and mhc class i binding peptide binding predictions for hla dr, dp and dq molecules a systematic assessment of mhc class ii peptide binding predictions and evaluation of a consensus approach immune epitope database and analysis resource (iedb) . . mhc-i processing predictions-tutorial netmhcpan- . : improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data prediction of peptide-mhc binding using profiles. immunoinformatics: predicting immunogenicity in silico prediction of mhc class i binding peptides using profile motifs a consensus epitope prediction approach identifies the breadth of murine t(cd +)-cell responses to vaccinia virus netmhcpan, a method for mhc class i binding prediction beyond humans gapped sequence alignment using artificial neural networks: application to the mhc class i system netmhc- . : accurate web accessible predictions of human, mouse and monkey mhc class i affinities for peptides of length - derivation of an amino acid similarity matrix for peptide: mhc binding and its application as a bayesian prior generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method quantitative peptide binding motifs for human and mouse mhc class i molecules derived using positional scanning combinatorial peptide libraries the pickpocket method for predicting binding specificities for receptors based on receptor pocket similarities: application to mhc-peptide binding netmhccons: a consensus method for the major histocompatibility complex class i predictions pan-specific prediction of peptide-mhc class i complex stability, a correlate of t cell immunogenicity information on syfpeithi. institute for cell biology-department of immunology-heidelberg accurate pan-specific prediction of peptide-mhc class ii binding affinity with improved binding core identification nn-align. an artificial neural network-based alignment algorithm for mhc class ii peptide binding prediction prediction of mhc class ii binding affinity using smm-align, a novel stabilization matrix alignment method generation of tissue-specific and promiscuous hla ligand databases using dna microarrays and virtual hla class ii matrices identifying mhc class i epitopes by predicting the tap transport efficiency of epitope precursors properties of mhc class i presented peptides that enhance immunogenicity predicting population coverage of tcell epitope-based diagnostics and vaccines allele frequency net update: new features for hla epitopes, kir and disease and hla adverse drug reaction associations modeling of proteinpeptide interactions using the cabs-dock web server for binding site search and flexible docking cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site human papillomavirus type e protein as a therapeutic target recombinant adeno-associated virus expressing human papillomavirus type e peptide dna fused with heat shock protein dna as a potential vaccine for cervical cancer immunotherapy of established (pre) malignant disease by synthetic long peptide vaccines more than one reason to rethink the use of peptides in vaccine design identification and characterization of enhancer agonist human cytotoxic t-cell epitopes of the human papillomavirus type prediction of mhc-peptide binding: a systematic and comprehensive overview prediction of epitope-based peptides for vaccine development from coat proteins gp and vp of ebola virus using immunoinformatics cd + ctl priming by exact peptide epitopes in incomplete freund's adjuvant induces a vanishing ctl response, whereas long peptides induce sustained ctl reactivity identification of immunotherapeutic epitope of e protein of human papillomavirus- : an in silico approach computational prediction of linear b-cell epitopes in the e oncoprotein of the human papillomavirus type using several bioinformatics tools multi epitope peptide vaccine prediction against sudan ebola virus using immuno-informatics approaches a systematic bioinformatics approach for selection of epitope-based vaccine targets a novel multi-epitope peptide vaccine against cancer: an in silico approach epitope-based vaccine target screening against highly pathogenic mers-cov: an in silico approach applied to emerging infectious diseases advances in peptide-based human papillomavirus therapeutic vaccines therapeutic vaccination with papillomavirus e and e long peptides results in the control of both established virus-induced lesions and latently infected sites in a pre-clinical cottontail rabbit papillomavirus model vaccination with cytotoxic t lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type -transformed cells cytotoxic t lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type -induced tumors scheme for ranking potential hla-a binding peptides based on independent binding of individual peptide side-chains cytotoxic t-lymphocyte responses to human papillomavirus type e and e proteins and hla-a* -restricted t-cell peptides in cervical cancer patients hla class i binding promiscuity of the cd t-cell epitopes of human papillomavirus type e protein novel oligomannose liposome-dna complex dna vaccination efficiently evokes anti-hpv e and e ctl responses identification of an hla-a -restricted cytotoxic t lymphocyte epitope from human papillomavirus type- e : the combined effects of bortezomib and interferon-gamma on the presentation of a cryptic epitope identification of human papillomavirus -e protein-derived peptides with the potential to generate cytotoxic t-lymphocytes toward human leukocyte antigen-a + cervical cancer human papillomavirus e -specific cd ra+ ccr + high avidity cd + t cells fail to control tumor growth despite interferon-gamma production in patients with cervical cancer a conserved e -derived cytotoxic t lymphocyte epitope expressed on human papillomavirus -transformed hla-a + epithelial cancers immunization with a poly (lactide co-glycolide) encapsulated plasmid dna expressing antigenic regions of hpv and results in an increase in the precursor frequency of t cells that respond to epitopes from hpv , , and identification in humans of hpv- e and e protein epitopes recognized by cytolytic t lymphocytes in association with hla-b and determination of the hla-b -specific binding motif up-regulation of hla class-i antigen expression and antigen-specific ctl response in cervical cancer cells by the demethylating agent hydralazine and the histone deacetylase inhibitor valproic acid human t-cell responses to hla-a-restricted high binding affinity peptides of human papillomavirus type proteins e and e synthetic peptides of human papillomavirus type e harboring hla-a . motif can induce peptide-specific cytotoxic t-cells from peripheral blood mononuclear cells of healthy donors establishment of an hla-a* human papillomavirus type tumor model to determine the efficacy of vaccination strategies in hla-a* transgenic mice different methods of identifying new antigenic epitopes of human papillomavirus type e and e proteins naturally processed and hla-b -presented hpv e epitope recognized by t cells from patients with cervical cancer human ctl epitopes encoded by human papillomavirus type e and e identified through in vivo and in vitro immunogenicity studies of hla-a* -binding peptides dendritic cell-based tumor vaccine for cervical cancer ii: results of a clinical pilot study in individual patients human ctl epitopes encoded by human papillomavirus type e and e identified through in vivo and in vitro immunogenicity studies of hla-a* -binding peptides identification of a naturally processed hla-a* hpv e t cell epitope by tumor cell mediated in vitro vaccination role of hla-a motifs in identification of potential ctl epitopes in human papillomavirus type e and e proteins prospects of combinatorial synthetic peptide vaccine-based immunotherapy against cancer experience with synthetic vaccines for cancer and persistent virus infections in nonhuman primates and patients mechanisms of peptide vaccination in mouse models: tolerance, immunity, and hyperreactivity therapeutic vaccination against human papilloma virus induced malignancies immunologic treatments for precancerous lesions and uterine cervical cancer therapeutic cancer vaccines the authors sincerely thank dr. ali namvar and miss elnaz agi for their valuable guidance and comments during preparation of the paper. key: cord- -xhpv y authors: land, kevin j.; boeras, debrah i.; chen, xiang-sheng; ramsay, andrew r.; peeling, rosanna w. title: reassured diagnostics to inform disease control strategies, strengthen health systems and improve patient outcomes date: - - journal: nat microbiol doi: . /s - - - sha: doc_id: cord_uid: xhpv y lack of access to quality diagnostics remains a major contributor to health burden in resource-limited settings. it has been more than years since assured (affordable, sensitive, specific, user-friendly, rapid, equipment-free, delivered) was coined to describe the ideal test to meet the needs of the developing world. since its initial publication, technological innovations have led to the development of diagnostics that address the assured criteria, but challenges remain. from this perspective, we assess factors contributing to the success and failure of assured diagnostics, lessons learnt in the implementation of assured tests over the past decade, and highlight additional conditions that should be considered in addressing point-of-care needs. with rapid advances in digital technology and mobile health (m-health), future diagnostics should incorporate these elements to give us reassured diagnostic systems that can inform disease control strategies in real-time, strengthen the efficiency of health care systems and improve patient outcomes. or in sequence. in case of discrepancies, a third test is used as a tiebreaker. an example of this approach is for hiv case detection. specificity. diagnostics should have low false positive rates. the ideal scenario is where the sensitivity and specificity achieved from the diagnostics used at the poc approach those of laboratory-based assays wherever possible. however, a lower specificity can be tolerated if the harm of overtreatment is much less critical than missing the diagnosis of an infection. screening syphilis during pregnancy is an example of this approach, as missing maternal syphilis can lead to stillbirths, preterm birth and congenital syphilis in a third of pregnant women compared to the harm of overtreatment with a single dose of penicillin . user friendliness. tests should be easy to perform in - steps and require minimal user training with no prior knowledge of diagnostic testing. typically, results should be available in - minutes after sample collection and enable patient management and treatment during the same visit. the benefits of a rapid test versus a more accurate test that requires patients to return for results have been previously demonstrated . robustness refers to the ability of the test to withstand the supply chain (temperature, humidity, time delays, mechanical stresses) without requiring additional (and often costly) transport and storage conditions (for example, refrigeration). ideally the test does not require any special equipment or can be operated in small portable devices that use solar or battery power. deliverable to end-users. delivery refers to the organizational structures and relationships established with the purpose of coordinating and steering the logistics of selecting, procuring, shipping, storing, distributing and delivering a new health technology to ensure it reaches the end-users in resource-constrained settings . compared to when assured was proposed, access to laboratories in resource-limited settings has improved dramatically both in numbers and in quality , , especially in areas such as hiv and malaria testing, cd counting and tb. however, there remains a critical need for a wider range of diagnostics that can be performed at the poc. from this perspective, we undertake a review of the assured benchmark over the last years, assess the factors contributing to the success and failure of the assured diagnostics over the past decade, identify areas that have remained difficult to address as well as lessons learned from implementation in resourcelimited settings, and highlight additional conditions that should be considered in addressing poc needs. since , several diagnostic tests that satisfy (or nearly satisfy) the assured criteria have been developed to identify major human pathogens. this includes tests for hiv, malaria, syphilis and tb ( table ) . the hiv rapid test is the first test to fulfil the assured criteria, followed closely by the malaria and syphilis rapid tests and, recently, a near-poc test for tb. much of this development was the result of advocacy by donors such as the bill & melinda gates hiv rdts. early detection of hiv infection has been a critical component of hiv control programmes worldwide since the early days of the epidemic. since most infected individuals do not show specific symptoms and the period of viraemia is short, screening for hiv antibodies in blood as a marker of exposure has been the most cost-effective means of identifying infected individuals. the who developed a process and criteria for validating the performance of hiv antibody detection assays more than a decade ago and showed that hiv rdts using finger-pricked whole-blood specimens has acceptable performance compared to laboratory-based immunoassays , . many donors -for example, global fund and implementation partners, including the who, unaids and pepfar -have helped affected countries with the procurement and introduction of rapid hiv tests and enable early detection and prevent onward transmission. as a result of funding from donors, increased accessibility of rdts, and the 'architecture' provided by national hiv/ aids control programmes, hiv tests successfully reached as many as million children and adults in low-and middle-income countries in (ref. ) . two major challenges remain: training and quality assurance. with thousands of poc testing sites across a country, even the most well-organized control programmes would find it difficult to provide adequate training on an ongoing basis. this problem is most acute in health facilities where there is high turnover of staff. the us centres for disease control and prevention (cdc) has developed a convenient means of external quality assurance by working with countries to generate thermally stable proficiency panels that can be shipped to every poc testing site to ensure competence of testing personnel. however, a study in south africa found that only % of hiv rdts were performed correctly . although not every error will result in an incorrect diagnosis, the alarming reality is that with million tests being performed annually worldwide, assuming a % accuracy rate, as many as . million incorrect results per year could potentially be generated. ongoing quality assurance efforts include developing key policy and quality documents for the implementation of hiv-related poc testing . for example, as poc technologies for hiv viral load and early infant diagnosis were being developed, there was tremendous emphasis on quality, given the complexity of the test and lessons learned from hiv rdts. malaria is estimated to be the cause of at least a million deaths a year worldwide, most of which are in sub-saharan africa. while microscopic identification of parasites in blood smears has been the traditional means of diagnosing malaria in patients presenting with fever, microscopy requires equipment, a source of electricity and trained laboratory technicians. malaria rdts were developed as many rural communities lack these resources, and to date there are over brands of malaria rdts made by approximately companies , which vary widely in performance, manufacturing quality and price. the who has set up a pre-qualification programme with the cdc and the foundation for innovative new diagnostics to evaluate these tests to inform test selection and procurement for national malaria control programmes . in countries that permit the sale of malaria rdts and medicines over the counter, the quality of these commodities cannot be guaranteed. the price of most rdt brands is between us$ . and us$ . per test , , and as with all diagnostics, it is important to caution that price pressure will ultimately affect quality. as with hiv, with the support of donors, selection of high-quality rdts and the architecture provided by national malaria control programmes, malaria rdts have reached millions of patients every year. this promising trend, coupled with the effectiveness of bednets for preventing transmission, has led to the call for malaria elimination in many parts of the world . however, challenges remain for the future of malaria poc testing and the global elimination of malaria. first, highly accurate tests are required for all malaria species affecting humans, but these panspecific tests are usually about % more expensive than rdts that only detect plasmodium falciparum. most p. falciparum rdts detect a malarial antigen, histidine-rich protein (hrp) , and the discovery of parasites that have a deletion in this gene has raised concerns about false negative results and ongoing transmission. also, the problems of providing adequate training and quality assurance of malaria tests and testing at remote poc sites are similar to those described for hiv rdts , . in the near future, as countries progress towards malaria elimination, funding for rdts may become an issue. as the intensity of transmission decreases due to lower parasite density in infected individuals, more sensitive tests will be needed, which can only be achieved with costlier amplification steps or with ultrasensitive platforms for antigen detection. also, decreasing numbers of cases mean that malaria tests are no longer cost-effective, and thus it is more difficult to justify funding in light of multiple competing priorities for limited health budgets. syphilis. syphilis, caused by the spirochete treponema pallidum, has a long latent period during which patients have no symptoms, but can remain infectious. syphilis in pregnancy can lead to adverse outcomes of stillbirths and miscarriage, and babies born with congenital syphilis in the developing world only have a % chance of survival during the first years of life , . despite the availability of simple diagnostic tests for antenatal screening and the effectiveness of treatment with a single dose of long-acting penicillin, syphilis is re-emerging as a global public health problem . it is estimated that , babies die each year as a result of syphilis-associated stillbirths and congenital syphilis, largely because of lack of access to antenatal screening , . rdts exist for detecting syphilis and are reported to have acceptable performance , and operational characteristics. the introduction of rdts was also acceptable to patients and health care providers and was shown to contribute to the improvement of antenatal care in low-resource settings . despite all this, syphilis rdts have not had the same success as hiv and malaria rdts. the main reasons for this are the lack of advocacy and political will to translate the evidence to national policy, lack of funding to make the tests affordable to those in need and the lack of a national control programmes to provide the architecture needed to coordinate all the different aspects of testing. ensuring adequate training for health care workers and supplies of commodities were cited as key implementation barriers in africa , . dual hiv and syphilis rdt testing in countries was prioritized by the who for the elimination of mother-to-child transmission (mtct) of hiv and syphilis by , , but a recent review showed that while prevention of mother to child transmission (pmtct) programmes for hiv have resulted in a dramatic decrease in the number of hiv-positive infants in sub-saharan africa, the rate of syphilis screening of pregnant women in the same countries has remained at unacceptably low levels of approximately % (ref. ). this is due largely to disparity in funding and lack of political will despite the global fund allowing countries to purchase syphilis rdts with hiv rdts since (ref. ). countries need to harness the architecture provided by hiv pmtct programmes to screen women for both infections using a single drop of blood in a single visit to a health care facility. dual rapid hiv-syphilis rdts with acceptable performance are now available and the who, as well as many countries, has adopted these hiv and/or syphilis rdts into their national guidelines , . tuberculosis. tb causes . million deaths a year, % of which occur in low-and middle-income countries. ending the tb epidemic by is among the health targets of the sustainable development goals . the tb lipoarabinomannan (lam) antigen test provides poc screening for active tb in hiv positive patients. this novel rapid test detects lam in urine samples, providing results in just minutes, enabling earlier treatment for patients . the lam test does not assess tb drug susceptibility, but multidrug-resistant tb is a threat to global health security with an estimated , new cases a year showing resistance to rifampicin (rif) -the most effective first-line drug . nucleic acid tests (nats) for tb are available and provide highly sensitive and specific means of diagnosis. the recent development of a sample-in-answerout automated testing device that allows for simultaneous detection of mycobacterium tuberculosis (mtb) and rif resistance in h min has improved case detection and could decrease transmission, though nats still require patients to make a return visit for test results and treatment. this test system is available in - modules, allowing for flexibility in throughput. the mtb/rif near-poc assay can improve time to diagnosis and treatment and increase the efficiency of the health system if introduced appropriately - , and as of june , two-thirds of high-burden mtb countries and half of countries with a high multidrug-resistant mtb had adopted this assay into their national tb programme guidelines. a wider adoption of such high-performing assays would allow countries to increase their case detection rates and potentially reach the milestones of the end tb strategy . although national tb programmes provide a robust architecture for the implementation of new technologies, challenges associated with the near-poc nat assay remain as barriers -affordability (molecular assays are device-based and costly, even with subsidy), expertise (more technically demanding than lateral flow rdts) and sustainability , in addition to power and per-test time. we expect that addressing these barriers would improve patient outcomes, but a true poc test is still needed to overcome remaining challenges such as sample preparation and demands on human resources . while culture has been a long-standing microbiological gold-standard and is highly specific, it is also the most technically demanding, costly and slowest of diagnostic options, requiring patients to return for another clinic visit to obtain their test results and treatment if necessary. thus, it does not conform well to the development of assured diagnostics. successful poc tests achieve high sensitivity, which is needed as screening tools to ensure that all true and suspect cases are brought to the attention of control programmes and appropriately managed. in particular, antibody-based detection tests, such as those for hiv and hepatitis c virus, are highly sensitive as antibodies are present in large quantities in blood, and blood samples can be collected with a finger prick and put directly into the test without any prior processing. also, antibody detection can be rapid and easy to perform with minimal training required (table ) . however, the choice of the antibody target affects a test's effectiveness -unless the diagnostic target is specific to the intended infection, there is the potential for false positive results due to cross-reactivity, which is the case with dengue and zika immunoassays , . therefore, antibody detection assays should be interpreted within the appropriate epidemiological context and findings from physical examination. the other major disadvantage of antibody-based tests is that antibodies are usually a marker of exposure to a pathogen and cannot be used to distinguish between those with active and past infection, which is the case of the rk tests for visceral leishmaniasis (vl) . a presumptive diagnosis of active vl can only be made using a combination of a positive rk test and more than weeks of fever and splenomegaly. the same is true of most syphilis rdts, which detect long-lived antibodies to treponemal antigens and thus do not diagnose active syphilis. antibodies to a non-treponemal antigen, which appears with infection and declines in the months after successful treatment, are used in a flocculation assay called the rapid plasma reagin assay, which can yield a result in minutes but requires electricity to operate, a centrifuge for processing serum from whole blood, a shaker and cardiolipin as an antigen. a combination treponemal and non-treponemal rapid test has been developed in an immunochromatographic test in a lateral flow format with acceptable performance characteristics . antigen detection poc tests, such as those for chlamydia and gonorrhoea, suffer from three major drawbacks (table ) . first, the specimen used for sexually transmitted infections are usually from urethral, cervical or vaginal swabs that require multiple steps to solubilize the bacteria and free antigen before reaction with the capture antibody. this sometimes requires a heating step and adds to the complexity and cost of the poc test. urine is the preferred specimen in men with such infections, and current poc tests require a urine centrifugation step to concentrate the bacteria before processing and reaction. another common drawback of antigen detection tests is the potential for false positive results due to cross-reactivity with antigens from closely related bacterial or viral species. this is especially true if polyclonal antibodies are used as capture antibodies. finally, a common disadvantage of current antigen detection poc assays is low sensitivity, often requiring to bacteria for the rdt to become positive. malaria rdts are an exception to these drawbacks in that the parasites are present in large quantities in blood and no pre-processing or concentration steps are required. for most antigen detection poc tests, the low sensitivity may be due to low concentration of target antigens, inefficiency of extraction or limited optimization of reagents, which has hampered chlamydia and gonorrhoea tests , . however, gift et al. showed that rapid chlamydia tests with a sensitivity of % can lead to more infected patients being treated compared to nats, which require patients to return for their test results . they called this the rapid test paradox, which was also recently seen with a p assay for early infant diagnosis of hiv, where a sensitivity of % can still lead to a higher 'diagnostic yield' than nats performed on dried blood spots sent from remote antenatal clinics . nats offer a more sensitive and specific alternative to antigen detection assays as nucleic acid targets can be selected from a genome sequence with high specificity, and the amplification process to increase sensitivity can be fairly rapid (table ) . for example, the near-poc tb diagnostic involves primer-based amplification of mtb dna specifically, including regions associated with rif drug resistance. thus, nat tests are generally highly sensitive and more specific than antigen-or antibody-based tests, and may help inform on drug susceptibilities. while instruments are available for highthroughput screening of patients and subsidies can bring test costs down to us$ , the equipment (capable of four tests at a time) costs more than us$ , , which may be prohibitive in high-incidence regions. thus, a truly poc diagnostic based on nats without needing any equipment is still a promise and not a reality as yet , . of all the criteria originally accepted, the requirement of equipment-free is perhaps the only one which is not as critical as originally defined. a wide range of near-poc nats have been developed for use outside of laboratory settings and most of these assays are automated, requiring only - minutes of hands-on time and minimal training. these near-poc devices come in sealed units with internal self-calibration making it easier to ensure quality of testing and most are also equipped with data transmission capabilities, which make these platforms very attractive in the context of disease surveillance and epidemic preparedness. most of these near-poc devices have a broad menu of diagnostic targets which makes the capital cost of purchasing the device less of an issue. reagents are pre-measured and dried ready for hydration with the introduction of the specimen. however, near-poc nat cartridges are expensive to manufacture and not affordable to most developing countries without subsidies. rapid advances in miniaturization, material science, electronics and data transmission in recent years have made minimal instrumentation a reality for new diagnostics, including the use of smart phones, which are widely available, custom developed and low cost. the use of a dongle to connect a smart phone to a microfluidic disc to detect hiv and syphilis antibodies in finger-pricked blood allows the phone to power the reaction, interpret the results and transmit the data to a central database. this smart phone-based poc assay is currently undergoing clinical trials . use of a smart phone to power nats is in development and will play an important role in future poc applications , . in particular, there has been considerable interest shown in utilizing mobile phones as readers and connectivity for rdts using rfid (radio frequency identification) to prevent errors in subjective interpretation and transcription [ ] [ ] [ ] [ ] . while phone-based diagnostics are attractive as an option, regulatory approval and rapid updates of phone software that can affect test performance are important challenges. defining and quantifying risks and benefits. while successes need to be celebrated, significant challenges remain. first, as no diagnostic is perfect, countries continue to struggle with defining acceptable risks of false positive or negative results when a novel diagnostic technology may provide incremental clinical benefits. in the developing world where over % of the population reside in rural areas, and the sustainable development goals urge countries to provide universal health coverage and leave no one behind, the trade-off between accuracy (sensitive and specific) and accessibility (user friendly, rapid and deliverable) becomes paramount . while it impossible to give absolute values across different tests, both sensitivity and specificity remain critical. in selecting an ideal test, the positive and negative predictive values, which are dependent on the prevalence of the disease as well as the characteristics of the diagnostic test, should be taken into account, but few studies have been performed to determine quantitatively what tradeoffs are acceptable. an analysis for syphilis screening compared the use of a laboratory-based immunoassay with treponemal rdts in tanzania . it showed that a test that has % sensitivity but can only be used in sophisticated laboratories can realistically only be accessed by - % of the population, while a rapid lateral flowbased antibody test that has only % sensitivity, but can be used at all levels of the health care system effectively gives a correct diagnosis to as many as % of the population, highlighting that tests should not be evaluated purely on technical performance, but rather on diagnostic performance and clinical impact. training and quality assurance. assuring the quality of tests and testing is the biggest challenge when testing is decentralized at poc. there are many rapid tests from various manufacturers available, and test quality may impact accurate and inconsistent diagnosis across different sites, with studies showing that high rates of errors were observed even in performance of simple rdts for hiv and malaria , . quality of testing requires proficiency panels be sent to all the poc testing sites by a national reference laboratory. including positive and negative controls with each box of tests will allow all tests to be approved once they arrive at their destination and before first use. recently, nine african countries developed a national system for assuring the quality of poc testing for hiv , . with data connectivity from each testing site, quality assurance results can be linked to test results at each poc testing site at a centralized database to trigger alerts for corrective action. supply chain software can also be linked through these connectivity solutions, avoiding stock-outs. demonstrating the value of a novel diagnostic technology. the value of a diagnostic goes beyond the technology, as each test needs to be matched to its 'testing environment' , which includes population characteristics, prevalence of target diseases, comorbidities/coinfections and health system characteristics. national programmes should take advantage of the rapid turnaround time of assured tests to streamline patient pathways and increase the efficiency of the health care system. the roll-out of poc diagnostics requires the use of implementation science to ensure success , taking into account the cultural, behavioural, socioeconomic and health systems contexts. this includes a careful assessment of acceptability and feasibility linked to possible increased stresses on the health system/provider when testing is introduced into settings where thus far no or limited testing was performed. studies using the genexpert mtb/rif tb test showed that new diagnostic tests would not have the expected impact on outcomes if test introduction is not accompanied by changes in patient pathways or practices , . these studies highlight the importance of programmatic monitoring of the impact of novel technologies beyond studies that are usually conducted under controlled conditions. likewise, same-day testing and treatment using a syphilis rdt strengthened health systems in the amazon forest and rural china when policy makers were involved and testing was introduced within the appropriate social and cultural contexts , . for example, in peru, women normally need to present six times to the largest maternal hospital in lima for their prenatal syphilis screening to be completed and treatment provided, but the introduction of the rapid syphilis test streamlined testing and treatment to one visit, reducing patient out-of-pocket expenses and increasing the efficiency of a busy health care facility . there are few detailed analyses which have been conducted to accurately determine the economic impact of many of the available tests, including the diagnostic cost versus the overall economic impact. while these benefits are intuitively important, they are often difficult to quantify . it would, therefore, be extremely useful to have economic analysis tools for each of the major diseases or conditions, so that developers have an understanding of cost implications and what cost structures would be acceptable to health service implementers. as an example, it may be necessary to accept cost trade-offs when addressing global health threats (for example, zika, ebola, and so on), where speed of intervention is critical. this having been stated, low-cost diagnostic tests remain critical in resource-limited settings. in the past two decades, the biggest drivers of diagnostic development were well-resourced diseases such as hiv, malaria and tb. in recent years, the increasing frequency and severity of global health emergencies caused by infectious diseases of epidemic potential, such as severe acute respiratory syndrome (sars) coronavirus, middle east respiratory syndrome coronavirus (mers) co-v, ebola and zika virus, have made the global community realize the need to accelerate assured test development, validation, manufacturing and deployment. there is a need for innovation in rapid detection technologies that are assured but allow multiplex detection of a panel of pathogens such as major causes of respiratory illness, haemorrhagic fevers or enteric infections in a single specimen. these tests should not only identify the cause of outbreak but also be used to process multiple specimens with high throughput throughout the outbreak. another major driver of test development is the need for cheaper, better and faster tests that can be used at poc to reduce inappropriate antimicrobial use. an estimated , people die from untreatable conditions due to antimicrobial resistance (amr) every year worldwide , and if amr continues to spread, by , million people may die from resistant infections annually at an economic cost estimated at $ trillion. at the primary care level, a simple rapid test that can be used to distinguish bacterial from nonbacterial causes of fever, respiratory and enteric infections would allow health care providers to reduce antibiotic use and preserve them for future generations. the o'neill report also pointed out that a test that can identify a pathogen and its antibiotic susceptibility at the poc would allow the safe use of first-line drugs or drug combinations with savings to the health care system. to stimulate interest in innovation in diagnostic tests that can be used at poc, several countries have set up challenge prizes. in , the united kingdom announced the longitude prize of £ million, which seeks an affordable, accurate, fast and easy-to-use test for bacterial infections that will allow health professionals worldwide to administer the right antibiotics at the right time. the challenge is currently ongoing, with final submission by . more information available at: https://longitudeprize.org. in september , the us department of health and human services announced a challenge prize competition in which up to $ million will be awarded for one or more novel and innovative poc diagnostics that would have clinical and public health value in combating the development and spread of antibiotic resistant bacteria. more information is available at: https://dpcpsi.nih.gov/amrchallenge. these prizes may catalyse new technologies, but there are still no guarantees to the sustainability of these tests in resource-limited countries given that cost is a critical factor woven into every step needed to bring about and sustain testing. donors and funders of diagnostics, such as the the bill & melinda gates foundation, global fund, unitaid and aid agencies from national governments such as pepfar and dfid, play a critical role in incentivizing diagnostic innovation and addressing market dynamics. technology advances to support innovation. technology , markets and medical devices have matured to enable connected diagnostics to become a useful tool for epidemiology, patient care and tracking, research, and amr and outbreak surveillance. the ability to digitize data from laboratory and poc platforms, including lateral flow rdt results, can standardize the interpretation of results and allows data to be linked to proficiency testing to ensure testing quality, reducing interpretation and transcription errors. remote monitoring of poc instrument functionality and utilization through connectivity allows programmes to optimize instrument placement, algorithm adoption and supply management. alerts can be built into the system to raise alarm at unusual trends such as outbreaks. the application of mobile devices and related technologies to health care is improving patients' access to health information and treatment, offering possibilities to diagnose, track and assess the impact of infectious disease interventions across the world , . smart devices can also be used to automatically add data to a central database and allow systems to assess likelihood of a diagnosis or predict disease outbreaks through machine learning, providing a route to smart diagnostics that incorporates historical or epidemiologic data to make diagnoses more accurate. finally, new engineering fields such as the internet of things (iot), industry . and printed electronics [ ] [ ] [ ] [ ] promise to add significantly to the technologies that can be chosen and incorporated into new diagnostic devices. many of these technologies are aimed at high-volume and low-cost distributable manufacturing, thus fitting in well with the goal of poc diagnostics. new detectors and light sources also suggest that incorporating these into tests could allow for more accurate result reading and the possibility of multiplexing. while it is clear that the original assured criteria remain relevant, opportunities exist to improve future diagnostics by incorporating new technological elements to provide real-time quality control for testing and treatment and overcoming the difficulties in specimen collection and/or processing , which currently limits scaling-up of diagnostics in resource-limited areas. we therefore propose two additional criteria of r (real-time connectivity) and e (ease of specimen collection and environmental friendliness) into the original assured, to create a new acronym of reassured (table ) . to use testing to effectively survey or treat patients, it is critical to obtain and analyse results at the poc . however, one of the main challenges to poc testing is ensuring that results are rapidly provided to the patient once testing has been completed ; this is formidable challenge when testing is decentralized at hundreds or thousands of different sites by health care workers who are not consistently skilled in reading test results, leading to risk that incorrect data is being recorded and/or transmitted. one solution would be to allow the analysis of test results at a centralized level for consistent diagnosis and epidemiological surveillance. many manufacturers are now embedding connectivity into poc and near-poc instruments (for example, the alere pima poc cd device ). other manufacturers have developed innovative connectivity solutions that can use mobile phones to read the results from lateral flow assays and provide electronic result exchange, while a third option would be to include connectivity directly onto the test . ideally, connectivity should not only include collection and transmission of test data, but also analysis to provide feedback for immediate patient treatment or for surveillance. with the addition of barcodes, two-dimensional codes or electronic storage into tests, a number of other data points can be collected, including manufacturing information (for example, lot numbers), dates, expiry dates, stock availability and possibly even environmental conditions such as temperature and humidity under which the tests have been manufactured, transported, stored and used. for instrumented tests, maintenance and other machine data can be collected. thus, connectivity solutions can increase quality assurance for poc tests and would allow for centralized and real-time decision-making, even across tiered laboratory systems and during outbreak investigations and global health emergencies. moving forward, it is critically important to make provisions for poor or non-existent internet availability to support sustainability standards and that systems are developed with compatibility so that they can be linked into central databases. ease of specimen collection. specimen collection and processing may need further in-depth development. first, it is ideal to have noninvasive specimen collection given that more tests become available as self or home tests. second, samples come in many different formats (blood, urine, sputum, stool, swab, breath, and so on) and require different preparation depending on the test to be performed. this may include concentrating the sample (or possibly titration), purification, lysing of cells, target amplification, and so on. while most of these processes can be easily performed in the laboratory, there remain few solutions for collecting and performing these tasks at the poc . oral tests for hiv and hepatitis c virus are good examples of advances in non-invasive sampling, although the collection device is more expensive than blood collection via disposable capillary tubes. related, given that rapid lateral flow antigen detection tests often have lower sensitivity, extra processing steps such as heating or other forms of extraction are required when using specimens other than blood. environmental friendliness. advances in technology have made the assured requirement for equipment somewhat less daunting. however, in light of more diagnostic tests now being performed in both urban and rural areas, there could be more future effort devoted to the environmental impact of these tests. the cumulative effect of many tens of thousands of tests performed at remote sites away from laboratory infrastructure may pose health and environmental risks and put a strain on limited resources. some examples include the plastics used in current rapid tests that cannot be recycled and give out toxic fumes when burned, and testing cartridges that may even contain harmful chemicals and need to be disposed of properly. recyclable materials should be used, when possible, for the housing, substrate matrix materials, and reagents used in tests. paper, an obvious choice of substrate material that has many inherent advantages over standard plastic materials, will see increased usage, not only in lateral flow formats but other paper based formats as well such as in micro paperbased analytical devices (μ pads) [ ] [ ] [ ] . and recently, cell-free synthetic gene networks for in vitro applications at poc, have been achieved by freeze-drying cell-free systems onto paper, which increases stability at room temperature and can be easily transported and stored, and simply re-activated by adding water . other factors also need to be considered, such as disposal of test reagents, clinical samples and materials used for active components such as electronic tracks and electrodes. the assured criteria have been a valuable framework for developing devices and methods to detect major human diseases in challenging poc contexts. however, over the last decade, new technologies, not envisaged or available at the time of the first assured criteria definition, have given rise to the possibility of including new considerations into the next generation of devices and tests. we propose the acronym reassured for the design of future diagnostic tests to address important priorities such as global health emergencies and amr. an ideal test would be one that combines the best of both existing diagnostic worlds (instrumented laboratory based tests with lateral flow tests) and technologies currently being developed (fig. ) , which would provide an important extension to diagnostic laboratory systems and fulfil the sustainable development goals of 'no one left behind' in terms of effective health care service delivery. such tests can only be created by forming strong collaborative partnerships across many disciplinary boundaries, and we look toward a future when data connectivity linking cost-effective assured diagnostics to laboratory systems will form the backbone of health care systems and provide real-time data for evidence-based disease control and prevention strategies, more efficient health systems and improved patient outcomes. diagnostics for the developing world mapping the landscape of diagnostics for sexually transmitted infections: key findings and recommandations the costs of accessible quality assured syphilis diagnostics: informing quality systems for rapid syphilis tests in a tanzanian setting reducing the burden of sexually transmitted infections in resource-limited settings: the role of improved diagnostics the rapid test paradox: when fewer cases detected lead to more cases treated: a decision analysis of tests for chlamydia trachomatis access: how do good health technologies get to poor people in poor countries laboratory medicine in low-income and middle-income countries: progress and challenges strengthening national health laboratories in sub-saharan africa: a decade of remarkable progress global health diagnostics requirements for high impact diagnostics in the developing world rapid point-of-care hiv testing in pregnant women: a systematic review and meta-analysis expanding the role of diagnostic and prognostic tools for infectious diseases in resource-poor settings ensuring quality who-find malaria rdt evaluation programme availability and price of malaria rapid diagnostic tests in the public and private health sectors in : results from nationally representative cross-sectional retail surveys plasmodium falciparum parasites lacking histidine-rich protein and : a review and recommendations for accurate reporting minimising human error in malaria rapid diagnosis: clarity of written instructions and health worker performance improving community health worker use of malaria rapid diagnostic tests in zambia: package instructions, job aid and job aid-plustraining the global elimination of congenital syphilis: rationale and strategy for action (who, department of reproductive health and research untreated maternal syphilis and adverse outcomes of pregnancy: a systematic review and meta-analysis global estimates of syphilis in pregnancy and associated adverse outcomes: analysis of multinational antenatal surveillance data accelerating worldwide syphilis screening through rapid testing: a systematic review are treponema pallidum specific rapid and point-of-care tests for syphilis accurate enough for screening in resource limited settings? evidence from a meta-analysis introduction of rapid syphilis testing in antenatal care: a systematic review of the impact on hiv and syphilis testing uptake and coverage scaling down to scale up: a health economic analysis of integrating point-of-care syphilis testing into antenatal care in zambia during pilot and national rollout implementation introduction of syphilis point-of-care tests, from pilot study to national programme implementation in zambia: a qualitative study of healthcare workers' perspectives on testing, training and quality assurance avoiding hiv and dying of syphilis elimination of mother-to-child transmission of hiv and syphilis (emtct): process, progress, and program integration global burden of maternal and congenital syphilis in and : a health systems modelling study celebrating the decline in syphilis in pregnancy: a sobering reminder of what's left to do a systematic review and meta-analysis of studies evaluating the performance and operational characteristics of dual point-of-care tests for hiv and syphilis who guideline on syphilis screening and treatment for pregnant women diagnostic accuracy of a low-cost, urine antigen, point-of-care screening assay for hiv-associated pulmonary tuberculosis before antiretroviral therapy: a descriptive study rolling out xpert mtb/rif® for tuberculosis detection in hiv-positive populations: an opportunity for systems strengthening advances in tuberculosis diagnostics: the xpert mtb/rif assay and future prospects for a point-of-care test perspectives on advances in tuberculosis diagnostics, drugs, and vaccines the impact of the roll-out of rapid molecular diagnostic testing for tuberculosis on empirical treatment in cape town drug-resistant tuberculosis: time for visionary political leadership implementation of xpert mtb/rif for routine point-of-care diagnosis of tuberculosis at the primary care level evaluation of diagnostic tests: dengue innovative and new approaches to laboratory diagnosis of zika and dengue: a meeting report visceral leishmaniasis: what are the needs for diagnosis, treatment and control? metaanalysis of the performance of a combined treponemal and nontreponemal rapid diagnostic test for syphilis and yaws systematic reviews of point-of-care tests for the diagnosis of urogenital chlamydia trachomatis infections performance and operational characteristics of point-of-care tests for the diagnosis of urogenital gonococcal infections the rapid test paradox: when fewer cases detected lead to more cases treated: a decision analysis of tests for chlamydia trachomatis sex point-of-care p infant testing for hiv may increase patient identification despite low sensitivity emerging technologies in point-of-care molecular diagnostics for resource-limited settings a simple, inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings expert review of molecular diagnostics portable devices and mobile instruments for infectious diseases point-of-care testing a smartphone dongle for diagnosis of infectious diseases at the point of care point-of-care diagnostics in low resource settings: present status and future role of microfluidics the present and future role of microfluidics in biomedical research novel developments in mobile sensing based on the integration of microfluidic devices and smartphones integrating biochemiluminescence detection on smartphones: mobile chemistry platform for point-of-need analysis lateral flow technology for field-based applications-basics and advanced developments using electronic readers to monitor progress toward elimination of mother-to-child transmission of hiv and syphilis: an opinion piece leaving no one behind: a neglected tropical disease indicator and tracers for the sustainable development goals the trade-off between accuracy and accessibility of syphilis screening assays ensuring quality and access for malaria diagnosis: how can it be achieved? assuring the quality of diagnostic testing: the future is now external quality assurance for hiv point-ofcare testing in africa: a collaborative country-partner approach to strengthen diagnostic services implementation science: the laboratory as a command centre cost-effectiveness of xpert mtb/rif for tuberculosis diagnosis in south africa: a real-world cost analysis and economic evaluation xpert mtb/rif versus sputum microscopy as the initial diagnostic test for tuberculosis: a cluster-randomised trial embedded in south african roll-out of xpert mtb/rif point-of-care tests to strengthen health systems and save newborn lives: the case of syphilis rapid syphilis tests as catalysts for health systems strengthening: a case study from peru existing and emerging technologies for point-ofcare testing tackling drug-resistant infections globally: final report and recommendations expert review of molecular diagnostics: the impact of digital technologies on point-of-care diagnostics in resource-limited settings mobile health to improve tuberculosis care and control: a call worth making bringing mhealth connected infectious disease diagnostics to the field integrating electronics and microfluidics on paper from smartphones to diagnostics : low cost electronics for programmable digital microfluidics and sensing a flexible future for paper-based electronics biosensors: sense and sensibility rapid diagnostic tests for neurosyphilis compounding diagnostic delays: a qualitative study of point-of-care testing in south africa data connectivity: a critical tool for external quality assessment functional screen printed radio frequency identification tags on flexible substrates, facilitating low-cost and integrated point-of-care diagnostics a low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami sensing approaches on paper-based devices: a review diagnostics for the developing world: microfluidic paper-based analytical devices recent developments in paper-based microfluidic devices paper-based synthetic gene networks printed microfluidic channels the authors declare no competing interests. reprints and permissions information is available at www.nature.com/reprints.correspondence should be addressed to x.-s.c. or r.w.p.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -repm vw authors: ungchusak, kumnuan; heymann, david; pollack, marjorie title: public health surveillance: a vital alert and response function date: - - journal: the palgrave handbook of global health data methods for policy and practice doi: . / - - - - _ sha: doc_id: cord_uid: repm vw ungchusak, heymann and pollack address the critical global issue of public health surveillance. they describe how epidemiologists collect and use surveillance data to detect unusual events or outbreaks and to guide control programmes. drawing on their combined international experience, the authors explain the vital role that data play in alerting authorities to respond to outbreaks such as severe acute respiratory syndrome, ebola, zika virus and avian influenza. they point to the importance of sharing information globally while ensuring equal benefits to providers of data, coordinating surveillance activities across sectors, building capacity for surveillance and coordinating national surveillance activities. the authors emphasise the need for enhanced global cooperation to prepare for future public health emergencies of international concern. a three-month delay in identifying the outbreak of ebola virus in rural guinea in late resulted in its rapid spread to urban areas and to neighbouring liberia and sierra leone [ ] . once local and international responders identified the virus, they took a year to interrupt its widespread transmission. by april , ebola had accounted for more than , cases and over , deaths. people around the world watched with increasing alarm, as this tragic course of events played out, and with concern that air travel could enable the virus to spread across continents. this epidemic highlighted not only the inadequacy of local health systems to recognise and respond but also that international organisations were not ready to provide timely expertise and resources to control the situation and ameliorate the virus's spread through the region. had health officials identified ebola in west africa promptly, they could have minimised its impact on the lives and livelihoods of the populations of west africa by implementing appropriate control procedures. public health officials coined the term surveillance to describe systems they set up to watch out for and control occurrence of health threats. just as police, for example, set up closed-circuit television devices and community watch programmes to detect and prevent crime, public health surveillance systems engage all possible means to detect unwanted health events and prevent them from escalating and damaging population health. while public health surveillance originated to control spread of infectious diseases such as plague and cholera, it has evolved to include some non-communicable diseases, occupational health and injuries as well as surveillance of biological, behavioural and social determinants of these conditions. we start by reviewing the public health need for surveillance and the development by the international community of regulations to control infectious diseases and other public health emergencies of international concern (pheic). we describe how epidemiologists use surveillance data to detect unusual events or outbreaks and to guide control programmes, and we provide guidance about maintaining data quality. we examine networks that contribute to global surveillance systems and highlight the role of social media and information technology in providing data to monitor new events of international importance. we consider challenges facing epidemiologists responsible for surveillance and describe efforts to address them. public health surveillance is vital to the functioning of national and global health systems. policymakers and health administrators need surveillance information to set priorities to address population health problems, allocate resources and monitor progress of prevention and control programmes; they need surveillance systems to alert them immediately of public health threats. emerging infectious diseases, such as avian influenza of different subtypes, severe acute respiratory syndrome (sars) coronavirus, pandemic influenza h n and the zika virus (zikv) have the potential to spread rapidly causing severe loss of life and to impact socio-economic activity, especially trade and travel [ ] . the outbreak of sars in november highlighted the importance of every country having functioning and connected surveillance systems (see box . ). surveillance requires high-level government support, well-trained health workers, strong health information systems, well-functioning laboratories, effective communication systems and operational health facilities. to be effective, surveillance systems also require a strong legal framework to ensure that individual data can be shared while maintaining confidentiality as far as possible. global cooperation between countries, with up-to-date international health agreements to build and maintain these capacities, is essential to decrease risk of international spread of infectious diseases and contain the risk of bio-terrorism. sars originated in wildlife and spread silently among humans as atypical pneumonia in guangdong province, china, two months before officials became aware of it. authorities began surveillance to identify atypical pneumonia cases but this, and the containment response, were too late to stop sars spreading. a chinese urologist who was infected travelled to hong kong and spread sars to another persons. within weeks, sars spread to countries with more than , reported cases ( fig. . ) [ ] . by the end of the epidemic in july , sars had killed people [ ] . although unable to contain the outbreak of sars, the international community was able to bring the epidemic under control within six months-by collaborating across countries to identify and isolate all probable cases. nevertheless, the asian development bank estimated that the economic loss due to sars in affected countries was up to us $ billion with us$ . billion on mainland china (approximately . per cent of its annual gross domestic product (gdp)) and us$ . billion in hong kong (approximately per cent of its annual gdp) [ ] . plague ravaged europe during the fourteenth century and although authorities had no cure, they realised it was important to swiftly identify and isolate cases to prevent and control this lethal condition. authorities understood that international spread of such diseases followed cross-border trade, pilgrimage and war; and so prevention of disease was a national security issue. in the city-state of venice, authorities instigated quarantine measures-keeping arriving ships in the harbour for days before docking, and holding people in isolation for days at land borders to prevent entry of plague [ ] . in the mid-nineteenth century, recognising that quarantine measures were not enough, governments agreed international conventions aimed at stopping spread of plague and cholera-and two other infectious diseases, yellow fever and smallpox. the conventions required each country to report outbreaks of these diseases to all signatories of the convention, and permitted application of certain public health measures at international borders once a country reported of one of the diseases. in the early twentieth century, governments in the americas and in europe set up regional conventions called international sanitary bureaus. in , the newly formed world health organization (who) led establishment of the international sanitary regulations (isr) to foster global cooperation in reporting and acting at international borders to guard against spread of cholera, plague, yellow fever and smallpox. in , the who replaced the isr with the international health regulations (ihr) which required countries to report any cases of cholera, plague, yellow fever and smallpox to who [ ] . if a country reported one of these diseases, other countries could apply pre-established control measures at international borders-such as a requirement of proof of vaccination against yellow fever of any passenger arriving from a country that reported yellow fever to who. some countries reported to who late, or not at all, because of lack of capacity for public health surveillance, or because of fear of stigmatisation and economic repercussions. after hiv spread across international borders before being identified in , the international community realised that infectious diseases could not be stopped at borders. diseases often cross borders while still being incubated in humans, or in non-human hosts-insects, animals, and food and agricultural goods. in , after the sars outbreak, who updated and revised the ihr as a legal framework to include more diseases, and developed real-time evidence-based recommendations for prevention and control of outbreaks. who evaluates each newly identified outbreak for its potential to become a pheic by the country in which it is occurring. the ihr mandate who member countries to report immediately the occurrence of a single case of four diseases (smallpox, poliomyelitis due to wild type poliovirus, human influenza caused by a new subtype, and sars) [ ] . even though the world eradicated smallpox in , the ihr still maintain it on the list to cover the risk of the virus escaping from a laboratory. each country has an additional list of diseases that it requires its health workers to report by law. diseases of greatest public health threat are reportable, meaning that health workers or laboratory technicians must report individual cases as they occur. reportable diseases include those required by ihr and, for example, anthrax, cholera, ebola, legionellosis, plague and the zikv. other conditions are notifiable, meaning that health workers should report the number of cases that have occurred in a given time period. the number, frequency of reporting and breakdown of reportable and notifiable diseases varies by country. diarrheal cases, influenza cases, tuberculosis, aids and other significant endemic diseases are usually required to be notified to local health authorities. in some countries the notifiable list can include non-infectious conditions such as maternal or infant deaths. the ihr require countries to develop core capacities in public health, including surveillance systems and epidemiology services, that can analyse and act on surveillance information to detect and respond to diseases where and when they occur so that their potential to spread internationally is decreased. the purpose of surveillance activities is to: ( ) detect at an early stage, acute public health threats from all hazards-biological, chemical, radiation, natural disaster and deliberate acts-which require rapid investigation and response; and ( ) guide control programmes by measuring disease burden, monitoring trends, describing disease distribution and evaluating public health programme effectiveness (see table . ). the structure of government responsibilities for public health surveillance varies across countries. most often, countries set up dedicated early warning and rapid response surveillance teams that work with or complement surveillance activities of vertical control programmes such as malaria, hiv/aids or tuberculosis. surveillance and response teams detect early stage public health threats while control programmes gather disease (or condition) specific information to plan activities. control programmes share information with surveillance teams as required. a national network of public health laboratories, often linked to international reference laboratories, confirms etiologic agents, genetic strains and antibiotic resistance patterns. surveillance activities are said to be active when health workers pro-actively seek out cases and passive when the system relies on patients to report themselves to a clinic. using standard case definitions, health workers report individual cases of reportable and notifiable diseases to the local or national surveillance centre where staff aggregates reports, and clean and analyse the data. in cleaning the data, staff look for coding and classification errors, and for duplicate reports. epidemiologists analyse the data to determine how many new cases have occurred during the past day or week and their distribution in time, place and by person to see whether the magnitude and pattern of the disease under surveillance is changing. they note any changes in frequency, clustering or distribution and flag them for verification and explanation. box . illustrates how careful data analysis led to malaysia identifying nipah virus in [ ] . reporting of specific information about cases or patients or behaviour of populations under surveillance produces indicator-based data, that is individual or aggregated data derived from patients diagnosed-by syndrome description, clinical or laboratory confirmation-and identified through routine collection or active case search. the surveillance unit will also use eventbased data about outbreaks, unusual events or changes in human exposure [ ] . rather than wait for official reports, the surveillance team gathers information and rumours through the media, internet and unusual events reported by the community, and investigates these reports. the team captures abnormal health events in real-time and confirms potential outbreaks by triangulating these data with indicator-based data. epidemiologists responsible for surveillance use standard epidemiological methods to analyse trends, identify clusters and investigate suspected risk factors (see chap. for an overview of epidemiological methods). for example, high numbers of reported cases of kaposi sarcoma among young men in new york and california during the early s led to an investigation which showed a japanese encephalitis commonly occurs in school-age children of both sexes. there is a seasonal pattern of disease related to the rainy season when transmission and therefore disease occurrence, increases; there is no difference in occurrence between ethnic or religious groups. from september to april , surveillance teams sent reports of cases of febrile encephalitis ( per cent fatal) to the malaysian ministry of health [ ] . initially, the ministry considered japanese encephalitis virus to be the probable etiologic agent for this outbreak, and instituted conventional interventions of vaccination and insecticide to control mosquitoes. when they examined the surveillance data closely, the epidemiological pattern of encephalitis cases was different to what they expected-the disease occurred mostly among male adults of chinese ethnic origin whose occupations related to pig farming. the ministry sought a different cause and found the etiologic agent to be a new paramyxovirus, later named nipah virus. common risk factor of homosexual behaviour and its relationship with hiv/ aids [ ] . using increasingly sophisticated technologies for data capture and analysis, surveillance teams can monitor real-time occurrence, in time and place, of unusual events such as cholera or legionella, or seasonal outbreaks such as malaria (see chap. for an introduction to spatial and spatio-temporal techniques and to chap. which discusses predicting climate-related health outcomes such as malaria). once epidemiologists have concluded their analyses (sometimes in realtime), they prepare reports which can trigger immediate action by a rapid response team to visit the site of the events, investigate the situation and contain the outbreak. the team also sends reports to clinicians in hospitals and to local and national programme managers. many countries publish weekly disease surveillance reports that are also available to the general public: for example, the us centers for disease control and prevention (cdc) publish the morbidity and mortality weekly report (mmwr) [ ] , the european centre for disease control (ecdc) publishes eurosurveillance [ ] , and the who publishes the weekly epidemiological record [ ] . box . shows how epidemiologists associated microcephaly with zikv which led who to declare zikv a pheic [ ] . public health surveillance guides control programmes by undertaking the following functions: in late , zikv spread rapidly through latin america especially in brazil and el salvador. surveillance of birth defects in brazil identified a major increase in microcephaly during the period when zikv transmission increased. this alerted policymakers and epidemiologists to study whether the increase in birth defects was associated with zikv infection during pregnancy. who declared the suspected increase in microcephaly in association with zikv infection of pregnant women a pheic and recommended pregnant women to protect themselves from mosquito bites and to avoid travel to areas with known zikv transmission. the observation that men who travelled to areas with known zikv transmission could sexually transmit zikv to their partners led who to recommend practising safer sex or abstinence for a period of six months for men and women returning from areas of active transmission. its epidemiological patterns disease in humans results from interactions between the human host and causative agents or hazards of all types. the natural and socio-economic environment influences these interactions. diseases usually occur in the same pattern when there is no change in the causative agent (such as mutation), in the human host (such as vaccination) or in the environment (such as climate change). a surveillance system can closely monitor any changes in these dynamic factors and their consequences, as illustrated by the case of nipah virus in malaysia (box . ). public health surveillance must also address risk. for example, surveillance of annual per capita cigarette consumption in the us showed an increased trend from cigarettes in to , cigarettes in . researchers related this trend to advertising and an expansion in the number of cigarette companies. in , after the first studies suggesting cigarette consumption was related to lung cancer, and the us surgeon general issued a warning, the annual per capita consumption decreased to , [ ] . with surveillance information, epidemiologists can forecast an increase in lung cancer without intervention thereby providing evidence for policy to implement effective interventions such as taxation to prevent smoking. evaluating performance of control programmes after they have implemented interventions, health authorities use surveillance data to see if disease incidence declines. for example, when vaccine coverage increases, the number of cases of vaccine preventable diseases is expected to decrease. increasing taxes on cigarettes is one way to reduce consumption. surveillance data can document a correlation between increasing taxes and decreasing trends in cigarette consumption. to achieve these functions, programme managers collect data through patient records, surveys, programme records or informal sources. types of data include determinants of the condition, behaviours or risk factors associated with the condition, morbidity and mortality associated with the condition, programme responses, and abnormal or unusual events associated with the condition. table . provides examples of these types of data for surveillance of an hiv/aids control programme. to ensure surveillance programmes have adequate resources and produce useful information, public health authorities regularly review their surveillance activities. in , the us cdc issued guidelines to evaluate surveillance systems which, with some updating, are still widely used [ ] . these guidelines focus evaluation of public health surveillance on three areas: ( ) the surveillance system itself, describing the system, its structure, diseases under surveillance, sources of data, and how data are processed, analysed and disseminated; ( ) the resources used to operate the system, including funding sources, adequately trained staff and information technology; and ( ) the usefulness and quality of surveillance information, using the following indicators: usefulness of data do the data and information disseminated to data providers and users contain comprehensible facts and findings and useful recommendations to improve control measures and guide programme management? has the system detected outbreaks? how many of the detected outbreaks were investigated and controlled in a timely manner? timeliness of data and other information is data dissemination timely and regular? for example, epidemic prone diseases require weekly summary, while other diseases require only monthly or quarterly summaries. are these requirements met? validity and completeness of data much of the data come from clinical diagnoses that do not have laboratory confirmation. it is useful to conduct studies to determine the accuracy of diagnoses using standard laboratory confirmation testing. this helps in preparing estimates of the proportion of confirmed cases among all reported cases. when undertaking field investigations, investigators can compare the number of actual cases they find with the number of cases reported through the system. this provides an estimate of reporting completeness of the system. global public health surveillance is the collection, analysis and use of standardised information about health threats or their risk factors from more than one country, and usually worldwide. while surveillance mainly focuses on infectious diseases, global systems also seek to identify deliberate use of biological agents or toxins to cause harm. who leads the global public health surveillance system, gathering information from formal and informal sources working through its country and regional offices. who extends its reach through the global outbreak and response network (goarn) [ ] which comprises over national technical institutions that support who to detect public health threats and respond to outbreaks. who uses the information for risk assessment and analysis as part of its routine disease control and prevention programme activities. when requested by countries for support, who works with goarn institutions to recruit suitable experts. goarn includes regional networks of countries that cooperate independently to prevent and control infectious diseases occurring in their regions, for example, the east african integrated disease surveillance network (eaidsnet), [ ] and the mekong basin disease surveillance network (mbds) [ ] . who leads global networks that work to control specific diseases. these networks depend on cooperation of governments, public health workers and scientists to report cases, provide specimens and share information so that specific diseases can be controlled globally. these include: networks to support influenza control through vaccine development the global influenza surveillance and response system (gisrs) consists of national sentinel centres and national and regional laboratories which annually collect , - , nasal swabs from patients presenting with influenza-like illness. their analyses provide information about the distribution of strains circulating each year and enable scientists to recommend the influenza vaccine composition for the following year based on predominant sequences. gisrs also uses flunet, a public web-based data collection and reporting tool that tracks movement of influenza viruses globally and provides epidemiological data about influenza outbreaks [ ] . initiative. clinical health workers and epidemiologists report all cases of acute flaccid paralysis (afp) in children under years of age from whom they have collected stool specimens for isolation and identification of the poliovirus. through its network of national, regional and specialised laboratories, gpln determines whether polio was the cause of the afp, genetically sequences viruses and compares them to a global database to understand their geographic source. if a polio virus is found, gpln informs the national authority and who regional office for appropriate action. project on anti-tuberculosis drug resistance surveillance [ ] is a common surveillance platform to which countries can provide data that are then used to monitor the evolution and spread of multi-drug resistant tuberculosis (mdr-tb) and extensively drug-resistant tuberculosis (xdr-tb). national laboratories provide susceptibility testing of tuberculosis organisms collected from patients, supported by a supranational tuberculosis reference laboratory network. the global project provides understanding of the prevalence and distribution of tuberculosis resistance worldwide. [ ] . its goal is to develop a standardised strategy to collect, analyse and share clinical, laboratory and epidemiological data globally, assess the burden and support local, national and global strategies to control amr. until recently, surveillance systems depended on paper-based reporting, compilation and analysis of data. computers and electronic reporting have made compilation and analysis of data much easier, and the world wide web (www) and the internet improve the comprehensiveness of reporting. digital and internet-based technology can retrieve information from medical records on a daily basis-but this must be done without infringing personal privacy. hospitals, especially private ones, may refuse to provide patient information to the public health sector unless privacy issues are addressed. cell phone technology has extended the scope of informal and event-based surveillance while social media has transformed exploring rumours of new events. some ground-breaking examples of the use of information and communication technology include: electronic reporting of events the programme for monitoring emerging diseases (promed-mail) is a fully moderated internet-based listserv, that receives and publishes reports of public health events in humans, animals, wildlife and plants from its subscribers and other traditional and nontraditional information sources [ ] . promed-mail uses information available on the www and from voluntary listserv reporters who actively search for and report public health events in realtime from the media, internet blogs and other sites. promed-mail editors and expert moderators review, analyse, evaluate and where possible validate reports, and then disseminate them to listserv members and post them on its website. using big data to identify events the subscription-based application global public health intelligence network (gphin) continuously scans the www gathering information from multiple source news aggregators in real-time [ ] . gphin searches in nine languages for key words that could indicate infectious disease outbreaks, or environmental, radioactive and natural disasters. analysts identify new events and inform subscribers-who are governmental and non-governmental agencies with an established public health mandate. every hours, analysts communicate new information to who which validates reports through its network of regional and country offices. who discusses events that it validates in confidence with health departments in the countries involved. mapping events in real-time healthmap, a fully automated application, utilises online informal sources for disease outbreak monitoring and real-time surveillance of emerging public health threats [ ] . healthmap trawls www sources of information (in nine languages) including online eyewitness reports, expert-curated discussions such as promed-mail, validated official reports, for example from who, or the food and agriculture organization of the united nations, and news aggregation services such as google news. using open source software, healthmap displays the events by time, geographic location and aetiology. participatory flu tracking diseases and abnormal events happen all the time in the community. only some patients, especially those presenting with severe disease manifestations, seek medical care. flu near you invites anyone living north america, over years of age, to report if they have an influenza-like illness [ ] . once registered, participants are asked weekly by e-mail to complete a brief survey that seeks information on ten symptoms linked to influ-enza, and other information such as whether or not the registered participant has had an influenza vaccination. other countries, including the uk, have adopted similar participatory influenza surveillance systems, thereby adding a greater understanding of the epidemiology of influenza around the world. participatory onehealth disease detection (podd) chang mai university in thailand, with support from the skool foundation, developed this mobile application which connects volunteers in local governments. when volunteers notice an abnormal event such as poultry dying off or sickness in animals or humans, they use podd to notify local authorities who dispatch a surveillance and rapid response team to investigate and contain the event. after months of implementation, podd has enabled the detection of , abnormal events, including chicken high-mortality outbreaks, four cattle disease outbreaks, three pig disease outbreaks and three fish disease outbreaks, all of which were detected and controlled [ ] . since revision of the ihr in , outbreaks due to infections, including the middle east respiratory syndrome coronavirus and ebola virus, have highlighted continued weaknesses in public health surveillance and response capacities in most countries, with international spread causing disruptions in trade and travel, and negatively impacting economies. we present some challenges and suggest some solutions. most countries have established disease control programmes each with a surveillance component reporting from grassroots through provincial and national levels. national surveillance units may have sufficient staff for each disease control programme, but at lower levels of the health system, the same individuals often manage more than one programme and are heavily burdened by reporting requirements. there is also duplication of effort in reporting between programmes. who supports countries to coordinate surveillance activities across departments, programmes and administrative levels through integrated disease surveillance and response (idsr) [ ] . idsr links surveillance with other health information activities and strengthens overall capacity of countries to maintain public health surveillance. the ihr obligates countries to develop comprehensive disease surveillance, detection and response when and where infectious diseases and other acute public health threats occur. in reality, national surveillance capacity in many countries is still not at expected and necessary levels. this may be, as the ebola epidemic demonstrated in west africa, that health systems are weak and under-funded, or that the surveillance system itself does not function efficiently. regular evaluation of the system, as we describe in sect. . , can identify which components need to be strengthened. an over-riding issue is for the system to deploy and maintain enough professionals throughout the system with the required skills-understanding the nature and limitations of the data they are working with and able to interpret and draw important findings from the analyses of the surveillance data. approximately per cent of newly identified human diseases are zoonotic in origin [ ] and per cent of these diseases have their origins in wildlife [ ] . since the outbreak of h n avian influenza in hong kong, animal surveillance and human surveillance units have begun to share information and alert each other of unusual events. environmental factors are also crucial to disease occurrence, for example, paralytic shellfish poisoning among people who consume shellfish affected by harmful algae growth in the sea [ ] . the one health approach involves sharing information between multiple health sectors and working together to identify and resolve outbreaks [ ] . during the avian influenza outbreak, who requested all affected countries to share the virus isolated from humans for further study and vaccine development. some governments expressed concern about potential negative economic consequences of sharing information and about possible inequities in the benefits of sharing. this led to the jakarta declaration on responsible practices for sharing avian influenza viruses and resulting benefits [ ] . this declaration underlined need for continued open, timely and equitable sharing of information, data and biological specimens related to influenza; it also emphasised need for more equitable sharing of benefits for example in the generation of diagnostics, drugs and vaccines. the jakarta declaration led to the pandemic influenza preparedness framework (pip) under which manufacturers of influenza vaccines, diagnostics and pharmaceuticals that use gisrs information make annual financial contributions to who. who uses approximately per cent of these contributions for pandemic preparedness activities and surveillance, and per cent for pandemic response including purchase of vaccines and antivirals at the time of a pandemic for countries without access to these supplies. in may , the chatham house centre on global health security, after a series of roundtable consultation with experts in public health surveillance, produced a guide on strengthening data sharing for public health surveillance. this guide facilitates both informal and formal data sharing. the guide proposes seven principles: building trust; articulating the value; planning; using quality data; understanding the legal context; coming to agreement; and evaluating. the guidelines help create the right environment for data sharing and to facilitate good practice in addressing technical, political, ethical, economic and legal concerns that may arise. the guidelines aim to ensure, to the greatest extent possible, that any benefits arising from use of the data are shared equitably [ ] . similar to clinical or public health practice, institutions or agencies responsible for public health surveillance need a set of ethical principles to guide their operations. the who guidelines on ethical issues in public health surveillance proposed guidelines [ ] . these guidelines fall into three major groups: first, the mandate and broad responsibility of the agency to undertake surveillance and subject it to ethical scrutiny; second, the obligation to ensure appropriate protection and rights of individuals under surveillance; and third, considerations in making decisions about how to communicate and share surveillance data to pursue common good and equity of population without harm to individual. the west african ebola outbreak provided a costly lesson that policymakers must commit to establishing, maintaining and advancing public health surveillance systems to protect and promote population health. to prepare for the next major outbreak, the world needs to invest in a strong warning and response system led by a global institution with sufficient authority and funding to react swiftly [ ] . who serves this role but is chronically underfunded. similar investment is needed in countries where a fully supported, well-functioning surveillance office or programme must coordinate different components of the surveillance system. surveillance information should be disseminated widely to alert the public and health programmes of outbreaks so that they can contain the disease at source before it spreads internationally. because the world urgently needs reliable and timely surveillance information, public health surveillance should continue to make innovative use of new technology to gather and share information strategically and fairly. • the ebola outbreak highlighted inadequacies of national and global surveillance systems to detect and respond to public health threats. • surveillance provides critical data and information to guide, improve and protect public health. • more trained staff are needed for effective and efficient surveillance especially in low-and middle-income countries. • innovative use of information technology and social media can aid detection of public health threats. one year into the ebola epidemic: a deadly, tenacious and unforgiving virus: world health organization emerging infectious diseases in . years after the institute of medicine report. mbio sars molecular epidemiology: a chinese fairy tale of controlling an emerging zoonotic disease in the genomics era the legacies of sars -international preparedness and readiness to respond to future threats in the western pacific region. western pacific surveillance and response journal sars economic impacts and implications. economic and research department policy brief no . asian development bank lessons from the history of quarantine, from plague to influenza a. emerging infectious diseases world health organization. the international health regulations world health organization early detection, assessment and response to acute public health events: implementation of early warning and response with a focus on event-based surveillance: interim version who/hse/gcr/lyo/ . world health organization world health organization. who director-general summarizes the outcome of the emergency committee regarding clusters of microcephaly and guillain-barré syndrome achievements in public health, - : tobacco use-united states updated guidelines for evaluating public health surveillance systems the global outbreak alert and response network. global public health east african integrated disease surveillance network mekong basin disease surveillance network global polio laboratory network world health organization. surveillance of drug resistance in tuberculosis global anti-microbial resistance surveillance system (glass) epidemic intelligence -systematic event detection podd: an innovative one health surveillance system preventing pandemics with animal origins training programs in epidemiology and public health interventions network risk factors for human disease emergence global trends in emerging infectious diseases woods hole oceanographic institution. harmful algae one health initiative. one health initiative will unite human and veterinary medicine jakarta declaration on virus sharing: a strategic step to more equitable and affordable avian flu vaccines distribution a guide to sharing the data and benefits of public health surveillance. london (uk): the royal institute of international affairs chatham house world health organization. who guidelines on ethical issues in public health surveillance the next epidemic-lessons from ebola key: cord- -a acr o authors: koch, r. m.; diavatopoulos, d. a.; ferwerda, g.; pickkers, p.; de jonge, m. i.; kox, m. title: the endotoxin-induced pulmonary inflammatory response is enhanced during the acute phase of influenza infection date: - - journal: intensive care med exp doi: . /s - - - sha: doc_id: cord_uid: a acr o background: influenza infections are often complicated by secondary infections, which are associated with high morbidity and mortality, suggesting that influenza profoundly influences the immune response towards a subsequent pathogenic challenge. however, data on the immunological interplay between influenza and secondary infections are equivocal, with some studies reporting influenza-induced augmentation of the immune response, whereas others demonstrate that influenza suppresses the immune response towards a subsequent challenge. these contrasting results may be due to the use of various types of live bacteria as secondary challenges, which impedes clear interpretation of causal relations, and to differences in timing of the secondary challenge relative to influenza infection. herein, we investigated whether influenza infection results in an enhanced or suppressed innate immune response upon a secondary challenge with bacterial lipopolysaccharide (lps) in either the acute or the recovery phase of infection. methods: male c bl/ j mice were intranasally inoculated with × ( ) pfu influenza virus (ph n , strain a/netherlands/ / ) or mock treated. after (acute phase) or (recovery phase) days, mg/kg lps or saline was administered intravenously, and mice were sacrificed min later. cytokine levels in plasma and lung tissue, and lung myeloperoxidase (mpo) content were determined. results: lps administration days after influenza infection resulted in a synergistic increase in tnf-α, il- β, and il- concentrations in lung tissue, but not in plasma. this effect was also observed days after influenza infection, albeit to a lesser extent. lps-induced plasma levels of the anti-inflammatory cytokine il- were enhanced days after influenza infection, whereas a trend towards increased pulmonary il- concentrations was found. lps-induced increases in pulmonary mpo content tended to be enhanced as well, but only at days post-infection. conclusions: an lps challenge in the acute phase of influenza infection results in an enhanced pulmonary pro-inflammatory innate immune response. these data increase our insight on influenza-bacterial interplay. combing data of the present study with previous findings, it appears that this enhanced response is not beneficial in terms of protection against secondary infections, but rather damaging by increasing immunopathology. patients with influenza infection often suffer from severe secondary bacterial infections, which are associated with high morbidity and mortality rates [ , ] . a striking example of this relationship was provided by bacteriological and histopathological analysis of infected lung tissue obtained from people who died of influenza during the - "spanish flu" pandemic, in whom bacterial pneumonia was found to be the predominant cause of death [ ] . these data suggest that an influenza infection profoundly influences the immune response upon a secondary bacterial infection. several studies have evaluated immunological interactions between influenza and bacterial infections, including infections with gram-negative bacteria [ ] . in vitro studies in which influenza-infected alveolar macrophages were subsequently stimulated with bacterial lipopolysaccharide (lps), a bacterial compound that induces a profound innate immune response, revealed increased levels of pro-inflammatory cytokines tumor necrosis factor (tnf) α, interleukin (il)- β, and il- [ ] [ ] [ ] [ ] [ ] , indicative of a priming effect on these cells by influenza. data from in vivo animal studies are ambiguous. similar to the in vitro data, some report enhanced responses. for instance, influenza infection in mice was shown to enhance the inflammatory response and neuropathogenicity resulting from lps administration on days and after influenza inoculation [ ] . likewise, murine influenza infection resulted in increased levels of pro-inflammatory cytokines in both plasma and lungs, and enhanced pulmonary neutrophil influx upon pneumococcal infection days later [ ] . similar results were observed in mice days after influenza infection [ ] . however, two otherwise largely comparable studies demonstrated reduced pulmonary pro-inflammatory cytokine concentrations upon streptococcus pneumoniae and staphylococcus aureus infections in mice infected with influenza days before, indicative of influenza-induced immunosuppression [ , ] . these equivocal results may be due to differences in the severity or kinetics of the influenza infection or the use of different bacteria as secondary challenges, thereby targeting various complex multi-receptor signaling pathways. also, the use of live bacteria could have contributed to these ambiguous results. for instance, if influenza would induce immunosuppression and thereby facilitate outgrowth of bacteria upon a secondary live infectious challenge, the increased bacterial burden can eventually result in fulminant inflammation, which would wrongfully suggest influenza-induced augmentation of the immune response. in the present study, we investigated whether influenza infection results in an enhanced or suppressed innate immune response upon a secondary challenge with lps. furthermore, we assessed the kinetics of these influenza-induced effects by performing the lps challenges in either the acute or the recovery phase of influenza infection. all procedures described were in accordance with the requirements of the dutch experiments on animals act, the ec directive / , and approved by the animal ethics committee of the radboud university nijmegen medical center (ru-dec - ). forty-eight male c bl/ j mice (charles river, sutzfield, germany) aged - weeks and weighing - g were used. mice were housed in individually ventilated cages, with five mice per cage at the central animal facility of the radboud university. at day , six groups of eight mice (total n = ) were anesthetized by isoflurane and intranasally inoculated with a sublethal dose of influenza virus (ph n , strain a/ netherlands/ / , × pfu) or mock treated (nacl . %) in a volume of μl. following infection, all mice were monitored and weighed daily. the temperature was recorded with an infrared thermometer on the skin, and physical condition was scored using a scoring and weight sheet (weight, body temperature, ruffled coat, hunched back, reduced mobility, and moribund). at either day (acute phase) or day (recovery phase), mice were placed in a temperature-controlled chamber to receive lps (e coli, serotype :b , mg/kg) or nacl . % by intravenous injection in the tail vein. ninety minutes after lps or nacl administration, mice were deeply anesthetized with isoflurane and exsanguinated through orbital extraction, followed by cervical dislocation after which organs were collected. ethylenediaminetetraacetic acid (edta)-anticoagulated blood was centrifuged at ×g for min at room temperature after which plasma was stored at − °c until analysis. subsequently, perfusion of the lungs was performed by intracardiac injection with phosphate-buffered saline (pbs), after which lung lobes were harvested and snap frozen in liquid nitrogen and stored at − °c until homogenization. lung tissue was placed in ml lysis buffer containing pbs, . % triton x- , and a protease inhibitor cocktail (complete edta-free tablets, roche, woerden, the netherlands, tablet per ml lysis buffer). subsequently, lung lobes were homogenized at hz, using a polytron homogenizer, and subjected to two rapid freeze-thaw cycles using liquid nitrogen. finally, homogenates were centrifuged ( min, , ×g, °c), and the supernatant was stored at − °c until cytokine analysis. concentrations of tnf-α, il- β, il- , and il- in plasma and lung homogenates were measured using a luminex assay (milliplex, millipore, billerica, ma) according to the manufacturer's instructions. the lower detection limit of the assay was pg/ml for all cytokines. plasma il- β levels were below the detection limit in the majority of animals. lung homogenate cytokine concentrations were normalized to total protein content determined by bicinchoninic acid assay (bca protein assay; thermo fisher scientific). myeloperoxidase (mpo) content was measured in lung homogenates using an enzyme-linked immunosorbent assay (hycult biotech, uden, the netherlands) according to the manufacturer's instructions. concentrations were normalized to total protein content as described above. all data were normally distributed according to the shapiro-wilk test. the grubbs test (extreme studentized deviate method) was used to exclude significant outliers from analysis (maximum of one exclusion per dataset). to determine the number of animals required per group, we performed a power calculation based on a minimal detectable difference of % in lps-induced plasma tnf-α levels between influenza-infected and non-influenza-infected mice. mean ± sd ( ± pg/ml) tnf-α plasma levels were obtained from previous work from our group, in which male c bl/ j mice were also injected intravenously with mg/kg lps and sacrificed min later [ ] . using a two-sided α of . and a power of % (β of . ) in an unpaired t test design, six animals per group were required. to account for potential loss of animals due to influenza infection, eight animals per group were used. the effect size was based on previous work [ ] , in which influenza infection modulated the plasma cytokine response to lps administration by at least %. comparisons were analyzed using unpaired student's t tests and repeated measures one-way analysis of variance (anova) as indicated in the figure legends. statistical analyses were performed in graphpad prism . for windows (graphpad software, san diego, ca). two-tailed p values < . were considered statistically significant. all influenza-inoculated mice showed clinical signs of infection, including weight loss, lethargy, and pyrexia. four influenza-infected mice were prematurely taken out of the experiment because of signs of severe infection. body weight decreased in all influenza-infected mice in the acute phase of infection, whereas it remained stable in mock-inoculated mice (fig. ) . from day onwards, body weight started to increase, marking the recovery phase of influenza infection (fig. ) . influenza infection by itself did not result in increased plasma levels of any of the cytokines measured at both and days post-infection (fig. ) . expectedly, lps fig. body weight of influenza-or mock-inoculated mice. data are presented as mean with sem. dagger indicates the two time points at which mice in the respective groups were sacrificed administration led to profoundly increased plasma concentrations of tnf-α, il- , and il- . although tnf-α and il- plasma levels appeared to be somewhat higher in mice challenged with lps days after influenza infection compared with mock-inoculated mice, this did not reach statistical significance (p = . and p = . , respectively). plasma concentrations of the anti-inflammatory cytokine il- were however significantly enhanced in mice challenged with lps days after influenza infection. no differences in any of the plasma cytokine levels were measured between influenza-infected and mock-inoculated mice at days. in lung homogenates, influenza by itself caused mildly elevated levels of tnf-α, il- β, il- , and il- at days post-infection and to a lesser extent at days after infection (fig. ) . similar to what was found in plasma, lps challenge also led to increased concentrations of all measured cytokines in lung tissue. a synergistic increase of all pro-inflammatory cytokines in the lungs was found in influenza-infected mice challenged with lps days later and, to a lesser extent, in mice challenged with lps days post-influenza infection. for il- , the potentiating effect was additive rather fig. plasma levels of tnf-α, il- , and il- in mice that received influenza/mock followed by lps/nacl or days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < . , **p < . , ***p < . (calculated by unpaired student's t tests) fig. levels of tnf-α, il- β, il- , and il- in lung homogenates of mice that received influenza/mock followed by lps/nacl or days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < . , **p < . , ***p < . , # p = . - . (calculated by unpaired student's t tests) than synergistic, only observed at days post-influenza infection, and reached a trend towards statistical significance. in accordance with pulmonary cytokine levels, influenza infection by itself led to increased mpo content in the lungs days after infection and tended to result in increased mpo content days post-infection (fig. ) . again, lps administration also resulted in increased mpo levels in lung tissue, and there was a trend towards enhanced mpo content in influenza-infected mice challenged with lps days after infection. in the present study, we demonstrate that a systemic lps challenge in the acute phase of influenza infection ( days post-infection) results in an enhanced pulmonary, but not systemic pro-inflammatory cytokine response. this effect was synergistic rather than additive, indicating that influenza infection actually modulates the immune response to a subsequent challenge with lps. furthermore, this effect remained present, although less pronounced, in the recovery phase of influenza infection ( days post-infection). the lps-induced increase in mpo content in lung homogenates, reflecting pulmonary neutrophil influx or sequestration, tended to be enhanced in the acute phase of influenza infection as well. these results suggest that influenza infection, especially in the acute phase, may cause a more pronounced pulmonary pro-inflammatory immune response upon a secondary bacterial infection. our results are in accordance with in vitro data reporting a cellular priming effect of influenza observed upon secondary stimulation with lps [ ] [ ] [ ] [ ] [ ] , as well as with other murine in vivo studies that report increased inflammation and pulmonary neutrophil influx or sequestration upon a secondary bacterial infection or lps challenge in the acute phase of influenza infection [ , ] . for example, a preceding influenza infection in mice gravely enhanced lung injury induced by a secondary infection with streptococcus pneumoniae days later, resulting in a severe necrotic pneumonia accompanied by increased mortality [ ] . also, fig. mpo content in lung homogenates of mice that received mock/influenza followed by nacl/lps or days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < . , **p < . , ***p < . , # p = . - . (calculated by unpaired student's t tests) the increased mpo content observed in our study is an important hallmark of acute respiratory distress syndrome (ards) [ , ] , a severe complication of influenza infection caused by excessive pulmonary inflammation. these and our study reveal that the enhancing effect on the pro-inflammatory innate immune response is most evident in the lungs, probably because the influenza-induced damage and consequent inflammatory effects are most pronounced at this site. in this context, our data are in line with the recommendation to use corticosteroids in patients with severe influenza infections in the intensive care unit to counteract the pulmonary hyperinflammatory response causing ards. several underlying mechanisms may contribute to the observed effects. at the cellular level, studies have shown that influenza and certain bacterial pathogens, such as haemophilus influenzae and streptococcus pneumoniae, utilize similar immunological pathways and that the overlap in the inflammatory mediators produced thereby creates augmentation of the immune response during sequential infection, in turn causing immunopathology [ , ] . furthermore, it has been hypothesized that influenza stimulates tnf-α gene transcription activators or may interfere with labile transcription repressor proteins and stabilizes tnf-α mrna by delaying its degradation [ ] . alternatively, the increased lung mpo levels observed do not necessarily reflect pmn infiltration into the lungs, but may (also) result from pmns trapped in the vasculature, as circulating activated neutrophils become rigid and can be trapped within the small capillaries of the lung [ ] . as such, increased entrapment of leukocytes in the pulmonary vasculature during influenza infection could also contribute to the enhanced inflammatory cytokine levels upon lps challenge. we can only speculate on this, because no histological data are available, which represents a limitation of this work. it may be argued that the enhanced pro-inflammatory immune response induced by influenza serves as a means to efficiently eliminate the primary pathogen and to enhance host defense towards a secondary infection. for instance, pro-inflammatory cytokines are induced in influenza-infected cells to limit viral replication and to initiate downstream immune responses [ ] . however, this is not supported by previous work, where an increased bacterial burden was observed irrespective of an enhanced or suppressed response [ ] [ ] [ ] . several explanations for this observation may be put forward. first, next to potentiating pro-inflammatory cytokine responses, the present study and work by others [ ] have shown that influenza infection also potentiates production of the key anti-inflammatory cytokine il- , which was demonstrated to be crucial in facilitating bacterial outgrowth upon secondary challenge with streptococcus pneumoniae [ ] . second, influenza may on the one hand prime for production of innate cytokines produced by myeloid cells, but impair t cell-derived cytokines that are instrumental for the adaptive immune response. this was elegantly demonstrated by kudva et al., who showed that, in line with our results, infection with staphylococcus aureus days after influenza resulted in increased pulmonary levels of innate cytokines such as il- and mcp- , and increased neutrophil influx to the lungs, but decreased concentrations of t-cell-derived il- and il- , which were demonstrated to play a pivotal role in fending off the staphylococcal infection [ ] . whereas the enhancing effects of influenza on pro-inflammatory innate immune parameters were less pronounced at days post-infection, a suppressed response was neither evident. this could be partly biased by the exclusion of two mice in both recovery groups due to severe influenza infection. however, it might also be argued that days post-infection is too soon for these effects to manifest. for example, profound desensitization towards lps and flagellin, another toll-like receptor (tlr) ligand, was observed in alveolar macrophages obtained from mice up to weeks after influenza infection [ ] . furthermore, the direction of the response upon a secondary challenge is probably highly dependent on the pathogen or stimulus used, each using distinct intracellular signaling pathways. with regard to this, it is well-known that influenza virus particularly predisposes to aspergillus fumigatus, which is present in % of all influenza patients [ , ] , causing infections such as invasive pulmonary aspergillosis that are associated with very high mortality rates. as different mechanisms may be important in host defense towards various pathogens, the specific response towards aspergillus fumigatus could be suppressed by a preceding influenza infection. the use of corticosteroids may be another important factor in the observed vulnerability towards particular secondary infections, as steroid use was shown to be independently associated with the presence of aspergillus fumigatus in sputum of cystic fibrosis patients [ ] and with a substantially increased risk of community-acquired staphylococcus aureus bacteremia [ ] . furthermore, a meta-analysis revealed that the use of corticosteroids was significantly associated with nosocomial infections [ ] . to the best of our knowledge, these putative detrimental effects of corticosteroid treatment during influenza on secondary infections have yet to be studied systematically in animal models. in any case, it remains to be determined whether the overall effects of corticosteroid treatment are beneficial or not, as they may lead to increased susceptibility in a subset of influenza virus-infected patients but may also provide health benefits in another subset of influenza virus-infected patients. an lps challenge in the acute phase of influenza infection results in an enhanced pulmonary pro-inflammatory innate immune response. these data increases our insight concerning viral-bacterial interplay. combined with previous findings, it appears that this enhanced pro-inflammatory response does not lead to protection against secondary infections but rather causes immunopathology leading to damage, and thereby to organ failure. predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness secondary bacterial infections associated with influenza pandemics influenza-induced type i interferon enhances susceptibility to gram-negative and gram-positive bacterial pneumonia in mice infection of macrophages by influenza a virus: characteristics of tumour necrosis factor-alpha (tnf alpha) gene expression the potentiating effect of lps on tumor necrosis factor-alpha production by influenza a virus-infected macrophages cytokine release from human peripheral blood leucocytes incubated with endotoxin with and without prior infection with influenza virus: relevance to the sudden infant death syndrome tumor necrosis factor-alpha production of influenza a virus-infected macrophages and potentiating effect of lipopolysaccharides influenza a virus infection of macrophages. enhanced tumor necrosis factor-alpha (tnf-alpha) gene expression and lipopolysaccharidetriggered tnf-alpha release lipopolysaccharide treatment and inoculation of influenza a virus results in influenza virus-associated encephalopathy-like changes in neonatal mice induction of pro-and anti-inflammatory molecules in a mouse model of pneumococcal pneumonia after influenza il- is an important mediator of the enhanced susceptibility to pneumococcal pneumonia after influenza infection inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from influenza infection influenza infection leads to increased susceptibility to subsequent bacterial superinfection by impairing nk cell responses in the lung spleen-derived ifn-gamma induces generation of pd-l (+)-suppressive neutrophils during endotoxemia cxcl -cxcr enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin contribution of neutrophil-derived myeloperoxidase in the early phase of fulminant acute respiratory distress syndrome induced by influenza virus infection insights into the interaction between influenza virus and pneumococcus patterns in bacterial-and viralinduced immunosuppression and secondary infections in the icu the neutrophil in vascular inflammation new fronts emerge in the influenza cytokine storm influenza a inhibits th -mediated host defense against bacterial pneumonia in mice sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory influenza infection invasive pulmonary aspergillosis is a frequent complication of critically ill h n patients: a retrospective study influenzaassociated aspergillosis in critically ill patients inhaled corticosteroids and aspergillus fumigatus isolation in cystic fibrosis use of glucocorticoids and risk of community-acquired staphylococcus aureus bacteremia. a population-based case-control study mayo corticosteroids for the treatment of human infection with influenza virus: a systematic review and meta-analysis. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases the authors thank fred van opzeeland, elles simonetti, francine van der poll, ilona van de brink, and jelle gerretsen for their help with the mouse experiments and laboratory analyses. this work was supported by an efro (dutch: "europees fonds voor regionale ontwikkeling," english: "european regional development fund") grant ( - ). efro had no role in the design of the study; in the collection, analysis, and interpretation of data; and in writing the manuscript. data sharing is not applicable to this article as no reusable datasets were generated or analyzed during the current study.authors' contributions rk designed and conducted the study, analyzed and interpreted the data, and drafted the manuscript. dd and gf aided in the study design and conduct, interpreted the data, and critically revised the manuscript. pp and mdj supervised the study, interpreted the data, and critically revised the manuscript. mk designed and supervised the study, interpreted the data, and critically revised the manuscript. all authors read and approved the final manuscript. all procedures described were in accordance with the requirements of the dutch experiments on animals act, the ec directive / , and approved by the animal ethics committee of the radboud university nijmegen medical center (ru-dec - ). not applicable. the authors declare that they have no competing interests. key: cord- -iryb v z authors: kao, kuo-chin; chang, ko-wei; chan, ming-cheng; liang, shinn-jye; chien, ying-chun; hu, han-chung; chiu, li-chung; chen, wei-chih; fang, wen-feng; chen, yu-mu; sheu, chau-chyun; tsai, ming-ju; perng, wann-cherng; peng, chung-kan; wu, chieh-liang; wang, hao-chien; yang, kuang-yao title: predictors of survival in patients with influenza pneumonia-related severe acute respiratory distress syndrome treated with prone positioning date: - - journal: ann intensive care doi: . /s - - - sha: doc_id: cord_uid: iryb v z background: patients with influenza complicated with pneumonia are at high risk of rapid progression to acute respiratory distress syndrome (ards). prone positioning with longer duration and lung-protective strategies might reduce the mortality level in ards. the aim of this study is to investigate the survival predictors of prone positioning in patients with ards caused by influenza pneumonia. methods: this retrospective study was conducted by eight tertiary referral centers in taiwan. from january to march in , all of the patients in intensive care units with virology-proven influenza pneumonia were collected, while all of those patients with ards and receiving prone positioning were enrolled. demographic data, laboratory examinations, management records, ventilator settings and clinical outcomes were collected for analysis. results: during the study period, patients with severe influenza pneumonia were screened and patients met the diagnosis of ards. totally, patients receiving prone positioning were included for analysis. the -day survivors had lower acute physiology and chronic health evaluation (apache) ii score, pneumonia severity index (psi), creatinine level and lower rate of receiving renal replacement therapy than non-survivors ( . ± . vs. . ± . , p = . ; . ± . vs. . ± . , p = . ; . ± . mg/dl vs. . ± . mg/dl, p = . ; and % vs. %, p < . ). multivariate cox regression analysis identified psi (hazard ratio . , % confidence interval . – . ; p < . ), renal replacement therapy (hazard ratio . , % confidence interval . – . ; p < . ), and increase in dynamic driving pressure (hazard ratio . , % confidence interval . – . ; p = . ) which were independent predictors associated with -day mortality. conclusions: in the present study, in evaluating the effect of prone positioning in patients with influenza pneumonia-related ards, pneumonia severity index, renal replacement therapy and increase in dynamic driving pressure were associated with -day mortality in patients with influenza pneumonia-related ards receiving prone positioning. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. severe complicated influenza including pneumonia, myocarditis and neurologic complications are still a burden on intensive care units (icu) nowadays, especially viral or secondary bacteria pneumonia-induced acute respiratory distress syndrome (ards) [ , ] . during the winter season in , there was an outbreak of influenza in taiwan. totally, subjects were admitted to icus due to severe complicated influenza pneumonia according to the data from the centers for disease control of taiwan [ ] . patients with influenza pneumonia needing mechanical ventilation were at high risk of rapid progression to ards. for the pandemic h n virus infection, - % of patients admitted to icus had complications with ards [ , ] . there are several therapeutic options for refractory hypoxemia in patients with severe ards [ , ] , but only a few options have been confirmed with clinical validity by previous studies, including higher positive endexpiratory pressure (peep) [ , ] , lower tidal volume [ ] , neuromuscular blocking agents [ ] and prone positioning [ ] . prone positioning was first suggested in [ ] ; however, the clinical benefit of prone positioning in patients with ards was not confirmed until when the proseva study showed decreased -day and -day mortality and increased ventilator-free days only when it was started early and there were sufficiently long sessions [ ] . further, meta-analysis by cochrane database also revealed that prone positioning would reduce the mortality rate when used with lung-protective strategies and longer duration in patients with severe ards [ , ] . few studies have explored the effect of prone positioning focused on influenza pneumonia-related ards patients. xu et al. [ ] studied h n influenza patients with prone positioning, and decrease in carbon dioxide retention was noted, but no clinical outcome was mentioned. moreover, what factors that can predict the efficacy of prone positioning in severe ards are not entirely clear [ ] . the aim of this study is to investigate the survival predictors of prone positioning in patients with severe ards caused by influenza pneumonia. this multicenter retrospective cohort study was conducted by the taiwan severe influenza research consortium (tsirc), which included eight tertiary referral centers (four hospitals in northern taiwan, two hospitals in central taiwan and two hospitals in southern taiwan). over a period of months from january to march in , all patients with the virology-proven influenza infection who were admitted to icus due to severe complicated influenza in these eight hospitals were collected and their data were analyzed. all patients diagnosed as severe ards according to berlin definition and also receiving prone positioning were collected for investigation [ ] . the berlin definition of ards was defined by acute onset within week, bilateral lungs opacities, no evidence of cardiac failure-related hydrostatic edema by echocardiography, and pao /fio ratio < mm hg with positive end-expiratory pressure (peep) ≥ cm h o. the demographic and laboratory data, treatment record, mechanical ventilation settings, and clinical outcomes were analyzed from the electronic medical records with a standardized case report form in each hospital. university hospital rind, tri-service general hospital - - - ). the need for informed consent was waived, and patients' data were anonymized and de-identified prior to analysis. influenza infection was confirmed by one of the following tests revealing as positive including the rapid antigen test, nucleic acid reverse transcriptase polymerase chain reaction (rt-pcr), viral culture sampling from nasopharynx swab, throat swab, sputum or bronchoalveolar lavage and positive serum antibody serologic test (antibody titers increased more than times from acute to convalescent stages). the usual practice in the units was that patients be ventilated with lung-protective strategy by low tidal volume - ml/kg of predict body weight plus low positive endexpiratory pressure (peep)-oxygen fraction in air (fio ) table for pressure-controlled or volume-controlled ventilation [ ] . ventilation was monitored by arterial blood gas measurements, with ventilator settings changed as needed. pulse oximetry (spo ) was used to monitor oxygenation, and ventilatory settings were adjusted to maintain spo > % or pao > mm hg and to avoid raising the plateau pressure > cm h o. the method of prone positioning complied with the proseva study [ ] . doses of neuromuscular blocking agent with intravenous cisatracurium and sedatives with intravenous midazolam were adjusted to maintain synchrony between the ventilator and the patient's breathing, as well as hemodynamics. the criteria for stopping prone positioning were any of the following: improvement in oxygenation (defined as a pao /fio ratio ≥ mm hg, with a peep of ≤ cm h o and an fio ≤ . ), a decrease in the pao /fio ratio ≥ % or complications happening during prone positioning such as spo ≤ % or pao /fio ratio ≤ mm hg, severe cardiac arrhythmia, systolic blood pressure ≤ mm hg and any other life-threatening condition for which the intensivist decided to stop the prone positioning. the laboratory data including baseline characteristics, underlying disease, complete blood count, differential count and biochemistry data were obtained when the patient was admitted to the icu. the mechanical ventilator settings were recorded such as peak inspiratory pressure, peep, artery blood gas, partial pressure of oxygen in arterial blood (pao ), pao /fio ratio, tidal volume, dynamic driving pressure and dynamic compliance of the respiratory system before and day after the first prone positioning. the above physiological data were recorded before prone positioning on the supine position and day after first prone positioning on the prone position. the dynamic driving pressure and dynamic compliance were computed as peak pressure minus peep and tidal volume divided by peak pressure minus peep. the severity scores including pneumonia severity index (psi) [ ] , acute physiology and chronic health evaluation ii (apache ii) score [ ] , curb- (confusion, urea > mmol/l, respiratory rate ≥ /min, blood pressure [systolic < mm hg or diastolic ≤ mm hg] and age ≥ years) pneumonia severity score [ ] and sequential organ failure assessment (sofa) score [ ] were collected on the icu admission day. statistical analyses and database management were performed using spss version . . (spss inc., chicago, il). the data were presented as number (percentages) for nominal variables, and as mean ± standard deviation for continuous variables. the chi square test was used to compare the nominal variables, and the student's t test was used to compare the continuous variables. cox proportional hazard models were used with covariates significantly different between survivors and non-survivors at the threshold of . and mortality at day as the dependent variable. calibration was assessed using hosmer-lemeshow goodness-of-fit test (c statistic, goodness of fit was defined as a p value > . ), and discrimination was assessed by the area under the receiver operating curves. even though peak airway pressure, dynamic driving pressure, and compliance are mathematically coupled, we planned to formally test the collinearity within them and, if verified, to use a specific cox model for each. we also included those collinear variables two-by-two into three additional cox regression models [ ] , besides the other covariates. one model pertained to peak airway pressure and dynamic driving pressure, one to peak airway pressure and compliance, and one to dynamic driving pressure and compliance. if both variables in the couple lacked significance, the conclusion could be that the same information was carried by each component of the couple. if one of the variables in the couple remained significantly correlated with survival, this variable would be more informative than the other in the couple. univariate and multivariate cox proportional hazard regression models were used to estimate the hazard ratio (hr). in this study, we used the two-tailed test, and the definition of significance was p value < . . in total, patients with virology-proven severe influenza pneumonia were admitted to icus and screened during the study period. there were patients with influenza a (including h n in patients and h n in patients), patients with influenza b, and patients with undetermined influenza type. of these patients, patients ( %) met the diagnosis of severe influenza pneumonia-related ards. the rates of mild, moderate and severe ards were % ( / ), % ( / ) and % ( / ), respectively. of these patients with ards, patients ( %) receiving prone positioning were included for analysis (fig. ) . the rate of receiving prone positioning was % ( / ) in mild, % ( / ) in moderate and % ( / ) in severe ards, respectively (p = . ). the characteristics of the subjects according to the -day survivors and non-survivors are summarized in table . the mean age was . ± . years, and patients ( %) were male. the duration of prone positioning of survivors and non-survivors was not significantly different ( . ± . days vs. . ± . days, p = . ). the survivors had lower apache ii score, psi, creatinine level and lower rate of receiving renal replacement therapy than did non-survivors ( . ± . vs. . ± . , p = . ; . ± . vs. . ± . , p = . ; . ± . mg/dl vs. . ± . mg/dl, p = . ; and % vs. %, p < . ). regarding the oxygenation, the mean pao /fio ratio of these patients before prone positioning was . ± . mm hg. before prone positioning, there were no significant differences in the pao /fio ratio, paco , tidal volume, peep, peak airway pressure, dynamic driving pressure and dynamic compliance between surviving and non-surviving patients. the data regarding the gas exchange and lung mechanics were recorded before prone positioning and after -day prone positioning (table ) . for the -day survivors, there were no significant differences in these parameters compared with -day non-survivors except for peak airway pressure. after prone positioning, the -day survivors had decreased peak airway pressure (− . ± . cm h o) and the -day non-survivors had increased peak airway pressure ( . ± . cm h o). compared with -day non-survivors, the peak airway pressure and dynamic driving pressure were both decreased in -day survivors (− . ± . univariate analysis was used to identify variables that have prognostic value for -day mortality, and multivariate cox regression analysis was used to identify variables that did have significant predictive value (table ) . pneumonia severity index (hazard ratio . , % confidence interval . - . ; p < . ), renal replacement therapy (hazard ratio . , % confidence interval . - . ; p < . ) and increased dynamic driving pressure (hazard ratio . , % confidence interval . - . ; p = . ) were identified as significant and independent predictors associated with -day mortality. as the collinearity between Δ dynamic driving pressure, Δ peak airway pressure and Δ dynamic compliance was statistically significant, a cox model was constructed for each of these variables. after multiple adjustments of coupled variables, three additional cox models were performed (additional file ). when Δ dynamic driving pressure and Δ peak airway pressure were analyzed two-by-two, Δ dynamic driving pressure remained significant but Δ peak airway pressure did not (model in additional file ). when Δ dynamic driving pressure and Δ dynamic compliance were analyzed two-by-two, Δ dynamic driving pressure remained significant but Δ dynamic compliance did not (model in additional file ). when Δ peak airway pressure and Δ dynamic compliance were analyzed two-by-two, both did not reveal significant (model in additional file ). receiver operating curves analysis and c statistic of variables of predictors revealed . in psi ( % confidence interval, . - . , p = . ), . in renal replacement therapy ( % confidence interval, . - . , p = . ) and . ( % confidence interval, . - . , p = . ) in delta dynamic driving pressure (fig. ). the aim of this multicenter retrospective study was to evaluate the effect of prone positioning focusing on patients with influenza pneumonia-related ards. after multivariate cox regression analysis, psi, renal replacement therapy and increased dynamic driving pressure were associated with -day mortality in patients with influenza pneumonia-related ards receiving prone positioning. most of the studies evaluating the effect of prone positioning were in ards patients with heterogeneous risk factors [ , ] . for specific conditions such as burns, prone positioning has been demonstrated to safely implement and improve oxygenation (in burn patients with severe ards) in a burn intensive care unit [ ] . the present study was more homogenous and specific to patients with ards caused by influenza pneumonia. systematic review and meta-analysis studies in prone positioning have revealed decreased mortality in patients with severe acute hypoxemic respiratory failure, but not in less severe hypoxemia. survival benefits were noted using a range of pao /fio ratio thresholds up to approximately mm hg [ ] or less than mm hg [ ] . in the present study, the pao /fio ratio was . ± . mm hg before prone positioning. however, the pao /fio ratio was not significantly different between -day survivors and -day non-survivors ( . ± . mm hg vs. . ± . mm hg, p = . ). in terms of the response of prone positioning to ards, the different entities of the risk factor possibly produce different outcomes. in addition to severity of hypoxemia, further clinical trials would assist in clarifying the survival benefits of prone positioning in the specific risk factors. some studies have shown that acute kidney injury (aki) was common and an independent risk factor for mortality in patients with influenza a [ ] [ ] [ ] [ ] . in patients with severe ards caused by h n influenza pneumonia, a recent study also revealed aki was common and demonstrated significantly increased mortality [ ] . the % mortality rate among the patients requiring renal replacement therapy was significantly higher than the % mortality rate among the patients not requiring renal replacement therapy. the present study in patients receiving prone positioning caused by influenza pneumonia-related ards demonstrated that the requirement for renal replacement therapy had nearly times the mortality rate (hazard ratio . ) than patients not requiring renal replacement therapy. in order to reduce the mortality in patients with severe ards caused by h n influenza pneumonia, it is important to prevent development ards acute respiratory distress syndrome, bmi body mass index, apache ii acute physical and chronic health evaluation, sofa sequential organ function assessment, psi pneumonia severity index, curb- curb- for pneumonia severity, wbc white blood cell count, paco atrial pressure of carbon dioxide in arterial blood, pao atrial pressure of oxygen in arterial blood, fio oxygen fraction in air, pbw predict body weight, peep positive end-expiratory pressure all values are expressed as the number of patients (percentage) or mean ± sd *p < . : survivors versus non-survivors of aki and need for renal replacement therapy by avoiding nephrotoxic agents and supplying sufficient renal perfusion and oxygenation. amato and colleagues analyzed randomized controlled trials in ards patients and demonstrated that driving pressure was the strongest predictor of mortality [ ] . a secondary analysis of data from ards patients enrolled in two independent randomized controlled trials revealed that when ventilating patients with low tidal volume, driving pressure was a risk factor for death in ards patients, as was plateau pressure or compliance of respiratory system [ ] . airway driving pressure was significantly related to lung stress and could detect lung over-stress with acceptable accuracy (r = . p < . and r = . p < . at and cm h o of peep) in ards patients [ ] . furthermore, the apronet study on prone positioning of ards patients found that prone positioning was associated with low complication rates, significant increase in oxygenation, and a significant decrease in driving pressure [ ( - cm h o) to [ ] [ ] [ ] [ ] [ ] [ ] [ ] cm h o, p = . ] [ ] . our previous study for severe ards patients with ecmo revealed that higher dynamic driving pressure [hazard ratio . ( . - . ), p = . ] during the first days of ecmo was one of the factors independently associated with icu mortality [ ] . the present study in influenza pneumonia-related ards patients receiving prone positioning also found that increased dynamic driving pressure (hazard ratio . , % confidence interval . - . ; p = . ) was identified as ards acute respiratory distress syndrome, Δ change between before and after prone positioning day, pao partial pressure of oxygen in arterial blood, paco atrial pressure of carbon dioxide in arterial blood, one of the independent predictors associated with -day mortality. it was suggested that ventilatory support with lung-protective strategy with low tidal volume and optimal peep level be applied, and these be then adjusted according to the driving pressure, ideally less than cm h o, although this limit should be addressed in future studies [ ] . despite some studies associating driving pressure with physiological and clinical outcomes, it is necessary to evaluate the driving pressure as a primary end point during ventilatory setting in ards patients in the near future. the lung safe study showed that the use of prone positioning actually depended on the severity of hypoxemia, from % in mild to . % in moderate and to . % in severe ards [ ] . a prospective international prevalence study (the apronet study, ards prone position network) found that the rates of prone positioning were up to . %, . % and . % in mild, moderate and severe ards [ ] . in our study, the rates of prone positioning were %, % and % in mild, moderate and severe ards, respectively. the substantially different rates in the use of the prone positioning may reflect the management bias of prone positioning in patients with ards between the different studies. furthermore, among our eight involved hospitals, the rate of prone positioning varied from % ( / ) to % ( / ) and the bias even existed between different hospitals in the same study. it is important to be homogenous on the indication and management in the selected prone position as one of the standard interventions in severe ards. this study has some limitations. firstly, since this study is retrospective, some patients or data might be missing. secondly, the primary end point of this study was -day mortality, and the value of computed power was . . this was a retrospective study, and patients with severe ards receiving prone positioning were analyzed. although more patients were needed to increase the power of this study, the limitation was from the nature of retrospective study within a -month period. thirdly, prone positioning is not a routine intervention in the management of ards and has no standard procedure such as how many hours a day, how to perform it or how to protect the patients. in this study, even though every patient had prone positioning for more than h a day, the exact duration showed little difference between each hospital. fourthly, the change in physiological measurements pertains to a difference between supine and prone position, and hence, the impact of chest wall is not taken into account. finally, in this study, we focused on influenza-related ards patients, and whether the result can be extrapolated to all patients with ards is unknown, requiring further investigation. to confirm the benefit of prone positioning in ards especially in influenza this study was designed to evaluate the effect of prone positioning in influenza pneumonia-related ards patients. after multivariate cox regression analysis, it was found that psi, renal replacement therapy and increased dynamic driving pressure were associated with -day mortality in patients with influenza pneumoniarelated ards receiving prone positioning. h n : viral pneumonia as a cause of acute respiratory distress syndrome pathogenesis of influenzainduced acute respiratory distress syndrome taiwan national infectious disease statistics system. taiwan centers for disease control critical care services and h n influenza in australia and new zealand critically ill patients with influenza a(h n ) infection in canada the berlin definition of ards: an expanded rationale, justification, and supplementary material european society of intensive care medicine, and society of critical care medicine an official american thoracic society/european society of intensive care medicine/society of critical care medicine clinical practice guideline: mechanical ventilation in adult patients with acute respiratory distress syndrome positive end-expiratory pressure setting in adults with acute lung injury and acute respiratory distress syndrome: a randomized controlled trial ventilation strategy using low tidal volumes, recruitment maneuvers, and high positive end-expiratory pressure for acute lung injury and acute respiratory distress syndrome: a randomized controlled trial ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome neuromuscular blockers in early acute respiratory distress syndrome prone positioning in severe acute respiratory distress syndrome conference on the scientific basis of respiratory therapy. pulmonary physiotherapy in the pediatric age group. comments of a devil's advocate prone position for acute respiratory failure in adults effect of prone positioning during mechanical ventilation on mortality among patients with acute respiratory distress syndrome: a systematic review and meta-analysis a multicenter retrospective review of prone position ventilation (ppv) in treatment of severe human h n avian flu treatment of ards with prone positioning acute respiratory distress syndrome: the berlin definition a prediction rule to identify lowrisk patients with community-acquired pneumonia apache ii: a severity of disease classification system defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine effect of driving pressure on mortality in ards patients during lung protective mechanical ventilation in two randomized controlled trials prone positioning improves oxygenation in adult burn patients with severe acute respiratory distress syndrome prone ventilation reduces mortality in patients with acute respiratory failure and severe hypoxemia: systematic review and meta-analysis prone position for acute respiratory distress syndrome: a systematic review and meta-analysis writing committee of the who consultation on clinical aspects of pandemic (h n ) influenza influenza a infection and acute kidney injury: incidence, risk factors, and complications acute kidney injury among critically ill patients with pandemic h n influenza a in canada: cohort study acute kidney injury in criticallyill adult patients with seasonal influenza infection outcomes of acute kidney injury in patients with severe ards due to influenza a(h n ) pdm virus driving pressure and survival in the acute respiratory distress syndrome airway driving pressure and lung stress in ards patients a prospective international observational prevalence study on prone positioning of ards patients: the apronet (ards prone position network) study dynamic driving pressure associated with mortality in acute respiratory distress syndrome with extracorporeal membrane oxygenation driving pressure: a marker of severity, a safety limit, or a goal for mechanical ventilation? epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in countries we thank professor meng-chih lin, the president of the taiwan society of pulmonary and critical care medicine, who organized and coached the tsirc team. on behalf of all authors, the corresponding author states that there is no conflict of interest. not applicable. not applicable. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - bxvenue authors: ashwell, m.; freire, m.; o’nan, a.t.; benito, j.; hash, j.; mcculloch, r.s.; lascelles, b.d.x. title: characterization of gene expression in naturally occurring feline degenerative joint disease-associated pain date: - - journal: vet j doi: . /j.tvjl. . . sha: doc_id: cord_uid: bxvenue degenerative joint disease (djd) associated-pain is a clinically relevant and common condition affecting domesticated cats and other species including humans. identification of the neurobiological signature of pain is well developed in rodent pain models, however such information is lacking from animals or humans with naturally occurring painful conditions. in this study, identification of housekeeping genes (hkg) for neuronal tissue and expression levels of genes considered associated with chronic pain in rodent models were explored in cats with naturally occurring osteoarthritic pain. fourteen adult cats were evaluated — seven without clinical signs of osteoarthritic pain, and seven with hind limb radiographic djd and pain. expression of an investigator-selected set of pain signaling genes (including asic , atf , cox , cx cl , nav . , nav . , nav . , ngf, nk r, tnfα, trka) in lumbar spinal cord dorsal horn and lumbar dorsal root ganglia tissues from clinically healthy cats and cats with djd were studied using quantitative rt-pcr (qpcr). hkg identified as the most stable across all tissue samples were many of the ribosomal protein genes, such as rpl and rps . qpcr results showed atf and cx cl up-regulated in djd-affected dorsal root ganglia compared to clinically healthy controls. in spinal cord, cx cl was up-regulated and ngf was down-regulated when djd-affected samples were compared to healthy samples. further work is needed to understand the neurobiology of pain in naturally occurring disease and what rodent models are predictive of these changes in more heterogeneous populations such as domestic cats. feline degenerative joint disease (djd) can be associated with mobility impairment in feline patients (gruen et al., (gruen et al., , lascelles, ) . this impairment is believed to be due to pain associated with the djd as mobility appears to be increased when pain relief is provided (gruen et al., (gruen et al., , lascelles, ) . the approach to pain management can be divided into two basic approachesto try analgesics known or thought to be effective in other conditions or other species, or to base the analgesic selection on a rationale evaluation of the neurobiological changes present in the target disease state in the target species. this latter approach could be described as making rationale analgesic choices on the neurobiological signature of the pain. presently, nothing is known about the neurobiology of feline djd pain. one approach to understanding the neurobiology of pain is to look at differences in gene expression between normal and phenotypically well-defined diseased states. limited work has been performed along these lines in dogs. hegemann et al. ( ) measured gene expression levels of interleukin (il)- α, il- ß, il- , il- , il- , il- , il- , il- , il- , interferon gamma (if-g), transforming growth factor beta (tgfß), and tumor necrosis factor alpha (tnfα) in synovial fluid collected from dogs with osteoarthritis and immune-mediated polyarthritis using semi-quantitative real-time pcr methods. they reported differences in gene expression levels for some of these genes when they compared affected and unaffected dogs, but did not relate the findings to the presence of painan important consideration given that common measures of disease, such as radiographic features, do not correlate well with pain levels. other investigators have evaluated the changes in gene expression in canine osteoarthritic cartilage (clements et al., (clements et al., , , but again, the results were not evaluated against whether or not there was a pain state present. quantitative real-time pcr (qpcr) studies have evaluated expression levels of various cytokines in feline allergic skin disease (taglinger et al., ) , feline chronic gingival stomatitis (harley et al., ) and feline coronavirus infection (gelain et al., ) . however, no studies have been performed in cats with djd to determine gene expression changes in relation to pain. we were interested in understanding the gene expression changes at crucial points in the nociceptive signal (pain) generation and transmission pathway in the dorsal root ganglia (drg) and the dorsal horn (dh) of the spinal cord. in this study, we first evaluated a panel of potential housekeeping genes to identify the most stable reference genes in djd and healthy cats in drg and dorsal horn as this had not been previously done. after the most stable reference genes were identified, a selection of genes previously associated with nociception in rodent models, and of interest to the authors, were examined using qpcr in the same samples to allow us to start characterizing the neurobiological signature of pain associated with djd in cats. identification of stable reference genes in pain-related tissues in felines is important because of the cat's use as a potential animal model for human conditions and the prevalence of aging cats in veterinary medicine. this study was approved by the ncsu institutional animal care and use committee (iacuc; approval no. - -o; approval date: october ). fourteen domestic cats euthanased at a local animal shelter, and of known pain status via pre-euthanasia examinations, were used for sample collection. the investigators evaluated the cats prior to euthanasia to determine whether they had pain associated with hind limb joints. cats were euthanased with an overdose of barbiturates for reasons unrelated to this study (population control) and the investigators had no input into which cats were euthanased. immediately after euthanasia orthogonal digital radiographs of the lumbar axial skeleton and appendicular joints of the hind limbs were performed and evaluated for the presence of radiographic signs indicative of naturally occurring djd as described previously (lascelles, ) . the tarsus, stifle and hip joints were opened and visually inspected for evidence of macroscopic lesions indicative of djd as described previously (freire et al., ) and scored as described previously . the spinal canal from the last thoracic vertebrae to the sacrum was opened and evaluated for macroscopic lesions indicative of intervertebral disc degeneration with disk prolapse and spinal cord compression. in summary, cats were considered 'pain positive' if there were signs of discomfort on manipulation of hind limb joints; there was gross evidence of djd in those joints; there was muscle atrophy in the hind limbs that was not seen elsewhere in the body; and there were no grossly visible indications of intervertebral disk prolapse or spinal cord compression. seven cats were considered free of musculoskeletal pain on pre-euthanasia evaluation, and of free of naturally occurring appendicular joint djd and spinal djd, and were included in the normal group (no abnormalities seen on digital radiographs and on macroscopic inspection of appendicular joints and axial skeleton). seven cats were included in the hind limb djd group and showed radiographic signs indicative of moderate-severe djd in one or multiple appendicular joints of the hind limbs. no evidence of spinal compressive lesions was present. the lumbar spine from mid body of third lumbar vertebrae to mid body of the fifth lumbar vertebrae (lumbar intumescence) was harvested and drg of fourth, fifth and sixth spinal segments from both sides were dissected and individually stored. right and left dh of the spinal cord segment harvested were dissected and individually stored (after initial exposure to rnalater for at least h). dissection was performed using a dissecting microscope (olympus szx , ). looking at the transverse cut surface of the spinal cord, the white and grey matters can be differentiated and the dorsal horn can easily be identified. the dorsal and ventral aspects of the spinal cord are identifiable by location of the ventral median fissure and the cranial and caudal aspects are determined by looking at the orientation of the nerve rootlets. the dura mater was opened and the spinal cord was divided into right and left segments following the ventral median fissure. another cut placed just dorsal to the central canal allowed the dorsal horn to be separated from the ventral. this cut was performed carefully to assure separation by identification of the grey matter corresponding to the dorsal horn on the transverse cut surface of the spinal cord (supplementary figs. s -s ). the same drg and dh segments of the spinal cord were sampled in normal cats for comparison. all tissue samples were stored in rnalater (qiagen) at À c for h and at À c thereafter until sample processing. total rna from drg and dh was extracted using the qiagen rneasy kit and an on-column dnase digestion to remove genomic dna. the manufacturer's protocol was followed through the chloroform extraction but instead of precipitating the rna in % ethanol, % ethanol was added and the mixture applied to the rneasy (qiagen) column, following the rneasy kit instructions through the end of that manufacturer's protocol. quantity of the extracted rna was measured on a nanodrop spectrophotometer (thermo scientific, wilmington, de) and quality was evaluated by running the extracted rna on a . % agarose gel to determine the integrity of the s and s ribosomal subunits. only samples showing a s: s rrna band intensity ratio of approximately (as determined visually) were used in this study. a high capacity cdna reverse transcription kit (applied biosystems inc., foster city, ca) was used to reverse transcribe ng of total rna per the manufacturer's protocol. subsequently, reactions were diluted : to provide enough template for all genes to be evaluated. primer sequences for gusb, hmbs, rpl , rps , rps and ywhaz were obtained from penning et al. ( ) . the remaining housekeeping and target gene primers were designed using beacon designer software (premier biosoft intl., palo alto, ca) to be compatible with sybr green i and to span an intron in order to detect genomic contamination. pcr primer sequences for the remaining housekeeping genes and pain-related target genes are given in table . quantitative pcr was performed in a volume of ml, consisting of ml of diluted cdna, nmol/l of forward and reverse primers, nmol/l fluorescein, and  power sybr green master mix (applied biosystems). a three-step amplification protocol was performed in an icycler iq (bio-rad). each reaction included one cycle at c for min, following by cycles at s at c, s at c- c for annealing, and extension for s at c. specificity of each reaction was assessed by melt curve analysis ( cycles starting at c with an increase of . c every cycle, with a dwell time of s) and one amplicon from each primer pair was dna sequenced to confirm identity. reactions were performed in duplicate, c t values were averaged for the replicates and negative controls were included to detect possible contamination. any duplicate measurements more than . c t apart were repeated for that sample or removed from the analysis. standard curves were evaluated for each primer pair by combining equal amounts of cdna from each specimen into a pool. the pool was then diluted : , : , : , : and : . dilutions were evaluated in duplicate to calculate amplification efficiencies which ranged from % to %, depending on the type of tissue and primer pair. bestkeeper (pfaffl et al., ) , genorm (vandesompele et al., ) and normfinder (ohl et al., ) were used to evaluate the potential reference genes and select the most stable in these particular tissues. fold changes between healthy and djd affected samples were computed via normalization to the geometric mean of the selected housekeeping genes. the fold change calculations incorporated corrections for reaction efficiency using the method of pfaffl et al. ( ) . changes in the expression of healthy and djd samples were evaluated for statistical significance using a linear mixed model derived from the work of steibel et al. ( ) . bayesian information criteria was used to select the best parameters to include in the model (those contributing to the lowest bic score). fixed factors included health status (healthy vs. djd), reproductive status (sterilized vs. intact), and radiographic score for djd. random factors included variables for position of the sample (left vs. right), age, and sex of the cats. the model was established with duplicate samples from a given cat nested within the cat. all analysis was conducted with spss software (version . , ibm) and an alpha (α) of . was used for all analyses. cats included in the djd-pain group were two castrated males, three intact males and two spayed females. mean age (aestandard deviation, sd) was ( . ) years old, and mean weight (aesd) was . ( . ) kg; median body condition score was / . the characteristics of the cats are detailed in table s (supplementary data), and all were designated 'pain positive'. bilateral hind limb appendicular joint djd in one or multiple joints were present in five of seven cats and samples from right and left dh of the spinal cord and right and left drg were analyzed. in the other two cats in the djd-pain group, only one hind limb had one or multiple joints affected with djd and samples from the nervous tissue ipsilateral to the affected side were collected. cats studied included in the healthy group were two castrated males, three intact males, one spayed female and one intact female. mean age (aesd) was . ( . ) years, and mean weight (aesd) was . ( . ) kg; median body condition score was / . samples were collected from right and/or left nervous tissues in these cats to match with the samples collected in the cats with djd. ten potential housekeeping genes for drg and dh collected from djd-affected and unaffected cats were examined using the three gene expression reference gene programs. the ten genes evaluated in this study were selected based on previous work by penning et al. ( ) that examined the stability of these genes in feline liver, kidney, dental roots, heart and mammary gland tissues but not in any central nervous system tissues. the genes examined were beta actin (actb); beta- -microglobulin (b m); beta glucuronidase (gusb); hydroxymethylbilane synthase (hmbs); tyrosine -monooxygenase/tryptophan -monooxygenase activation protein-zeta polypeptide (ywhaz); and ribosomal proteins l (rpl ), l (rpl ), s (rps ), s (rps ) and s (rps ). due to dissection errors, two drg were missing from the sample set; however drg representing all cats were used to evaluate the reference gene panel using bestkeeper and norm-finder, as those programs allow for missing data. only paired samples from cats were able to be evaluated using genorm as that program requires complete data. all genes amplified in the majority of the samples examined but b m had a sd > and was removed as a potential reference gene based on the recommendation from the bestkeeper software. based on bestkeeper results, actb, rps and rps were the most stable housekeeping genes in the drg samples. using results from genorm, rps , rps and actb were the most stable. and norm-finder produced results that differed from both genorm and bestkeeper, finding rpl , hmbs and gusb as the most stable genes in the drg samples (table ). an additional feature of genorm is the determination of the optimum number of reference genes needed for accurate normalization (fig. s a and s b ). the number of reference genes associated with an expression stability value of . or lower is considered the optimum number of housekeeping genes needed. for each of the tissues examined in the feline samples, the variation in normalization factor with two vs. three reference genes was < . , indicating the optimum table reference gene rankings for each tissue type using bestkeeper, genorm and normfinder. reference gene ranked order (most stable to least stable) bestkeeper number of genes needed was two. however, since the three programs did not always agree on the two most stable genes, three were selected for each tissue type. based on these results, the geometric mean of actb, rps and rpl was used as the reference value in each of the drg samples. a total of dh samples ( from cats with djd and from healthy cats) were used to evaluate potential housekeeping genes in this tissue. all ten reference genes amplified and had sd < so all were included in the reference gene analyses. determination of the most stable reference genes in dh was very consistent across the three evaluation programs, with hmbs being the most stable gene ( table ) . actb was the next most stable using bestkeeper and normfinder and this gene was placed as the third most stable using genorm. based on these results, the geometric mean of actb, hmbs and rpl was used as the reference value in each of the dh samples. after a set of stable reference genes were identified for each tissue type, genes associated with pain in rodents were selected (based on current knowledge of genes involved in pain states (foulkes and wood, ) and their expression levels compared in the drg from djd-affected and healthy samples. we hypothesized that these genes would show increased expression in djd cats. the selected genes were as follows: acid sensing ion channel (asic ); activating transcription factor (atf ); calcitonin gene related peptide (cgrp); cyclooxygenase (cox ); chemokine (c-x -x motif) ligand (cx cl ); sodium channel, voltage-gated, alpha subunit types ix (nav . ), x (nav . ) and xi (nav . ); nerve growth factor (ngf); tachykinin receptor (nk r); substance p (tac ); tumor necrosis factor alpha (tnfα); and tropomyosin receptor kinase a (trka). although two primer pairs were designed for each target gene, no amplification was detected for cgrp and tac , so they were excluded from this study. when djdaffected cats were compared to healthy control cats for the remaining target genes, atf and cx cl were significantly upregulated in the drg ( . -fold and . -fold, respectively; table ). ten target genes were selected for examination in dh samples-asic , cox , cx cl , nav . , nav . , nav . , ngf, nk r, tnfα, and trka. asic , nav . and trka were excluded because their pcr efficiencies were outside the acceptable range of - % and two genes (nav . and nav . ) did not amplify in any of the dh samples. of the five remaining genes, cx cl was up-regulated ( . -fold) and ngf was down-regulated ( . -fold) in the djd affected samples (table ). with quantitative pcr, multiple target genes may be evaluated to measure changes in expression. however, to accurately determine the relative expression levels, a reference is used to normalize the expression results for differences in cdna quantity between different samples, enabling comparisons between target genes across disease states. bestkeeper, genorm, and normfinder provide three different approaches for examining potential genes to select as the most stable genes for a given set of conditions. at least three other studies have identified suitable gene expression housekeeping genes in various feline tissues. penning et al. ( ) found that most of the ribosomal genes they tested appeared to be suitable reference genes in the different feline tissues. wensman et al. ( ) tested six potential housekeeping genes in different brain tissues and found that hprt, ywhaz and rps were the most stable. kessler et al. ( ) tested potential reference genes in neoplastic, endocrine, blood, liver, intestine and lymphoid tissues in healthy cats and found that rps , actb and abl were the most stably expressed housekeeping genes. while none of these other feline studies examined central nervous tissues, our results are similar, with several of the ribosomal genes and actb being stable reference genes. in rat dorsal root ganglia and dorsal horn samples piller et al. ( ) found that actb and hprt and also rpl and rpl a were suitable housekeeping genes in drg and dh samples, respectively, and wan et al. ( ) found rpl , rpl and actb most stable in drg samples isolated from a rat model of neuropathic pain. based on these other studies, our findings that many of the ribosomal genes and actb are the most stable in feline drg and dh samples are reasonable. the target genes selected in this study were chosen based on results from work on skeletal pain conducted in rodents (e.g. mantyh, ) and knowledge of genes involved in pain states (foulkes and wood, ) in an attempt to characterize the neurobiology of djd-associated pain in the cat. they were also chosen because of therapeutics that are available, or in development, or because of their involvement in neuropathic pain states. mrna levels of cx cl , also known as fractalkine, were higher in the drg of djd-affected cats, suggesting that these cats were experiencing a neuropathic pain state (clark & malcangio, ) . in studies examining changes in gene expression in drgs, the expression of activating transcription factor (atf ) is increased in the drg after peripheral nerve injury (shortland et al., ) and more recently has been shown to be increased also in models of oa in rodents (thakur et al., ) . atf is considered indicative of neuronal-damage and neuropathic pain. similar results were identified in the drgs isolated from the djd-affected cats used in this study, where the expression of atf was increased . -fold, and further suggests that cats with djd may be experiencing neuropathic pain like states. very little other work has been performed in drg from nonrodent species, but in horses with laminitis, immunohistochemical analysis of drg from hind limb laminitic horses, comparing lumbar drg with cervical drg showed an increased expression of atf , and the authors suggested this may be indicative of neuropathic pain (jones et al., ) . in many experimental pain models the expression of cox has been found to be increased in the spinal cord in response to inflammation and injury (vardeh et al., ) and contributes to pain, but we did not observe a significant difference in cox when we compared djd and healthy samples. the transcription factor atf has been shown to negatively regulate cox levels during acute inflammation in mice (hellmann et al., ) and our results may indicate the same negative regulation is occurring in cats with djd-pain. however, it is important to remember that our results reflect a single crosssectional chronic time point. the mechanisms of pain likely change over time, and the time course of the rodent models tends to be very short, and may not reflect mechanisms at later stages of the pain state. however, considering again our findings around cox , investigators have struggled to show clinical benefit of nsaids in cats with djd-associated pain (gruen et al., ) . in the djd-affected cats, we found significant increased expression of cx cl in dh, similar to what we detected in the drg samples. studies in rodents have shown that cx cl and its receptor cx cr are an important signaling pathway involved in microglial contributions to chronic pain. interestingly the fractalkine protein is membrane-bound until it is cleaved by different proteases, including cathepsin s. the cleaved, soluble form of fractalkine is associated with neuropathic pain, not the membrane-bound form (clark & malcangio, ) . we also found ngf to be significantly differentially expressed in dh, but not in the expected direction of that reported in other pain models. interestingly, in a study of a monoclonal antibody to nerve growth factor (anti-ngf mab) (gruen et al., ) , the anti-ngf mab produced robust and long-lasting improvements in activity and mobility, presumably due to a decrease in pain. this may illustrate how simply evaluating the expression of genes may not give the whole picturefor a comprehensive understanding of the neurobiology of pain, gene expression, translation into protein and post-translational modifications need to be evaluated. taken together, our results do point to increased expression of genes considered to be involved in neuropathic pain, and may be evidence of a neuropathic like pain state in cats with djdassociated pain. we expected that several of the genes we selected (which have all been shown to play a role in pain in rodent models of arthritis) would show altered expression between the djd-pain and healthy groups. there are several reasons for the relative lack of change across the genes we selected. rodent models are just thatinduced models, and these models may not reflect the neurobiology of pain in naturally occurring pain states. indeed, this is one of the reasons that has been suggested for the lack of translation of basic pain research into effective new therapeutics (lascelles et al., ) . another aspect of our study that should be factored into the interpretation of the results is that the tissues we collected contained varying types of cells and indeed tissue, and so we cannot directly infer that changes or lack of changes are due to any particular tissue type. we made every attempt to only use cats that definitely had djd-pain or were healthy, however, we did not have detailed history on each cat, and our pain status was simply designated as 'yes' or 'no'. future studies should endeavor to collect tissues from more highly phenotyped animals (with respect to pain status). this is particularly important as the neurobiology of pain probably varies over time and with features of the phenotype (e.g. excessive sensitivity present or not). the lack of changes in our study may reflect an underpowered study and the heterogeneity of the samples assessed. despite the shortcomings, we believe our study points to the need to look more closely at the naturally occurring pain states and such investigations may lead to novel therapeutic targets. however, there is a danger of missing novel, relevant targets if the starting point is already biased to what is known from rodent models, as our study was. selection of appropriate stable housekeeping genes is extremely important and has not been previously done for feline nervous tissues. as rodent species continue to illustrate they are not appropriate models for different human conditions, other animal models with naturally occurring disease, such as the cat, will become more and more prevalent and appropriate reference genes will be needed for accurate gene evaluation studies. our approach of evaluating neurobiological changes (gene expression) in nervous system tissue from cats with naturally occurring pain appears feasible. the results of this small study point to increased expression of genes considered to be involved in neuropathic pain, and may be evidence of a neuropathic like pain state in cats with djd-associated pain. further work should be undertaken to confirm our results, and expand on these studies. funding for this work was provided by private donations to translational research in pain program from individuals concerned about the lack of effective therapeutics for cats with chronic pain. jb was employed on a morris animal foundation grant, d fe- , working to develop an assessment tool for chronic pain in cats. none of the authors has any other financial or personal relationships that could inappropriately influence or bias the content of the paper. fractalkine/cx cr signaling during neuropathic pain analysis of normal and osteoarthritic canine cartilage mrna expression by quantitative polymerase chain reaction cartilage gene expression correlates with radiographic severity of canine elbow osteoarthritis pain genes radiographic evaluation of feline appendicular degenerative joint disease vs. macroscopic appearance of articular cartilage whole blood cytokine profiles in cats infected by feline coronavirus and healthy non-fcov infected specific pathogen-free cats detection of clinically relevant pain relief in cats with degenerative joint disease associated pain criterion validation testing of clinical metrology instruments for measuring degenerative joint disease associated mobility impairment in cats a feline-specific anti-nerve growth factor antibody improves mobility in cats with degenerative joint disease-associated pain: a pilot proof of concept study cytokine mrna expression in lesions in cats with chronic gingivostomatitis cytokine profile in canine immune-mediated polyarthritis and osteoarthritis atf negatively regulates ptgs /cox expression during acute inflammation neuropathic changes in equine laminitis pain quantitative taqman real-time pcr assays for gene expression normalisation in feline tissues feline degenerative joint disease crosssectional study evaluating the prevalence of radiographic degenerative joint disease in domesticated cats spontaneous painful disease in companion animals can facilitate the development of chronic pain therapies for humans the neurobiology of skeletal pain identification and validation of suitable endogenous reference genes for gene expression studies of human bladder cancer a validation of feline reference genes for gene expression measurements in snap-frozen tissues relative expression software tool (rest©) for group-wise comparison and statistical analysis of relative expression results in real-time pcr determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestkeeper -excel-based tool using pair-wise correlations reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search atf expression in l dorsal root ganglion neurons after l spinal nerve transection a powerful and flexible linear mixed model framework for the analysis of relative quantification rt-pcr data quantitative real-time rt-pcr measurement of cytokine mrna expression in the skin of normal cats and cats with allergic skin disease characterisation of a peripheral neuropathic component of the rat monoiodoacetate model of osteoarthritis accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes cox in cns neural cells mediates mechanical inflammatory pain hypersensitivity in mice identification and validation of reference genes for expression studies in a rat model of neuropathic pain development of a real-time rt-pcr assay for improved detection of borna disease virus supplementary data associated with this article can be found, in the online version, at https://doi.org/ . /j.tvjl. . . . key: cord- -k g r lw authors: maykowski, philip; smithgall, marie; zachariah, philip; oberhardt, matthew; vargas, celibell; reed, carrie; demmer, ryan t.; stockwell, melissa s.; saiman, lisa title: seasonality and clinical impact of human parainfluenza viruses date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: k g r lw background: widespread availability of rapid diagnostic testing for respiratory viruses allows more in‐depth studies of human parainfluenza viruses (hpiv). objectives: this study aimed to assess seasonality of hpiv types ‐ , clinical outcomes by hpiv type, and risk factors for illness severity. patients/methods: this retrospective study was performed from january to december in children and adults with hpiv, detected by multiplex reverse transcription polymerase chain reaction, participating in a community surveillance study of acute respiratory infections (aris) in new york city and patients admitted to a tertiary care center in the same neighborhood. seasonality trends by hpiv type were compared between the community and hospital groups. the associations between hpiv type, demographics, clinical characteristics, and illness severity were assessed. results: hpiv was detected in ( %) of community surveillance participants (median age . years) and hospitalized patients (median age . years). seasonality for hpiv types ‐ agreed with previously described patterns; hpiv‐ occurred annually in late summer and fall. in the community cohort, ( %) participants sought medical care, ( %) reported antibiotic use, and ( %) reported ≥ day of missed work or school. among hospitalized patients, % had ≥ chronic conditions. multivariable ordinal logistic regression demonstrated that increased severity of illness was significantly associated with hpiv‐ and chronic cardiovascular and respiratory conditions in children and with age ≥ years and chronic respiratory conditions in adults. conclusions: hpiv‐ presented late summer and early fall annually and was associated with increased severity of illness in hospitalized children. human parainfluenza viruses (hpiv) types - are common respiratory pathogens that cause both upper and lower respiratory tract illnesses, especially in young children. hpiv- and hpiv- are more common in children and more often associated with croup. hpiv- is more frequently associated with bronchiolitis, bronchitis, and pneumonia. hpiv- , while least well-characterized, has been described as associated with both mild illness in children and lower respiratory tract disease. hpiv types have varying seasonality, prevalence, and clinical manifestations. in the united states, hpiv- usually occurs in the fall of odd numbered years, hpiv- occurs every fall, and hpiv- occurs in spring and summer. , the seasonality of hpiv- has not been as well defined, often due to its low prevalence. a recent study conducted in colorado found a year-round prevalence of hpiv- with peaks in the fall of odd numbered years. however, estimates of hpiv- prevalence are increasing, potentially due to improved and increased diagnostic testing, but few recent studies have assessed this. , widespread availability of multiplex reverse transcription polymerase chain reaction (rt-pcr) assays allows for more in-depth studies of the epidemiology and impact of hpiv types. the population of this study included both a community-based cohort and hospitalized children and adults with laboratory-detected hpiv. the inclusion of the community cohort allowed for a broader evaluation of the impact of hpiv than has been previously assessed. the study's objectives were to (a) assess the seasonality of hpiv types - , (b) determine clinical outcomes by hpiv type, and (c) identify risk factors for increased severity of illness. a retrospective study of participants in a community surveillance cohort and hospitalized patients with hpiv types - detected from january , to december , was performed. the community cohort was derived from the mobile surveillance for acute respiratory infections (aris) and influenza-like illness (ili) in the community (mosaic) study, a -year community-based surveillance ordinal logistic regression demonstrated that increased severity of illness was significantly associated with hpiv- and chronic cardiovascular and respiratory conditions in children and with age ≥ years and chronic respiratory conditions in adults. conclusions: hpiv- presented late summer and early fall annually and was associated with increased severity of illness in hospitalized children. epidemiology, parainfluenza, respiratory, seasonality, viruses f i g u r e flowcharts depicting the overall number of respiratory viral panel (rvp) tests ordered which yielded the final number of human parainfluenza virus (hpiv) types in the community cohort ( a) and in hospitalized patients ( b) study in new york city (nyc) that includes households annually. , households were identified by contacting a random sample of participants who had taken part in a large population-based survey of an urban, primarily immigrant latino community. for this study, eligible households had or more members with at least member under years of age, were spanish-or english-speaking, and had a cellular telephone with text messaging. text messages were sent twice weekly inquiring about possible respiratory illness among household members. nasal swabs were collected from ill participants by research staff if at least two of the following were reported to be present: fever/feverishness, runny nose/congestion, sore throat, cough, and/or myalgia. for infants, either runny nose or congestion prompted collection of a swab. hospitalized adults and children with laboratory-detected hpiv admitted to a university-affiliated medical center in nyc located in the same neighborhood as the community cohort were included. testing was requested by treating clinicians, and as per the standard of care, is generally recommended for all hospitalizations to evaluate acute respiratory symptoms and guide transmission precautions. patients hospitalized within calendar-days of hpiv detection were included. those with hpiv detected > days before admission, > days after admission and those with a second positive test for the same hpiv type within weeks of the first positive test were excluded. nasopharyngeal swabs for both the community cohort and the hospitalized patients were tested for respiratory pathogens using multiplex rt-pcr (biofire diagnostics, inc. salt lake city, utah) which has been reported to have %- % sensitivity and %- % , swabs from the community were tested in a research laboratory at columbia university medical center (cumc). swabs from hospitalized patients were tested in the clinical microbiology laboratory at newyork-presbyterian hospital at cumc. blood, urine, and respiratory tract cultures for bacteria were sent by treating clinicians as per the standard of care for suspected infections and also processed by the clinical microbiology laboratory. for the community cohort, days of missed school or work; medical care in primary care clinics, urgent care, or emergency departments; hospitalizations; and use of antibiotics were collected by follow-up calls at least days after the resolution of respiratory illness. for hospitalized patients, demographic and clinical characteristics were obtained from electronic medical records (emr). primary icd- diagnoses were categorized as respiratory or nonrespiratory. patients' primary and secondary diagnoses were categorized into groups of chronic conditions previously described as risk factors for severe respiratory disease in adults and children with respiratory viruses. [ ] [ ] [ ] conditions were excluded if they could not be classified within a chronic condition category, had < occurrences across all patients, or have not been associated with severe respiratory disease, for example bipolar disorder. manual chart review of readmitted patients was performed to determine whether readmissions within weeks were due to respiratory illness. hospital course and healthcare utilization data were obtained from the emr including radiographs within days of hpiv detection; all antibiotics within days prior to or days after hpiv detection; icu admission and -day mortality. respiratory support including use of continuous positive airway pressure (cpap), mechanical ventilation, or extracorporeal membrane oxygenation (ecmo) was determined by billed procedure codes. use of bilevel positive airway pressure (bipap) or oxygen supplementation was not assessed, as accurate usage data were unavailable in structured electronic sources. ta b l e comparison of demographic characteristics, symptoms, and outcomes of participants in a community cohort, by human parainfluenza viruses (hpiv) type to assess seasonality, epidemiologic curves that modeled the detection of each hpiv type in the community cohort versus hospitalized patients were created. demographic and clinical characteristics of the two groups were analyzed as proportions, means, and/or medians; associations between hpiv type and clinical course were examined using chi-square, fisher's exact, and anova tests, as appropriate. ta b l e comparison of demographic and clinical characteristics of hospitalized patients with different human parainfluenza viruses (hpiv) in the community cohort, swabs were obtained, of which ( %) of participants were positive for at least one hpiv type ( figure a ). the hpiv-positive swabs were from households; hospital community at least one hpiv type ( figure b) the mean length of hospitalization was . (sd ± . ) days and varied by hpiv type ( were associated with higher odds of increased severity of illness. among the adults, ( %) had severe, ( %) had moderate, and ( %) had mild illness (table ). in the final adjusted model for adults, age ≥ years and respiratory conditions were associated with higher odds of increased severity of illness. we had a unique opportunity to compare the epidemiology and clinical impact of hpiv types among individuals in a communitybased surveillance study and among hospitalized patients during the same time and in the same geographic area. the seasonality of hpiv types - was consistent with published trends, , but we found that hpiv- occurred annually in late summer and fall, suggestive of a local epidemiologic trend that has not been previously described. we found that hpiv- was most common among hospitalized patients ( %) while hpiv- was most common in the community cohort ( % we modified a previously validated severity of illness score to further explore the predictors of illness severity. in children, increased severity of illness was associated with respiratory conditions, cardiovascular conditions and hpiv- while in adults, increased severity of illness was associated with age ≥ years and cardiovascular conditions. the association of comorbid conditions and severe illness in those with hpiv is consistent with other respiratory viruses, most notably rsv and influenza, which both can exacerbate underlying cardiac and pulmonary conditions. [ ] [ ] [ ] , this may also explain why % of hospitalized patients had a nonrespiratory primary diagnosis. the rate of co-detection was similar across hpiv strains and as others have shown, viral co-detections did not impact severity of disease. however, due to limited sample size, we could not assess the impact of specific co-infections. while only % of hpiv-positive patients had a positive bacterial culture, % received at least one dose of an antibiotic between days before and days after hpiv detection. thus, most antibiotic usage represented empiric therapy, suggesting an opportunity for antibiotic stewardship. this study had limitations. the community cohort was % hispanic/latino, reducing the study's generalizability, and small, limiting our ability to find differences in severity of illness or symptomatology associated with hpiv types. our hospital is a referral center and cares for many patients with underlying conditions. furthermore, its referral status could impact accurate interpretation of "local" epidemiology as only % of the hospitalized patients lived in the same zip codes as the mosaic study participants (m. stockwell, personal communication). testing of hospitalized patients relied on clinicians' judgement. we did not review radiographic findings or capture bipap use which may have biased assessment of severity of illness. the severity of illness score was developed for children and not previously tested in adults. use of administrative data restricted our ability to assess patients' symptomatology. although most patients had a respiratory diagnosis, we may have misclassified hpiv infections that actually represented prolonged hpiv shedding from previous illnesses. we did not classify positive bacterial cultures as consistent with infection vs. colonization vs. contamination. in this study, each hpiv type demonstrated seasonality, but each was similar in the community and among hospitalized patients. we found that hpiv- had distinct epidemiology, not previously described, and was associated with increased severity of illness in hospitalized children. as demonstrated for other respiratory viruses, older age and underlying conditions, particularly respiratory and cardiac conditions, were associated with increased severity of illness. additional studies exploring hpiv incidence and severity in both children and adults could help reinforce the need for vaccine development. the authors thank alexandra hill-ricciuti for analytic assistance. seasonal trends of human parainfluenza viral infections: united states estimates of parainfluenza virus-associated hospitalizations and cost among children aged less than years in the united states epidemiology and clinical presentation of parainfluenza type in children: a -year comparative study to parainfluenza types - epidemiology of parainfluenza infection in england and wales, - : any evidence of change? human parainfluenza type infections mosaic: mobile surveillance for acute respiratory infections and influenza-like illness in the community community -and hospital laboratory-based surveillance for respiratory viruses. influenza other respir viruses filmarray respiratory panel information sheet multicenter evaluation of the eplex respiratory pathogen panel for the detection of viral and bacterial respiratory tract pathogens in nasopharyngeal swabs respiratory syncytial virus infection in elderly and high-risk adults children with complex chronic conditions in inpatient hospital settings in the united states pediatric deaths attributable to complex chronic conditions: a population-based study of washington state predicting severe pneumonia outcomes in children human parainfluenza virus types - in hospitalized children with acute lower respiratory infections in china a prospective study of parainfluenza virus type infections in children attending daycare severe pneumonia after cardiac surgery as a result of infection with parainfluenza virus type human parainfluenza virus outbreak and the role of diagnostic tests defining the risk and associated morbidity and mortality of severe respiratory syncytial virus infection among infants with chronic lung disease influenza virus infections among patients attending emergency department according to main reason to presenting to ed: a -year prospective observational study during seasonal epidemic periods single-and multiple viral respiratory infections in children: disease and management cannot be related to a specific pathogen seasonality and clinical impact of human parainfluenza viruses key: cord- - no e authors: jeannoël, m.; lina, g.; rasigade, j. p.; lina, b.; morfin, f.; casalegno, jean sebastien title: microorganisms associated with respiratory syncytial virus pneumonia in the adult population date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: no e respiratory syncytial virus (rsv) has been recognized as responsible for severe respiratory illness in adults, especially in the elderly. while pneumonia is commonly observed during rsv infection, the burden and epidemiology of bacterial superinfection is poorly understood. the aim of this study was to identify microorganisms associated with rsv-positive pneumonia in adults. a retrospective study was conducted during three consecutive winters (october to april – ) in the university hospital of lyon, france. during rsv circulation periods, a systematic rsv screening was performed by reverse-transcription pcr on all respiratory samples collected from adults. records of rsv-positive patients were subsequently analyzed to identify radiologically confirmed pneumonia cases. bacteria were identified by standard bacteriology cultures or urinary antigen screening and classified as potentially causative of pneumonia if quantification was above the specific threshold as defined by the european manual of clinical microbiology. overall, , adult respiratory samples were screened for rsv detection by pcr. in total, had a positive rsv detection ( . %) among which presented with pneumonia including bacterial superinfections ( . %) with streptococcus pneumonia, haemophilus influenza, staphylococcus aureus, pseudomonas aeruginosa, and moraxella catarrhalis. most patients were elderly ( . %) and patients with comorbidities ( . %). a more severe outcome was observed for rsv-bacteria-associated pneumonia compared with rsv pneumonia: length of stay was significantly longer ( days vs days) and icu hospitalization more frequent ( . % vs . %) (p < . ). in conclusion, we did not observe major differences in the epidemiology of bacterial superinfections in rsv-positive pneumonia compared to reports on post-influenza pneumonia. respiratory syncytial virus (rsv) is now recognized as an important cause of respiratory illness in adults, especially in the elderly [ ] . secondary bacterial pneumonia after or during an rsv infection is among the more severe complications reported in this population [ ] . few studies have described superinfection or coinfection with bacteria among adults with rsv-positive pneumonia in inpatient setting [ , ] . diagnostic testing for rsv is usually not routinely performed in this population. however burden and epidemiology of rsv-positive pneumonia in adults is important to assess in regards of the current antibiotic therapy recommendations [ ] . the aim of our study was to gain insights in the epidemiology of bacterial superinfections associated with rsv pneumonia in adults. we conducted a retrospective, monocenter, and observational study on three successive winter periods (october to april, april, - . in the university hospital of lyon (hospices civils de lyon, france), upper and lower respiratory tract samples from patients with respiratory symptoms are systematically screened for rsv and metapneumovirus (hmpv) using a reverse transcriptase polymerase chain reaction (rt-pcr). we included all adult patients (≥ years old) positive for rsv with radiologically confirmed pneumonia as diagnosed in the medical chart. hospital-acquired pneumonia (hap) was defined as radiologically confirmed pneumonia that manifests ≥ h after the patient's admission to the hospital, and not present at the time of intubation. data collected included laboratory results, demographics (age, sex), underlying conditions (cardiovascular disease, pulmonary disease, diabetes mellitus, and immunodeficiency), as well as clinical outcomes (length of stay, acute respiratory distress syndrome (ards), intensive care unit (icu) admission, in-hospital death). for virus detection, clinical specimens comprised nasopharyngeal swabs, endotracheal aspirates, and broncho-alveolar lavages. respiratory specimens were extracted using easymag (biomerieux, marcy l'étoile, france). rsv detection was performed using a rt-pcr (abi ; thermofisher, illkirch, france), or duplex rt-pcr detecting rsv and hmpv (r-gene detection kit, biomérieux-argene, france) as previously described [ ] . for bacteria detection, pathogens were classified as potentially causative of pneumonia if identified in pleural fluid or blood, or if identified within a sputum specimen, endotracheal aspirate, and broncho-alveolar lavage with good-quality criteria and if quantification was above the appropriate emcm decision threshold. semi-quantitative bacterial culture process and interpretation was performed following the european manual of clinical microbiology (emcm) guidelines [ ] . maldi-tof (vitek-ms) was used for species identification. statistical analyses were performed using graphpad prism software (version . , graphpad software, la jolla, ca, usa). univariate analysis was used to compare rsvbacteria-associated pneumonia and rsv-positive pneumonia. quantitative variables (age and length of stay) were expressed as median and interquartile range (iqr). difference between the age groups was assessed by student's t test. difference between lengths of stay was assessed by non-parametric mann-whitney test. qualitative variables were expressed as number and percentage. a fisher's exact test was used for univariate comparisons. a p value < . was considered significant. during the study period, viral rt-pcr was performed on , respiratory samples collected from adult patients. influenza was detected in patients ( . % of all respiratory samples), rhinovirus in patients ( . % of all respiratory samples), rsv in patients ( . % of all respiratory samples), and hmpv in patients ( . %). the median age of patients tested positive for rsv was years (interquartile range to ), with a sex ratio of ( . % were men). among rsv-positive patients, . % of case records were retrieved (n = / ). primary discharge diagnoses in patients without respiratory symptoms were as follows: . % (n = / ) of systematic sampling without acute respiratory infection in immunosuppressed patients, . % (n = / ) of acute heart failure, . % (n = / ) of sepsis, and . % (n = / ) of neurological disorders. . % (n = / ) patients presented with respiratory disorders, including . % (n = / ) of respiratory failure, . % (n = / ) of acute respiratory infection, . % (n = / ) of acute respiratory distress syndrome, and . % (n = / ) radiologically confirmed pneumonia. among these pneumonia, cases were associated with bacteria classified as potentially causative (rsv-bacteria-associated pneumonia) and cases had no bacterial documentation (rsv-positive pneumonia). among these rsvpositive pneumonia, cases were associated with another virus ( rsv and influenza coinfections, rsv and picornavirus coinfection, rsv and bocavirus coinfection, and rsv and cmv coinfection). among the rsv-bacteria associated pneumonia, hospital-acquired pneumonia (hap) was observed mostly in adults ≥ years old (n = / ) whereas community-acquired pneumonia (cap) occurred mostly in to -year-old adults (n = / ). most of the patients presenting a pneumonia were elderly (median age years (interquartile range to ) and had underlying conditions ( . %) ( table ). only . % were resident of nursing homes. demographic characteristics did not differ between patients with an rsv-bacteria-associated pneumonia and patients with an rsv-positive pneumonia. among these pneumonia, patients received an empiric antibiotic treatment and immunosuppressed patients received an antiviral treatment: patient received ribavirin, patient received polyvalent immunoglobulins, and patient received ribavirin and polyvalent immunoglobulins. in total, patients presented an ards, patients were admitted to icus and in-hospital mortality occurred in adults of which all were ≥ years old (n = / ) or immunosuppressed (n = / ). rsv and bacteria coinfection was statistically associated with a more severe outcome than rsv-positive pneumonia as length of stay was significantly longer ( days vs days) and icu hospitalization more frequent ( . % vs . %) (p < . ). however no significant difference was observed regarding in-hospital mortality ( table ) . among the cases of rsv-positive pneumonia with superinfection, the most frequent bacteria were streptococcus pneumoniae, enterobacteriaceae ( kleb siella p neu monia , en teroba cte r cloa ca e , citrobacter koseri), pseudomonas aeruginosa, haemophilus influenza, staphylococcus aureus, and moraxella catarrhalis. in two patients, more than one bacterial pathogen was detected. in patients hospitalized f o r c a p, t he m o s t f r e qu en t ba ct er i a w e re s . pneumoniae, h. influenzae, and s. aureus whereas in hap the most frequent bacteria were p. aeruginosa and enterobacteriacae (table ). in this study, detection rates of the screened viruses were consistent with previous reports describing the epidemiology of respiratory viruses in hospitalized adults [ ] [ ] [ ] . rsv detection in our study was low ( . %) compare to rates of rsv detection reported in previous studies ranging from % of hospitalized adult patients to % of hospitalization for acute respiratory illness in an elderly population [ ] [ ] [ ] . it is probably due to the systematic testing strategy associated to a species distribution of pathogenic bacteria involved in rsv-positive pneumonia (cap) and hospital-acquired pneumonia (hap) sampling bias toward influenza-like illness. those symptoms are indeed poorly predictive of an rsv infection [ ] . among rsv infected patients, . % presented a radiologically confirmed pneumonia and . % had an rsv-bacteriadocumented pneumonia. despite the large number of rsv testing among the adult population, we identified only a few rsv radiologically confirmed pneumonia. although this is a low prevalence, it is in agreement with the current literature in which to % of rsv-bacteria co-infections were reported [ ] . in our study, a higher prevalence was noted in the elderly ( / ), immunosuppressed, and patients with comorbidities ( / ). as it has been previously described, we observed that rsv-bacteria-associated pneumonia have a more severe outcome than rsv-positive pneumonia [ ] . enterobacteriaceae and pseudomonas aeruginosa were the most frequently detected pathogens in hap and did not differ from the pathogens usually responsible for hap in icu [ ] . s. pneumonia and h. influenza were the most frequently detected pathogens in cap. in a previous study in adults with cap, s. pneumonia and h. influenza were also mainly reported in rsv-bacteria coinfections [ ] . we did not observed major differences to influenza-bacterial coinfections [ ] . those results suggest that there is no main difference between the microbiological epidemiology of rsv-bacterial coinfections and influenza-bacterial coinfections. this observation needs to be confirmed by other studies. further research should focus on estimating the rsv cap burden in adult as well as understanding the underlying mechanisms of copathogenesis. conflict of interest the authors declare that they have no conflict of interest. ethical approval this article does not contain any studies with human participants performed by any of the authors. informed consent for this type of study formal consent is not required. risk of mortality associated with respiratory syncytial virus and influenza infection in adults viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia respiratory syncytial virus infection in elderly and high-risk adults incidence and characteristics of viral community-acquired pneumonia in adults community-acquired pneumonia genetic characterization of respiratory syncytial virus highlights a new ba genotype and emergence of the on genotype in lyon european manual of clinical microbiology. basel: european society for clinical microbiology and infections diseases : société française de microbiologie can analysis of routine viral testing provide accurate estimates of respiratory syncytial virus disease burden in adults? prevalence of respiratory viruses among adults, by season, age, respiratory tract region and type of medical impact of viral multiplex real-time pcr on management of respiratory tract infection: a retrospective cohort study respiratory syncytial virus hospitalization in middle-aged and older adults rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults respiratory syncytial virus infection in adults clinical characteristics and outcome of respiratory syncytial virus infection among adults hospitalized with influenza-like illness in france elucidation of bacterial pneumonia-causing pathogens in patients with respiratory viral infection hospital-acquired pneumonia in icu the co-pathogenesis of influenza viruses with bacteria in the lung key: cord- - e xrd y authors: watanabe, tokiko; iwatsuki-horimoto, kiyoko; kiso, maki; nakajima, noriko; takahashi, kenta; jose da silva lopes, tiago; ito, mutsumi; fukuyama, satoshi; hasegawa, hideki; kawaoka, yoshihiro title: experimental infection of cynomolgus macaques with highly pathogenic h n influenza virus through the aerosol route date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: e xrd y several animal models are used to study influenza viruses. intranasal inoculation of animals with a liquid inoculum is one of the main methods used to experimentally infect animals with influenza virus; however, this method does not reflect the natural infection with influenza virus by contact or aerosol route. aerosol inhalation methods have been established with several influenza viruses for mouse and ferret models, but few studies have evaluated inoculation routes in a nonhuman primates (nhp) model. here, we performed the experimental infection of nhps with a highly pathogenic h n influenza virus via the aerosol route and demonstrated that aerosol infection had no effect on clinical outcome, but caused broader infection throughout all of the lobes of the lung compared with a non-aerosolized approach. aerosol infection therefore represents an option for inoculation of nhps in future studies. influenza viruses cause annual epidemics and periodic pandemics. we have experienced three pandemics in the th century (i.e., spanish flu in - , asian influenza in , and hong kong influenza in ) and one in the st century (i.e., pandemic influenza a (h n ) in ). these influenza pandemics have a huge impact on public health and the global economy . additionally, recent sporadic human infections with h n and h n avian influenza viruses have raised the pandemic threat of these viruses [ ] [ ] [ ] [ ] . the continued circulation of h n viruses in birds provides opportunities for them to infect humans. indeed, human cases of h n infections have been reported in several countries, with a total of confirmed cases and fatalities as of oct , which is a case fatality rate of approximately % (http://www.who.int/influenza/human_animal_interface/ _ _ _tableh n .pdf?ua= ). therefore, concern over the pandemic potential of h n viruses is clearly justified. influenza virus can be transmitted from person to person through several transmission modes, including direct contact, indirect contact with a contaminated object (fomite), droplet transmission (droplets of > µm in diameter), and droplet nuclei transmission, also referred as airborne or aerosol transmission (droplet nuclei of < µm in diameter, which can remain suspended in the air for prolonged periods). experimental infection of animal models with influenza viruses is an essential component of the characterization of influenza viruses. mice, ferrets, and nonhuman primates (nhps) are commonly used to examine the pathogenicity, replicative ability, and transmissibility of influenza viruses, although each model has its advantages and limitations. nhps have been frequently used as a model to characterize the virulence of various influenza viruses, such as highly pathogenic h n , and the and pandemic viruses, because of their close genetic relationship to humans [ ] [ ] [ ] . division of virology, department of microbiology and immunology, institute of medical science, university of tokyo, tokyo, - , japan. department of pathology, national institute of infectious diseases, shinjuku-ku, tokyo, - , japan. intranasal and/or intratracheal inoculation with a liquid inoculum of influenza viruses has been the primary method used to infect nhps; however, a liquid inoculum does not reflect the natural infection. aerosol inhalation methods for inoculation of mice and ferrets have been established with several influenza viruses, including h n viruses [ ] [ ] [ ] [ ] [ ] . in the ferret model, these studies demonstrated that the inoculation of animals with highly pathogenic avian influenza h n virus via the aerosol route led to higher nasal wash virus titers, earlier onset of clinical signs, and/or a broader spectrum of disease compared with infection via intranasal inoculation despite no difference in lethality [ ] [ ] [ ] . in a murine model, belser et al. recently established aerosol infection with highly pathogenic h n influenza virus in the nhp model; however, they have not compared the outcomes in the nhps infected via the aerosol route with those in nhps infected via the conventional method of liquid virus inoculation. therefore, in this study, we compared the virus distribution, pathogenicity, and disease outcome in nhps infected via the aerosol route with these parameters in nhps infected through multiple routes (e.g., the intranasal and intratracheal routes). establishment of an h n virus infection system in nonhuman primates through an aerosol route by using a commercial nebulizer. a wide range of commercial nebulizers are available for clinical and research use. in this study, we used the ultrasonic nebulizer ne-u from omron healthcare co, ltd, japan. this was chosen because it is easy to use and could reduce the treatment time needed compared with other nebulizer devices , thereby making the procedure less stressful for the animals. the particle size is also critical for aerosol inhalation because aerosol transmission (also referred to as droplet nuclei transmission) occurs when small particles (< µm) are inhaled. a previous study demonstrated that the ne-u nebulizer generates a large fine particle fraction of aerosol mass < µm (the mean percentage of the fine particle fraction was %) . we used -to -year-old male cynomolgus macaques that weighed - kg each. four animals were inoculated with ml of a pfu/ml solution of the highly pathogenic h n avian influenza virus a/vietnam/ ut / strain (vn ) through the aerosol route by using the ultrasonic nebulizer ne-u . these animals were defined as 'the aerosol method group' . as a control, ml of the virus solution was administered through multiple routes by the conventional method, that is, by the intranasal ( . ml each to the left and right nostril), intraoral ( . ml each to the left and right tonsil), intraocular ( . ml per eye), and intratracheal ( . ml using a tracheal catheter) routes (defined as 'the conventional method group'). the virus dose of × pfu of influenza virus was chosen for inoculation because a similar virus dose has been widely used in previous studies to infect nhps to ensure the greatest likelihood of virus infection and replication in the inoculated animals , , [ ] [ ] [ ] . upon virus infection, none of the animals exhibited noticeable clinical signs (i.e., no appreciable body weight loss or fever was observed in any of the infected animals) except for a slight decrease in appetite among all of the infected animals. to examine virus shedding, nasal swab samples were collected from the infected animals on days and post-infection for virus titration. on day post-infection, vn virus was recovered from four animals in the conventional method group, whereas viruses were detected from three of four animals in the aerosol method group (table and fig. ). the mean virus titers were not significantly different between the conventional and the aerosol method groups [ . and . log (pfu/ml), respectively] (fig. ). on day post-infection, vn virus was recovered from nasal swabs of two and three animals in the conventional and aerosol method groups, respectively, and the mean virus titers were comparable between the two groups. to compare the distribution of h n virus in animals infected via the conventional method with that in animals infected via the aerosol method, animals from each group were euthanized on day post-infection and organ samples were collected for virus titration. relatively high virus titers were detected in the tonsils of all infected animals in both groups ( table ) . viruses were recovered from the nasal mucosa, trachea, and bronchus of some of the infected animals in both groups (table ). additionally, virus was recovered from the mediastinal lymph nodes of two and one of animal id virus titers (log pfu/ml) of animals infected with vn virus via the: aerosol method # - # - # - # - # - # - # - # - table . virus titers in nasal swabs from cynomolgus macaques infected with the h n virus via the conventional or aerosol method a . a cynomolgus macaques were inoculated with ml of a pfu/ml solution of a/vietnam/ut / virus (vn ) by the conventional method (through a combination of the intratracheal, intranasal, ocular, and oral routes) or via the aerosol method by using a nebulizer. b -, virus not detected (detection limit: . log pfu/ml). virus titers in respiratory swabs from infected cynomolgus macaques. cynomolgus macaques were inoculated with ml of a pfu/ml solution of the highly pathogenic h n avian influenza virus a/vietnam/ ut / strain (vn ) through the aerosol route by using the ultrasonic nebulizer ne-u (defined as "the aerosol method group"). as a control, ml of the virus solution was administered through multiple routes, which is the conventional method; by intranasal ( . ml each to the left and right nostril), intraoral ( . ml each to the left and right tonsil), intraocular ( . ml per eye), and intratracheal ( . ml using a tracheal catheter) routes (defined as "the conventional method group"). nasal swab samples were collected on days and postinfection for virus titration. red and blue horizontal bars show the mean titers for the aerosol and conventional method groups, respectively. virus titers (log pfu/g) of animals infected with vn virus via the: four animals in the conventional and aerosol method groups, respectively. also, virus was detected in the duodenum and brain of one of four animals in the conventional and aerosol method groups, respectively (table ) . interestingly, the virus distribution in the lower respiratory tract differed between the conventional and aerosol method groups. vn virus replicated efficiently in all of the lung lobes of the infected animals in the aerosol method group, whereas no virus was recovered from some of the lung lobes of some animals in the conventional method group ( table ). the mean virus titers in right upper, right middle, left upper, and left middle lobes of the lungs of the infected animals in the aerosol method group were significantly higher than those of the infected animals in the conventional method group [the mean virus titers in the aerosol method group were . , . , . , and . log (pfu/g), whereas those in the conventional method group were . , . , . , and . log (pfu/g), respectively] (fig. ) . in contrast, vn replicated well in the right-and left-lower lung lobes of the infected animals in the conventional method group [the virus mean titers were . and . log (pfu/g), respectively]. presumably, the introduction of virus as a liquid via the intratracheal and intranasal routes led to a large influx of virus solution into the right-and left-lower lobes. we assume that this also occurs in ferrets because we often observe similar macroscopic pathological changes in the lower lung lobes of infected ferrets (unpublished data). taken together, these findings demonstrate that a productive h n virus infection in nhps can be induced via the aerosol route by using the commercial ultrasonic nebulizer ne-u , and inoculation of nhps with aerosolized h n virus can result in the development of virus infection across a large area of the lower respiratory tract and the distribution of virus in all of the lung lobes of the infected animals, whereas inoculation via the conventional method can result in some lung lobes not being exposed to or infected with virus. histopathologic changes in the respiratory tract of animals infected with h n virus via the conventional and aerosol methods. next, we compared histopathological changes in the infected animals in the aerosol method group with those in the conventional method group. for this histopathologic analysis, we focused on two animals for each group in which virus was recovered from a variety of organs (i.e., # - and # - from the conventional method group, and # - and # - from the aerosol method group). nasal cavity, trachea, lung, epiglottis, heart, liver, spleen, kidney, small intestine, large intestine, and brain (i.e., olfactory bulb, cerebrum, cerebellum, and brainstem) were examined using hematoxylin-and-eosin-stained sections obtained from the selected animals. we also examined the distribution of influenza virus antigen by immunohistochemistry. we found no obvious changes and no viral antigens in most of the tissues tested, except for the tracheal and lung tissues. in the trachea, we observed mild inflammatory cell infiltration but no virus-antigen-positive cells. pulmonary edema and inflammatory cell infiltration into the alveolar spaces were observed in all of the infected figure . replication of h n virus (vn ) in the lung lobes of infected monkeys. cynomolgus macaques were inoculated with ml of a pfu/ml solution of vn through the aerosol route or conventional multisite routes (i.e., intranasal, intraoral, intraocular, and intratracheal routes) by using the aerosol or conventional method, respectively. four animals from each group were euthanized on day post-infection for virus titration. red and blue horizontal bars show the mean titers for the aerosol and conventional method groups, respectively. in the aerosol method group, the virus titers in some of the lung lobes were significantly higher than those in the conventional method group on day post-infection (p < . in the right-upper, right-middle, and left-middle lobes, and p < . in the left-upper lobe). detection limit = . log pfu/ml/tissue sample. scientific reports | ( ) : | doi: . /s - - - animals tested. the degree of inflammation, which had spread from around the bronchi, was different for each part of the lungs of each animal. the total pathologic scores appear to be higher in the animals infected by the conventional method than those by the aerosol method, although no statistical analysis was conducted because of the small number of animals used ( table ). the parts of the lung section with a pathologic score of are shown in fig. . neutrophils and mononuclear cell infiltration with fibrinous exudates were observed in the interstitial areas and alveolar spaces. edema and desquamation of alveolar epithelial cells into the alveolar spaces were also observed. these observations are identical to those reported in human patients who died as a result of h n infection [ ] [ ] [ ] [ ] . the conventional method group showed more severe inflammation in the lungs compared with the aerosol method group (table ) . all animals showed virus-antigen-positive signals in the bronchial and alveolar epithelium (table ). the use of animal models is essential for studying the biological properties of influenza viruses such as pathogenicity, replicative ability, and disease outcome. in most cases, intranasal administration with a liquid containing the virus is used to infect animals with influenza virus; however, a liquid inoculum does not reflect the natural influenza virus infection. recently the aerosol inhalation method has been tested in several mammalian models, including mice, ferrets, and nhps due to its resemblance to human exposure to influenza virus. here, we inoculated nhps with a highly pathogenic h n influenza virus via the aerosol route and showed that aerosol infection did not affect clinical outcome, but did cause more widespread infection throughout the lower respiratory tract compared with the conventional approach. the uniform infection in all of the lung lobes and the bronchial epithelium afforded by the aerosol infection system offers an advantage in some experimental settings. for example, when researchers take lung tissues for multiple purposes (e.g., virus titration, histopathology, and various omics analyses), they need to take samples from different parts of the lungs. therefore, infection via the aerosol route would minimize experimental variability. recently, wonderlich et al. demonstrated that aerosol infection of nhps with a highly pathogenic h n avian influenza virus caused fulminant pneumonia that rapidly progress to acute respiratory distress syndrome, and some of the infected animals died or were humanely sacrificed due to respiratory failure. the same phenomenon has also been observed in the ferret model [ ] [ ] [ ] ; however, findings by wonderlich et al. are not consistent with our findings here. the differences in disease outcomes between their study and ours may be caused by the different conditions used for the aerosol infection; that is, they used a head-only exposure chamber (into which the nhp's head was inserted), which allowed the animals to inhale the aerosolized h n virus with an exposure duration ranging from to min. in contrast, we used a standard inhalation mask, which is not a tight-fitting mask and allows the animals to breathe the aerosol mist through the nose and mouth spontaneously; the exposure duration was approximately min, potentially leading to virus infection with a lower dose compared to wonderlich's study. in addition, other parameters, such as origin, gender, and age of the macaques, as well as the conditions for virus stock generation (i.e., egg-grown virus vs. mdck-grown virus (our study), which could table . pathologic scores and detection of influenza virus antigen in animals infected with h n virus via the conventional or aerosol method a . a cynomolgus macaques were inoculated with ml of a pfu/ ml solution of vn virus by using the conventional method (through a combination of intratracheal, intranasal, ocular, and oral routes) or via the aerosol method by using a nebulizer. b pathologic severity scores for infected animals. to represent comprehensive histological changes, respiratory tissue samples were evaluated by scoring pathologic changes. pathologic scores were determined for each lung lobe in summary, here we established a system for aerosol infection of nhps with highly pathogenic h n influenza virus. we found that experimental infection with aerosolized h n virus led to a wider distribution of virus in the lower respiratory tract compared with liquid virus inoculation via the conventional method. however, no difference in clinical outcome was observed between the two inoculation methods. a highly pathogenic h n avian influenza virus, a/vietnam/ut / (h n ; vn ), was isolated in our laboratory from the same human specimen from which a/vietnam/ / (h n ; vn ) was isolated. there are no amino acid differences between vn and vn although there are two nucleotide differences, one in the na gene at position and the other in the m gene at position (in both cases, vn has adenine at these positions and vn has guanine). all experiments with vn virus was performed in enhanced biosafety level (bsl ) containment laboratories at the university of tokyo and kyoto university, japan, which are approved for such use by the ministry of agriculture, forestry, and fisheries, japan. cells. madin-darby canine kidney (mdck) cells were maintained in eagle's minimal essential medium (mem) containing % newborn calf serum. the genetic origin of the mdck cells was confirmed by dna fingerprinting (using random amplified polymorphic dna) conducted by takara bio japan. mdck cells were used for plaque assays to titrate viruses. experimental infection of nonhuman primates. two-to three-year-old male cynomolgus macaques, weighing . - . kg and serologically negative by neutralization assays for currently circulating human influenza viruses, were obtained from cambodia (obtained from japan laboratory animals, inc., tokyo, japan). all experiments with macaques were performed in accordance with the regulation on animal experimentation guidelines at kyoto university and were approved by the committee for experimental use of nonhuman primates in the institute for virus research, kyoto university. for the aerosol inoculation (aerosol method group), animals were anesthetized with ketamine via intramuscular injection and, by using the ultrasonic nebulizer ne-u (omron healthcare co, ltd, japan), ml of pfu/ figure . pathological findings in the lungs of infected monkeys. shown are representative pathological findings in the lungs of monkeys infected with vn by using the conventional (a,c; right-lower lobe of animal # - , whose pathologic score and score of detection of antigen-positive cells were and +, respectively) or aerosol (b,d; right-middle lobe of animal # - , whose pathologic score and score of detection of antigen-positive cells were and +, respectively) method at day post-infection with hematoxylin-eosin (he) staining (a,b) and immunohistochemistry for influenza viral antigen (np) (c,d). the animal id numbers correspond to those shown in tables , , and . scale bar is μm. scientific reports | ( ) : | doi: . /s - - - ml of virus was aerosolized and administered to each animal via an inhalation mask, which is not a tight-fitting mask and allows the individual to breathe the aerosol mist through the nose and mouth spontaneously. for virus inoculation through multiple routes, that is, the conventional method (the conventional method group), animals were anesthetized with ketamine via intramuscular injection and inoculated with a suspension containing a total of × pfu of virus through a combination of the intratracheal ( . ml), intranasal ( . ml per nostril), ocular ( . ml per eye) and oral ( . ml each to the left and right tonsil) routes. body temperature was monitored at , , , and days post-infection by use of a rectal thermometer. at the indicated timepoints post-infection, two or four macaques per group were euthanized for virologic and pathologic examinations. the virus titers in various organs and nasal swabs were determined by using plaque assays in mdck cells. pathologic examination. animal tissues were fixed in % phosphate-buffered formalin for pathologic examination. they were then processed for paraffin embedding and cut into -µm-thick serial sections. one section from each tissue sample was subjected to standard hematoxylin-and-eosin staining, while another was processed for immunohistochemistry with a mouse monoclonal antibody for type a influenza virus np antigen that reacts comparably with all of the test viruses. this antibody was produced in our laboratory and used for immunohistochemistry at : dilution. specific antigen-antibody reactions were visualized by use of , ′-diaminobenzidine tetrahydrochloride staining and a dako envision system (dako co. ltd., tokyo, japan). for statistical analyses, we used r (www.r-project.org) and lme to fit a linear mixed effects model to the virus titer data. different models were fitted for each biological question, namely, (i) the differences in virus titers between the groups over time (table ) , and (ii) infection of different organs (table ) , and infection of different lung parts (table and fig. ). in the first case, we used as fixed effects the different groups (i.e., the aerosol or conventional infection methods), and the time of the measurement. in the second case, we used as fixed effects the different groups (i.e., the aerosol or conventional infection methods), and the different organs in which the virus titers were measured. in both models, as random effects, we had intercepts for the different animals in the study. finally, we used the lsmeans package , to compare the groups for each model separately, and the p-values were adjusted using holm's method. p-values were considered significant if they were less than . . modified organisms of the university of tokyo and kyoto university, and, when required, by the competent minister of japan. all experiments were approved by the respective committees at the university of tokyo and kyoto university. all experiments with h n viruses were performed in enhanced biosafety level laboratories at the university of tokyo (tokyo, japan), and kyoto university (kyoto, japan), which are approved for such use by the ministry of agriculture, forestry and fisheries, japan. staff working in enhanced bsl- and bsl- ag wear disposable overalls and powered air-purifying respirators. the enhanced bsl- facilities at the university of tokyo and kyoto university include controlled access, effluent decontamination, negative air-pressure, double-door autoclaves, hepa-filtered supply and exhaust air, and airtight dampers on ductwork connected to the animal cage isolators and biosafety cabinets. the structure is pressure-decay tested regularly. all personnel complete biosafety and bsl- training before participating in bsl- -level experiments. human infection with a novel avian-origin influenza a (h n ) virus epidemiology of human infections with avian influenza a(h n ) virus in china a. h n influenza-continuing evolution and spread pandemic influenza as a current threat early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus pathogenesis of influenza a (h n ) virus infection in a primate model aberrant innate immune response in lethal infection of macaques with the influenza virus ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus a/vietnam/ / (h n ) influenza virus aerosol exposure and analytical system for ferrets comparison of the levels of infectious virus in respirable aerosols exhaled by ferrets infected with influenza viruses exhibiting diverse transmissibility phenotypes comparison of traditional intranasal and aerosol inhalation inoculation of mice with influenza a viruses experimental transmission of influenza virus infection in mice. i. the period of transmissibility influenza a virus challenge models in cynomolgus macaques using the authentic inhaled aerosol and intra-nasal routes of infection widespread virus replication in alveoli drives acute respiratory distress syndrome in aerosolized h n influenza infection of macaques identification and validation of nebulized aerosol devices for sputum induction lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes massive mobilization of dendritic cells during influenza a virus subtype h n infection of nonhuman primates experimental infection of macaques with a wild water bird-derived highly pathogenic avian influenza virus (h n ) molecular biology, and pathogenesis of avian influenza a (h n ) infection in humans the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h n -a review apoptosis and pathogenesis of avian influenza a (h n ) virus in humans h n infection of the respiratory tract and beyond: a molecular pathology study fitting linear mixed-effects models using lme least-squares means: the r package lsmeans we thank susan watson for scientific editing. we also thank naomi fujimoto, mikiko tanaka, kazue goto, yuko sato, hiromi sakawaki, and tomoyuki miura for technical assistance. competing interests: y.k. has received grant support from chugai pharmaceuticals, daiichi sankyo pharmaceutical, toyama chemical, and ttsumura co., ltd; royalties from medimmune; and is a co-founder of flugen.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -gcfx authors: ianevski, aleksandr; zusinaite, eva; kuivanen, suvi; strand, mårten; lysvand, hilde; teppor, mona; kakkola, laura; paavilainen, henrik; laajala, mira; kallio-kokko, hannimari; valkonen, miia; kantele, anu; telling, kaidi; lutsar, irja; letjuka, pille; metelitsa, natalja; oksenych, valentyn; bjørås, magnar; nordbø, svein arne; dumpis, uga; vitkauskiene, astra; Öhrmalm, christina; bondeson, kåre; bergqvist, anders; aittokallio, tero; cox, rebecca j.; evander, magnus; hukkanen, veijo; marjomaki, varpu; julkunen, ilkka; vapalahti, olli; tenson, tanel; merits, andres; kainov, denis title: novel activities of safe-in-human broad-spectrum antiviral agents date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: gcfx according to the who, there is an urgent need for better control of viral diseases. re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified compounds that target at least three viral diseases. we tested of these compounds against eight different rna and dna viruses. we found novel activities for dalbavancin against echovirus , ezetimibe against human immunodeficiency virus and zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against rift valley fever virus. thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases. dozens of viruses, such as fluav, hsv- , vzv, cmv and nov, constantly infect human population and represent substantial public health and economic burden (dalys and collaborators, ; disease et al., ) . emerging and re-emerging viruses, such as ebov, marv, lasv, chikv, zikv, denv, rvfv, mers-and sars-cov, surface from natural reservoirs approximately one each year and also represent global threats (howard and fletcher, ; who, ) . according to who, there is an urgent need for better control of these viruses, including drug-resistant and vaccine immunity escaping viral strains (bekerman and einav, ; de clercq and li, ) . antiviral drugs and vaccines are the most powerful tools to combat viral diseases. most drugs and vaccines, however, selectively target a single virus, thereby providing a "one drug-one bug" solution. by contrast, broad-spectrum antivirals (bsas) can cover multiple viruses and genotypes and reduce the likelihood of development of resistance. therefore, some bsas can be used for rapid management of new or drug-resistant viral strains, for treatment of viral co-infections reducing therapy complexity, as well as a first-line treatment or the prophylaxis of acute virus infections. thus, to overcome time and cost issues associated with the development of virus-specific drugs and vaccines, the development of bsas should be prioritized (bekerman and einav, ) . nucleotide and nucleoside analogues are excellent examples of bsas. they inhibit transcription and/or replication of different rna and dna viruses (de clercq, ) . in particular, valaciclovir inhibits replication of different herpesviruses and hbv (laube et al., ; vere hodge and field, ) . cidofovir and its lipid conjugate brincidofovir also inhibit replication of dsdna viruses, such as herpesviruses, adv, bkv, and hpv (andrei et al., ) . ribavirin blocks viral rna synthesis of fluav, hcv and rsv (hong and cameron, ) . favipiravir and bcx also inhibit replication of different rna viruses (mckimm-breschkin et al., ) . however, viruses are able to develop resistance to some of these nucleotide and nucleoside analogues. other examples of bsa agents include inhibitors of cellular pathways, which are exploited by different viruses for efficient viral replication (debing et al., ) . these agents overcome the problem of antiviral drug resistance. for example, lipid-lowering statins (atorvastatin, lovastatin, simvastatin, and fluvastatin) inhibit cellular hmg-coa reductase and attenuate replication of some enveloped viruses (bernal et al., ; enserink, ) . anti-malaria quinolones (chloroquine and hydroxychloroquine) inhibit acidification of endosomes, which is an essential process for uncoating of ssrna viruses (al-bari, ). anticancer kinase inhibitors dasatinib, imatinib, gefitinib, nilotinib, erlotinib and sunitinib impair intracellular viral trafficking and exert bsa effects (bekerman et al., ; schor and einav, ) . the anti-duchenne muscular dystrophy agent, alisporivir, targets cellular cyclophilin and inhibits the folding of hcv, hiv, mers-and sars-cov proteins, and, therefore, prevents formation of infectious virus particles (boldescu et al., ; de wilde et al., ; soriano et al., ) . thus, both host-directed antivirals and nucleotide/nucleoside analogues could possess bsa activity. here, we hypothesised that some of the identified safe-in-human bsas could possess novel antiviral activities and, therefore, could be used for treatment of many different viral infections. to prove this hypothesis, we reviewed safe-in-man approved, investigational and experimental antiviral agents. we identified compounds that target at least three viral diseases. we tested of the compounds against different viruses and found novel activities for of these agents. we conclude that the spectrum of antiviral activities for existing bsa agents could be expanded towards other viral diseases. information on the viruses and associated human diseases is summarized in table s . information on approved, investigational and experimental safe-in-human antivirals is summarized in tables s -s . this information was extracted from drugbank ( ), clinical trials websites ( ) and pubmed. information on approved, investigational, and experimental antivirals, which target ≥ viral diseases, is summarized in table s . eye diagrams and interaction network plots were created with javascript library d .js v ( ). a structural similarity plot for the drugs was constructed and visualized using a c-spade web application (ravikumar et al., ) . the compounds used in this study, their suppliers and catalogue numbers are summarized in table s . to obtain mm stock solutions compounds were dissolved in % dimethyl sulfoxide (dmso, sigma-aldrich) or milli-q water. the solutions were stored at − °c until use. bhk- cells (baby hamster kidney fibroblasts) were grown in glasgow's minimal essential medium (gmem) containing . % fetal bovine serum (fbs; gibco, paisley, uk), % tryptose phosphate broth (tpb), mm hepes, u/ml penicillin and . mg/ml streptomycin (penstrep, lonza basel, switzerland). ach- cells, a model for chronic hiv- infection, which possesses a single integrated copy of the provirus hiv- strain lai (nih aids reagent program), were grown in rpmi- medium supplemented with % fbs and penstrep. madin-darby canine kidney epithelial (mdck) cells, human embryonic kidney cells (hek t) and african green monkey kidney epithelial cells (vero) were grown in dulbecco modified eagle's medium (dmem; sigma-aldrich, st. louis, mo, usa) supplemented with mm l-glutamine (lonza; basel, switzerland), u/ml penstrep and % fbs. human telomerase reverse transcriptase-immortalized retinal pigment (rpe) cells were grown in dmem-f medium supplemented with u/ml penstrep, mm l-glutamine, % fbs, and . % sodium bicarbonate (sigma-aldrich). tzm-bl cells were grown in dmem supplemented with % heat-inactivated fbs and penstrep. human lung adenocarcinoma epithelial cells, a , were cultured in dmem containing . g/l nahco , mm hepes (euroclone, milan, italy), penstrep, and % fetal bovine serum (fbs, gibco) at °c. all cell lines were grown in humidified incubator at °c in the presence of % co . human influenza a/wsn/ (h n ) virus was generated using eight-plasmid reverse genetics system in hek and vero-e cells, as described previously (hoffmann et al., ) . ev strain was propagated in a monolayer of vero cells, as described earlier (myllynen et al., ) . hsv- was amplified in vero cells, as described previously (nygardas et al., ) . zikv fb-gwuh- strain was cultured in vero e cells, as described earlier (driggers et al., ) . for production of hiv- , × ach- cells were seeded in ml of full culture media, and hiv- production was induced by the addition of nm phorbol -myristate -acetate (viira et al., ) . the induced cells were incubated for h, and subsequently, the hiv- containing media was collected and filtered. the amount of hiv- in the stock was estimated by quantification of p protein in the media. quantity of p was measured using a reference recombinant purified p protein and anti-p -elisa, which was developed in-house. chikv- sg-nanoluc strain was generated from icdna clone of picres representing lr opy strain belonging to east/central/ south african genotype (utt et al., ) . rrv- sg-nanoluc strain derived from rrv-t strain (jupille et al., ) . sequence encoding nanoluc protein was placed between non-structural and structural regions of chikv or rrv genomes under the control of native subgenomic promoters of the viruses. to ensure expression of viral structural proteins, a copy of subgenomic promoter (residues − to + for chikv and residues − to + for rrv, positions given with respect to start site of subgenomic rna) was inserted immediately downstream of sequence encoding for nanoluc. recombinant rvfv expressing the far-red fluorescent protein katushka instead of the deleted nss protein (rrvfvΔnss::katushka) was used in this study (islam et al., ) . the virus stocks were stored at − °c. all the experiments with viruses were performed in compliance with the guidelines of the national authorities using appropriate biosafety laboratories under appropriate ethical and safety approvals. fluav virus was titrated in mdck cells using plaque assay as described previously (denisova et al., ) . ev titers were determined by plaque assay on a cells, as described earlier (marjomaki et al., ) . zikv titers were determined by plaque assay on vero-e cells, as described earlier (kuivanen et al., ) . hsv- titers were determined by plaque titration in vero cells in the presence of human immunoglobulin g ( μg/ml), as described earlier (paavilainen et al., ) . chikv- sg-nanoluc and rrv- sg-nanoluc were titrated in bhk- cells using plaque assay, as described previously (oo et al., ; taylor et al., ) . for testing the viability and death of compound-treated mock-, fluav-, ev -, chikv- sg-nanoluc-, rrv- sg-nanoluc-, zikv-and hsv -infected cells, approximately × rpe cells were seeded in each well of a -well plate. the cells were grown for h in cell growth medium. the media was replaced with virus growth medium (vgm) containing . % bsa, mm l-glutamine, . % nahco , and μg/ml l- -tosylamido- -phenylethyl chloromethyl ketone-trypsin (tpck)-trypsin (sigma-aldrich) in dmem-f . in the case of zikv, the media was replaced with dmem-f medium supplemented with u/ ml penstrep, mm l-glutamine, % fbs, and . % sodium bicarbonate. the compounds were added to the cells in three-fold dilutions at seven different concentrations starting from μm. no compounds were added to the control wells. the cells were mock-or virus-infected at a multiplicity of infection (moi) of one. when virus induced a cytopathic effect in cells (typically - hpi), celltox green express cytotoxicity reagent (ctxg, : dilution in the assay well, promega, madison, wi, usa) was added in vgm and the fluorescence was measured with a pherastar fs plate reader (bmg labtech, ortenberg, germany) or hidex sense microplate reader (hidex oy, turku, finland). the media was removed from the cells and stored at − °c. celltiter-glo viability reagent (ctg, promega, madison, wi, usa) containing firefly luciferase and luciferin was added ( μl per well). the luminescence was measured with a pherastar fs plate reader. for testing virus titers in compound-treated and non-treated fluav-, ev -, zikv-and hsv -infected cells the media was collected, serially diluted in pbs, and added to mdck (fluav), a (ev ), and vero-e (zikv and hsv- ) cells. the media was changed, and the cells were overlaid with plaque assay media. the cells were fixed, and viral titers were calculated. the titers were expressed as plaque-forming units per ml (pfu/ml). the presence of chikv- sg-nanoluc and rrv- sg-nanoluc in the media from non-or drug-treated rpe cells was evaluated by passaging the viruses in fresh rpe cells. the viruses expressed nano-luciferase from viral promoter, which was measured from lysed cells h post infection using renilla luciferase assay (promega, madison, wi, usa). for testing compound toxicity and efficacy against hiv- , approximately × tzm-bl cells were seeded in each well of a -well plate. tzm-bl cells express firefly luciferase under control of hiv- ltr promoter allowing quantitation of the viral infection (tat-protein expression by integrated hiv- provirus) using firefly luciferase assay. the cells were grown for h in cell growth medium. compounds were added to the cells in three-fold dilutions at seven different concentrations starting from μm. no compounds were added to the control wells. the cells were infected with hiv- (media that contained ng of hiv- p was used per well) or mock. at hpi, celltox green express cytotoxicity reagent (ctxg, : dilution in the assay well) was added and the fluorescence was measured with a pherastar fs plate reader. the media was removed from the cells, the cells were lysed, and firefly luciferase activity was measured using the luciferase assay system (promega, madison, wi, usa) and pherastar fs plate reader. to determine the efficacy of compounds against rvfv, the fluorescent intensity of individual infectious cell foci was quantified. briefly, a cells ( × /well) were grown in -well black plates with transparent bottoms (greiner bio-one international). compounds were serially diluted in dmso in two-fold steps from μm to . μm and mixed with pfu of rrvfvΔnss::katushka virus in a total volume of μl medium (moi, . ). the final concentration of dmso in the assay was %. the growth medium was removed from the wells and μl of compound and virus mixture was added to the cells for each compound concentration. at hpi the medium was removed, and cells were fixed with % paraformaldehyde (pfa) for h and washed with phosphate-buffered saline (pbs). μl pbs was added to each well and the plate was analyzed in the trophos plate runner hd (trophos, roche group) to count the number of virus infected cells per well, by identifying all individual cells expressing the far-red fluorescent protein katushka (islam et al., ) . the toxicity of compounds was analysed using ctg assay. the half-maximal cytotoxic concentration (cc ) and the halfmaximal effective concentration (ec ) for each compound were calculated after non-linear regression analysis with a variable slope using graphpad prism software version . a (graphpad software la jolla, ca, usa) or using hill curve fit with sigmastat . software (spss inc., chicago, il, usa). the relative effectiveness of drugs were quantified as the selectivity indexes (si = cc /ec ). there is limited information available on approved, investigational and experimental bsas. to identify all potential bsas, we reviewed approved, investigational and experimental safe-in-human antiviral agents in drug bank, clinical trials websites and pubmed, respectively. first, we searched for drugs, which have been approved for treatment of viral diseases in human. by excluding vaccines and discontinued drugs, we identified active antiviral substances. these compounds target viral, human and unknown factors and inhibit viruses (table s ). the approved antivirals include neutralizing antibodies, interferons, antisense oligonucleotide, and small molecules. only of the molecules target viruses or host factors associated with ≥ viral diseases. fig. shows bsas and other approved antiviral drugs linked to viral and host targets through viruses they inhibit. next, we reviewed all investigational antivirals and their safety profiles. by excluding vaccines from the search parameters, we found active compounds. these compounds target viral, human and unknown factors and inhibit viruses (table s ). the drug candidates include neutralizing antibodies, antisense oligonucleotides, interferons, enzyme and small molecules. only of the small molecules target ≥ viral diseases. fig. shows these and other investigational antiviral agents linked to cellular and host factors through targeted viruses. next, we searched for drugs, which have been approved for treatment of non-viral diseases, but for which antiviral activity has been reported. we found active compounds, which are all small-molecules. these compounds target viral, human and unknown factors, and inhibit human viruses (table s ) . thirty-nine of the antiviral agents target ≥ viral diseases. fig. shows these and other experimental antiviral agents connected to their host and viral targets through viruses they inhibit. altogether, we identified antiviral agents with available safety information in humans. these agents target host, viral and unknown factors and inhibit different viruses, belonging to viral families. fifty-nine agents have bsa activity (table s ). these agents target of the reported viruses (except andv, emcv and rabv) (fig. s ). structural analysis of bsa revealed that several drugs (such as, acyclovir, famciclovir and valacyclovir; imatinib and disatinib; minocyclin and doxycycline; ritonavir and lopinavir; hydroxychloroquine and chloroquine; esomeprazole and omeprazole) have similar scaffolds (fig. s ) . these drugs target a limited number of host and viral factors (fig. s ). in particular, statins (fluvastatin, lovastatin and simvastatin) target human hmg-coa; dalbavancin, oritavancin, teicoplanin and telavancin target human cyp a ; acyclovir, valacyclovir, famciclovir, azacitidine, brincidofovir, cidofovir, foscarnet, trifluridine, and vidarabine inhibit viral dna polymerases; bcx , favipiravir and ribavirin inhibit viral rna polymerases; and lamivudine and tenofovir disoproxil inhibit viral reverse transcriptases. we hypothesized that approved, investigational and experimental antiviral agents, which target ≥ viral diseases, could inhibit other viral infections. as a proof of concept, we tested of the identified bsas against fluav, ev , chikv, rrv and hsv- infections in rpe cells (alisporivir, cyt , sunitinib and thymalfasin were excluded; table s ). we also tested of the bsas against zikv infection in rpe cells (alisporivir, cyt , sunitinib, thymalfasin, dalbavancin, pentosan polysulfate and rapamycin were excluded). we evaluated viability and death of virus/mock-infected cells using ctg and ctxg assays, respectively. the ctg assay quantifies atp, an indicator of metabolically active living cells, whereas ctxg assay uses fluorescent cyanine dye that stains the dna of dead cells. after initial screening we found several hits, which kept infected cells alive or rescued infected cells from virus-mediated death. these hits are gemcitabine, gefitinib and vibarabine (fluav); gemcitabine, pirlindole dibucaine, fluoxetine and dalbavancin (ev ); gemcitabine, imatinib, ivermectin, lopinavir, lovastatin, ezetimibe, fluoxetine, bcx , chloroquine and hydroxychloroquine (zikv); chloroquine and mycophenolic acid (chikv); chloroquine, mycophenolic acid, dibucaine and itraconazole (rrv); as well as -azacitidine, gemcitabine, trifluridine and vidarabine (hsv- ). we repeated the ctg and ctxg assays with selected compounds and titrated fluav, ev , zikv and hsv- produced in drug-treated and non-treated cells. the presence of chikv and rrv viruses in the media collected from non-or drug-treated rpe cells was evaluated by infecting fresh rpe cells and measuring reporter protein expression from viral promoter (nanoluciferase activity). these experiments confirmed antiviral activity of gemcitabine against fluav (fig. s ) ; gemcitabine, pirlindole, dibucaine, fluoxetine, and dalbavancin against ev (fig. s ) ; gemcitabine, lopinavir, ezetimibe, bcx , chloroquine, and hydroxychloroquine against zikv (fig. s ) ; chloroquine and mycophenolic acid against chikv (fig. s ) ; mycophenolic acid against rrv (fig. s ) ; and gemcitabine against hsv- (fig. s ) . importantly, dalbavancin and ezetimibe demonstrated novel antiviral activities against ev and zikv, respectively. in particular, they rescued infected rpe cells from virus-mediated cell death, and lowered production of infectious virus particles without detectable cytotoxicity (table ) . we also examined toxicity and antiviral activity of of the bsa agents (excluding alisporivir, cyt , sunitinib and thymalfasin) against hiv- infection in tzm-bl cells. our primary screen identified ezetimibe, minocycline and rapamycin as anti-hiv- agents. validation experiment confirmed all three hits (fig. s , table ). interestingly, ezetimibe is a novel inhibitor, whereas minocycline and rapamycin are known anti-hiv- agents (heredia et al., ; singh et al., ) . in addition, we examined antiviral activity and toxicity of of the bsa agents (alisporivir, cyt , sunitinib, thymalfasin, dalbavancin, pentosan polysulfate and rapamycin were excluded) against rvfv expressing the far-red fluorescent protein katushka in a cells. our screen identified azacitidine, bortezamibe, cyclosporine, doxycycline, ezetimibe, fluoxetine, gefitinibe, minocycline, oritavancin, ritonavir and topotecan. azacitidine, cyclosporine, minocycline, oritavancin, ritonavirand and bortezamib remained after excluding compounds with si < ( fig. s ; table ). interestingly, azacitidine, cyclosporine, minocycline, oritavancin and ritonavirand are novel, whereas bortezamib is a known inhibitor of rvfv infection (keck et al., ) . thus, we tested several known bsa agents against (−)ssrna, (+) ssrna, ssrna-rt and dsdna viruses and identified novel activities for dalbavancin against ev , ezetimibe against zikv and hiv- , as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against rvfv. fig. shows known, validated and novel interactions between bsas and viruses. several approved and investigational agents, as well as safe in man chemical probes, were discovered for potential treatment of various viral diseases (bekerman and einav, ; de clercq, ; de clercq and li, ) . re-purposing such therapeutics from one viral disease to another could save resources and time needed for development of novel drugs. in this study, we tested approved, investigational and experimental bsa agents against fluav, rvfv, ev , zikv, chikv, rrv, hiv- and hsv- in vitro. we identified novel antiviral activities for dalbavancin (against ev ), ezetimibe (against hiv- and zikv), azacitidine, cyclosporine, minocycline, oritavancin and ritonavir (against rvfv) (fig. ) . dalbavancin is a lipoglycopeptide antibiotic, which is approved by the fda for the treatment of acute bacterial skin infections caused by staphylococcus aureus and streptococcus pyogenes. dalbavancin also inhibits mers-cov and sars-cov infections. it binds cathepsin l in the late endosomes/lysosomes and blocks the entry of ebov (zhou et al., ) . our study demonstrates that it also inhibits replication of ev , which also enters the cells via an endocytic route (krieger et al., ) . ezetimibe is an fda-approved medication that lowers plasma cholesterol by decreasing its absorption in the small intestine. ezetimibe also inhibits hbv and hdv infections by impairing viral entry mediated by pres -specific receptor hntcp (blanchet et al., ; lempp and urban, ; monrroy-bravo et al., ) . our study shows that ezetimibe also inhibited hiv- and zikv infections. however, the entry of these viruses into the cell is mediated by other receptors (hamel et al., ; wilen et al., ) . most probably, the anti-hiv- , ev , as well as hcv action of ezetimibe is associated with depletion of cholesterol which is required for entry of these viruses into the host cells (sainz et al., ) . importantly, this drug was successfully tested in combination with antiretroviral therapeutics to lower cholesterol levels in serum of hiv-infected patients (saeedi et al., ; wohl et al., ) . the question remains whether ezetimibe alone reduces hiv titers in these patients. azacitidine is a chemical analogue of cytidine, which is used in the treatment of myelodysplastic syndrome. it also inhibits fluav, adv, hiv- and hiv- replication by blocking viral rna or dna synthesis (beach et al., ; rawson et al., ) . cyclosporine is an immunosuppressive agent used for the treatment of rheumatoid arthritis, psoriasis, crohn's disease, nephrotic syndrome and keratoconjunctivitis sicca. in addition, cyclosporine is used to prevent graft rejection in organ transplant recipients. cyclosporine also inhibits hcv, fluav, wnv and zikv replication through blocking interaction of cellular cyclophilins with viral proteins and attenuating viral rna synthesis (barrows et al., ; firpi et al., ; qing et al., ) . minocycline is a broad-spectrum antibiotic and antiviral agent, which possesses activity against denv, hiv- and wnv (leela et al., ; quick et al., ; singh et al., ) . oritavancin is a semisynthetic glycopeptide antibiotic used for the treatment of gram-positive bacterial skin infections. it also inhibits ebov, mers-cov, and sars-cov infections (zhou et al., ) . ritonavir is an antiretroviral medication. it has also antiviral activity against mers-cov (chan et al., ) . we showed that azacitidine, cyclosporine, minocycline, oritavancin and ritonavir are active against rvfv. we also confirmed previously reported activities of chloroquine against chikv and zikv, mycophenolic acid against chikv and rrv, fluoxetine, pirlindole and dibucaine against ev , bcx , lopinavir and hydroxichloroquine against zikv, as well as gemcitabine against ev , fluav, zikv and hsv- (cao et al., ; delogu and de lamballerie, ; delvecchio et al., ; denisova et al., ; julander et al., ; kang et al., ; khan et al., ; kuivanen et al., ; lee et al., ; soderholm et al., ; ulferts et al., ; yuan et al., ) (fig. ) . the number of compounds with novel and confirmed antiviral properties may have been higher if we had used other cell lines, other viruses and viral strains, concentration ranges and purity of compounds, as well as endpoint measurements. also testing other compounds, which target < viral diseases, could reveal novel antiviral properties of these compounds and increase the number of potential bsas. for conducting this properly, a harmonized antiviral bioactivity data annotation, standardization, curation, and intra-resource integration is needed. excellent antiviral profiles from cell-line-based assays might not be reflected in vivo because systemic mechanisms may compensate the blocked target effect. therefore, the identification of bsa targets that are essential for viral replication but redundant for the cell is critical for reducing putative toxicities associated with blocking cellular pathways. thus, follow-up mechanistic studies and in vivo experiments are needed to validate our in vitro results. in conclusion, repositioning of safe-in-man agents from one viral disease to another could play a pivotal role in development of broadly acting antivirals. our study demonstrated the potential value of such approach with some examples, such as dalbavancin, ezetimibe, azacitidine, cyclosporine, minocycline, oritavancin and ritonavir, confirming a principle that "rich becomes richer". effective bsa treatment may shortly become available, pending the results of further pre-clinical studies and clinical trials. the most effective and tolerable compounds will expand the available therapeutics for the treatment of viral diseases. some of these compounds could be used as first-line therapeutics to combat emerging and re-emerging viral threats, having global impact by improving preparedness and the protection of the general population from viral epidemics and pandemics. the authors declare no financial and non-financial competing interests. half-maximal cytotoxic concentration (cc ), the half-maximal effective concentration (ec ) and minimal selectivity indexes (si) for selected broad-spectrum antivirals. ctxg -celltox green express cytotoxicity assay, ctg -celltiter-glo viability assay, otherplaque assay or reporter gene expression assay, n.a.not available. fig. . the interaction network between viruses and bsas, which are safe in man. drug-like shapes represent antiviral agents. blue spheres represent viruses. the diameter of spheres corresponds to the number of interactions between the viruses and the drugs. novel interactions between bsas and viruses are shown in red, validatedin blue, and known -in grey. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases insights into the mechanism of action of cidofovir and other acyclic nucleoside phosphonates against polyoma-and papillomaviruses and non-viral induced neoplasia a screen of fda-approved drugs for inhibitors of zika virus infection novel inhibitors of human immunodeficiency virus type infectivity infectious disease. combating emerging viral threats anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects statins in hiv-infected patients: potential beneficial effects and clinical use use of fda approved therapeutics with hntcp metabolic inhibitory properties to impair the hdv lifecycle broad-spectrum agents for flaviviral infections: dengue, zika and beyond inhibition of autophagy limits vertical transmission of zika virus in pregnant mice treatment with lopinavir/ritonavir or interferon-beta b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset clinical trial resources global, regional, and national disability-adjusted life-years (dalys) for diseases and injuries and healthy life expectancy (hale) for countries and territories, - : a systematic analysis for the global burden of disease study curious (old and new) antiviral nucleoside analogues with intriguing therapeutic potential approved antiviral drugs over the past years alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model the future of antivirals: broad-spectrum inhibitors chikungunya disease and chloroquine treatment chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection global, regional, and national incidence, prevalence, and years lived with disability for diseases and injuries for countries, - : a systematic analysis for the global burden of disease study zika virus infection with prolonged maternal viremia and fetal brain abnormalities infectious disease. old drugs losing effectiveness against flu; could statins fill gap? cyclosporine suppresses hepatitis c virus in vitro and increases the chance of a sustained virological response after liver transplantation biology of zika virus infection in human skin cells targeting of mtor catalytic site inhibits multiple steps of the hiv- lifecycle and suppresses hiv- viremia in humanized mice a dna transfection system for generation of influenza a virus from eight plasmids pleiotropic mechanisms of ribavirin antiviral activities emerging virus diseases: can we ever expect the unexpected? anti-rift valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir- , a broadacting antiviral compound efficacy of the broad-spectrum antiviral compound bcx against zika virus in cell culture and in a mouse model mutations in nsp and pe are critical determinants of ross river virus-induced musculoskeletal inflammatory disease in a mouse model synergistic antiviral activity of gemcitabine and ribavirin against enteroviruses characterizing the effect of bortezomib on rift valley fever virus multiplication cellular impdh enzyme activity is a potential target for the inhibition of chikungunya virus replication and virus induced apoptosis in cultured mammalian cells echovirus entry into polarized caco- cells depends on dynamin, cholesterol, and cellular factors associated with macropinocytosis obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism valaciclovir for chronic hepatitis b virus infection after lung transplantation gemcitabine, a broadspectrum antiviral drug, suppresses enterovirus infections through innate immunity induced by the inhibition of pyrimidine biosynthesis and nucleotide depletion drug repurposing of minocycline against dengue virus infection inhibitors of hepatitis b virus attachment and entry internalization of echovirus in caveolae prevention and treatment of respiratory viral infections: presentations on antivirals, traditional therapies and host-directed interventions at the th isirv antiviral group conference effect of ezetimibe in hcv viral load after liver transplantation a novel open and infectious form of echovirus a herpes simplex virus-derived replicative vector expressing lif limits experimental demyelinating disease and modulates autoimmunity deciphering the potential of baicalin as an antiviral agent for chikungunya virus infection inhibition of clinical pathogenic herpes simplex virus strains with enzymatically created sirna pools cyclosporine inhibits flavivirus replication through blocking the interaction between host cyclophilins and viral ns protein minocycline has anti-inflammatory effects and reduces cytotoxicity in an ex vivo spinal cord slice culture model of west nile virus infection c-spade: a web-tool for interactive analysis and visualization of drug screening experiments through compound-specific bioactivity dendrograms synergistic reduction of hiv- infectivity by -azacytidine and inhibitors of ribonucleotide reductase lipid lowering efficacy and safety of ezetimibe combined with rosuvastatin compared with titrating rosuvastatin monotherapy in hiv-positive patients identification of the niemann-pick c -like cholesterol absorption receptor as a new hepatitis c virus entry factor repurposing of kinase inhibitors as broad-spectrum antiviral drugs minocycline attenuates hiv- infection and suppresses chronic immune activation in humanized nod/ltsz-scidil- rgamma(null) mice immuno-modulating properties of saliphenylhalamide, sns- , obatoclax, and gemcitabine directly acting antivirals against hepatitis c virus effects of an in-frame deletion of the k gene locus from the genome of ross river virus screening of a library of fda-approved drugs identifies several enterovirus replication inhibitors that target viral protein c versatile transreplication systems for chikungunya virus allow functional analysis and tagging of every replicase protein antiviral agents for herpes simplex virus design, discovery, modelling, synthesis, and biological evaluation of novel and small, low toxicity s-triazine derivatives as hiv- non-nucleoside reverse transcriptase inhibitors who publishes list of top emerging diseases likely to cause major epidemics hiv: cell binding and entry. cold spring harb ezetimibe alone reduces low-density lipoprotein cholesterol in hiv-infected patients receiving combination antiretroviral therapy structure-based discovery of clinically approved drugs as zika virus ns b-ns protease inhibitors that potently inhibit zika virus infection in vitro and in vivo glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) this study was supported by the european regional development fund, the mobilitas pluss project mobtt (to denis e. kainov), jane and aatos erkko foundation grant # (to the vejo hukkanen). we thank qiuwei abdullah pan from the department of gastroenterology and hepatology, erasmus mc, university medical center rotterdam for advice. we thank ritva kajander for the hsv- culture. supplementary data related to this article can be found at http://dx. doi.org/ . /j.antiviral. . . . 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. ( e ) ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. microb combined effects of water activity, temperature and chemical treatments on the survival of salmonella and escherichia coli o : h on alfalfa seeds survey of current water use practices on fresh fruit and vegetable farms and evaluation of microbiological quality of surface waters intended for fresh produce production survival of pathogenic and indicator organisms in ground water the-survival-characteristics-of-a-non-toxigenic-strain-of-escherichia coli-o : h seafood and freshwater toxins: pharmacology, physiology, and detection microbiological spoilage and pathogens in minimally processed refrigerated fruits and vegetables incidence, contributing factors, and control of bacterial pathogens in produce comparison of survival of campylobacter jejuni in the phyllosphere with that in the rhizosphere of spinach and radish plants fitness of human enteric pathogens on plants and implications for food safety plant lesions promote the rapid multiplication of escherichia coli o : h on postharvest lettuce salmonella interactions with plants and their associated microbiota human pathogens associated with raw produce and unpasteurized juices, and difficulties in decontamination extended survival and persistence of campylobacter spp. in water and aquatic biofilms and their detection by immunofluorescent-antibody and-rrna staining water microbiology. bacterial pathogens and water multistate outbreaks of salmonella serotype poona infections associated with eating cantaloupe from mexicoeunited states and canada list of selected multistate foodborne outbreak investigations listeria outbreaks list of selected multistate foodborne outbreak investigations waterborne escherichia coli o environmental factors affecting escherichia coli and salmonella typhimurium numbers on land used for effluent disposal survival of campylobacter jejuni and escherichia coli in groundwater during prolonged starvation at low temperatures colonization of arabidopsis thaliana with salmonella enterica and enterohemorrhagic escherichia coli o : h and competition by enterobacter asburiae incidence and tracking of escherichia coli o : h in a major produce production region in california persistence of escherichia coli and salmonella in surface soil following application of liquid hog manure for production of pickling cucumbers microbial ecology of foodborne pathogens associated with produce current methods for extraction and concentration of enteric viruses from fresh fruit and vegetables: towards international standards investigation of rotavirus survival in different soil fractions and temperature conditions the influence of manure slurry irrigation on the survival of fecal organisms in scranton fine sand microbial quality of irrigation water used in leafy green production in southern brazil and its relationship with produce safety internalization of e. coli o : h and salmonella spp. in plants: a review prevalence and trends of bacterial contamination in fresh fruits and vegetables sold at retail in canada. food contr. , e viruses in the environment and their potential hazards. canadian centre for inland waters multinational salmonella paratyphi b variant java (salmonella java) outbreak, augustedecember isolation of animal viruses from farm livestock waste, soil and water grain feeding and the dissemination of acid-resistant escherichia coli from cattle risk profile on the microbiological contamination of fruits and vegetables eaten raw kinetics and strain specificity of rhizosphere and endophytic colonization by enteric bacteria on seedlings of medicago sativa and medicago truncatula survival and retention of escherichia coli o : h and campylobacter in contrasting soils from the toenepi catchment. nzjar (n survival and transport of selected bacterial pathogens and indicator viruses under sandy aquifer conditions listeria monocytogenes survival in soil and incidence in agricultural soils bacillus cereus, the causative agent of an emetic type of food-borne illness could flies explain the elusive epidemiology of campylobacteriosis? fate of pathogens in soils receiving animal wastesda review microbial risks associated with cabbage, carrots, celery, onions, and deli salads made with these produce items infrequent internalization of escherichia coli o : h into field-grown leafy greens internalization of escherichia coli o : h following spraying of cut shoots when leafy greens are regrown for a second crop european food safety authority (efsa), . scientific opinion on the risk posed by pathogens in food of non-animal origin. part : outbreak data analysis and risk ranking of food/pathogen combinations retention of quality and nutritional value of freshcut vegetables treated with low dose radiation listeria monocytogenes persistence in food-associated environments: epidemiology, strain characteristics, and implications for public health fecal source tracking, the indicator paradigm, and managing water quality the diversity and biogeography of soil bacterial communities different behavior of enteric bacteria and viruses in clay and sandy soils after biofertilization with swine digestate escherichia coli survival in lettuce fields following its introduction through different irrigation systems faecal contamination and hygiene aspect associated with the use of treated wastewater and canal water for irrigation of potatoes (solanum tuberosum) major outbreak of hepatitis a associated with orange juice among tourists effects of cattle feeding regimen and soil management type on the fate of escherichia coli o : h and salmonella enterica serovar typhimurium in manure, manureamended soil, and lettuce manure amended soil characteristics affecting the survival of e. coli o : h in dutch soils ecology of e. coli o : h and salmonella enterica in the primary vegetable production chain variability of escherichia coli o strain survival in manure-amended soil in relation to strain origin, virulence profile, and carbon nutrition profile persistence of shiga toxin-producing escherichia coli o in cow slurry leaching of escherichia coli o : h in diverse soils under various agricultural management practices persistence of escherichia coli o : h in soil and on plant roots on-farm and postharvest processing sources of bacterial contamination to melon rinds influence of temperature and predation on survival of salmonella enterica serovar typhimurium and expression of inva in soil and manure-amended soil fresh produceeassociated listeriosis outbreaks, sources of concern, teachable moments, and insights hospital-acquired listeriosis outbreak caused by contaminated diced cele-rydtexas fate of wastewater bacteria and viruses in soil the role of water and water testing in produce safety. microb. saf. fresh prod. e persistence of zoonotic pathogens in surface soil treated with different rates of liquid pig manure development of a risk-based index for source water protection planning, which supports the reduction of pathogens from agricultural activity entering water resources persistence of mycobacterium avium subsp. paratuberculosis and other zoonotic pathogens during simulated composting, manure packing, and liquid storage of dairy manure a review of the use of water in uk agriculture and the potential risks to food safety. food standards agency isolation and characterization of various microorganisms from composts; its value addition to control fusarium wilt of solanum lycopersicum l. & cloning and expression of heat stress resistance inducing candidate gene, thpil of trichoderma harzianum (doctoral dissertation survival of salmonellae on and in tomato plants from the time of inoculation at flowering and early stages of fruit development through fruit ripening flies and campylobacter infection of broiler flocks outbreaks associated with fresh produce: incidence, growth, and survival of pathogens in fresh and fresh-cut produce a framework for developing research protocols for evaluation of microbial hazards and controls during production that pertain to the quality of agricultural water contacting fresh produce that may be consumed raw rationing in europe? implementation of a national electronic reporting system in lithuania vacsatc (vaccine safety: attitudes, training and communication): why such a project? heat inactivation of e. coli during manure composting salmonella in animal slurry can be destroyed by aeration at low temperatures a community outbreak of travel-acquired hepatitis a transmitted by an infected food handler survival of escherichia coli o : h and salmonella typhimurium in cow manure and cow manure slurry. fems (fed enteric viral interactions on fresh produce human enteric pathogen internalization by root uptake into food crops four outbreaks of norovirus gastroenteritis after consuming raspberries outbreak of cyclosporiasis in british columbia associated with imported thai basil survival in manure-treated soils during simulated seasonal temperature exposure plant pathogens in irrigation water: challenges and opportunities mobility and survival of salmonella typhimurium and human adenovirus from spiked sewage sludge applied to soil columns development of quantitative methods for the detection of enteroviruses in sewage sludges during activation and following land disposal effect of length of time before incorporation on survival of pathogenic bacteria present in livestock wastes applied to agricultural soil escherichia coli contamination of vegetables grown in soils fertilized with noncomposted bovine manure: garden-scale studies factors controlling long-term survival and growth of naturalized escherichia coli populations in temperate field soils persistence of enterohemorrhagic escherichia coli o : h in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or irrigation water fate of salmonella enterica serovar typhimurium on carrots and radishes grown in fields treated with contaminated manure composts or irrigation water assessing microbial risks and management strategies in vegetables interactions of escherichia coli o : h , salmonella typhimurium and listeria monocytogenes plants cultivated in a gnotobiotic system overview of microbial hazards in fresh fruit and vegetables operations. microb. hazard ident. fresh fruit veg movement and persistence of fecal bacteria in agricultural soils and subsurface drainage water: a review resuspension of sediment-associated in a natural stream modern food microbiology what is the risk from wild animals in food-borne pathogen contamination of plants fate of escherichia coli o : h in manureamended soil regrowth of potential opportunistic pathogens and algae in reclaimed-water distribution systems potential uptake of escherichia coli o : h from organic manure into crisphead lettuce microbial antagonists of escherichia coli o : h on fresh-cut lettuce and spinach the effect of temperature, solids content and ph on the survival of salmonellas in cattle slurry epidemiological studies of risks associated with the agricultural use of sewage sludge three die in us outbreak of hepatitis a effect of the food production chain from farm practices to vegetable processing on outbreak incidence the effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria leaching of genetically modified pseudomonas fluorescens through organic soils: influence of temperature, soil ph, and roots reducing microbial contamination on wastewater irrigated lettuce by cessation of irrigation before harvesting use of fecal streptococci as indicators of pollution in soil the growth potential of escherichia coli o : h , salmonella spp. and listeria monocytogenes in dairy manure-based compost in a greenhouse setting under different seasons water in food production and processing: quantity and quality concerns water quality and sanitation status of improved drinking water sources in selected rural areas of southern nations nationalities peoples region, ethiopia (doctoral dissertation presence and persistence of salmonella enterica serotype typhimurium in the phyllosphere and rhizosphere of spray-irrigated parsley pre-harvest strategies to ensure the microbiological safety of fruit and vegetables from manure-based production systems. handbook of organic food safety and quality two outbreaks of norovirus infections associated with the consumption of imported frozen raspberries food hygiene and toxicology in ready to eat foods foodborne outbreaks in canada linked to produce interactions of salmonella enterica with lettuce leaves analysis of escherichia coli o : h survival in ovine or bovine manure and manure slurry friend or foe: genetic and functional characterization of plant endophytic pseudomonas aeruginosa outbreak of shigella sonnei infection traced to imported iceberg lettuce colonization of barley (hordeum vulgare) with salmonella enterica and listeria spp. fems (fed influence of soil type, moisture content and biosolids application on the fate of escherichia coli in agricultural soil under controlled laboratory conditions persistence of foodborne pathogens and their control in primary and secondary food production chains microbial threats to health: emergence, detection, and response virus retention and survival in sand control of enteric pathogens in ready-to-eat vegetable crops in organic and 'low input'production systems: a haccp-based approach flagellar motility is critical for listeria monocytogenes biofilm formation outbreaks of shigellosis in denmark and australia associated with imported baby corn microbiological quality and safety of fresh produce in west virginia and kentucky farmers' markets and validation of a post-harvest washing practice with antimicrobials to inactivate salmonella and listeria monocytogenes factors responsible for the emergence of arboviruses; strategies, challenges and limitations for their control analysis of native microflora and selection of strains antagonistic to human pathogens on fresh produce the interaction of human enteric pathogens with plants black soldier fly (diptera: stratiomyidae) larvae reduce escherichia coli in dairy manure bacteriological quality of organically grown leaf lettuce in norway effects of temperature, nutrients, organic matter and coral mucus on the survival of the coral pathogen, serratia marcescens pdl stabilization of confined beef cattle manure: characteristics of produced fertilizers the effect of soil type and climate on hookworm (necator americanus) distribution in kwazulu-natal, south africa microbiology of organic and conventionally grown fresh produce an international outbreak of salmonella infections caused by alfalfa sprouts grown from contaminated seeds commercial watercress as an emerging source of fascioliasis in northern france in : results from an outbreak investigation detection of human norovirus from frozen raspberries in a cluster of gastroenteritis outbreaks enteric human pathogens associated with fresh produce: sources, transport and ecology. microb. saf. fresh prod an overview of the control of bacterial pathogens in cattle manure microorganisms associated with fruits and vegetables. in microbiology of fresh produce the produce contamination problem: causes and solutions enteric pathogen-plant interactions: molecular connections leading to colonization and growth and implications for food safety factors that affect proliferation of salmonella in tomatoes post-harvest: the roles of seasonal effects, irrigation regime, crop and pathogen genotype effects of community versus single strain inoculants on the biocontrol of salmonella and microbial community dynamics in alfalfa sprouts survival of verocytotoxigenic escherichia coli o in soil, water and on surfaces pathogens in livestock waste, their potential for movement through soil and environmental pollution predicting salmonella populations from biological, chemical, and physical indicators in florida surface waters diversity of salmonella isolates from central florida surface waters the survival of escherichia coli o : h in slurry from cattle fed different diets seasonal variation in accurate identification of escherichia coli within a constructed wetland receiving tertiary-treated municipal effluent factors affecting survival of listeria monocytogenes and listeria innocua in soil samples role of stomata in plant innate immunity and foliar bacterial diseases role of rodents in transmission of salmonella and campylobacter fresh produce-associated outbreaks: a call for haccp on farms? alternative disinfection methods to chlorine for use in the fresh-cut industry the rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. fems (fed effect of route of introduction and host cultivar on the colonization, internalization, and movement of the human pathogen escherichia coli o : h in spinach influence of organic waste type and soil structure on the bacterial filtration rates in unsaturated intact soil columns transfer of escherichia coli o : h from soil, water, and manure contaminated with low numbers of the pathogen to lettuce plants impact of soil type, biology and temperature on the survival of non-toxigenic escherichia coli o carriage of microorganisms by domestic cockroaches and implications for food safety mortality of escherichia coli o : h in two soils with different physical and chemical properties experimental studies on the survival of pathogenic and indicator bacteria in aerated and non-aerated cattle and pig slurry disability-adjusted life years (dalys) for diseases and injuries in regions, e : a systematic analysis for the global burden of disease study the influence of soil ph on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria bacteriological study of irrigated vegetables. sewage ind. wastes e a widespread outbreak of yersinia pseudotuberculosis o: infection from iceberg lettuce inactivation of escherichia coli o : h and salmonella typhimurium in manure-amended soils studied in outdoor lysimeters fate and survival of salmonella typhimurium and escherichia coli o : h in repacked soil lysimeters after application of cattle slurry and human urine antimicrobial properties of glycerol monolaurate either alone or combined with selected organic acids against listeria monocytogenes (doctoral dissertation presence and survival of escherichia coli o : h on lettuce leaves and in soil treated with contaminated compost and irrigation water survival of escherichia coli o : h and salmonella enterica serovar typhimurium in manure and manure-amended soil under tropical climatic conditions in sub-saharan africa fate of escherichia coli o : h and salmonella enterica in the manureamended soil-plant ecosystem of fresh vegetable crops: a review studies on survival of salmonella typhimurium in different types of soils under outdoor climatic conditions (author's transl). zentralblatt fur bakteriologie assessing the impact of manure application in commercial swine farms on the transmission of antimicrobial resistant salmonella in the environment bacteria and fungi can contribute to nutrients bioavailability and aggregate formation in degraded soils survival of escherichia coli o : h in wastewater from dairy lagoons behavior and transport of microbial pathogens and indicator organisms in soils treated with organic wastes changing structure of global food consumption and trade: an introduction. changing structure of global food consumption and trade. anita regmi , e effect of land use and hydrological processes on escherichia coli concentrations in streams of tropical escherichia coli o : h induces stronger plant immunity than salmonella enterica typhimurium sl infectious disease potential of land application of wastewater radiation processing for elimination of salmonella typhimurium from inoculated seeds used for sprout making in india and effect of irradiation on germination of seeds epidemiological potential of excretion and regurgitation by musca domestica (diptera: muscidae) in the dissemination of escherichia coli o : h to food effect of anaerobic digestion and application method on the presence and survivability of e. coli and fecal coliforms in dairy waste applied to soil a new shiga toxin variant (stx f) from escherichia coli isolated from pigeons an introduction to soil concepts and the role of soils in watershed management microbial antagonists of foodborne pathogens on fresh, minimally processed vegetables mediterranean fruit fly as a potential vector of bacterial pathogens influence of aerobic and anaerobic conditions on survival of escherichia coli o : h and salmonella enterica serovar typhimurium in luriaebertani broth, farmyard manure and slurry foodborne viruses and fresh produce a novel approach to investigate the uptake and internalization of escherichia coli o : h in spinach cultivated in soil and hydroponic medium survival and persistence of nonpathogenic escherichia coli and attenuated escherichia coli o : h in soils amended with animal manure in a greenhouse environment importance of soil amendments: survival of bacterial pathogens in manure and compost used as organic fertilizers future trends in electron beam technology for food processing. electron beam pasteurization compl. food process fate of escherichia coli o : h during on-farm dairy manureebased composting determining thermal inactivation of escherichia coli o :h in fresh compost by simulating early phases of the composting process transmission of escherichia coli o : h from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization use of fluorescent microspheres as a tool to investigate bacterial interactions with growing plants how do plants achieve immunity? defence without specialized immune cells risk factors associated with salmonella and listeria monocytogenes contamination of produce fields landscape and meteorological factors affecting prevalence of three food-borne pathogens in fruit and vegetable farms application of microbial risk assessment to the development of standards for enteric pathogens in water used to irrigate fresh produce influence of soil mineral colloids on metabolic processes, growth, adhesion, and ecology of microbes and viruses future threats to agricultural food production posed by environmental degradation, climate change, and animal and plant diseasesea risk analysis in three economic and climate settings association of escherichia coli o : h with filth flies (muscidae and calliphoridae) captured in leafy greens fields and experimental transmission of e. coli o : h to spinach leaves by house flies (diptera: muscidae) cultural and environmental factors affecting the longevity of escherichia coli in histosols microbial hazards and emerging issues associated with producey a preliminary report to the national advisory committee on microbiologic criteria for foods the survival of escherichia coli and salmonella dublin in slurry on pasture and the infectivity of s. dublin for grazing calves persistence of poliovirus in soil and on vegetables grown in soil previously flooded with inoculated sewage sludge or effluent salmonella elimination during composting of spent pig litter straindependent variability in growth and survival of escherichia coli in agricultural soil the roles of ammonia, water activity, and ph in the salmonellacidal effect of long-used poultry litter outbreak of listeria gastroenteritis in italy caused by contaminated corn salad plants as a habitat for beneficial and/or human pathogenic bacteria microbial hazards in irrigation water: standards, norms, and testing to manage use of water in fresh produce primary production fate of escherichia coli and escherichia coli o in soils and drainage water following cattle slurry application at sites in southern scotland. soil use manag isolation of vero cytotoxin-producing escherichia coli o from wild birds fate of enterohemorrhagic escherichia coli o : h in bovine feces an overview of the environmental effects of land application of farm effluents. nzjar (n the tricks learnt by human enteric pathogens from phytopathogens to persist within the plant environment biological control of soilborne plant pathogens in the rhizosphere with bacteria survival of listeria monocytogenes in soil outbreak of salmonella stanley in sweden associated with alfalfa sprouts outbreak of salmonella stanley in sweden associated with alfalfa sprouts zoonotic infections in europe in : a summary of the efsa-ecdc annual report abundance and diversity of viruses in six delaware soils survival of escherichia coli o : h in the rhizosphere of maize grown in waste-amended soil examination of microbiological quality of in-field leafy vegetables and identification of on-farm generic escherichia coli transmission dynamics (doctoral dissertation who report on global surveillance of epidemicprone infectious diseases world health organization world health organization overview of recent events in the microbiological safety of sprouts and new intervention technologies the effect of indigenous bacteria on virus survival in ground water survival of salmonella enterica serovar newport in manure and manure-amended soils diversity and ecology of viruses in hyperarid desert soils plant stomata: a checkpoint of host immunity and pathogen virulence mortality rates of fecal bacteria in subsoil amended with poultry manure lack of internalization of escherichia coli o : h in lettuce (lactuca sativa l.) after leaf surface and soil inoculation survival of escherichia coli o : h in soils from jiangsu province, china identification and characterization of integron-mediated antibiotic resistance among shiga toxin-producing escherichia coli isolates 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- -mr authors: oh, myoung-don; park, wan beom; park, sang-won; choe, pyoeng gyun; bang, ji hwan; song, kyoung-ho; kim, eu suk; kim, hong bin; kim, nam joong title: middle east respiratory syndrome: what we learned from the outbreak in the republic of korea date: - - journal: korean j intern med doi: . /kjim. . sha: doc_id: cord_uid: mr middle east respiratory syndrome coronavirus (mers-cov) was first isolated from a patient with severe pneumonia in . the korea outbreak of merscov involved cases, including fatalities. a total of % of transmission events were due to five superspreaders, and % of the mers cases were the patients who had been exposed in nosocomial transmission at hospitals. the epidemic lasted for months and the government quarantined , individuals for days to control the outbreak. this outbreak provides a unique opportunity to fill the gap in our knowledge of mers-cov infection. therefore, in this paper, we review the literature on epidemiology, virology, clinical features, and prevention of mers-cov, which were acquired from the korea outbreak of merscov. middle east respiratory syndrome coronavirus (mers-cov) was first isolated from a patient with severe pneumonia in september [ ] . since then, as of january , the world health organization (who) has been notified of , laboratory-confirmed cases of mers-cov infection, including at least deaths, from countries (fig. ) [ ] . most of mers-cov human infections have occurred in the arabian peninsula. saudi arabia reported , cases, including deaths (fatality rate, . %) [ ] . a recent article summarized the current knowledge on the epidemiology, virology, human pathogenesis, clinical management, and prevention of mers-cov infection [ ] . another comprehensive review article on risk factors and determinants of transmission of mers-cov from human to human in the community and hospital settings has been published recently [ ] . public health england also published an extensive review of literature for clinical decision-making support for treatment of mers-cov patients [ ] . the korea outbreak of mers-cov is the largest outside the arabian peninsula and provides a unique opportunity to fill the gap in our knowledge about mers-cov infection. since it has now been almost years since the mers outbreak in the republic of korea, we reviewed the literature to summarize the lessons we have learned from the korea outbreak. the korean journal of internal medicine vol. , no. , march fields. as of january , a total of articles were retrieved, and of them were written in english. the full texts of the english articles were reviewed and critically assessed. the korea outbreak of mers-cov resulted in cases including fatalities [ ] . the index case was a returning traveler from the middle east. the infection had spread within the hospital, and subsequently to other hospitals because of patient movement, resulting in nosocomial transmission at clinics and hospitals. the epidemic lasted for months, with the government declaring a "virtual" end to the epidemic on july , . in order to control the outbreak, the government quarantined , individuals for days, and the economic loss was estimated at . trillion korean won ( . billion us dollars) [ ] . the first patient (index case) with mers-cov infection was a -year-old korean man returning from the middle east. he ran a commercial greenhouse business in bahrain. he flew from seoul to bahrain on april , , and traveled through the united arab emirate and the kingdom of saudi arabia, and came back to seoul on may , . during his -day business trip, he did not visit a camel farm or a hospital. he did not eat camel meat or any other camel products. he did not remember meeting any persons with respiratory symptoms [ ] [ ] [ ] [ ] . seven days after returning home, on may , he developed fever and myalgia. he visited a local clinic on may , , and . his symptoms, however, did not improve and a new non-productive cough developed on th may. he visited pyeongtaek st. mary's (ptsm) hospital ( km south from seoul), a secondary hospital in gyeonggi province. his chest x-ray showed a consolidation in the upper zone of right lung, and he was admitted to the hospital for pneumonia on may . ceftriaxone and amikacin were started, but his pneumonia progressed despite the antibiotic treatment. he was transferred to , reported to who as of november , republic of korea other countries saudi arabia the chain of transmission was identified in all mers patients [ , ] . of the healthcare institutions that had taken in mers-cov-infected patients, nosocomial transmission occurred in hospitals and four clinics (plus two ambulances) [ , , ] . the epidemiological characteristics are summarized in table [ ] . the epidemic curve is shown in fig. . during his admission from may to at ptsm hospital, the first patient was unknowingly spreading mers-cov. patients, visitors, and healthcare workers (hcws) were exposed to mers-cov. therefore, by may , when the korea cdc noticed the importation of mers-cov into the republic of korea, individuals staying on the same floor as the first case had already been exposed to mers-cov. as a result, a total of mers cases occurred at ptsm hospital. of them, patients developed symptoms within days after exposure, suggesting they were first-generation cases; the remaining patients may be second-generation cases [ , ] . the mode of transmission of mers-cov at ptsm hospital remains uncertain. in addition to family members of the first patient, only one patient shared the same room (# ) with him; all the other cases stayed in other rooms, which were located > m apart from room # . all patient rooms were equipped with an air ventilating system except for room # . a recent study using a multi-agent modeling framework suggested the transmission occurred via a long-range airborne route or via a combination of a long-range airborne route and direct contact transmission [ ] . a chest computed tomography (ct) scan taken in the early stages (day or ) of illness showed very small, ground glass nodules in the periphery of the lungs, suggesting inhalation of airborne particles [ ] . many individuals had been exposed to the superspreader- at the er, and contact tracing identified patients, an estimated visitors, and hcws as contacts of superspreader- . of them, were quarantined and were under active monitoring. as a result of exposure to superspreader- , had laboratory-confirmed mers-cov infection, and an additional were secondarily infected from them [ , ] . the index case (superspreader- ) for the daejeon ( km south from pyeontaek city) outbreak was infected at ptsm hospital. he developed symptoms on may and was admitted to daechung hospital on may . as his pneumonia progressed despite the treatments, he was transferred to geonyang hospital on may . a total of secondary cases ( in daechung hospital and in geonyang hospital) were detected. although superspreader- caused the two hospital outbreaks, the me- dian time of incubation ( . days vs. . days), secondary attack rates ( . % vs. . %), and case fatality rate ( . % vs. . %) were different between the two outbreaks. the high fatality rate was associated with pre-existing pneumonia or poor underlying pulmonary function [ ] [ ] [ ] . a -year-old male, who had been exposed to the first index case on may , developed back pain on may . he is a son of the third mers case. his sister had also been exposed to mers- epidemiological characteristics have been reported by several groups (table ) [ , , , , , [ ] [ ] [ ] [ ] . the defining epidemiological characteristics were superspreading events at hospitals [ ] [ ] [ ] . early superspreading events generated a disproportionately large number of second-ary infections, and the transmission potential decreased sharply in subsequent generations [ , ] . a total of % of transmission events were due to five superspreaders, and % of the mers cases were in-patients who had been exposed within the hospitals [ ] . the spreaders transmitted mers-cov from days to of their illness (median, days), and the number of patients infected by each spreader ranged from to (interquartile range, to ) [ ] . the median incubation period and serial interval was days and . days, respectively [ , ] . r ranged from . to . , higher than the previous estimate of < [ ] . it was estimated at . for the outbreak cluster in ptsm hospital, and . for the outbreak in smc [ ] . a mathematical modeling study demonstrated that although r < overall, cluster sizes of over cases are not unexpected for mers-cov infection [ ] . compared with the non-spreaders, the spreaders had a higher frequency of fever (≥ . °c) and chest infiltrates in more than three lung zones, and longer non-isolated in-hospital days [ ] . the spreaders had higher viral load in sputum samples with the cycle thresholds for the upe and orf a genes of . vs. . and . vs. . , respectively. the spreaders with more than three transmissions had higher numbers of contacts and er visits [ ] . a brief exposure of a -minute stay and minutes of talking was enough for the transmission of mers-cov [ ] . the whole genome sequences of mers-cov isolated from the korea outbreak were reported [ , [ ] [ ] [ ] [ ] . the virus most similar to the korean isolates was a camel virus (camel/riyadh/ry / ) that belonged to lineage , a recombinant of lineage and [ , ] . viruses from lineage had been predominant in saudi arabian camels since november , but human viruses of this lineage were reported from february [ ] . a phylogenetic study of spike glycoprotein genes also showed the korean strains were closely related to the viral strains from riyadh, saudi arabia [ ] . in a molecular study, of mers-cov genome had an i t mutation and one d g mutation in the receptor binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, cd [ ] . heterogeneity analysis revealed the combined frequency of i t and d g was high ( . %), although the frequency of both mutations varied greatly among specimens [ ] . another genome sequence study also reported deletions of and nucleotides between orf and the e protein, suggesting that the virus might be defective in its ability to package mers cov [ ] . a microevolution study found no evidence of changes in the evolutionary rate [ ] . however, whether the transmissibility and virulence of the korean isolates are different from those of the previous isolates from the kingdom of saudi arabia remains to be determined by phenotypic assays. a viral shedding study showed that the copies of mers-cov rna detected by real-time reverse transcription polymerase chain reaction (rrt-pcr) in respiratory samples peaked during week , and the median value was . log in the severe group and . log in mild cases [ ] . the study also showed that viral titers were higher in throat samples than in nasopharyngeal samples. another study also demonstrated higher viral loads were associated with severity and mortality [ ] . after mers-cov infection, antibody responses developed by the third week of illness in most patients [ ] . seroconversion rates increased with the increase of dis-ease severity. in a serologic study of mers patients, the seroconversion rate was % in asymptomatic infection, . % in symptomatic infection without pneumonia, . % in pneumonia without respiratory failure, and % in pneumonia with respiratory failure [ ] . the serological response remained detectable for > months in all survivors ( / ) who had severe disease. antibody titers were not detectable in four of six patients who had mild pneumonia, suggesting that mers-cov seroepidemiological studies may underestimate the extent of mild and asymptomatic infection [ ] . the korean society of infectious diseases compiled the clinical data of the mers patients [ ] . the median age was years (range, to ). the male to female ratio was : . the most common coexisting medical conditions were hypertension ( . %), diabetes ( . %), solid organ malignancy ( . %), and chronic lung disease ( . %). patients with mers-cov infection developed a spectrum of illnesses, ranging from asymptomatic to fulminant pneumonia with fatal outcome. the respiratory symptoms were similar to other acute viral respiratory infections. at the time of presentation, fever had developed in . %, cough in . %, and sputum in . % of patients. symptoms of upper respiratory-tract infections were infrequent: sore throat was present in . % and rhinorrhea in . % of patients. gastrointestinal symptoms were also observed: diarrhea was present in . %, nausea or vomiting in . %, and abdominal pain in . % of patients. diarrhea may be due to the side effect of lopinavir/ritonavir, as % of the patients received antiviral drugs for mers-cov treatment [ ] . in the early stages of illness, cough and sputum were not prominent even in patients who later developed overt pneumonia [ ] . at admission, % ( / ) of patients had abnormalities on chest radiographs, while . % ( / ) of them developed the abnormalities during the course of the disease [ ] . sudden progression of pneumonia occurred around day of illness [ , ] . predictive factors for development of pneumonia included older age, high fever, thrombocytopenia, lymphopenia, c-reactive protein (crp) ≥ mg/dl, and a high viral load www.kjim.org https://doi.org/ . /kjim. . in sputum (threshold cycle value of rrt-pcr < . ) [ ] . forty-five patients ( . %) were treated with mechanical ventilation and patients ( . %) needed extracorporeal membrane oxygenation [ ] . the typical course of pneumonia is shown in fig. . acute kidney injury (aki) developed in . % of the patients, . % ( / ) had proteinuria, and . % ( / ) had hematuria. fifteen patients were treated with renal replacement therapy [ ] . old age is an independent risk factor for the occurrence of aki [ ] . neuromuscular manifestations were not uncommon; hypersomnolence, weakness and tingling in the extremities were reported during the treatment of mers, suggesting guillain-barré syndrome or virus-related sensory neuropathy [ ] . serial changes in chest radiographs was reported in five patients [ ] . chest ct scans revealed rapidly developed multifocal nodular consolidations with ground-glass opacity halo and mixed consolidation, mainly in the dependent and peripheral areas [ ] . there are few laboratory findings specific to mers-cov infection, although monocytosis with normal white blood cell count and low crp level were more common in mers patients at initial presentation [ , ] . the guidelines for the molecular diagnosis of mers-cov infection have been published by the korean society for laboratory medicine [ , ] . specimen type and quality is important in the laboratory diagnosis of mers-cov infection. lower respiratory-tract samples, such as sputum and tracheal aspirates, have higher viral loads than upper respiratory-tract samples [ ] . however, mers patients may not shed the virus during the early stage of their illness. therefore, initial negative results should not rule out the possibility of mers [ ] , and patients suspected of having mers-cov infection should be retested using a lower respiratory-tract sample [ , ] . when sputum cannot be obtained, throat swabs may be an alternative source of diagnostic samples [ ] . special attention should be given to diagnosing mers-cov infection in immunocompromised patients, as they may present with atypical features, such as a longer incubation period, a longer period from initial pcr positivity to symptom onset, and persistent viral shedding [ ] . an antiviral treatment guideline has been published by the writing committee with support from the korean society of infectious diseases and the korean society for chemotherapy [ ] . the guideline recommended a tri- ple combination regimen of type interferon, ribavirin, and lopinavir/ritonavir for to days. however, the guideline was mostly based on expert opinions and further study for the optimal antiviral treatment in mers is warranted. the median duration of fever was days. the median time to negative conversion of mers-cov in sputum samples by rrt-pcr was days (range, to ) [ ] . the median time from symptom onset to death was days. the case fatality rate was . % ( / ) (fig. ) . the in-hospital mortality, -day mortality, and -day (from symptom onset) mortality were . % ( / ), . % ( / ), and . % ( / ), respectively [ ] . host factors associated with mortality were old age (> years), smoking history, pre-existing pneumonia, abnormal renal function, and comorbid conditions [ , [ ] [ ] [ ] . low albumin, altered mentality, and high pneumonia severity index score at admission were risk factors for mortality [ ] . mers-cov rna in blood samples was detected in % ( / ) of patients at presentation, and it was associated with a worse clinical outcome [ ] . a shorter incubation period was also associated with an increased risk of death [ , ] . a pregnant woman infected with mers-cov in her weeks of gestational age developed dyspnea and her chest radiograph showed diffuse infiltrates in the left lower lung zone. she recovered from the infection and delivered a healthy full-term baby without vertical mers-cov transmission [ ] . a patient developed organizing pneumonia days after the resolution of mers-cov infection. he was successfully treated with corticosteroids [ ] . in a mental health study, . % of , patients who were quarantined showed symptoms of anxiety, and . % reported feelings of anger during the weeks of quarantine [ ] . mental health support, accurate information, and appropriate supplies, including food, clothes, and accommodation, should be provided to isolated persons [ ] . a case of possible transfusion-related acute lung injury following transfusion of convalescent plasma was also reported [ ] . a comprehensive "mers infection prevention and control guideline for healthcare facilities" was drafted by the guideline development committee with generous support from korean academic societies [ ] . the guideline included practical aspects of infection control and prevention based on the experience from the korea outbreak, such as the composition of members for the mers emergency committee, a space plan for an anteroom for donning and doffing, isolation of in-patients and hcws in a hospital affected by an outbreak, and management of the deceased and autopsy [ ] . the hemodialysis unit may become an epicenter for mers-cov outbreak because hemodialysis patients must receive renal replacement therapy even during the outbreak, and the risk of exposure to mers-cov continues. during the korea outbreak, a hemodialysis unit was found to have a mers patient, and a total of patients and hcws were exposed to mers-cov. with support from the korean society of nephrology, the dialysis unit could continue to operate while the exposed patients were isolated in the unit. fortunately, no further transmission occurred at the unit [ ] . after this successful experience, the society published a clinical practice guideline for hemodialysis facilities dealing with mers patients [ ] . the korea outbreak was driven by the superspreaders who visited multiple healthcare facilities; thus, generating a large number of secondary cases. limiting unnecessary contacts with patients with respiratory symptoms in healthcare settings, especially in emergency departments, is of critical importance [ ] . mers-cov could survive for longer than hours at °c and % of relative humidity, suggesting contact or fomite transmission might occur in healthcare settings [ ] . mers-cov was detected by rrt-pcr in specimens taken from the medical equipment [ ] [ ] [ ] . mers-cov was also isolated by cell culture of the air and swab samples taken from a mers isolation unit, suggesting extensive contamination of the isolation unit [ , ] . of the cases, % were infected by undocumented contact between cases (i.e., indirect transmission of mers-cov via environmental contact) [ ] . therefore, fomites with possible mers-cov contamination should be sanitized, and a minimum room ventilation rate of six air changes per hour should be implemented to minimize recirculation of pathogen-bearing droplets. meticulous environmental cleaning may be important for preventing transmission in healthcare settings. there was a case of a -year-old female nurse who was infected with mers-cov during a cardiopulmonary resuscitation lasting hour for a mers patient who had pneumonia and hemoptysis [ ] . serological surveillance conducted after the mers outbreak for asymptomatic infection among hcws involved in the direct care of mers patients showed that . % ( / ) of them were mers-cov igg positive by an indirect immunofluorescent assay. among the hcws who did not use appropriate personal protective equipment (ppe), seropositivity was . % ( / ) compared with % ( / ) in hcws with appropriate ppe use [ ] . another serological survey also showed that none of the hcws were positive for mers-cov immunoglobulin g, although . % ( / ) of the hcws reported experiencing mers-like symptoms while caring for the mers patients [ ] . in a third study, of hcws showed seroconversion by a plaque reduction neutralization test, although % to % of hcws reported mers-like symptoms [ ] . during the outbreak in smc, all hcws assigned to mers patients were screened for mers-cov, regardless of the presence or absence of symptoms. of the hcws, three ( . %) asymptomatic hcws (two nurses and one physician) were found to be mers-cov (+), but they did not transmit the virus to others [ ] . in another study, an asymptomatic nurse without ppe contacted hcws, including close contacts who were exposed within m from the index nurse and were not using ppe. none of the exposed hcws were infected [ ] . however, the potential for transmission from asymptomatic rrt-pcr positive individuals is still unknown. therefore, asymptomatic hcws who are rrt-pcr-positive for mers-cov should be isolated and should not return to work until two consecutive respiratory-tract samples test negative on rrt-pcr [ ] . the government requested the korean society of infectious diseases to participate in the control of the mers outbreak. on june , , the society organized the rapid response team, consisting of infectious-disease doctors and two infection-control professionals [ ] . critical suggestions to prevent future epidemics were made regarding a rapid alerting system for index cases, provision of a sufficient airborne infection isola- the korean journal of internal medicine vol. , no. , march tion facility, education and training of hcws for important infectious diseases, and overcrowding and visitor control in the hospital [ ] . the korea outbreak was the largest outbreak outside of the middle east. researchers had a unique opportunity to compare the nature of mers-cov infection with the middle east experience. the outbreak unveiled the weak points of infrastructure in our medical system, especially of preparedness for emerging global infectious diseases. nosocomial transmission was one of the core features of mers-cov infection. the introspection for loose medical referral systems, overcrowding at the er, a lack of expert resources and infection control infrastructure, and lack of organized preparedness for medical crises prompted the government to reform the healthcare system, and healthcare sectors to invest further in infectious diseases and infection control. although the improvement is still ongoing, the speed and content are still currently insufficient. international cooperation to prepare for and defeat emerging infectious diseases should also be emphasized. lessons we learned from the outbreak are summarized in table . a single, missed case may trigger a huge, nationwide outbreak the first line of defense is not the thermal scanner at the airport. it is doctors in the community clinics/hospitals. due to sudden deterioration of pneumonia around day of illness, patients may visit emergency department, or be admitted to intensive care unit. superspreading events may occur in healthcare settings, especially at the emergency department. patients may transmit mers-cov as early as days after symptom onset. early detection and isolation is of critical importance. aggressive strategy for quarantine maybe necessary, especially when large number of individuals are exposed in the healthcare settings. huge socioeconomic impact with an estimated . billion us dollars isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) [internet]. geneva: world health organization ministry of health kingdom of saudi arabia. mers-cov daily update ministry of health kingdom of saudi arabia, c middle east respiratory syndrome middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission treatment of mers-cov: information for clinicians. clinical decision-making support for treatment of mers-cov patients uk): public health england middle east respiratory syndrome coronavirus outbreak in the republic of korea the korean middle east respiratory syndrome coronavirus outbreak and our responsibility to the global scientific community the clinical and virological features of the first imported case causing mers-cov outbreak in south korea epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital middle east respiratory syndrome in persons, south korea the first case of the korean middle east respiratory syndrome outbreak south korea coronavirus mers case list: including imported and exported cases. ministry of health & who confirmed data only fl): flutrackers, c mers-cov infection: current status-interactive and infographics probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, : epidemiology, characteristics and public health implications ministry of health and welfare. the mers outbreak in the republic of korea: learning from mers (the white paper) a study of the probable transmission routes of mers-cov during the first hospital outbreak in the republic of middle east respiratory syndrome coronavirus superspreading event involving persons, korea mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon hospital outbreaks of middle east respiratory syndrome high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea imported case of mers-cov infection identified in china origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis clinical and epidemiologic characteristics of spreaders of middle east respiratory syndrome coronavirus during the outbreak in korea epidemiologic parameters of the middle east respiratory syndrome outbreak in korea epidemiological investigation of mers-cov spread in a single hospital in south korea transmission characteristics of mers and sars in the healthcare setting: a comparative study understanding and modeling the super-spreading events of the middle east respiratory syndrome outbreak in korea mers outbreak in korea: hospital-to-hospital www transmission identifying determinants of heterogeneous transmission dynamics of the middle east respiratory syndrome (mers) outbreak in the republic of korea, : a retrospective epidemiological analysis comparison of incubation period distribution of human infections with mers-cov in south korea and saudi arabia estimating and modelling the transmissibility of middle east respiratory syndrome coronavirus during the outbreak in the republic of korea high reproduction number of middle east respiratory syndrome coronavirus in nosocomial outbreaks: mathematical modelling in the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission risk factors for transmission of middle east respiratory syndrome coronavirus infection during the outbreak in south korea transmission among healthcare worker contacts with a middle east respiratory syndrome patient in a single korean centre complete genome sequence of middle east respiratory syndrome coronavirus kor/knih/ _ _ , isolated in south korea complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china isolation of middle east respiratory syndrome coronavirus from a patient of the korean outbreak microevolution of outbreak-associated middle east respiratory syndrome coronavirus, south korea co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia the recent ancestry of middle east respiratory syndrome coronavirus in korea has been shaped by recombination variations in spike glycoprotein gene of mers-cov spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd during the south korean outbreak analysis of intrapatient heterogeneity uncovers the microevolution of middle east respiratory syndrome coronavirus two deletion variants of middle east respiratory syndrome coronavirus found in a patient with characteristic symptoms viral load kinetics of mers coronavirus infection comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity kinetics of serologic responses to mers coronavirus infection in humans serologic responses of mers-coronavirus-infected patients according to the disease severity mers-cov antibody responses year after symptom onset, south korea clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea clinical implications of cases of middle east respiratory syndrome coronavirus infection in a south korean outbreak clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients renal complications and their prognosis in korean patients with middle east respiratory syndrome-coronavirus from the central mers-cov designated hospital neurological complications during treatment of middle east respiratory syndrome middle east respiratory syndrome-coronavirus infection: a case report of serial computed tomographic findings in a young male patient differential cell count and crp level in blood as predictors for middle east respiratory syndrome coronavirus infection in acute febrile patients during nosocomial outbreak guidelines for the laboratory diagnosis of middle east respiratory syndrome coronavirus in korea korean society for laboratory medicine practice guidelines for the molecular diagnosis of middle east respiratory syndrome during an outbreak in korea in importance of specimen type and quality in diagnosing middle east respiratory syndrome an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) atypical presentations of mers-cov infection in immunocompromised hosts antiviral treatment guidelines for middle east respiratory syndrome predictors of mortality in middle east respiratory syndrome (mers) mortality risk factors for middle east respiratory syndrome outbreak, south korea real-time characterization of risks of death associated with the middle east respiratory syndrome (mers) in the republic of korea viral rna in blood as indicator of severe outcome in middle east respiratory syndrome coronavirus infection association between severity of mers-cov infection and incubation period mers-cov infection in a pregnant woman in korea successful treatment of suspected organizing pneumonia in a patient with middle east respiratory syndrome coronavirus infection: a case report mental health status of people isolated due to middle east respiratory syndrome possible transfusionrelated acute lung injury following convalescent plasma transfusion in a patient with middle east respiratory syndrome middle east respiratory syndrome infection control and prevention guideline for healthcare facilities middle east respiratory syndrome coronavirus transmission in dialysis unit and infection control interventions in korea middle east respiratory syndrome clinical practice guideline for hemodialysis facilities stability of middle east respiratory syndrome coronavirus (mers- www cov) under different environmental conditions environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea environmental contamination and viral shedding in mers patients viral shedding and environmental cleaning in middle east respiratory syndrome coronavirus infection extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards transmissibility of middle east respiratory syndrome by the airborne route nosocomial amplification of merscoronavirus in south korea healthcare worker infected with middle east respiratory syndrome during cardiopulmonary resuscitation in korea surveillance of the middle east respiratory syndrome (mers) coronavirus (cov) infection in healthcare workers after contact with confirmed mers patients: incidence and risk factors of mers-cov seropositivity seroprevalence of middle east respiratory syndrome coronavirus among healthcare personnel caring for patients with middle east respiratory syndrome in south korea serologic evaluation of mers screening strategy for healthcare personnel during a hospital-associated outbreak middle east respiratory syndrome coronavirus transmission in dialysis unit and infection control interventions in korea management of asymptomatic persons who are rt-pcr positive for middle east respiratory syndrome coronavirus (mers-cov): interim guidance [internet]. geneva: world health organization, c collaborative intervention of middle east respiratory syndrome: rapid response team institutional preparedness to prevent future middle east respiratory syndrome coronaviruslike outbreaks in republic of korea no potential conflict of interest relevant to this article was reported. key: cord- -yj r rwt authors: jain, richa; gupta, kirti; bhatia, anmol; bansal, arun; bansal, deepak title: hepatic sinusoidal-obstruction syndrome and busulfan-induced lung injury in a post-autologous stem cell transplant recipient date: - - journal: indian pediatr doi: . /s - - - sha: doc_id: cord_uid: yj r rwt veno-occlusive disease of the liver is mostly encountered as a complication of hematopoietic stem cell transplantation with myeloablative regimens with an incidence estimated to be . %. it is clinically characterized by tender hepatomegaly, jaundice, weight gain and ascites. strong clinical suspicion and an early recognition of clinical signs are essential to establish the diagnosis and institute effective regimen. another complication of cytotoxic drugs given for cancers, is development of busulfan-induced lung injury. a strong index of suspicion is needed for its diagnosis, especially in setting where opportunistic fungal and viral infections manifest similarly. we illustrate the clinical and autopsy finings in a ½-year-old boy who received autologous stem-cell transplantation following resection of stage iv neuroblastoma. he subsequently developed both hepatic veno-occlusive disease and busulfan-induced lung injury. the autopsy findings are remarkable for their rarity. volume __ september , jain, et al. hepatic sinusoidal-obstruction syndrome increased efforts, hypoxia (saturation on room air: %; increasing to % on % fio ). tachycardia was present (heart rate /minute); however, circulatory parameters were normal (bp / mm hg, normal capillary refill time and pulse pressure, warm extremities). pallor was present, with no evidence of skin or mucosal bleeding. systemic examination was consistent with pneumonitis as the patient had tachypnea, bilateral equal air entry, and presence of coarse crepitations in bilateral lung fields. abdominal examination showed dark brown pigmentation over abdomen with no tenderness, guarding or rigidity. hepatomegaly was present with liver palpable cm below costal margin (span cm). spleen was not palpable. there was no free fluid. cardiac and neurological examinations were essentially normal. the index case was managed as a case of pneumonitis post auto-sct. respiratory support was initially provided with nasal prongs-continuous positive airway pressure. intravenous antibiotics were started cefoperazone-sulbactam, amikacin and azithromycin. due to rapidly progressive respiratory distress, child was transferred to pediatric intensive care unit where mechanical ventilation was provided. there was single episode of fever on the day of admission ( . o c) with a subsequent afebrile period throughout the hospital stay. progressive respiratory distress worsened into acute respiratory distress syndrome (ards). ventilation strategy was modified accordingly. on day , there was development of hypotensive shock, initially responding to fluid boluses. on day , the shock necessitated inotropic support (dopamine, adrenaline, noradrenaline and pre-terminally, and vasopressin). there was development of left sided pneumothorax followed by cardiac arrest on the same day. the patient could be revived and pneumothorax was drained. multi-organ dysfunction developed with acute kidney injury (onset day ), requiring peritoneal dialysis. significant transaminitis with elevated bilirubin levels was documented. antibiotics were changed to vancomycin and meropenam on day ; azithromycin was continued. intravenous co-trimoxazole and gancyclovir were added in therapeutic, renal modified doses. platelet concentrates were transfused to maintain a platelet count above × /mm . there was development of refractory shock on day . the child suffered another cardiac arrest on day , and could not be revived. the fig. a-d) . with these chest radiographs, possible etiologies considered were infective, including fungal pneumonia and pcj pneumonia, cmv disease and miliary tuberculosis (tb). non-infective etiologies under consideration included pulmonary graft-versus host disease (gvhd) and pulmonary veno-occlusive disease (vod). this is a case of neuroblastoma, stage iv, day post auto-sct, presenting with fever, pneumonia, hypoxia, and investigations showing polymorphic leucocytosis with deranged liver function tests (lft). in a posttransplant patient, complications can be divided according to the duration subsequent to transplant. in the initial days, there is presence of neutropenia; between - days is the early post-engraftment phase and beyond days is late post-engraftment phase. the index case developed symptoms in the early post engraftment phase. common complications seen in early post-engraftment phase can be divided into infective and non-infective. infective etiologies include cmv which can explain both pneumonia and hepatitis. it is a common pathogen causing disease weeks post sct. india is an endemic country for cmv. in the index case, eta demonstrated polymerase chain reaction (pcr) positivity volume __ september , jain, et al. hepatic sinusoidal-obstruction syndrome for cmv along with radiological findings which were supportive of the diagnosis. however, the child deteriorated despite administration of gancyclovir from day onwards, which is unusual. typically blood pcr is positive in such cases, though not mandatory for diagnosis of cmv pneumonia. fungal infections are the next possibility, supported by the presence of candida in eta on two occasions. presence of normal neutrophil count and a normal serum galactomannan are odd points. galactomannan < . has shown a good negative predictive value for aspergillus infection [ ] . other viral infections that are important in post-sct scenario include respiratory syncytial virus (rsv), para-influenza virus, influenza, metapneumovirus, and coronavirus. multiorgan failure and lymphopenia is common in these patients. patients with rsv often require ventilation. associated co-infection with fungus, especially aspergillus can be seen. in the absence of investigations directed towards the myriad respiratory viruses, it is difficult to rule in or rule out these infections. tuberculosis should be considered in an immunocompromised patient in an endemic country; however, the rapid onset of disease, absence of a contact and negative evaluation make it unlikely. in our case other bacterial infections typically seen in an immunocompromised child are also unlikely in view of sterile cultures, complete absence of fever and normal creactive protein (crp).though this clinical presentation can be caused by infection with pcj, it is an uncommon infection. other atypical infections like nocardia and cryptococcus are rarer still. the non-infective etiologies causing respiratory symptoms in a post-transplant setting can be pulmonary gvhd, idiopathic pneumonia syndrome (ips), bronchiolitis obliterans syndrome (bos), cryptogenic organising pneumonia (cop) and sos. ips is a very common disease in this situation, but is typically seen post allo-sct and hepatitis is not an associated feature. on-going hepatic sos is unlikely as there was no weight gain or tender hepatomegaly. gvhd and bos are also typically diseases seen in allo-sct setting. pulmonary sos is very rare and normal echo findings negate this possibility. the clinical presentation is consistent with cop, though it is more common in females undergoing allogenic transplant. the final diagnosis is neuroblastoma stage iv, day + post auto-sct (bu-mel) with pneumonitis, ards and multi-organ failure; likely etiology being fungal pneumonia or cmv pneumonia and hepatitis secondary to ischemia with underlying sos. pediatric hemato-oncologist : ips occurs post sct day+ to . this child had typical bilateral basilar infiltrates and hypoxia. moreover, ips has a relationship with use of busulfan and pre-existing sos. presence of cmv positivity in eta is of questionable significance as it is a common organism. histopathological evidence from lung biopsy is essential to prove cmv pneumonia. liver dysfunction in the form of transaminitis was likely due to shock and ischemia. pediatric hemato-oncologist : bacterial and fungal infections cannot be excluded despite absence of fever, several sterile cultures and continued normal values of crp, though less likely. however, both cmv and pcj infections are possible with normal crp. absence of adventitious lung sounds at initial presentation, along with presence of hypoxia may be a pointer towards pcj pneumonia. immunocompromised state, lymphopenia and the fact that the child was not on pcj prophylaxis are important here. moreover, cmv is ubiquitous in our volume __ september , jain, et al. hepatic sinusoidal-obstruction syndrome pediatric population, and in pediatric oncology patients, we have seen a near % seropositivity. reactivation of cmv can occur at any point of time in these patients. important non-infective possibilities are ips and cryptogenic pneumonia. pulmonary sos is quite unlikely given the normal echo findings. adult hematologist: immune reconstitution posttransplant takes typically to months. this child was immunocompromised. adenovirus infection can be considered. it can be rarely seen in association with hepato-pulmonary syndrome. the excised tumor on histology was categorized as differentiating neuroblastoma ( fig. a-c) . autopsy revealed normal serous cavities. liver weighed g and revealed irregular areas of sinusoidal congestion, confluent at places with necrosis of adjoining parenchyma involving both right and left lobe (fig. d) . no thrombi were identified in right and left hepatic vein or inferior vena cava. microscopically, areas of centrizonal congestion were identified (fig. e) . furthermore, the dominant pathology was seen in the central vein and terminal hepatic venule (thv). there was varying degree of obliterative changes with subendothelial fibrosis and laying down of reticulin fibres and collection of extracellular matrix in subintimal zone. at places, the thv was completely obliterated with wipe out of centrizonal hepatocytes while the periportal hepatocytes were preserved (web fig. a-e) . in other regions, extravasated rbcs and areas of hemorrhage were noted in centri-zonal regions. besides acute obliterative changes, subacute changes in form of deposition of collagen around the hepatic venule and collection of hemosiderin laden macrophages were also noted. loss of hepatic parenchyma resulted in approximation of central veins structures (web fig. a) . both lungs weighed g with dull pleura. microscopy revealed features of busulfan-induced lung injury with marked prominence of type ii pneumocytes; many of them demonstrated nuclear atypia and hyperchromasia. marked thickening of interstitium with fibrosis was also noted (web fig. a-b) . other regions showed patchy acute bronchopneumonia and alveolar haemorrhages. features of pulmonary arteriopathy were also noted with prominence of intra-acinar arterioles. there were no features of vod in the pulmonary veins. an occasional focus of septic emboli with candida infiltration into parenchyma was noted. no cmv inclusions were noted in lungs. pcr carried out on lung tissue for adenovirus, rsv and metapneumovirus were negative. acute ulcers with candida infiltration were noted in stomach and small intestine. candida had disseminated to heart causing mural endocarditis, myocardial abscess and tiny ( - mm) vegetations on left atrial wall (webfig. c-f). both tricuspid and mitral valves were normal. dissemination with formation of fungal abscesses were also detected in psoas muscle and omental fat. subsequent to septic emboli, infarcts were detected in right kidney (upper pole) with thrombi within the branches of renal vessels and spleen. right kidney also revealed features of acute tubular necrosis in noninfracted regions. no residual tumor was detected in lymph nodes, thymus and bone marrow. the autopsy diagnosis is concluded as follows: in a known case-of neuroblastoma, undifferentiated (adrenal) post-autologous stem-cell transplant: • features of busulfan-induced lung injury with organizing bronchopneumonia and pulmonary arterial hypertension; • veno-occlusive disease in liver. • fungal (candida) ulcers in git with extensive dissemination to heart (mural endocarditis and myocardial abscess), lungs, skeletal muscle and omental fat producing embolic infarcts in right kidney and spleen. • no residual disease in bone marrow. hepatic sinusoidal obstruction syndrome (sos) is an obliterative venulitis of thv which occurs as a result of cytoreductive therapy prior to hematopoietic stem cell transplantation (hsct), ingestion of pyrrolizidine alkaloids, or radiation therapy [ ] [ ] [ ] [ ] . the primary pathogenetic event is the endothelial injury of sinusoids and small hepatic veins. following which, there is deposition of fibrin-related aggregates and oedema in the subendothelial zone [ ] . accumulation of these aggregates and entrapment of fluid and cellular debris progressively occlude the hepatic venous flow and leads to post-sinusoidal intrahepatic hypertension. this is accompanied by necrosis of perivenular hepatocytes. histologically, acute, sub-acute and chronic forms of sos have been described depending upon collagenization and fibrosis of terminal hepatic venule. incidence of sos varies from - %, as it depends on the conditioning regimen used as well as upon the patient's risk factors [ ] [ ] [ ] . sos occurs more often after allo-than after auto-hsct ( v/s %, respectively), suggesting a role of immune reactions in this disorder [ ] . few independent studies have documented increase in circulating levels of plasminogen activator inhibitor- (pai- ), a molecule released by the endothelial cells, in patients developing sos [ , ] . increased pai- levels might be of clinical utility in challenging clinical situations in patients with hyperbilirubinemia occurring after hsct. it forms one of the therapeutic targets for defibrotide, which reduces circulating pai- levels along with other actions. other endothelial markers, like intercellular adhesion molecule- (icam- ), e-selectin, von willebrand factor (vwf), and thrombomodulin may also be helpful in early identification of patients at risk of sos who may benefit from early introduction to therapies [ ] . diagnosis of sos is based on constellation of signs and symptoms and serum bilirubin levels. hepatic sos is clinically characterized by jaundice caused mainly by conjugated hyperbilirubinemia, tender hepatomegaly, fluid accumulation manifested as rapid weight gain and ascites [ ] . most commonly used diagnostic criteria for sos includes the seattle criteria [ ] , the modified seattle criteria [ ] , and the baltimore criteria [ ] . because of its high incidence and mortality, prophylaxis for hepatic sos is widely practiced, using different regimens in different centres. when hepatic sos is established, specific therapy is usually given in addition to general supportive care, especially in moderate or severe cases. hepatic sos is a formidable challenge both for patients undergoing stem cell transplantation and for their physicians. the second pathology in this child which significantly contributed to his downhill course was busulfan induced lung injury. intriguingly, in the present clinical setting, busulfan induced lung injury remains an diagnosis of exclusion, particularly with respect to considering usual and atypical infections. its clinical presentation includes a spectrum ranging from acute, rapidly progressive respiratory distress to chronic, interstitial lung disease with insidious onset [ , ] . the pathophysiology of drug-induced lung injury is not fully understood but direct toxicity of the drug to parenchymal cells, cell-mediated immune reactions and release of cytokines are believed to contribute to the lung injury. the pathologic findings consist of mainly diffuse interstitial pneumonitis, organizing alveolitis and cellular atypia within type ii pneumocytes. the injury pattern with busulfan is diffuse alveolar damage (dad) either in acute exudative phase with alveolar and interstitial oedema and hyaline membranes; or late reparative phase, which is characterized by proliferation of type ii pneumocytes and interstitial fibrosis [ ] . marked atypia of the type ii pneumocytes is a morphological clue in favour of busulfan induced lung injury in contrast to organizing bacterial pneumonia. moreover, pcr for cmv is helpful in excluding viral pneumonia. the prevalence of drug-induced pulmonary toxicity is increasing, and more than drugs are now known to cause lung injury. because this lung injury can be progressive and fatal, early recognition is important. the diagnosis of pulmonary drug toxicity should be considered in any patient with a history of drug therapy who presents with new or progressive respiratory complaints. the superadded fungal ulcer which developed preterminally with extensive dissemination to heart causing mural endocarditis and myocardial abscess eventually led to the demise of the child. hepatic sos contributes considerably to transplantation-related morbidity and mortality. recognition of this disease in the post-transplantation setting remains a challenge in the absence of specific diagnostic features as many other more common conditions can mimic it. a high index of suspicion is needed to identify patients with sos. while hepatic sos and busulfan induced lung injury are commonly reported as isolated findings following autologous sct, the coexistence of these are extremely rare and have not been documented in the literature thus far. the present case adds observational data to the existing literature and highlights the importance of keeping high index of suspicion for these two entities in patients following hsct, and early institution of effective therapy. veno-occlusive disease of the liver and multiorgan failure after bone marrow transplantation: a cohort study of patients serum galactomannan assay for the diagnosis of invasive aspergillosis in children with haematological malignancies sinusoidal obstruction syndrome (hepatic veno-occlusivedisease) hepatic veno-occlusive disease (sinusoidal obstruction syndrome) after hematopoietic stem cell transplantation sinusoidal obstruction syndrome vascular disorders of the liver. american association for the study liver diseases incidence and outcome of hepatic venoocclusive disease after blood or marrow transplantation: a prospective cohort study of the european group for blood and marrow transplantation. european group for blood and marrow transplantation chronic leukemia working party the relevance of plasminogen activator inhibitor (pai- ) as a marker for the diagnosis of hepatic veno-occlusive disease in patients after bone marrow transplantation endothelial dysfunction after bone marrow transplantation: increase of soluble thrombomodulin and pai- in patients with multiple transplant-related complications prediction of veno-occlusive disease using biomarkers of endothelial injury venocclusive disease of the liver after bone marrow transplantation: diagnosis, incidence, and predisposing factors venoocclusive disease of the liver following bone marrow transplantation busulphan lung in childhood lung function yrs after allogeneic bone marrow transplantation conditioned with busulphan and cyclophosphamide interstitial pneumopathies caused by busulfan. histologic, developmental and bronchoalveolar lavage analysis of cases key: cord- -ltx w zh authors: zhu, liqian; jiang, xinyi; fu, xiaotian; qi, yanhua; zhu, guoqiang title: the involvement of histone h acetylation in bovine herpesvirus replication in mdbk cells date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ltx w zh during bovine herpesvirus (bohv- ) productive infection in cell cultures, partial of intranuclear viral dna is present in nucleosomes, and viral protein vp associates with histones and decreases histone h acetylation, indicating the involvement of histone h acetylation in virus replication. in this study, we demonstrated that bohv- infection at the late stage (at h after infection) dramatically decreased histone h acetylation [at residues k (h k ac) and k (h k ac)], which was supported by the pronounced depletion of histone acetyltransferases (hats) including cbp/p (creb binding protein and p ), gcn l (general control of amino acid synthesis yeast homolog like ) and pcaf (p /cbp-associated factor). the depletion of gcn l promoted by virus infection was partially mediated by ubiquitin-proteasome pathway. interestingly, the viral replication was enhanced by hat (histone acetyltransferase) activator ctpb [n-( -chloro- -trifluoromethylphenyl)- -ethoxy- -pentadecylbenzamide], and vice versa, inhibited by hat inhibitor anacardic acid (aa), suggesting that bohv- may take advantage of histone acetylation for efficient replication. taken together, we proposed that the hat-dependent histone h acetylation plays an important role in bohv- replication in mdbk (madin-darby bovine kidney) cells. bovine herpesvirus (bohv- ) is an important pathogen that causes pneumonia, conjunctivitis, genital disorders, and abortions in cattle [ ] . bohv- infection induces severe inflammatory response though diverse mechanisms, such as by overexpression of pro-inflammatory cytokines and reactive oxidative species [ ] [ ] [ ] . the suppression of host immune response by virus infection may render secondary infection by diverse pathogens, such as bovine viral diarrhea viruses (bvdv), bovine respiratory syncytial virus (brsv), parainfluenza- virus (pi v), bovine coronaviruses, mannheimia haemolytica, pasteurella multocida, histophilus somni and mycoplasma spp. [ , ] , and consequently lead to a life-threatening pneumonia known as bovine respiratory disease complex (brdc), one of the costliest ailments in cattle feeding [ , ] . in eukaryotes, dna is packaged into a protein-dna complex called chromatin, with nucleosome as monomeric subunit containing a core of histone proteins (h a, h b, h , and h ) surrounding by~ bp of genomic dna [ ] . the chromatin is dynamically organized into regions of either loosely packaged actively transcribed chromatin (euchromatin) or highly condensed cbp/p rabbit mab (monoclonal antibody) (cat# , : ), pcaf rabbit mab (cat# , : ), gcn l rabbit mab (cat# , : ), histone h rabbit mab (cat# , : ), acetyl-histone h (lys ) rabbit mab (cat# , : ), acetyl-histone h (lys ) rabbit mab (cat# , : ), ubiquitin mouse mab(cat# , : ), hdac (histone deacetylas) mouse mab (cat# , : ), hdac mouse mab (cat# , : ), hdac mouse mab (cat # , : ), hdac rabbit mab (cat # , : ), β-actin rabbit mab(cat# , : ), hrp (horseradish peroxidase) labeled anti-mouse igg (cat# , : ) and hrp labeled anti-rabbit igg (cat# , : ), were purchased from cell signaling technology (beverly, ma, usa). bohv- vp antibody ( : ) is kindly provided by prof. vikram misra at the university of saskatchewan [ ] . anacardic acid (aa) (cat#a ), trichostatin a (tsa) (# ). mg (cat# - ), ammonium chloride (nh cl) (cat# ), were ordered from sigma-aldrich (st. louis, mo, usa). bortezomib (#s ) was obtained from selleckchem.com (houston, tx, usa). n-( -chloro- -trifluoromethyl-phenyl)- -ethoxy- -pentadecyl-benzamide (ctpb) (cat# - - ) was provided by santa cruz biotechnology (dallas, tx, usa). cytotoxicity of indicated chemicals in mdbk cells was assessed by trypan-blue exclusion test, as described by fiorito et al. [ , ] , with modification. in brief, mdbk cells in -well plates were treated with or without chemicals at indicated concentrations for h. then the cells were collected by trypsinization, and an aliquot of the cell suspension was mixed with an equal volume of . % trypan-blue ( . %) (bio-rad, hercules, ca, usa, # ). after incubation for min, cells were counted using a burker chamber under a light microscope. the percentage of cell viability in the chemical treatment groups was calculated by normalization of the number of live cells to that in the control samples. the value of cell viability in the control was arbitrarily set to %. confluent mdbk cells in mm dishes were infected with bohv- (moi = ) for , and h. cell lysates were prepared using lysis buffer ( % triton x- , mm sodium chloride, mm edta, mm egta, mm sodium fluoride, mm sodium pyrophosphate, mm phenylmethylsulfonyl fluoride, . g/ml leupeptin, mm benzamidine, and mm sodium orthovanadate in mm tris-hcl, ph . ). to test the effects of certain inhibitors on the designated signaling, mdbk cells were infected for h along with treatment with indicated chemicals at the designated concentrations. cell lysates were prepared using lysis buffer as described above. cell lysates were separated on or % sds-polyacrylamide gels, and proteins were transferred to a polyvinylidene difluoride (pvdf) membrane (bio-rad, hercules, ca, usa). targeted proteins were detected using respective antibodies. the intensity of immune reactive bands was analyzed with free software image j (https://imagej.nih.gov/ij/download.html). to calculate the relative protein expression levels, the band intensity of target proteins was firstly normalized to β-actin, then normalized to the control lane. for ip studies, mdbk cells in -mm dishes were infected with bohv- at an moi of . at h after infection, cells were lysed with ml of ripa buffer ( × pbs, % np- , . % sodium deoxycholate, . % sds) supplemented with protease inhibitor as described above in western blots analysis. cell lysates were clarified by centrifugation at , rpm for min, and incubated with dynabeads ® (life technologies, carlsbad, ca, usa, cat. no. d), which have been incubated with µl of acetyl-histone h (lys ) rabbit mab (cell signaling technology, cat# ) or gcn l rabbit mab (cell signaling technology, cat# ) for h at room temperature with rotation. after overnight incubation at • c with rotation, the beads were collected with the help of a magnet (dynamag™) (life technologies, cat. no. d). after three washing with pbs, beads were boiled in sds loading buffer and western blots were performed to detect the designated proteins. mdbk cell in -well plates were infected with bohv- (moi of ) along with the treatment of indicated chemicals(paa, anacardic acid, tsa, and ctpb) at the designated concentration for h at • c, after three washing with pbs, fresh medium with designated chemicals was added to each well. at h after infection, viral yields were titrated in mdbk cells. the cell cultures treated with dmso was used as a control. the results are expressed as tcid /ml calculated using the reed-muench formula. confluent mdbk cells in mm dishes were infected with bohv- using an moi of . at , and h post infection(hpi) total rna was purified with trizol ls reagent (ambion, thermo fisher scientific, waltham, ma, usa, cat# ) following the manufacturers' instructions. freshly prepared rna ( µg) was used as a template for the synthesis of the first-strand cdna with commercial random hexamer primers for viral mrna detection using thermoscript™ rt-pcr system kit (invitrogen, carlsbad, ca, usa, cat# - ). the cdna products were used as templates for relative qrt-pcr to measure levels of viral mrna of bicp , and bicp as well as cellular gene glyceraldehyde- -phosphate dehydrogenase (gapdh) with specific primers as previously described in the reference [ ] . analysis of gapdh mrna was used as an internal control. relative qrt-pcr was carried out using the abi fast real-time system (applied biosystems, foster city, ca, usa). separate gapdh amplification was used to normalize gene expression. the data were analyzed using the equation −∆∆ct method. acetylation of histone h is involved in transcription activation, and h k ac is an epigenetic marker for histone acetylation. to test whether bohv- infection alters histone h acetylation, we evaluated the expression levels of h k ac, h k ac as well as h in virus infected mdbk cells at , , and hpi. as a result, virus infection consistently decreased the expression levels of h k ac, h k ac and h , and peaked at hpi ( figure a ). quantitative analysis indicated that the levels of h k ac, h k ac and h were decreased to . %, . % and . % relative to the control, respectively( figure b ), suggesting that bohv- infection reduced histone h acetylation (h k ac and h k ac). histone acetylation and deacetylation are reversible processes regulated enzymatically by hats and histone deacetylases (hdacs). hats such as cbp/p , gcn l , and pcaf, are enzymes that ultraviolet (uv) light-inactivated viruses are replication deficient because it could bind to the receptors and enter the cells, but are unable to express viral genes [ , ] . to further understand whether complete viral replication cycle was required to affect the histone h acetylation, uv-inactivated viral particles were employed for further investigation. as shown in figure c ,d, uv-inactivated virus had no effects on histone h acetylation. thus, these results suggested that de novo viral protein production and/or dna replication seems to be associated with the decreased acetylation of histone h . histone acetylation and deacetylation are reversible processes regulated enzymatically by hats and histone deacetylases (hdacs). hats such as cbp/p , gcn l , and pcaf, are enzymes that acetylate conserved lysine residues on histones. to understand the mechanisms underlying the decreased histone h acetylation by virus infection, we initially detected the protein levels of cbp/p , pcaf, and gcn l following bohv- infection at , , and hpi. virus infection altered the expression of cbp/p , pcaf, and gcn l , only at hpi, all of them were robustly decreased in comparison to the mock infected control (figure a ). the protein levels of cbp/p , pcaf, and gcn l were reduced to approximately . %, . %, and . % relative to the control, respectively ( figure b ). the global decease of hats expression at h after infection may reflect their reduced capability for histone acetylation, which is in agreement with the decreased histone h acetylation. hdacs are a family of enzymes controlling deacetylation of histones. the family of mammalian hdacs is comprised of at least members which are classified into four classes: class i (hdac , , and ), class ii (hdac , , , , and ), class iii (sirt to ) and class iv (hdac ) [ ] . in this study, the expression of hdac - in response to virus infection was detected using western blots. as can be seen in figure c ,d, virus infection altered the expression of both hdac and hdac with distinct manners, while neither hdac nor hdac were apparently affected. at hpi, the expression levels of hdac and hdac were decreased to approximately . % and . % relative to the control, respectively. the unexpected decreased expression levels of both hdac and hdac may undermine the finding that virus infection decreased the acetylation of histone h . taken together, virus infection altered the expression of hats and hdacs with distinct manners. relative to hdacs, the prominently decreased expression of hats strongly supported the reduced acetylation of histone h at hpi. hdacs are a family of enzymes controlling deacetylation of histones. the family of mammalian hdacs is comprised of at least members which are classified into four classes: class i (hdac , , and ), class ii (hdac , , , , and ), class iii (sirt to ) and class iv (hdac ) [ ] . in this study, the expression of hdac - in response to virus infection was detected using western blots. as can be seen in figure c ,d, virus infection altered the expression of both hdac and hdac with distinct manners, while neither hdac nor hdac were apparently affected. at hpi, the expression levels of hdac and hdac were decreased to approximately . % and . % relative to the control, respectively. the unexpected decreased expression levels of both hdac and hdac may undermine the finding that virus infection decreased the acetylation of histone h . taken together, virus infection altered the expression of hats and hdacs with distinct manners. relative to hdacs, the prominently decreased expression of hats strongly supported the reduced acetylation of histone h at hpi. our foregoing results demonstrated that virus infection differentially altered hats and hdacs expression, particularly the depletion of hats correlated with the reduced histone h acetylation. therefore, the role of hats and hdacs in bohv- productive infection was independently investigated using hat inhibitor anacardic acid (aa) and hdac inhibitor trichostatin a (tsa), respectively. aa specifically inhibits the enzymatic activity of hats, such as cbp/p and pcaf, and thereby affects hat-dependent gene transcription [ ] . also, aa has been reported to have multiple other biological effects such as antitumor activity and antioxidant activity [ ] . in this study, we found that the treatment of virus-infected cells with hat inhibitor aa at a concentration of µm and µm resulted in a . -and . -log reduction of the virus titer comparing to that in the mock-treated control, respectively ( figure a) . indeed, µm of aa treatment could inhibit histone h acetylation as demonstrated by the reduced levels of h k ac relative to the control, but aa increased the levels of h k ac in the context of virus infection in comparison to the mock treated but infected cells ( figure e ,f). maybe the significantly decreased progeny virus by aa treatment led to rescuing the depletion of h k ac attributed to virus infection. tsa is an hdac-specific inhibitor that can selectively inhibit the enzymatic activities of class i and ii hdacs, but not class iii hdacs [ ] . interestingly, it was reported that influenza virus infection decreased hdac expression in a cells, and the treatment of infected cells with and µm of tsa resulted in . -fold and . -fold increase of progeny virus relative to the control, respectively [ ] . in this study, we used much fewer concentrations of tsa to investigate its role in bohv- replication. we found that the treatment with nm of tsa could restore histone h acetylation ( figure g ,h), but had no impact on the viral replication because the virus titer was only increased~ . log (equal to -fold), with a difference not statistically significant (p > . ) ( figure b ). of note, all the concentrations used for indicated inhibitors had no cytotoxicity to mdbk cells ( figure e ). but we noticed that in the context of virus infection the cell viability was reduced to approximately . % by tsa ( nm) treatment ( figure i ), which is a possible reason for why tsa treatment could not evidently booster viral replication. these results suggested that the maintenance of hat activities was essential for virus productive infection. ctpb is a potent activator of cbp/p , but not of pcaf activities. to further confirm the role of hat played in the virus infection, ctpb, was employed for further investigation. ctpb at a concentration of µm showing no cytotoxicity to mdbk cells can significantly promote virus productive infection ( figure c ,e). the virus titer was increased~ . log by the treatment with µm of ctpb in comparison with the mock-treated control ( figure c ). this observation further suggested that the histone acetylation by hat played an essential role in bohv- productive infection. considering that histone acetylation regulated by hat is an important factor controlling gene expression, we further investigated the effects of chemical inhibition of hats on viral gene expression. for this purpose, the virus-infected cells were treated with µm of aa or dmso as a control, and the mrna levels of immediate early (ie) genes including bicp and bicp were detected with relative qrt-pcr. in aa treated virus-infected cell cultures, the mrna levels of bicp considering that histone acetylation regulated by hat is an important factor controlling gene expression, we further investigated the effects of chemical inhibition of hats on viral gene expression. for this purpose, the virus-infected cells were treated with µm of aa or dmso as a control, and the mrna levels of immediate early (ie) genes including bicp and bicp were detected with relative qrt-pcr. in aa treated virus-infected cell cultures, the mrna levels of bicp decreased to . % and . % relative to the mock-treated control, at and hpi, respectively, but at hpi the chemical treatment showed minor effects ( figure a ). bicp mrna levels were reduced to . %, . % and . % by aa treatment relative to the control samples, at , and hpi, respectively ( figure b ). though these ie proteins were not detected due to the unavailability of given antibodies, these results of qrt-pcr indicated that the hat inhibitor aa affected the transcription of these ie genes. an additional study was performed to examine the effects of aa on the expression of viral tegument protein vp by western blots using an antibody against vp . as demonstrated in figure c , at hpi vp protein expression levels decreased approximately . -fold by the treatment with aa relative to that in the mock-treated control, indicating that aa affected vp expression. in summary, these findings further suggested that this hat inhibitor affected bohv- productive infection in mdbk cells, and therefore the maintaining of hat activity is essential for virus efficient replication. viruses , , x for peer review of decreased to . % and . % relative to the mock-treated control, at and hpi, respectively, but at hpi the chemical treatment showed minor effects ( figure a ). bicp mrna levels were reduced to . %, . % and . % by aa treatment relative to the control samples, at , and hpi, respectively ( figure b ). though these ie proteins were not detected due to the unavailability of given antibodies, these results of qrt-pcr indicated that the hat inhibitor aa affected the transcription of these ie genes. an additional study was performed to examine the effects of aa on the expression of viral tegument protein vp by western blots using an antibody against vp . as demonstrated in figure c , at hpi vp protein expression levels decreased approximately . -fold by the treatment with aa relative to that in the mock-treated control, indicating that aa affected vp expression. in summary, these findings further suggested that this hat inhibitor affected bohv- productive infection in mdbk cells, and therefore the maintaining of hat activity is essential for virus efficient replication. the virus infected cells were treated with dmso or aa ( µm). total rna was prepared at indicated time points, and qrt-pcr was performed to determine the mrna levels of bicp (a) and bicp (b). (c) mdbk cells in mm dishes were infected by bohv- at an moi of with the treatment of dmso or aa ( µm), at hpi, cell lysate was prepared and subjected to western blots to detect vp protein. the band intensity was analyzed with software image j. and analysis was compared with that of untreated but infected control. data represent three independent experiments. significance was assessed with the student t test (* p < . , ** p < . ). ns: not significant. generally, there are two potential pathways to control protein degradation in eukaryotic cells, one mediated by ubiquitin-proteasome and other mediated by lysosome [ ] , which can be efficiently inhibited by chemical inhibitors, such as mg and nh cl, respectively. to identify whether the ubiquitin-proteasome and/or lysosome pathways are involved in the reduced acetylation of histone h at h after infection, the virus-infected mdbk cells were treated with either proteasome inhibitor mg or lysosome inhibitor nh cl throughout infection as determined elsewhere [ , ] . the treatment of mg ( µm) reversed the depletion of both h k ac and h k ac attributed to virus infection, and rescued their expression to a level higher than that in the uninfected control, whereas nh cl treatment did not show any effect ( figure a,b) . the increased levels of ubiquitinated proteins . total rna was prepared at indicated time points, and qrt-pcr was performed to determine the mrna levels of bicp (a) and bicp (b). (c) mdbk cells in mm dishes were infected by bohv- at an moi of with the treatment of dmso or aa ( µm), at hpi, cell lysate was prepared and subjected to western blots to detect vp protein. the band intensity was analyzed with software image j. and analysis was compared with that of untreated but infected control. data represent three independent experiments. significance was assessed with the student t test (* p < . , ** p < . ). ns: not significant. generally, there are two potential pathways to control protein degradation in eukaryotic cells, one mediated by ubiquitin-proteasome and other mediated by lysosome [ ] , which can be efficiently inhibited by chemical inhibitors, such as mg and nh cl, respectively. to identify whether the ubiquitin-proteasome and/or lysosome pathways are involved in the reduced acetylation of histone h at h after infection, the virus-infected mdbk cells were treated with either proteasome inhibitor mg or lysosome inhibitor nh cl throughout infection as determined elsewhere [ , ] . the treatment of mg ( µm) reversed the depletion of both h k ac and h k ac attributed to virus infection, and rescued their expression to a level higher than that in the uninfected control, whereas nh cl treatment did not show any effect ( figure a,b) . the increased levels of ubiquitinated proteins in mg -treated cells but not in nh cl-treated cells confirmed the efficiency of mg as an inhibitor for the proteasome pathway in mdbk cells ( figure c ). though the concentration of mg used in this study did not show obvious cytotoxicity to mdbk cells ( figure f ), the chemical may have off-target effects. so, another proteasome inhibitor bortezomib was employed to validate the rescued effects of mg . as expected, bortezomib at a concentration of nm showing no cytotoxicity to mdbk cells could reverse bohv- -reduced expression of histone acetylation marker h k ac to a level a little bit higher than that in the uninfected control ( figure d-f) , though bortezomib showed relative lower capability than mg . these results suggested that the ubiquitin-proteasome pathway may contribute to the decreased acetylation of histone h in the virus infected cells. viruses , , x for peer review of in mg -treated cells but not in nh cl-treated cells confirmed the efficiency of mg as an inhibitor for the proteasome pathway in mdbk cells ( figure c ). though the concentration of mg used in this study did not show obvious cytotoxicity to mdbk cells ( figure f ), the chemical may have off-target effects. so, another proteasome inhibitor bortezomib was employed to validate the rescued effects of mg . as expected, bortezomib at a concentration of nm showing no cytotoxicity to mdbk cells could reverse bohv- -reduced expression of histone acetylation marker h k ac to a level a little bit higher than that in the uninfected control ( figure d-f) , though bortezomib showed relative lower capability than mg . these results suggested that the ubiquitinproteasome pathway may contribute to the decreased acetylation of histone h in the virus infected cells. in view that the proteasome inhibitors of both mg and bortezomib could rescue the depletion of h k ac (a marker for histone h acetylation) attributed to virus infection, we investigated whether h k ac was ubiquitinated in the cells with or without infection by ip assay. unexpectedly, when we performed ip with the h k ac specific monoclonal antibody, the ubiquitinated protein bands could not be detected in the cells with or without infection using ubiquitin specific antibody ( figure a ), indicating that h k ac is not ubiquitinated in mdbk cells. so it was highly possible that the depletion of acetylated histone h due to virus infection was not caused by the proteasomemediated h k ac degradation. in view that the proteasome inhibitors of both mg and bortezomib could rescue the depletion of h k ac (a marker for histone h acetylation) attributed to virus infection, we investigated whether h k ac was ubiquitinated in the cells with or without infection by ip assay. unexpectedly, when we performed ip with the h k ac specific monoclonal antibody, the ubiquitinated protein bands could not be detected in the cells with or without infection using ubiquitin specific antibody ( figure a ), indicating that h k ac is not ubiquitinated in mdbk cells. so it was highly possible that the depletion of acetylated histone h due to virus infection was not caused by the proteasome-mediated h k ac degradation. our foregoing results indicated that both pcaf and gcn l were significantly decreased by the virus infection ( figure a) . interestingly, the treatment of virus-infected cells with µm of mg could significantly reverse the depletion of gcn l but not pcaf ( figure b,c) . we speculated that virus infection may promote gcn l degradation via proteasome pathway. therefore, ip was performed with gcn l specific monoclonal antibody, and ubiquitin specific antibody was used to detect the ubiquitination of gcn l in the cell cultures. as a result, the ubiquitinated gcn l could be detected from the ip sample by the immunoblot using ubiquitin specific antibody ( figure d ). this result indicated that virus infection might target gcn l for ubiquitin-mediated degradation, which would partially account for the depletion of acetylated histone h and correlated with the reversed depletion of h k ac by the treatment of mg in the virus-infected cells. our foregoing results indicated that both pcaf and gcn l were significantly decreased by the virus infection ( figure a) . interestingly, the treatment of virus-infected cells with µm of mg could significantly reverse the depletion of gcn l but not pcaf ( figure b ,c). we speculated that virus infection may promote gcn l degradation via proteasome pathway. therefore, ip was performed with gcn l specific monoclonal antibody, and ubiquitin specific antibody was used to detect the ubiquitination of gcn l in the cell cultures. as a result, the ubiquitinated gcn l could be detected from the ip sample by the immunoblot using ubiquitin specific antibody ( figure d ). this result indicated that virus infection might target gcn l for ubiquitin-mediated degradation, which would partially account for the depletion of acetylated histone h and correlated with the reversed depletion of h k ac by the treatment of mg in the virus-infected cells. acetylation is one of the best-characterized covalent modifications of histones. hyperacetylation of histones is associated with an "open chromatin" conformation and transcriptional activation, whilst hypoacetylation of histones is associated with condensed chromatin and gene silencing [ , ] . for some dna viruses, such as hsv- and canine parvovirus, much is known about the effects of histone modification on virus replication. during hsv- productive infection, the viral genomes are associated with histones immediately after injection into the nucleus, and viral proteins icp and vp are required to enhance histone acetylation on the viral genome to enable efficient viral gene expression [ , ] . during canine parvovirus infection, cellular histones are associated with viral dna, and histone acetylation on parvoviral dna is essential for viral gene expression [ ] . bohv- tegument protein vp associates with histones and thereby decreased histone h acetylation in infected cells or vp transfected cells [ ] . in this study, for the first time we demonstrated that histone h acetylation (h k ac and h k ac) was significantly decreased during bohv- infection in mdbk cells, and peaked at h after infection (figure ) , which suggested the involvement of histone h acetylation in bohv- infection. to further elucidate the potential mechanisms for the decreased histone h acetylation in response to the virus infection, the steady state expression of certain hats and hdacs were investigated because they regulate enzymatically histone acetylation with opposite effects. we found that the virus infection led to a global decrease of all the detected hats, including cbp/p , gcn l and pcaf (figure a) , which was clearly in favor of the finding that virus infection decreased histone h acetylation (figure ). however, it seems that downregulation of both hdac and hdac by virus infection was not correlated with the decreased levels of histone h acetylation, which emphasized the complexity of the mechanisms for the regulation of histone acetylation following virus infection. it has been reported that hdac regulates influenza a virus (iav) replication independent of its deacetylation activity [ ] . hdac stimulates host type i interferon antiviral response, therefore iav infection decreases hdac expression for efficient replication [ ] . the treatment with tsa, an hdac inhibitor, increases iav infection via the inhibition of signal transducer and activator of transcription i (stat ) pathway, and the depression of interferon-stimulated genes including ifitm , isg , and viperin in iav-infected cells [ ] . hdac is required for inflammatory gene expression in response to lps stimulation [ ] . so, hdac and hdac can stimulate host antiviral response or inflammatory response, which tend to be depressed by virus infection. moreover, hdac inhibitors promote hsv- productive infection in neural cells [ , ] . likewise, we found that tsa enhanced bohv- virus yield approximately -fold, but the difference was not statistically significant ( figure ). therefore, we assumed that the downregulation of hdac and hadc would be beneficial for bohv- efficient replication independent of their deacetylation activity, which needs further extensive studies, in the future. it has been reported that the ubiquitin-proteasome pathway, generally known to mediate protein degradation, is involved in bohv- productive infection and immune invasion [ , ] . here, we found that the ubiquitin-proteasome inhibitors could reverse the depletion of acetylated h in bohv- infected cells (figure ) . mechanistically, the virus infection targeted gcn l but not acetylated histone h for proteasome-mediated degradation (figure ), which may consequently reduce the acetylation of histone h . previous studies have reported that some bohv- -encoded viral proteins promote the proteasome-mediated degradation of certain cellular proteins. for example, both interferon response factor (irf ) and promyelocytic leukemia (pml) are targeted by viral proteins bicp for proteasome-dependent degradation [ , ] . viral ul . protein is involved in the proteasome-mediated degradation of the transporter associated with antigen presentation [ ] . the bohv- host shutoff protein ul destabilizes the expression of immune responses related genes by ubiquitin-proteasome pathway [ ] . taken together, the above evidence indicates that virus-encoded proteins may target specific cellular proteins for proteasome-dependent degradation. whether a specific viral protein(s) is involved in the depletion of gcn l through proteasome pathway is an interesting subject which is remained to be determined in the future. in this study, we found that bohv- replication was positively correlated with hat activity because the treatment with hat activator (ctbp) increased the virus yield, and vice versa, it was significantly decreased by hat inhibitor (aa) partially through affecting virus gene expression (figures and ) . this finding is supported by a previous report that cbp/p , an hat, enhances bohv- productive infection and transactivation of late viral protein gc promoter [ ] . taken together, these findings suggested that bohv- infection may take advantage of histone acetylation for efficient replication. we speculated that hat-dependent histone h acetylation plays an important role in bohv- replication in mdbk cells. in this study, the expression status of acetylated h including h k ac and h k ac was investigated in the context of virus infection. we demonstrated that histone acetylation played an important role in bohv- replication, while the virus infection decreased histone h acetylation by differentially altered expression of hats and hdacs. in addition, virus infection targeted gcn l for degradation via the proteasome pathway, which correlated well with the reversed depletion of h k ac by the treatment of mg in the virus-infected cells. we suggested that the hat-dependent histone (h ) acetylation plays an important role in bohv- replication in mdbk cells in summary, we provided evidence that bohv- infection decreased histone h acetylation in mdbk cells, while histone acetylation played an important role in bohv- replication. we suggested that hat-dependent acetylation of histone h plays a vital role in bohv- replication. this finding might add our knowledge on understanding the mechanism for the viral pathogenesis. bovine herpesvirus (bhv- ): biology, pathogenesis, and control active genes are tri-methylated at k of histone h response of proinflammatory and anti-inflammatory cytokines in calves with subclinical bovine viral diarrhea challenged with bovine herpesvirus- bhv- induced oxidative stress contributes to mitochondrial dysfunction in mdbk cells regulation of innate immune responses by bovine herpesvirus and infected cell protein (bicp ). viruses a review of the biology of bovine herpesvirus type (bhv- ), its role as a cofactor in the bovine respiratory disease complex and development of improved vaccines susceptibility loci revealed for bovine respiratory disease complex in pre-weaned holstein calves detection and characterization of viruses as field and vaccine strains in feedlot cattle with bovine respiratory disease chromatin structure: a repeating unit of histones and dna histone exchange, chromatin structure and the regulation of transcription regulation of histone modifying enzymes by the ubiquitin-proteasome system histone h a variants in nucleosomes and chromatin: more or less stable? regulation of chromatin by histone modifications linking histone acetylation to transcriptional regulation deciphering the transcriptional histone acetylation code for a human gene during lytic infection herpes simplex virus type is associated with histones bearing modifications that correlate with active transcription vp -dependent association of chromatin-modifying coactivators and underrepresentation of histones at immediate-early gene promoters during herpes simplex virus infection herpes simplex virus icp promotes both histone removal and acetylation on viral dna during lytic infection chromatin control of herpes simplex virus lytic and latent infection promoter-targeted histone acetylation of chromatinized parvoviral genome is essential for the progress of infection c , a novel p /creb-binding protein-specific inhibitor of histone acetyltransferase, attenuates influenza a virus infection bovine herpesvirus tegument protein vp interacts with histones, and the carboxyl terminus of vp is required for nuclear localization occurrence of bovine herpesvirus- dna in nucleosomes and chromatin of bovine herpesvirus- -infected cells: identification of a virion-associated protein in chromatin of infected cells protein composition of the bovine herpesvirus . virion the role of phospholipase c signaling in bovine herpesvirus infection protein and dna elements involved in transactivation of the promoter of the bovine herpesvirus (bhv) ie- transcription unit by the bhv alpha gene trans-inducing factor tetrachlorodibenzo-p-dioxin regulates bovine herpesvirus type induced apoptosis by modulating bcl- family members mg- reduces virus release in bovine herpesvirus- infection the high mobility group at-hook protein stimulates bovine herpesvirus productive infection the cellular protein p ipk regulates influenza virus mrna translation and replication through a pkr-mediated mechanism histone deacetylase is a component of influenza a virus-induced host antiviral response -alkylsalicylates are selective tip inhibitors and target the acetyl-coa binding site inhibition of histone acetyltransferase activity by anacardic acid sensitizes tumor cells to ionizing radiation trichostatin a and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function influenza a virus dysregulates host histone deacetylase that inhibits viral infection in lung epithelial cells the ubiquitin-proteasome pathway and proteasome inhibitors influenza a virus-induced degradation of eukaryotic translation initiation factor b contributes to viral replication by suppressing ifitm protein expression histone acetylation increases chromatin accessibility histone modification patterns during gene activation herpes simplex virus vp , but not icp , is required to reduce histone occupancy and enhance histone acetylation on viral genomes in u os osteosarcoma cells histone deacetylase plays an acetylation-independent role in influenza a virus replication requirement for the histone deacetylase hdac for the inflammatory gene expression program in macrophages histone deacetylase inhibition enhances oncolytic viral replication in glioma histone deacetylase inhibitors induce reactivation of herpes simplex virus type in a latency-associated transcript-independent manner in neuronal cells the infected cell protein encoded by bovine herpesvirus (bicp ) induces degradation of interferon response factor and, consequently, inhibits beta interferon promoter activity regulation of promyelocytic leukemia (pml) protein levels and cell morphology by bovine herpesvirus infected cell protein (bicp ) and mutant bicp proteins that do not localize to the nucleus varicelloviruses avoid t cell recognition by ul . -mediated inactivation of the transporter associated with antigen processing the ul -encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of mhc class i molecules by bovine herpesvirus bovine herpesvirus immediate-early protein (bicp ) interacts with the histone acetyltransferase p , which stimulates productive infection and gc promoter activity key: cord- - gq e f authors: staroverov, sergey a.; volkov, alexei a.; mezhenny, pavel v.; domnitsky, ivan yu.; fomin, alexander s.; kozlov, sergey v.; dykman, lev a.; guliy, olga i. title: prospects for the use of spherical gold nanoparticles in immunization date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: gq e f recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. we used spherical gold nanoparticles (average diameter, nm) as a platform for the antigen for swine transmissible gastroenteritis virus (tgev). the literature data demonstrate that immunization of animals with the tgev antigen coupled to gold nanoparticles (gnps) not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. the contents of γ-ifn, il- β, and il- in animals immunized with gnp-antigen conjugates were found to be higher than those in intact animals or in animals given the antigen alone. the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. the increased concentration of il- indicates that the injected preparations are stimulatory to cellular immunity. immunization with the tgev antigen conjugated to gnps as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to the activation of antibody generation. furthermore, the use of this conjugate allows marked improvement of the structure of the animals’ immune organs and restores the morphological–functional state of these organs. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction. the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. these results can be used to develop vaccines against this infection by employing tgev antigen coupled to gold nanoparticles as a carrier. swine transmissible gastroenteritis is an acute, rapidly spreading, highly contagious enteric disease of pigs of all ages. it is characterized by vomiting, watery and yellow diarrhea, weight loss, and rapid dehydration, leading to a high mortality rate, especially in piglets (mortality decreases with age). swine transmissible gastroenteritis is caused by transmissible gastroenteritis virus (tgev) of the genus coronavirus in the family coronaviridae. the major route of virus transmission is fecal-oral. infected animals cannot digest food properly and die from dehydration. the disease causes great economic losses in pig farms all over the world, and the primary means of animal protection against it is vaccination (cheng and niu ; straw et al. ) . therefore, the development of new vaccines and the implementation of large-scale vaccination programs are important in pig breeding (moxley and olson ( ) . in several countries, vaccination against swine transmissible gastroenteritis is conducted effectively but only with inactivated monovalent and combined vaccines not recognized internationally. traditional inactivated vaccines have high contents of contaminant substances (up to %), causing side effects, and/or may contain, as inactivating compounds, toxic agents such as phenol and formaldehyde, normally used to inactivate microbial cells. moreover, traditional vaccines are usually low immunogenic, because killed microorganisms cannot induce fully functioning cellular immunity. therefore, there is a need to develop new, more effective, and less costly vaccines against swine transmissible gastroenteritis (mout et al. ) . recent years have seen extremely fast development of new viral nanovaccines and diagnostic agents using nanostructures prepared by biological and chemical synthesis. in particular, this can be attributed to the development of biocompatible inorganic nanomaterials-gold nanoparticles. gold nanoparticles are stable metallic nanoparticles which have excellent surface functional chemistry, high biocompatibility, and nil toxicity. in addition, gold nanoparticles can be easily synthesized, giving them shapes such as spheres, rods, cubes, stars, and pyramids. several reports have described the development and use of nanostructures for the delivery of biologically active agents to target cells (mout et al. ; bhattacharya and mukherjee ; staroverov et al. ). to reach their target cells in vivo, nanoparticles must bypass many barriers that protect animals against antigens (dreaden et al. ) . gold nanoparticles (gnps) have been used widely as carriers of antigens and therapeutic agents in human medicine, veterinary practice, and biology . of particular interest is the ability of gnps to induce humoral immune response to weakly immunogenic antigens and haptens (dykman et al. a; dykman et al. b ). m o r e o v er, g n p s c on j u ga t ed t o t g e v h a v e a n immunostimulatory effect (dykman et al. b) . we studied the immunological variable and morphological changes in the immune organs of guinea pigs immunized with tgev antigen conjugated to gnps as a nanocarrier. antigen tgev strain vn- was taken from the museum of strains of the laboratory of virology (scientific research veterinary institute, russian academy of sciences, saratov) and deposited in the all-russian state collection of microbial strains used in veterinary and animal husbandry by the number bvn- ^(patent ru a ). tgev strain vn- with a titer of . - . lg tcd ml − was used. test cultures were inoculated with the same strain at . lg tcd ml − after h of cultivation. tgev was purified in a sucrose gradient according to horzinek et al. ( ) . the resultant virus suspension was used for protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) at a constant voltage of v for min and then at v for h (maniatis et al. ) . for molecular m a s s d e t e r m i n a t i o n s , p r e c i s i o n p l u s p r o t e i n ™ kaleidoscope™ prestained protein standards no. were used. the results of protein separation were processed with the gel-imager and gel explorer, version . , programs (dna-technology, moscow). genome identity was checked by polymerase chain reaction (pcr) using a specific primera conservative part of tgev's s genome, containing base pairs (bp) (herrington and mcgee ) . rna was isolated from tgev by using a nucleos+tm suspension silicate sorbent (biokom, moscow) according to the manufacturer's instructions. pcr results were recorded on a transilluminator with a dna-analyzer video system. pcr products obtained from a preparation of the tgev strain vn- were analyzed by electrophoresis in agarose gel ( . %) using dna ladder as a molecular size marker and dna of bacteriophage λ as a negative control. as viral nucleic acid is responsible for virulence, it was inactivated, after being isolated from tgev and purified, with ribonuclease (rnaase), a hydrolase enzyme that catalyzes rna hydrolysis. the inactivation was done by the procedure described by fedorova et al. ( ) . the antigen resulting from nucleic acid inactivation was used for animal immunization. spherical gold nanospheres (average diameter, nm) were made by the citrate method of frens ( ) . for reduction, . ml of . % aqueous tetrachloroauric acid (haucl ; sigma-aldrich) was heated under reflux to °c on a magnetic stirrer in an erlenmeyer flask. this was followed by the addition of . ml of % aqueous sodium citrate to the flask. the particle diameter was found as described earlier (khlebtsov and dykman ) , by using a libra transmission electron microscope (carl zeiss, germany), a specord s spectrophotometer (analytik jena, germany), and a zetasizer nano-zs particle size and zeta potential analyzer (malvern). to prepare antigen-gold nanoparticle conjugates, we found the bgold number^(minimal amount of antigen that protects the sol against salt aggregation) for the antigen solution. to this end, μl of an antigen solution in pbs (initial concentration, mg ml − ) was titrated twofold on a -well microtiter plate. each well received μl of gnps and μl of . m nacl. the minimal stabilizing concentration for the antigen was . μg ml − . conjugation was done by simple mixing (without the use of coupling agents) (dykman et al. ). thirty six male guinea pigs, weighing - g at the time the experiment started, were used. the animals were cared for and handled in compliance with the guide for the care and use of laboratory animals, the european convention for the protection of vertebrate animals used for experimental and other scientific purposes, and the legislation of the russian federation. for immunobiological studies, the guinea pigs were divided into four groups of nine each. the animals were immunized subcutaneously along the spinal column by two injections with an interval of days between. the guinea pigs in group were injected with . ml of tgev antigen-gnps (antigen concentration, . μg per animal). group received ml of tgev antigen solution ( . μg per animal). group served as the control, which received ml of physiological saline. group served as the control gnps, which received ml of gnps solution. gnp-antigen group was in comparison with group which received only physiological saline (control group). the animals were killed days after the last injection, and blood serum samples were taken for immunological studies. immediately after removal, all pieces of tissues and organs were placed in a fixing solution ( % aqueous neutral formalin), and then -μm-thick histological sections were prepared on a freezing microtome, model (reichert wien, austria). all histological samples were stained with hematoxylin-eosin. for isolating peritoneal macrophages, the animals were killed and then fixed on their backs. an incision was made along the midline of the anterior abdominal wall, and the skin flap was carefully separated, with care taken to keep the peritoneum intact. after a puncture had been made with a needle connected to a syringe, ml of pbs, ph . , was injected into the peritoneal cavity. the anterior abdominal wall was then gently massaged, and after - min, peritoneal fluid was collected with a pasteur pipet through a cut made in the peritoneum and was filtered into a test tube through a nylon filter. the cells were washed three times by centrifugation in pbs at g, after which they were resuspended in ml of pbs and counted in a goryaev chamber. peritoneal macrophages were cultured by standard procedures (leiter ) . for isolating splenic lymphocytes, an incision was made along the white line of the peritoneum after peritoneal macrophages had been isolated, and the spleen was removed. the spleen was minced with scissors, and the tissue pieces were mashed through a fine sieve into a petri plate containing sterile pbs. the resulting suspension was subjected to ficoll-urografin density-gradient centrifugation. the lymphocyte ring was collected in a new test tube. the lymphocytes were washed three times by centrifugation in pbs, ph . , at g for min, and the cell sediment was resuspended in ml of pbs. the lymphocytic cells were counted with a haemascreenvet hematology analyzer (hospitex diagnostics, italy). the titer of antibodies in the sera was estimated by enzyme-linked immunosorbent assay (elisa) with horseradish peroxidase-labeled antibodies against guinea pig igg (jackson immunoresearch, uk). the synthetic peptide was used as the immobilized antigen. the reaction results were recorded on a plate screen microplate spectrophotometer. the interleukin concentration in the sera was measured with a plate screen analyzer (hospitex diagnostics, italy) and using reagents of il- β, il- , and inf-γ (vector-best, russia). respiratory activity was measured conventionally (bernas and dobrucki ) by the ability of cells to reduce nitrotetrazolium blue [ -( , -dimethylthiazol- -yl)- , diphenyl tetrazolium bromide, mtt (sigma-aldrich)] to formazan. briefly, suspensions of known concentrations of isolated animal cells (macrophages and lymphocytes) were centrifuged at g for min, and the sediment was resuspended in ml of . % mtt and incubated at °c for h. after incubation, the cells were centrifuged at g for min and the sediment was resuspended in . ml of dimethyl sulfoxide (fluka, switzerland). the amount of reduced formazan was measured with a genesys s uv-vis spectrophotometer at nm. to construct the calibration curve, we used commercial formazan (sigma-aldrich) at . , . , . , and mg ml − . for studying possible morphological changes in the spleen as an important part of the macrophage system, the animals were immunized with tgev antigen-gnps. the control group comprised of nonimmunized animals. we used the spleens of guinea pigs of the same physiological age. longitudinal and transverse spleen sections ( -μm-thick) were prepared on a freezing microtome, model (reichert wien, austria), by following standard procedures. histological sections were differentially stained with hematoxylin-eosin. the results were statistically processed by the standard procedures using student's t test to evaluate the reliability of the differences between samplings in the experimental and control studies. after the arithmetic mean and the standard deviation for a given data sample were found, we determined the standard error of the arithmetic mean and its confidence limits by student's t coefficient (n, p) at a significance level of % (p = . ). the obtained results were statistically processed by the standard procedures integrated in excel software (microsoft corp., usa). after the arithmetic mean and the standard deviation for a given data sample had been found, we determined the standard error of the arithmetic mean and its confidence limits with account of student's t coefficient (n, p) [number of measurements n = , significance level = % (p = . )]. these results are presented as histograms. the significance of differences between individual samples was evaluated by a two-sample unpaired student's t test with unequal variances. differences were considered significant when the experimentally found p exp value was ≤ . . the reliability of the changes recorded in the results of each of the experiments described above for the entire set of antigen formulations examined was assessed by one-way analysis of variance (anova) by using fisher's ratio test. the dependences found were considered significant at f > f crit , where the critical value f crit at n = and m = - (number of data sets) corresponded to p = . (with the number of degrees of freedom (df) lying between and ) and the effective value p eff was < . . swine transmissible gastroenteritis is caused by an rna virus belonging to group а of the genus alphacoronavirus in the family coronaviridae. the virion has a spherical shape with a diameter of - nm (martins et al. ) . the viral nucleocapsid is a flexible spiral containing single-stranded rna and a large number of nucleocapsid protein molecules (delmas et al. ; sturman et al. ) . the virus replicates in the cytoplasm of mature epithelial cells located at the ends of the intestine villi. a general scheme of the experiment is depicted in fig. . after the antigen was isolated and its protein composition was analyzed by sds-page, we found that the molecular masses of the separated proteins were identical to those of the tgev proteins. the antigen contained the following proteins: p protein (large surface glycoprotein s; molecular mass, - kda), n protein (basic nucleocapsid protein, - kda), and m protein (matrix membrane protein, - kda) (fig. ) . a protein fraction of - kda was also present. according to wesley et al. ( ) , kda is the size of an unglycated structural protein of tgev. thus, all known tgev proteins were separated. the tgev nucleic acid was identified by pcr using a specific primer-a conservative part of tgev's s genome, containing bp. analysis of the pcr products from a purified preparation of the tgev strain vn- , which was done by agarose gel electrophoresis with dna ladder , demonstrated sufficiently high separation of cdna (fig. ) . the presence of only a characteristic cdna fraction (size, bp) indicated the presence of the tgev genome in the sample. after the virus's nucleic acid was inactivated with ribonuclease, the resultant antigen (a mixture of viral capsid proteins) was used for conjugation with gnps and for subsequent animal immunization. the gnp diameter was measured by tem, the average particle size was . nm. analysis of the antibody titer data showed that the titer produced by immunization with gnp-antigen conjugates ( : . ; p = . ) was higher than that produced by immunization with unconjugated tgev antigen ( : . ). in immunology, the mononuclear phagocyte system (mps) (also known previously as the reticuloendothelial or macrophage system) is part of the immune system that consists of phagocytic cells located in reticular connective tissue. the cells are primarily monocytes and macrophages, which accumulate in lymph nodes and in the spleen. the effectiveness of interaction of the gnp-antigen conjugate with cells of the reticuloendothelial system was evaluated using the mtt test. on day after immunization, an increase in the respiratory activity of macrophages was noted (fig. ) . after immunization with the gnp-antigen conjugate, the activity increased by % (p = . ) as compared with the control and by % (р = . ) as compared with the immunization with tgev antigen and % (р = . ) as compared with gnp. a study of the respiratory activity of splenic lymphoid cells (fig. ) showed that after immunization with the conjugate, the activity increased . -fold, as compared to the control, whereas after immunization with tgev antigen alone, it did not change much. the data demonstrate that immunization of animals with tgev antigen-gnps not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid (antibody-forming) cells. this indicates that gnps can present antigens to the organs of the endothelial system. the effect of the conjugate on humoral immunity was assessed by comparing changes in the cytokine concentration between the control and experimental animals. there was a statistically significant increase in the content of the γ-ifn in animals immunized with gnp-antigen conjugates (fig. ) . these animals had the highest content of γ-ifn ( . ± . pgml - ; p = . ) this group was comparison with group which received only physiological saline (control group). we determined the p value error probability when deviating from the null hypothesis (errors of the first kind). we determined that for our research, p was ≤ . .when the animals were given the native antigen and gnps, no appreciable changes in γ-ifn content were found. the content of il- β in animals immunized with gnpantigen conjugate was . ± . pgml - (p = . ), which is higher than it was in intact animals ( . ± . pgml - ) or in animals given the antigen alone ( . ± pgml - ; p = . ) and gnps ( ± . pgml - ; p = . ) (fig. ) (p value for gnp-antigen group was comparison with group which received only physiological saline (control group). gnp-antigen group was comparison with group which received only physiological saline (control group). the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. this point is so important, since it is desirable to avoid an inflammatory process during immunization, which results in the appearance of granulomas. similar data were obtained with il- ( fig. ) . this fact indicates that the injected preparations are stimulatory to cellular immunity. it is known that interferons, which possess protective properties, are produced in the body only when the virus enters or tumor diseases. that is why the analysis of the effect of immunization with the help of gold nanoparticles on the change in the level of interferon was made. it was found that in the immunized group, the concentration of interferon increased by % (compared to the control) against % for the group immunized with the antigen. when immunizing animals with native antigen, no significant changes in the concentration of interferon in blood serum of the immunized animals were observed. thus, an increase in the concentration of interferon indicates the stimulation of the introduced drugs of the cell link of immunity. the results obtained correlate with research in this area by other scientists. for example, in work (zhang et al. ) showed that tgev n protein induced er stress and activated nf-κb, which was responsible for the upregulation of il- markers protein markers (kda) protein p protein and bcl- expression. tao et al. ( ) also revealed an increase in the concentration of cytokines including interferon gamma and interleukin in response to immunization aunp-m e + scpg. but he mainly considered the reaction of splenocytes to a given complex (their reaction, which was expressed in the production of cytokines, on the effect of aunp) was studied. in addition, aunp-m e was administered together with scpg which is a strong immunomodulator. it was interesting for us to look at the production of these cytokines in the body as a whole and to reveal the general clinical changes that can develop on the introduction of antigen and aunp. one can speculate that gnps facilitate antigen expression with the major histocompability complex on the surface of antigen-presenting cells. this subsequence ensures that the viral peptides are effectively presented to cytotoxic t lymphocytes and natural killers. our data for the increase in γ-ifn activity are in agreement with those for the increase in the respiratory activity of splenocytes on day after the last injection, another indication that colloidal gold can stimulate the release of γ-ifn by t cells, thereby enhancing the activity of lymphoid immunity. in guinea pigs, the spleen forms leucocytes and removes old red blood cells (erythrocytes). the spleen framework is formed by trabeculae, which consist of reticular connective tissue, smooth muscle cells, and blood vessels (at the same time, the spleen contains only efferent lymphatic vessels). the reticular connective tissue includes both red and white pulp. the white pulp is a lymphoid tissue that produces immune and blood cells. the red pulp functions to filter blood for antigens, microorganisms, and defective red blood cells; therefore, it contains various blood corpuscles, including large and small lymphocytes, immature leucocytes, leucocytes at the final stage of graininess, and disintegrating erythrocytes. the spleen is also important for the development of humoral immunity and is a b-system organ. in the animals immunized with gnp-antigen conjugates, the lymphoid follicles had distinct boundaries and contained large number of densely spaced lymphoid cells (fig. c . the lymphoid tissue was well structured and was located in a compact manner. there was a clear boundary between the red and white pulp regions, and there were full of cellular elements. for comparison, we analyzed morphological changes in the spleens of the animals immunized with tgev antigen alone. the observed changes were characterized by the existence of perivascular edema (fig. d) , sometimes accompanied by proliferation and desquamation of the vessels' endothelium. moreover, the lymphoid tissue of the follicles became sparse, and the number and density of lymphoid cells decreased. in spleen tissue sections from the animals immunized with tgev antigen alone, the boundary between red and white pulp was less clear, the size and total number of follicles in the microscopic field decreased, and no germinative centers could be detected. these results point to degradative processes in the immune organs of the animals. in the control group of guinea pigs and gnps, injected with physiological saline, no morphological changes in spleen tissue were observed (fig. a, b) . gold nanoparticles are stable metallic nanoparticles which have excellent surface functional chemistry, high biocompatibility, and nil toxicity (pissuwan et al. ( ) . in addition, gold nanoparticles can be easily synthesized, giving them shapes such as spheres, rods, cubes, stars, and pyramids. this quality allows the use of gold nanoparticles for a variety of purposes, including vaccine delivery (versiani et al. ). there are a series of works devoted to the use of gold nanoparticles as carriers of antigens for pathogens such as the virus grippe (tao et al. ) , virus foot-and-mouth (chen et al. ) , tetanus toxoid (barhate et al. ) , and yersinia pestis (gregory et al. ) . in our work, we used spherical gold nanoparticles (average diameter, nm) as a platform for the antigen for tgev. the data demonstrate that immunization of animals with tgev antigen-gnps not only activates antigen-presenting cells but also increases the proliferative activity of splenic lymphoid a b c d fig. a spleen tissue section from the animals immunized gnps. hematoxylin-eosin staining; magnification, × . b spleen tissue section from the animals control group. hematoxylineosin staining; magnification, × . c spleen tissue section from the animals immunized with tgev antigen-gnps. hematoxylin-eosin staining; magnification, × . lymphoid follicles have distinct boundaries. d spleen tissue section from the animals immunized with tgev antigen alone. hematoxylineosin staining; magnification, × . perivascular edema and sparse lymphoid tissue in the follicles can be observed fig. concentration of il- in animals immunized with gnpantigen conjugates (tgev antigen р = . ; gnp-antigen conjugate р = . ; gnps р = . ) (antibody-forming) cells. the contents of γ-ifn, il- β, and il- in animals immunized with gnp-antigen conjugates were higher than those in the intact animals or in animals given the antigen alone. the increased concentration of il- β in the immunized animals directly correlated with the activity of macrophages and stimulated b cells, which produce this cytokine when activated. the increased concentration of il- indicates that the injected preparations are stimulatory to cellular immunity. several reports have described the development and use of nanostructures for stimulating cellular immunity. for example, xu et al. ( ) showed that gold nanorods associated with the dna vaccine may cause increased formation of γ-ifn in hiv-infected mice. assis et al. ( ) showed that gold nanorods, functionalized with cysteamine and associated with the protein rsm , are stimulatory to il- . in a work, tao et al. ( ) showed that the use of nanostructured vaccine led to an increase in the concentration of igg a, iga, and il- a, il- , il- , tnf-и rantes. our research correlates with these studies but in the present work we used gold nanoparticles for the first time as a platform for antigen for tgev. also, we did nonspecific adsorption of the antigen and did not perform a chemical surface functionalization. this made it possible to simplify the conjugation process, as well as to remove additional procedures related to the purification of the drug from toxic components. additionally we analyzed the morphological changes in the spleens of the animals immunized with gnp-antigen conjugates and tgev antigen. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction (galaktionov, ) . the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. in conclusion, immunization with tgev antigen conjugated to gnps as a carrier activates the respiratory activity of lymphoid cells and peritoneal macrophages, which is directly related to their transforming activity and to activation of antibody generation. furthermore, the use of this conjugate allows marked improvement of the structure of the animals' immune organs and restores the morphological-functional state of these organs. the results of this study can be used in the further development of nanovaccines against swine transmissible gastroenteritis. the microanatomical changes (increased number of follicles) indicate the activation of the b-dependent zone of the spleen and, consequently, the development of a humoral-type immunological reaction (galaktionov, ) . the degradative processes observed in the animals immunized with tgev antigen alone are evidence of weak resistance to pathogen attack. funding information this study was funded by the russian science foundation (project no. - - ). conflict of interest the authors declare that they have no conflict of interest. ethical statement all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. the use of gold nanorods as a new vaccine platform against schistosomiasis quillajasaponaria extract as mucosal adjuvant with chitosan functionalized gold nanoparticles for mucosal vaccine delivery: stability and immunoefficiency studies the role of plasma membrane in bioreduction of two tetrazolium salts, mtt, and ctc biological properties of bnakedm etal nanoparticles assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide investigation on the porcine epidemic diarrhea prevalent on qinhai antigenic structure of transmissible gastroenteritis virus. ii. domains in the peplomer glycoprotein the golden age: gold nanoparticles for biomedicine gold nanoparticles in biomedical applications: recent advances and perspectives adjuvant properties of gold nanoparticles gold nanoparticles as an antigen carrier and an adjuvant. nova science publishers gold nanoparticles: synthesis, properties, biomedical applications inactivation of a non-enveloped rna virus by artificial ribonucleases: honey bees controlled nucleation for the regulation of the particle size in monodisperse gold suspensions conjugation of y. pestis f -antigen to gold nanoparticles improves immunogenicity diagnostic molecular pathology: a practical approach antigenic relationships among homologous structural polypeptides of porcine, feline, and canine coronaviruses optical properties and biomedical applications of plasmonic nanoparticles the nod mouse: a model for insulin dependent diabetes mellitus //current protocols in immunology diagnosis to detect porcine transmissible gastroenteritis virus (tgev) by optical and transmission electron microscopy techniques surface functionalization of nanoparticles for nanomedicine clinical evaluation of transmissible gastroenteritis virus vaccines and vaccination procedures for inducing lactogenic immunity in sows prospects for gold nanorodparticles in diagnostic and therapeutic applications usage of phage mini-antibodies as means of detecting ferritin concentration in animal blood disease of swine proteolytic cleavage of the e glyco protein of murine coronavirus by trypsin and separation of two different k cleavage fragments gold nanoparticle-m e conjugate coformulated with cpg induces protective immunity against influenza a virus consensus m e peptide conjugated to gold nanoparticles confers protection against h n , h n and h n influenza a viruses gold nanoparticles and their applications in biomedicine genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus surface-engineered gold nanorods: promising dna vaccine adjuvant for hiv- treatment transmissible gastroenteritis virus n protein causes endoplasmic reticulum stress, upregulates interleukin- expression and its subcellular localization in the porcine intestinal epithelial cell key: cord- - vp fwv authors: simonsen, lone; higgs, elizabeth; taylor, robert j; wentworth, deborah; cozzi-lepri, al; pett, sarah; dwyer, dominic e; davey, richard; lynfield, ruth; losso, marcelo; morales, kathleen; glesby, marshall j; weckx, jozef; carey, dianne; lane, cliff; lundgren, jens title: using clinical research networks to assess severity of an emerging influenza pandemic date: - - journal: clinical infectious diseases doi: . /cid/ciy sha: doc_id: cord_uid: vp fwv background: early clinical severity assessments during the influenza a h n pandemic (ph n ) overestimated clinical severity due to selection bias and other factors. we retrospectively investigated how to use data from the international network for strategic initiatives in global hiv trials, a global clinical influenza research network, to make more accurate case fatality ratio (cfr) estimates early in a future pandemic, an essential part of pandemic response. methods: we estimated the cfr of medically attended influenza (cfr(ma)) as the product of probability of hospitalization given confirmed outpatient influenza and the probability of death given hospitalization with confirmed influenza for the pandemic ( – ) and post-pandemic ( – ) periods. we used literature survey results on health-seeking behavior to convert that estimate to cfr among all infected persons (cfr(ar)). results: during the pandemic period, . % ( . %– . %) of ph n -positive outpatients were hospitalized. of ph n -positive inpatients, . % ( . %– . %) died. cfr(ma) for ph n was . % ( . %– . %) in the pandemic period – but declined -fold in young adults during the post-pandemic period compared to the level of seasonal influenza in the post-pandemic period – . cfr for influenza-negative patients did not change over time. we estimated the pandemic cfr(ar) to be . %, -fold lower than cfr(ma). conclusions: data from a clinical research network yielded accurate pandemic severity estimates, including increased severity among younger people. going forward, clinical research networks with a global presence and standardized protocols would substantially aid rapid assessment of clinical severity. clinical trials registration: nct and nct . in , uncertainty about the emerging ph n virus' clinical severity hindered the early global response. although the rapid spread of the virus around the world fulfilled the traditional pandemic definition, its global mortality impact in the end proved to be smaller than any th century pandemic [ , ] . however, its relative mildness was not known in the early months of the outbreak. the earliest estimate of the case fatality ratio (cfr) was on par with the rating for the catastrophic pandemic, and a june assessment put it in the pandemic range (table ) [ ] . an evaluation of the pandemic response ordered by the world health organization's (who) director general [ ] found that a systematic way to assess both transmissibility and clinical severity-also known as its "seriousness" [ ] -is needed in the early phase of a future pandemic to assess the level of threat accurately and to mobilize resources appropriately. cfr is one important measure of clinical severity; others include the risk of admission to the intensive care unit (icu) and the need for mechanical respiratory support. a who task force is currently developing the data inputs and study designs needed to generate timely estimates of clinical severity [ ] . the centers for disease control and prevention has proposed a scheme for comparing pandemic and seasonal influenza graphically, plotting attack rates against clinical severity [ ] . in , uk public health england spearheaded what has become a standard first-line approach to assessing the clinical severity of a pandemic, known as the "first few hundred" (ff ) [ ] . these and similar studies gather data on the earliest cases that come to medical attention through outpatient facilities and hospitals and provide important descriptive data about symptoms, risk factors, and risk of progression to severe illness or death [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these data can in turn be combined with other data on population attack rates to forecast national and global hospitalization and mortality estimates using a pyramid modeling strategy [ , ] . standard ff studies, however, lack historic controls in the form of a baseline from recent seasonal influenza seasons. they are also subject to selection bias, as the first cases that come to attention are likely to be more severe [ ] . unless an ff study is set in an existing surveillance system or ongoing clinical research data collection scheme, there is no obvious seasonal influenza baseline against which to compare the clinical severity of the pandemic virus. moreover, unless the pandemic is severe, an ff study in the outpatient setting alone will not have the statistical power to accurately estimate the cfr unless many thousands of patients are enrolled. global clinical research networks that study mild and severely ill influenza patients could be used to overcome many of these problems. two ongoing clinical cohort studies of influenza are conducted under the international network for strategic initiatives in global hiv trials (insight) umbrella, sponsored by the national institutes of health. since , insight has undertaken cohort studies- outpatient (flu ) and inpatient (flu )-specifically to address gaps in clinical research on the emerging influenza pandemic, including factors linked to disease progression and severe outcomes [ ] . insight annually enrolls hundreds of patients with suspected or confirmed influenza, with intake sites in countries. at these sites, experienced teams use a standardized protocol to collect extensive clinical data, perform long-term follow-up (at and days for inpatients, days for outpatients), and bank patient samples for further study. several articles on influenza have been published using insight data, including protocol descriptions and preliminary data [ ] , an exploration of biomarkers of influenza case severity [ ] , patient outcomes after ph n infection [ ] , and phylogeography of the ph n virus [ ] . we used insight data collected in the pandemic period ( - ) to retrospectively demonstrate how clinical research networks can provide essential early insights into pandemic clinical severity and other epidemiological parameters. to "leverage" the cfr computation, we multiplied the conditional probability of progression from outpatient to hospitalization by that of progression from hospitalization to death. to underscore the importance of having baseline data, we compared the estimated ph n clinical severity to that of seasonal influenza types and subtypes and noninfluenza respiratory patients in the post-pandemic period ( ) ( ) ( ) ( ) . our cfr estimates were in reasonable agreement with final global cfr estimates based on excess mortality estimates from time series of nationwide vital statistics data and seroepidemiology data-final estimates of a type that would only be available several years after the next pandemic emerges [ , , ] . here, we discuss what it would take to move a clinical research network like insight from routine research operation into emergency mode to generate timely and robust clinical severity assessments. the national institute of allergy and infectious diseases (niaid)-funded insight network initially focused solely on hiv but expanded first to include ph n and then all influenza types and subtypes and emerging respiratory pathogens such as middle east respiratory syndrome and severe acute respiratory syndrome. sites, located in of world regions (figure ), consecutively enroll adult patients aged ≥ years with suspected influenza. flu recruits patients who present at a physician's office or clinic with influenza-like illness (ili), defined as fever with either cough or sore throat. flu recruits patients with known or suspected influenza who require hospitalization. at enrollment, patient medical history and demographic information are recorded, and blood and oropharyngeal swabs are analyzed and stored. testing for influenza is done both locally and at an insight central laboratory. all patients are followed up, regardless of influenza test result, at days after enrollment in flu and at and days in flu . we extracted insight data on demographics, illness onset, medical history, and vital status at follow-up visit from the protocol databases. we defined the pandemic period as the to follow-up were treated as missing and removed from the analysis. we identified relevant case series in the literature reporting data on patients aged > years. after excluding studies with fewer than patients or with a specialty population (such as high-risk patients), we chose outpatient studies, set in the united states [ ] and in the united kingdom [ ] , and inpatient studies [ , ] , both set in the united states, for comparison with flu and flu ph n laboratory-confirmed patients during the pandemic period (table ) . we calculated the medically attended cfr (cfr ma ) from the probability that a medically attended ili (flu ) patient would progress to hospitalization by day and the probability that a hospitalized (flu ) patient would die by day : where h = hospitalization and d = death to estimate cfr among all infected persons (cfr ar ), we used findings from a uk health behavior survey that found that % of patients aged ≥ years with ili sought care for their illness [ ] and a uk serology study that found that % of influenza-infected adults aged - years were symptomatic [ ] . assuming that the nonmedically attended and asymptomatic influenza cases would not progress to severe illness, we have: , where "infection" is defined as a person who responded immunologically. the % confidence intervals (cis) on the cfr estimate were generated from the variance of the product of the proportions, p(h/ili) × p(d/h), using the delta method or a first-order taylor series expansion. we assumed the proportions were independent. in small samples with large variability, this may not be a good approximation. in some cases, negative values for the cis may be obtained. data analysis was done using sas, version . , and excel. the flu and flu protocols were approved by the institutional review boards or institutional ethics committees at the university of minnesota and at each of the participating clinical sites. all patients (or their proxies) gave signed informed consent prior to enrollment. during the pandemic period (october through september ), ili and hospitalized patients tested influenza ph n positive. of these, . % of ph n -infected flu outpatients were aged - years compared to only % of the flu inpatients. during the post-pandemic period (october through september ), ili and hospitalized patients were ph n positive; of these, % of ili outpatients and % of hospitalized patients were aged - years. in the pandemic period about / of outpatients and / of inpatients were from european sites, while during the post-pandemic period, after the network expanded to sites in world regions, these figures were / of outpatients and / of inpatients. we found that demographic and clinical characteristics of insight pandemic period ph n patients were similar to those described in published ff -like studies of adult ph n patients [ ] with respect to mean age, prevalence of symptoms and underlying diseases, mortality rates, and other characteristics ( table ) . five percent of ph n -confirmed ili patients were hospitalized, and . % of ph n -positive inpatients died (table , figure ). this yielded a ph n cfr ma of . % ( . %- . %) both for all adults and for adults aged - years. the cfr ma for patients aged ≥ years could not be established with confidence due to the small number of older outpatients in the study. as a nonhistoric control, the all-ages cfr ma of influenza testnegative patients was . % during the pandemic period, albeit with wide cis. it was not possible to establish a seasonal influenza comparison for the pandemic period because non-ph n influenza cases (h n , b) in the pandemic period were rare. the cfr ma for ph n cases in the post-pandemic period was . % for patients aged - years, -fold lower than the value for the pandemic period and comparable to the influenza-negative patients of the same age. we could not reliably assess ph n cfr ma for the ≥ years age group due to small numbers in the post-pandemic period; however, cfr ma was . % for seniors aged ≥ years positive for any influenza virus in the post-pandemic period vs . % for younger adults positive for any influenza virus. for the post-pandemic period (any subtype), we also estimated the conditional probabilities and the cfr ma by region (table ) . because the final who cfr estimate from the pandemic was based on attack rates as revealed by serology data, we sought to convert our medically attended cfr to one based on the attack rate. to do so, we used data from a study that indicated that approximately % of all cases are asymptomatic [ ] and from survey data that indicate that approximately % of adult ili cases sought medical attention [ ] . we found the cfr ar to be . % ( . %- . %; table ), or -fold lower than the cfr ma . who has recently expanded its pandemic definition to include clinical severity. this means that rapid and accurate estimates of pandemic clinical severity are needed to characterize the threat level and guide the global response. our analysis combining data from inpatient and outpatient insight cohorts demonstrates how preestablished global research networks could immediately begin rigorous studies to estimate the cfr, a key parameter of clinical severity of an emerging pandemic. assessments of the clinical severity in the pandemic became less dire as time passed [ ] . the earliest estimate of cfr, an ff -like case series of hospitalized patients in mexico, was a disturbing % of influenza-positive patients. however, as studies of the first (summer) wave in the united states, the complete southern hemisphere season in new zealand, and further studies from mexico were completed, it became clear that the pandemic would be relatively mild (table ) . several factors contributed to the early confusion in . the most important was probably selection bias toward sicker patients in the earliest ff -type case series studies [ ] . another factor was simply that studies reported on different types of cfr-either as a proportion of medically attended cases (cfr ma ) or as a proportion of all infected individuals (cfr ar ). most early assessments were of the cfr ma type, but these were not directly comparable. our method, retroactively applied to insight databases, yielded a cfr ma estimate of . %. using literature values that indicated that the probability of symptomatic people seeking medical treatment was % [ ] and that the probability of infected individuals being asymptomatic was also % [ ] , our cfr ma value would be equivalent to a cfr ar of . %, which data are for the pandemic and post-pandemic periods, computed as the product of the risk of flu influenza-like illness outpatients getting hospitalized and the flu hospitalized patients having died at day . abbreviations: p (d|h), probability of death given hospitalization; p (h|ili) , probability of hospitalization given influenza-like illness. *case fatality rate not calculated when fewer than outpatients or inpatients contained in any stratum. is in reasonable agreement with the final global who cfr ar estimate of . % [ , , ] . in addition to an absolute measurement of cfr, data from previous seasons can provide a relative comparison of pandemic to seasonal influenza severity; even if the absolute estimate of cfr is uncertain, it would be useful to know if an emerging pandemic has a cfr far higher than previous seasonal influenza experiences. thus, we also estimated cfrs for influenza patients from seasonal influenza epidemics - , as a surrogate for pre-pandemic baseline seasons. age greatly influences both seasonal and pandemic clinical severity estimates. in all influenza pandemics since , mortality was higher than normal in younger people and lower than normal in seniors, sometimes dramatically so [ ] . in the post-pandemic period ( - ) we found that the cfr ma of ph n for patients aged - years had fallen -fold from the pandemic period value, becoming similar to that of a/h n and b. this suggests that the emerging virus had settled into a seasonal epidemic pattern due to accumulated population immunity. moreover, in the post-pandemic period patients aged ≥ years with any influenza virus had a cfr ma approximately -fold higher than patients aged < years. these results corroborate a previous metaanalysis of ff studies that concluded that age is an important confounder of cfr estimates for ph n pandemic influenza [ ] . they also show how important it is to take into account both the age group and the type of cfr being calculated when comparing across regions and time. it is also possible that discrepancies in early assessments of cfr may in fact have reflected true geographical differences. for example, a comprehensive study of pandemic mortality that applied a uniform methodology to different regions found the mortality impact in central and south american countries table was approximately -fold higher than in europe [ ] . this indicates that early reports of higher severity in mexico than in new zealand may not solely have been the result of ascertainment bias. clinical severity can even increase substantially over time, as was seen in the influenza pandemic when a milder summer wave preceded the severe autumn waves [ ] . the best way around the measurement problems that occur early in a pandemic would be to compute the same type of cfr with the same protocol in multiple geographical settings. if possible, estimates should be stratified by risk factors, such as pregnancy and chronic illness, and baseline data should be collected during seasonal epidemics. while some countries have created ff protocols since the pandemic, a global standard along the lines we have outlined here would be helpful. we recognize limitations to our approach to computing cfr by multiplying conditional probabilities of disease progression. first, we used distinct groups of outpatients and inpatients who were recruited under different circumstances at different sites, often in different countries. it is therefore possible the cohorts differed in age composition, health status, or other important respects that could bias the result. however, we argue that the approach, while not ideal, would nonetheless supply timely and useful data, especially if it could be compared to baseline seasons. we also note that the characteristics of the insight ph n outpatients and inpatients in the pandemic period - are reassuringly similar in terms of age, symptoms, comorbidities, and outcomes to published uk and us ff studies of adult ph n influenza outpatients and inpatients ( table ) . a second possible caveat-that insight inclusion criteria might have varied over time and explained the drop in cfr ma over time-could be dismissed on the grounds that the influenza-negative patients did not have a significant drop in cfr ma between the pandemic and post-pandemic period. this means that the measured decrease in ph n clinical severity was real and not due to ascertainment or other bias. our retrospective analysis of pandemic clinical severity indicates that it is possible to use research networks to assess both the absolute magnitude of the clinical severity of a future pandemic and the relative increase compared to a seasonal influenza baseline. even if the seroepidemiology and health-seeking behavior surveys needed to convert cfr ma to cfr ar could not be done rapidly, comparison of cfr ma to previous seasons would reveal much about the relative magnitude of the emerging threat. to be useful in a prospective scenario, however, it would be necessary to ramp up the network's pace of operations from routine to emergency mode. for insight, that would mean, at a minimum, enhancing enrollment in sites located in areas initially affected by the emerging pandemic and increasing the tempo of laboratory processing of specimens and data analysis. in addition to assessing clinical severity, global research networks could play other key roles in pandemic response including studies of comorbidity patterns, risk factors, hospital and icu utilization, and mortality risk of hospitalized patients. moreover, protocols that enroll children could be used to understand the pathogen in this key age group. once a future pandemic outbreak begins, studies set in these networks could both characterize pathophysiology to optimize clinical management and provide a platform for rigorous clinical trials of new therapeutics. we suggest, therefore, that a specific role for clinical research networks carrying out ongoing rigorous research compliant with international standards be added to the international health regulations that govern international and national responsibilities for public health emergencies of international concern. notes acknowledgments. we thank and acknowledge all the patients who participated in this study. disclaimer. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. potential conflicts of interest. l. s. and r. j. t. report earning consulting fees from sage analytica, llc, during the conduct of the study. r. l. reports serving as co-editor on a book on infectious disease surveillance published by wiley blackwell, with royalties donated to the minnesota department of health. all remaining authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. global mortality estimates for the influenza pandemic from the glamor project: a modeling study estimated global mortality associated with the first months of pandemic influenza a h n virus circulation: a modelling study case fatality risk of influenza a (h n pdm ): a systematic review report of the review committee on the functioning of the international health regulations ( ) in relation to pandemic (h n ) infection fatality risk of the pandemic a(h n ) virus in hong kong novel framework for assessing epidemiologic effects of influenza epidemics and pandemics pandemic (h n ) influenza in the uk: clinical and epidemiological findings from the first few hundred (ff ) cases human infection with new influenza a (h n ) virus: clinical observations from mexico and other affected countries pandemic potential of a strain of influenza a (h n ): early findings epidemiologic analysis of the laboratory-confirmed cases of influenza a(h n )v in colombia pandemic influenza a(h n )v in new zealand: the experience from the severity of pandemic h n influenza in the united states estimating age-specific cumulative incidence for the influenza pandemic: a meta-analysis of a(h n ) pdm serological studies from countries. influenza other respir viruses pandemic influenza a (h n ) in saudi arabia: description of the first one hundred cases critically ill patients with influenza a(h n ) in mexico insight flu : an anti-influenza virus hyperimmune intravenous immunoglobulin pilot study hospitalized patients with h n influenza in the united states systematic review of clinical and epidemiological features of the pandemic influenza a (h n ) factors associated with death or hospitalization due to pandemic influenza a (h n ) infection in california utility of the first few approach during the influenza a(h n ) pandemic in the netherlands optimizing the precision of case fatality ratio estimates under the surveillance pyramid approach potential biases in estimating absolute and relative case-fatality risks during outbreaks surveillance of illness associated with pandemic (h n ) virus infection among adults using a global clinical site network approach: the insight flu and flu studies the association between serum biomarkers and disease outcome in influenza a(h n )pdm virus infection: results of two international observational cohort studies outcomes of influenza a(h n )pdm virus infection: results from two international cohort studies extensive geographical mixing of human h n influenza a virus in a single university community emergence of a novel swine-origin influenza a (h n ) virus in humans using an online survey of healthcare-seeking behaviour to estimate the magnitude and severity of the h n v influenza epidemic in england comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study preliminary estimates of mortality and years of life lost associated with the a/h n pandemic in the us and comparison with past influenza seasons epidemiologic characterization of the influenza pandemic summer wave in copenhagen: implications for pandemic control strategies flu clinical site investigators: argentina: laura barcan key: cord- -zvwy bgt authors: chapman, christie; delahanty, kim title: convergencia mundial de las enfermedades infecciosas emergentes: a tan solo un viaje en avión de distancia date: - - journal: nan doi: . /j.nursi. . . sha: doc_id: cord_uid: zvwy bgt nan el de abril de , un macaco de la india, utilizado con fines de investigación en un estudio sobre la fiebre amarilla en la selva zika de uganda, enfermó con fiebre de , °c. los científicos tomaron muestras de sangre y se le realizaron análisis. el macacus rhesus n.º estaba aquejado de algo desconocido. con el tiempo, el virus emergente se bautizaría así por el lugar donde se descubrió por primera vez. a principios de , los científicos aislaron también el virus del zika en mosquitos aedes africanus capturados en la misma selva. a pesar de que el virus del zika despertaría una atención científica esporádica y limitada durante las siguientes décadas, había surgido una nueva amenaza microbiana para la salud humana . aunque hoy día estamos familiarizados con la neumonía atípica asiática (o síndrome respiratorio agudo grave [sars, severe acute respiratory syndrome]), la enfermedad del Ébola y la infección por el virus de la inmunodeficiencia humana (vih), no hace mucho eran consideradas enfermedades infecciosas emergentes. según los centers for disease control and prevention (cdc), una enfermedad infecciosa emergente es aquella cuya incidencia ha aumentado en los últimos años y podría aumentar en un futuro próximo . curiosamente, en la bibliografía se informa de que el % de las enfermedades que en la actualidad se consideran "emergentes" son zoonóticas, lo que significa que se originaron en animales . ningún factor por sí solo desencadena la aparición de enfermedades. los seres humanos y los microbios se hallan en el centro de un conjunto complejo de elementos en continua evolución, los cuales originan esta tormenta perfecta. el modelo de convergencia elaborado por el institute of medicine es una forma útil de visualizar la aparición de enfermedades (v. el cuadro visualización de una enfermedad emergente según el modelo de convergencia). esta ilustración muestra cómo varios factores en continua evolución influyen en la interacción entre el ser humano y los microbios. de acuerdo con este modelo, la aparición de una amenaza microbiana para la salud del ser humano se origina a partir de la convergencia de cuatro ámbitos: factores genéticos y biológicos. factores medioambientales físicos factores ecológicos. factores sociales, políticos y económicos. en la intersección entre el ser humano y los microbios hay un cuadrado que representa la convergencia de los factores que conducen a la aparición de una enfermedad infecciosa. el interior de esta caja es un gradiente en movimiento hacia el interior que pasa del color blanco al negro. los bordes exteriores blancos representan lo que sabemos acerca de los factores en la aparición. el centro negro representa lo desconocido. al igual que ocurre con la búsqueda de la "caja negra" de un avión después de un accidente para tratar de entender por qué se produjo, los investigadores trabajan continuamente para arrojar luz en la "caja negra" de la aparición de nuevas enfermedades. este conocimiento podría ayudar a evitar la próxima amenaza microbiana global. reproducido con el permiso de national academies press, copyright , national academy of sciences. los rinovirus, causantes del resfriado común. sin embargo, de vez en cuando descubrimos un microbio patógeno que puede hacernos mucho daño o incluso puede poner en peligro nuestra existencia. a pesar de la tecnología de que se dispone, podemos identificar solo una pequeña parte de los microbios que habitan en la tierra y en la biosfera humana. todavía no entendemos muchas cosas sobre el mundo invisible de los microbios y sus efectos en el cuerpo humano. los microbios son la forma de vida más abundante y se han desarrollado durante millones de años. durante este largo tiempo de vida, se han vuelto muy hábiles en adaptarse para así evitar la aniquilación por el sistema inmunológico humano . desafortunadamente, los seres humanos les ayudan, sin saberlo, en sus esfuerzos por imponerse. hemos sido tan generosos en el uso de antibióticos que los microbios han aprendido a crear versiones resistentes de sí mismos y a propagar esta resistencia a otros microbios. mediante la creación de entornos para que estos "súper" microbios proliferen, los seres humanos les están quitando el poder a las mejores armas que tenemos en nuestro arsenal para luchar contra las peligrosas infecciones bacterianas . el acto de viajar es una fuerza poderosa en la aparición y transmisión de enfermedades. hoy día, las personas podemos viajar hasta el otro extremo de nuestro planeta en menos tiempo del que se necesita para que se manifiesten los síntomas y signos de una enfermedad en personas infectadas, las cuales, a su vez, pueden infectar a cualquier otra persona que entre en contacto con ellas durante su recorrido . por ejemplo, se ha descrito el efecto de los viajes en la transmisión de una enfermedad. en , los investigadores publicaron datos empíricos que sugerían que la propagación de la gripe en estados unidos en se retrasó debido al descenso de los viajes en avión después de los ataques del de septiembre de aquel año . las repercusiones de los desplazamientos en la transmisión del virus del zika después de los juegos olímpicos de en brasil es un ejemplo similar. el período de incubación estimado del virus del zika desde la exposición hasta la aparición de los primeros síntomas dura entre y días , . el deportista olímpico o el espectador desprevenido que viaja de vuelta de río dispone de mucho tiempo para propagar el contagio. muchas personas viajan por negocios o por placer, pero otras se ven obligadas a abandonar su hogar para escapar de la guerra, el hambre, la opresión o la pobreza. este tipo de emigración trae consigo muchos más elementos que pueden contribuir a la aparición de enfermedades infecciosas: hacinamiento, condiciones de vida y fuentes de agua insalubres, así como prácticas nocivas de obtención y preparación de alimentos . millones de personas en aldeas y campamentos de explotación forestal de África oriental y meridional carecen de suficientes animales domesticados para el consumo de carne y deben depender de la caza de animales salvajes para satisfacer esta necesidad. aproximadamente, un millón de toneladas métricas de carne de animales salvajes se consume cada año en África central. también es alarmante la entrada anual de miles de libras de carne de primates, antílopes y otros animales salvajes en estados unidos y europa como objeto de contrabando para actos culturales . su caza, preparación y consumo están vinculados con varias pandemias y epidemias, sobre todo con el vih, la enfermedad del Ébola y el sars . aunque el aumento del riesgo de transmisión de enfermedades debido al comercio internacional es un fenómeno no muy bien entendido, podría ser un peligro potencial para las personas. los cambios en la forma en que los seres humanos utilizan la tierra y el medio ambiente son motivos habituales de la aparición de enfermedades (v. el cuadro motivos de la aparición de enfermedades en los seres humanos). el desarrollo territorial para vivienda o para uso agrícola y la construcción de embalses y presas para crear fuentes de agua para uso agrícola y público en áreas geográficas previamente no desarrolladas aumentan las posibilidades de que los seres humanos entren en contacto con mosquitos, garrapatas, roedores y otros animales que pueden ser portadores de una enfermedad infecciosa . la incursión humana en estas áreas vírgenes aumenta el acceso de los seres humanos a zonas remotas e incorpora más vectores y reservorios de infección a nuevos huéspedes . en respuesta a cada amenaza de enfermedad emergente, por lo general, empleamos recursos sanitarios a nivel mundial en la búsqueda de vacunas para eludir la capacidad de estos microbios para hacer daño a gran escala. las vacunas han evitado un número aproximado de millones de muertes solo en estados unidos y continúan salvando , millones de vidas en todo el mundo cada año . un avance reciente y apasionante en el desarrollo de vacunas es la finalización de la fase de los ensayos clínicos de la rts,s/as , una vacuna contra la malaria que se experimentó en los países subsaharianos, donde el número de muertes por malaria en apenas pasó de . . el trabajo va avanzando rápidamente en vacunas para otras amenazas que se plantean actualmente, como la enfermedad del Ébola, el dengue y el zika. las vacunas son un arma potente contra la amenaza de una enfermedad conocida, pero ¿no sería maravilloso que pudiéramos contar con la tecnología necesaria para localizar y prevenir estas enfermedades emergentes antes que tuvieran la oportunidad de causar estragos? esto todavía no es realidad, pero grupos como el global disease detection program (gdd) de los cdc y la global outbreak alert and response network de la oms están vigilando sin descanso la evolución de la interacción entre microbios y seres humanos en todo el planeta, creando tecnología de seguimiento y respondiendo a los informes sobre el aumento de enfermedades y otras emergencias sanitarias . cada uno de nosotros puede colaborar para disminuir la amenaza de las enfermedades infecciosas saliendo de nuestro refugio doméstico y adoptando una mentalidad global. tenemos que ver la realidad de nuestra interrelación y ser conscientes de que lo que está sucediendo en otras partes de nuestro mundo puede encontrar la manera de que ocurra en nuestra ciudad. podemos hacernos menos vulnerables a las amenazas de enfermedades (conocidas y desconocidas) mediante la comprensión de los hechos básicos acerca de cómo se transmiten las enfermedades, la importancia de la higiene de las manos y de mantenerse al día de las vacunas que corresponden a cada edad. las enfermedades infecciosas emergentes son una amenaza para todos y cada uno de los habitantes del planeta y todo el mundo puede desempeñar un papel, ya sea en el retraso de la transmisión o la prevención. después de todo, la próxima enfermedad emergente podría estar a tan solo un viaje en avión de distancia. ■ bibliografÍa zika virus outside africa what we do microbial threats to health: emergence, detection, and response. institute of medicine of the national academies about antimicrobial resistance empirical evidence for the effect of airline travel on interregional influenza spread in the united states zika virus: symptoms, testing and treatment zika virus infection: an overview tourism and the health effects of infectious diseases targeting transmission pathways for emerging zoonotic disease surveillance and control. vector borne zoonotic dis breakthroughs in bioscience: vaccinesessential weapons in the fight against disease world health organization. malaria vaccine development the viral storm christie chapman es especialista en prevención de infecciones y kim delahanty es directora administrativa de epidemiología clínica de prevención de infecciones las autoras declaran no tener ningún conflicto de intereses económicos relacionado con este artículo key: cord- - r hg ld authors: pawliw, rebecca; farrow, rebecca; sekuloski, silvana; jennings, helen; healer, julie; phuong, thuan; sathe, pri; pasay, cielo; evans, krystal; cowman, alan f.; schofield, louis; chen, nanhua; mccarthy, james; trenholme, katharine title: a bioreactor system for the manufacture of a genetically modified plasmodium falciparum blood stage malaria cell bank for use in a clinical trial date: - - journal: malar j doi: . /s - - -x sha: doc_id: cord_uid: r hg ld background: although the use of induced blood stage malaria infection has proven to be a valuable tool for testing the efficacy of vaccines and drugs against plasmodium falciparum, a limiting factor has been the availability of good manufacturing practice (gmp)—compliant defined p. falciparum strains for in vivo use. the aim of this study was to develop a cost-effective method for the large-scale production of p. falciparum cell banks suitable for use in clinical trials. methods: genetically-attenuated parasites (gap) were produced by targeted deletion of the gene encoding the knob associated histidine rich protein (kahrp) from p. falciparum strain d . a gap master cell bank (mcb) was manufactured by culturing parasites in an fda approved single use, closed system sterile plastic bioreactor. all components used to manufacture the mcb were screened to comply with standards appropriate for in vivo use. the cryopreserved mcb was subjected to extensive testing to ensure gmp compliance for a phase investigational product. results: two hundred vials of the gap mcb were successfully manufactured. at harvest, the gap mcb had a parasitaemia of . %, with % of parasites at ring stage. testing confirmed that all release criteria were met (sterility, absence of viral contaminants and endotoxins, parasite viability following cryopreservation, identity and anti-malarial drug sensitivity of parasites). conclusion: large-scale in vitro culture of p. falciparum parasites using a wave bioreactor can be achieved under gmp-compliant conditions. this provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials. the use of controlled human malaria infection (chmi) for testing the efficacy of vaccines and drugs against plasmodium falciparum is now well established. induced blood stage malaria (ibsm) infection was pioneered in the s [ ] [ ] [ ] , and was subsequently refined [ ] [ ] [ ] [ ] . a limiting factor in the ibsm system has been the restricted number of defined p. falciparum strains available. this is in part due to difficulties obtaining suitable material for ibsm studies. until recently it has been necessary to rely on access to deliberately infected volunteers or malariainfected travellers returning from overseas. in both cases, ethical approval is required to collect, store and use this material and there are concerns about contaminating infectious agents. a process for culturing sufficient volumes of p. falciparum in tissue culture flasks for in vitro production of good manufacturing practice (gmp) grade blood stage malaria cell banks was recently described [ ] . this provides a culture-based alternative to sourcing parasites from individuals who have either experimental or natural infection (travellers returning from malaria endemic areas). this allows more flexibility in that new banks can be created as required, and not only when a suitable donor becomes available. it also allows for tight control over all components used in the cell bank manufacture and ensures that cell banks are renewable resources. however, bulk preparation of p. falciparum cultures is both time consuming and technically demanding; yields can be low and parasite quality can be variable when grown in static culture. multiply-infected erythrocytes (containing two or more parasites) are also observed more frequently than in suspension cultures [ ] . the presence of erythrocytes containing multiple parasites is undesirable for ibsm studies where the parasite dose should be carefully controlled. dalton et al. have developed a method for production of large scale suspension cultures of asexual and sexual blood stage p. falciparum using a wave bioreactor (wave bioreactor ™ / eht system) [ , ] . growth of p. falciparum in the wave bioreactor system is superior to static flask cultures because parasites retain synchrony over at least cycles of invasion. additionally, the development of multiply infected erythrocytes is reduced; this was shown to be both consistent and reproducible [ , ] . disposable plastic bioreactors also allow for careful control and monitoring of the cellular environment, and have been widely used for the culture of plant, animal and microbial cells, and for the production of proteins, antibodies and other biologics conforming to gmp standards [ ] . here, the manufacture and release of a cell bank that complies with the gmp requirements for a phase investigational product is described. the cell bank was manufactured using a wave bioreactor and consists of erythrocytes infected with genetically modified p. falciparum parasites. this cost-effective methodology using appropriate gmp compliant conditions allows for the production of sufficient quantities of material for ibsm studies and whole blood-stage vaccine trials, with sufficient material available for quality control and ongoing stability testing. the manufacture of the master cell bank was conducted in accordance with the united states food and drug administration (fda) guidance for industry: cgmp for phase investigational drugs. genetically attenuated parasites (gap) were produced by targeted deletion of the gene encoding the knobassociated histidine-rich protein (kahrp) from p. falciparum strain d , in a similar manner as previously described for this and other genes [ , ] . briefly, deletion of kahrp was achieved by a positive-negative selection strategy [ ] whereby the entire kahrp gene was replaced through double cross-over homologous recombination with a cassette encoding human dihydrofolate reductase (dhfr), a selectable marker that renders transfectants resistant to wr (jacobus pharmaceuticals, nj). the loxp-dhfr-loxp-pcc plasmid was created by insertion of a loxp-hdhfr-loxp cassette into the pcc plasmid backbone [ ] , as previously described [ ] . the kahrp-ko plasmid was created by cloning kahrp ′ and ′ sequences into this plasmid. plasmid dna was transfected into p. falciparum d by electroporation. following wr positive selection, parasites were grown without wr to deselect those containing the wr-containing plasmid, thereafter wr was reapplied to select those with integration of the dhfr cassette into the genome. these were then placed under negative selection with -fluorocytosine ( -fc; sigma) to select for homologous recombination. -fc resistant parasites were genotyped by pcr. kahrp-ko parasites were cloned by limiting dilution in -well flat bottom plates and the resulting clonal line, d -kahrp-ko, was characterized by whole genome sequencing (accession number prjeb ). the p. falciparum d parasites used for transfection with the kahrp knockout vector were created from the d parental strain described previously [ ] . this strain was used for the original ibsm studies [ ] and has been extensively used for subsequent studies. the gap seed bank was produced by thawing the contents of one vial of the gap pre-seed bank following a standard protocol [ ] and expanding the parasites in culture (rpmi media supplemented with % heat-treated pooled human serum and % human erythrocytes) using standard methods [ ] . at each passage, fresh complete media and erythrocytes were added and the culture was expanded to achieve the required volume for cell banking. when the culture contained a minimum of % ring-stage parasites at - % parasitaemia, the seed bank was cryopreserved using glycerolyte (fenwal inc.) in a : ratio, aliquoted into ml cryovials, and stored in vapour phase liquid nitrogen. the final gap seed bank was transferred to a therapeutic goods administration (tga) licensed gmp facility (q-gen; qimr berghofer medical research institute [qimrb]) for further expansion and production of the gap master cell bank. the d kahrp-ko gap master cell bank (gap mcb) was manufactured in a iso class biological safety cabinet within an iso class clean room using tissue culture flasks and a bioreactor (ge wave ™ system). the gap mcb dossier was reviewed by the united states food and drug administration (fda) under the investigational new drug (ind) program. a summary of the process is shown in fig. and described in detail below. a vial of the d kahrp-ko gap seed bank was thawed at °c with the stepwise addition of and . % sodium chloride following a standard protocol [ ] . the resulting red blood cell (rbc) pellet was washed in . % sodium chloride and resuspended in ml of complete rpmi-hepes media (life technologies) containing µl of % haematocrit (hct) washed rbcs, and the cell suspension was transferred to a t cm flask. parasites were cultured in flasks under standard malaria culture conditions [ ] in an atmosphere of % carbon dioxide, % oxygen and % nitrogen at °c and media was changed every h. parasitaemia was monitored by examination of giemsa-stained thin blood films. additional media and fresh rbcs were added as required to maintain the culture at a parasitaemia of between . and %, and with a - % hct. the parasite culture was expanded to a volume of approximately ml and the parasites were synchronized by a single treatment with % sorbitol [ ] prior to transfer to a l cellbag of the wave ™ bioreactor. the ready to process wave ™ system (ge healthcare) was used for this study and configuration of the equipment is shown in fig. . the system consists of a pre-sterile single use cultivation container (cellbag) made of fda approved plastic. each cellbag has builtin inlet and outlet air filters, and ports that allow the addition of culture medium and extraction of samples (fig. a) . the cellbag is placed on a temperature-controlled rocking platform (fig. b) ; gas transfer and mixing of the cell culture is facilitated by movement of the rocker unit which induces a wave motion in the culture. temperature, rocking speed, rocking angle and atmospheric conditions (fig. c ) are monitored and controlled by the cellbag control unit (cbcu). the system is controlled from a pc running unicorn software version . . or later. the synchronous parasite culture was transferred into a l cellbag and maintained under conditions controlled electronically by the bioreactor system (fig. c ). parasitaemia was monitored by examination of giemsastained thin blood films. the culture medium was changed daily, with washed rbcs added as required to maintain a parasitaemia of . - . % and a - % hct. when the culture volume reached ml it was transferred to a l cellbag and the parasite culture was expanded to a final volume of approximately l in line with the manufacturers recommended culture volumes for and l cellbags. to facilitate media change and transfer between cellbags the cell culture was removed from the in use cellbag under sterile conditions, centrifuged in ml conical bottles (corning) and the supernatant removed. the required volume of fresh media was pumped into the destination cell bag under the control of the cbc unit and the required volume of inoculum added under gravity flow. the culture was monitored to ensure synchronicity and was maintained such that the % of ring stage parasites (as a % of the total parasites) present in the culture did not fall below %. when the required volume was reached, the culture was maintained for one further invasion cycle before parasites were harvested for banking at a parasitaemia target of > % and a minimum of % ring stage parasites. progression through the final multiplication cycle was monitored by examination of giemsa-stained thin blood films every - h. the cell culture supernatant was tested for microbial contamination prior to culture in the wave ™ bioreactor system and prior to harvesting. at harvest the entire cell culture was removed under sterile conditions and centrifuged in ml conical bottles to pellet the rbcs prior to freezing. rpmi-hepes media (life technologies) was supplemented with % heat inactivated (hi) pooled human serum and hypoxanthine (ht) supplement ( . mm sodium hypoxanthine and . mm thymidine). complete media was used immediately or dispensed in aliquots and stored at a temperature of between and °c for a maximum of days. fig. wave bioreactor configuration. a each cellbag has built-in inlet and outlet air filters, and ports that allow the addition of culture medium and extraction of samples. b the ready to process wave ™ system (ge healthcare) was used for this study. c temperature, rocking speed, rocking angle and atmospheric conditions are monitored and controlled by the cellbag control unit (cbcu) the serum used was supplied, screened and tested by key biologics (memphis usa), an fda registered blood collection and processing establishment. whole blood units were collected from donors who were screened and found acceptable for donation of transfusable blood components based on all applicable fda requirements. infectious disease testing as required for qualification of blood components for transfusion to humans was carried out and was required to be non-reactive or negative. serum from individual donors was tested (using fda approved test kits) for human immunodeficiency virus (hiv)-i and hiv-ii, human t-lymphotropic virus (htlv) types i and ii, hepatitis b virus (hbv), hepatitis c virus (hcv), trypanasoma cruzi and syphilis (by serology), and underwent nucleic acid testing for hbv, hcv, hiv, and west nile virus (wnv). serum was then pooled from the multiple donors, frozen, stored and shipped under controlled conditions to qimrb. on arrival the serum was thawed, heat inactivated at °c, filtered and stored at − °c in ml aliquots until required. the pooled serum was additionally tested for epstein barr virus, parvovirus b and herpes virus type by pcr at pathology queensland. endotoxin testing and sterility testing was performed at q-gen. leukodepleted packed rbcs (group o rh (d) negative) used for in vitro cultivation of p. falciparum parasites were obtained from the australian red cross blood service (blood service). all supplied rbcs were screened according to blood service protocols to eliminate individuals with risk of exposure to transmissible spongiform encephalopathy and blood borne agents. the blood was collected and leukodepleted at the blood service and the donor's blood sample was tested and certified nonreactive for hiv, hbv, hcv, htlv and syphilis using diagnostic kits approved by the tga. a blood sample was additionally tested by pathology queensland for infectious diseases that are not included in mandatory testing for blood and blood products including anti-flavivirus igm antibodies, trypanasoma cruzi igg antibodies, barmah forest virus igm and igg antibodies, and ross river virus igm and igg antibodies. samples were also screened (by pcr testing) for epstein barr virus (ebv), cytomegalovirus (cmv), parvovirus b , human herpes virus type and type (hhv- and hhv- ), and wnv. on receipt at qimrb the cells were transferred from the blood service pack to a sterile container and a ml aliquot was removed for sterility testing. aliquots of rbcs were washed three times in rpmi and resuspended in complete rpmi to give a hct of %. blood was washed as required in aliquots not exceeding ml and stored at - °c for up to days after washing. percentage parasitaemia and parasite life cycle stage were assessed by microscopic examination of thin blood films and when cultures achieved > % parasitaemia with at least % ring-stage parasites the cell bank was cryopreserved using glycerolyte in a : ratio (i.e. two volumes of glycerolyte were added to each volume of prbc) as previously described [ ] . the cell bank was dispensed in ml aliquots in cryovials, cryopreserved using a controlled rate freezer as previously described [ ] and stored in vapour phase liquid nitrogen at - °c. a total of vials were produced; two vials were allocated as retention samples. table . parasite viability post-freezing was determined by measurement of cloning efficiency via a limiting dilution assay [ , ] with positivity determined by production of p. falciparum histidine-rich protein ii (hrp-ii). in order to determine post-freeze viability, a vial of the gap mcb was thawed following a standard protocol [ ] and a sample was removed for rbc counting using a haemocytometer. the number of parasitized/infected rbcs (prbcs) was determined via microscopic examination of a giemsa-stained thin blood smear. dilutions of prbcs were prepared in complete media and dispensed in μl aliquots in -well flat bottom plates at % hct at theoretical concentrations of , , , , . , . , . and . parasites/well. thirty-two replicates of each concentration were plated out and non-parasitized rbcs were included as a negative control. plates were incubated in an atmosphere of % o , % co and % n at °c for days and media was replenished on day . on day , supernatant was removed from each well and processed immediately or frozen for future testing. the supernatant was tested for p. falciparum hrpii using the sd malaria antigen pf elisa kit, according to the manufacturer's instructions. briefly, supernatants from each well were transferred to the corresponding well of an uncoated microplate and incubated for min following the addition of µl . a µl sample was then transferred from each well of the uncoated microplate to the corresponding well of the pre-coated assay microplate and incubated. plates were washed and µl of tetramethylbenzidine substrate added to each well. after a min incubation, the reaction was stopped by the addition of µl of ml/l ( n) hydrochloric acid. the absorbance was read at nm with reference wavelength at nm. the mean absorbance values (optical density [od]) for the positive and negative controls were calculated in order to ensure the validity of the assay. the cut off value for parasite positivity was calculated using the absorbance values of the two wells containing the rbc negative control. the cut off value was the mean plus standard deviations and this value was applied to the test samples to calculate the number of positive and negative wells. limiting dilution analysis was performed to determine the frequency of viable parasites as previously described [ ] . anti-malarial drug sensitivity testing was carried out on the gap mcb parasites using the standard [ h]-hypoxanthine uptake inhibition assay [ ] to ensure that the drug resistance phenotype of the parasites had not changed during culture. the in vitro sensitivity of the gap mcb parasites to nine anti-malarial drugs was assessed (chloroquine, dihydroartemisinin, piperaquine, lumefantrine, amodiaquine, atovaquone, pyronaridine, mefloquine, quinine). the parental d line (chloroquine sensitive) and w (chloroquine resistant) were included as controls. the assays were performed at the army malaria institute, queensland, australia. briefly, aliquots of parasites from the gap mcb were grown in vitro and synchronized to give % ring stage parasites. the parasite culture was dispensed into a -well plate containing twofold dilutions of each antimalarial drug for testing. [ h]-hypoxanthine was added and the amount incorporated into parasites was measured after a -h incubation. the ic values for each drug were determined by nonlinear regression analysis. this calculation was performed using graphpad prism software (graphpad prism version . ). drug threshold values were expressed as mean ic ( % confidence interval) in nmol/l. the results were compared to published threshold values for drug resistance [ ] . the genotype of the gap mcb was determined by pcr using primers to confirm kahrp gene deletion and hdhfr integration. in vitro culture samples were taken at the beginning of the process (day ) and at harvest (day ) and placed in qiagen lysis buffer. genomic dna was extracted from paired samples using a qiagen dnamp dna blood mini kit and gdna from the parent strain d was used as a control. standard pcr was set up using pairs of primers ( to further validate the successful genetic attenuation of p. falciparum d and verify hdhfr integration, a real time pcr assay was set up using primers designed at various regions of the p. falciparum d gap (fig. ) . primer sequences are listed in table . to determine limit of detection of possible contamination with wildtype p. falciparum d , two single copy genes (pfmdr- and pfldh) were amplified and used as reference for copy number estimation. a standard curve containing five ten-fold serial dilutions of wildtype p. falciparum d was prepared, starting from . ng/μl to amplify kahrp excised region and mdr and ldh genes. triplicate pcr reactions were set up using the roche fast start essential dna green master mix (cat. no. ), μm of each primer ( table ) and μl of each gdna sample in a μl reaction volume. quantitative pcr was performed using a roche light cycler with the following cycling conditions: °c for min; in vitro assays for the detection of viral contaminants and inapparent viruses was also performed. a cell bank consisting of erythrocytes infected with genetically-modified p. falciparum parasites was successfully manufactured using a wave bioreactor. two hundred vials of the gap mcb were produced that met the requirements for use in phase i clinical trials. gap parasites maintained an asexual blood stage cycle of approximately h during the in vitro culture period in the bioreactor, and showed a normal maturation pattern. the percent parasitaemia and percent ring stage parasites of the culture over time is illustrated in fig. . parasitaemia was maintained between . and % by the addition of rbcs during the expansion phase (day - ). during the banking phase (day - ) the parasitaemia was maintained below %. at harvest the culture had a parasitaemia of . %, with % of parasites at ring stage (high percentage of ring stage parasites is illustrated in fig. ) . thus, the pre-defined acceptable criteria of > % parasitized rbc and ≥ % ring stage parasites (table ) were met. pcr assays confirmed kahrp gene deletion and hdhfr integration in parasite samples collected at the initiation of culture (day ) and at harvest (day ) (fig. ) demonstrating that the genotype of the parasites was maintained during the culture and banking process. amplification of the kahrp excised region on serially diluted wildtype p. falciparum d and comparing ct values obtained from serially diluted gap showed undetectable contamination of the gap starting from . ng/ μl dna concentration up to as low as . × − ng/ μl. this is equivalent to a single copy or less using single copy gene references pfmdr- and pfldh. the in vitro sensitivity of the gap mcb parasites to a panel of anti-malarial drugs is shown in table . the mean ic ( % confidence interval) results for each drug are presented in table along with the corresponding results for the control p. falciparum strains d and w . gap master cell bank parasites demonstrated sensitivity to eight of the anti-malarial drugs tested (chloroquine, artemisinin, piperaquine, lumefantrine, amodiaquine, atovaquone, pyronaridine, quinine) and resistance to mefloquine. parasite viability was tested using samples taken approximately h (time ) after freezing and at months postproduction. the gap mcb was determined to contain % viable parasites at time and % viable parasites at months post-production. these values conform to the pre-defined acceptable criteria of > % viable parasites. the variability between the two time points is within normally observed limits for this assay. our aim was to produce a gmp compliant cell bank consisting of kahrp deficient p. falciparum parasites using the wave bioreactor system. previous studies have shown that the wave bioreactor system is suitable for the production of large volumes of high quality p. falciparum cultures [ ] . we have taken this method further to demonstrate that the wave bioreactor can be used to culture parasites for use in phase clinical trials. the use of a wave bioreactor system has a number of significant advantages over traditional culture of p. falciparum in flasks. foremost is that it allows close control of culture conditions that can be monitored and maintained electronically by the wave bioreactor system; the cellbags used are single use closed systems approved by the fda. significant savings in time and labour were also achieved; ml of culture was transferred to a cellbag on day , expanded to ml on day , and a culture volume of l was achieved on day with harvest/ cryopreservation on day . during this rapid expansion phase the labour required to maintain the culture (monitor parasitaemia, change media, add fresh rbcs) was less than would be required if growing in multiple tissue culture flasks. in addition, the use of multiple flasks can result in cultures spending more time in less than the continual wave motion of the wave bioreactor ensures gentle movement of cells in culture resulting in a high proportion of single infections [ ] . this allows for accurate calculation of parasite numbers which is essential for defined dose delivery in clinical trials. results confirmed that wave bioreactor cultures contained high quality ring stage parasites with a low number of multiply infected red cells (< . %). additionally, the effective cryopreservation of the mcb was demonstrated by the fact that parasite viability was over % after months. the ability to use research grade starting material, including genetically-modified parasites, to produce a gmp grade product offers significant opportunities. it allows for genetically-modified parasites to be used as vaccine candidates and for the number of p. falciparum strains available for ibsm studies to be expanded. the availability of multiple antigenically and geographically diverse p. falciparum strains for ibsm studies is essential drug threshold values are expressed as mean ic ( % confidence interval) in nmol/l the assay was repeated twice and reproducible results were obtained a third assay was performed for lumefantrine as ic values were not detectable in the first two assays to allow testing of new anti-malarials against multiple strains before field testing begins. the methodology described here is highly cost effective, with each of the vials produced containing sufficient material for doses at an approximate cost of usd . /dose. although the method requires use of a specialist item of equipment (wave bioreactor system), the fact that these units are becoming widely used for a range of applications (including tissue engineering, cellular and gene therapies, the production of therapeutic proteins and human vaccines) means that they are frequently available within research facilities or hospitals. the use of sterile, single use closed system cell bags means that a wave bioreactor system can be shared between groups without risk of cross contamination. measurement of plasmodium falciparum growth rates in vivo: a test of malaria vaccines effect of vaccination with recombinant asexual-stage malaria antigens on initial growth rates of plasmodium falciparum in non-immune volunteers immunity to malaria after administration of ultra-low doses of red cells infected with plasmodium falciparum a phase trial of msp -c , a blood-stage malaria vaccine containing isoforms of msp formulated with montanide(r) isa a phase ii pilot trial to evaluate safety and efficacy of ferroquine against early plasmodium falciparum in an induced blood-stage malaria infection study a pilot randomised trial of induced blood-stage plasmodium falciparum infections in healthy volunteers for testing efficacy of new antimalarial drugs blood-stage challenge for malaria vaccine efficacy trials: a pilot study with discussion of safety and potential value development of cultured plasmodium falciparum blood-stage malaria cell banks for early phase in vivo clinical trial assessment of anti-malaria drugs and vaccines large-scale growth of the plasmodium falciparum malaria parasite in a wave bioreactor the development of sexual stage malaria gametocytes in a wave bioreactor bag bioreactor based on wave-induced motion: characteristics and applications targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress preerythrocytic, live-attenuated plasmodium falciparum vaccine candidates by design negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in plasmodium falciparum for targeted gene deletion by double crossover recombination exported proteins required for virulence and rigidity of plasmodium falciparum-infected human erythrocytes gene deletion from plasmodium falciparum using flp and cre recombinases: implications for applied site-specific recombination convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc genetic analysis of the human malaria parasite plasmodium falciparum human malaria parasites in continuous culture synchronization of plasmodium falciparum erythrocytic stages in culture an improved method for undertaking limiting dilution assays for in vitro cloning of plasmodium falciparum parasites elda: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays estimation of plasmodium falciparum drug susceptibility by the h-hypoxanthine uptake inhibition assay. worldwide antimalarial resistance network (wwarn) field application of in vitro assays sensitivity of human malaria parasites antimalarial drugs. geneva: world health organization we would like to thank the australian red cross blood service for providing human erythrocytes. this methodology may represent a cost effective strategy by allowing extensive testing in a controlled environment (phase trials) before deciding to move to field studies (phase trials) which can be very expensive due to logistic and administrative challenges associated with carrying out clinical trials in a distant research site. the limiting factor for this methodology is that it is currently only useful for p. falciparum as other human malaria species are not easily grown in vitro. the large scale in vitro culture of p. falciparum parasites using a wave bioreactor can be achieved under gmp compliant conditions. this provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials. the authors declare they have no competing interests. all data generated or analysed during this study are included in this published article. not applicable. not applicable. the study was funded by nhmrc development grant app and nhmrc fellowship support app (ls) and nhmrc fellowship support app (jm). springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -tu xrt x authors: li, cui; gao, fei; yu, lei; wang, ruoke; jiang, yisheng; shi, xuanling; yin, chibiao; tang, xiaoping; zhang, fuchun; xu, zhiheng; zhang, linqi title: a single injection of human neutralizing antibody protects against zika virus infection and microcephaly in developing mouse embryos date: - - journal: cell rep doi: . /j.celrep. . . sha: doc_id: cord_uid: tu xrt x zika virus (zikv) is a mosquito-transmitted flavivirus that is generally benign in humans. however, an emergent strain of zikv has become widespread, causing severe pre- and post-natal neurological defects. there is now an urgent need for prophylactic and therapeutic agents. to address this, we investigated six human monoclonal antibodies with zikv epitope specificity and neutralizing activity in mouse models of zikv infection and microcephaly. a single intraperitoneal injection of these antibodies conveyed distinct levels of adult and in utero protection from zikv infection, which closely mirrored their respective in vitro neutralizing activities. one antibody, zk b , showed the most potent neutralization activity, completely protected uninfected mice, and markedly reduced tissue pathology in infected mice. thus, zk b is a promising candidate for the development of antibody-based interventions and informs the rational design of zikv vaccine. zika virus (zikv) is a mosquitotransmitted flavivirus that can cause severe neurological defects in humans. li et al. have identified a human monoclonal antibody capable of protection against zikv infection and related diseases when tested in mouse models. this antibody serves as a promising candidate for clinical development against zikv. the recent, widespread neurological deficits caused by an emergent strain of zika virus (zikv) have caught the world off guard wikan and smith, ) . zikv was first identified in the forests of uganda, and infection was generally benign in humans (dick et al., ) . however, this new strain of zikv is far more virulent and causes a range of clinical anomalies rasmussen et al., ; wikan and smith, ) . most notable are microcephaly and other congenital defects in infants born to mothers infected with zikv during pregnancy (mlakar et al., ; petersen et al., ; rasmussen et al., ; wikan and smith, ) . although the exact mech-anism of neuropathogenesis remains uncertain, clinical abnormalities have been linked to the aberrant development and loss of neural progenitor cells (npcs) (cugola et al., ; gabriel et al., ; garcez et al., ; li et al., a li et al., , b tang et al., ) . the contemporary strain of zikv has enhanced replication capacity and a specialized tropism for npcs (cugola et al., ; dang et al., ; garcez et al., ; li et al., b; tang et al., ) , although other types of cells are susceptible (tabata et al., ; weisblum et al., ) . the infection inhibits npc proliferation and differentiation and can trigger apoptosis or autophagy. critically, the highest rates of birth defects occur in pregnant mothers who are infected during their first and second trimesters. this is presumably because, during the early stages of gestation, npcs have a greater susceptibility to zikv infection, and there is more viral transfer across the placental barrier (mlakar et al., ; petersen et al., ; rasmussen et al., ; wikan and smith, ) . to fully protect the developing fetuses, an intervention must occur before this period or, ideally, prior to infection (marston et al., ) . neutralizing antibodies are the essential mediator of immunity against viral infection (burton and hangartner, ; corti and lanzavecchia, ) . for zikv and other flaviviruses, human neutralizing monoclonal antibodies target the surface envelope glycoprotein (e) that facilitates infection (dejnirattisai et al., ; fernandez et al., ; fibriansah et al., ; heinz and stiasny, ; magnani et al., ; pierson and diamond, ; pierson and graham, ; robbiani et al., ; rogers et al., ; sapparapu et al., ; stettler et al., ; wang et al., wang et al., , b zhao et al., ) . we previously reported on a panel of monoclonal antibodies (mabs) derived from the longitudinal samples of a zikv-convalescent individual and characterized their neutralizing activities, epitope specificities, and development timeline over the course of infection . we also reported on mouse models of zikv infection and microcephaly, with enhanced specificity for neurological infection figure . experimental design to evaluate the prophylactic potential of six human mabs against zikv infection in developing fetuses and ag mice (a) timeline for mab injection, zikv inoculation, infant delivery, and the monitoring of a complementary set of clinical, virological, and neuropathological outcomes from embryonic day . to p . the six mabs tested are shown alongside their ic values and epitope specificities. zikv-inoculated fetuses and neonates are indicated by zikv + in red; those left unexposed are indicated by zikv À in blue. the cartoon mice on p include zikv + (small and large indicated in red) and zikv À (large indicated in blue); the size representations reflect potential body weight outcomes. each mab was tested, and the outcomes were monitored in three littermate neonates of three pregnant mice on p and p . (b) different levels of protection conferred by the six mabs, shown with the number (n) of neonates monitored for survival in each mab treatment group. (c) the body weight of neonates among the different mab groups. zikv + neonates indicated in red are presented as a percentage of the blue zikv À littermates. the zk b -treated group had no discernable differences between the zikv + and zikv À littermates and is, therefore, shown as a single red/blue checkered bar. the number of neonates for each analysis is indicated above the respective mab, with zikv + in red and zikv À in blue. (legend continued on next page) using a contemporary zikv asian strain (gz ). in the model of microcephaly, the virus was inoculated directly into the lateral ventricles of the fetal mouse brain (li et al., a) . zikv replicated in the fetal brain, with preferential infection of npcs. infection resulted in cell-cycle arrest, differentiation defects, and a large number of cell deaths, as well as clinical presentations of microcephaly (li et al., a) . here, we use the mouse models of zikv infection and microcephaly to analyze the in vivo protective activities of six human mabs and compare the findings with our reported in vitro neutralization activity, as measured by plaque reduction neutralization test (prnt). our results offer compelling evidence that the in vivo protection is directly associated with in vitro neutralization. one antibody, zk b , provides complete protection in pre-exposure treatment and partial protection in post-exposure therapy, with markedly reduced tissue pathology. we believe that zk b is a promising candidate for the development of antibody-based interventions and informs vaccine design specific to zikv infection. in vivo protection correlates with in vitro neutralizing activity of mabs we evaluated and compared the protective potential of six representative human mabs following the protocol highlighted in figure a . all these (zk - , zk - , zk f , zk c , zk c , and zk b ) were isolated by our team. two additional zikv domain iii (diii)-specific mabs (zv and z ) isolated by others were included for comparative analysis (robbiani et al., ; zhao et al., ) . a human antibody, middle east respiratory syndrome- (mers- ), targeting the middle east respiratory syndrome coronavirus (mers-cov), was used as a negative control. our mabs were initially isolated from the peripheral b cells of a convalescent chinese man who acquired zikv while traveling to venezuela during the outbreak in . specimens were derived from sequential blood samples collected on day (zk - ), day (zk - ), day (zk f ), or day (zk c , zk c , and zk b ) after symptom onset. they targeted the zikv e with varying degrees of binding and neutralizing activities and epitope specificities . for example, zk b and zk c were strictly zikv specific and demonstrated the most potent neutralizing activities, as measured by prnt. of these, zk b recognized diii, while zk c was specific for domain i/domain ii (di/dii). in comparison, zk - , zk - , zk f , and zk c were much less potent against zikv but cross-neutralized other members of the flavivirus family, such as dengue virus serotypes denv and denv . on the zikv envelope, they targeted either di/dii (zk - , zk - , and zk f ) or di/dii/diii (zk c ). in terms of genetic analyses, these antibodies demonstrated diverse heavy-chain variable regions. two, zk - and zk f , fell into the immunoglobulinheavy variable (ighv) - family, while zk - and zk c belonged to the ighv - family. finally, zk b and zk c belonged to the ighv - and ighv - families, respectively. to test prophylactic activity against zikv infection, mg/kg of each mab was administered intraperitoneally to a group of three pregnant mice on embryonic day . ( figure a ). the following day (e . ), the developing littermate fetuses in each pregnant mouse were either left untouched (zikv À ; figure a , blue) or inoculated with plaque-forming units (pfus) of zikv asian strain gz directly into the lateral ventricles of the brain (zikv + ; figure a , red). the neonates were closely monitored after delivery and analyzed for a complementary set of clinical, virological, and neuropathological outcomes on post-natal day (p) and for survival up to p . as shown in figure b , the levels of protection conferred by the different mabs varied considerably but clearly correlated with their neutralizing activities, as measured by half-maximal inhibitory concentrations (ics ) in the prnt . the most potently neutralizing mab, zk b , conferred complete protection. survival rates of infected fetuses were as high as % on p . the next potent, zk c , however, conferred only partial protection with median survival of days. the two antibodies (zv and z ) isolated by others, together with the rest of the mabs isolated by us, offered negligible protection and were virtually similar to the isotype control mers- . we also assessed impact on developing body weights and found weights measured on p closely correlated with the neutralizing activity. the zikv + neonates treated with the most potent mabs, zk b and zk c , had weight gain similar to that of their zikv À littermates. the zikv + neonates treated with zv , z , and the remaining mabs failed to do so. the control zikv + mers- group had the greatest loss in body weight. next, we studied whether the protection pattern observed in the pregnant mice also occurred in non-pregnant mice. we administered mg of each mab (zk - , zk - , zk f , zk c , zk c , zk b , zv , and z ) or isotype control mers- to a group of four -to -week-old ag mice via the intraperitoneal (i.p.) route. the following day, animals were challenged with pfus of zikv asian strain gz via the i.p. route. survival rates, body weight, and viral rna copies in the blood were monitored for up to days ( figure d ). consistent with the outcomes for pregnant mice, the level of protection in nonpregnant mice correlated with antibody neutralizing activities as measured by half-maximal inhibitory concentrations (ics ) in the prnt ( figure e ). for example, the most potent and protective mab in pregnant mice, zk b , provided complete protection in non-pregnant mice, with survival rates of % days after the challenge. the next potent and protective, zk c , conferred only partial protection, with median survival of days. the rest of the tested mabs, including the diii-specific ones (zv and z ) isolated by others (robbiani et al., ; zhao et al., ) , offered even lower levels of protection. zk - was virtually identical to the isotype control mers- . (d-g) shown here: (d) timeline for mab injection, zikv inoculation, and monitoring for (e) survival, (f) body weight, and (g) blood zikv rna up to days in ag mice. the body weight and zikv rna in the whole blood derived from a single measurement showed distinct results among the study groups. the number of animals used in each group was four. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. similarly, changes in body weight measured over the -day follow-up also correlated with the neutralizing activities. the zikv + mice treated with zk b maintained relatively stable body weight throughout the study, although there was a noticeable decline after the first blood sample collection on day (figure f ). in contrast, zikv + mice treated with zk c lost substantial body weight beginning on day post-challenge. animals treated with the remaining mabs had severe and rapid losses in body weight that coincided with a clinical deterioration (figure e) . lastly, for zikv + mice treated with zk b , blood levels of viral rna were indistinguishable from those measured in uninfected animals on both day ( . ± . versus . ± . ) and day ( . ± . versus . ± . ) after challenge ( figure g ). however, rna levels measured in zk c -treated mice were, on average, log greater on day ( . ± . versus . ± . ) and more than logs greater on day ( . ± . versus . ± . ), compared to zk b . the remaining mabs failed to suppress viral replication. the measured zikv rna load on day ranged from . to . logs higher than that of zk b , depending on the mab used ( figure g ). taken together, these results demonstrated that the protective patterns of each mab were similar in both pregnant and non-pregnant mice and closely mirrored their respective in vitro neutralizing activities. the protective effects of each mab on body weight and overall survival mirrored their respective virological and neuropathological outcomes in treated mice. for instance, rna loads measured in zikv + neonates treated with zk b were suppressed in the blood ( . ± . log copies per milliliter) and the brain ( . ± . log copies per gram) to levels indistinguishable from those in zikv À littermates. in fact, zk b was able to reduce the zikv rna by . logs in the blood and . logs in the brain, relative to that in the zikv + mers- group (figures a and b ). in comparison, zk c was equally effective at suppressing replication in blood ( . ± . log copies per milliliter) but only moderately protective in the brain ( . ± . log copies per gram). the mabs zv and z only moderately suppressed zikv replication in the blood ( . ± . and . ± . log copies per milliliter, respectively) and the brain ( . ± . and . ± . log copies per gram, respectively). none of the remaining mabs notably suppressed replication in either blood or brain tissue. all of the zikv + neonates treated with these mabs had high levels of zikv rna, which were similar to levels in the zikv + mers- control group and significantly higher than those in their zikv À littermates (figures a and b ). importantly, low levels of zikv rna were associated with the healthier development of the neonatal brains. of the six representative mabs selected for in-depth neuropathological study, zikv + mice treated with either zk b or zk c maintained normal brain growth and structure. there were no notable differences in cerebral size, cortical thickness, or lateral ventricle area between these zikv + neonates and their zikv À littermates ( figures d- h ). however, zikv + mice treated with zv and z received only moderate protection, while zk f and mers- failed to provide any protection. low protection was measured by reduced cerebral size, thinner cortices, and enlarged lateral ventricles in zikv + neonates compared to their zikv À littermates ( figures d- h ). it is interesting to note that the e-specific human immunoglobulin g (igg) levels in the brain were higher in mice treated with zk b and zk c compared to the other mab groups. in particular, the levels of zk b ( . ± . mg/kg, which is translated into . ± . mg/ml) in zikv + neonates were well above the ic neutralization values measured in the prnt, whereas igg levels in the other mab groups measured less than or similar to those in the negative control pbs group ( figures a and c) . these results indicate that the tested mabs were able to transport across both the maternal-fetal placental barrier and the primitive blood-brain barrier of developing fetuses (saunders et al., ) . we also conducted immunohistochemical analyses of brain sections collected from p neonates in each group. as shown in figure a , in zk b and zk c groups, no zikv-infected cells (green) or apoptotic cells (cas + , red) were detected in the cortices of either zikv + neonates or their zikv À littermates. the mature neurons (neun; figure a , gray) and nuclei (dapi; figure a , blue) appeared healthy, with a tightly packed structure throughout the tissue sections. in distinct contrast, a large number of zikv-positive cells were identified throughout the cortices of the zikv + neonates in zv , z , zk f , and mers- groups. this high prevalence of infection was associated with significant cell death ( figure a , red) and degradation of the cortical structure ( figure a , gray and blue). we also quantitatively assessed the protective activity of zk b and zk c using marker cells from multiple tissue sections. as shown in figures b and c , treatment with zk b and zk c in zikv + neonates reduced zikv infection and cell death to levels indistinguishable from those of their zikv À littermates. furthermore, in the zk b and zk c groups, counts of mature neurons were equivalent between zikv + neonates and their zikv À littermates ( figure d ). however, for the zikv + neonates treated with either zv , z , zk f , or mers- , zikv-positive cell and apoptotic cell counts were as high as , cells and , cells, on average, per square-millimeter tissue section, respectively ( figures b and c) . counts of mature neurons were also significantly reduced in these groups ( figure d ). these results support the enhanced therapeutic activity of zk b and zk c at the cellular and tissue levels relative to zv , zv , zk f , and mers- and also offer an explanation for their impressive in vivo protection of developing fetuses. as zk b demonstrated the greatest prophylactic efficacy against zikv infection, we went on to evaluate its treatment efficacy after zikv infection of the developing fetuses. specifically, after the littermate fetuses were either left untouched (zikv À ) or inoculated with pfus of zikv gz in the lateral ventricle (zikv + ) on e . , we administered mg/kg zk b i.p. to the pregnant mice. this was done either on the same day (e . , day ), day later (e . , day ), or days later (e . , day ). the littermate neonates were closely monitored after delivery and analyzed for the same outcomes measured in the prevention experiments ( figure a ). as shown in figure a , delaying treatment from day to either day or day reduced the survival percentages. treatment on day offered the most effective treatment, with a median survival of about days. this is signif-icantly higher than that of mice treated on either day ( days) or day ( days). the survival advantage conferred by treatment on day corresponded with a clear reduction of viral load in the brain ( figure d ) and less damage to the cortical thickness and lateral ventricles (figures f and g) . however, the effect was less obvious when measured by body weight or each red dot represents a zikv + ; each blue dot represents a zikv À neonate. the red dots with blue coating represent no discernable differences between zikv + and zikv À littermates in zk b -and zk c -treated groups. the numbers of neonates for each mab analyses are indicated above each group, with zikv + in red and zikv À in blue. in (g) and (h), each dot represents the mean value of at least two slices from one neonate. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. blood serum viral loads on p (figures b and c ). considering that, in our mouse model, the brain is the initial and active site of viral replication, we expected that antibody treatment effects would be more sensitive and immediate in this organ and would be apparent before effects on other organs and body weight as a whole. this hypothesis was supported by immunohistochemical analyses of brain sections derived from p neonates. as shown in figure a , zikv-positive cells (green) were only sporadically detected in the day- treatment group. however, they were detected throughout the cortices if treatment was delayed to day or day . a greater level of infection was associated with increased cell death ( figure a , red) and cortical abnormalities ( figure a , gray and blue). in particular, the tightly packed and well-organized structure of matured neurons in the cortices became loosely connected and disorganized. furthermore, a quantitative assessment of marker cells derived from multiple each red dot represents a zikv + neonate; each blue dot represents a zikv À littermate. red dots with blue coating represent no discernable differences between zikv + and zikv À littermates in zk b -and zk c treated groups. each dot represents the mean value of at least two slices from one neonate analyzed. the numbers of neonates for each analysis are indicated above each group, with zikv + in red and zikv À in blue. scale bar, mm. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . . tissue sections supported the evidence that the time of treatment administration impacted treatment efficacy. for instance, in the zikv + neonate group, treatment on day significantly suppressed the number of zikv-positive cells to ± and apoptotic cells to ± per square millimeter of tissue section. treatment on day , however, allowed widespread cell infection and cell death counts as high as , ± , and ± per square-millimeter tissue section, respectively ( figures b and c ). however, although tissue samples from all three temporal treatment groups showed structural abnormalities in the cortices, there were no obvious reductions in the total number of mature neurons ( figure d ). treatment with the isotype mers- control was worst among all groups, with no signs of figure . evidence of therapeutic potential of zk b against zikv infection in developing fetuses (a) decreased survival when treatment administration is delayed from day (e . ) to day (e . ) or to day (e . ). the number (n) of neonates monitored for survival is indicated. (b-d) after treatment with zk b on day , day , or day , we analyzed zikv + neonates (indicated in red) and zikv À littermates (indicated in blue) at p for (b) body weight, (c) zikv rna copies in the blood, and (d) zivk rna copies in the brain. the number of neonates analyzed for each treatment time point is indicated above each group. (e-g) shown here: (e) the representative coronal sections of the neonatal brains at p was visualized with nissl staining and analyzed for (f) cortex thickness and (g) ventricular area. in (f) and (g), each dot represents the value of one slice. the total numbers of slices analyzed for zikv À , day , day , day , and mers- were , , , , and , respectively. the number of neonates analyzed for each treatment time point is indicated above each group. scale bar, mm. all data are presented as mean ± sem. *p < . ; **p < . ; ***p < . ; ns, no significant. protection (figures and ) . these results highlight the critical role of zk b in attenuating disease progression by inhibiting cell infection and death at the cellular and tissue levels. furthermore, the time of administration relative to infection impacts efficacy. treatment has the greatest protective effect when initiated immediately after infection. we report here the systematic analyses of the prophylactic and therapeutic potential of a panel of human mabs against zikv infection in a mouse model of microcephaly and non-pregnant ag mice. we showed that different mabs demonstrated distinct protective activity against zikv infection and that the major determinant of efficacy is their intrinsic neutralizing activities. a single i.p. injection of pregnant and non-pregnant mice with the most potent mab, zk b , provided developing fetuses and adult animals with a complete protection against zikv infection. treatment with zk b markedly reduced fetal infection and tissue pathology and significantly delayed mortality. thus, zk b is a promising candidate for the development of antibody-based intervention and informs rational design of zikv vaccine. two unique aspects of our study are worth highlighting here. one is based in the tested mabs that are all derived from the same zikv convalescent individual at different time points in the course of natural infection. each has distinct neutralizing activity, epitope specificity, and lineage ancestry . this diversity not only allows us to pinpoint the major determinants of protection in the mouse model but also provides evidence for when protective potential arises during the course of a natural infection in humans. our results clearly show that neutralizing activity, rather than other features, is the critical biomarker for protection against zikv infection, although the role of fc-mediated antiviral functions still need further investigation (dejnirattisai et al., ; pierson and graham, ; sapparapu et al., ; stettler et al., ). among the mabs tested here, zk b is the most potent, and its epitope is located in the lateral ridge region within the diii of e . mabs that target diii with potent neutralizing activity have also been isolated by other groups, derived from either infected humans or mice, and have been shown to be effective in various models of zikv pathogenesis (fernandez et al., ; magnani et al., ; robbiani et al., ; stettler et al., ; wang et al., b; zhao et al., ) . some weakly neutralizing mabs have also been reported against the same domain (robbiani et al., ; sapparapu et al., ; stettler et al., ; zhao et al., ) . it would be hard to determine whether this convergence on diii is a purely random event or whether diii epitopes are somehow more exposed and, therefore, more accessible by the mabs. it would be interesting to compare the exact epitopes of the highly potent mabs to see whether the vulnerable spots are widely spread or confined to restricted regions on the diii. this information would undoubtedly contribute to our understanding of the immune recognition mechanisms and assist in the rational design of vaccines against zikv infection. similar to other reported diii-specific mabs, zk b was isolated late after the onset of symptoms ( days), when the production of diii-specific antibodies had become more prevalent the total numbers of slices analyzed for zikv À , day , day , day , and mers- were , , , , and in (b) and (d), and , , , , and in (c), respectively. the total number of neonates analyzed is indicated above each group. all data are presented as mean ± sem. **p < . ; ***p < . ; ns, not significant. . however, why the diii-specific response seems to be delayed relative to di/ii-specific responses, and whether a vaccine could induce the preferred diii-specific response ahead of those targeting di/ii, requires more in-depth study. nevertheless, the potent prophylactic and therapeutic activities demonstrated by zk b and other diii-specific mabs will serve as the key criteria for future antibody-based intervention against zikv infection. the other unique aspect of our study was the mouse model of microcephaly, in which the virus was directly inoculated into the lateral ventricles of the fetal brain. despite the surgical sophistication required with this technique, the model has been standardized with remarkable reproducibility and captures the phenotypic features that are key to zikv infection in humans (li et al., a wang et al., a; yuan et al., ) . direct inoculation eliminates the need for immune-deficient pregnant mice and allows for control over the developmental stage at which infection occurs. as a result, both pathogenesis and intervention strategies can be evaluated with more precision. critically, it is likely the most stringent model for brain infection, as it tests the immediate protective activity of mabs at the injection site, as well as their capacity in preventing subsequent dissemination throughout the body. in this regard, the levels of protection conveyed by zk b are, indeed, remarkable. furthermore, direct inoculation on e . also allows for the study of immediate and long-term impacts of zikv infection in the developing fetuses. in this study, neonates were followed up to p , but this could have been extended to investigate the long-term sequelae of zikv infection, be they physiological, cognitive, or behavioral. this feature is complementary to the existing model, in which zikv infection was initiated earlier in embryonic development and was frequently associated with fetal demise before or shortly after delivery (fernandez et al., ; morrison and diamond, ; sapparapu et al., ) . in the future, it would be interesting to test the panel of mabs identified here in the early infection model in order to evaluate their protective potential. in conclusion, our study provides compelling evidence that the protective potential of tested mabs against zikv infection in pregnant and non-pregnant mice was directly associated with their neutralizing activities measured in the prnt. the most potent neutralizing antibody, zk b , demonstrated impressive prophylactic and therapeutic activities and, therefore, could serve as a promising candidate for antibody-based interventions and inform rational vaccine design against zikv infection. we previously reported on the isolation and characterization of a panel of human mabs from longitudinal samples of a zikv convalescent individual and showed their distinctions in neutralizing activities, epitope specificities, and time of development during nature infection . we also reported on one of the most stringent mouse models of microcephaly, in which a contemporary zikv asian strain is inoculated directly into the lateral ventricles of the fetal brains (li et al., a) . the present study combines these methodologies in order to evaluate the protective potential of six representative human mabs against zikv infection and microcephaly. using a single i.p. injection of mabs in pregnant mice prior to zikv infection, we showed that these antibodies convey distinct levels of protection to the developing fetuses and newborns and that these levels closely mirrored their respective in vitro neutralizing activities. the most potent neutralizing antibody tested, zk b , provided a complete protection from zikv infection in pre-exposure treatment and partial protection in post-exposure therapy, as measured by markedly reduced tissue pathology. the tested mabs (zk b , zk c , zk f , zk c , zk - , and zk - ) targeted the zikv e and were initially isolated from a zikv convalescent chinese traveler visiting venezuela during the viral outbreak in . the details of their neutralizing activity, epitope specificity, lineage ancestry, and the time of isolation during natural infection were previously reported . two zikv diii-specific mabs, zv and z , were isolated by others (robbiani et al., ; zhao et al., ) and used here for comparative analyses. all mabs were in the human igg backbone, manufactured in f cells (atcc) by transient transfection, and purified by affinity chromatography using protein a agarose (thermo scientific). the mab concentration was determined using the bca protein assay kit (thermo scientific). we previously isolated the human mab mers- , which targets against the mers-cov and was used as a negative control (jiang et al., ) . approximately mg/kg of each tested mab or isotype control mers- was administered i.p. to a group of three pregnant mice at e . . all icr pregnant mice were purchased from beijing vital river laboratory animal technology. the experimental protocol and procedure were approved by the institutional animal care and use committee at tsinghua university ( -zlq ). the pregnant mice were kept in separate cages and maintained on a -hr/ -hr light/dark cycle throughout the experiment. for each tested mab, a group of three pregnant mice were included, and their littermate neonates were monitored for a complementary set of clinical, virological, and neuropathological outcomes on p and for survival up to p ( figure a) . specifically, the first (i) and second (ii) groups of littermate neonates were sacrificed on p . brain and blood samples were immediately collected, processed, and frozen at À c until use. group i was used to measure for viral loads in the blood serum and the brain, as well as human igg in the brain. group ii was used for blood serum viral loads, brain size, and section analysis. the third (iii) group littermate neonates were monitored for body weight at p and for survival up to p . non-pregnant c bl/ mice deficient in interferon (ifn)a, -b, and -g receptors (ag mice) were purchased from institute pasteur of shanghai, chinese academy of sciences. the mice were bred and maintained in a pathogenfree animal facility at tsinghua university. specifically, mg of each tested mab (zk - , zk - , zk f , zk c , zk c , zk b , zv , and z ) or isotype control mers- was administered to a group of four -to -weekold ag mice via the i.p. route. the following day, the animals were challenged with pfus of zikv (gz strain) via i.p. injection and monitored for survival, body weight, and viral rna copies in the blood up to days. on day and day after challenge, blood was collected from each animal for viral load measurement. microinjection of zikv into the brain ml zikv asian strain gz (genbank: ku and virus stock concentration . pfu/ml) was injected into the right side of the lateral ventricle of the embryonic mouse brains at e . , as described previously (li et al., a) . approximately half of the littermate neonates were injected with zikv (zikv + ), while the rest remained untouched (zikv À ). gz was derived from the same chinese traveler from whom the tested mabs were isolated . phylogenetic analysis of the complete coding sequences indicated that gz was tightly clustered with those circulating in the americas and belonged to the asian lineage, including those identified from french polynesia in . the whole blood ( ml) and right brain of the neonates were collected on p , immediately transferred to an eppendorf tube containing lysis buffer (qiagen), and kept in À c until use. each brain sample was weighted and homogenized using a stainless steel blender (next advance). total rna from the homogenized brain and the whole blood was extracted using an rneasy mini kit ( , qiagen) and reverse-transcribed into cdna using an iscript cdna synthesis kit ( - , bio-rad). as previously described, viral rna copies were quantified through taqman qpcr amplification of zikv envelope genes and expressed as log viral rna copies per gram for the brain or per millimeter for the blood samples calculated against the standard curve. the sequences for the primers and probes used for the analysis are shown as follows: zikv-fccgctgcccaacacaag, zikv-r ccactaa cgttcttttgcagacat, zikv-probe agcctaccttgacaagcartcagac actcaa ( fam, tamra). the neonatal right brains were collected on p and immediately frozen at À c until use. the frozen brain samples were weighted and homogenized with a stainless steel blender (next advance) before being mixed with ml pbs and allowed to stand at À c for hr. the tested mabs, suspended in the pbs, were collected by centrifugation at , g for min and quantified by serial dilution and application to elisa plates pre-coated with recombinant full-length e glycoprotein of zikv (gz , ku ), as previously described . bound mabs were detected using goat antihuman igg (fc specific) conjugated with horseradish peroxidase (promega, madison, wi, usa) and , , , -tetramethylbenzidine (tmb) substrate (cwbio, beijing, china). the mabs levels in the neonatal brains were calculated against a curve that was standardized using the corresponding mab suspended in pbs and expressed as milligrams per kilogram or micrograms per milliliter. brains were fixed in % pfa, dehydrated in % sucrose, and frozen in tissuefreezing medium before being sliced into -mm-thick tissue sections. for nissl staining, brain sections were stained with . % toluidine blue for min; dehydrated, in turn, by %, %, and % ethanol ( s, twice for each); and then hyalinized by xylene for more than min. for immunohistochemical staining, sections were blocked for hr at room temperature (rt) and incubated with the primary antibody for one night at c. after three rounds of extensive washing with phosphate-buffered saline with . % tween- (pbst), the secondary antibody was added and incubated at rt for hr. the section was then washed once more with pbst. the tissue sections were then imaged on an lsm (carl zeiss) confocal microscope and analyzed with imaris and imagej, as described previously (li et al., a) . the antibodies used for immunohistochemical analysis were caspase- (abcam, ab , : , ) and neun (abcam, ab , : , ). zikv serum ( : , ) was derived from the same convalescent patient. nuclei were stained with dapi (invitrogen). for experiments involving pregnant and non-pregnant mice, - mice were included in each assessment group to ensure representation and consistency of the data. all data were analyzed using prism software (graphpad). statistical evaluation was performed by student's unpaired t test. data were presented as mean ± sem. *p < . ; **p < . ; and ***p < . . broadly neutralizing antibodies to hiv and their role in vaccine design broadly neutralizing antiviral antibodies the brazilian zika virus strain causes birth defects in experimental models zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor tlr a new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus zika virus. i. isolations and serological specificity antibody-mediated neutralization of flaviviruses: a reductionist view a dynamic landscape for antibody binding modulates antibody-mediated neutralization of west nile virus human antibodies to the dengue virus e-dimer epitope have therapeutic activity against zika virus infection a highly potent human antibody neutralizes dengue virus serotype by binding across three surface proteins recent zika virus isolates induce premature differentiation of neural progenitors in human brain organoids zika virus impairs growth in human neurospheres and brain organoids flaviviruses and their antigenic structure potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein zika virus disrupts neural progenitor development and leads to microcephaly in mice zika virus infects neural progenitors in the adult mouse brain and alters proliferation -hydroxycholesterol protects host against zika virus infection and its associated microcephaly in a mouse model neutralizing human monoclonal antibodies prevent zika virus infection in macaques considerations for developing a zika virus vaccine zika virus associated with microcephaly animal models of zika virus infection, pathogenesis, and immunity zika virus molecular mechanisms of antibodymediated neutralisation of flavivirus infection zika virus: immunity and vaccine development zika virus and birth defects-reviewing the evidence for causality recurrent potent human neutralizing antibodies to zika virus in brazil and mexico zika virus activates de novo and cross-reactive memory b cell responses in dengue-experienced donors neutralizing human antibodies prevent zika virus replication and fetal disease in mice the rights and wrongs of blood-brain barrier permeability studies: a walk through years of history specificity, crossreactivity, and function of antibodies elicited by zika virus infection zika virus targets different primary human placental cells, suggesting two routes for vertical transmission zika virus infects human cortical neural progenitors and attenuates their growth molecular determinants of human neutralizing antibodies isolated from a patient infected with zika virus transfer of convalescent serum to pregnant mice prevents zika virus infection and microcephaly in offspring a human bi-specific antibody against zika virus with high therapeutic potential zika virus infects early-and midgestation human maternal decidual tissues, inducing distinct innate tissue responses in the maternal-fetal interface zika virus: history of a newly emerging arbovirus delineating antibody recognition against zika virus during natural infection a single mutation in the prm protein of zika virus contributes to fetal microcephaly excretion of infectious zika virus in urine structural basis of zika virus-specific antibody protection we are grateful to the zikv convalescent patient for donating his blood samples and drs. jiang wang, wenxin hong, and lingzhai zhao for providing the patient with treatment and care. we thank drs. cheng-feng qin and gong cheng for providing the zika virus isolate gz and for assisting with evaluations of antibody protective activity in ag mice. we also thank ms. angela fan for editing the manuscript. all authors contributed to the present study, including intellectual input, results analysis, and actual writing and editing of the manuscript. specifically, c.l., f.g., r.w., y.j., l.y., and x.s. performed the experiments. c.y., x.t., f.z., z.x., and l.z. designed the study, and c.l., f.g., z.x., and l.z. analyzed the data and wrote the paper. patents have been filed for the isolated antibodies, and they are all available for collaboration in research and development through material transfer agreement. key: cord- -vox xsgd authors: deng, xufang; baker, susan c title: an “old” protein with a new story: coronavirus endoribonuclease is important for evading host antiviral defenses date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: vox xsgd here we review the evolving story of the coronavirus endoribonuclease (endou). coronavirus endou is encoded within the sequence of nonstructural protein (nsp) , which was initially identified as a component of the viral replication complex. biochemical and structural studies revealed the enzymatic nature of nsp /endou, which was postulated to be essential for the unique replication cycle of viruses in the order nidovirales. however, the role of nsp in coronavirus replication was enigmatic as endou-deficient coronaviruses were viable and replicated to near wild-type virus levels in fibroblast cells. a breakthrough in our understanding of the role of endou was revealed in recent studies, which showed that endou mediates the evasion of viral double-stranded rna recognition by host sensors in macrophages. this new discovery of nsp /endou function leads to new opportunities for investigating how a viral endou contributes to pathogenesis and exploiting this enzyme for therapeutics and vaccine design against pathogenic coronaviruses. we provide a brief outline of the major research findings related to coronavirus (cov) endoribonucleases (endou) in table . in the text below, we describe the experimental approaches that led to these findings and compare the activity of cov endou with reports of other viral and host ribonucleases. initial studies focused on identifying the products of the cov replicase polyprotein, pp ab (depicted in fig. a ). heusipp et al. used a murine monoclonal antibody ( g ) that recognizes amino acid residues - of human cov e pp ab (heusipp et al., ) . they identified a -kda polypeptide in virus-infected cells and found that this polypeptide was a product of the viral c-like proteasemediated cleavage of pp ab. this protein localizes to the perinuclear region, as detected by immunofluorescence assay, similar to other pp ab-derived polypeptides (heusipp et al., ) . shi and coworkers obtained similar findings while studying mouse hepatitis virus (mhv) (shi et al., ) . they generated a rabbit antiserum against amino acids - of mhv pp ab and found that this antiserum detected a -kda product in infected cells. they also found that this protein colocalized with de novo synthesized viral rna, and therefore postulated that this viral protein associated with the viral rna replication/ transcription machinery (shi et al., ) . later, the corresponding polypeptides that these antibodies recognized were defined as the th cleavage product of pp ab (called nsp ), counting from the aminoterminus to the carboxyl terminus of pp ab (ziebuhr et al., ; snijder et al., ) . after the outbreak of severe acute respiratory syndrome (sars) in [ ] [ ] , and once a cov had been confirmed as the etiological agent of sars, researchers intensively scrutinized cov genomic sequences to better understand this novel human pathogen. by comparative genomic characterization of cov replicases, snijder et al. reported that the c-terminus of nsp has high sequence similarity to the xenopus laevis poly(u)-specific endoribonuclease and therefore predicted that nsp possesses endou activity (snijder et al., ) . based on the available sequence information of viruses from the order nidovirales at that time, the endou was considered a nidovirus-specific marker (called nendou) (fig. b) (snijder et al., ) . the members of the family arteriviridae, including equine arteritis virus (eav) and porcine respiratory and reproductive syndrome virus (prrsv), also have endou domains within nsp . however, it was later discovered that the presence of the endou domain is not universal in all nidoviruses. nam dinh virus, the first insect nidovirus belonging to the family mesoniviridae, and roniviruses that infect invertebrates, do not encode an endou domain (nga et al., ; lauber et al., ) . these findings suggest that the endou domain may only serve as a signature for vertebrate nidoviruses, including covs and arteriviruses (fig. ). previous reviews have comprehensively summarized the biochemical and structural features of nidovirus ribonucleases (ulferts and ziebuhr, ; snijder et al., ) . here, we highlight the key experiments with respect to nidovirus endou and provide recent updates. studies performed by ivanov et al. ( ) and bhardwaj et al. ( ) first demonstrated the endou activity of sars-cov nsp in vitro (ivanov et al., ; bhardwaj et al., ) . the wild-type (wt) nsp and its mutants with alanine (ala) substitutions of the putative catalytic residues were expressed in e. coli. the recombinant nsp -wt, but not the mutants, could efficiently cleave single-stranded (ss) and doublestranded (ds) rnas. in contrast, neither ssdna nor dsdna molecules could be processed by nsp , demonstrating its predicted ribonuclease-as opposed to deoxyribonuclease-activity. blocking either the ′ or the ′ terminus of the rna substrates by covalent modifications with fluorescein or puromycin, respectively, had no effect on the rna cleavage (bhardwaj et al., ) , confirming that nsp is an endorather than an exoribonuclease. similar endou activity was detected in other cov nsp s, including human cov e, mhv, infectious bronchitis virus (ibv), and turkey cov (ivanov et al., ; bhardwaj et al., ; cao et al., ) , and in the nsp s of arteriviruses eav and prrsv (nedialkova et al., ) . hexamerization of nsp is critical for its endou activity. guarino and coworkers found that cov nsp could be present in solution as either monomers or hexamers in a protein concentration-dependent manner. the hexamer is the fully active form of endou that binds rna and executes optimal endou activity (guarino et al., ) . the residues in the n-terminal domain of nsp are critical for hexamer formation (guarino et al., ) . in addition, nsp requires divalent metal ions as a co-factor for rna cleavage and prefers mn + over mg + or other divalent cations (ivanov et al., ; bhardwaj et al., ) . addition of mn + significantly affects the protein conformation, enhances rna binding, and increases endou activity (ivanov et al., ; bhardwaj et al., ) . in contrast to cov nsp , arterivirus nsp forms dimers in solution and does not require divalent cations as a cofactor for activity in vitro (nedialkova et al., ; shi et al., ) . instead, a concentration of mn + that greatly stimulated the activity of cov nsp inhibited the endou activity of eav and prrsv nsp s. cov endou was revealed biochemically to hydrolyze the ′ end of pyrimidines, with a preference for uridylates, and release products with ′, ′-cyclic phosphate and ′-hydroxyl ends (ivanov et al., ; bhardwaj et al., ) . this finding seems to implicate a broad range of targets; however, the endou activity of cov nsp can be affected by secondary structure and modification of the rna substrate. bhardwaj et al. found that nsp preferentially cleaved unpaired uridylates in hairpin-structured rnas and that the neighboring nucleotides of targeted sites also influenced hydrolysis (bhardwaj et al., ) . on the other hand, ivanov et al. found that ′-o-ribose methyl groups present on the substrate rna blocked endou-mediated cleavage (ivanov et al., ) . these data suggest that multiple factors might limit the range of endou targets. this is reasonable because the endou activity of nsp is likely to be tightly regulated during infection in cells to avoid unwanted cleavage on viral and/or cellular targets. for example, cov nsp is a ′-o-ribose methyltransferase, whose function could theoretically block the endou-mediated cleavage of viral rnas. similar to cov nsp , arterivirus nsp also prefers ′ of uridylates for cleavage and yields products with ′, ′-cyclic phosphate ends (nedialkova et al., ) . further studies are needed to address whether the endou activity of arterivirus nsp is restricted by rna modifications or secondary structures. guarino and coworkers visualized single particles of sars-cov nsp using electron microscopy (guarino et al., ) and the same group later reported an . Å structure of nsp by cryoelectron microscopy (bhardwaj et al., ) . they reported that the nsp hexamer comprises a dimer of trimers and proposed that the rna substrate binds to the inter-trimer interface. x-ray crystal structures of sars-cov and mhv nsp s were first solved by ricagno et al. and xu et al., respectively (ricagno et al., ; xu et al., ) . these high-resolution structures define cov nsp as a separate endou family with unique folds that differ from cellular endoribonucleases (fig. a) . the monomer of sars-cov nsp consists of three domains: a small nterminal domain (ntd) (residues - , cyan), a middle domain (residues - , magenta), and a large c-terminal domain (ctd) (re-sidues - , green). the endou is located in the ctd. the monomeric structure of mhv nsp can be superimposed onto that of sars-cov nsp , except for a flexible loop structure in the middle domain of mhv nsp (fig. b ). this flexible loop is encoded by a viral rna packaging signal sequence, which is present in mhv nsp but not in sars-cov nsp (kuo and masters, ) . these structural studies demonstrated that the presence of the flexible loop did not alter the overall folding of mhv nsp relative to sars-cov nsp . in addition, through these and other structural studies (ricagno et al., ; xu et al., ; joseph et al., ; bhardwaj et al., ) , the nsp hexamer was again confirmed to be a dimer of trimers. as shown in fig. c , three monomers form a trimer and two trimers interact headto-head to form a hexamer. the ntds line up in the center of the hexamer, while the ctds face outward. this architecture allows the nsp hexamer to possess six active sites. the extensive contact between subunits through the ntd and middle domain is critical for hexamerization. the crystal structure of arterivirus nsp was also recently reported (shi et al., ; zhang et al., ) . these structural studies revealed that the monomer of nsp contains only two domains: ntd and ctd (no middle domain). the monomeric structures of cov nsp and arterivirus nsp could not be superimposed except in the ctd (catalytic domain). distinct from the hexameric structure of cov nsp , nsp monomers assembled into an asymmetric dimer (shi et al., ) . virus-encoded endoribonuclease is a genetic signature of nidoviruses that infect vertebrates. a phylogenic tree of representative nidoviruses was generated based on a conserved region of rdrp (sequnces and genbank assession numbers are available upon request). multiple sequence alignment and phylogeny analyses were conducted with the programs muscle and phyml, respectively (available at http:// www.phylogeny.fr/). the phylogenic tree was generated using dendroscope software version with default parameters. virology ( ) - since the crystallographic studies of cov nsp revealed that the active site of endou is structurally similar to that of rnase a, researchers evaluated small molecule inhibitors of rnase a for their ability to inhibit nsp activity. ortiz-alcantara et al. tested several commercially available rnase a inhibitors (benzopurpurin b, congo red, and others) and reported that their % inhibitory concentration (ic ) values for inhibiting the endou activity of sars-cov nsp ranged from . μm to μm. benzopurpurin b was shown to have a broad-spectrum activity and could inhibit nsp orthologs from mhv and ibv with ic values of . μm and . μm, respectively (ortiz-alcantara et al., ). these rnase a inhibitors had variable effects on cov replication in cell cultures. in plaque formation assays, treatment with congo red resulted in -fold and -fold reductions in mhv and sars-cov titers, respectively, while benzopurpurin b led to marginal inhibition of both covs (ortiz-alcantara et al., ) . although the impact of these rnase a inhibitors on cov replication requires more comprehensive investigation, these early results suggest that the similarity between nsp /endou and rnase a may provide a basis for exploiting small molecule inhibitors to modulate viral endou activity. due to its unique enzymatic activity and co-localization with the replicating viral rna, nsp /endou was initially thought to play an important role in virus replication. however, mhv encoding catalyticdefective endou exhibited only a subtle defect in rna synthesis and a slight reduction in viral titers (~ log) compared to wt virus when evaluated in fibroblasts (kang et al., ) . similar results were obtained for sars-cov and hcov- e nsp mutants generated using either reverse genetics or replicon systems (ulferts and ziebuhr, ) . these data indicate that the endou activity of nsp is not essential for cov replication, as was initially proposed (snijder et al., ; ivanov et al., ; bhardwaj et al., ) . intriguingly, nsp may indirectly affect virus replication through other mechanisms. ivanov et al. demonstrated that when the conserved aspartic acid (asp)- of hcov- e nsp was replaced with an ala, its endou activity was eliminated, viral rna synthesis was completely abolished, and no viable virus was recovered (ivanov et al., ) . similar phenotypes were observed for an mhv nsp mutant with an ala substitution of asp- (equivalent to asp- of hcov- e) (kang et al., ) . it is unclear how the asp residues affect viral rna synthesis. kang et al. predicted that asp- is critical for an ionic-bond network and observed that the ala substitution resulted in an insoluble protein when mhv nsp was expressed in e. coli (kang et al., ) . these data suggest that the ala substitution may prevent the nsp protein from folding correctly. since the proteins adjacent to nsp are critical replicative components, any protein-folding issue with nsp may have an effect on the proteolytic processing of the neighboring components, thereby leading to a nonviable phenotype. overall, current evidence indicates that the endou activity of cov nsp is dispensable for viral rna synthesis and virus replication in cell culture. further work is required to address any non-endou-mediated role of nsp protein in virus replication. mutagenesis of arterivirus nsp revealed pleiotropic effects of endou on the viral life cycle (posthuma et al., ; sun et al., ) . similar to cov nsp , mutations in the asp- and asp- residues (corresponding to mhv nsp asp- and asp- , respectively) in eav nsp resulted in a nonviable phenotype. compared with the mild effect of mutating the catalytic histidine (his) residues (hi-s ala and his ala) of mhv nsp , eav infectious clones with catalytic residue mutations (his ala/gln and his ala/gln) exhibited smaller plaque sizes, reduced rna synthesis, and dramatic titer reductions up to log units. other substitutions at conserved, noncatalytic residues of eav nsp resulted in an intermediate phenotype: intermediate plaque sizes and~ - log reduction in titers. similar results were obtained with the prrsv nsp mutant viruses (sun et al., ) . these data indicate that arterivirus nsp may be involved in virology ( ) - viral rna synthesis. however, similar to the asp ala mutation of mhv nsp , both asp ala and asp ala mutations in eav nsp rendered the protein insoluble, again raising the question of whether or not these mutations influence viral rna synthesis through interfering with proteolytic processing of the orf b polyprotein. the absence of an endou domain in insect nidoviruses and invertebrate roniviruses further indicates that endou activity is not required for the unique rna synthesis strategy of nidoviruses (nga et al., ; lauber et al., ) . the aforementioned studies highlight the extensive efforts of the field to investigate the characteristics of nsp /endou and its role in virus life cycle. the endou activity of cov nsp was found to play a non-essential role in viral rna synthesis and replication in immortalized fibroblast cells. recent work with different cell types and in vivo experiments revealed a novel function of nsp /endou in virus replication and pathogenesis and provided a new direction of study with respect to this "old" protein. cov nsp was first suggested to possess interferon (ifn) antagonism capabilities through ectopic expression experiments. frieman et al. used an alphavirus replication-defective vector (vrp) to screen sars-cov proteins that suppress vrp-induced ifn responses. one of the identified ifn antagonists of sars-cov was nsp (frieman et al., ) . later, arterivirus nsp was also identified as an ifn antagonist and its endou activity was found to mediate inhibition of ifn-beta induction (beura et al., ; shi et al., ) . several other studies also reported that cov nsp and arterivirus nsp inhibit cellular innate responses in ectopic expression experiments (lei et al., ; wang et al., ) . these data seem to indicate that these two proteins function as ifn antagonists; however, the endou activity of overexpressed nsp /nsp may unexpectedly affect the activities of the reporters used in these assays. we found that transfected mhv nsp reduced the signal of both ifn-reporter firefly luciferase and the internal control renilla luciferase (hackbart m, deng x, and baker s, unpublished data). a similar result was obtained with the overexpressed prrsv nsp (shi et al., ) . these observations imply that nsp /nsp may execute non-specific cleavage when ectopically expressed. since cov nsp is part of the viral replicase/transcriptase complex (rtc), it is reasonable to predict that its endou activity is tightly regulated during viral infection to avoid unwanted cleavage. in line with this prediction, we and others reported a specific, punctate, perinuclear localization of cov nsp during viral infection (heusipp et al., ; shi et al., ; deng et al., ; athmer et al., ) , while ectopically expressed nsp was distributed throughout the cytoplasm (cao and zhang, ) . hence, we advise caution when interpreting the results of overexpression studies, as the nature of the endou activity was only revealed after studying nsp /nsp in the context of viral infection. it was first discovered that the endou activity of nsp mediates the evasion of host recognition of viral dsrna by infecting primary macrophages with endou-deficient covs (deng et al., ; kindler et al., ) . infection with mhv endou-deficient mutants stimulated mouse bone-marrow derived macrophages (bmdms) to produce a remarkably high level of type i ifns during the early phase of infection compared to wt infection. this ifn response is mda -dependent, as both the ifn mrna and protein levels were not elevated in mda -deficient bmdms. moreover, the replication of endou-deficient covs was severely impaired in primary macrophages. interestingly, the ifn-induced antiviral response is not the only player responsible for this replication defect, as the titers of the endou-deficient covs were not completely restored in bmdms that lack critical genes (e.g. mda , mavs, and irf / / ) involved in the ifn response (deng et al., ; kindler et al., ) . these data suggest that other antiviral pathways may also contribute to the observed replication defect. in support of this, it was found that the infection of endou-deficient covs also activate the pkr and the oas-rnase l pathways (deng et al., ; kindler et al., ) , which both execute potent antiviral functions, discussed further below. indeed, the replication of endou-deficient covs could be partially restored in pkr/ rnase l-double knockout cells (kindler et al., ) . replication was only fully restored in type i ifn receptor-knockout macrophages, as these cells not only have a defect in ifn signaling but also express very low basal levels of pkr and oas relative to wt cells (deng et al., ; birdwell et al., ) . taken together, these studies using live viruses in primary cells effectively illustrated the ifn antagonistic properties of cov endou. as mentioned above, infection with endou-deficient covs also activates the pkr and oas-rnase l pathways in macrophages (deng et al., ; kindler et al., ) . pkr is a dsrna-activated protein kinase and serves as a dsrna sensor. activated pkr phosphorylates eukaryotic initiation factor α (eif α), resulting in inhibition of host and viral mrna translation. thus, pkr-mediated translation shutoff plays an important role in the host antiviral defense (barber, ) . the endou-deficient cov-infected macrophages exhibited increased levels of phosphorylated eif α and decreased levels of translation (deng et al., ; kindler et al., ) , indicating that pkr was activated during infection. another piece of evidence of pkr activation was that endou-deficient covs induced rapid apoptotic cell death in infectedmacrophages (deng et al., ) . it has been shown that pkr-mediated translation shutoff leads to apoptosis in macrophages (hsu et al., ) . when the endou-deficient cov-infected macrophages were treated with a pkr-specific inhibitor (c ), the level of apoptosis was significantly reduced (deng et al., ) . this result further supports the hypothesis that endou-deficient covs activate pkr. interestingly, loss of pkr expression or inhibition of its activity only partially restored the replication of endou-deficient covs in macrophages (kindler et al., ) . moreover, treatment with the pkr inhibitor did not affect ifn induction or rnase l-mediated ribosomal rna degradation in the endou-deficient cov infected-macrophages (deng et al., unpublished data) . these results imply that the infection of endou-deficient cov activates multiple host dsrna sensors independently, including mda , pkr, and oas. oas is a protein family of ′, ′-oligoadenylate ( - a) synthetases. upon activation, oas can synthesize - a, which binds to and activates rnase l. rnase l is a host ribonuclease that executes global degradation of host and viral rnas. thus, oas and rnase l constitute a potent host antiviral system. macrophages infected by the endou-deficient covs exhibited an early, rnase l-mediated degradation of ribosomal rna, demonstrating that the oas-rnase l system was activated (deng et al., ; kindler et al., ) . lack of mda expression or treatment with the pkr inhibitor did not affect virus-induced rna degradation (deng et al., ; kindler et al., ) , suggesting that the nsp -mediated blockage of oas-rnase l activation is independent of the mda -ifn and pkr pathways. loss of rnase l expression does not restore the replication of endou-deficient covs in macrophages. taken together, these results suggest again that multiple antiviral pathways, including mda -ifn, pkr, and the oas-rnase l system, were activated during infection with endou-deficient covs. the antiviral defense executed by these pathways contribute together to the replication defect of endou-deficient covs in macrophages. interestingly, some covs encode a ′, ′-phosphodiesterase (pde) (e.g. mers-cov orf b and mhv ns ) (thornbrough et al., ; zhao et al., ) . this cov pde also prevents rnase l-mediated rna degradation through digesting the - a produced by oas. thus, the presence of two antagonists of the oas-rnase l system in some cov genomes represents a functional redundancy or tissue-specific roles for these antagonists. in fact, not all covs encode a pde, and it has been reported that loss of the pde activity mitigated mhv pathogenicity in the mouse liver but not in the brain, suggesting a liver-specific effect of cov pde activity in vivo (roth-cross et al., ; zhao et al., ) . several endou-deficient covs have been evaluated in fibroblast cell lines and no marked phenotypes were obtained (mild reduction of viral rna synthesis and replication) (discussed above). we speculate that the activation of an ifn response and apoptosis in macrophages is due to high levels of basal expression of host sensors, such as mda , pkr, and oas (deng et al., ; birdwell et al., ) . indeed, when the expression of oas was induced by pre-treatment with ifn, the immortalized fibroblast cells also exhibited rna degradation upon infection with endou-deficient cov (kindler et al., ) . in line with this, without the stimulation of ifn, constitutively expressed oas and pkr in the mda -deficient macrophages is capable of sensing the endou-deficient cov infection and implementing antiviral processes (deng et al., ) . although the production of ifn is dispensable for the activation of the oas-rnase l system and pkr, type i ifn receptors or a direct signal is required for maintaining the high basal expression of these ifn-inducible genes (deng et al., ; birdwell et al., ) . this is biologically relevant because macrophages and other myeloid cells are quick responders to early virus infection, responding even before ifn is highly induced. it is unclear whether other cell types, such as epithelial cells, behave similar to macrophages, but at least mouse embryonic fibroblasts have been shown to display the nsp /endoumediated effects (kindler et al., ) . due to robust activation of antiviral responses, mhv endou-deficient mutants also exhibited a marked attenuation in vivo relative to mhv-wt. depending on the inoculation route, mhv infection can result in hepatitis or encephalitis in c /bl mice. strikingly, regardless of which infection route (intraperitoneal or intracranial injection) was used, we found that mhv endou-deficient mutants were highly attenuated (deng et al., ) . when mice were inoculated intraperitoneally using a high dose of the mutant virus, there was no detectable viral titer in the liver or spleen at day post-infection and only a minimal detection of viral mrna in mesenteric lymph nodes at day post-infection. when a sensitive encephalitis model was used, mice infected with mhv endou-deficient mutants exhibited only a transient loss of body weight and recovered completely. this significant attenuation is attributed to the loss of endou-mediated evasion of host antiviral defenses. we found that endou-deficient mutants maintained wt-level virulence in type i ifn receptor knockout (ifnar -/-) mice (deng et al., ) . similar results obtained by kindler et al. showed that the mutant virus was only detected in the organs from ifnar -/mice but not wild-type or other gene-deficient (mda -/-, tlr -/-, and mda -/-/tlr -/-) mice at day post-infection (kindler et al., ) . interestingly, even though mhv endou-deficient mutants were highly attenuated and exhibited limited replication in vivo, the pre-infected mice were protected from subsequent lethal challenges of wild-type mhv in both disease models (deng et al., ) . these results demonstrate that nsp plays an important role in virus pathogenesis and illustrate the potential use of endou-deficient covs as vaccine candidates. . . how does nsp mediate the evasion of host sensors? as an endou, it is plausible that nsp may degrade viral dsrna to prevent host recognition. previous studies of pestivirus envelope glycoprotein (e rns ) and lassa virus nucleoprotein linked viral ribonuclease activity to type i ifn antagonism through degrading viral dsrna (python et al., ; qi et al., ; hastie et al., ) , although no direct evidence for this linkage has been obtained. for cov nsp , kindler et al. detected an increased level of dsrna in endou mutantinfected cells by flow cytometry using a dsrna-specific monoclonal antibody (mab) (kindler et al., ) . this mab recognizes dsrna molecules longer than bp in length, regardless of the sequence, meaning that a long dsrna could potentially bind multiple mab molecules. to saturate the mab-dsrna binding, we tested serial concentrations of antibody but did not detect any significant change of dsrna levels by either flow cytometry or immunofluorescence analysis (deng et al., ) . the discrepancy between these two studies may be ascribed to the experimental settings in respective experiments. however, it is also possible that the methods used in these studies may not be sensitive enough to detect changes in dsrna levels if the wt-nsp produces dsrna cleavage products that are > bp, such that the uncleaved and cleaved dsrnas are indistinguishable for binding by the anti-dsrna antibody. importantly, it has been demonstrated that the endou activity of cov nsp is influenced by secondary structures and modifications of dsrna (ivanov et al., ; bhardwaj et al., ) . because these factors may limit the number of cleavage events and/or the targets of endou, nsp -mediated cleavage may produce few overall cleavage products that are < bp. consequently, nsp mediated cleavage may not reduce the level of total dsrna in the cell, but rather may hydrolyze long dsrnas into shorter cleavage products that are sufficiently short to evade recognition by host sensors (e.g. mda ) but not by the anti-dsrna antibody. it has been documented that cov dsrnas form cytoplasmic aggregated foci during replicating in cells (becares et al., ; hagemeijer et al., ) . these dsrna foci co-localize with the viral rtcs early during infection. interestingly, we noted that during the early stages of infection, dsrna foci were not co-localized with the rtcs in the endou-deficient cov-infected cells. this decrease in colocalization relative to wt infection resulted in a dispersed pattern of dsrna foci, such that some dsrnas did not appear to associate with the rtcs. it is not known whether these "free" dsrnas trigger host sensors, but this dispersed distribution of dsrna does notably coincide with early activation of the ifn response and other dsrna sensors (e.g. pkr and oas). overall, additional studies with new methods are needed to characterize the intracellular localizations and the fates of dsrna in cov-infected cells. the detailed strategy used by cov endou to evade host recognition remains enigmatic. more studies are needed to address several key questions: (i) what is the natural target of endou? (ii) how does the endou activity of nsp alter the fate of dsrna? (iii) how is this endou activity regulated to avoid unwanted cleavage events? (iv) do any interaction partners (viral or cellular) of nsp participate in regulating its endou activity (bhardwaj et al., ; athmer et al., ) ? answers to these questions will be essential for understanding the mechanism of the endou-mediated evasion of host dsrna sensors. additionally, it is possible that endou serves as a conserved antagonist in vertebrate nidoviruses. a similar phenotype has been observed in human blood-derived macrophages infected with the hcov- e nsp mutant virus (kindler et al., ) , which is a representative alphacoronavirus. whether arterivirus nsp also functions as an ifn antagonist during infection is still unknown. one obstacle to studying arterivirus nsp mutants in cell culture is that mutations of catalytic residues have been reported to severely affect viral replication even in ifn-deficient cells (posthuma et al., ; sun et al., ) . nonetheless, given the striking roles of cov endou in macrophages and in vivo, it will be important to understand the underlying mechanism of endou-mediated evasion of host antiviral defenses in order to advance an ultimate goal of exploiting this enzyme as a target for therapeutics and vaccine design against existing and emerging pathogenic covs. in situ tagged nsp reveals interactions with coronavirus replication/transcription complex-associated proteins the dsrna-dependent protein kinase, pkr and cell death mutagenesis of coronavirus nsp reveals its potential role in modulation of the innate immune response porcine reproductive and respiratory syndrome virus nonstructural protein beta modulates host innate immune response by antagonizing irf activation structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease nsp the severe acute respiratory syndrome coronavirus nsp protein is an endoribonuclease that prefers manganese as a cofactor rna recognition and cleavage by the sars coronavirus endoribonuclease the coronavirus endoribonuclease nsp interacts with retinoblastoma tumor suppressor protein activation of rnase l by murine coronavirus in myeloid cells is dependent on basal oas gene expression and independent of virus-induced interferon comparative in vivo analysis of the nsp endoribonuclease of murine, porcine and severe acute respiratory syndrome coronaviruses turkey coronavirus non-structure protein nsp -an endoribonuclease coronavirus nonstructural protein mediates evasion of dsrna sensors and limits apoptosis in macrophages severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf and nf-kappab signaling mutational analysis of the sars virus nsp endoribonuclease: identification of residues affecting hexamer formation visualizing coronavirus rna synthesis in time by using click chemistry crystal structure of the lassa virus nucleoprotein-rna complex reveals a gating mechanism for rna binding identification and subcellular localization of a kda, polyprotein ab processing product in human coronavirus e-infected cells the protein kinase pkr is required for macrophage apoptosis after activation of toll-like receptor major genetic marker of nidoviruses encodes a replicative endoribonuclease crystal structure of a monomeric form of severe acute respiratory syndrome coronavirus endonuclease nsp suggests a role for hexamerization as an allosteric switch biochemical and genetic analyses of murine hepatitis virus nsp endoribonuclease early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication functional analysis of the murine coronavirus genomic rna packaging signal mesoniviridae: a proposed new family in the order nidovirales formed by a single species of mosquito-borne viruses mavs-mediated apoptosis and its inhibition by viral proteins biochemical characterization of arterivirus nonstructural protein reveals the nidovirus-wide conservation of a replicative endoribonuclease discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest rna virus genomes small molecule inhibitors of the sars-cov nsp endoribonuclease site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle efficient sensing of infected cells in absence of virus particles by blasmacytoid dendritic cells is blocked by the viral ribonuclease erns cap binding and immune evasion revealed by lassa nucleoprotein structure crystal structure and mechanistic determinants of sars coronavirus nonstructural protein define an endoribonuclease family organ-specific attenuation of murine hepatitis virus strain a by replacement of catalytic residues in the putative viral cyclic phosphodiesterase ns colocalization and membrane association of murine hepatitis virus gene products and de novo-synthesized viral rna in infected cells endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp was essential for nsp to inhibit ifn-β induction a dimerization-dependent mechanism drives the endoribonuclease function of porcine reproductive and respiratory syndrome virus nsp unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage the nonstructural proteins directing coronavirus rna synthesis and processing nonstructural protein of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production nidovirus ribonucleases: structures and functions in viral replication the nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits nf-κb signaling by means of its deubiquitinating activity new antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp structural biology of the arterivirus nsp endoribonucleases antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus virus-encoded proteinases and proteolytic processing in the nidovirales we thank robert c. mettelman and aaron volk for assistance with editing the manuscript. our studies are supported by national institutes of health r ai (to scb). key: cord- - owcqt d authors: iketani, sho; shean, ryan c.; ferren, marion; makhsous, negar; aquino, dolly b.; des georges, amedee; rima, bert; mathieu, cyrille; porotto, matteo; moscona, anne; greninger, alexander l. title: viral entry properties required for fitness in humans are lost through rapid genomic change during viral isolation date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: owcqt d human parainfluenza viruses cause a large burden of human respiratory illness. while much research relies upon viruses grown in cultured immortalized cells, human parainfluenza virus (hpiv- ) evolves in culture. cultured viruses differ in their properties compared to clinical strains. we present a genome-wide survey of hpiv- adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates (zero passage virus). nonsynonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins—the receptor binding protein hemagglutinin-neuraminidase (hn) or the fusion protein (f). we recovered genomes from hpiv- primary culture isolates and hpiv- strains directly from clinical samples. hn mutations arising during primary viral isolation resulted in substitutions at hn’s dimerization/f-interaction site, a site critical for activation of viral fusion. alterations in hn dimer interface residues known to favor infection in culture occurred within days (h and n ). a novel cluster of residues at a different face of the hn dimer interface emerged (p and r ) and imply a role in hpiv- -mediated fusion. functional characterization of these culture-associated hn mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured hpiv- ; the hn-f complex showed enhanced fusion and decreased receptor-cleaving activity. these results utilize a method for identifying genome-wide changes associated with brief adaptation to culture to highlight the notion that even brief exposure to immortalized cells may affect key viral properties and underscore the balance of features of the hn-f complex required for fitness by circulating viruses. altered to suit cultured cells and could no longer be considered clinical strains. there has been a relative dearth of sequence information available for hpiv compared to other respiratory viruses ( ) , and more importantly a lack of information about strains prior to passaging in the laboratory and thereby being subjected to selective pressure. to address the question of which features associated with fitness in humans may be lost in routine viral culture, we sought to understand the genomic changes associated with brief exposure of hpiv- from clinical samples to culture during the isolation process. we used metagenomic next-generation sequencing (mngs) of field strains of hpiv- isolated from humans to analyze the diversity and commonalities of circulating hpiv- strains at the genomic level, and we simultaneously sequenced paired specimens that were subjected to culture for viral isolation. the sequencing strategy we used to sequence clinical strains directly without passage in immortalized cells does not rely on designing sequence-specific primers that may amplify only a subset of samples (in case of mutations) ( ) . deep genomic sequencing of nine sets of paired clinical samples (primary nasal swabs in viral transport medium) and culture isolates (culture harvest from zero passage virus) led to discovery of a number of hn mutations associated with rapid evolution in culture. to assess the frequency of mutations identified earlier, we also performed deep sequencing of hpiv- clinical samples and culture isolates from the university of washington virology laboratory, allowing us to confirm that the alterations associated with brief exposure to culture for viral isolation were almost entirely found in the sequences of culture isolates and found commonly within populations of viruses in those isolates. functional characterization of the effects of several of the specific hn gene mutations found in the cultured isolates in a clinical isolate (ci) background-in expressed hn-f complexes or recombinant viruses bearing the mutated genes-reveals that the culture-associated mutations all increase fusion promotion by the hn-f complex. these results support the notion that clinical strains differ from cultured viruses in the balance of fusion properties. the locations of the residues that differ after brief culture of clinical viruses point to specific domains that are relevant to the control of viral entry in vivo. the sequencing data, when correlated to functional data, help define the flexibility and constraints of the hpiv- fusion complex. these data provide the first characterization of culture-associated hn mutations in the background of hpiv- clinical strains directly from patient samples. we show that, even during transient exposure to culture ("zero passage" virus), the act of isolating a virus is associated with genomic evolution that may significantly affect viral protein function and not reflect in vivo dynamics. mutation and mixed allele frequency comparison between clinical samples and their matched cultured specimens. to compare hpiv- sequenced directly from patients with hpiv- sequenced after brief growth in culture, nine hpiv- -positive (hpiv- ϩ) nasal swabs were sequenced metagenomically, and their paired viral isolates were sequenced after initial isolation in rhesus monkey kidney (rhmk) cells. we investigated the variant alleles with a minor allele frequency of Ͼ % that arose in culture. the nine swabs included one clinical sample that was independently cultured twice, such that ten viral isolates were sequenced. clinical respiratory samples that tested positive for hpiv- by quantitative reverse transcription-pcr (qrt-pcr) with cycle thresholds under were either sequenced directly by metagenomic nextgeneration sequencing (mngs) without exposure to culture or grown on rhmk cells and viral isolates were harvested and sequenced. the choice of rhmk cells for this study was based on the fact that these are the standard cells used for viral isolation by many or most diagnostic microbiology laboratories and would allow us to answer the question of whether this standard process leads to alteration in the viruses. seven of the nine cultured isolates were harvested before day of culture, and no viruses were passaged beyond initial isolation. sequencing reads for the cultured viruses were analyzed for mutational changes relative to the hpiv- consensus genome obtained from metagenomic sequencing of the original clinical sample (without expo-sure to any cell culture), and both assemblies were analyzed for variants with Ͼ % minor allele frequency (maf) ( ) . a total of nonsynonymous mutational changes with maf change of Ͼ % in response to exposure to cell culture were found across the nine sets of hpiv- viral isolates, with of the in the hn protein (p Ͻ . by fisher's exact test) (fig. ) . of the ten mutations found outside the hn protein, four were found in the p protein, four in the l protein, and two in the f protein. of the two l variants that increased in allele frequency in cultured specimens, one (g d) was not found in a separate viral isolation performed on the same clinical sample, while the other (p l) was found at a high allele frequency in the original clinical sample (fig. d to g). evolution of the polymerase complex will be explored separately, as the focus of this current paper is on the entry complex. during viral isolation from sample sc , we noted the appearance of the previously described hn h q mutation at % maf after only days in culture (fig. g) . hn h q has previously been isolated in laboratory-adapted hpiv- strains and is possible cpe day -cpe, had+ sc l n h f m p n r c % r , , , , , , , , , , , , , , mutational changes and allele frequencies in matched clinical samples and cultures. blue genomes represent clinical samples, and the green genomes below the blue genomes represent the same sample after it was inoculated into culture and harvested several days later. the number of days cultured, hemagglutinin adsorption assay results (had), and observed cytopathic effect (cpe) are shown for each viral isolate. virus from sample sc in panel g was isolated twice. coverage is averaged across the entire genome and depicted underneath each genome. allele frequencies are plotted to scale above the genome organization. mutational changes are described relative to the amino acid in the consensus genome of the original patient sample. variants indicated in italics were found exclusively on separate reads and are considered unlinked. iketani et al. ® associated with enhanced fusion promotion as a result of hn's f-triggering function ( , ( ) ( ) ( ) . two independent viral isolations in rhmk cells of this clinical sample resulted in selection for the hn h q variant allele. during viral isolation in rhmk cells from sample sc , an hn n d mutation emerged from % to % allele frequency in only days, the greatest change in allele frequency during the brief exposure to culture (fig. e ). previous comparison of direct clinical isolates with laboratory-adapted strains showed that every clinical strain bore asparagine (n) at position , while our laboratory strains bore aspartic acid (d) at position ( ). the hns derived from clinical isolates (cis) showed from -to -fold-higher neuraminidase activity than hns derived from the lab-adapted strains, and the substitution of the aspartic acid at this position in the laboratory strains led to an approximately -fold-lower receptor cleavage in the laboratory-adapted strains, suggesting that the asparagine at position is essential for the high propensity for receptor cleavage in field strains. the rapid emergence of this adaptation to culture by reducing receptor cleavage is remarkable. during viral isolation from both samples sc and sc , an hn l s mutation, adjacent to residues and mentioned above, arose to % and % maf ( fig. a to i). during isolation of sample sc , the same site contained an l f mutation that was unlinked to the h q mutation that also arose during that isolation (i.e., was found exclusively on separate reads). the sc isolation also resulted in selection of novel mutations hn s n and hn t i, comprising a cluster from positions to with significant alterations in the cultured viruses (fig. h) . while the region of hn corresponding to the cluster at positions to above was known to be of interest for entry in vivo (based on features of h and n ), during independent viral isolations from sample sc , a set of mutations emerged in hn at residues previously unrecognized to be relevant to fitness: p and r (hn p l and hn r k). these mutations were not linked to each other. the p l mutation described above was also recovered during viral isolations of sc and sc ( fig. c to h). adjacent to these residues, another previously undescribed hn mutation l f was selected at a high allele frequency ( %) during isolation of sc and at a low level during isolation of sc ( % maf in k and % maf in cul ). as discussed further below, these three residues describe a region of interest based on our previous structural analysis of the hn dimer interface ( ) . phylogeny and hn mutation history in original samples. to dissect the influence exerted by background hn sequence of the clinical virus on subsequent culture adaptation, we generated a phylogenetic tree based on amino acid substitution distance in the hn protein of our clinical samples (fig. ) . none of the phylogenydetermining mutations among direct clinical sample strains were located in hn's receptor binding site ii or second dimer interface domain that emerged during rapid culture adaptation. independent viral isolates from sample sc ( k and cul ) produced p l, r k, and h q. the hn mutations l f, p l, s n, and l s also arose among separate lineages. large-scale whole-genome sequencing of hpiv- direct-and culture-exposed strains shows that culture-associated mutations are rarely found in clinical strains. to place the alleles that arose in the paired clinical-culture specimens above in the broader context of hpiv- sequence information, we used mngs to obtain whole genomes from an additional hpiv- strains, of which were primary viral isolates (passage zero virus; brief exposure to culture only during viral isolation) and the remaining were directly from patient samples. all specimens were collected in or (see table s in the supplemental material). recombination analysis using rdp ( , ) revealed no recombination among these strains similarly to other nonsegmented negative-strand rna viruses ( , ) . phylodynamic analysis of the concatenated coding regions of the hpiv- sequenced strains revealed a mean mutation rate of . eϪ substitutions/site/year ( % confidence interval, . eϪ to . eϪ ). analysis of concatenated coding regions from all hpiv- sequences with complete coding sequences (cds) in the genbank nt database as of september revealed a similar mean mutation rate of . eϪ substitutions/site/year ( % confidence interval, . eϪ to . eϪ ) similar to those of other paramyxoviruses ( ) . phylogenetic analysis of the newly sequenced hpiv- coding sequences revealed multiple lineages of hpiv- present in - clinical samples and clustering by time of collection as the dominant feature rather than by sequence obtained from viral isolate versus clinical sample (fig. ) . however, examination of the deep sequencing data for of the viral isolates revealed mutations with Ͼ % maf in residues and and residues to selected during the paired sample-isolate analysis above. while the original clinical samples for these isolates are not available, none of the clinical samples sequenced metagenomically contained variant residues at these loci. of the strains with variant residues, only viral isolates and laboratoryadapted strains contained variant residues with Ͼ % maf that would be expected to be reflected in consensus sequence available publicly. in addition to the newly derived hpiv- genome sequences, we also examined all complete and partial consensus hpiv- hn sequences available in the genbank nt database as of august -a total of , sequences-in a search for the culture-associated mutations isolated here. of the culture-associated mutations in the hn protein described above, p l ( ), s n ( ), l f ( ), l s ( ), n d ( ) , and t i ( ) appeared exclusively in sequences from cultured isolates ( , ( ) ( ) ( ) ( ) ( ) ( ) . none of the consensus sequences in ncbi with a p l mutation had a mutation in the to residue range (consensus sequence, -hkslnt- ). the h q mutation appeared previously in sequences, with the vast majority of these ( / ) occurring in culture isolates; note that as above, even sequences listed as clinical may have experienced brief exposure to culture conditions. the l f mutation has not been reported in any consensus hpiv- hn sequence in ncbi. three instances of the r k alteration were found in ncbi genbank ( , ) . one of these was recovered using rt-pcr directly from clinical specimens during an hpiv- outbreak in a children's hospital cancer ward in barcelona, spain, in summer ( ) . the remaining two r k mutations reported were from a likely culture-adapted college of american pathologists proficiency testing sample (strain f , genbank accession no. ky ) and a primary culture isolate from japan (ddbj accession no. ab ) ( ) . hpiv- culture adaptations localize to the dimer interface of hn. we mapped all the residue changes in the hn protein that arose during adaptation to brief culture onto the hpiv- hn protein crystal structure wef (fig. ). all alterations localized to the dimer interface. we previously described critical residues at the hn dimer interface for hn's roles in receptor engagement and activation of f at h -which we identified as the secondary sialic acid binding site-and in receptor cleavage at n , shown at the top of the hn globular head dimer interface in the diagram (fig. ) . the diagram highlights the fact that n likely makes contact with the nac modification of n , and glycosylation could therefore have a significant effect on binding. we have shown that alterations that enhanced dimer contacts were associated with culture adaptation, while host fitness was correlated with reduced dimer contacts at these residues ( ) . alterations in residues and have been associated with other culture conditions parainfluenza virus fitness for host tissues ® as well ( - ), supporting the notion that this site is intimately involved in the mechanism whereby hn activates f in the natural host. the mutations p l and r k have not been described before and appear in a cluster at the opposing side of the dimer interface. the rapid emergence of alterations here during adaptation of a patient's hpiv- to culture suggests a role in viral entry in the natural host. of note, p does not make specific contact with the opposing monomer, but a mutation would change the conformation of the loop significantly, thereby altering the contacts that the loop can have. hn mutations in site ii and dimer interface modulate fusion promotion. to understand the selection forces that affect hpiv- fitness, we next analyzed the biological properties conferred by the culture-associated mutations in hn. here we characterized the influence of each specific residue on the function of the hn-f fusion complex. to investigate whether the identified mutations altered hn's fusion promotion ability we had previously observed in clinical isolates of hpiv- , we introduced seven of these rapidly emerging mutations into the background of a well-characterized clinical isolate hn, clinical isolate (ci- ) ( , ). we paired these mutated hn proteins with a lab-adapted f to ensure that we could observe adequate levels of fusion to compare function, since clinical isolate hn and f pairs result in nearly unobservable levels of fusion ( ) . fusion promotion by each of these mutant hn-f pairs was quantified with a ␤-galactosidase complementation assay and compared to the wildtype clinical isolate hn (ci wt) paired with the lab-adapted f (fig. ) . we included an influenza a virus hemagglutinin (ha) and f pair as a positive control for fusion, as it had been previously shown that hn has a stabilizing property that prevents f triggering; that is, an increase in fusion relative to the ci wt would be expected and was seen ( ) . the fusion exhibited by all mutant hn-f pairs was considerably higher than that of the ci wt hn-f pair, ranging from a -fold increase (hn s n) to a -fold increase (hn l f). neuraminidase activity is reduced by hn's adaptations to culture except for h q. after observing that fusion promotion was increased by all hn mutants, we examined several interacting properties of hn (see introduction) that are involved in this process. we assessed the neuraminidase (receptor cleavage) activity and also quantified the release of receptor-bearing erythrocytes (rbcs) from hn-expressing cells as a measure of the balance between avidity and receptor-cleaving properties. neuraminidase activity was measured through the cleavage of =-( -methylumbelliferyl)-␣-d-n-acetylneuraminic acid, sodium salt hydrate ( -munana) by hn (fig. a) . as previously described, almost all the culture-adapted hn mutants displayed lower neuraminidase activity than ci wt, ranging from roughly % (l s) to % (r k) relative activity ( ). the h q mutant had increased neuraminidase activity, with roughly % activity relative to ci wt. we have previously shown that this mutation at h on hn confers both higher receptor avidity and intrinsically enhanced triggering of f and thereby augments fusogenicity, an effect that overrides the increase in receptor cleavage (see the release assay below) ( ) . release of bound receptor-bearing rbcs by cell surface hns reflects the balance of neuraminidase activity, which would release the bonds tethering the cells, and avidity of the hns, which retain the bound cells (fig. b) . we quantified the amount of rbcs released at several time points when incubated at ph . and °c. differences between the hns was most readily observed at the earliest time point of min, when ci wt had the most release (~ %), whereas the mutant hns all had less release, reflecting either their higher receptor avidity, lower neuraminidase activity, or both. we observed two separate groups of mutants in their capability to release rbcs. a more rapidly releasing group-p l, r k, and s n-had roughly % release at min and complete release (~ %) after min. in contrast, the more slowly releasing group, consisting of h q, l f, l s, and n d, had to % release at min and to % release after min. as noted above, the higher receptor avidity of hn can override the higher-than-wt neuraminidase activity. (fig. b, yellow squares) , producing no detected progeny compared to a titer of to pfu/ml for each of the viruses bearing a single adaptive hn mutation. the growth in cv- cells reveals that while the clinical virus is severely handicapped in monolayer culture, the mutations that emerged during the rhmk culture isolation process confer growth in these monolayer cultures. while the wt ci shows negligible fitness in monolayer culture compared to the adapted viruses on day , by day infectious virus is released into the supernatant fluid of the virus to cell culture. at day however, adaptive mutations have appeared (table ) and correlate with increased production of infectious particles (fig. b, yellow squares) . in hae tissue, the wt ci- has a growth advantage over all the mutated viruses, showing that the individual culture-adaptive hn mutations modestly impair infection of hae (fig. b, orange circles) . at day after hae infection, wt ci releases . ϫ infectious particles into the hae apical surface, which is between and times more particles than released by the mutant viruses. as expected based on our previous studies ( ), growth in hae did not result in emergence of mutations (table ) . mechanisms that affect respiratory virus-cell interplay in the context of humans are finely tuned to the environment of the host ( , ) . to understand natural viral infection and the features that govern infectivity and transmissibility in humans, we have endeavored to analyze field strains in airway tissues. we have shown for human parainfluenza virus type that conclusions drawn from laboratory strains can be misleading with regard to the receptor interaction and viral fusion properties that govern entry and fitness in vivo ( , ) . analysis of clinical strains suggests that the hn-f fusion pairs of circulating hpiv- viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo; the fusion we have previously used whole-genome sequencing of field strains of hpiv- isolated from humans and grown only in airway cultures to analyze the diversity and the commonalities of circulating strains of hpiv- ( ). the sequences were used to define functional and structural properties of proteins of circulating strains in order to identify the genetic basis for properties that confer fitness in the field. while laboratoryadapted and ci airway-grown strains shared the basic properties of the hn-f fusion complex-receptor binding, fusion triggering, receptor cleavage-the balance between these properties at each step is shared across all cis we studied and different from all laboratory strains. the ci fusion pairs all possessed lower fusogenicity and decreased f activation compared to those of laboratory strains. here we carried out deep genomic screens of paired hpiv- clinical samples; one portion of each sample was sequenced directly from the patient with no culture step, while the other portion was exposed to brief culture for viral isolation in immortalized rhesus monkey kidney (rhmk) cells, a standard cell line used in clinical laboratories for virus identification. we hypothesized that even during such brief periods of culture, mutations arising in the hn-f fusion complex could be informative about features essential for growth in humans that would be stringently selected against in culture. hpiv- evolution upon exposure to culture was extraordinarily rapid and almost entirely limited to the hn protein. bioinformatic analyses of existing hn sequences revealed that hn mutations that arose during brief exposure to culture are almost uniformly found in consensus sequences from hpiv- that are either adapted to culture or exposed to culture-and not found in hpiv- sequenced directly from clinical samples. deep sequencing of newly derived hpiv- isolates and clinical samples revealed that more than one-third of hpiv- isolates had subpopulations of hn dimer interface variants that were not reflected in consensus sequences and were not present in hpiv- sequences obtained directly from clinical samples. the sequences were used to define functional and structural properties that differ from laboratory-selected circulating strains in order to identify the genetic basis for properties that may confer fitness in the field. the hn mutations that we identified in response to exposure to culture included both novel and previously observed and characterized adaptations to immortalized cells. remarkably, all the mutations map to the dimer interface and receptor binding site ii of hn. these results point to the critical role of this region in the hpiv- entry mechanism and for fitness in vivo, as we have deduced from smaller-scale and mechanistic studies ( - , ). the h residue at receptor binding site ii at the dimer interface of hpiv- hn influences hn's dimerization and its activation of f ( , ). we have shown that the presence of the glutamine at position at the dimer interface of the hpiv- hn increases hn oligomerization and thereby modulates f triggering, with this hn more actively triggering f ( , ). when the virus bearing this mutated hn was subjected to growth in human airway, it rapidly acquired a compensatory mutation, q r, which reduced fusion triggering and reduced hn dimerization. structural analysis of footprints of the dimer interfaces showed that when hn h q is present in receptor binding site ii, more of the interface residues are in contact with the opposing monomer face. however, in the hn with the q r compensatory mutation that permits growth in the airway, the dimer interface footprint shows decreased points of contact. the dimer interface footprint pointed to several residues and domains whose contact with the opposing monomer may be critical for the key functions governed by hn's receptor binding site ii. the emergence of the h q mutation after brief culture highlights both the flexibility of hn's site ii to adapt to new conditions and also the exquisite constraint of the virus's fusion complex with respect to alterations in the environment. adjacent to the h residue in the dimer interface, but not making contact in the dimer interface footprint mentioned above, is n , a residue we have previously shown to be highly conserved in all clinical strains. this residue was previously found to be an aspartic acid in laboratory-adapted strains, associated with a dramatic decrease in neuraminidase activity consistent with the overall high-receptor-contact/highfusion laboratory-adapted phenotype. in the present study, the n d mutation emerged during the very brief exposure to culture, highlighting the advantage of lowering neuraminidase activity to reduce receptor cleavage for culture conditions versus the importance of neuraminidase for human infection. within the same cluster, s was altered during culture adaptation; this is a residue that contacts the opposing monomer in the dimer interface cluster ( , ) . the precise role of this residue in the functions of the dimer region will be explored. strikingly, several mutations emerged in residues at the opposite side of the hn dimer interface from the cluster at positions to : residues , , and . the points of contact of n and r with the opposing hn monomer in the dimer interface footprint had been noted to correlate with fusion promotion and airway adaptation; however, the function of these residues had been unexplored ( ) . the selection for l f, p l, and r k in the absence of mutations in the domain from positions to suggests that the newly identified important region in the hn dimer interface may act independently of receptor binding site ii, and the functional relationships between these two sites will be explored in future studies. numerous deep sequenced viral isolates also demonstrated p l and r k hn variants in the absence of mutations in the domain from positions to . the finding that this second dimer interface domain undergoes rapid selection in the background of authentic clinical strains suggests that it may be relevant to fitness in the airway. to investigate the biological significance of these adaptations, we generated singly mutated hn molecules in the background of our well-characterized hpiv- clinical virus hn, ci- ( ), and generated recombinant viruses bearing the hn mutations on the genetic background of ci- . brief exposure to culture, through this diversity of strategies, resulted in increased fusion promotion for all the hn variants tested: h q, s n, l f, l s, n d, p l, and r k. while perhaps not unexpected based on our previous hypotheses, the absolute consistency of this finding-that through a range of specific mutations and specific functional and structural differences, the virus arrives at increased fusion-is remarkable. each individual hn mutation conferred fitness for recombinant virus infection of immortalized monolayer culture cells and for spread of virus through the culture, compared to the parent wt ci. to explore the components of hn's properties that contribute to the enhanced fusion promotion and infection properties for all the emerging hn mutants, we assessed neuraminidase activity and hn's release of receptor-bearing target cells, a measure of the balance between receptor avidity and cleavage ( ) . we have shown that clinical viruses, compared to laboratory strains, have a combination of high neuraminidase, low avidity, low f triggering, resulting in lower fusogenicity. the hn mutants that emerged here all except for hn h q had decreased neuraminidase activity. it is of interest that mutations at site ii affect neuraminidase enzymatic activity, since this site is not known to possess enzymatic activity, which resides in the bifunctional active site at t /d ( , ( ) ( ) ( ) . one mechanistic conjecture that will be explored is that alterations or interactions at one hpiv- hn site (site i or ii) modulate the function of the other site, as we have shown for another paramyxovirus, newcastle disease virus, where receptor interaction at hn site i leads to the activation of site ii ( , ) . these data may suggest an unexplored cross talk between site i and site ii that could alter neuraminidase activity allosterically. all the mutants-including hn h q-showed reduced release of rbcs compared to the clinical strain. the hn h q mutant's decreased release of rbcs in the face of increased neuraminidase activity relative to the ci wt is in agreement with our previous finding that this mutation confers increased receptor avidity ( ) that balances the receptor-cleaving activity for an overall retention of contact. interestingly, the h q mutation in the lab-adapted hn background does not have higher neuraminidase activity ( ), supporting the notion that the phenotype conferred by these sites may be modified in the context of the genetic background. collectively, the pattern is of enhanced receptor engagement and an increase in fusogenicity that confers fitness in vitro. the genetically engineered viruses bearing the individual alterations in hn show that each is fit for growth in monolayer culture compared to the wt parent and less fit for growth in hae. the mutations emerging during the brief viral isolation process thus led directly to fitness for growth in monolayer culture. interestingly, there is no single mutation or minor allele required for successful growth in culture, although all samples in this study did produce at least one nonsynonymous minor allele on either the f or hn proteins. the minor alleles were extremely biased toward mutations in the hn or f protein, with eight of ten cultures presenting minor alleles on the hn protein and four presenting a minor allele on the f protein, supporting the critical nature of the hn-f fusion complex for host adaptation ( ). intriguingly, single mutations in hn did not confer fatal growth disadvantage in the airway model, despite conferring fitness in monolayer culture. it will be of interest to determine whether an accumulation of several fusion-skewing mutations is necessary for an absolute inability to grow in airway and whether a more sensitive experimental system (i.e., infection in vivo) would be responsive to single alterations or only to a greater dimer interface disturbance. the genome sequences presented here provide a significant resource for future hpiv- genomic epidemiology and biochemical characterization, increasing the number of publicly available genomes by more than % ( , ) . we obtained full genome sequence coverage from as few as an estimated , starting copies ( ) and found that relatively uniform whole viral genome coverage can be achieved with , paired-end reads, suggesting that many samples could be sequenced in a single miseq run. whole-genome viral sequencing from clinical samples should be both technically and economically feasible, permitting investigators to avoid the use of laboratoryadapted strains of virus in immortalized cultured cell lines. the deep genome-wide screens of paired clinical samples and viral isolates allow for simple, rapid interrogation of viral evolution in the context of multiple clinical strain backgrounds. the methods pursued here allowed for discovery of novel hn residues of interest that heretofore had not been identified based on consensus sequence. deep sequencing after rapid selection pressure may allow for discovery of a greater number of novel mutations that may be removed from the population by drift or competition with other variant subpopulations after longer selection periods yet provide clues to function. other techniques that can more comprehensively profile single mutations, such as deep mutational scanning, are often limited by their use of singular backgrounds, often in the context of culture-adapted strains, and focus on singular proteins. our results are consistent with an "uncertainty principle" for virology: the act of attempting to study a clinical viral sample through commonly performed laboratory procedures, such as viral culture in cell lines, affects the authenticity of the viral biology even before the first passage. that viruses evolve uniquely in culture has been shown for hpiv- ( ) ; however, the extraordinarily rapid nature of evolution in culture, even without passage, is notable. most of the adaptive genotypes could be found as minor alleles only by deep sequencing of clinical samples and viral isolates. with the increased provision of deep coverage viral genomes obtained directly from clinical samples due to diagnostic mngs, experiments similar to the one outlined here will be critical for potentially pathogenic viruses in order to assess whether previous viral characterization studies apply to newly sequenced clinical samples ( , ) . for hpiv- , our results are consistent with the notion that use of authentic culture models that better reflect hpiv- growth in vivo is critical if we are to understand and characterize pathogenesis ( ) . the specific mutations that emerged in the cultured samples, while disturbing from the perspective of the "uncertainty" described above, provide a wealth of information about hn domains that may be critical for fitness in humans. in our focus on the correlation of emerging hn mutations to function, we did not characterize all combinations of hn mutations, and for functional studies, we paired hns with a lab-adapted more fusogenic f protein in order to readily discern differences in hn properties; ongoing studies will explore the potential for epistatic interactions in hpiv- evolution. of particular interest is the cluster of residues around p and r that were uncovered as critical in this study. the interactions of these residues with the opposing monomer, and these points of contact of this domain within the interface, were suggested based on structural data to be relevant for fusion promotion ( ) . future studies will address the function of this hn dimer interface domain in hpiv- fusion and cell entry. sample description. the term "clinical sample" is used to describe primary nasal swabs in viral transport media, while "viral isolate" or "culture isolate" is used to describe culture harvest from zero passage virus (i.e., brief exposure to culture only during viral isolation). for paired clinical sample/culture isolate sequencing, l of nasal swab viral transport medium from nine patients from to with an hpiv- cycle threshold (c t ) of Ͻ was inoculated onto primary rhesus monkey kidney (rhmk) cells (quidel diagnostics) and incubated on a roller drum at °c. the tubes were assessed for cytopathic effect daily and confirmed via hemadsorption with % guinea pig red blood cells (rbcs). positive cultures were harvested and stored at Ϫ °c. deidentified bronchial alveolar lavage (bal) fluid and nasal swab samples from patients from used for prior clinical testing were stored at Ϫ °c and subjected to mngs. in addition, culture harvests for which viral isolation was performed for prior clinical testing of bal fluid samples, nasal swabs, and sputum samples from to at the university of washington virology laboratory were also stored at Ϫ °c and subjected to mngs. culture harvests from to were performed using the same protocol detailed above for the paired sequencing experiment. use of excess deidentified clinical specimens was approved by the local university of washington institutional review board (irb) (protocol study ). sample metadata, including genbank accession numbers for consensus hpiv- genomes, are available in table s in the supplemental material and associated with ncbi bioproject . mngs and amplicon sequencing. mngs was performed as previously described ( ) . rna was extracted from l of thawed culture harvests and clinical samples using the zymo viral rna kit (zymo research) and treated with turbo dnase i (thermo fisher) for min at °c ( ) . doublestranded cdna was made using random hexamers, superscript iii reverse transcriptase (thermo fisher), and sequenase v . (thermo fisher) and cleaned using zymo- dna clean and concentrator (zymo research). mngs libraries were produced by adding one-third volume of nexteraxt (illumina) and finished with cycles of dual-indexed pcr amplification and cleaned using . ϫ ampure beads (beckman coulter) ( ) . libraries were sequenced using one -bp and two -bp runs on an illumina miseq system. libraries from the matched viral isolates were prepared twice in independent library preparations and sequencing runs to control for artifacts of library preparation. amplicon sequencing was performed to investigate linkage between mutations recovered during mngs. reverse transcription-pcr (rt-pcr) was performed using cycles of the default parameters (melting temperature [t m ], °c) in the one-step rt-pcr kit (qiagen) with pcr primers hpiv - f (f stands for forward) ( =-tacagatagggataataactgtaaa- =) and hpiv - r (r stands for reverse) ( =-gctttgctcctaagttttttatatt- =). amplicons were purified using . ϫ ampure beads, and dualindexed truseq libraries were prepared using the kapa hyperprep kit with eight cycles of pcr amplification ( ) . sequence data alignment and analysis. sequencing reads were adapter and quality trimmed using cutadapt and then aligned to the hpiv- reference sequence (nc_ ) with geneious v . . . consensus genomes were extracted for original patient samples, and culture sample sequencing reads were aligned to the sample consensus. consensus genomes were visualized and mutations and minor alleles with coverage of Ͼ ϫ and minor allele frequency (maf) of Ͼ % were called using geneious . . . the known locus of rna editing in the phosphoprotein gene was not included among the variant nonsynonymous alleles, nor were adenosine deaminase-like changes present on the same strand in p virus (affecting minor variants at r g and i v in ci- in table ). phylodynamic analysis. for phylodynamic analysis, coding nucleotide sequences from the newly generated hpiv- genomes sequenced were extracted, concatenated, and aligned using genious v . . and mafft ( ) . the resulting alignment was used to generate a bayesian skyline tree with an uncorrelated relaxed clock and hasegawa, kishino, and yano (hky) substitution model with gamma plus invariant site heterogeneity model (four gamma categories) and three codon partitioning in beast v . . with a markov chain monte carlo (mcmc) length of , , iterations ( ) . tree generation trace information and evolutionary clock rates were gathered and analyzed using tracer v . . ( ). the resulting trees were annotated and compiled using treeannotator v . . and then annotated and scaled in figtree v . . . the same phylodynamic protocol was also performed on all complete hpiv- genomes present in ncbi nt as of august . recombination detection was performed using rdp 's rdp, chimaera, maxchi, genecov, siscan, and seq algorithms with default settings ( ) . to determine whether culture-associated hn amino acid changes were present in our paired sample sequencing, we downloaded all partial and complete hpiv- hn sequences from ncbi nt as of august . metadata on whether specific sequences originated from clinical sample or culture material were gathered from the ncbi genbank sequence record and associated manuscripts in pubmed. however, it is not always possible to determine whether there was brief exposure to culture conditions prior to sequencing. to show how the background of hn protein sequence could influence the amino acid mutations selected during culture, a majority consensus amino acid sequence of hn protein from each putative clinical sample was extracted and aligned using mafft. a neighbor-joining tree was created using the genious tree building algorithm, jukes-cantor distance method, and , replicates. functional characterization of hpiv- hn. the hn mutants examined in this study were generated through site-directed mutagenesis of the previously constructed pcaggs mammalian expression vector with the ci- hn gene ( ) . plasmids were sanger sequence confirmed prior to usage. transfections with plasmids were performed in hek t cells using lipofectamine in accordance with the manufacturer's instructions. cell surface expression of each hn was measured through standard immunofluorescence assay procedures using monoclonal antibodies against hpiv- hn and used for normalization as appropriate. two monoclonal anti-hn antibodies used here (vt- g -b and vt- f -b ) were custom-made by aldevron using dna immunization. briefly, a cdna encoding hpiv- hn was cloned into expression plasmids (pegfp) ( ) . groups of laboratory rats (wistar) were immunized by intradermal application of dna-coated gold particles using a handheld device for particle bombardment (gene gun). serum samples were collected after a series of immunizations and tested in flow cytometry on hek cells transiently transfected with the above expression plasmids. antibody-producing cells were isolated and fused with mouse myeloma cells (ag ) according to standard procedures. measurement of fusion promotion. the ability of individual hns to promote fusion was measured through a ␤-galactosidase complementation assay as previously described ( , ) . hns were paired with the f protein from a lab-adapted strain ( ), transiently transfected in hek t cells, and then cell fusion was quantified. lab-adapted f partner molecules were used as previously described ( , ) , since fusion is minimal when ci hn and f are expressed in pairs, and does not allow for quantitative comparisons. measurement of neuraminidase activity. neuraminidase activity of hns were measured as previously described ( , ) . hek t cells were transiently transfected with individual hns, collected through detachment with mm edta, washed with dulbecco phosphate-buffered saline (dpbs) supplemented with g/liter d-glucose and mg/liter sodium pyruvate (gibco), then resuspended in co -independent medium (ph . ) (gibco). cells were then added to =-( -methylumbelliferyl)-␣-d-n-acetylneuraminic acid, sodium salt hydrate ( -munana) at a : ratio for a final concentration of . mm. fluorescence was measured every min for h at °c with a microplate reader (tecan). measurement of rbc release. the ability of hns to release bound rbcs was measured as described previously ( , ) . rbcs were bound to cells for min at °c and washed to remove unbound rbcs. co -independent medium (ph . ) was then added to the cells, and the cells were incubated at °c for the appropriate times before being collected and replaced for the next time point. after min, rbcs that remained bound to the cells were lysed with ack (ammonium-chloride-potassium) lysing buffer (gibco) and quantified by measurement of absorbance at nm. immortalized cell culture. cv- (african green monkey kidney) and t (human kidney epithelial) cells were grown in dulbecco's modified eagle's medium (dmem) (cellgro; mediatech) supplemented with % fetal bovine serum (fbs) and antibiotics at °c in % co . recombinant virus production and analysis. rhpiv clinical isolate (ci- )-egfp is a recombinant virus generated with a plasmid coding for the majority consensus sequence of hpiv- ci- ( , , ) obtained by deep sequencing and containing an enhanced green fluorescent protein (egfp) expression cassette between p and m viral genes. all mutants were derived from this unique sequence. recombinant viruses were generated by reverse genetics in the rhpiv -ci- -egfp-f k background using a modification of methods previously described ( ) . briefly, t cells were seeded in six-well plates. the following day, the cells were transfected using profection mammalian transfection system, calcium phosphate kit (promega) according to the manufacturer's recommendations with g of pflc plasmid encoding the full-length viral genome, ng of pcaggs-hpiv p, ng of pcaggs-hpiv l, ng of pcaggs-hpiv n, ng of pcaggs-t polymerase, ng of pcaggs-hpiv lab-adapted hn, and ng of pcaggs-hpiv lab-adapted f ( ). the following day, supernatant fluids were replaced with dmem supplemented with % fbs. two days after transfection, the cells were mechanically detached by pipetting up and down and overlaid onto cv- cells in a -cm culture dish. cultured cells were monitored by fluorescence microscopy until % of the cells were infected. the medium was replaced with dmem without fbs, and cells were incubated for up to days at °c in % co . supernatant fluid was collected, centrifuged at ϫ g for min at °c, aliquoted, and kept at Ϫ °c until use. all recombinant viruses were sequenced at this stage using the same mngs methods detailed above on l of supernatant fluid to confirm the correct ci backbone and hn mutation sequences before use in experiments. viral propagation in human airway epithelia. the hae epiairway air- system (mattek corporation) consists of normal human-derived tracheo/bronchial epithelial cells that have been cultured to form a pseudostratified, highly differentiated mucociliary epithelium closely resembling that of epithelial tissue in vivo. upon receipt from the manufacturer, human airway epithelium (hae) cultures were transferred to six-well plates (containing ml medium per well) with the apical surface remaining exposed to air and incubated at °c in % co . hae cultures were infected with , pfu of recombinant clinical isolate egfp ( ) (referred to as ci- -egfp-f k ) at the apical surface for min at °c. the wild type (wt) ci- -egfp-f k or ci- -egfp-f k harboring single mutations in hn (i.e., p l, r k, h q, s n, or l f) was used. at min, the medium containing the inoculum was removed, and cultures were placed at °c and fed each day with ml medium via the basolateral surface. viruses were harvested by adding l of ϫ phosphate-buffered saline (pbs) with calcium and magnesium chloride (mattek corporation) per well to the hae cultures' apical surface and equilibrating for min at °c. the suspension was then collected, and viral titers were determined as previously described ( ) . this viral collection was performed sequentially with the same wells of cells on each indicated day postinfection. viral propagation in cv- . cv- cultures were seeded in -well plates. seventy percent confluent cells were infected with , pfu of recombinant ci- -egfp-f k (wt or harboring single mutations in hn [i.e., p l, r k, h q, s n, l f]) at the apical surface for min at °c. after min, the medium containing the inoculum was replaced with ml of dmem supplemented with % fbs. the supernatant fluid was then collected, aliquoted, and replaced with the same volume of dmem supplemented with % fbs, and viral titers were determined as previously described ( ) . this viral collection was performed sequentially with the same wells of cells on each indicated day postinfection. supplemental material for this article may be found at https://doi.org/ . /mbio . - . table s , pdf file, . mb. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis control of an outbreak of human parainfluenza virus in hematopoietic stem cell transplant recipients epidemiology and clinical presentation of the four human parainfluenza virus types features of circulating parainfluenza virus required for growth in human airway circulating clinical strains of human parainfluenza virus reveal viral entry requirements for in vivo infection interaction between the hemagglutinin-neuraminidase and fusion glycoproteins of human parainfluenza virus type iii regulates viral growth in vivo adaptation of human parainfluenza virus to airway epithelium reveals fusion properties required for growth in host tissue unity in diversity: shared mechanism of entry among paramyxoviruses triggering of human parainfluenza virus fusion protein (f) by the hemagglutininneuraminidase (hn) protein: an hn mutation diminishes the rate of f activation and fusion mechanism of fusion triggering by human parainfluenza virus type iii: communication between viral glycoproteins during entry regulation of paramyxovirus fusion activation: the hemagglutinin-neuraminidase protein stabilizes the fusion protein in a pretriggered state the hemagglutininneuraminidase of human parainfluenza virus type : role of the neuraminidase in the viral life cycle a structural explanation for the low effectiveness of the seasonal influenza h n vaccine effects of egg-adaptation on receptor-binding and antigenic properties of recent influenza a (h n ) vaccine viruses cell culture adaptation of hepatitis c virus and in vivo viability of an adapted variant sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture antigenic drift of the pandemic a(h n ) influenza virus in a ferret model clinical sequencing uncovers origins and evolution of lassa virus quantification of bk virus standards by quantitative real-time pcr and droplet digital pcr is confounded by multiple virus populations in the who bkv international standard copy number heterogeneity of jc virus standards cooperating h n influenza virus variants are not detectable in primary clinical samples prolonged shedding of human coronavirus in hematopoietic cell transplant recipients: risk factors and viral genome evolution global epidemiology of non-influenza rna respiratory viruses: data gaps and a growing need for surveillance a decade of rna virus metagenomics is (not) enough measurements of intrahost viral diversity are extremely sensitive to systematic errors in variant calling a second receptor binding site on human parainfluenza virus type hemagglutininneuraminidase contributes to activation of the fusion mechanism relative affinity of the human parainfluenza virus type hemagglutinin-neuraminidase for sialic acid correlates with virus-induced fusion activity molecular evolution of the haemagglutininneuraminidase gene in human parainfluenza virus type isolates from children with acute respiratory illness in yamagata prefecture, japan rdp : detection and analysis of recombination patterns in virus genomes homologous recombination in negative sense rna viruses the evolutionary and epidemiological dynamics of the paramyxoviridae a recombinant, infectious human parainfluenza virus type expressing the enhanced green fluorescent protein for use in high-throughput antiviral assays nucleotide and deduced amino acid sequence of hemagglutinin-neuraminidase genes of human type parainfluenza viruses isolated from to contribution of cysteine residues in the extracellular domain of the f protein of human respiratory syncytial virus to its function genetic characterization of parainfluenza virus derived from guinea pigs fixation of oligosaccharides to a surface may increase the susceptibility to human parainfluenza virus , , or hemagglutinin-neuraminidase a prototype recombinant vaccine against respiratory syncytial virus and parainfluenza virus type multiplex real-time pcr for prompt diagnosis of an outbreak of human parainfluenza virus in children with acute leukemia engineered intermonomeric disulfide bonds in the globular domain of newcastle disease virus hemagglutinin-neuraminidase protein: implications for the mechanism of fusion promotion fusion promotion by a paramyxovirus hemagglutinin-neuraminidase protein: ph modulation of receptor avidity of binding sites i and ii the anti-influenza virus agent -gu-dana (zanamivir) inhibits cell fusion mediated by human parainfluenza virus and influenza virus ha inhibition of parainfluenza virus type and newcastle disease virus hemagglutinin-neuraminidase receptor binding: effect of receptor avidity and steric hindrance at the inhibitor binding sites paramyxovirus receptor-binding molecules: engagement of one site on the hemagglutinin-neuraminidase protein modulates activity at the second site second sialic acid binding site in newcastle disease virus hemagglutinin-neuraminidase: implications for fusion database resources of the national center for biotechnology information clinical metagenomic identification of balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis rapid metagenomic next-generation sequencing during an investigation of hospital-acquired human parainfluenza virus infections a metagenomic analysis of pandemic influenza a ( h n ) infection in patients from north america rule-out outbreak: -hour metagenomic next-generation sequencing for characterizing respiratory virus source for infection prevention myeloablation-associated deletion of orf in a human coronavirus e infection mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform bayesian phylogenetics with beauti and the beast . tracer v mechanism of interference mediated by human parainfluenza virus type infection infection of ciliated cells by human parainfluenza virus type in an in vitro model of human airway epithelium human parainfluenza virus infection of the airway epithelium: the viral hemagglutinin-neuraminidase regulates fusion protein activation and modulates infectivity we thank the patients from western washington state and staff of the university of washington virology laboratory who contributed to the project. this work was supported by nih/national institute of allergy and infectious diseases (niaid) r ai and r ai to a.m. r.c.s. received support from the mary gates endowment for students. key: cord- -d hsguds authors: coudert, pascal title: les principales maladies du porc date: - - journal: actualités pharmaceutiques doi: . /j.actpha. . . sha: doc_id: cord_uid: d hsguds résumé plusieurs pathologies autrefois mortelles au sein des élevages porcins ont disparu de notre territoire, mais de nombreuses maladies polysystémiques subsistent. si l’antibiothérapie s’avère parfois efficace, la vaccination permet de prévenir un certain nombre de pathologies alors qu’il n’existe aucun traitement préventif ni curatif pour d’autres. par ailleurs, tous les organes du porc sont susceptibles d’être la cible d’agents pathogènes. là encore, des vaccins et des traitements antibiotiques sont disponibles. summary several pathologies which were once mortal within pig farms have disappeared from our territory, numerous polysystemic diseases remain. if antibiotherapy is sometimes effective, vaccination can prevent a certain number of pathologies while there is no preventive of curative treatment for others. moreover, all the pig's organs are susceptible to being targeted by pathogenic agents. here again, vaccines and antibiotic treatments are available. a vec l'amélioration des conditions d'élevage, notamment grâce aux contrôles vétérinaires réguliers, l'état sanitaire des élevages porcins s'est nettement amélioré au fil des années. c'est ainsi qu'en france, certaines affections ont quasiment disparu. À l'inverse, des pathologies, comme la maladie de glässer, sont encore susceptibles d'engendrer d'importantes pertes économiques dans des élevages à haut niveau sanitaire. l'élevage en flux continu sur paille ou en plein air associé à des insuffisances de prophylaxie favorise l'apparition de pathologies comme le rouget, la gale et l'ascaridose. en engraissement, les maladies respiratoires et les entérites sont fréquemment retrouvées. enfin, l'âge des animaux constitue un élément à prendre en compte : la diarrhée colibacillaire peut entraîner une forte mortalité chez le porcelet. en , six maladies et infections à déclaration obligatoire (encéphalite à virus nipah, gastro-entérite transmissible, cysticercose, peste porcine classique et africaine, syndrome dysgénésique et respiratoire) ont été recensées par l'organisation mondiale de la santé animale pour les suidés . un certain nombre de maladies polysystémiques causent encore aujourd'hui des pertes économiques significatives pour les éleveurs [ ] [ ] [ ] . peste porcine ✦ causée par un virus de la famille des flaviridae, la peste porcine classique n'est actuellement plus présente en france tant dans les cheptels porcins domestiques que sauvages. les porcs peuvent se contaminer au contact d'un animal malade, par ingestion de viande de porc contaminée ou de produits dérivés infectés. les sangliers vétérinaire pratique sauvages constituent un réservoir de virus en europe de l'est. ✦ les animaux infectés présentent de la fièvre, une perte d'appétit, une conjonctivite, une constipation suivie de diarrhées et des lésions hémorragiques cutanées, l'ensemble pouvant être fatal dans les formes aiguës. À l'inverse, il existe des formes bénignes sans symptômes apparents. aucun traitement spécifique n'est disponible et la vaccination préventive des cheptels porcins n'est plus autorisée dans l'union européenne depuis suite à la mise en place d'un programme d'éradication de la maladie. ✦ la peste porcine africaine, dont l'agent responsable est un virus à acide désoxyribonucléique (adn) de la famille des asfarviridés, est apparue en europe en mais ne touche actuellement que les pays de l'est (lituanie, pologne, lettonie, estonie, république tchèque) tout en progressant régulièrement vers l'ouest. la symptomatologie de cette maladie est très proche de celle de la peste porcine classique. fièvre aphteuse et maladie vésiculeuse ✦ la fièvre aphteuse est causée par un aphtovirus, virus à arn de la famille de picornaviridae. la symptomatologie de la maladie est caractérisée par de la fièvre à laquelle s'associent des aphtes dans la bouche et des cloques sur les pieds. il n'existe aucun traitement spécifique. si les porcs adultes guérissent généralement spontanément, une mort soudaine suite à des lésions de la musculature cardiaque est possible chez les porcelets. l'europe est considérée indemne mais des épizooties peuvent apparaître suite à l'introduction d'animaux malades, d'objets ou de produits contaminés. ainsi, en , plusieurs foyers ont été recensés en france après utilisation, en angleterre, de déchets de cuisine d'un navire comme aliments pour porcs. ✦ la maladie vésiculeuse, impossible à distinguer d'un point de vue clinique de la fièvre aphteuse, provoque également de la fièvre, des vésicules se transformant en ulcères superficiels sur les pieds, le groin, les mamelles ou dans la bouche. cette affection, pour laquelle il n'existe ni traitement ni vaccin, résulte d'une infection par des entérovirus de la famille des picornaviridae. il est donc nécessaire de procéder à des analyses en laboratoire pour établir un diagnostic de certitude. salmonellose ✦ plusieurs espèces de salmonelles sont à l'origine de la salmonellose porcine, en particulier salmonella derby, s. typhimurium et s. choleraesuis. si l'infection d'un élevage est souvent inapparente, une multiplication importante de ces bactéries au niveau intestinal peut engendrer une diarrhée aqueuse brun jaunâtre avec amaigrissement, pratique vétérinaire des sténoses rectales, une septicémie, voire une toux en cas d'atteinte pulmonaire. ✦ la thérapeutique fait appel à la colistine ou la gentamycine par voie injectable car les animaux malades ne boivent plus et cessent de s'alimenter. néanmoins même à la suite d'un traitement antibiotique, les porcs peuvent rester porteurs sains pendant des semaines, voire des mois. ✦ due à la bactérie haemophilus parasuis, dont il existe sérotypes différents plus ou moins virulents, la maladie de glässer est une affection dont le symptôme principal est une polysérosite fibrineuse. streptococcus suis, mycoplasma hyorhinis peuvent être à l'origine d'un tableau clinique similaire chez le porc. d'autres troubles sont susceptibles d'apparaître tels une polyarthrite, une méningite, une septicémie, une myosite et des difficultés respiratoires. le regroupement d'animaux d'origines différentes et les facteurs de stress (sevrage, transport) favorisent l'appa rition de la maladie au sein de grandes exploitations. ✦ l'antibiothérapie fait appel aux bêtalactamines (amoxicilline, ampicilline, ceftiofur) ou aux associations pénicilline-streptomycine et triméthoprime-sulfamide. toutefois, du fait de l'existence d'un nombre élevé de souches d'h. parasuis multirésistantes, la réalisation d'un antibiogramme est recommandée pour la mise en place d'un traitement. les vaccins disponibles stimulent le développement d'une immunité active uniquement contre les sérotypes les plus prévalents et (suvaxyn ® m. hyo-parasuis) ou seulement (porcilis ® glässer). ✦ l'agent du rouget est une bactérie à gram positif dénommée erysipelothrix rhusiopathiae. les principales voies d'inoculation sont cutanées et intradermiques (écorchures). la maladie peut présenter des évolutions variées : suraiguë ou aiguë, parfois fatales en quelques heures, chronique, septicémique ou localisée. les symptômes sont donc divers : lésions cutanées sous forme de plaques d'urticaire rectangulaires, endocardites, néphrites et arthrites surtout au niveau des jarrets. ✦ les pénicillines a ou g s'avèrent en général efficaces, mais la doxycycline ou les macrolides peuvent être utilisés en cas d'allergie aux bêtalactamines. des vaccins existent en prévention du seul rouget (eryseng ® , ruvax ® ) ou couplés à la parvovirose porcine (eryseng parvo ® , parvoruvax ® , porcilis ® ery + parvo). ces derniers ne protègent pas les porcs contre l'évolution chronique de cette affection et favorisent même parfois l'apparition d'arthrites. ✦ les infections à streptococcus suis font partie des infections majeures dans les élevages porcins. elles affectent surtout les porcelets sevrés et les porcs à l'engraissement. la pathologie peut revêtir plusieurs aspects selon la virulence de la souche et l'organe atteint : hyperthermie, septicémie, méningite, pneumonie, endocardite ou atteinte articulaire. ✦ le traitement fait appel à des antibiotiques : l'ampicilline ou l'amoxicilline. reproduction, mise bas et lactation ✦ un certain nombre d'affections rencontrées au cours de la reproduction est consécutif à des problèmes alimentaires tel le syndrome de la truie maigre qui apparaît en cas de sous-alimentation en gestation, puis en lactation. il est souvent difficile de convaincre les éleveurs que, dans cette situation, un parasitisme externe est la conséquence et non la cause de la maladie. À l'inverse, il existe un syndrome de la truie grasse due à une suralimentation modérée continue, appréciée par des replis graisseux entre les cuisses. ✦ par ailleurs, la présence de problèmes locomoteurs avec une faiblesse des membres en fin de gestation, d'un allongement de la durée moyenne de la mise bas et d'écoulements vulvaires caractérise une truie grasse à la mise bas et maigre au sevrage ("truie accordéon"). il est aujourd'hui admis qu'il est possible d'améliorer le déroulement des mises bas et de la lactation par l'alimentation afin de dynamiser la vitalité des porcelets nouveau-nés. ✦ si les écoulements vulvaires sont physiologiques chez la truie en chaleurs, ils ont pour origine, chez la cochette non saillie, des infections ascendantes provenant de l'environnement et traduisant une immaturité du système immunitaire. chez la truie nullipare, un épisode infectieux peut apparaître à jours après une contamination lors de la saillie. classiquement l'endométrite postpartum se rencontre chez la truie multipare après une colonisation de l'endomètre par des staphylocoques peu fréquente, mais transmissible à l'homme, la brucellose porcine concerne les élevages de plein air contaminés par la faune sauvage. maladies d'élevage des porcs veterinary microbiology and microbial disease merck sharp & dohme corp. msd veterinary manual key: cord- -ru ubss authors: roingeard, philippe; raynal, pierre‐ivan; eymieux, sébastien; blanchard, emmanuelle title: virus detection by transmission electron microscopy: still useful for diagnosis and a plus for biosafety date: - - journal: rev med virol doi: . /rmv. sha: doc_id: cord_uid: ru ubss transmission electron microscopy (tem) is the only imaging technique allowing the direct visualization of viruses, due to its nanometer‐scale resolution. between the s and s, tem contributed to the discovery of many types of viruses and served as a diagnostic tool for identifying viruses directly in biological samples, either in suspension or in sections of tissues or mammalian cells grown in vitro in contact with clinical samples. the diagnosis of viral infections improved considerably during the s, with the advent of highly sensitive techniques, such as enzyme‐linked immunosorbent assay (elisa) and pcr, rendering tem obsolete for this purpose. however, the last years have demonstrated the utility of this technique in particular situations, due to its “catch‐all” nature, making diagnosis possible through visualization of the virus, without the need of prior assumptions about the infectious agent sought. thus, in several major outbreaks in which molecular techniques failed to identify the infectious agent, tem provided the answer. tem is also still occasionally used in routine diagnosis to characterize infections not diagnosed by molecular assays. it is also used to check the microbiological safety of biological products. many biopharmaceuticals are produced in animal cells that might contain little‐known, difficult‐to‐detect viruses. in this context, the “catch‐all” properties of tem make it possible to document the presence of viruses or virus‐like particles in these products. however, the last years have demonstrated the utility of this technique in particular situations, due to its "catch-all" nature, making diagnosis possible through visualization of the virus, without the need of prior assumptions about the infectious agent sought. thus, in several major outbreaks in which molecular techniques failed to identify the infectious agent, tem provided the answer. tem is also still occasionally used in routine diagnosis to characterize infections not diagnosed by molecular assays. it is also used to check the microbiological safety of biological products. many biopharmaceuticals are produced in animal cells that might contain little-known, difficult-to-detect viruses. in this context, the "catch-all" properties of tem make it possible to document the presence of viruses or virus-like particles in these products. microscope had a much higher resolution than any of the light microscopes available at the time and promised to revolutionize many aspects of science, including cell biology and virology. ernst ruska was a physicist ( nobel prize winner in physics), but his younger brother, helmut ruska, who had trained in medicine, rapidly recognized the potential of this microscope for investigating the nature of viruses. in the early s, viruses were classified according to their hosts and the clinical symptoms they caused. despite the lack of established methods of biological sample preparation for transmission electron microscopy (tem) at this time, helmut ruska was able to characterize the morphology of several viruses and he developed a rough viral classification based on the size and shape of the viral particles. tem was rapidly adopted for its first major use in clinical virology: the differential diagnosis of smallpox, caused by the variola virus abbreviations: elisa, enzyme-linked immunosorbent assay; em, electron microscopy; emea, european medicines agency; fda, food and drug administration; fpert, fluorescent product-enhanced reverse transcription; lcmv, lymphocytic choriomeningitis virus; pcr, polymerase chain reaction; sars, severe acute respiratory syndrome; sfts, severe fever with thrombocytopenia syndrome; tem, transmission electron microscopy from the poxvirus family, and chickenpox, caused by the varicellazoster virus of the herpes family, based on investigations of fluid samples from the vesicles on the patients' skin. the chickenpox virus appeared to be spherical and to nm in diameter, with a central body, a structure clearly different from that of the much larger, brick-shaped smallpox virus. "dirty" clinical samples, such as plasma, urine and feces in the s constituted a major breakthrough for studies of these viruses. the etiologic agents of hepatitis b and a were detected in plasma and stool samples, respectively. the bk virus, a polyomavirus, was identified for the first time in the urine of patients undergoing renal transplantation. rotaviruses were also identified by tem as the main cause of epidemic gastroenteritis in humans and animals. , however, many other viruses were also found to cause gastroenteritis. the first of these viruses was the norwalk virus, identified during an outbreak of gastroenteritis in norwalk, ohio, usa. , viruses with a similar morphology were subsequently discovered elsewhere and called "norwalk-like" viruses, to reflect the similarity of their appearance on tem, before being officially renamed "noroviruses." other viruses from the adenovirus, astrovirus, , and calicivirus families were also identified in the stool samples of children with gastroenteritis. tem was, thus, widely used on negatively stained samples for routine diagnosis, as a rapid, "catch-all" method for distinguishing between the diverse viruses potentially implicated in human gastroenteritis, providing a diagnosis within minutes of the arrival of the sample in the laboratory. although more time-consuming, due to the need to embed a sample in resin and cut ultrathin sections with an ultramicrotome, the tem has also proved useful in medical virology, in searches for viruses in tissues. panels e and f in figure illustrate a parapoxvirus (orf virus) visualized in a skin biopsy specimen from a patient with a severe finger ulcer this was the case, in particular, for the diagnosis of viral gastroenteritis, for which molecular techniques capable of identifying most of the virus families involved in human gastroenteritis have been established. [ ] [ ] [ ] [ ] a similar shift in practice occurred in veterinary medicine, with elisas and pcr progressively replacing tem for the routine diagnosis of viral infections. [ ] [ ] [ ] [ ] in human medicine, the use of tem to differentiate between smallpox virus and the other viruses present in the fluids of cutaneous vesicles is no longer required, since the successful eradication of the variola virus in thanks to a worldwide vaccination program. it has been argued that tem remains potentially useful for this application in a context of bioterrorism. , however, the risk of smallpox reappearing is extremely small, and even in the unlikely event of this happening, molecular techniques would undoubtedly outperform tem for this diagnosis. consequently, the number of laboratories making use of tem for diagnostic purposes has decreased considerably. sometimes revealing unexpected infectious agents, while molecular methods require previous knowledge of the virus to be tested. tem has also the advantage of being able to potentially identify double or multiple infections caused by more than one virus, which could be missed by molecular or antigen tests. moreover, the nature of the samples to be analyzed can be diverse, from body fluids or biopsies analyzed directly or after cell culture. in some cases, tem has also been used to confirm a diagnosis previously established with molecular techniques. [ ] [ ] [ ] [ ] figure illustrates most of the viral particles produced by these cells such as intracytoplasmic or intracisternal a-type particles are defective and noninfectious ( figure ) . however, other particles such as c-type particles bud at the cell surface and may infect nonrodent cells ( figure ). some murine retroviruses have been shown to be tumorigenic in primates, and cases of leukemia have been reported in children with severe combined immunodeficiency treated by gene therapy involving the use of murine retroviral vectors. all these elements demonstrate the relevance of tracking the presence of retroviruses in biological products derived from rodent cells. reverse transcriptase assays performed on bulk harvests are often hampered by high background levels due to cell-derived dna polymerases. tem may, therefore, help to document the presence of retrovirus-like particles in these bulk harvests ( figure ). tem can also be used to gauge the concentration of viral particles to validate the clearance of retroviruses or any other virus suspected to be present in the master cell bank. fortunately, the endogenous retroviruses present in the chinese hamster ovary (cho) cell line, the main rodent cell line used to produce biological products in the biotech industry, have been shown to be noninfectious. testing requirements are now lower for this well-characterized cell line than for other cell lines with which experience is more limited. nevertheless, detection of retrovirus-like particles by transmission electron microscopy (tem) with negative staining in bulk harvests of rodent cells used for the production of biological products. scale bars represent nm in both panels novel cell substrates, including insect cell lines in particular, are now being introduced into the biotech industry. their use will carry new concerns about unknown viruses for which there is a potential risk of contamination, and tem will undoubtedly be useful for documenting the presence of viruses or virus-like particles in these cells and the products derived from them. in conclusion, although tem is sometimes seen as a somewhat "oldfashioned" technique, it still has an important role to play in virus detection. it is particularly useful for identifying unknown agents involved in particular outbreaks or transmission clusters. in routine diagnosis, it may be useful to confirm or even, in some cases, to guide the diagnosis of a viral infection. tem can also be used to check the viral safety of biopharmaceutical products. this technique has several disadvantages, such as the cost of electron microscopes and their maintenance, the need for well-trained microscopists, and timeconsuming analysis, particularly if the samples must be embedded in resin for the cutting of ultrathin sections. however, all the techniques available have benefits and disadvantages, and their complementary natures mean that there are advantages to be gained by using them in combination. in this respect, the principal advantage of tem is its ability to provide an image of the virus, providing additional confidence in the result. nobel lecture: the development of the electron microscope and of electron microscopy die bedeutung der ubermikroskopie fur die virusforschung versuch zu einer ordnung der virusarten the use of the electron microscope in diagnosis of variola, vaccinia, and varicella a negative staining method for high resolution electron microscopy of viruses direct electron-microscopy of organ culture for the detection and characterization of viruses viruses in the stools virus-like particles in serum of patients with australia antigen-associated hepatitis hepatitis a: detection by immune electron microscopy of a virus-like antigen associated with acute illness new human papovavirus (b.k.) isolated from urine after renal transplantation virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis virus particles in gastroenteritis visualization by immune electron microscopy of a -nm particle associated with acute infectious nonbacterial gastroenteritis the discovery of the -nm norwalk virus: an historic perspective the electron microscopical and physical characteristics of small round human fecal viruses: an interim scheme for classification a summary of taxonomic changes recently approved by ictv epidemic viral enteritis in a long-stay children's ward viruses and gastroenteritis in infants nm particles in faeces in infantile gastroenteritis caliciviruses in man orf skin ulcer bk virus infection in a renal transplant recipient broadly reactive nested reverse transcription-pcr using an internal rna standard control for detection of noroviruses in stool samples real-time reverse transcription-pcr for detection of rotavirus and adenovirus as causative agents of acute viral gastroenteritis in children detection of human sapovirus by real-time reverse transcription-polymerase chain reaction real-time reverse transcription pcr detection of norovirus, sapovirus and astrovirus as causative agents of acute viral gastroenteritis development of a ssrna internal control template reagent for a multiplex rt-pcr to detect turkey astroviruses an elisa optimized for porcine epidemic diarrhoea virus detection in faeces epidemiology of norwalk-like virus infections in cattle in the netherlands detection and molecular characterization of cultivable caliciviruses from clinically normal mink and enteric caliciviruses associated with diarrhea in mink countering the posteradication threat of smallpox and polio bioterrorism and electron microscopic differentiation of poxviruses from herpesviruses: dos and don'ts application of transmission electron microscopy to the clinical study of viral and bacterial infections: present and future a morbillivirus that caused fatal disease in horses and humans fatal encephalitis due to nipah virus among pig-farmers in malaysia role of electron microscopy in nipah virus outbreak investigation and control a novel coronavirus associated with severe acute respiratory syndrome ultrastructural characterization of sars coronavirus the detection of monkeypox in humans in the western hemisphere fever with thrombocytopenia associated with a novel bunyavirus in china transmission of lymphocytic choriomeningitis virus by organ transplantation fatal hemorrhagic fever caused by west nile virus in the united states life-threatening cache valley virus infection a new phlebovirus associated with severe febrile illness in missouri nosocomial outbreak of parechovirus infection among newborns diagnosis of progressive multifocal leukoencephalopathy in the absence of dna from the jc virus in the cerebrospinal fluid new adenovirus species found in a patient presenting with gastroenteritis discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin complete genome sequence of a sapporo virus gv. variant from a outbreak of gastroenteritis in sweden clinicopathologic analysis of coxsackievirus a new variant induced widespread mucocutaneous bullous reactions mimicking severe cutaneous adverse reactions novel poxvirus infection in an immune suppressed patient novel poxvirus infection in patients from the united states novel orthopoxvirus infection in an alaska resident novel human reovirus isolated from children with acute necrotizing encephalopathy isolation and molecular biological investigations of avian poxviruses from chickens, a turkey, and a pigeon in croatia lethal outbreak of disease associated with feline calicivirus infection in cats an outbreak of the polyomavirus infection in budgerigars and cockatiels in slovakia, including a genome analysis of an avian polyomavirus isolate fatal poxvirus outbreak in a colony of new world monkeys identification and phylogenetic analysis of orf virus from goats in taiwan an outbreak of teat papillomatosis in cattle caused by bovine papilloma virus (bpv) type and unclassified bpvs poxvirus infection in a great tit (parus major) outbreak of cutaneous form of poxvirus on a commercial turkey farm caused by the species fowlpox an outbreak of pseudocowpox in fattening calves in southern brazil fatal outbreak in tonkean macaques caused by possibly novel orthopoxvirus, italy the use of convalescent sera in immuneelectron microscopy to detect non-suspected/new viral agents guidance for industry: a viral safety evaluation of biotechnology products derived from cell lines of human or animal origin adventitious agent issues one-step fluorescent probe productenhanced reverse transcriptase assay retroviruses produced by hybridomas genomic organization and expression of endogenous retrovirus-like elements in cultured rodent cells helper virus induced t cell lymphoma in nonhuman primates after retroviral mediated gene transfer lmo -associated clonal t cell proliferation in two patients after gene therapy for scid-x evaluation of a quantitative productenhanced reverse transcriptase assay to monitor retrovirus in mab cell-culture characterisation of endogenous retrovirus in rodent cell lines used for production of biologicals virus detection by transmission electron microscopy: still useful for diagnosis and a plus for biosafety we wish to thank fabienne arcanger, sonia georgeault, christine the authors have no competing interest.orcid philippe roingeard http://orcid.org/ - - - key: cord- -ji emajn authors: zhou, jie‐ying; peng, ying; peng, xiao‐you; gao, han‐chun; sun, ya‐ping; xie, le‐yun; zhong, li‐li; duan, zhao‐jun; xie, zhi‐ping; cao, you‐de title: human bocavirus and human metapneumovirus in hospitalized children with lower respiratory tract illness in changsha, china date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: ji emajn background: lower respiratory tract illness is a major cause of morbidity and mortality in children worldwide, however, information about the epidemiological and clinical characteristics of lrtis caused by hmpv and hbov in china is limited. objectives: human bocavirus (hbov) and human metapneumovirus (hmpv) are two important viruses for children with lower respiratory tract infections (lrti). we aimed to assay the correlation between viral load and clinical characteristics of hbov and hmpv with lrti in changsha, china. methods: nasopharyngeal aspirates (npas) from children with lrti were collected. real‐time pcr was used to screen hbov and hmpv. analyses were performed using spss . software. results: pneumonia was the most frequent diagnosis. there was no significant difference between hbov‐ and hmpv‐positive patients in age (p = . ) or hospitalization duration (p = . ); . % and . % were positive for hbov and hmpv. hbov infections peaked in summer ( . %), and hmpv infections peaked in winter ( . %). the hbov‐positive patients had a shorter hospitalization duration than the hbov‐negative patients (p = . ), and the hmpv‐positive patients had a higher prevalence of fever than the hmpv‐negative patients (p = . ). the hbov viral load was significantly higher among patients aged < year (p = . ). the mean hbov and hmpv viral loads were not significantly different between patients with single infections and coinfections. patients infected with hbov only were older than those coinfected with hbov and other respiratory viruses (p = . ). no significant difference was found in the clinical characteristics of patients infected with hmpv only and those coinfected with hmpv and other respiratory viruses. conclusion: pneumonia was the most frequent diagnosis caused by hbov and hmpv. neither hbov nor hmpv viral load was correlated with disease severity. hmpv, which was originally isolated in the netherlands and is closely related to avian metapneumovirus, was the first human disease-causing pathogen identified in the genus metapneumovirus. hmpv causes various clinical symptoms, such as cough, wheezing, and fever. young children and the elderly are more susceptible to hmpv infection. , indeed, hmpv mainly infects children under years of age and causes upper respiratory tract and severe lower respiratory tract infections. [ ] [ ] [ ] [ ] [ ] hbov was discovered in sweden and classified in the family parvoviridae, and causes various clinical symptoms, including cough, wheezing, and fever. hbov infections are largely confined to infants and young children (< months). globally, the average prevalence of hbov in respiratory tract samples ranges from . (ci: . - . ) to . % (ci: . - . ). however, information about the epidemiological and clinical char- (table ) . to standardize quantification of hbov and hmpv, known numbers of dna or rna transcripts containing the primer targets were used in -fold serial dilutions ( to copies/μl). any amplification detected before cycles was considered positive. quantification of > copies/μl ( copies/ml) was considered a positive result. an internal positive control gene (gapdh), positive control, and negative (water) control were included in all reactions. viral loads were expressed as initial copy numbers per real-time pcr reaction, and quantitative results were transformed as the log number of viral copies/μl. continuous variables are expressed as means ± standard deviation (sd) and were compared by independent samples t test. categorical variables are expressed as frequencies or percentages. comparisons were performed by chi-squared or fisher's exact test. analyses were performed using spss . software. two-sided p-values <. were considered to indicate statistical significance. in total, we analyzed samples collected from lrti patients patients were divided into two groups: those with and without hbov or hmpv infections (table ) (table ). according to the british thoracic society guidelines for the management of community-acquired pneumonia in children and the clinical diagnosis, lrtis were classified as mild to moderate or severe. the mean hbov and hmpv viral loads did not differ significantly between the two groups (p = . and p = . ) (figure ). in this study, we used real-time pcr to explore the etiology and ex- with lrtis. this is higher than previous study ( . %) in changsha that used traditional pcr, and lower than that a study in lanzhou that used real-time pcr. on the one hand, the high detection rate in our study could be caused by the greater sensitivity of real-time pcr compared with traditional pcr. on the other hand, it is possible that the detection rate differs geographically. rsv was the most frequently virus detected in patients with respiratory infections, followed by piv , hbov, adv, and hmpv. our data support previous reports of rsv, piv , hbov, adv, and hmpv as the major agents associated with lrtis among children in a hospital setting. , [ ] [ ] [ ] [ ] the hbov infection rate ( . %) in the present study is consistent with earlier reports ( . %- . % , , , [ ] [ ] [ ] [ ] , and the incidence of hmpv ( . %) in patients with respiratory tract infections is similar to that in other regions ( . %- % , , , , [ ] [ ] [ ] . therefore, hmpv and hbov are major causes of lrtis worldwide. the seasonal peaks of hbov and hmpv infections vary among countries because of differences in climatic and geographic factors. in this study, hbov activity peaked in summer, in agreement with the report by jiang. in contrast, detection of hbov in lanzhou peaked in december and april, possibly due to the dry, cold weather in lanzhou and the warm, humid weather in changsha. hmpv detection peaked in winter, which is in line with previous studies. , , , in contrast, in hong kong, hmpv detection peaks in spring/summer. the seasonality of hbov differed geographically, possibly due to climatic factors. the most frequent symptoms of hbov-and hmpv-positive patients were cough and fever, in accordance with previous reports. , , however, deng reported that in chongqing, wheezing was the most frequent symptom exhibited by hbov-positive patients with severe lrtis. there was no difference between the hbov-and hmpv-positive patients in the incidence of fever, tachypnea, cyanosis, o therapy, or wbc count. the hbov-positive patients had a shorter hospitalization duration than hbov-negative patients (p = . ). in contrast, deng reported that hbov-positive patients had a longer hospitalization duration. the longer hospitalization duration of hbov-negative patients in our study may have been caused by the presence of other viruses (such as rsv and piv ) in many of them. also, hospitalization duration was significantly associated with age (≤ months), maternal smoking during pregnancy, and a family history of asthma. a high hmpv viral load contributes to development of fever. , indeed, hmpv-positive patients had a higher incidence of fever than hmpv-negative patients (p = . ) in our study. we assessed the relationship between viral load and clinical features (age, gender, respiratory rate, temperature, cyanosis, hospitalization duration, and wbc count). the only significant association of hbov-or hpmv-positive patients was a higher viral load in < -year-old hbov-positive patients. this is in agreement with jiang's report that patients with a high viral load were significantly younger. in contrast, the duration of wheezing and hospitalization was longer in children with a high than a low hbov viral load in the study by deng, possibly due to inclusion of only patients with severe lrtis. hmpv and hbov coinfections with other viruses are common in this study has the following strengths: a considerable duration, large number of patients, and comparison of the most common viruses. its limitation includes the lack of a control group without clinical evidence of illness. further studies should compare a symptomatic group with a control group and a symptomatic period with an asymptomatic period. in summary, we present a prospective study of lrtis caused by hbov and hmpv. a further comprehensive and in-depth study of the role of hbov and hmpv in lrtis in china is warranted. a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in germany outbreak of human metapneumovirus infection in a severe motor-and-intellectual disabilities ward in japan viral load and acute otitis media development after human metapneumovirus upper respiratory tract infection clinical disease and viral load in children infected with respiratory syncytial virus or human metapneumovirus clinical characteristics and viral load of respiratory syncytial virus and human metapneumovirus in children hospitaled for acute lower respiratory tract infection detection of human metapneumovirus and respiratory syncytial virus by real-time polymerase chain reaction among hospitalized young children in iran incidence, morbidity, and costs of human metapneumovirus infection in hospitalized children high human bocavirus viral load is associated with disease severity in children under five years of age human bocavirus: current knowledge and future challenges british thoracic society guidelines for the management of community acquired pneumonia in children: update etiology, seasonality and clinical characterization of viral respiratory infections among hospitalized children in beirut, lebanon infections and coinfections by respiratory human bocavirus during eight seasons in hospitalized children abdel baset rw. respiratory syncytial virus and human metapneumovirus in severe lower respiratory tract infections in children under two human metapneumovirus infection in jordanian children: epidemiology and risk factors for severe disease high viral load of human bocavirus correlates with duration of wheezing in children with severe lower respiratory tract infection burden of human metapneumovirus infection in young children clinical features of human metapneumovirus genotypes in children with acute lower respiratory tract infection in changsha severe lower respiratory tract infection in infants and toddlers from a non-affluent population: viral etiology and co-detection as risk factors viruses in community-acquired pneumonia in children aged less than years old: high rate of viral coinfection associations between co-detected respiratory viruses in children with acute respiratory infections multiple viral respiratory pathogens in children with bronchiolitis viral etiologies of hospitalized acute lower respiratory infection patients in china bocavirus in children with respiratory tract infection 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 viral coinfection in acute respiratory infection in mexican children treated by the emergency service: a cross-sectional study comparison of severe pneumonia caused by human metapneumovirus and respiratory syncytial virus in hospitalized children detection of human bocavirus and human metapneumovirus by real-time clinical significance of different virus load of human bocavirus in patients with lower respiratory tract infection human bocavirus infection in young children with acute respiratory tract infection in lanzhou the prevalence of human bocavirus, human coronavirus-nl , human metapneumovirus, human polyomavirus ki and wu in respiratory tract infections in kuwait children with respiratory disease associated with metapneumovirus in hong kong human metapneumovirus (hmpv) infection in immunocompromised children human metapneumovirus and human bocavirus associated with respiratory infection in apulian population real-time pcr for diagnosis of human bocavirus infections and phylogenetic analysis human metapneumovirus viral load is an important risk factor for disease severity in young children association between high nasopharyngeal viral load and disease severity in children with human metapneumovirus infection development and assay of rna transcripts of enterovirus species a to d, rhinovirus species a to c, and human parechovirus: assessment of assay sensitivity and specificity of real-time screening and typing methods application of taqman lowdensity arrays for simultaneous detection of multiple respiratory pathogens human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays impact of human coronavirus infections in otherwise healthy children who attended an emergency department real-time quantitative pcr detection of four human bocaviruses human bocavirus and human metapneumovirus in hospitalized children with lower respiratory tract illness in changsha, china we thank li-li li, na-liu, jie-mei yu, li-li pang, and other staff members in department of viral diarrhea (national institute for viral disease control and prevention) for detection of virus pathogens. the authors have no competing interests to report. the process obtained families' informed consent, and the study protocol was approved by the first affiliated hospital of hunan normal university, changsha, china. http://orcid.org/ - - - key: cord- -tdswzj r authors: freitas, andré ricardo ribas; donalisio, maria rita title: excess of mortality in adults and elderly and circulation of subtypes of influenza virus in southern brazil date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tdswzj r purpose: in the elderly population, the influenza infection and its clinical complications are important causes of hospitalization and death, particularly, in longer-lived age. the objective of this study is to analyze the impact of influenza virus circulation on mortality in the elderly and adults, in years with different predominant virus strains. methods: we performed a time trend study to evaluated excess of mortality for pneumonia and influenza, respiratory disease, and all-causes in southern region of brazil, from to . after considering other models, we opted for serfling regression. excess of death rates per , inhabitants were analyzed in specific age groups ( – , – , – , ≥ years) and by year of occurrence. mortality information were taken from brazilian mortality information system and etiological data were accessed in sentinel virological surveillance database, getting the weekly positivity of the immunofluorescence tests for influenza a (h n , h n ), and b. results: in southern brazil, there is an evident seasonal pattern of all death outcomes among different age groups in the dry and cold season (april–september). the highest excess mortality rates occurs among older, particularly in years of circulation of influenza ah n , especially among people ≥ years, in and —years of great severity of influenza activity. after , with the introduction of the pandemic influenza ah n , we observed a lower impact on the mortality of the elderly compared to < years. discussion: a cross reactivity antibody response from past exposure probably provided protection against disease in the elderly. despite not controlling for comorbidities, climate, and vaccination, for the > years, ratio of respiratory diseases excess mortality rates between ah n ( ) and severe year of h n ( ) shows protection in the pandemic year and great vulnerability during ah n virus predominance. conclusion: the reduced immune response to infection, and to vaccination, and presence of comorbidities recommend a special attention to this age group in brazil. besides medical assistance, the timeliness of vaccine campaigns, its composition, and etiological surveillance of respiratory diseases are some of the preventive and public health measures. introduction human influenza viruses can cause diseases through many direct and indirect pathological effects. consequences are destruction of infected cells, release of cytokines leading to fever, malaise, damage to respiratory epithelium and pulmonary parenchyma, and pneumonia. it includes secondary bacterial infections because of tissue damage and exacerbation of preexisting comorbidities such as cardiovascular and renal diseases, diabetes, or chronic lung disease ( ) ( ) ( ) . the rates of hospitalization and mortality associated with influenza are higher among patients with chronic diseases, children under year and after years of age ( , ) . with the aging population in recent decades, the raw number of hospitalizations and deaths related to pneumonia and influenza tends to increase ( ) , this phenomenon has been observed also in brazil ( , ) . however, the impact and severity of influenza virus circulation depend in part, on the strain that predominates in the season each year. due to the lack of laboratory confirmation, influenza-associated morbidity and mortality are often classified as pneumonia, other respiratory diseases, or other causes. given the difficulty of directly measuring influenza morbidity and mortality, time series models are used to elucidate disease patterns in various age groups. trends are usually determined by means of statistical inference, based on seasonal coincidence of the occurrence of certain diseases or death and laboratory confirmation of the viral circulation ( , ) . different approaches, with and without the quantification of the proportion of viral isolates, can produce average estimates of excess deaths associated with the circulation of certain viral variants ( ) ( ) ( ) . viral surveillance data, hospitalization, or death indicators are particularly useful for the study of influenza in the tropics, as seasonality may be less evident ( ) ( ) ( ) . serfling regression has been used to analyze excess of mortality related with respiratory virus circulation ( , ( ) ( ) ( ) . despite some limitations ( ) , the inclusion of sinusoidal terms in weekly regression may reduce spurious correlation between influenza occurrence and death ( , ) . it is particularly useful when no other covariables are available, and with small samples of viral sentinel surveillance data ( ) . poisson regression and the generalized linear model (glm) can produce more specific estimates and support adjustments for variables (temperature, humidity, comorbidities, other circulations of viruses), although they require a more robust and consistent virological surveillance and cannot be used for pandemics ( ) . in brazil, surveillance for influenza syndromes was implemented in , monitoring the occurrence of respiratory viruses (influenza a and b, parainfluenza , , and , respiratory syncytial virus, adenovirus). the brazilian ministry of health provides vaccination coverage annually since for seniors and some risk groups, with vaccine coverage of the elderly population at around % in southern brazil, the region with the highest coverage of the country. despite the adequate coverage, protective titers after vaccination (hi ≥ ) are consistently lower with poorer cell mediate and antibody responses in the elderly comparing to adults ( ) . considering the vulnerability of the elderly to influenza virus infection, and the lack of studies on its repercussion in brazil, the objective of this study was to analyze the impact of different strains of influenza a virus circulation. we analyzed particularly the most predominant variants (ah n and ah n ) on excess of mortality in the adults and elderly of different age groups in a region with marked seasonality of respiratory diseases in brazil. this is a time trend study to evaluated excess of mortality from to in southern region of brazil (states of paraná, santa catarina e rio grande do sul), total area is , , km , population is , , inhabitants with subtropical climate (köppen-geiger classification cfa). we choose these states for analysis because of the consistent seasonal pattern of influenza, as well as the availability and quality of etiological data from the virological surveillance system in that region. for the mortality rates of specific age groups ( - , - , - , and ≥ years) and death causes, we took data from brazilian mortality information system. causes are classified according to international causes of death icd- revision, pneumonia, and influenza (icd j to j . ), respiratory diseases (icd j to j ), and all-cause (excluding external causes of mortality). we obtained population of each year and age group from instituto brasileiro de geografia e estatística-ibge from the census- , and population estimates for the following years. etiologic information of flu-like syndrome was accessed in database of the national sentinel virological surveillance system. it has data from sentinel units distributed in all regions of the country-north ( units), northeast ( units), southeast ( units), south ( units), and central west ( units). surveillance is performed through the systematic collection of weekly samples of nasopharyngeal secretions from patients who present flu-like syndrome. reference laboratories process samples by using indirect immunofluorescence (iif), with tests for influenza a and b, parainfluenza , , and , respiratory syncytial virus, and adenovirus. a portion of the samples is submitted to polymerase chain reaction tests to identify the virus genotype. we calculated the laboratory positivity indicator using weekly positive results of iif divided by the total of weekly valid tests, i.e., excluding the results within inadequate samples (not enough biological material, improper storage, incorrect material in the sample) or inconclusive results (no valid results). influenza vaccination coverage (%) of southern region from to was obtained from brazilian national program of immunization data base (datasus). the criteria used to define the period of increase of influenza activity was when the positivity of the samples tested exceeded twice the annual mean of the weekly positivity of samples processed by surveillance, during two consecutive weeks. in the year , we consider the period officially recognized by the brazilian ministry of health as epidemic by the influenza ah n pmd strain, due to irregularity of the sample collection by the sentinel surveillance system at the end of epidemic. we calculated the weekly mortality rates by age group using the number of deaths per group of causes divided by the estimated population in the middle of the year multiplied by , . we constructed a serfling cyclical regression model ( ) for weekly data applied to each age group and causes of death (pneumonia and influenza, respiratory diseases, and all causes), as seen in others studies ( , ) , to estimate baseline of predicted deaths in the absence of influenza epidemics. to fit regression, we used period of years (from to ), excluding the weeks of epidemics periods. a cyclical linear regression was adjusted with the equation: where y is the mortality rate, β is the coefficients of regression, t is time in weeks, and t and t are variables for adjusting the secular trend of the disease. we used of sine and cosine for adjust of annual and semiannual periodic components. after adjusting a linear regression and define the expected mortality rate, we delimited % upper confidence limit of the baseline as the reference threshold in the absence of influenza epidemics. we calculated the excess of deaths as the observed mortality minus the expected mortality in the periods when mortality was above % of the confidence interval during epidemics periods. we also present ratios of excess mortality rates among years of predominant circulation of influenza strains ah n (mean and years of severity), ah n pre-pandemic, and ah n postpandemic for each age group. for data compilation, we used microsoft office excel , and for statistical analysis, spss for windows, version . . results table shows the proportion of positivity of the iif nasopharyngeal samples and the annual prevalence of strains of influenza in the period. before , the year of entry of the pandemic strain ah n pmd , there was a predominance of influenza ah n in the years to . after , there is alternation of strains in the southern brazil. annual elderly vaccination coverage in southern region is high and homogeneous, around %, and even higher in the recent years. there is an evident seasonal pattern of deaths from pneumonia and influenza, respiratory diseases, and all-causes among the elderly in different age groups in the dry, cold months (april-september) in southern region (figure ) . we note a progressive increase in the rates of excess deaths (of all outcomes) with increasing age, especially among those older than years. in the pre-pandemic years with dominance of the ah n strain, the excess of mortality rates associated with influenza were relatively low, compared to years of prevalence of ah n strain ( table ) . among those over years, the ratio of excess mortality rates between and the years with dominium of h strains was less than one. this ratio suggests that this age group was spared in the pandemic. however, in years of predominance of strain h , excess of mortality rate of all causes in this group were . per , (corresponding to , obits), , and . times greater than the same rate in years of circulation of h n in pre-and post-pandemic period, respectively. among adults ( - years), we observe a large excess of deaths rates during the pandemic ( obits), which correspond to . excess deaths from all causes, and excess mortality from respiratory diseases associated with viral infection in every , individuals of the age group. the ratio between excess mortality rates due to pneumonia/influenza in the pandemic year ( ) and the mean rate of the period was times higher among the youngest ( table ) . rates of excess mortality by pneumonia and influenza and respiratory diseases are lower than all causes in all age groups, but particularly high in older than years ( table ) . the results highlight the great vulnerability of elderly to influenza ah n , especially among older than years in severe years of influenza activity, like and . the study also shows the lower impact of influenza ah n pdm in this age group compared to younger. risk of dying among the elderly in years of circulating ah n influenza has been reported in several parts of the world ( , , , ) ; however, in brazil, there are no recent estimates available. few studies analyze the circulation and impact of influenza in tropical and subtropical regions ( , , ( ) ( ) ( ) . influenza b virus is also associated with severe disease ( ); however, this variant did not circulate with intensity during the study years in brazil. although the elderly are the most vulnerable group to viral respiratory infections, we found relative small excess of deaths in years of circulating ah n pre pandemic ( and ) . study comparing excess deaths from respiratory diseases in the elderly in latin america shows stable rates (mean of . per , inhabitants) in southern brazil between and (prepandemic flu a-h n ), although higher in brazil than in other countries ( ) . in the usa and in european countries, influenza seasons dominated by subtype ah n are typically associated with mortality two to three times higher than in seasons with predominance of ah n (prior to pandemic strain ) and of influenza b viruses ( , , , ) . when all causes of death are studied, the overall mortality associated with influenza among elderly exceeds that observed in younger age group. it should be considered that all causes mortality is a non-specific measure and a distant outcome of influenza infection. however, it is difficult to determine which group of causes of death could better characterize the influenza burden in mortality. by choosing only the respiratory causes, we may underestimate clinical complications of pulmonary viral infection (e.g., cardiovascular). therefore, in this study, we analyzed all causes, respiratory, and pneumonia and influenza deaths. the unfavorable evolution of infection in the elderly is possibly due to the prevalence of comorbidities, deficiencies in defense mechanisms, and poor antibody response to vaccination, as cellmediated and humoral responses limit severity of disease ( ) . patients with chronic diseases are more susceptible to infection due to decline of the immune function through inflammatory mechanisms, hindering the mucosal barrier, and the adaptive and innate immunological defense mechanisms ( ) . the immune response to infection in the elderly tend to be delayed and weak, with prolonged inflammatory responses, which involves different types of host reaction, mainly to clearance virus. the exacerbations of these mechanisms may induce immunemediated pathology causing tissue damage ( ) . cytokine high serum levels of il- , tnf-a, ifn-g and sil- r, chemokines ip- , mcp- , and monokine induced by ifn-g (mig), are associated with severe clinical cases and lung damage ( ) . immunological abnormalities in people with diabetes, chronic respiratory diseases, cardiopathy, or other chronic diseases have increased risk of severe infection and bad prognosis ( ) . for example, there is the consistent association of influenza infection with cardiovascular mortality, particularly acute myocardial infarction ( ) . in part, it is attributed to altering endothelial function due to an acute inflammatory and procoagulant stimulus during viral infection ( , ) . clinical complications of diabetes triggered by influenza infection cause impairment of leukocyte function and increase post-infection colonization rates resulting in poor prognosis in the elderly ( , ) . in young people and adults, in , the emerging influenza ah n strain had a notable impact on the mortality of people up to years in various parts of the world, including brazil ( , , , ) . excess mortality of individuals aged - years in the state of são paulo, brazil was identified during the pandemic ah n virus ( ) . pregnant women adults with metabolic conditions, including obesity, chronic respiratory disease, and other chronic diseases were significantly associated with severe acute respiratory syndrome and the lethality in brazil ( ) . our study showed a . -fold higher rate of mortality from pneumonia and influenza in adults ( - years) in the pandemic year ah n than the average of years with predominance of ah n circulation in southern region. in addition to the clinical severity and the large portion of the affected population, pandemics affect age groups in different ways ( ) . while only % of deaths from seasonal influenza occur among those under years of age, in the pandemics of , - , and , this proportion was , , and %, respectively ( ) . therefore, pandemics tend to affect a larger proportion of young people than seasonal influenza. in this study, higher rates of death due to pneumonia, influenza, respiratory, and all causes were observed among those aged - years in . one explanation for the higher mortality observed among the youngest is that they would be more prone to the situation known as "cytokine storm, " i.e., a dysfunctional overproduction of cytokines that would lead to diffuse damage to the respiratory tract with severe and potentially lethal systemic repercussions ( ) . viral replication and production of inflammatory mediators seem to be involved in the pathogenesis of infection with influenza a h n pmd , hindering the clearance of virus in lung tissue and leading to pathologic lesions ( ) . another explanation for the lower mortality in the elderly is that they were exposed previously to antigens of the pandemic virus. hancock et al. ( ) suggested a cross-reactive antibody response to pandemic ah n . similarities between ah n antigen from and were detected. this last virus strain has not circulated since ( ) , when the ah n strain was displaced by ah n (asian flu). at that time, ah n viral circulation occurred mainly in children, the current elderly of . the emergence of the ah n strain in the pandemic year (hong kong flu) affected several age groups. this new strain resulted from a large genetic mutation (shift) recombining virus material of the circulating ah n with the avian h , of asian origin, resulting in the new variant ah n ( ) . in - , under selective pressure an antigenic small mutation (drift), resulted in a/fujian/ / (h n ) a strains emerged after a "jump" in genes evolution of hemagglutinin and neuraminidase proteins of virus surface ( , ) . the circulation of the fujian strain had a great impact on the mortality from pneumonia in several parts of the world in - and - ( ) and in brazil ( ) . in , a new drift resulted in influenza ah n detected in south brazil ( ) also affecting hospitalizations and deaths in various parts of the world ( ) . we observed high rates of excess mortality in the elderly, in the years of and . limitations of this study refer mainly to the ecological analysis of pooled data. we did not analyze individual information regarding comorbidities and history of vaccination that could be important confounders influencing mortality ( ) . we just had the overall annual vaccination coverage which were in general, around % in the period. estimates of the number of deaths (all causes, respiratory, and pneumonia-influenza) supposedly related to influenza may be inaccurate in inferring the impact of respiratory viruses. correlations in time series studies may produce spurious associations, especially between all causes of death and influenza infection, due to the distance between cause and outcome, and to multiple components of the obits. serfling addresses part of this limitation by introducing sinusoidal terms in equation, since non-influenza mortality is not expected to coincide exactly with sinusoidal pattern ( , ) . moreover, excess mortality of pneumonia, respiratory diseases, and all causes can be considered as an alert to surveillance of viral respiratory diseases, such as a sentinel indicator to be investigated ( , ) . although all causes mortality is a non-specific indicator, it does not underestimate the complications of chronic diseases associated with influenza ( ). despite the influenza component in all causes mortality is small, the indicator can be considered an indirect measure, a warning, useful in epidemiological monitoring. another limitation is the lack of robust etiologic data from virological surveillance in the years - , which could lead to imprecision in the analyses; however, the data on the predominance strains in the southern region are reliable, and influenced the composition of the vaccine of each season. considering the option for the analysis model, serfling linear regression may produce different estimates when compared with other models (poisson, arima, and glm) ( , ); poisson and arima models produce higher mortality estimates than serfling, and serfling higher than glm, especially among the elderly ( , , ) . we chose serfling model because we do not have robust virological surveillance data, before , and the study period includes a pandemic year ( ) . besides, in this study, we did not analyze climatic variables (minimum temperatures and relative air humidity) that could also interfere with viral transmission and increase the impact of the disease, particularly in the elderly. in conclusion, probably previous exposures to influenza ah n in the past influenced the mortality of brazilian elderly in , despite the vulnerability of this age group to clinical complications. for the > years, we observe higher excess mortality rates (of all outcomes) in severe year of ah n circulation ( , ) . it is also worth noting that vaccination has been associated with the prevention of death particularly at age ( ) . therefore, the high elderly vaccination cover in southern brazil may have attenuated excess of mortality estimated, although the immune response is limited among those. more attention should be given to the circulation of influenza ah n in subtropical regions in brazil. the reduced immune response to infection and to vaccination, and associated comorbidities recommend a special attention to this age group. besides medical assistance, the timeliness of vaccine campaigns, its composition, and etiological surveillance of respiratory diseases in the region are some of the preventive and public health measures. both authors made contributions to the conception of the work, acquisition, analysis, interpretation of data, and writing the manuscript. center for disease control and prevention. prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) influenza and the winter increase in mortality in the united states, - impact of respiratory virus infections on persons with chronic underlying conditions epidemiology of seasonal influenza: use of surveillance data and statistical models to estimate the burden of disease influenza-related hospitalizations among children in hong kong trends in mortality from respiratory diseases among the elderly and the influenza vaccine intervention mortality associated with influenza in tropics, state of sao paulo, brazil, from to : the pre-pandemic, pandemic, and post-pandemic periods influenza-related deaths -available methods for estimating numbers and detecting patterns for seasonal and pandemic influenza in europe excess mortality associated with influenza epidemics in portugal time series methods for obtaining excess mortality attributable to influenza epidemics influenza associated mortality in the subtropics and tropics: results from three asian cities influenza in tropical regions seasonality of influenza in brazil: a traveling wave from the amazon to the subtropics methods for current statistical analysis of excess pneumonia-influenza deaths impact of influenza vaccination on seasonal mortality in the us elderly population influenza-related mortality in spain is influenza-like illness a useful concept and an appropriate test of influenza vaccine effectiveness? estimates of us influenza-associated deaths made using four different methods mortality associated with influenza and respiratory syncytial virus in the united states role of humoral and cell-mediated immunity in protection from influenza disease after immunization of healthy elderly mortality burden of the a/h n influenza pandemic in france: comparison to seasonal influenza and the a/h n pandemic the global circulation of seasonal influenza a (h n ) viruses comparing clinical characteristics between hospitalized adults with laboratory-confirmed influenza a and b virus infection trends in mortality from respiratory disease in latin america since and the impact of the influenza pandemic estimated global mortality associated with the first months of pandemic influenza a h n virus circulation: a modelling study t cell mediated immunity to influenza: mechanisms of viral control t-cell immunity to influenza in older adults: a pathophysiological framework for development of more effective vaccines a question of self-preservation: immunopathology in influenza virus infection fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia influenza as a trigger for acute myocardial infarction or death from cardiovascular disease: a systematic review acute respiratory tract infections: a potential trigger for the acute coronary syndrome influenza and atherosclerosis: vaccination for cardiovascular disease prevention multiple immunological abnormalities in patients with type (insulin dependent) diabetes mellitus use of influenza and pneumococcal vaccines in people with diabetes mortality attributable to influenza in england and wales prior to, during and after the pandemic risk factors for severe outcomes following influenza a (h n ) infection: a global pooled analysis pandemic h n influenza in brazil: analysis of the first , notified cases of influenza-like illness with severe acute respiratory infection (sari) pandemic versus epidemic influenza mortality: a pattern of changing age distribution epidemiology of influenza and its control aberrant innate immune response in lethal infection of macaques with the influenza virus cytokine and chemokine profiles in lung tissues from fatal cases of pandemic influenza a (h n ): role of the host immune response in pathogenesis crossreactive antibody responses to the pandemic h n influenza virus the evolution of human influenza a viruses from to : a complete genome study molecular evolution of human influenza a/h n virus in asia and europe from to molecular characterization of influenza viruses collected from young children in uberlandia, brazil-from virus influenza detectados no estado do rio grande do sul durante center for disease control and prevention. influenza activity -united states and worldwide, - season the impact of influenza epidemics on mortality: introducing a severity index deaths averted by influenza vaccination in the us during the seasons / through / 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. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - awng h authors: goggin, rachel k.; bennett, catherine a.; bassiouni, ahmed; bialasiewicz, seweryn; vreugde, sarah; wormald, peter-john; psaltis, alkis j. title: comparative viral sampling in the sinonasal passages; different viruses at different sites date: - - journal: front cell infect microbiol doi: . /fcimb. . sha: doc_id: cord_uid: awng h background: with the emergence of the microbiome as an important factor in health and disease in the respiratory tract standardised, validated techniques are required for its accurate characterisation. no standardised technique has been reported specifically for viral sampling in the sinonasal passages. aim: to optimise viral sampling techniques from the sinonasal cavity. methods: sterile cytology brushes were used under endoscopic guidance to sample the sinonasal mucosa at time of endoscopic sinus surgery at both the middle and inferior meatuses (mm and im). dna and rna were extracted from the samples and underwent pcr or rt-pcr testing, respectively, for a panel of common upper respiratory tract viruses. results: twenty-four adult patients were recruited for this study. / ( %) patients were positive for virus in at least one site, while / ( %) were positive for virus at both sites. the mean number of viruses identified at the two sites were similar ( . ± . at the mm vs. . ± . at the im). / ( %) of patients showed no virus at either site, while / ( . %) demonstrated the same viral species at both sites. conclusion: although the number of viruses present at different sites with the nasal cavity are similar, discord exists in the viral species between sites. it is therefore recommended that both sites are sampled in the clinical and research setting better to characterise the viral species within the nasal cavity. the role of the healthy human microbiome in prevention and eradication of disease is an area of burgeoning interest in recent years. the interplay between various colonising organisms, their relative abundance, and the importance of a fine microbial balance has been shown to be essential for normal functioning of multiple organ systems, not least respiratory (lloyd-price et al., ; mitchell and glanville, ) . conversely, disruption of this balance between viruses, bacteria, and single-celled eukaryotes has been implicated in numerous disease processes, including acute infective processes as well as many chronic inflammatory diseases (lloyd-price et al., ) . microbial dysbiosis (specifically bacterial) has been implicated in several respiratory diseases, including asthma (fazlollahi et al., ) and chronic rhinosinusitis (crs) (cleland et al., ) . persistent nasal and paranasal sinus inflammation characteristic of crs affects up to % of the western population (fokkens et al., ) and manifests as nasal congestion, facial pain or pressure, anterior or post-nasal drainage, and reduction or loss of smell (benninger et al., ) . although the exact aetiopathogenesis of this condition remains elusive, it is considered multifactorial in origin. current theories include the fungal hypothesis, the bacterial hypothesis (implicating dysbiosis with staphylococcus aureus overgrowth, superantigen production, and biofilm formation), and an overactive immune response (resulting in chronic inflammation and defective mechanical and innate immune barriers to infection in the crs population) (lam et al., ) . an area that is anecdotally suggested to play a role in crs pathogenesis is a viral dysbiosis (jang et al., ; cho et al., ). this is due to self-reports by many crs patients that their symptoms almost invariably developed after an initial viral upper respiratory tract infection (urti) . research into the ideal method to sample the sinonasal bacterial microbiome is ongoing (copeland et al., ) , however similar efforts to investigate and standardise sampling of the virome have not been made. studies attempting to investigate the upper respiratory virome are limited. the lack of standardisation in sampling has led to conflicting results regarding the presence of virus and the composition of the virome. collection techniques employed thus far include nasal washes, aspirates, brushings, and traditional viral swabs, with viral analysis performed by pcr (cheung et al., ; tao et al., tao et al., , ramadan et al., ; jang et al., ; zaravinos et al., ; wood et al., ; cho et al., ; costa et al., ; liao et al., ; abshirini et al., ; lima et al., ; rowan et al., ) . few studies have compared sampling methods; heikkinen et al. found no difference in the detection of childhood influenza comparing nasal swabs and aspirates (heikkinen et al., ) . spyridaki et al. found a higher detection of rhinovirus (rv) in nasal lavages compared with nasal brushings, but found no difference in any other viruses tested when comparing these to nasal aspirates and swabs (spyridaki et al., ) . to date there have been no studies that have compared different sites within the sinuses and nasopharynx in terms of viral detection. the aim of the study here presented was to establish differences in viral detection and species sampled from different sinonasal sites, in an effort to validate and standardise viral collection techniques, and facilitate further investigation of the sinonasal virome. patients for this study were recruited from the tertiary rhinologic practices of the two senior authors (pjw and ajp). this study was carried out in accordance with the recommendations of the central adelaide local health network ethics committee (hrec/ /tqeh/ ). the protocol was approved by the same. all subjects gave written informed consent in accordance with the declaration of helsinki. patients were included in this study if they were older than years of age and were undergoing endoscopic surgery. control patients consisted of patients with an absence of clinical or radiologic evidence of crs. crs patients fulfilled the diagnostic criteria for crs as outlined in the american guidelines (rosenfeld et al., ) . the radiological severity of disease was scored for all patients using a lund-mackay score (lms) (lund and mackay, ) . using an aseptic technique, endoscan cytology brushes (mcfarlane medical, melbourne, australia) were used to sample the sinonasal mucosa (figure ) of the left and right middle meatuses (mm) and inferior meatuses (im) of each patient. this was done under endoscopic visualisation with caution to avoid cross-contamination from neighbouring tissue. the samples were then placed in a viral transport medium [ % roswell park memorial institute medium supplemented with % foetal bovine serum, % amphotericin b, and % penicillin streptomycin (all gibco by thermofisher, waltham, usa)] and immediately transported on ice to the laboratory for processing. sample material was removed from the brushes and centrifuged at • c and , rpm for min in order to isolate cellular material. the supernatant was discarded, after which samples were stabilised with µl rpe buffer (qiagen, hilden, germany) and . µl beta-mercaptoethanol (gibco by thermofisher, waltham, usa) and stored at − • c. samples were thawed in batches to undergo rna and dna extraction using an allprep dna/rna mini kit (qiagen, hilden, germany). this yielded dna samples of µl (average concentration . ng/µl, range . - . ng/µl) and rna samples of µl (average concentration . ng/µl, range - . ng/µl). extracted dna and rna were stored at − • c until batch testing for a range of upper respiratory tract viruses using realtime pcr. the panel included rv, influenza a-c, parainfluenza (piv) - , respiratory syncytial virus (rsv) a and b, coronavirus (cov) hku- , oc , nl , and e, enterovirus (env), metapneumovirus (hmpv), adenovirus (adv), bocavirus (bov), polyomaviruses wupyv and kipyv, epstein-barr virus (ebv), cytomegalovirus (cmv), herpes virus (hhv ), herpes simplex virus (hsv) and , and varicella zoster virus (vzv). all dna extracts first underwent an endogenous retrovirus (erv ) assay (present as two copies per human diploid cell) in order to confirm respiratory sample collection quality. briefly, dna extracts were screened for erv , adv, bov, wupyv, kipyv, cmv, ebv, vzv, hsv and , and hhv using an identical set of conditions previously optimised so as not to compromise sensitivity ( table ) . said conditions were pmol of each primer, . pmol of the respective probe(s), and µl of template, made up to a final reaction volume of µl using the bioline sensi mix ii probe pcr mix kit (bioline australia). details of the target genes, primer, and probe sequences are summarised in tables , . samples then underwent the following cycling conditions: • c for min, followed by cycles of • c for s and • c for s. the rna extracts were tested for rv, influenza a-c, piv - , rsv a and b, covs hku- , nl , oc , and e, env, and hmpv (table ) using identical quantities of primer, probe, and template to the dna reactions but with the bioline sensifast probe one-step rt-pcr kit (bioline, sydney, australia). there were two exceptions to these quantities; the iv a/b duplex where asymmetric probe amounts were used ( . and . pmol, respectively) and the rv assay where pmol of probe was used. samples then underwent the following "+" indicates a locked nucleic acid (e.g., +a is a locked nucleic adenine analogue). frontiers in cellular and infection microbiology | www.frontiersin.org cycling conditions: • c for min, and cycles of • c for s and • c for s. all samples were run with both positive and negative controls; the positive controls were either previously established clinically positive samples, or synthetic controls specific for each assay. all cycling was conducted on viia instruments (thermofisher, scoresby, australia). viral detection was defined as a cycle threshold (ct) of forty or less. statistics were performed using software from scientific python, namely scipy and pandas through the jupyter notebook interface (oliphant, twenty-four patients were recruited at time of endoscopic surgery; this included men and women, with an age range of - years, and a mean age of years. seven patients had crs without polyps (crssnp), eight had crs with polyps (crswnp), and nine were controls. demographics and patient characteristics are summarised in table . all patients in the crs groups underwent functional endoscopic sinus surgery (fess), while those in the control group underwent trans-sphenoidal resections of pituitary masses. erv was detected in all patient samples, with a median ct of . (range . - . ), showing adequate cellular material was captured throughout the collection and dna extraction phases. eighteen patients were positive for at least one virus in at least one site ( / , %), while six ( / , %) were negative for any of the viruses for which the samples were screened ( a standardised, validated technique for viral sampling in the sinonasal passages has not yet been described. this study shows a significant discrepancy in viral presence and species between just two of the sites commonly sampled, highlighting the need for such a standardisation. this indicates that viral sampling needs to be conducted with a cytobrush in both the im and mm. collection variability has the potential to impact respiratory viral detection significantly. the sample volume and location, as well as the documented uneven distribution of viruses within the nasal cavity, can all contribute to false negatives (van wesenbeeck et al., ) . given that clinically relevant, actively replicating viruses of the upper respiratory tract are intracellular and largely reside in the upper epithelial layers of the mucosa (vareille et al., ) , adequate cell sampling is an important consideration when searching for viruses. traditional viral sampling brushes have the advantages of causing less trauma to the delicate mucosa and thus less discomfort to a conscious patient, but risk sampling largely secreted elements rather than the cells themselves (spyridaki et al., ) . viruses do certainly reside in these secretions, but this may not necessarily represent actively replicating virus causing disease. for these reasons we elected to use cytology brushes for this study. cytology brushes are designed specifically for cell sampling due to their larger and more rigid design than traditional viral sampling brushes. although this may potentially increase the risk of trauma or discomfort to the awake patient, when used in the anaesthetised patient, as was the case in this study, they have the significant advantage of increased cellular sampling yield (stokes et al., ) . as mentioned viral yields are also difficult to compare in respiratory samples, as sample volume can vary widely. the samples here averaged a dna concentration of . ng/µl and an rna concentration of . ng/µl, but with ranges of . - . and - . ng/µl, respectively. to minimise the impact of this variability on results all samples underwent an erv assay prior to pcr. this has been identified previously as a positive indicator of respiratory sample quality, and all samples were wellwithin previously published target ranges (alsaleh et al., ; sarna et al., ) . viral sampling is traditionally performed from the inferior nasal septum and anterior nasal floor as they are easily accessible and cause minimal patient discomfort. the posterior nasopharyngeal wall is also traditionally endorsed, but confirmation of access to this site is difficult without endoscopic equipment. there is no evidence however that these three sites are any more or less appropriate. these areas may indeed be less than ideal due to their relative proximity to airborne pathogens (and therefore risk of contamination), their distance from areas of particular interest (such as the paranasal sinuses), and the tendency for pooling of potentially contaminating secretions in these areas. the mm (sampled in our study) remains relatively simple to access but is further away from potential sources of contamination, and receives drainage from a much wider area including the maxillary, frontal, and anterior ethmoid sinuses. there are indeed a number of other sites in the nasopharynx not investigated here, for example the superior meatus, the sphenoethmoidal recess, and the post-nasal space, however these are difficult to reach without endoscopic equipment that is not readily available in the primary care setting, and can be subject to contamination from other more anterior sites during insertion and removal of sampling instruments. should these areas demonstrate greater viral presence than the mm and im the specialist input required to access the sinuses themselves would likely delay or miss altogether the diagnosis and window for anti-viral treatment. large-scale economic viability of the collection method here proposed also warrants mention; pooling of viral samples from the same patient prior to analysis and limitation of viral testing to a smaller panel of more prevalent, clinically relevant pathogens would be prudent, however selection of such a panel requires further investigation. common, clinically relevant upper respiratory viruses are largely of the rna subtype, and include rv, influenza, rsv, and hmpv, and to a lesser extent cov, piv, and env. of the dna viruses here investigated adv is certainly a notable urt pathogen. bov has been linked largely with lower respiratory illness (gottlieb, ) . the other dna viruses here investigated were chosen not primarily for their clinical relevance in viral respiratory disease, but instead for either their near-ubiquity, their ability to remain latent in the respiratory tract, or both. ebv and hhv have here shown themselves to be particularly useful in testing viral sampling methods as they are almost omnipresent in the adult sinonasal passages, and are rarely entirely cleared after first infection. effort was made in this study to identify any correlation between control/crssnp/crswnp status and viral presence. patient reports of recent viral infection, sinonasal outcome test (snot- ) scores, lund-mackay computed tomography scores, lund-kennedy endoscopic scores, and rt-pcr cycle threshold values were collected for all patient and samples, but the sample size here was too small to demonstrate any significant differences. the inclusion of the extremely common herpesviruses (seen, as expected, in many of our controls) also skewed any such results. this is an area that requires significant further investigation. neither of the sites from whence samples were taken were more or less likely to be positive than the other. our observation that the mm and im only completely agree in terms of viral presence or absence as well as viral species in % of cases indicates a significant proportion of viruses present would not be identified were only one site to be sampled. our findings suggest viral sampling from the sinonasal passages should be taken from both sites in both nasal cavities. the sampling method here described has significant implications for further research into a field of emerging importance in both rhinologic and also respiratory disease on a larger scale. the raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. rg contributed to study design, sample collection and processing, data analysis, and writing of the manuscript. cb contributed to sample collection. ab contributed to statistical analysis. sb contributed to sample processing and writing of the manuscript. sv contributed to study design and review of the manuscript. p-jw and ap contributed to study design, sample collection and review of the manuscript. this study was funded by the university of adelaide, adelaide, australia. prevalence of rhinovirus and respiratory syncytial virus among patients with chronic rhinosinusitis nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control exploring the prevalence of ten polyomaviruses and two herpes viruses in breast cancer newly identified human rhinoviruses: molecular methods heat up the cold viruses adult chronic rhinosinusitis: definitions, diagnosis, epidemiology, and pathophysiology latent sites of epstein-barr virus infection high rates of detection of respiratory viruses in the nasal washes and mucosae of patients with chronic rhinosinusitis the bacterial microbiome in chronic rhinosinusitis: richness, diversity, postoperative changes, and patient outcomes chronic rhinosinusitis: potential role of microbial dysbiosis and recommendations for sampling sites detection of herpesviruses - and community-acquired respiratory viruses in patients with chronic rhinosinusitis with nasal polyposis human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays the nasal microbiome in asthma european position paper on rhinosinusitis and nasal polyps community-acquired respiratory viruses real-time rt-pcr detection of respiratory viral infections in four triplex reactions nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses rapid and quantitative detection of human adenovirus dna by real-time pcr detection of rhinovirus in turbinate epithelial cells of chronic sinusitis quantitative analysis of epstein-barr virus load by using a real-time pcr assay the etiology and pathogenesis of chronic rhinosinusitis: a review of current hypotheses comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction respiratory viral infection in the chronic persistent phase of chronic rhinosinusitis the seasonality of respiratory viruses in patients with chronic rhinosinusitis the healthy human microbiome staging in rhinosinusitus real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages the human respiratory microbiome: implications and impact comparative evaluation of taqman real-time pcr and semi-nested vp pcr for detection of enteroviruses in clinical specimens python for scientific computing adenovirus and respiratory syncytial virus in chronic sinusitis using polymerase chain reaction quantitative detection and differentiation of human herpesvirus subtypes in bone marrow transplant patients by using a single real-time polymerase chain reaction assay clinical practice guideline (update): adult sinusitis. otolaryngol the role of viruses in the clinical presentation of chronic rhinosinusitis viruses causing lower respiratory symptoms in young children: findings from the orchid birth cohort comparison of four nasal sampling methods for the detection of viral pathogens by rt-pcr-a ga( )len project comparison of three different brushing techniques to isolate and culture primary nasal epithelial cells from human subjects evidence for lytic infection by epstein-barr virus in mucosal lymphocytes instead of nasopharyngeal epithelial cells in normal individuals detection of epstein-barr virus-infected mucosal lymphocytes in nasal polyps detection of human bocavirus in respiratory, fecal, and blood samples by real-time pcr frequent detection of human coronaviruses in clinical specimens from patients with respiratory tractinfection by use of a novel real-time reverse-transcriptase polymerase chain reaction applicability of a real-time quantitative pcr assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults sampling variability between two mid-turbinate swabs of the same patient has implications for influenza viral load monitoring the airway epithelium: soldier in the fight against respiratory viruses a novel duplex real-time pcr for hpiv- detects co-circulation of both viral subtypes among ill children during real-time quantitative pcr assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients rapid detection of herpes simplex virus and varicella-zoster virus infections by real-time pcr a '-nuclease real-time reverse transcriptase-polymerase chain reaction assay for the detection of a broad range of influenza a subtypes, including h n is chronic rhinosinusitis caused by persistent respiratory virus infection? int a quantification of human cells using an erv- real time pcr assay prevalence of human papilloma virus and human herpes virus types - in human nasal polyposis the authors thank prof. eric gowans for his assistance in establishing the methodology and editing this work. the authors also acknowledge the contributions of dr. mian ooi and mr. aden lau in the collection of samples. key: cord- - syjr k authors: kanno, toru; ishihara, ryoko; hatama, shinichi; uchida, ikuo title: a long-term animal experiment indicating persistent infection of bovine coronavirus in cattle date: - - journal: j vet med sci doi: . /jvms. - sha: doc_id: cord_uid: syjr k a long-term animal experiment involving inoculation with bovine coronavirus (bcov) was conducted to verify its persistent infection in cattle. three colostrum-deprived holstein calves were housed separately in individual rooms of a high-containment facility and inoculated with the bcov strain kumamoto/ / . until the end of the experiment ( , , and days, respectively), viral rnas were detected sporadically by rt-pcr and nested pcr from plasma, nasal discharge, and feces. seroconversion and titer changes were validated by hemagglutination inhibition tests and neutralization tests. among the samples, nasal discharge showed a higher viral positivity than feces, which seemed to be associated with positive detection in the plasma. these data demonstrate the existence of persistent infection of bcov in the respiratory tissues of cattle. bovine coronavirus (bcov) is a member of the order nidovirales, family coronaviridae, subfamily coronavirinae, genus betacoronavirus, species betacoronavirus- . this species also includes human coronavirus oc , porcine hemagglutinating encephalomyelitis virus, equine coronavirus, and canine respiratory coronavirus [ ] . bcov infects the respiratory and digestive organs of cattle and causes neonatal calf diarrhea, bloody diarrhea in adult cattle (winter dysentery), and respiratory symptoms, including shipping fever in feedlot cattle [ , , ] . bcovs have spread widely across cattle farms all over the world, and thus, nearly all adult cattle have antibodies against the virus. outbreaks typically occur during autumn and winter [ ] and are associated with the housing period; however, several cases have also been reported during warmer seasons [ , , ] . although the mortality of this disease is low, it causes substantial economic losses owing to a reduction in milk production in dairy farms [ , ] and meat production in beef farms [ , ] . the main transmission route of bcovs is horizontal infection, i.e., ingestion or inhalation of virus from feces or nasal discharges into the mouth or nasal cavity [ , ] . there has been no report of vertical infection so far. bcov antigens are detected in the feces of clinically healthy cattle [ ] , and studies on bcov shedding in feces showed the virus to be detected over a long period on farms, even though the number of cattle shedding the virus decreased [ ] . this suggests the existence of persistently infected cattle, which might also be the origin of transmission. to verify this hypothesis, we conducted a longitudinal animal experiment. the bcov used in this experimental study was obtained from a clarified suspension of feces from a cow on a farm where a bcov case had been confirmed in by using hrt- g cells (isolate kumamoto/ / ) [ ] . the culture supernatant of the isolate was inoculated into cattle by the oral route. feces were collected when the cattle showed diarrhea. the isolate was passaged in colostrum-deprived holstein calves twice, and feces of the infected calves were harvested as inoculum for this study (data not shown). three colostrum-deprived holstein calves were used in this study. each animal was maintained in a separate room of a highcontainment facility at our institute. in this facility, the animal room area is separated from the preparation room by a shower room area. to enter the animal room area, the investigator undresses and puts on work clothes and boots in this area. the animal room area has independent animal rooms and a corridor. each animal room has a ventilator with hepa filers to prevent the doi: . /jvms. - reintroduction of the virus from outside to the experimental cattle. during the animal experiment, we adhered to the strict high hygiene and biosecurity measures to prevent reinfection from other cattle. to enter the animal room, boots were removed at the corridor, and the "inside" boots and waterproof clothes kept in the animal room were worn. to exit the room after husbandry and sampling, put off the boots and waterproof clothes were taken off after disinfection with sodium hypochlorite using a spraying device. the animal care and use committee of the national institute of animal health approved all animal procedures prior to the initiation of this study (authorization number: - ). one calf ( days old, referred to as "calf ") was inoculated orally using a catheter that delivered a sample of the centrifuged supernatant at , × g for min from a mixture of g feces as mentioned above and ml phosphate-buffered saline (pbs) containing µg/ml gentamicin into the esophagus. samples of nasal discharge, feces, plasma, and sera were collected daily until days post inoculation (dpi), followed by weekly collection until dpi and then twice-weekly collection until the end of the experiment ( , dpi). the nasal discharge samples were collected by inserting cotton into the nasal cavity for a few min and extracting the discharge using sterilized disposable syringes. feces were collected directly from the rectum and prepared as a % suspension in pbs containing µg/ml gentamicin and µg/ml trypsin for virus isolation and rna extraction. the same experiment was carried out on another calf ( days old, "calf ") using the centrifuged supernatant from a mixture of ml pbs and g feces of calf collected at dpi (after it showed diarrhea). samples of nasal discharge, feces, plasma, and sera were collected daily until dpi, followed by weekly collection until dpi and twice-weekly collection until the end of the experiment ( dpi). the same experiment was carried out on yet another calf ( days old, "calf ") using centrifuged supernatant from a mixture of ml pbs and g feces of calf collected at dpi, when it showed diarrhea. nasal discharge, feces, plasma, and serum samples were collected daily until dpi, followed by weekly collection until dpi and twice-weekly collection until the end of the experiment ( dpi). viral rna was extracted from plasma, nasal discharge, and % fecal suspensions in pbs using the high pure viral rna kit (roche diagnostics, tokyo, japan) according to the manufacturer's instructions. bcov-specific genes were detected from the extracted rna by the rt-pcr assay using a titan one tube rt-pcr kit (roche diagnostics), with the following thermal cycling profile: min at °c; min at °c; cycles of denaturation at °c for sec, annealing at °c for sec, and extension at °c for sec; and completion of amplification with a -min extension step at °c [ ] . the oligonucleotide primers used for nested pcr were designed from the nucleotide sequence of the kakegawa strain (genbank accession no. ab ) in this study. the primers were as follows: bcov-nu, ʹ-tgctacttctcagcaaccatcag- ʹ (nt , - , , sense primer), and bcov-nd, ʹ-ttggcatgcggtcctgttccaag- ʹ (nt , - , , antisense primer). the size of the amplification fragment is bp. nested pcr was performed by using takara ex taq (takara-bio., kusatsu, japan), and the thermal cycling profile was as follows: min at °c; cycles of denaturation at °c for sec, annealing at °c for sec, and extension at °c for sec; and completion of amplification with a -min extension step at °c. to prevent laboratory contamination in rt-pcr and nested pcr, we set up mixtures for these reactions in a laminar flow cabinet equipped with an uv lamp, wore fresh gloves at each step, used pipette tips with aerosol filters. negative controls were also included in every pcr experiment to detection of contamination. the rt-pcr and nested pcr products were visualized on . % agarose gels stained with ethidium bromide. the hemagglutination inhibition (hi) test was conducted by the microtiter method [ ] . sera were treated with kaolin and chicken erythrocytes and heat inactivated. as antigen for the hi test, the kakegawa strain was used in this assay because the hemagglutinating (ha) activity of the kumamoto/ / strain was low and unstable with chicken and mouse erythrocytes, similar to recent japanese isolates (kanno et al., unpublished data), whereas the ha activity of the kakegawa strain was sufficient. briefly, twofold dilutions of serum were prepared in duplicate and reacted with the virus. the hi titers were expressed as the reciprocal of the highest serum dilution that completely inhibited the ha activity of ha units of the virus. serum samples were also tested in a virus neutralization test by preparing serial twofold dilutions of serum were reacted in duplicate with of % tissue culture infective doses (tcid ) of kumamoto/ / , followed by incubation for hr at °c. after incubation, the serum-virus mixture was transferred onto monolayered human rectal tumor cells (hrt- g) cultured in microplates and incubated for days at °c. the neutralization antibody titers were expressed as the reciprocal of the highest serum dilution that completely inhibited the cytopathic effect (cpe). all calves showed clinical signs, such as loose or diarrheal stool, up to - dpi. viral rna was detected from the plasma during the period of onset (fig. a-c) . after recovery from the disease, viral rna was sporadically detected by rt-pcr and nested pcr from plasma. final detection days (last day when a sample showed a positive result) by rt-pcr were , , and dpi, and by nested pcr, , , , and dpi, respectively, in the calves. as with the plasma, viral rna was detected from nasal discharge on the basis of viral titers, and was detected sporadically by pcr-based experiments. final detection days by rt-pcr were , and dpi, and by nested pcr, , and dpi, respectively, in the calves. viral rna was not detected from the feces after the disease onset in calves and . however, nested pcr was positive for the virus at dpi in calf . in calf , viral rna from feces was detected sporadically by the pcr-based experiments. in this calf, the final detection day was dpi by rt-pcr and , dpi by nested pcr. virus isolation was conducted from the samples showing positive results by rt-pcr and nested pcr. the samples were inoculated into hrt- g monolayer cells in -well plates and incubated for days at °c and % co . if a cpe was not observed in the incubation period, passaging into fresh cells was conducted twice using the inoculum after freeze-thawing the cultures thrice. however, the virus was not isolated, except in the samples from the period of onset. the hi and neutralization titers in the calves increased several days after disease onset and peaked at , and weeks post doi: . /jvms. inoculation, and at , and weeks post inoculation, respectively, in the calves. thereafter, the titers gradually decreased. however, both titers showed a rapid rise at dpi in calf ( and , respectively) (fig. a ). ten days before the sampling, calf showed diarrhea at and dpi (data not shown). therefore, we surmised that the virus was reactivated in the cattle, probably in the respiratory tissues, causing diarrhea followed by this booster effect. the virus in the digestive tract might have been quickly inactivated and excreted; therefore, viral rnas were not detected from nasal discharge and feces at dpi, days after the onset. unfortunately, feces samples had not been collected at the onset, so no virus was isolated. a rapid rise in these titers was also found in calf at dpi, just after the detection of viral rna from the plasma at dpi by both rt-pcr and nested pcr (fig. b) . this finding might also be attributed to virus reactivation in respiratory tissues and quick inactivation by host immunity without clinical signs. this study showed that the bcov rna was long-lasting, having been detected from the nasal discharge of cattle that had been maintained in an isolated room of a high-containment facility to prevent virus intrusion from outside. this indicated the existence of persistent infection of bcov in the respiratory tissues of cattle, although further investigations are warranted because the virus was not isolated. other coronaviruses, such as transmissible gastroenteritis virus, canine coronavirus, and feline coronavirus, have also been reported to cause long-term infection [ , , ] . thus, coronavirus tends to cause persistent infection in host animals. often, calf diarrhea caused by bcov repeatedly occurs at the same farm every year. this might be explained by the presence of persistently infected cattle. conflicts of interest. the authors have no potential conflicts of interest to declare with respect to the research, authorship, and/or publication of this article. ratification vote on taxonomic proposals to the international committee on taxonomy of viruses bovine coronavirus shedding of enteric coronavirus in adult cattle monoclonal antibody capture enzyme-linked immunosorbent assay for detection of bovine enteric coronavirus severe outbreak of bovine coronavirus infection in dairy cattle during the warmer season isolation of bovine respiratory coronaviruses from feedlot cattle and comparison of their biological and antigenic properties with bovine enteric coronaviruses a longitudinal study of bovine coronavirus enteric and respiratory infections in dairy calves in two herds in ohio coronavirus-like particles in the feces of normal cats hemagglutination with nebraska calf diarrhea virus molecular analysis of the s glycoprotein gene of bovine coronaviruses isolated in japan from to phylogenetic studies of bovine coronaviruses isolated in japan association between infection of the respiratory tract attributable to bovine coronavirus and health and growth performance of cattle in feedlots detection and characterization of bovine coronaviruses in fecal specimens of adult cattle with diarrhea during the warmer seasons bovine respiratory coronavirus experimentally induced coronavirus infections in calves: viral replication in the respiratory and intestinal tracts coronavirus isolation from nasal swab samples in cattle with signs of respiratory tract disease after shipping morbidity in swedish dairy calves from birth to days of age and individual calf-level risk factors for infectious diseases risk factors for calf mortality in large swedish dairy herds experimental reproduction of winter dysentery in lactating cows using bcv -comparison with bcv infection in milk-fed calves experimental inoculation of adult dairy cows with bovine coronavirus and detection of coronavirus in feces by rt-pcr recovery of transmissible gastroenteritis virus from chronically infected experimental pigs astrovirus-like, coronavirus-like, and parvovirus-like particles detected in the diarrheal stools of beagle pups acknowledgments. we are grateful to mr. keiji itoh, mr. kazuhiko takase, mr. mitsutoshi noi, mr. yoshihiro himoro, and mr. kiyoshi tanaka for their skilled handling of the animals at the institute of animal health, hokkaido research center. key: cord- -xxc vdnt authors: ahmed, anwar e.; al-jahdali, hamdan; alshukairi, abeer n.; alaqeel, mody; siddiq, salma s.; alsaab, hanan; sakr, ezzeldin a.; alyahya, hamed a.; alandonisi, munzir m.; subedar, alaa t.; aloudah, nouf m.; baharoon, salim; alsalamah, majid a.; al johani, sameera; alghamdi, mohammed g. title: early identification of pneumonia patients at increased risk of middle east respiratory syndrome coronavirus infection in saudi arabia date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: xxc vdnt background: the rapid and accurate identification of individuals who are at high risk of middle east respiratory syndrome coronavirus (mers-cov) infection remains a major challenge for the medical and scientific communities. the aim of this study was to develop and validate a risk prediction model for the screening of suspected cases of mers-cov infection in patients who have developed pneumonia. methods: a two-center, retrospective case–control study was performed. a total of patients with confirmed pneumonia who were evaluated for mers-cov infection by real-time reverse transcription polymerase chain reaction (rrt-pcr) between september , and june , at king abdulaziz medical city in riyadh and king fahad general hospital in jeddah, were included. according to the rrt-pcr results, patients were positive for mers-cov and were negative. demographic characteristics, clinical presentations, and radiological and laboratory findings were collected for each subject. results: a risk prediction model to identify pneumonia patients at increased risk of mers-cov was developed. the model included male sex, contact with a sick patient or camel, diabetes, severe illness, low white blood cell (wbc) count, low alanine aminotransferase (alt), and high aspartate aminotransferase (ast). the model performed well in predicting mers-cov infection (area under the receiver operating characteristics curves (auc) . ), on internal validation (auc . ), and on a goodness-of-fit test (p = . ). the risk prediction model, which produced an optimal probability cut-off of . , had a sensitivity of . and specificity of . . conclusions: this study provides a simple, practical, and valid algorithm to identify pneumonia patients at increased risk of mers-cov infection. this risk prediction model could be useful for the early identification of patients at the highest risk of mers-cov infection. further validation of the prediction model on a large prospective cohort of representative patients with pneumonia is necessary. middle east respiratory syndrome coronavirus (mers-cov) was first identified in saudi arabia in . the diagnosis of this infection remains complex (al johani and hajeer, ; sung et al., ; ahmed, a) and it has a high fatality rate (ahmed, b, c; aleanizy et al., ; sherbini et al., ; kim et al., ) . the early detection and identification of individuals at high risk of a disease is the most effective strategy to improve patient clinical outcomes (ahmed, a) and reduce the costs of testing, both physical and economic (ahmed et al., (ahmed et al., , . the real-time reverse transcription polymerase chain reaction (rrt-pcr) has been found to be valid and accurate for the evaluation of respiratory swabs, sputum, and other endotracheal aspirate material (corman et al., a, b) . however, although rrt-pcr is the most accurate and sensitive test available at the authors' centers, absolute identification of mers-cov may require multiple clinical specimens from different sources and take days (corman et al., a, b; anon, a) . the saudi ministry of health (moh) has developed mers-cov visual triage guidelines to identify suspected cases (anon, b) . the current guidelines include fever, respiratory symptoms, gastrointestinal symptoms, chronic diseases, and risk of exposure to mers-cov. in clinical practice, identifying high-risk individuals can be challenging, since most laboratory-confirmed mers-cov patients report common clinical risk indices to patients with other respiratory conditions. for instance, respiratory and gastrointestinal symptoms are common for both mers-cov and non-mers-cov patients (mohd et al., ) . thus, further exploration must take place to reduce the risk of mers-cov infection. a risk prediction model is urgently needed to stratify patients with suspected mers-cov. this may decrease the risk of virus transmission to others who are in close contact, for example healthcare workers, patients, and hospital visitors, by allowing the careful management of those who are potentially infected at an early stage of infection. developing a mers-cov prediction model may more efficiently aid physicians in identifying individuals at high risk and selecting the necessary test(s) to improve prevention and control measures. several methodological studies have shown that combining demographic characteristics with clinical, radiological, and laboratory information can improve risk assessment and diagnostic accuracy (ahmed et al., (ahmed et al., , sidransky, ; etzioni et al., ) . these previous studies used a linear combination to develop an algorithm that combines demographic characteristics, symptoms, and clinical, radiological, and laboratory findings to identify the highly suspected mers-cov cases. mers-cov was initially identified in a patient being treated for pneumonia in (zaki et al., ) , and since then, pneumonia and its symptoms have remained common characteristics in mers-cov patients (saad et al., ; choi et al., ) . the aim of this study was to develop and validate a reliable risk prediction model for the screening of suspected cases of mers-cov infection in patients who have developed pneumonia. a two-center, retrospective case-control study was conducted utilizing data from king abdulaziz medical city in riyadh (kamc-r) and king fahad general hospital in jeddah (kfgh-jed), saudi arabia. the data were obtained between september , and june , . kfgh-jed experienced a mers outbreak between march and may (oboho et al., ) , and kamc-r experienced a large mers outbreak between june and august (al-dorzi et al., ) . both study centers applied the saudi moh case definitions (anon, b) to identify suspected individuals in the population studied, and rrt-pcr was used as the gold standard test to diagnose mers-cov in multiple and different clinical specimens when necessary. mers-cov-related symptoms were common at both centers. the project received ethical approval from two independent ethics committees: the saudi moh (irb log number - e) and king abdullah international medical research center (study number rc / ), riyadh saudi arabia. during the study, the medical records of subjects who were being assessed by rrt-pcr for suspected mers-cov were reviewed. the suspicion of mers-cov infection at both kamc-r and kfgh-jed was determined based on the saudi moh guidelines (anon, b) . two groups were compared: patients who were positive and patients who were negative for mers-cov infection. in an effort to reduce heterogeneity between the study groups, only subjects with a lung infiltrate on chest x-ray and/or computed tomography (ct) scan of the chest were included in the analysis. thus, subjects who had no available results of a chest x-ray or ct scan of the chest were excluded. the initial screening for suspected mers-cov patients includes pneumonia (anon, b) . most of the patients studied were evaluated for pneumonia immediately after presentation. the study excluded subjects who were less than years of age (pediatrics/children), as defined in the saudi moh guidelines (anon, b) , and excluded subjects who had upper respiratory tract infections (respiratory symptoms, positive or negative mers-cov rrt-pcr, and normal chest x-ray and ct scan of the chest). the final sample comprised a cohort of subjects who had a lung infiltrate on chest x-ray and/or a ct scan of the chest, who were classified according to the results of mers-cov rrt-pcr. the case group consisted of pneumonia patients who were positive for mers-cov infection, and the control group consisted of pneumonia patients who were negative for mers-cov infection. cases were defined as patients with mers-cov pneumonia who had positive mers-cov rrt-pcr on nasopharyngeal aspirate and/ or bronchoalveolar lavage in addition to a lung infiltrate on chest xray and/or ct scan of the chest. controls were defined as patients with respiratory symptoms, a lung infiltrate on chest x-ray and/or ct scan of the chest, pneumonia, and negative mers-cov rrt-pcr result of nasopharyngeal aspirate and/or bronchoalveolar lavage. nineteen predictive variables were included: age, sex, fever (temperature ! c), one composite respiratory symptom (the presence of cough, bloody cough, shortness of breath, or chest pain), one composite gastrointestinal symptoms (the presence of diarrhea, vomiting, or nausea), seven mers-cov potential risk factors (contact with sick patients or camels, severe illness (defined according to the patient's clinical status, 'yes/no', which is based on clinical judgment), diabetes, lung disease, liver disease, renal disease, and heart disease), and seven laboratory measurements (white blood cell (wbc) count ( /l), platelets ( /l), creatinine (mmol/l), bilirubin (mmol/l), alanine aminotransferase (alt; u/l), aspartate aminotransferase (ast; u/l), and albumin (g/ l)). the reference ranges for the laboratory measures were as follows: wbc count, -  /l; platelets, -  /l; creatinine, - mmol/l; bilirubin, . - . mmol/l; alt, - u/l; ast, - u/l; albumin, - g/l. no serological tests were available at the centers for these patients. stata statistical software release , (statacorp. llc, college station, tx, usa) was used for the data analysis. the sample characteristics were recorded as the frequency and percentage, or as the mean ae standard deviation (sd). laboratory measurements were summarized as the median and th- th percentiles. a subgroup analysis (chi-square test, independent samples t-test, or mann-whitney u-test) was used to identify unadjusted associations between demographic, clinical, and laboratory measurements according to mers-cov status. the performance of each bivariate predictor was further assessed by unbiased estimate, the area under the curve (auc), and its % confidence interval (ci). stepwise binary logistic regression was employed to develop a mers-cov risk prediction model and identify factors that could be used to estimate the probabilities of mers-cov infection. the goodness-of-fit of the final model was tested by hosmer-lemeshow procedure: a large p-value indicates that a model has a good fit. the discrimination ability between high and minimal risk of mers-cov infection of the final model was assessed by the auc and its % ci. a receiver operating characteristics (roc) curve was generated for the risk prediction model. two hundred random samples were drawn with replacements from the original study sample (n = ). the model internal validity was assessed in these samples by the auc and its % ci. optimal cut-off values of the probabilities for each model were determined using a generalized youden index (youden, ) . at these thresholds, it was possible to achieve the best balance between specificity and sensitivity. a total of pneumonia patients were included in the analysis: . % were confirmed mers-cov-positive and . % were confirmed mers-cov-negative. the mean age at presentation was . years, with a standard deviation of . years; age ranged between and years. of the total sample, . % had been in contact with a sick patient or camel, % had fever, . % had at least one respiratory symptom, and . % had at least one gastrointestinal symptom. the two groups were similar in the distribution of age (p = . ) and sex (p = . ). the characteristics of the sample can be found in table . subgroup analyses are presented in tables and . the chisquare test or the independent samples t-test revealed that sex (p = . ) and age (p = . ) were similar in the two groups. the risk of mers-cov infection was similar in patients with and without fever (p = . ), respiratory symptoms (p = . ), or gastrointestinal symptoms (p = . ). severe illness (p = . ), contact with a sick patient or camel (p = . ), diabetes (p = . ), heart disease (p = . ), and renal disease (p = . ) were significantly associated with an increased risk of mers-cov infection. the independent samples mann-whitney u-test revealed that the wbc count (p = . ) and platelet count (p = . ) were significantly lower in patients who were positive for mers-cov than in those who were negative for mers-cov infection. in contrast, creatinine (p = . ), bilirubin (p = . ), ast (p = . ), and albumin (p = . ) were significantly higher in patients who were positive for mers-cov than in those who were negative for mers-cov infection. alt (p = . ) was insignificantly higher in patients who were positive for mers-cov than in those who were negative for mers-cov infection. according to the individual roc curve analysis (table ) , severe illness, diabetes, wbc count, creatinine, bilirubin, albumin, and ast were the most important predictors of mers-cov infection. a risk prediction model was developed using stepwise binary logistic regression (p . ). the model retained seven independent variables that were associated with increased odds of mers-cov (table ). male sex (adjusted odds ratio (aor) . , p = . ), contact with a sick patient or camel (aor . , p = . ), diabetes (aor . , p = . ), severe illness (aor . , p = . ), low wbc count (aor . , p = . ), high ast (aor . , p = . ), and low alt (aor . , p = . ) were found to have a significant impact on the prediction of mers-cov. the hosmer-lemeshow test indicated that this model fitted the data well (p = . ). this model showed substantial discrimination, with an auc of . and a % ci of . - . ( figure ). the prediction model was validated using the bootstrap procedure. a total of random samples were drawn with replacements from the original sample, and the model showed a substantial ability to assess mers-cov infection in this study population (auc . , % ci . - . ). the findings in table were used to create a risk-probability model. the risk prediction for the model can be expressed by the following equation: predicted probability = [ + exp( . -( .  male) À ( .  sick patient or camel contact) À ( .  diabetes) À ( .  severe illness) + ( .  wbc count) À ( .  ast) + ( .  alt))] À . a calculator was developed to calculate the potential risk of mers-cov infection in pneumonia patients. we determined the optimal cut-off or threshold values of the probabilities to mark the differences between the high-risk and low-risk groups. when an equal weight was given for sensitivity and specificity (m = ), the optimal cut-off value (probability ! . ) produced sensitivity and specificity of . and . , respectively. when more weight was given for sensitivity than specificity (m = . ), the optimal cut-off value (probability ! . ) produced sensitivity and specificity of . and . , respectively. when more weight was given for specificity than sensitivity (m = . ), the optimal cut-off value (probability ! . ) produced sensitivity and specificity of . and . , respectively. based on data from the two largest hospitals in saudi arabia, a risk prediction model was developed for mers-cov infection in pneumonia patients. this model was generated from a retrospective study and should be assessed prospectively for external validation. seven variables were identified as having a great impact on the mers-cov risk assessment prediction. the risk prediction model highlights the strong potential impact of male sex, contact with a sick patient or camel, severe illness, diabetes, low wbc count, high ast, and low alt on mers-cov infection. these few important parameters could be part of routine medical examinations to be performed (for the purpose of identifying highly suspected individuals) in daily clinical practice in order to make a prompt and timely clinical decision. according to the model, high ast was associated with an increased risk of being infected with mers-cov. this finding is similar to that of mohd et al., who noted high ast levels in mers-cov patients (mohd et al., ) . unlike their findings, it was noted in the present study that the impact of alt became significantly negative after controlling for several confounders. however, this type of association should be evaluated further in a prospective study in the presence of other unmeasured confounders. although sex was found to have no impact on mers-cov infection in the unadjusted analysis, the multivariate analysis revealed that the risk of mers-cov infection was . % times higher in males than in females. this may be because other factors are playing a role in the development of mers-cov in males, such as camel contact, since males are more likely than females to have contact with camels. in agreement with the recent saudi moh mers-cov visual triage guidelines for the identification of suspected cases (anon, b) , it was found that the odds of being infected with mers-cov were higher in patients with diabetes as compared to those with no diabetes. this also supports the findings of previous studies (badawi and ryoo, ; assiri et al., ; al-tawfiq et al., ; alraddadi et al., ) in which researchers systematically recognized that diabetes is a risk factor for mers-cov infection. these findings indicate that more attention should be given to assessing the risk of mers-cov infection in diabetic patients and whether the risk depends on a specific diabetes type or condition in these patients. as asserted in the saudi moh mers-cov visual triage guidelines and many other studies (muhairi et al., ; younan et al., ; reeves et al., ; sabir et al., ; azhar et al., a, b) , contact with a sick patient or camel was identified as an independent predictor of mers-cov infection, according to the risk prediction model. it must be noted that the finding in the present study could have been limited by combining camel contact and sick patient contact due to the small sample size of each category. this study shows the importance of incorporating various types of information to improve the performance of the risk prediction. according to the linear combination model, it was found that several of the parameters highlighted in the saudi moh mers-cov visual triage guidelines were not able to distinguish between 'highrisk' and 'low-risk' groups, nor did they help in predicting mers-cov infection. for instance, fever, respiratory symptoms, gastrointestinal symptoms, heart disease, and renal disease were noted to have an insignificant impact on mers-cov infection. however, in agreement with the saudi moh mers-cov guidelines and two other reports (mohd et al., ; arabi et al., ) , the odds of being infected with mers-cov were associated with a significant risk reduction of . % for each unit increase in wbc count. these results suggest that demographic, clinical, radiological, and laboratory data should be used in routine practice to identify suspected mers-cov cases, as such data could serve as the first line of prevention strategies. it was found that the accuracy of prediction (figure ) was further improved when combining various medical and patient variables as opposed to relying on a single factor (table ). this has been proven theoretically and in application (ahmed et al., (ahmed et al., , (ahmed et al., , etzioni et al., ; shen, ; pepe and thompson, ; su and liu, ; thompson, ) , where a linear combination has been used to maximize the diagnostic accuracy of a disease of interest. the strength of this study lies in the fact that a simple and applicable predictive model was developed that incorporates demographic, clinical, radiological, and laboratory data, where these were functionally associated and contributed greatly to stratifying and distinguishing between a high and a minimal risk of mers-cov infection. this simple evaluation of suspected mers-cov cases appears promising and could be implemented easily in routine clinical practice. this model could be used as a risk stratification method or a triage tool to help physicians in making an informed decision and planning the next step when deciding whether an rrt-pcr or further investigation is necessary. it was possible to derive a risk probability algorithm (range - ), a generalized youden index (choi et al., ) was used to determine an optimal cut-off to stratify the risk, and a risk probability of ! . was identified as being the optimal cut-off, with a sensitivity of . and specificity of . . several limitations to the proposed risk prediction model were identified. the study findings were based on a retrospective design; therefore this prediction model should be interpreted with caution. limited information was available on patient variables, clinical variables, and transmission routes. for example, information on primary cases and secondary cases may be supplemented by the results of clinical, radiological, and laboratory data. in this study, 'contact with a sick patient' and 'contact with a sick camel' were combined into one variable due to the small number in each category. severe illness was based on a subjective judgment. an additional potential limitation was that the duration of symptoms was not available for these patients. this study investigated a very specific population (pneumonia) at only two centers, which could compromise the applicability and generalizability of the risk prediction model. moreover, the prediction model may not be generalizable to patients who do not fulfill the moh guidelines. further validation of the prediction model on an external sample and prospective cohort of representative patients with pneumonia is necessary, specifically in relevant settings: emergency, outpatient, inpatient, and community. despite these limitations, the model developed shows promise for the identification of suspected mers-cov cases in clinical practice. this model could be applicable in various healthcare settingsinpatient, outpatient, and emergency departmentsbecause no extensive laboratory testing is required and samples may be available within short turnaround times. this may allow rapid evaluation and improve clinical decision-making. in conclusion, this study provides a simple, practical, and valid algorithm to identify individuals at increased risk of mers-cov infection among patients who have developed pneumonia. this risk model is not only useful for risk stratification and decisionmaking in clinical practice, but it could also be useful in preventing and managing the possible spread of mers-cov. the usefulness of this newly developed tool most be validated in an external prospective study. the project received ethical approval from two independent ethics committees: the saudi ministry of health (irb log number - e) and king abdullah international medical research center (study number rc / ), riyadh saudi arabia. none. none declared. accuracy and cost comparison in medical testing using sequential testing strategies reducing cost in sequential testing: a limit of indifference approach believe the extreme (be) strategy at the optimal point: what strategy will it become diagnostic delays in symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia estimating survival rates in mers-cov patients and days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study the predictors of -and -day mortality in mers-cov patients mers-cov diagnosis: an update the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients outbreak of middle east respiratory syndrome coronavirus in saudi arabia: a retrospective study risk factors for primary middle east respiratory syndrome coronavirus illness in humans laboratory testing for middle east respiratory syndrome coronavirus (mers-cov) case definition and surveillance guidance -updated critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient evidence for camel-to-human transmission of mers coronavirus prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction combining biomarkers to detect disease with application to prostate cancer middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, : epidemiology, characteristics and public health implications predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia epidemiological investigation of middle east respiratory syndrome coronavirus in dromedary camel farms linked with human infection in abu dhabi emirate mers-cov outbreak in jeddah-a link to health care facilities combining diagnostic test results to increase accuracy mers-cov geography and ecology in the middle east: analyses of reported camel exposures and a preliminary risk map clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia on the principles of believe the positive and believe the negative for diagnosis using two continuous tests middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data emerging molecular markers of cancer linear combinations of multiple diagnostic markers comparative evaluation of three homogenization methods for isolating middle east respiratory syndrome coronavirus nucleic acids from sputum samples for real-time reverse transcription pcr assessing the diagnostic accuracy of a sequence of tests index for rating diagnostic tests mers and the dromedary camel trade between africa and the middle east isolation of a novel coronavirus from a man with pneumonia in saudi arabia the authors acknowledge the saudi ministry of health and king abdullah international medical research center for approving this research project. the authors would like to thank the leaders of king abdulaziz medical city in riyadh and king fahad general hospital in jeddah for their support and understanding. supplementary data associated with this article can be found, in the online version, at https://doi.org/ . /j.ijid. . . . key: cord- -narre e authors: aziz, muhammad abdul; khan, amir hasan; adnan, muhammad; ullah, habib title: traditional uses of medicinal plants used by indigenous communities for veterinary practices at bajaur agency, pakistan date: - - journal: j ethnobiol ethnomed doi: . /s - - - sha: doc_id: cord_uid: narre e background: the pastoral lifestyle of indigenous communities of bajaur agency is bringing them close to natural remedies for treating their domestic animals. several studies have been conducted across the globe describing the importance of traditional knowledge in veterinary care. therefore, this study was planned with the aim to record knowledge on ethnoveterinary practices from the remote areas and share sit with other communities through published literature. methods: data was gathered from community members through semi-structured interviews and analyzed through informant consensus factor (fic) to evaluate the consent of current ethnoveterinary practices among the local people. results: in total, medicinal plants were recorded under the ethnoveterinary practices. most widely used medicinal plants with maximum use reports (urs) were visnaga daucoides gaertn., foeniculum vulgare mill., solanum virginianum l., withania somnifera (l.) dunal, glycyrrhiza glabra l., and curcuma longa l. new medicinal values were found with confidential level of citations for species including heracleum candicans and glycerhiza glabra. family apiaceae was the utmost family with high number ( species) of medicinal plants. maximum number of medicinal plants ( ) was used for gastric problems. high fic was recorded for dermatological ( . ) followed by reproductive ( . ) and gastrointestinal disorders ( . ). the main route of remedies administration was oral. conclusions: current study revealed that the study area has sufficient knowledge on ethnoveterinary medicinal plants. this knowledge is in the custody of nomadic grazers, herders, and aged community members. plants with new medicinal uses need to be validated phytochemically and pharmacologically for the development of new alternative drugs for veterinary purposes. the historical utilization of plants as health remedies both for human and animal is centuries old. it has been recognized that plants have the capacity to combat several types of diseases ethnoveterinary medicines, a term generally used for folk skills, beliefs, knowledge, practices, methods related to animals' health, and cure of various ailments in the rural areas [ ] . ethnoveterinary practices have achieved immense significance for the last decade owing to the discovery of some effective ethnoveterinary products [ ] . the utilization of traditional remedies poses a cheaper, easier, and sustainable alternative to synthetic drugs and pharmaceuticals [ ] . it has been reported that due to lack of proper animal husbandry practices, about - % of the losses occur in the animals' breeding sectors especially in developing countries [ ] , where the rural people are heavily dependent on livestock farming for their livelihood activities [ ] . the indigenous communities living in rural and mountainous territories of developing world consider livestock a vital source for economy, social security, and food and is thought to be a symbol of prestige for a particular family [ ] . livestock being as a subsector contributes around % of value addition in the agriculture sector and approximately % towards the gross domestic product (gdp). about million people living in the rural areas of the country are involved with the livestock subsector [ ] . hence, livestock raring plays a significant role in poverty reduction strategies. according to the report of economic survey of pakistan [ ] , the national herd of pakistan includes . million goats, . million cattle, . million buffalos, . million sheep, and . million camels. people residing in the remote areas utilize medicinal plants for livestock's health. particularly, the conventional lifestyle of nomadic and pastoralists makes it difficult for them to reach veterinary extension services due to high costs and less availability of allopathic medicines [ ] . in south asia, several ethnoveterinary studies have been conducted [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] including pakistan [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, scarce studies on ethnoveterinary medicines have been reported from the federally administrated tribal areas (fata) of the country. the tribal areas mainly comprised of mountainous territories where people use medicinal plants to treat livestock's diseases. traditional ethnoveterinary knowledge is mainly transmitted orally from one generation to another generation in the form of folk remedies, drawing stories, poems, drawing stories, folk myths, songs, and proverbs. this transmission of indigenous knowledge through oral way faces critical threats of extinction. therefore, it is necessary to record, document, and encourage the ethnoveterinary medication and integrate them into the existing animal health care services [ ] . bajaur agency is among one of the federally administrated tribal areas (fata) of pakistan having diversity of medicinal plants being used for the livestock's healthcare services. due to remote nature and lack of quality education, the area has been little explored for the scientific documentations of traditional knowledge. there is a dire need to explore the folk knowledge about the utilization of herbal remedies for veterinary practices prior to being extinct. hence, the current study was planned to investigate and document the traditional ethnoveterinary knowledge and practices and release it from the custody of knowledge bearers for sharing it with other communities through publish literature. bajaur (khar: headquarter) is the smallest agency of the fata having a total area of km . it shares km border with afghanistan, which is of great importance to pakistan and the region. the study area lays at an altitude of m above the sea level and geographically exists between °- °and °- °latitudes and °- °and °- °longitudes. the agency is surrounded to the west by kunar valley of afghanistan being separated by the rugged hindukush hills and other mountain passes known as nawa pass, ghakhi pass, and letai sar being the notable ones. the agency borders on south with mohmand agency, on east with lower dir district and the panjkora river, and on north with the watershed between bajaur agency and district dir. moreover, the agency is situated at the extreme end of the himalayan range. the areas dominated by agricultural lands are receiving about mm of average rain fall per annum. the two main tribes of bajaur agency known as tarkani and utman khel are mainly populated into seven tehsils including barang, nawagai, khar, mamund, salarzai, utman khel, and chamarkand. by profession, mostly, the people are farmers, teacher, drivers, and doing small scale businesses and jobs inside/outside the country. almost every household has a herd of domestic animals for socioeconomic gains. there are only three degree-level colleges and five higher secondary schools. moreover, there are only two government hospitals in the study area, while most people are deprived of modern health facilities, which justify their reliance on local herbalists (hakims). the study area consists of one veterinary hospital and small dispensaries to treat the domestic cattle. however, the local people still rely on traditional recipes due to larger distances from the aforementioned health centers. the dominant vegetation in the area is comprised of ailanthus altissimo, eucalyptus camaldulensis, ficus carica, melia azedarach, morus indica, morus nigra, olea ferruginea, pinus roxburghii, quercus baloot, and rumex hastatus. in the month of april, respondents were targeted based on their strong reputation in the field of ethnomedicinal knowledge while field survey was conducted from may to august . field visits were carried out prior to medicinal data collection in order to acknowledge the cooperation of the indigenous communities. mr. amir hasan khan, the local occupant of the area, visited different sites with his volunteer team including a taxonomist and a pharmacist. he arranged several meetings with the local representatives known as maliks, to whom objectives of the study were presented. a semistructured questionnaire was developed to gather knowledge on ethnoveterinary plants by following the method adopted by martin [ ] . mostly, the folk knowledge was gathered from nomads, farmers, and aged community members. the interviews were conducted at various places and in the local language called "pashto." each informant was acknowledged by presenting the main theme of the study to them in order to gain their consent and trust, which allowed the informants to talk more freely and openly. the recorded information was once again redisplayed to the informants to avoid errors and falsification. data was collected from different sites known as pashat, tali, inayat kali, ghar shamozai, loe sum, barang, mandal, khar, mamund, and salarzai. accordingly, the sites were categorized into foot hill villages and mountainous villages (fig. ) . a total of key respondents were selected belonging to different age groups, i.e., males and females ( table ). the selection of respondent was based on their high reputation with respect to traditional knowledge on ethnoveterinary plants. continuous relationships were maintained with the indigenous communities throughout the course of survey for the strong validation of traditional knowledge. surveyed ethnoveterinary medicinal plants were collected and identified by taxonomist at the department of botany, shaheed benazir bhuto university sheringal, district dir (upper), khyber pakhtunkhwa, pakistan. species botanical names and their family names were corrected and verified through the website www.kew.org/mpns. after collection, plants were pressed and dried under the shade, were poisoned ( % hgcl solution), and were mounted properly on the herbarium sheets for future reference. each herbarium sheet was labeled with a voucher number and submitted to the aforementioned department [ , ] . for each of the specie, use reports (urs) (citations) were counted. ur may be defined as the utilization of part of a plant species for a particular disease mentioned by an informant. to determine the informant consensus factor (fic), the reported species were arranged in various groups according to the ailment treated [ ] . ten ailment categories were prepared from the data. to calculate the fic, we used the formula, i.e., fic = nur − nt/ nur − . here, nur indicates the number of citations in each use category and nt represents the number of species cited. prospects and challenges to traditional ethnoveterinary knowledge indigenous communities play significant role in reporting traditional uses of medicinal flora. indigenous knowledge can be used as a tool to conserve and maintain the green diversity, and could be further utilized for scientific validation [ ] . during the nd session of united nations educational, scientific and cultural organization (unesco), traditional knowledge on ethnoveterinary medicines was declared an important part of cultural heritage, which is required to be brought under study, sustenance, and protection [ ] . indigenous communities at bajaur agency are dependent on livestock for supporting their livelihood. medicinal plants have a pivot role in the treatment of livestock's ailments in the area. usually, this treatment process depends either on the traditional knowledge being orally transmitted to the current generation of local people from their ancestors or through personal experiences. previous scientific literature has focused on the correlation of traditional medical expertise to ethnobotanical knowledge for the treatment of human ailments [ , ] , although the same plants may be used to treat livestock [ , ] . in our study, we have observed that the herders, farmers, and older community members are more equipped with traditional knowledge and familiar with veterinary medications, diagnosis process, and treatment. indigenous people of the study area are rich in traditional knowledge on veterinary medicines, which may be due to their close observation on domestic animals being considered as an important part of traditional lifestyle. most commonly, the male community member grazes herds of animal, while females take part in households' management. figures and showed some of the images of the grazed domestic animals, which are treated with medicinal plant in the area. other studies have explained this in a different way that men due to close proximity tend to know more about the animal behavior than women [ ] . people of the study area use plants not only for medicinal purposes to treat their domestic animals but also as a fodder. local community also prevents their animals from such nutrition, which is not healthy in certain conditions and seasons. one may consider this prevention to be a part of ethnoveterinary practices. nutrition is playing an important role in ethnoveterinary practices in both prevention and cure of domestic animals [ ] . livestock usually ingests some extra and non-important food substances in the green fodder, which could be termed as food medicines or medicinal food [ ] . studies have highlighted the importance of "food as medicines" in the context of local traditional knowledge; however, possible health advantages of food in ethnoveterinary methods need further attention [ ] . testing the nutritional status of each traditional ethnoveterinary remedy is not necessary; however, it is essential to evaluate the biological efficacy from the phytochemical, pharmacological, toxicological, and clinical perspectives for wider application. a considerable proportion of the documented uses of plant taxa in our study are in accordance with the established pharmacological effects [ ] . the prevailing indigenous ethnoveterinary knowledge in the study area is facing certain constrains leading it towards extinction. as an example, the nature of traditional knowledge is making it more difficult to learn and then transfer it in an accurate way. furthermore, practicing traditional therapies are not being respected by the new generation. other challenges include low literacy rate in the study area, no proper documentation of indigenous knowledge, and introduction of modern allopathic medicines, rapid technological advancement, and environmental degradation. similar kinds of threats have also been reported in other communities across the world [ ] [ ] [ ] . informants with little education were found less familiar to the traditional knowledge while people having no formal education were more responsive in this regard. some studies have found that education can be correlated with expertise either positively [ , ] or negatively [ ] , while others found no relationship [ ] . moreover, it is also ambiguous to determine the effect of "modernity" on the loss of ethnomedicinal knowledge. modernity has an established association with greater medicinal competence in dominica [ ] but appeared unrelated to variation in expertise among tsimane horticulturalists in bolivia [ ] . furthermore, it is also unclear whether correlation of expertise exists between ethnomedicinal knowledge and ethnoveterinary approaches; however, livestock keepers hold extensive knowledge related to disease prevention, diagnosis, and both traditional and novel biomedical treatments [ ] . in summary, despite maintaining knowledge on ethnoveterinary practices by the locals, the tendency to utilize modern pharmaceuticals is increasing day by day. hence, the conservation of ethnomedicinal knowledge by the local communities is extremely important for the livestock's health in the remote areas. the use of plants for medical purpose to treat a wide array of maladies emanates traces since the recorded history and even before. in our study, plant species belonging to families were documented. table presents details on the documented medicinal plants including their botanical names, vernacular names, family names, specimen numbers, parts used, medicinal uses, and use reports. family apiaceae ( species) has the high number of individual species used in ethnoveterinary practices followed by fabaceae ( species). other studies have also reported apiaceae as the dominant plant family being used in traditional medications [ , ] . the rationale of high use of apiaceae species in the current study, though based on traditional evidence, may be referred to their chemical constituents such as phenolics, poly phenolics, lectins, alkaloids, terpenoids, and essential oils, which carry antimicrobial potential [ ] . due to the predominance of sheep, goats, cows, and donkeys in the study area, we have specifically recorded the ethnoveterinary practices used for the treatment of these four types of domestic animals. key informants declared extensive uses of visnaga daucoides gaertn. table ) . medicinal plants with high urs strengthen the concept that such species are more significant to the local population and useful in sharing the traditional knowledge with one another in the area. in our study, v. daucoides is used to treat diarrhea, abdominal pain, and retained placenta in domestic animals. a whole plant is subjected to powder and is combined with flour and black tea to treat digestive problems especially in cow and buffalo. amaryllidaceae bulb of the herb is crushed and added milk for orally given to animals ( - days) for curing digestive complaints. bulb is crushed and mixed with way to administered orally for several days in order to rate of fertility in domestic animals. narcissus tazetta l. "sbbu- " gul-e-nargas leaves along with gurr and flour, fresh leaves (¼ kg) are boiled and orally given to livestock for the retained placental removal. apiaceae tea is prepared from its fruit (¼ kg), and then, it is combined with flour and given to cattle for days in order to treat gastric problems. cuminum cyminum l. "sbbu- " half kilogram of fruit is boiled in black tea and orally given for - days on daily basis for the expulsion of intestinal worms and treated gastric problems. eryngium biehersteinianum (m. bieb.) nevski "sbbu- " stem and leaves and stem powder of its stem and leaves is orally given for the treatment of liver problems up to the duration of - days. foeniculum vulgare mill. "sbbu- " fruit, leaves decoction is made from it fresh leaves and fruit ( - g), and t hen, it is combined with gurr, given orally to livestock for appetite and as sedative for the duration of to days. wall. ex dc. "sbbu- " fresh root of the plant ( g) is combined with wheat flour and made to paste which is orally given to goat, cow, and sheep as sexual tonic and to enhance the rate of fertility up to days. trachyspermum ammi (l.) sprague "sbbu- " seeds (¼ kg) of the plant, allium cepa, wheat flour, and foeniculum vulgare are thoroughly mixed. the resultant blend is then orally given ( days) and is considered as good appetizer. tea is made from its fruit and given orally to sheep, goat, cow, and buffalo while treating diarrhea, abdominal pain, and retained placenta. the remedy is constantly utilized for the duration of days. calotropis procera (aiton) dryand. "sbbu- " plants' fresh leaves are taken and decoction is made, and after that, the decoction is combined with "ajuga integrifolia" and is used for dermal parasites for to days. nerium oleander l. "sbbu- " to relieve the external parasite, the decoction of its leaves is used for animal bathing especially goat and cow. the decoction obtained from its bark and is combined with butter which is administered orally to all type of domestic animals to treat skin problems and as blood purifier. cassia fistula l. "sbbu- " amaltas fruit fruit of the plant is subjected to boiling along with milk and administered orally up to days to all sort of domestic cattle to relieve fever and gastric complexities. glycyrrhiza glabra l. "sbbu- " khwaga waly roots root (¼ kg) is subjected to paste which is mixed with flour and oil and then is given to goat, sheep, cow, and buffalo to increase milk production and enhance the rate of fertility. the remedy is used for the duration of to days. lotus corniculatus l. "sbbu- " stem and leaves, stem and leaves are crushed in weight of ¼ kg and orally given to cattle along with bread or dough for to days as sexual tonic and for urinary tract infections (uti). trigonella foenum-graecum l. "sbbu- " malkhoozi seeds seeds ( g) are crushed and given in dough to animals ( - days) against gastric disorders. decoction is made from the leaves and then gurr is added. this remedy is given orally to cattle for blood purification and as vormifuge. the water is applied topically to treat skin ailments. mentha spicata l "sbbu- " powder is made and decoction is made and then mixed with gurr and taken by animals to cure digestive problems. ocimum basilicum l. "sbbu- " kashmaly leaves seed plant leaves and seeds are subjected to decoction and used topically for skin problems. salvia moorcroftiana wall. ex benth. "sbbu- " kharghwag leaves decoction of its leaves is given orally daily for the treatment of digestive problems. gossypium arboreum l. "sbbu- " pomba kal about ¼ kg of its powder is mixed with gurr and used for to days. this remedy is administered orally on daily basis as galactagogue. grewia optiva j.r.drumm. ex burret "sbbu- " whole plant dried plant powder is subjected to oil ( ml), administered orally and topically twice a day for to days for wound healing process. the usage mode of ethnoveterinary plant species by one ethnic community is different from other communities due to difference in traditional knowledge [ , ] . previous literature has shown that decoction of the fruit of v. daucoides is used during abdominal pain, which is used to enhance body temperature in the study area [ , ] . in the same way, the f. vulgare is considered as a strong appetite and sedative. in other cultures across the globe, f. vulgare is used for various livestock problems. for instance, this plant is effective in digestion and diarrhea, when mixed with camellia sinensis, trachyspermum ammi, ghee, and sugar [ , ] . pneumonia is also being treated by giving its seeds to the animals [ ] , while other uses include galactagogue and ruminative [ ] . various parts of s. virginianum are taken for the treatment of cough, fever, milk production, and pain. there is scarce literature on the use of s. virginianum as galactagogue, which shows the unique use of this plant species in the study area and familiarity of local population through longtime experiences. published literature has indicated that the plant is also used for wound healing process [ ] fever, cough, and intestinal infections [ ] . roots and leaves of w. somnifera are given to sheep, cow, and buffalo for milk production and used as antipyretic and sexual tonic. indigenous populations comprising of various cultures residing in lesser himalayas (pakistan) use w. somnifera for bovine mastitis [ ] , while in ethiopia, this plant is being used to protect animals from bad evils [ ] . the plant has carminative effects and is used to remove the flatulence [ ] . additionally, this plant is used as refrigerant and for abdominal pain, digestion, jaundice, skeletonmuscular ailments, and wound healing against sunstroke [ ] ; for treating diarrhea [ ] ; for trypanosomiasis [ ] ; and for anorexia [ ] . informant reported g. glabra as galactagogue and enhances the rate of fertility. mussarat et al. [ ] reported that this plant is culturally used for the treatment of cough by the indigenous communities residing near the indus river, pakistan. however, from the literature, no conclusive evidence was found on the reported uses of g. glabra in our study. such evidence-based observations could justify the idea of cultural diversity across the regional level in plant remedies. previous studies related to the human's uses of g. glabra have demonstrates its effectiveness in the treatment of sex hormone imbalances and menopausal symptoms in women [ ] . in the current investigation, rhizome of c. longa is used as antiparasitic and treating genital infection and problems. in other cultures, across the country, the dried rhizome of c. longa is mixed with eggs and given for mastitis [ ] , jaundice, and skeleton muscular ailments [ ] . decoction of its leaves is mixed sugar, which is used as wound healing agent [ , ] . a root of c. longa is used for hoof problems and sore joints [ ] . in our study, the mustard oil is mixed with whey and is taken orally to relieve abdominal pain. the cultural ethnoveterinary uses from the lesser himalayas (pakistan) include that the oil extracted from b. rapa seeds is utilized for stomach disorders, eye infection, and skin diseases [ ] . furthermore, brassica rapa l. seeds are used for the retention of fetal membrane, while its oil is effective in treating genital prolepses and sores [ ] . this plant is also used in placental retention and mastitis and as antiparasitic [ ] ; myiasis, mange, and helminthiasis [ ] ; and flatulence [ ] . all these researchbased findings showed that the same medicinal plants are being used in different parts of the country; however, their uses differ from area to area and from culture to culture [ ] . the ethnoveterinary plants use by one ethnic community is almost different from other communities due to several reasons. to make a comprehensive comparative cultural diversity analysis of plant utilization in ethnoveterinary practices, we have selected a study conducted by aziz et al. [ ] in the fata region of pakistan. in comparison, we have found that most widely used medicinal plant species in our study are v. daucoides, f. vulgare, s. virginianum, w. somnifera, g. glabra, and c. longa. while according to aziz et al. [ ] , the ethnic communities in south waziristan agency are widely utilizing plant species such as b. rapa, punica granatum, capparis decidua, mentha longifolia, withania coagulans, and c. longa, during comparative analysis, it was found that only medicinal plants were commonly used in both regions for ethnoveterinary practices, which include acacia modesta wall, allium cepa l., allium sativum l., b. rapa, calotropis procera (aiton) dryand., cannabis sativa l., chenopodium album l. c. longa, f. vulgare, juglans regia l., nicotiana tabacum l., peganum harmala l., quercus oblongata d. don, trachyspermum ammi (l.) sprague, and v. daucoides. certain variations in the utilization of these plants and their parts were observed in both areas. for instance, the bulb of a. cepa is used as galactagogue by waziristanian communities while in bajaur, it is used to treat digestive problems. a. sativum is utilized for genital prolapsed while the same plant is used as sexual tonic for animals in bajaur agency. the seeds of b. rapa are widely used as appetizer and tonic and for cough, seasonal allergies, stomach disorders, and skin infections in south waziristan agency, while in the other region, it is used only against gastro-intestinal disorders. the indigenous communities at south waziristan agency consider the leaves of c. procera useful in joint pain while on the other side, the residents of bajaur agency used the latex against skin problems. c. album is used for wound healing and flatulence at waziristan while as stomachic at bajaur agency. j. regia is given for the retention of placenta at waziristan while gastric problems in bajaur. p. harmala is extensively used for gastrointestinal problems, as antiparasitic, and for skin diseases by waziristanian communities, while it is used only for the riddance of external parasites in bajaur. the possible reason for low consensus of the two regions in ethnoveterinary medicinal plants may be due to unique vegetation and distinct socio-cultural values. according to a survey, out of plant-derived pure compounds, % ( plant species) were having the same potential as indicated in traditional medications [ ] . as an example, galegine is obtained from galega officinalis l. and is used in the production of metformin and other bisguanidine-type anti-diabetic drugs [ ] ; khellin, extracted from v. daucoides., led to the development of cromolyn in the form of sodium cromoglycate, which is used as a bronchodilator; and papaverine isolated from papaver somniferum forms the baseline for verapamil, which is generally utilized for hypertension [ ] . survey participants did not describe the standardized dosage and recovery time like other previous ethnoveterinary documentations. the main problem highlighted in other studies is the lack of accuracy in such ethnoveterinary practices, which also push the locals towards modern allopathic drugs for livestock health maintenance [ , ] . the main reason that veterinarian has always complained is the non-standardized dosage in traditional medicines. though this is an accusation, one ethnomedicine does not mean that they lack efficacy but require standardization, which could benefit the traditional system by minimizing risks and toxicities. according to kearns [ ] , ethnoveterinary medicines are facing a great intellectual challenge from social theory and postmodernism, and this challenge was focused while detecting variations in animal health practices, beliefs, and experiences of various social groups. generally, it is not possible for all ethnoveterinary practices to be effective and, at the same time, they have certain weakness in terms of their efficacy as compared to modern medications [ ] . though it is convincing that most of the traditional veterinary medications have clear and sound health effects, many modern allopathic drugs are based on these medicines [ ] . certain plants in our study were used in single form for more than one disease. for example, cedrus deodara (roxb. ex d. don) g. don is used in a condition, in which milk obtained from the cattle gives bad smell, then the oil is given orally to the cattle. it is also used as a cooling agent and in treating digestive problems. in large quantity, the oil have the potential to depress the sexual power of male animals [ ] . monteiro et al. [ ] also reported similar findings from pakistan and brazil, respectively, where they described multiple uses of a single medicinal plant. utilization of certain plant species for multiple diseases is a widespread practice in ethnoveterinary medications. in contrast, some ethnoveterinary remedies (polyherbal formulations) are being made by combing two or more plants and additives such as whey, ghee, and sugar. this addition is generally followed in remedies to counteract the astringent taste, dilute, and reduce the relative potency of the remedy [ ] . in the study area, a total of plants were reported for gastrointestinal problems with maximum use reports of (table ) , which is regarded as the most common disease category in domestic animals being represented by abdominal pain, diarrhea, and digestive problems. these health issues can be easily detected by the respondents and may explain the fact that why the gastric problem category is high in ours as well as in others studies. different ailments were categorized into groups such as dermatological, gastrointestinal, galactagogue, reproductive, respiratory disorders, tonic, wound healing fever, and miscellaneous. those medical conditions, which were not fully described by the interviewees, were placed into the miscellaneous category. these include eye problems, weakness, and abnormal conditions related to various organ systems of animal bodies. highest fic values were recorded for dermatological problems ( . ) followed by reproductive ailments ( . ) and gastric disorders ( . ) ( table ) . fic value is an indicator of showing the consent of the local people on a specific plant species and efficacy of a certain taxa [ ] . sharma et al. [ ] declared that when fic becomes , it means that the local population is exchanging their view, ideas, and information about traditional medications, while on the other side, if the fic value is , then it is vice versa. fic value in the current study was recorded in between . and . for various livestock ailments (table ). these findings indicate the highest consent among the local people on traditional herbal therapies. previous research studies conducted in other areas also agreed to high consent of local people on traditional animal therapies. for instance, the reported fic values for dermatological problems were . , . , and . [ , , ] ; for reproductive disorders, . and . [ , ] ; for gastric problems, . , . , . , . , and . [ , [ ] [ ] [ ] ; for galactagogue, . and . [ , ] ; and for wound healing, . and . [ , ] . heinrich et al. [ ] has submitted the idea that high fic values can be used as a tool to target the plants for the isolation of biologically active components. in our study, most livestock's ailments were mentioned to be seasonal and epidemic due to change in fodder. furthermore, the concept of hot and cold food is also famous in order to prevent animals from diseases. the local residents change the relative fodder in different seasons in order to minimize the chances of various health problems in cattle. as an example, the seeds of the nigella sativa l. and kernels of q. oblongata are given to the cattle to energize them during the cold season. similarly, the fruits of the streblus asper lour. produce cooling effects and considered to be a better remedy during hot summer season. in the same manner, local communities tend to give the infusion of cannabis sativa l to their livestock in the summer season. quinlan [ ] and raziq et al. [ ] has also mentioned the concept of hot and cold food in traditional veterinary medications. drugs derived from plants or their extracts have certain therapeutic properties. to replace antibiotics by suitable therapeutic agents, plants can play an important role in combating with bacterial pathogens. there are several essential oils, which can be used as alternate of antibiotics. these oils can be easily isolated, having low toxicity on mammalian cells, and can be easily degraded in soil and water [ ] . in this section, we will analyze the pharmacological evidences of the most utilized studied medicinal plant species in order to check their therapeutic efficacy. in f. vulgare, phenols, phenolic glycosides, and volatile aroma compounds such as transanethole, estragole, and fenchone are reportedly the key phytoconstituents and responsible for its antioxidant activity. f. vulgare is pharmacologically validated (in vitro and in vivo) in demonstrating activities such as antibacterial, antifungal, antioxidant, antithrombotic, and hepatoprotective [ ] . by investigation, it was found that the leaf extracts of s. virginianum is more active against candida albicans, salmonella typhi, staphylococcus aureus, and nematodes [ , ] . for various extracts obtained in alcohol and water, it was found that w. somnifera has antibacterial potential, antihypercholesterolemic activities as well as diuretic potential [ , ] . it has been reported that alcoholic and aqueous extracts of c. longa have shown antibacterial activity [ ] while its ethanol, petroleum, water, and chloroform extracts are effective against certain strains of viruses, bacteria, and fungi and also have shown anti-inflammatory effects [ ] . researchers have claimed that plant-derived medicines used in traditional systems across the globe can be used as an indicator to consider them more effective than modern drugs [ ] . livestock keepers are using several plant-derived remedies for various acute as well as chronic disorders of cattle. plant-derived medicines have been used by physicians for hundreds of years in traditional systems, and most of the world population rely on these products for health care systems [ ] . there are several thousand plants across the globe being utilized for various therapeutic purposes both animals and humans [ ] . out of these medicinal plants, very low proportion has been investigated and proved scientifically for their indigenous uses [ ] . the essential oils in medicinal plants are having strong antimicrobial potential. as an example, essential oils of cinnamon, thyme, and oregano are therapeutically effective [ ] . antibiotic resistance is an emerging global concern related to veterinary and human medications [ ] . hence, it is necessary to search for new compounds to combat antibiotic resistant bacteria. improper therapeutic utilization of antimicrobial medicines in fishery, poultry, agriculture, and animal farming facilitates the emergence and production of drug resistant strains. additionally, poor prevention and control of unhygienic practices contribute in resistance emergence. the world health organization, food and agriculture organization, and world organization for animal health are stressing to promote best practices to avoid the emergence and spread of antibacterial resistance. continuous attempts are in progress to promote the moderate use of antibiotics in human as well as in animals to tackle the problem of antimicrobial drug resistance [ ] . in general, plants should be used as an alternative to synthetic drugs and investigated for their therapeutic efficacy. certain plants in our study including boerhavia erecta l., celtis australis l., chamaecyparis obtusa (siebold & zucc.) endl., eryngium biehersteinianum (m. bieb.), gossypium arboreum l., h. candicans wall. ex dc., narcissus tazetta l., opuntia littoralis (engelm.) cockerell, and s. asper need comprehensive phytochemical, pharmacological, and toxicological investigations. current study, one health concept, and changing environment current study reports that there are several ailments being treated with medicinal plants by the indigenous populations. most prevalent disease categories were dermatological, reproductive, and gastric problems. the dominance of these diseases not only poses threats to the domestic animals but also increases the chances of zoonoses. local population uses various animal products; hence, there are maximum chances of the migration of infectious diseases from these animals to humans. linkage of the ethnoveterinary studies with the researches of other disciplines may form an interdisciplinary approach to combat several types of health issues in both animals and plants. this approach mainly led to the concept of one health, which contributes towards understanding the complexities in health problems of living beings [ ] . a recent surge in emerging infectious diseases and their putative associated costs to society have reignited interest in the drive of disease emergence. a number of pathogens have emerged in the last years, including the severe acute respiratory syndrome virus, hendra virus, and nipah virus. however, there is a growing concern about the h n influenza virus, which fuelled much of the recent debate around emerging infectious diseases (eids) [ ] . one of the benefits that accrued from the attention on eids has been an increased recognition across a range of disciplines that the health of animals (including humans) and the health of the broader ecosystem are inextricably linked, which certainly given momentum to one health movement. one health is not all about eids, but it also covers important issues of food security and food safety [ ] . there is a strong consensus that the climate is changing now and that human activities are the primary cause [ ] . however, it is clear that climate change will alter the distribution and incidence of a wide range of diseases either directly or indirectly (e.g., diseases with a development stage outside the host) [ , ] . the pathways by which climate change can affect host pathogen vector interactions have recently been well described by gallana et al et al. [ ] . one health initiative task force (ohitf) [ ] defines one health as "the promotion, improvement, and defense for the health and well being of all species by enhancing cooperation and collaboration between physicians, veterinarians, and other scientific health professionals and by promoting strengths in leadership and management to achieve these goals". the one health approach plays a significant role in the prevention and control of zoonoses. approximately % of new emerging human infectious diseases are defined as zoonotic [ , ] . of the infectious diseases, approximately % are caused by multi-host pathogens, characterized by their movement across various species [ ] . this gives significant credence to the importance of examining health effects across species, in order to fully understand the public health and economic impact of such diseases and to help implement treatment and preventive programs. the application of one health approach has been recognized as a critical need by international organizations as well as the preferred approach to address global health issues. it is also noted that knowledge in veterinary medicine and animal nutrition and husbandry could provide insights into human nutrition and growth. it is a widespread phenomenon that natural resources including plants are always prone to threats in their natural habitat due to rapid human intervention and destructions of natural resources. the collection process of medicinal plants for ethnic practices and other anthropogenic practices is not only destructing the indigenous flora but also posing a threat to the traditional knowledge. unesco has emphasized on the documentation and preservation of traditional knowledge in south asia generally and pakistan and india particularly. however, efforts are going on but they are not sufficient for the conservation of traditional knowledge persistent since several centuries, which can lead to valuable discoveries in modern healthcare system. the local perception of indigenous communities regarding the threats being faced to the ecological resources especially the medicinal plants was examined in the current study. the lack of awareness has been observed as a major threat to the conservation of plant resources. it was also observed that different factors including time of collection, processing, storage, and herbal preparations are important and necessary steps to be considered for both economic returns and conservation. mainly, the local healers are involved in the collection of medicinal plants. a study in the swat region of pakistan has shown that higher economic outcomes can be obtained from proper harvesting of wild medicinal plants as compared to the standard cash crop [ ] . other studies are supporting our results by showing an enormous potential in improving the harvesting, storage, use, preparation, and marketing of the herbal product as a source of income [ ] . in the remote areas of the study region, local inhabitants obtained significant economic advantages from forest products. similar advantages have been reported for other mountainous communities in the northern parts of pakistan [ ] . there are certain other threats to the medicinal plant resources of the study area, which include deforestation, heavy grazing pressure, uncontrolled collection of fodder, and other non-timber forest products by the local people and traders. several studies have reported a decrease in the number of medicinal plants due to over exploitation and environmental degradation [ , ] . it is therefore a dire need to manage and design the overall grazing system to encourage the sustainable regeneration and protection of medicinal plants. keeping the observation and findings of the current investigation, proper management steps should be taken with the active participation from the indigenous communities to conserve this precious flora. it is also important to aware the local people about the market value and sustainable harvesting of medicinal plants. rapid modernization and urbanization is not only a threat for plant species' degradation but also a threat for the associated folk knowledge. that is why that the disappearance of folk knowledge has been declared more in danger than the natural resources themselves [ ] . therefore, we present a strong recommendation that ethnobotany as a subject should be included into the curriculum to help students in recognizing the endangered and medicinally important species of their respective regions. in addition, incentives may be given to farmers for the cultivation of medicinal plants on marginal lands and home gardens. indigenous communities at bajaur agency are dependent on medicinal plants for ethnoveterinary practices. knowledge about the traditional medicinal system is restricted to the herders, farmers, and elder community members. the younger generation is unaware of this traditional treasure and takes no interest due to modernization. hence, this study is an attempt towards the preservation of traditional ethnoveterinary knowledge from being extinct. there are several medicinal plants, which are being used in traditional herbal system of veterinary disorders. some of the important are v. daucoides, f. vulgare, s. virginianum, w. somnifera, g. glabra, and c. longa. new ethnoveterinary uses used at the study area were found for h. candicans and g. glabra. apiaceae is utmost plant family being in use for various livestock ailments. thorough phytochemical and pharmacological investigations are required by isolating the active compounds and testing the in vitro or in vivo efficacy of the abovementioned plants against the targeted veterinary diseases. furthermore, critical toxicological investigations are also required to ensure the safe and secure use of documented ethnomedicines. in order to share and further maintain this knowledge, it is direly needed to aware the rural population about the significance of traditional ethnoveterinary knowledge and to motivate them on the conservation of natural flora. intersectoral health care delivery ethnoveterinary medicines used for ruminants in british columbia documentation of ethnoveterinary practices for mastitis in dairy animals in pakistan meas. contribution livest. househ. livelihoods: a livest. module multi-topic househ. surv botanical ethnoveterinary therapies in three districts of the lesser himalayas of pakistan dairy industry in pakistan: a scenario economic survey of pakistan. government of pakistan (gop), finance division, economic advisor wing ethnoveterinary treatments by dromedary camel herders in the suleiman mountainous region in pakistan. an observation and questionnaire study ethnoveterinary medicinal practices of the villagers of usilampatti taluk of madurai district, india ethnoveterinary medicines used by tribals of tadgarh-raoli wildlife sanctuary cattle wounds and ethnoveterinary medicine: a study in polasara block ethnoveterinary practices of aborigine tribes in odisha ethnoveterinary medicinal practices in tribal regions of andhra pradesh, india plants used as ethnoveterinary medicines in sikkim himalayas ethnoveterinary uses of medicinal plants among traditional herbal healers in alaknanda catchment of uttarakhand, india ethno-veterinary practices for ephemeral fever of yak. a participatory assessment by the monpa tribe of arunachal pradesh plants used in ethnoveterinary medicine by malayalitribals in salem district of tamil nadu ethnoveterinary study of medicinal plants in a tribal society of sulaiman range ethno veterinary practices for the treatment of parasitic diseases in livestock in cholistan desert pakistan ethnoveterinary study of medicinal plants in malakand valley ethnopharmacological assessment of medicinal plants used against livestock infections by the people living around indus river ethnoveterinary medicinal uses of plants of poonch valley azad kashmir documentation of ethnoveterinary practices used for treatment of different ailments in a selected hilly area of pakistan ethno veterinary uses of medicinal plants of district karak, pakistan ethnoveterinary medicinal plant knowledge and practice among the tribal communities of thakht-e-sulaiman hills, west pakistan a methods manual. london: chapman and hall flora of pakistan. nos. - flora of pakistan. nos. - ethnoveterinary health management practices using medicinal plants in south asia-a review do pharmaceuticals displace local knowledge and use of medicinal plants? estimates from a cross-sectional study in a rural indigenous community ethnomedicine of menstruation in rural dominica, west indies understanding interrelationships among predictors (age, gender, and origin) of local ecological knowledge a survey of plants and plant products traditionally used in livestock health management in buuri district xuan dung nn. food, feed or medicine: the multiple functions of edible wild plants in vietnam ethnoveterinary herbal remedies used by farmers in four north-eastern swiss cantons circum-mediterranean cultural heritage and medicinal plant uses in traditional animal healthcare: a field survey in eight selected areas within the rubia project medicinal plants used by tibetans in shangri-la discussion on the institutionalization of participatory livestock technological development comparison of health conditions treated with traditional and biomedical health care in a quechua community in rural bolivia market economy and the loss of folk knowledge of plant uses: estimates from the tsimane' of the bolivian amazon school and local environmental knowledge, what are the links? a case study among indigenous adolescents in oaxaca schooling and local environmental knowledge: do they complement or substitute each other secular changes of indigenous knowledge of useful plants: separating age and cohort effects modernization and medicinal plant knowledge in a caribbean horticultural village pharmaceutical ethnobotany in the montseny biosphere reserve (catalonia, iberian peninsula). general results and new or rarely reported medicinal plants curing animals with plants, traditional usage in tuscany cross-cultural analysis of medicinal plants commonly used in ethnoveterinary practices at south waziristan agency and bajaur agency, federally administrated tribal areas (fata) plant products as antimicrobial agents tobacco smoke is a source of toxic reaction glycation products preliminary evaluation of antinephritis and radical scavenging activities of glabridin from glycyrrhiza glabra ethnoveterinary medicines used for horses in trinidad and in british columbia an inventory of the ethno veterinary practices for reproductive disorders in cattle and buffaloes, sargodha district of pakistan medicinal plants in therapy the value of plants used in traditional medicine for drug discovery ethnoveterinary knowledge and practices at colares island, pará state, eastern amazon, brazil medical geography: making space for difference ethnoveterinary medicine an annotated bibliography iowa state university research foundation ethno veterinary medicine: harnessing its potential ethnoveterinary knowledge of the inhabitants of marajo' island, eastern amazonia an inventory of the ethnobotanicals used as anthelmintics in the southern punjab (pakistan) ethnobotanical study of medicinal plants used by people in zegie peninsula. northwestern ethiopia ethnoveterinary remedies of diseases among milk yielding animals in kathua ethnoveterinary plants for the treatment of camels in shiwalik regions of kathua district of jammu & kashmir, india traditional arabic palestinian ethnoveterinary practices in animal health care: a field survey in the west bank (palestine) quantitative traditional knowledge of medicinal plants used to treat livestock diseases from kudavasal taluk of thiruvarur district ethnoveterinary plants of ankober district medicinal plants in mexico, healer's consensus and cultural importance ethnomedicine and ethnobotany of fright, a caribbean culture bound psychiatric syndrome selected antimicrobial essential oils eradicate pseudomonas spp. and staphylococcus aureus biofilms foeniculum vulgare: a comprehensive review of its traditional use, phytochemistry, pharmacology and safety antimicrobial activity of certain indian medicinal plants used in folklore medicine antihelmintic properties of some indigenous plants screening of some indian medicinal plants for their antimicrobial properties new delihi: campus books international herbal medicine are medicinal plants a potential alternative for conventional antibiotics in animal husbandry antimicrobial activity spectra of pelargonium graveolens l. and cymbopogon winterianus jowitt oil constituents and acyl derivatives global action plan on antimicrobial resistance antimicrobial drug resistance in bacteria isolated from sick animals and their environment in year - at central disease diagnostic laboratory, indian veterinary research institute, izatnagar. noto-are med ( ) a review of the metrics for one health benefits infectious disease emergence and global change: thinking systemically in a shrinking world ecosystem sustainability and health: a practical approach the critical decade : climate change science, risks and response. common wealth of australia department of industry, innovation, climate change, science climate change effects on animal disease systems and implications for surveillance and control. in climate change: impact on the epidemiology and control of animal diseases climate change and infectious diseases of wildlife: altered interactions between pathogens, vectors and hosts a new professional imperative-one health initiative task force. american veterinary association the animal-human interface and infectious disease in industrial food animal production: rethinking biosecurity and biocontainment institute of medicine microbial threats to health, emergence detection, and response medicinal and aromatic plant cultivation in the swat valley, north-western pakistan, for economic development and biodiversity conservation ethnomedicinal survey of important plants practiced by indigenous community at ladha subdivision, south waziristan agency ethnobotanical profile of plants of shawar valley, district swat, pakistan an ethnobotanical study of medicinal plants in high mountainous region of chail valley (district swat-pakistan) conserving indigenous knowledge as the key to the current and future use of traditional vegetables the authors extend their high appreciation and acknowledgment towards the local communities by providing moral support to the authors. this research study did not receive any grant from any organization. data gathered during the course of the study has been included in the article.authors' contributions ahk and hu conducted the field work. maa wrote the draft manuscript. ahk and hu helped in the compilation of data. ma gave technical comments on the draft and indicated the language and grammatical mistakes. maa and ma supervised all the stages. all the authors read and approved the manuscript.ethics approval and consent to participate not applicable the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - voi y authors: han, hui-ju; liu, jian-wei; yu, hao; yu, xue-jie title: neutralizing monoclonal antibodies as promising therapeutics against middle east respiratory syndrome coronavirus infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: voi y since emerging in , middle east respiratory syndrome coronavirus (mers-cov) has been a global public health threat with a high fatality rate and worldwide distribution. there are no approved vaccines or therapies for mers until now. passive immunotherapy with neutralizing monoclonal antibodies (mabs) is an effective prophylactic and therapeutic reagent against emerging viruses. in this article, we review current advances in neutralizing mabs against mers-cov. the receptor-binding domain (rbd) in the spike protein of mers-cov is a major target, and mouse, camel, or human-derived neutralizing mabs targeting rbd have been developed. a major problem with neutralizing mab therapy is mutant escape under selective pressure, which can be solved by combination of neutralizing mabs targeting different epitopes. neutralizing mabs are currently under preclinical evaluation, and they are promising candidate therapeutic agents against mers-cov infection. middle east respiratory syndrome (mers) emerged in in saudi arabia with the death of a man with pneumonia; the causative agent was subsequently identified as mers-cov, which belonged to lineage c betacoronaviruses [ ] . with dromedary camels (camelus dromedarius, also known as arabian camel) as direct sources and bats as potential reservoirs [ ] , mers-cov has been frequently introduced into human populations. once mers-cov is introduced into a person, person-to-person transmission might occur, and is responsible for approximately % of mers cases globally [ ] . mers-cov has been a consistent threat to humans. as of october , mers-cov has caused laboratory-confirmed human cases, including deaths in countries, with the fatality rate as high as % (http://www.who.int/emergencies/mers-cov/en/). although mers cases are primarily reported in the middle east, facilitated by international travelling, mers-cov can also be a worldwide threat, which is well illustrated by the mers outbreak in south korea in [ ] . given the potential risk of causing worldwide public health emergencies and the absence of licensed vaccines and antiviral therapeutics, the world health organization has listed mers-cov in the "list of blueprint priority diseases" (http://www.who.int/blueprint/priority-diseases/en/). vaccines are the most important approach against viral infections, but usually take a long time to develop. they are also unable to provide either immediate prophylactic protection or treat ongoing viral infections. neutralizing monoclonal antibodies (mabs) have recently emerged as a powerful tool to provide prophylactic and therapeutic protection against emerging viruses [ ] . potent neutralizing mabs can be achieved by various technologies, such as hybridoma technology, humanized mouse, phage or yeast display, and single b cell isolation [ ] . mers-cov is a single, positive-stranded rna virus of about kb, which encodes four major viral structural proteins-including spike (s), envelope (e), membrane (m) and nucleocapsid (n)-as well as several accessory proteins [ ] . the s protein ( aa) plays an important role in virus infection and consists of a receptor-binding subunit s (aa - ) and a membrane-fusion subunit s (aa - ). s mediates viral attachment to host cells and s mediates virus-cell membrane fusion [ ] . the s subunit contains a receptor-binding domain (rbd) (aa - ) [ ] that can bind to cell receptor dipeptidyl peptidase (dpp , also known as cd ), and mediates viral attachment target cells [ ] . the rbd consists of a core subdomain and a receptor-binding motif (rbm) (aa - ). the schematic representation of mers-cov s protein is shown in figure a . neutralizing mabs binding to the s protein of mers-cov can prevent viral attachment to the cell receptor and inhibit viral entry [ ] . the s protein of mers-cov is a key target for antivirals, and rbd is the most popular focus. in this study, we review the current knowledge on neutralizing mabs targeting the rbd of mers-cov. stable hybridoma cell lines were generated by fusing myeloma cells with splenocytes of mice that were immunized with mers-rbd protein. two neutralizing mabs, c and e , had high affinity for the rbd of mers-cov and blocked both pseudovirus and live mers-cov entry into cells with high efficacy [ ] . humanized c showed similar neutralizing activity in cell entry tests. in vivo tests indicated that c could significantly reduce the virus titers in the lungs of ad -hcd -transduced mice which were infected with mers-cov, highlighting its potential application in humans not only for preventing but also treating mers-cov infection. crystallization of the c fab/mers-rbd complex showed that the c recognized conformational epitopes (y -n , k , l -k , p , v -s , w -e , and d -q ), which were partially overlapped the receptor-binding footprint in the rbd of mers-cov. the c complex interfered with mers-cov binding to dpp by both steric hindrance and interface-residue competition. e competed with c to bind to mers-rbd, indicating that they recognized proximate or overlapping epitopes [ ] . neutralizing mab mersmab was obtained by fusing myeloma cells with splenocytes of a mouse that was immunized with recombinant mers-cov s [ ] . mersmab effectively blocked the entry of pseudovirus and live mers-cov into cells. structural analysis showed that mersmab bound to the rbd of mers-cov through recognizing conformational epitopes, and all of the residues critical for mersmab binding were located on the left ridge of rbm. mersmab neutralized mers-cov by competitively blocking the binding of mers-cov rbd to dpp . based on escape mutant analysis of the key residues on the rbd, it was found that residue l , d , r , e , and w were critical for mersmab binding to the rbd, while mutation of e , d , or e did not affect the interaction of mersmab and the rbd at all [ ] . an ultra-large nonimmune human antibody-phage display library was constructed with b cells of unimmunized donors. with a unique spanning strategy, seven human neutralizing mabs with varying neutralization efficacy to mers-cov were identified [ ] . binding detection demonstrated that the epitopes of these mabs lay within aa - of the s protein, which overlapped a large part of the rbd of mers-cov. binding competition assays showed that these mabs recognized at least three distinct epitope groups, which was further confirmed by escape studies. with no cross-epitope resistance, these mabs neutralized mers-cov by competitively blocking the binding of the rbd of mers-cov to dpp . escape mutant assays showed that five residues were critical for neutralization of these mabs, namely l , t , y , r , and p . of the seven mabs, b exhibited the best neutralization activity against both pseudovirus and live mers-cov infectivity in cells. moreover, under the selective pressure of these mabs, the igg form of b was superior, since it did not induce neutralization escape [ ] . in vivo tests demonstrated that b reduced lung pathology in rhesus monkeys infected with mers-cov [ ] . with its high neutralizing activity and suppression of mutant escape, b in the igg form is a promising therapeutic mab against mers-cov. three human mabs-m , m , and m -were identified from a large naïve human phage display antibody library, which was constructed with peripheral blood mononuclear cells from healthy volunteers [ ] . the binding sties of the three mabs were within the rbd of mers-cov (aa - ), therefore they neutralized mers-cov by competing with dpp binding to the rbd. the three mabs also competed with each other to bind to the rbd of mers-cov, and mutant analysis showed that the three mabs possessed overlapping but distinct epitopes. of the three mabs, m neutralized both pseudovirus and live mers-cov infectivity in cells with exceptional potency (m inhibited % mers-cov pseudovirus infection at a concentration of . g/ml, and neutralized live mers-cov with ic of g/ml and ic of . g/ml). residues in the rbd crucial for m binding were l , d , e , d , w , and v [ ] . in vivo study demonstrated that prophylaxis with m reduced virus titers in the lung of rabbits infected with mers-cov [ ] , and m also provided transgenic mice expressing human dpp with full prophylactic and therapeutic protection from mers-cov [ ] . however, another study with a non-human primate, the common marmoset showed that m could only alleviate the severity of the disease, and did not provide complete protection against mers-cov [ ] . igg+ memory b cells were isolated from a mers patient, and were subsequently immortalized with epstein-barr virus. a neutralizing mabs, lca , was identified, and was the first fully human neutralizing mab with naïve heavy and light chain pairs [ ] . lca efficiently neutralize mers-cov infectivity in cells. in vivo study showed that lca provided balb/c mice transduced with adenoviral vectors expressing human dpp (hdpp ) with both prophylactic and postexposure protection against mers-cov. furthermore, the neutralizing efficacy of lca was evaluated in ifn-α/β receptor-knockout mice that were more stringent models of mers-cov infection. after transducing with hdpp , these mice showed more profound clinical symptoms when challenged with mers-cov [ ] ; administration of lca reduced mers-cov titer in the lungs of these mice more effectively (lung viral titer reduced by three logs in one day for ifn-α/β receptor-knockout mice vs. three days for balb/c mice) [ ] . with naïve heavy and light chain pairs, lca was more potent than b and comparable to m . cross-competition experiment demonstrated that lca competed with b to bind to the rbd. lca interacted with rbd residues around k , and the lca footprint on the rbd was partially overlapped with that of dpp . four residues in the rbd affected the binding of lca -namely t , k , e , and e -which were conserved in all mers-cov isolates. moreover, compared with dpp , the binding affinity of lca to rbd was significantly higher (~ -fold). therefore, one major neutralization mechanism of lca was to competitively inhibit the interaction of the rbd with dpp . interestingly, virus escape studies demonstrated that under the selective pressure of lca , a mutant variant (v a) in the n-terminal domain (ntd) of mers-cov s subunit was also generated [ ] . a gmp-approved cell line (lca . . ) that expresses lca in high concentrations has been established, highlighting its application as promising therapeutics against mers-cov infection [ ] . hybridoma b cells producing neutralizing mabs against the s protein of mers-cov were generated by immunizing humanized transgenic mice (velocimmune mice) with dna encoding the mers-cov s protein. two fully human neutralizing mabs, regn and regn , were obtained [ ] . the two mabs bound with high affinity to distinct epitopes on the rbd of mers-cov, which were conserved during the natural evolution of mers-cov. mutation as a result of selective pressure by one mab should not affect the binding of the other mab. regn neutralized a broad range of mers-cov isolates, the prototype emc/ strain and all clinical mutants including a p, s g, s f, a v, l f, d g, and v a. with the exception of v a variant, regn achieved similar neutralizing activity. in vivo study demonstrated that regn and regn reduced mers-cov replication in humanized dpp mice in both prophylactic and therapeutic settings [ ] . when evaluated in the common marmoset, both mabs seemed to be more effective for prophylaxis rather than for treatment of mers-cov infection [ ] . an anti-mers-cov phage display antibody library was constructed with the peripheral b cells of a mers survivor, and a human neutralizing mab against mers-cov, mca , was identified [ ] . mca showed potent neutralizing activity against mers-cov in cell entry tests. in vivo, mca completely inhibited the replication of mers-cov in common marmosets when administrated prophylactically or therapeutically. structure analysis of the mca fab-rbd complex showed that mca formed direct contacts with the receptor-binding site (rbs) subdomain on the rbd. epitopes on the rbs critical for mca binding were d , w , e , d , y , r , and q . superimposed structure analysis of mca -rbd and hdpp -rbd complexes showed that the binding interface of mca was largely overlapped with that of hdpp . therefore, the neutralizing mechanism of mca was achieved by competing with dpp for binding to the rbd [ ] . two potent human neutralizing mabs, mers- and mers- , were derived from a nonimmune human yeast display antibody library, which was constructed with spleen and lymph node polyadenylated rna from normal humans [ ] . [ ] . further structural analysis showed that mers- bound to unique epitopes and caused conformational changes in the rbd interface critical for accommodating dpp , therefore indirectly disrupting the interaction between the two. moreover, mers- also demonstrated synergistic effects with m and f (a ntd-specific mab). the special neutralizing mechanism made mers- a valuable addition for the combined use of mabs against mers-cov infection [ ] . thirteen ultrapotent neutralizing mabs, which all targeted the rbd of mers-cov were generated following a protocol for the rapid production of antigen-specific human mabs [ ] . briefly, antibody-secreting b cells were isolated from the whole blood of a mers patient, and the antibody genes were amplified and cloned into vectors to transfect human cell lines for mab production. of the mabs, mers-gd and mers-gd exhibited the strongest neutralizing activity against both pseudovirus and live mers-cov in cell infection tests. mers-gd directly competed with dpp to bind to the rbd to dpp , and the crystal structure of mers-gd showed that its epitopes were almost completely overlapped with dpp -binding sites. mers-gd and mers-gd recognized distinct epitopes on the rbd, and had a low level of competing activity. the combined use of the two mabs demonstrated synergistic effects in neutralization against pseudotyped mers-cov. mutant analysis demonstrated that residues l , d , v , e , and a on rbd were important for the neutralizing activity of mers-gd , and residue r was critical for mers-gd [ ] . moreover, in vivo study found that mers-gd could provide both prophylactic and therapeutic protection for hdpp -trangenic mice against mers-cov infection [ ] . dromedary camels exposed to mers-cov showed mild clinical signs but developed exceptionally potent neutralizing antibodies. camelid species naturally produced heavy chain-only antibodies (hcabs) [ ] , which are dimeric and devoid of light chains, and their antigen recognition region is solely formed by the variable heavy chains (vhhs) (also called nanobodies, nbs). vhhs or nbs have long complementarity-determining region (cdr ) loops and are capable of binding to unique epitopes not accessible to conventional antibodies [ ] . notably, camelid vhhs are relatively stable and can be produced with high yields in prokaryotic systems [ ] . because of their small size; good tissue permeability; and cost-effective production, storage, and transportation [ ] [ ] [ ] , vhhs or nbs have been gaining acceptance as antiviral agents. a vhh complementary dna library was constructed with the bone marrow of dromedary camels infected with mers-cov. four vhhs (vhh- , vhh- , vhh- , and vhh- ) with high neutralizing activity were identified by direct cloning and screening of the phage display antibody library [ ] . the four vhhs competed for a single epitope that partially overlapped with the rbd-dpp interface. mutant analysis showed that the four vhhs did not bind to the d n variant, which was a critical residue on the rbd for dpp binding [ , ] . therefore, these vhhs most likely neutralized mers-cov by blocking its binding to dpp . of the vhhs, vhh- showed the best neutralizing activity and epitope recognition. vhh- efficiently blocked the entry of mers-cov into cells, and it also prophylactically protected k transgenic mouse expressing hdpp from mers-cov infection. to extend the half-life of vhh- in serum, it was linked to a human fc domain lacking the ch exon to construct the chimeric camel/human hcab- , which showed similar neutralizing activity as vhh- . the chimeric camel/human hcab- was highly stable in mice and provided k mice with fully prophylactic protection against mers-cov infection [ ] . alpacas were immunized with recombinant mers-cov rbd-containing a c-terminal human igg fc tag, and vhhs were amplified from their peripheral blood mononuclear cells to construct a vhh phage display library. a neutralizing nb, nbms , which bound with high affinity to the rbd of mers-cov and blocked the binding of rbd to dpp , was identified [ ] . to extend its in vivo half-life, the human-fc-fused version, nbms -fc, was constructed. nbms competed with dpp to bind to rbd, indicating that the binding site of nbms on rbd overlapped with that of dpp . the binding site of the nbms on the rbd was mapped to be around residue d , which is part of a highly conserved conformational epitope at the receptor-binding interface in almost all the natural mers-cov published to date. nbms did not neutralize psuedotyped mers-cov bearing a mutation in d , confirming that residue d was critical for nbms binding. nbms efficiently neutralized the cell entry of live mers-cov. moreover, nbms showed potent prophylactic and therapeutic efficacy in protecting hdpp -transgenic mice against mers-cov infection [ ] . for their exceptionally high neutralization activity in vitro and in vivo, these newly identified neutralizing mabs are promising candidate therapeutics against the infection of mers-cov. however, the use of a single neutralizing antibody bears the risk of selecting escape mutants, a fact that has been observed for lca and other described antibodies [ , , ] . notably, the majority of these escape mutations had little impact on viral fitness and the interaction of dpp with the rbd [ ] . moreover, mutants of mers-cov during natural infection have also been reported [ ] . escape from neutralization is a major concern with therapeutic neutralizing mabs, however, this potential problem can be solved by combining mabs that target distinct epitopes and show different neutralizing mechanisms [ ] . this strategy can take advantage of the synergistic effects while decreasing the possibility of viral escape. currently, most of the mers-cov neutralizing mabs compete with dpp binding to the rbd, and residues on the rbd critical for mab neutralization are identified by mutant analysis. almost all of the residues identified critical for mab neutralization are located in rbm, and overlap with those critical for dpp binding ( figure b) . with the availability of crystal structure of mab fab-rbd complex, the neutralization mechanism of these mabs will be better illustrated. based on the crystal structure of rbd-dpp , it was found that several conserved residues in the rbd are critical for the interaction of the rbd with dpp (y , l , d , e , e , d , d , y , r , w , and v ) [ , ] . development of therapeutic neutralizing mabs targeting those critically conserved residues might be important for combating mers-cov. moreover, a study found a mouse-derived neutralizing mab, f , which bound to a possible linear epitope in the ntd of the mers-cov s subunit, exhibited efficient neutralizing activity against pseudovirus and live mers-cov in cell entry tests. this study highlighted the important role of ntd during the infection process of mers-cov. ntd might have significant implications for the development of prophylactic and therapeutic mabs against mers-cov infection [ ] . although the in vitro neutralizing potency of f was approximately -fold lower than that of the rbd-targeting neutralizing mabs [ ] , it may provide an alternative for the immunotherapy against mers-cov, once the virus mutates and is no longer susceptible to rbd-specific mabs. so far, there is a lack of appropriate animal models to mimic the pathology of merd-cov in humans. commonly-used laboratory animals-such as wild-type mouse, ferret, hamster, and guinea pig-are not susceptible to mers-cov infection due to differences in critical amino acids in the s-binding domain of their dpp [ ] [ ] [ ] . new zealand rabbits, hdpp -transduced/transgenic mice, camelids and non-human primates (rhesus macaque and common marmoset) are susceptible to mers-cov infection, however, rabbits showed asymptomatic infection [ ] ; dromedary camels displayed different clinical manifestations to that of humans [ ] ; rhesus macaque only showed transient lower respiratory infection [ ] , while common marmoset developed progressive pneumonia [ ] ; hdpp -trangenic mouse expressed hdpp extensively, and resulted in multiple organ damage [ ] ; hdpp -transduced mouse only exhibited mild transient clinical diseases [ ] . with robust animal models, the protective effects of these neutralizing mabs will be better evaluated. furthermore, ongoing efforts on developing therapeutic neutralizing mabs against mers-cov should also consider the different target populations (dromedary camels and humans) and their protective efficacy. isolation of a novel coronavirus from a man with pneumonia in saudi arabia evidence for zoonotic origins of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea human monoclonal antibodies as candidate therapeutics against emerging viruses. front genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans mers-cov spike protein: a key target for antivirals structure of mers-cov spike receptor-binding domain complexed with human receptor dpp dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution b -n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n / . virology exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection efficacy of antibody-based therapies against middle east respiratory syndrome coronavirus (mers-cov) in common marmosets prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus rapid generation of a mouse model for middle east respiratory syndrome rapid generation of a human monoclonal antibody to combat middle east respiratory syndrome pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection prophylactic and therapeutic efficacy of mab treatment against mers-cov in common marmosets human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein structural definition of a unique neutralization epitope on the receptor-binding domain of mers-cov spike glycoprotein ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient a novel human mab (mers-gd ) provides prophylactic and postexposure efficacy in mers-cov susceptible mice naturally-occurring antibodies devoid of light-chains molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies nanobodies: natural single-domain antibodies application of camelid heavy-chain variable domains (vhhs) in prevention and treatment of bacterial and viral infections nanobodies(r) as inhaled biotherapeutics for lung diseases generation and characterization of alx- , a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection chimeric camel/human heavy-chain antibodies protect against mers-cov infection molecular basis of binding between novel human coronavirus mers-cov and its receptor cd a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape severe respiratory illness caused by a novel coronavirus identification of residues on human receptor dpp critical for mers-cov binding and entry a novel neutralizing monoclonal antibody targeting the n-terminal domain of the mers-cov spike protein wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase -mediated entry of the middle east respiratory syndrome coronavirus asymptomatic middle east respiratory syndrome coronavirus infection in rabbits experimental infection of dromedaries with middle east respiratory syndrome-coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase an animal model of mers produced by infection of rhesus macaques with mers coronavirus infection with mers-cov causes lethal pneumonia in the common marmoset multi-organ damage in human dipeptidyl peptidase transgenic mice infected with middle east respiratory syndrome-coronavirus funding: this study was supported by a grant from national natural science funds of china (nos. ). the authors declare that they have no conflict of interest. key: cord- -rdlinzrn authors: gralinski, lisa e.; sheahan, timothy p.; morrison, thomas e.; menachery, vineet d.; jensen, kara; leist, sarah r.; whitmore, alan; heise, mark t.; baric, ralph s. title: complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: rdlinzrn acute respiratory distress syndrome (ards) is immune-driven pathologies that are observed in severe cases of severe acute respiratory syndrome coronavirus (sars-cov) infection. sars-cov emerged in to and led to a global outbreak of sars. as with the outcome of human infection, intranasal infection of c bl/ j mice with mouse-adapted sars-cov results in high-titer virus replication within the lung, induction of inflammatory cytokines and chemokines, and immune cell infiltration within the lung. using this model, we investigated the role of the complement system during sars-cov infection. we observed activation of the complement cascade in the lung as early as day following sars-cov infection. to test whether this activation contributed to protective or pathologic outcomes, we utilized mice deficient in c (c (–/–)), the central component of the complement system. relative to c bl/ j control mice, sars-cov-infected c (–/–) mice exhibited significantly less weight loss and less respiratory dysfunction despite equivalent viral loads in the lung. significantly fewer neutrophils and inflammatory monocytes were present in the lungs of c (–/–) mice than in c bl/ j controls, and subsequent studies revealed reduced lung pathology and lower cytokine and chemokine levels in both the lungs and the sera of c (–/–) mice than in controls. these studies identify the complement system as an important host mediator of sars-cov-induced disease and suggest that complement activation regulates a systemic proinflammatory response to sars-cov infection. furthermore, these data suggest that sars-cov-mediated disease is largely immune driven and that inhibiting complement signaling after sars-cov infection might function as an effective immune therapeutic. from coronaviruses circulating in animal markets in china ( ) . emergence of this novel virus led to a global outbreak of respiratory disease, with over , human cases and % mortality ( , ) . in , a new, related zoonotic coronavirus was identified in the middle east, designated middle east respiratory syndrome coronavirus (mers-cov), causing severe respiratory disease with greater than % mortality (www .who.int/emergencies/mers-cov/en) ( ) . both sars-cov and mers-cov cause a range of disease from asymptomatic cases to severe acute respiratory distress syndrome (ards) and respiratory failure ( ) . notably, metagenomics and synthetic virus recovery strategies have since revealed the existence of large pools of preepidemic sars-like bat coronaviruses which replicate in primary human airway epithelial cells. these viruses are poised for emergence because they both efficiently use human ace entry receptors and resist existing vaccines and immunotherapeutics ( , ) . due to the ongoing threat and continued emergence of new, highly pathogenic coronaviruses from animal reservoirs, a thorough understanding of the host-virus interactions that drive sars-cov pathogenesis will aid the public health response to current and future coronavirus outbreaks ( ) . the importance of complement in sars-cov pathogenesis is controversial. previous studies have investigated the role of known polymorphisms in the mannose-binding lectin (mbl) and mbl-associated serine protese- (masp ) genes in sars-cov infection outcome following the outbreak but with conflicting results. one retrospective analysis showed that people with low or deficient serum mbl levels were more likely to become infected with sars-cov ( ) than those with high mbl levels, suggesting that mbl and complement activation play a role in protecting the host from infection. however, a second study found no association between mbl haplotype and sars-cov infection status ( ) . additionally, it was shown mbl can bind to the sars-cov spike protein in vitro by some groups ( ) but not by others ( ) . examination of the role of the downstream complement gene masp found no association between genotype and sars susceptibility ( ) . together, the results leave a general uncertainty about the role of complement in response to sars-cov infection. despite the existing body of literature, the role of complement in sars-cov pathogenesis has never been directly assessed in vivo. the complement system is an ancient arm of the innate immune response comprised of multiple proteins whose reactive cascade of cleavage products can coordinate the inflammatory response at the sites of infection and can be directly antimicrobial. consisting of more than soluble and cell surface-associated proteins, complement is a major component of innate immunity that functions to recognize and eliminate invading pathogens ( ) . activation of the complement system occurs through multiple mechanisms that include three welldescribed pathways, the classical, lectin, and alternative complement activation pathways ( ) , and results in proteolytic processing of various components of the complement system, including c , c , and c . proteolytic processing of c generates an array of cleavage products that are involved in amplification of complement activity through formation of c and c convertases, opsonization of pathogens, and attraction and activation of leukocytes of both the innate and adaptive arms of the immune response. several studies, including a recent study showing that complement blockade results in reduced disease in a mers-cov human dpp transgenic (hdpp -tg) mouse model ( ) , have elucidated protective and pathogenic roles for the complement system following infection by a variety of viral pathogens ( ) . furthermore, the complement system has well-described roles in other pulmonary diseases ( ) , especially after influenza virus and respiratory syncytial virus infection ( ) ( ) ( ) . in this study, we assessed the role of the complement system in the pathogenesis of sars-cov infection. building from a systems biology analysis that suggested that complement was modulated during sars-cov infection, we confirmed that complement was activated upon sars-cov challenge. mice deficient in c (c -/-), the central protein of the complement signaling pathway, were protected from sars-cov-induced weight loss and had reduced pathology, improved respiratory function, and lower levels of inflammatory cytokines/chemokines in the lung and periphery. importantly, the kinetics and magnitude of virus replication in c -/and wild-type mice were the same, showing that complement does not play a role in controlling virus replication. we observed complement deposition in the lungs of sars-cov-infected mice, suggesting that complement activation results in immune-mediated damage to the lung. additionally, serum activation indicates that complement-mediated systemic inflammation may drive the pathogenic response to sars-cov infection. together, the results indicate that complement plays a critical role in sars-cov pathogenesis and that inhibition of the complement pathway might be an effective therapeutic to coronavirus-mediated disease. complement is activated in sars-cov ma -infected mice. while work by other laboratories has shown that c -deficient mice are extremely susceptible to both h n and h n influenza virus infection ( ) , the role of complement in sars-cov infection has not yet been evaluated in vivo. using a systems biology-based approach, we identified the complement pathway as a high-priority target for control of sars-cov ma (the mouse-adapted sars-cov) pathogenesis based on weighted gene correlation network analysis (wgcna) of rna transcripts in the lungs of mice infected with a lethal versus a sublethal dose of sars-cov ma ( ) . because the complement signaling cascade is activated through proteolytic cleavage events, we also assessed lung proteomics samples for complement protein abundance. c b, cfb, and c all had significantly higher abundances in the lungs of mice infected with a lethal dose of sars-cov ma than in those of mice infected with a sublethal dose (fig. a) . complement activation is measured by detection of pathway component cleavage products. c , the master regulator of complement signaling, is cleaved into c a and c b following creation of c convertase. c activation products (c fragments c a, c b, ic b, c dg, and c c) were detected by western blotting in lung tissue of sars-cov ma -infected mice, but not in control mice, as early as day postinfection (dpi) (fig. b) , confirming that sars-cov ma infection activates the complement pathway. to test the importance of the complement signaling pathway in sars-cov pathogenesis, we infected c -/mice and c bl/ j controls with sars-cov ma . control mice exhibited approximately % transient weight loss, with peak weight loss at day postinfection (fig. a) . in contrast, the c -/mice were significantly protected from infection, with no significant weight loss evident at any time point. surprisingly, viral titers in the lung were similar in c -/and c bl/ j controls (fig. b) , indicating that the lack of disease in c -/mice is uncoupled from viral replication efficiency and that complement signaling is not necessary for sars-cov ma clearance from the lung. we further measured sars-cov ma -induced disease by assessing respiratory function using whole-body plethysmography following infection of c bl/ j and c -/mice. enhanced pause (penh) is a calculated measure of airway resistance that we have associated with airway debris following sars-cov ma infection ( ) . the % exhalation force (ef ) measures the exhalation force midbreath, which increases as breathing becomes more difficult. finally, the ratio of peak expiratory flow (rpef) is the time to peak expiratory flow and has been associated with wheezing following infection. all three metrics have been shown to change significantly following sars-cov ma infection, with penh and ef increasing following infection and rpef decreasing. combined, these measurements show that sars-cov ma -infected animals have altered exhalation patterns in their breathing. c -/mice exhibit a decreased change in penh and ef levels following sars-cov ma infection relative to those of infected c bl/ j control mice; however, rpef values were similar between infection conditions ( fig. c to e). together, these data indicate that despite the lack of weight loss in c -/mice, the absence of the complement pathway did not alter host control of viral the respiratory function of sars-cov ma -infected c bl/ j and c -/mice and mock-infected mice was measured using a buxco whole-body plethysmography system for penh, a measure of calculated airway resistance (c), ef , midbreath expiratory flow (d), and rpef, the rate of peak expiratory flow (e). *, p Ͻ . between mock-infected mice and a given condition. (c to e) three mice were used for each infection group, and two mock-infected mice were used. replication or completely abolish respiratory disease following sars-cov ma infection. in order to determine which arm of the complement pathway contributes to sars-cov ma pathogenesis, we infected knockout mice lacking components upstream of c . c -deficient mice lack the ability to signal through both the classical and lectin pathways, while fb-deficient mice lack the ability to signal through the alternative pathway. both mouse strains showed reduced weight loss relative to that of infected control mice (see fig. s in the supplemental material) at days postinfection; however, neither c -/nor fb -/mice reproduced complete protection from weight loss observed in c -/controls. together, the results suggest that multiple arms of the complement pathway may be activated and contribute to sars-cov-mediated disease through c activation. reduced lung pathology in c -/mice. analysis of sars-cov ma -infected lung sections showed that the absence of c resulted in reduced, but still significant, lung pathology. at day postinfection, only minor effects on lung disease were observed with airway denudation and debris, the main histopathological phenotypes at this early time point; levels of pathology were similar between wild-type and knockout mice ( fig. ; table s ). c -/mice exhibited more airspace inflammation, including eosino- phils, at dpi than their wild-type controls, although this relationship was reversed later in infection. at dpi, c bl/ j mice displayed pronounced lung pathology, including inflammatory cells in the large airway and parenchyma, perivascular cuffing, thickening of the interstitial membrane, and low levels of intra-alveolar edema. in contrast, c -/mice showed reduced scores in these areas, consistent with the improved respiratory function observed in fig. . notably, the lung pathology results were not as pronounced as the complete absence of weight loss, suggesting a possible distinction between lung disease and overall pathogenesis. we also considered whether the decrease in sars-cov ma pathogenesis in c -/mice was due to reduced lung damage in the absence of complement pathway signaling. to investigate this possibility, we looked for signs of complement deposition on sars-cov ma lung tissue. at both and dpi, we observed scattered positive staining for complement in the lungs of sars-cov ma -infected mice, suggesting that local tissue damage might contribute to sars-cov pathogenesis (fig. ) . interestingly, staining was consistently found in the parenchyma of the lung and not in the large airways, which are the other main site of sars-cov ma replication. no positive staining was observed in the lungs of c -/mice. diminished infiltration of the lungs of a select immune population of infected c -/mice. in order to identify and quantitate inflammatory cells in the lung, we performed flow cytometry at dpi. in parallel with humans exhibiting lung pathology, c -/mice exhibited significant pulmonary infiltration following sars-cov ma infection, but this inflammation was reduced relative to that observed in wild-type mice. sars-cov ma -infected c bl/ j and c -/mice had similar total cell counts as well as similar percentages of cd -positive cells in their lungs (data not shown). consistently with what was observed in human sars-cov patients ( ) , lymphopenia was observed in the lungs of both sars-cov ma -infected c bl/ j and c -/mice with reduced percentages of b cells (fig. a ) and cd t cells relative to those in mockinfected mice following infection. despite similar overall lymphocyte levels, small but significant differences were observed in levels of t cell activation between infected c bl/ j and c -/mice; both cd and cd t cells in c -/mice expressed more ki- ( fig. c and d) , an intracellular marker of proliferation, than those in c bl/ j controls. analysis of myeloid cells in the lung showed that infected c bl/ j mice had significantly higher levels of neutrophils, particularly nonactivated neutrophils, in the lung than infected c -/mice ( fig. b and e) . furthermore, inflammatory monocytes, which have previously been associated with increased sars-cov ma pathogenesis ( ), were significantly increased in the lungs of wild-type mice but not c -/mice (fig. b) . finally, we observed significantly more dendritic cells and alveolar macrophages in the lungs of sars-cov ma -infected c -/mice than in the lungs of infected c bl/ j mice. together, although c -/mice produced a robust immune cell infiltration following sars-cov infection, they had significant reductions in both inflammatory monocytes and neutrophils relative to controls; both cell types that are associated with sars-cov pathogenesis ( ) . conversely, the presence of activated t cells is associated with recovery following infection ( ) . in addition to examining inflammatory cells, we evaluated the vascular integrity of the lung following sars-cov ma infection in the presence and absence of c . we observed no differences in the numbers of platelets present in the bronchoalveolar lavage (bal) fluid between c bl/ j and c -/mice at either or days postinfection, indicating that the absence of c does not appear to significantly alter vascular permeability following infection with sars-cov (fig. s b) . cytokine and chemokine levels are significantly decreased in the lungs of c -/mice. to further investigate the inflammatory response to sars-cov ma infection, we measured cytokine and chemokine protein levels in the lung in the presence and absence of complement signaling. multiple protein expression patterns were observed in response to infection. mip a, mip b, and mcp are all highly expressed in the lung following sars-cov ma infection of both c bl/ j and c -/mice (fig. a) , indicating that some inflammatory signaling remains intact in the absence of c . granulocyte colony-stimulating factor (g-csf), interleukin (il- ), tumor necrosis factor alpha (tnf-␣), and il- a comprised a group of cytokines and chemokines that were more highly produced in the lungs of c bl/ j mice than in c -/mice (fig. b) , all peaking at days postinfection. notably, these cytokines all have a role in the production, recruitment, or differentiation of neutrophils, consistent with the flow cytometry results in fig. b . with the exception of rantes, all inflammatory cytokines and chemokines were measured at the highest levels at dpi, indicating that the host immune response is triggered quickly following infection with sars-cov ma . together, these results indicate that the absence of complement has an impact on the magnitude of some cytokines and chemokines in the lung; however, robust production can occur in either the presence or the absence of c . sars-cov ma induces systemic complement activation. the absence of complement signaling resulted in reduced sars-cov ma pathogenesis, as measured by weight loss and a partial reduction of respiratory dysfunction, pathology, immune infiltration, and cytokine responses in the lung. we hypothesized that systemic disease coupled with no change in viral titer might also drive important elements of complement-mediated disease. therefore, we examined sera from wild-type and c -/mice for signs of systemic disease following infection. western blot analysis showed increased levels of c a-derived fragments in the serum, indicating systemic complement activation in sars-cov ma -infected mice at dpi (fig. a) . given this result, we next examined cytokine and chemokine protein levels for markers of inflammation in the sera of sars-cov ma -infected mice. both mcp- and rantes levels were elevated in the serum following infection, regardless of mouse genetic background (fig. b) . however, numerous cytokines and chemokines, such as il- , g-csf, and kc (keratinocyte chemoattractant or cxcl ) were present in significantly higher abundance in the lungs of c bl/ j mice than in those of c knockout mice (fig. c) . we further examined the possibility that sars-cov ma infection leads to complement deposition outside the lung and found no signs of increased complement staining in the kidney (fig. d) . although no complement deposition was seen, the presence of both activated complement and inflammatory cytokines in the sera likely contributes to a systemic inflammatory response that drives sars-cov ma -mediated weight loss following infection. the complement system is a critical part of the host immune response to bacterial and viral infection. originally identified in the s as a heat-sensitive, nonspecific complement to the more specific adaptive immune pathways ( ), the complement system is one way that the innate immune system detects and responds to foreign antigens. because of its potential to damage host tissues, the complement system is also tightly regulated through a number of inhibiting proteins that are constitutively present in the serum ( ). it has previously been shown that complement pathway signaling is critical for the protective host immune response to various bacterial infections ( ) as well as some influenza virus and flavivirus infections ( , , ) . furthermore, viruses, including herpesviruses, poxviruses, astroviruses, flaviviruses, and retroviruses, encode genes to help them evade detection by the complement system ( ) , strong evidence that complement is important in the host antiviral response. the host factors that drive protective ( ) or pathogenic ( ) complement-associated responses in viral infection are not well understood. of particular concern, the anaphylatoxins c a, c a, and c a are produced during activation of the complement signaling cascade; they have potent proinflammatory properties and can trigger inflammatory cell recruitment and neutrophil activation ( ) . c a and c a blockade has been proposed as a treatment for acute lung injury ( ) , and anti-c a antibody has been shown to protect mice from infection with influenza virus ( ) and, more recently, mers-cov ( ) . complement recognition is important for the control of paramyxoviruses ( ), dengue virus ( ) , and human t lymphotrophic virus type (htlv- ) ( ), and many more viruses have developed means of evading detection by the complement system ( ) . in contrast, the data presented here, in conjunction with recent findings for ross river virus ( , ) , influenza virus ( ) , and well-established autoimmune disease ( ) , demonstrate that complement system activation can also lead to exacerbated disease. previous reports clearly established the ability of mannose-binding lectin (mbl) to bind to the sars-cov spike protein ( ), dependent on an n-linked glycosylation site; however, the role of complement signaling in sars-cov pathogenesis was unclear ( , , , ) . in this study, we demonstrate that the complement system is activated following sars-cov ma infection (fig. b) . however, we did not observe any change in viral titer in c -/mice (fig. b) , indicating an important difference between in vivo and in vitro studies and the use of viral pseudoparticles. the absence of complement signaling resulted in protection from sars-cov ma -induced weight loss, as shown through the use of both c -deficient mice ( fig. a) and activation pathway-specific knockout mice (fig. s ). respiratory function in c knockout mice was improved relative to that of control mice, although significant changes in penh and rpef were still observed, indicating that the elimination of complement signaling did not completely remove the effects of sars-cov ma infection. while analysis of the cellular inflammatory response to sars-cov ma infection revealed modest changes in histopathology and overall inflammatory cell recruitment to the lungs, significant differences were observed in pathogenic inflammatory monocyte and neutrophil populations, indicating that complement signaling contributes to the broader immune response to infection. immunohistochemical staining revealed that sars-cov ma infection induced complement deposition in the lung (fig. ) , similar to that associated with pathogenesis in ross river virus-infected mice ( ) and some influenza virus infections ( ) , and it is likely that complement deposition contributes to pulmonary disease and inflammatory cell recruitment. the cytokines and chemokines il- , il- , kc (cxcl ), and g-csf have higher abundances in the lungs of sars-cov ma -infected wild-type mice than in c -/mice and are all known to promote neutrophil recruitment. indeed, significantly more neutrophils were observed in the lungs of sars-cov ma -infected c bl/ j mice than in the c -/mice (fig. b) . interestingly, while there were fewer neutrophils present, the neutrophils found in the lungs of c -/mice infected with sars-cov ma had significantly more staining of major histocompatibility complex class ii (mhc ii) and the costimulatory molecules cd and cd (fig. e) , indicating a state of activation ( ) . unpublished data from our laboratory have consistently demonstrated higher neutrophil counts in the lungs of mice with severe disease than in those of mice with only mild pathogenesis. additionally, neutrophilia in human sars-cov patients was associated with a poor outcome of infection ( ) , and studies of native rat coronavirus ( ) found both a protective and a pathogenic role for neutrophils following infection. combined, these data demonstrate that the absence of complement provides significant improvements in pulmonary disease following sars-cov ma infection and suggest that a nonpulmonary cause might also contribute to the lack of weight loss in c -/mice. importantly, our data demonstrate that sars-cov ma infection activates the complement system systematically as well as in the lung (fig. a and b) . wild-type c bl/ j mice exhibited an increased abundance of serum cytokines and chemokines in response to sars-cov ma infection (fig. c ) in comparison to c -/mice. in particular, the pyrogenic cytokine il- ( ) is present at higher abundance in the lungs and sera of c bl/ j mice than in those of c -/mice. il- ␣ and tnf-␣ are also more abundant in the lungs of c bl/ j mice, suggesting that wild-type but not c -/mice develop a fever response to infection that contributes to weight loss and respiratory dysfunction phenotypes. while the precise mechanism of complement activation following sars-cov ma infection is still unclear, it is likely through recognition of the viral spike glycoprotein and partially mediated by mbl (see fig. s in the supplemental material). the anaphylatoxins produced by the activated complement pathway, c a, c a, and c a, have important immunostimulatory roles in vascular permeability and inflammatory cell recruitment ( , ) . c a and c a in particular are noted for their roles in causing mast cell degranulation, initiating a cytokine storm, promoting vascular permeability, and contributing to acute lung injury ( , , ) . furthermore, a c a antibody blockade was recently shown to protect in a model of highly susceptible mers-cov mice ( ) . although there are no reports of mast cell activation following sars-cov or mers-cov infection, activation has been observed both in vitro and in vivo following infection with influenza virus ( ) and may occur following other severe respiratory infections, including coronaviruses. mast cells release cytokines, including il- , il- , and tnf-␣, consistent with the inflammatory profile observed following sars-cov ma infection. while this work cannot definitively conclude that the complement anaphylatoxins and mast cell activation contribute to sars-cov ma pathogenesis, the data are consistent with this possibility, and the concept warrants further investigation. complement pathway activation is a hallmark of bacterial infection, and genetic deficiencies in the complement pathway result in enhanced susceptibility to streptococcus pneumoniae, neisseria meningitidis, and haemophilus influenzae infections ( ), as well as to sepsis. interestingly, it has also been shown that sars-cov ma infection stimulates tlr ( , ) , which is classically known as the lipopolysaccharide (lps) receptor ( , ) and important for recognition of many bacterial infections. combined, these data suggest that host recognition of sars-cov ma infection may activate similar pathways recognizing a bacterial infection, leading to immune signaling cascades that cause systemic disease and enhance viral pathogenesis. while systemic activation of the complement pathway may be useful during a bacterial infection, it is less so during a localized acute viral infection, such as sars-cov. furthermore, complement activation, in conjunction with the presence of neutrophils, is known to cause increased vascular permeability, a condition that is also observed following sars-cov infection ( ) ( ) ( ) and was associated with poor outcome. baseline complement activation also increases with age ( , ), consistent with increased sars-cov morbidity and mortality in aged populations. finally, it has previously been reported that serum c a levels are predictive of ards development ( ) and that, in the absence of complement, animals are protected from bacterially induced "shock lung" ( ) , data consistent with the pathogenic role that we have found for complement following sars-cov ma infection. in this work, we demonstrate that sars-cov ma infection activates the complement pathway and that complement signaling contributes to disease following infection. this disease is likely mediated by complement protein deposition in the lung as well as systemic complement activation and inflammation. notably, the absence of c has no impact on viral titer, unlike what has been observed following influenza virus infection. despite these differences, it is notable that mers-cov and h n influenza virus-induced acute lung injury and pulmonary inflammation are reduced in mice that are treated with either a c a receptor (c ar) antagonist or antibodies to c a ( , ) . a similar treatment might be effective in mitigating sars-cov ma -induced disease, as sars, mers, and influenza have common disease manifestations, including development of acute lung injury. given the large array of zoonotic strains poised for cross-species transmission, broad-based inhibitors of emerging coronavirus infections are a high priority ( ) . pinpointing the precise arms of the complement pathway that contribute to sars-cov will help further identify therapeutic targets while minimizing unnecessary inhibition of the immune response. this work suggests that investigation of anticomplement drugs for treatment of coronavirus infections is warranted and would pair well with direct antiviral therapeutics. stocks of recombinant mouse-adapted sars-cov (ma ) ( ) were propagated and their titers were determined in vero e cells and stored as single-use aliquots at Ϫ °c as previously described ( ) . tissue titers of ma were determined by plaque assay on vero e cells as previously described ( , ) , with a limit of detection of pfu. all experiments using live virus were performed in a class ii biological safety cabinet in a certified biosafety level laboratory with negative air pressure and redundant exhaust fans; personnel wore personal protective equipment, including tyvek suits, hoods, and powered air-purifying respirators. mouse experiments. c bl/ j (stock number ) and c -/-(stock number ) mice were purchased from jackson laboratories. fb Ϫ/Ϫ mice were generously provided by charles jennette (unc), and c -/mice were provided by mark heise (unc). all animal husbandry and experiments were performed in accordance with all university of north carolina at chapel hill institutional animal care and use committee guidelines. age-matched ( -to -week-old) female mice were anesthetized with a mixture of ketamine-xylazine and intranasally inoculated with l of phosphate-buffered saline (pbs) or pfu of sars-cov ma diluted in pbs. mice were monitored for disease signs and weighed at -h intervals. microarray and proteomics analysis. microarray and proteomics analyses were performed on a time course and according to a dose-response study published by gralinski et al. by following the same methods ( ) . the proteomics data (experiment sm ) are publically available through the pnnl (http://omics.pnl.gov) web portal. briefly the mean intensity (abundance) for each protein was then graphed as an average ( mice for each infection, mice for mock infection) for each group at each time point. missing or absent values were not scored; however, if no value was observed in any of the samples at a time point, the sample was registered with a single , representing "not detected." histological analysis. at the times indicated in the figures, mice were euthanized using an overdose of isoflurane, and lung tissue was fixed in % formalin. tissues were embedded in paraffin, and -m sections were prepared. to determine the extent of inflammation and tissue pathology, tissues were stained with hematoxylin and eosin and scored in a blind manner from (no sign of phenotype) to (widespread and severe phenotype). platelet counts. for bronchoalveolar lavage (bal), immediately following euthanasia, ml of pbs was injected into the lung through the trachea by using a -gauge exel safelet catheter tip (fisher). this fluid was then drawn back out and used for subsequent analysis. two hundred fifty microliters of bal fluid was used for absolute counting of gross cell types using an abaxis vetscan hm analyzer. complement deposition staining. lung sections from sars-cov ma -, ross river virus-, or mock-infected mice were stained for the presence of c by the animal histopathology and laboratory medicine core at the university of north carolina. sars-cov ma lung samples were tested from -week-old mice at , , , and days after infection with pfu of virus. staining was performed using a goat anti-mouse c primary antibody (mp biomedicals). immunoblot analysis. mice were perfused with pbs, and then lung tissue was dissected and homogenized in lysis buffer ( mm tris [ph . ], mm nacl, % nonidet p- , . % deoxycholate, and . % sodium dodecyl sulfate [sds] supplemented with complete protease inhibitor cocktail [roche]). total protein concentrations were determined by using the coomassie plus assay kit (pierce). dilutions of serum or -to -g aliquots of protein were diluted in an equal volume of ϫ sds sample buffer, and sds-polyacrylamide gel electrophoresis was performed. proteins were transferred onto polyvinylidene fluoride membranes (bio-rad). membranes were blocked in ϫ pbs- % milk- . % tween and incubated with goat anti-mouse c antibody ( : , ; cappel) overnight at °c. membranes were washed in pbs- . % tween and incubated with rabbit anti-goat-horseradish peroxidase ( : , ; sigma) for h at room temperature. after a washing step, proteins were visualized by enhanced chemiluminescence (amersham) according to the manufacturer's instructions. flow cytometry. following euthanasia at days postinfection, mice were perfused with ml of pbs via cardiac puncture. lungs were dissected, minced, and incubated for min with vigorous shaking at , and cd -percp-efluor (clone e ; ebioscience). after being stained, cells were fixed in % paraformaldehyde overnight and then stored in pbs until acquisition within h. a minimum of , events were collected using an lsrii cytometer (becton, dickinson), and analysis was completed using flowjo software version (treestar). all samples were first evaluated through subsequent gates for (i) mononuclear cells, (ii) doublet exclusion, (iii) dead-cell exclusion based on uptake of a fixable live/dead cell discriminator (invitrogen), and cd -lca expression before downstream analyses. reported cell frequencies were normalized to the percentage of total cd -lca ϩ events, where appropriate. whole-body plethysmography. respiratory function was measured using whole-body plethysmography as described by menachery et al. ( ) . briefly, mice were loaded into individual chambers and allowed to acclimate for min before a -min measurement window. measurements were recorded every s for a total of measurements per time point per mouse. cytokine and chemokine protein analysis. the small center lung lobe of each mouse was homogenized in ml of pbs and briefly centrifuged to remove debris. fifty microliters of homogenate was used to measure cytokine and chemokine protein abundance using a bio-plex pro mouse cytokine -plex assay (bio-rad) according to the manufacturer's instructions. statistical analyses. percent starting body weights, viral titers, and inflammatory cell numbers were evaluated for statistically significant differences by the mann-whitney test or student's t test using graphpad prism software. accession number(s). the microarray data were previously deposited in the geo database under accession number gse ( ). supplemental material for this article may be found at https://doi.org/ . /mbio . - . research was supported by grants from the niaid of the nih (ai to r.s.b. and m.t.h., ai to r.s.b., ai to r.s.b., and k ag to v.d.m.). animal histopathology was performed by the animal histopathology and laboratory medicine core at the university of north carolina, which is supported in part by an nci center core support grant ( p ca - ) to the unc lineberger comprehensive cancer center. the unc flow cytometry core facility is supported in part by cancer center core support grant p ca to the unc lineberger comprehensive cancer center. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. molecular evolution of the sars coronavirus during the course of the sars epidemic in china characterization of a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe acute respiratory syndrome vs. the middle east respiratory syndrome a sars-like cluster of circulating bat coronaviruses shows potential for human emergence isolation and characterization of a bat sars-like coronavirus that uses the ace receptor severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection mannose-binding lectin in severe acute respiratory syndrome coronavirus infection influence of fc␥riia and mbl polymorphisms on severe acute respiratory syndrome a single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms the sars coronavirus spike glycoprotein is selectively recognized by lung surfactant protein d and activates macrophages lack of association between polymorphisms of masp and susceptibility to sars coronavirus infection molecules great and small: the complement system complement: a key system for immune surveillance and homeostasis blockade of the c a-c ar axis alleviates lung damage in hdpp -transgenic mice infected with mers-cov complement and viral pathogenesis complement in lung disease lack of the pattern recognition molecule mannosebinding lectin increases susceptibility to influenza a virus infection interaction of c q and mannan-binding lectin with viruses th cytokines are critical for respiratory syncytial virus-associated airway hyperreponsiveness through regulation by complement c a and tachykinins a protective role for complement c protein during pandemic h n and h n influenza a virus infection mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury new metrics for evaluating viral respiratory pathogenesis a major outbreak of severe acute respiratory syndrome in hong kong dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-covinfected mice the pathology and pathogenesis of experimental severe acute respiratory syndrome and influenza in animal models t cell responses are required for protection from clinical disease and for virus clearance in sars-cov-infected mice the complement system: history, pathways, cascade and inhibitors complement system part i-molecular mechanisms of activation and regulation. front immunol : bacteria under stress by complement and coagulation direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin protective immune responses against west nile virus are primed by distinct complement activation pathways inhibition of complement activation alleviates acute lung injury induced by highly pathogenic avian influenza h n virus infection role of c , c and anaphylatoxin receptors in acute lung injury and in sepsis the role of c a in acute lung injury induced by highly pathogenic viral infections differential mechanisms of complement-mediated neutralization of the closely related paramyxoviruses simian virus and mumps virus complement-mediated neutralization of dengue virus requires mannose-binding lectin human complement component c q inhibits the infectivity of cell-free htlv-i complement contributes to inflammatory tissue destruction in a mouse model of ross river virus-induced disease mannose binding lectin is required for alphavirus-induced arthritis/myositis the complement system in systemic autoimmune disease association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory coronavirus infection mouse neutrophils are professional antigen-presenting cells programmed to instruct th and th t-cell differentiation severe acute respiratory syndrome: clinical outcome and prognostic correlates neutrophils are needed for an effective immune response against pulmonary rat coronavirus infection, but also contribute to pathology fever and the thermal regulation gralinski et al. of immunity: the immune system feels the heat c a: the third anaphylatoxin of the human complement system role of c a in inflammatory responses the role of anaphylatoxins c a and c a in regulating innate and adaptive immune responses mast cell-induced lung injury in mice infected with h n influenza virus infections of people with complement deficiencies and patients who have undergone splenectomy allelic variation in the toll-like receptor adaptor protein ticam contributes to sars-coronavirus pathogenesis in mice toll-like receptor signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection defective lps signaling in c h/hej and c bl/ sccr mice: mutations in tlr gene cutting edge: toll-like receptor (tlr )-deficient mice are hyporesponsive to lipopolysaccharide: evidence for tlr as the lps gene product genome wide identification of sars-cov susceptibility loci using the collaborative cross complete protection against severe acute respiratory syndrome coronavirusmediated lethal respiratory disease in aged mice by immunization with a mouse-adapted virus lacking e protein lung pathology of severe acute respiratory syndrome (sars): a study of autopsy cases from singapore complement c q activates canonical wnt signaling and promotes aging-related phenotypes inflammation, the complement system and the diseases of aging association of complement activation and elevated plasma-c a with adult respiratory distress syndrome. pathophysiological relevance and possible prognostic value role of complement activation in a model of adult respiratory distress syndrome broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses a mouse adapted sars coronavirus causes disease and mortality in balb/c mice reverse genetics with a full length infectious cdna of the severe acute respiratory syndrome coronavirus mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range expansion in human airway epithelium flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung key: cord- -adaow w authors: asensio martín, m. j.; hernández bernal, m.; yus teruel, s.; minvielle, a. title: infecciones en el paciente crítico date: - - journal: medicine - programa de formación médica continuada acreditado doi: . /j.med. . . sha: doc_id: cord_uid: adaow w resumen introducción las infecciones son muy frecuentes en los pacientes que se encuentran ingresados en los servicios de medicina intensiva, siendo unas veces motivo de ingreso y en otras la infección se adquiere durante el ingreso. epidemiologia las causas más frecuentes de infección adquirida en la comunidad que precisa ingreso en la uci son las infecciones respiratorias, infecciones urinarias y las infecciones del sistema nervioso central. dentro de las infecciones adquiridas en la uci, las asociadas a dispositivos son las más frecuentes. etiología los gérmenes más frecuentes en la uci son los gram negativos. etiopatogenia. en el paciente crítico se aúnan factores, haciéndolos especialmente vulnerables a las infecciones. manifestaciones clínicas dependerán de la localización de la infección. diagnóstico debe ser precoz dada su alta mortalidad. pronóstico las infecciones nosocomiales se asocian con un aumento de la mortalidad y la estancia. tratamiento el retraso en el tratamiento se asocia con un aumento de la mortalidad. abstract introduction infections are very frequent in patients who are admitted to intensive care units, sometimes being a reason for admission and in others the infection is acquired during icu stay. epidemiology the most frequent causes of acquired infection in the community that require admission to the icu are respiratory infections, urinary tract infections and infections of the central nervous system. among the infections acquired in the icu, devices-associated infections are the most frequent. etiology the most frequent in icu are gram negative pathogens. etiopathogenesis in the critical patient, several factors are combined making them especially vulnerable to infections. clinical manifestations depends on the location of the infection. diagnosis it must be early due to its increased mortality. prognosis nosocomial infections are associated with an increase in mortality and in the length of stay. treatment the delay in treatment is associated with an increase in mortality. la prevalencia de la patología infecciosa en los servicios de medicina intensiva (smi) es elevada, siendo unas veces el motivo de ingreso en las unidades de cuidados intensivos (uci) y, en otras, la infección se adquiere durante su estancia en la misma. no obstante, existen marcadas diferencias, tanto en la frecuencia como en la etiología y la patogenia entre estas dos entidades infecciosas: la patología infecciosa comunitaria grave que requiere ingreso en la uci y la infección nosocomial (in) adquirida durante la estancia en la uci. la infección comunitaria grave es una de las principales causas de ingreso en los smi, tanto por las necesidades terapéuticas inherentes a la gravedad intrínseca del cuadro infeccioso como por el requerimiento de una adecuada monitorización del proceso. dentro del amplio campo de infecciones comunitarias que pueden requerir ingreso en los smi, destacan por su gravedad y frecuencia las neumonías, las infecciones del sistema nervioso central (snc) y las infecciones del tracto urinario (itu). la neumonía adquirida en la comunidad (nac) es una enfermedad infecciosa respiratoria aguda con una incidencia que oscila entre y casos por . habitantes por año, aumentando esta incidencia con la edad y las comorbilidades. el % de los pacientes con nac requiere ingreso hospitalario, y alrededor del % necesitan ser admitidos en una uci . la mortalidad global de la nac se sitúa alrededor del %, si bien en el subgrupo de pacientes que precisan ingreso en la uci la mortalidad es del - % . la prevalencia de los diferentes microorganismos causantes de la nac va a depender de diversos factores como la edad, la presencia de determinadas comorbilidades, los criterios diagnósticos utilizados o las pruebas diagnósticas empleadas. en la mayoría de los estudios, en cerca del % de los casos de nac no se puede demostrar la etiología. de los organismos aislados, el más frecuente es streptococcus pneumoniae, seguido de legionella pneumophila (cuya incidencia ha disminuido en los últimos años probablemente debido al uso extendido de las nuevas quinolonas y los macrólidos) y hamophilus influenzae. el tabaquismo y el tratamiento con corticoides son factores de riesgo para nac por legionella. las enterobacterias (sobre todo kebsiella pneumoniae) suelen provocar nac en pacientes con comorbilidades o antibioterapia previa. pseudomonas aeruginosa se identifica en un - % de las nac y generalmente en relación con la presencia de bronquiectasias, fibrosis quística, enfermedad pulmo-nar obstructiva crónica (epoc) grave, neoplasia o neutropenia y staphylococcus aureus se suele presentar tras infecciones víricas. los microorganismos atípicos, mycoplasma pneumoniae y clamydophila pneumoniae, a menudo son patógenos y pueden ocasionar cuadros graves . la mejoría en las pruebas diagnósticas con técnicas de biología molecular ha dado lugar a un aumento en el aislamiento de virus como causa etiológica, principalmente rinovirus e influenza . generalmente en adultos se presentan como copatógenos asociados a neumococo, h. influenzae y s. aureus. hay que resaltar que en una tercera parte de los casos de nac se aíslan dos o más patógenos, generalmente una combinación de bacterias y virus. en los últimos años, nuevos patógenos han emergido como causa de nac. el metapneumovirus, aislado por primera vez en el año , aunque típicamente se asocia con una enfermedad más leve, se ha relacionado con casos mortales de neumonía. los coronavirus también han surgido como grandes amenazas epidémicas, primero como un síndrome respiratorio agudo severo (sras) y, más recientemente, con el síndrome respiratorio de oriente medio (mers). el virus influenza también continúa siendo una amenaza, con la posibilidad de que varias cepas de influenza aviar, particularmente h n y h n , muten lo suficiente como para permitir una transmisión sostenida de ser humano a ser humano con el resultado de pandemias . en pacientes que precisan ingreso en la uci como patógenos causantes de la nac son frecuentes el neumococo resistente, s. aureus y legionella pp.. las manifestaciones clínicas habituales son fiebre mayor de ºc y afectación del estado general, acompañada de tos, expectoración, dolor torácico, disnea o taquipnea. el diagnóstico se basa en la existencia de clínica compatible, junto con presencia de ocupación del espacio alveolar en la radiografía de tórax. el hallazgo de un infiltrado en la radiografía es el patrón oro para establecer el diagnostico ( fig. ). para el diagnóstico etiológico es necesaria la realización de hemocultivos, tinción de gram y cultivo de esputo determinación de antígeno en orina de s. pneumoniae y l. pneumophila. la serología está indicada para el diagnóstico de neumonía por m. pneumoniae y clamydophila pneumoniae (título elevado de anticuerpos igm en el suero de la fase aguda y/o seroconversión del título de igg en el suero de la fase de seroconversión). igualmente la serología es la técnica diagnóstica en caso de que por contexto epidemiológico se sospeche infección por coxiella burnetti (fiebre q) o francisella tularensis (tularemia). las técnicas de biología molecular están indicadas en nac graves, en las que no se ha logrado establecer el diagnóstico etiológico por los medios habituales. asimismo, en determinados periodos epidémicos está indicada la detección de virus respiratorios, como el virus de la gripe en aspirado nasofaríngeo mediante inmunofluorescencia e inmunocromatografía, aunque el patrón oro sigue siendo el cultivo vírico. la toracocentesis diagnóstica con cultivo para aerobios y anaerobios está indicada en pacientes hospitalizados con derrame pleural significativo, no solo para el diagnóstico sino también porque el desarrollo de empiema es uno de los principales factores asociados a mala evolución de la nac . en pacientes con nac retrasar el ingreso en la uci en los pacientes que lo precisan se acompaña de un incremento considerable de la mortalidad. la sociedad americana del tórax (ats) y la sociedad americana de enfermedades infecciosas (idsa) proponen el ingreso en la uci de los pacientes que presenten shock séptico o insuficiencia respiratoria que precise ventilación mecánica (un criterio mayor), así como de los pacientes que presenten al menos de los criterios menores de la tabla . recientemente, las recomendaciones de la european respiratory society and european society for clinical microbiology and infections diseases , aconsejan el ingreso en la uci ante la presencia de al menos dos de las siguientes circunstancias: presión arterial sistólica menor de mm hg, presencia de insuficiencia respiratoria con pao /fio < o la afectación de o más lóbulos en la radiografía de tórax, o bien uno de los siguientes: necesidad de ventilación mecánica o necesidad de vasopresores durante más de horas (shock séptico), con un nivel de recomendación a . la administración precoz y efectiva de antibióticos puede disminuir la mortalidad de la nac grave, por lo que debe ser prioritario su inicio precoz. el espectro del tratamiento antibiótico debe ser amplio, capaz de cubrir los gérmenes etiológicos más probables, valorando la gravedad del cuadro clínico, factores de riesgo para microorganismos específicos, los patrones epidemiológicos propios de cada área geográfica y los patrones de resistencia (tabla ). etiología. la incidencia de meningitis bacteriana comunitaria es de - casos por . habitantes, siendo streptococcus pneumoniae y neisseria meningitidis responsables del % de los casos. son factores de riesgo la asplenia (neumococo y meningococo), agammaglobulinemia (neumococo) y el déficit del complemento (meningococo). en personas mayores de años, alcohólicos o jóvenes con alteración de la inmunidad celular, listeria monocytogenes suele ser el agente etiológico. en cuanto a las meningitis víricas, suelen tener una incidencia estacional, verano y otoño, y suelen estar causadas por enterovirus . manifestaciones clínicas. la clínica habitual cursa con fiebre, dolor de cabeza, rigidez de nuca y alteración de la conciencia, pero hasta un % presenta focalidad neurológica o convulsiones ( %). diagnóstico. la base del diagnóstico es el análisis del líquido cefalorraquídeo (lcr). en caso de existir focalidad neurológica, papiledema o convulsiones, debe realizarse una tomografía computadorizada (tc) craneal previa a la punción para descartar la existencia de efecto masa. en las meningitis bacterianas se observa pleocitosis ( . - . ) de predominio polimorfonuclear ( - %), glucosa baja (menos de mg/dl) y una ratio glucosa en lcr/glucosa en sangre igual o inferior a , y proteínas elevadas (más de mg/dl). por el contario, en las meningitis víricas, la pleocitosis es menor ( - células/mm ) de predominio linfocitario, glucosa normal y proteínas elevadas . ante la sospecha de meningitis debe realizarse de forma urgente tinción de gram del lcr, ya que permitirá una rápida identificación del germen en el - % de los casos, aunque su sensibilidad se reduce en pacientes que están recibiendo tratamiento antibiótico. tratamiento. el retraso en el tratamiento aumenta la morbimortalidad, por lo que este no debe demorarse por la realización de pruebas diagnósticas. se recomienda en las últimas guías clínicas administrar el tratamiento en la primera hora . el tratamiento debe incluir una cefalosporina de tercera generación (ceftriaxona g/ horas por vía intravenosa) asociada a vancomicina ( g por vía intravenosa/ horas) debido a que el neumococo presenta un % de resistencia a las penicilinas y un % a las cefalosporinas. si existen factores de riesgo para listeria se añadirá al tratamiento anterior ampicilina g por vía intravenosa cada horas y, en caso de sospecha de etiología vírica, añadir aciclovir mg/kg por vía intravenosa cada horas en adultos. debe administrarse en caso de sospecha de meningitis bacteriana, previo o de forma simultánea al tratamiento antibiótico, dexametasona en dosis de , mg/kg cada horas (adultos mg cada horas) durante días . tras los resultados del cultivo del lcr se modificará el tratamiento según antibiograma. etiología. hasta en un % de los casos de encefalitis no se logra identificar el agente etiológico, siendo los virus, sobre todo enterovirus, herpex simple tipo i y virus del nilo occidental, los gérmenes principalmente implicados. en las encefalitis existe afectación del parénquima cerebral, por lo que a la fiebre y la ce-falea se asocia una alteración del nivel de conciencia, junto con convulsiones y déficit motor. diagnóstico. el análisis del lcr es la base del diagnóstico, mostrando pleocitosis mononuclear moderada, glucosa normal y un aumento moderado de las proteínas, pero hasta en un % de los casos el lcr es normal. para el diagnóstico etiológico, la determinación de reacción en cadena de la polimerasa (pcr) en el lcr es la base del diagnóstico para herpes virus, virus de la varicela zoster (vvz), citomegalovirus (cmv) y virus de epstein-barr. la neuroimagen es el segundo pilar diagnóstico. la presencia en la rm cerebral de hemorragia y edema cerebral a nivel del lóbulo temporal es muy sugestiva de encefalitis por herpes simple. tratamiento. un retraso en el tratamiento se asocia a un mal pronóstico, por lo que ante su sospecha debe iniciarse el tratamiento de forma precoz, con aciclovir mg/kg cada horas en caso de sospecha de infección por el virus herpes o vvz. en caso de sospecha de infección por cmv, el tratamiento de elección es ganciclovir mg por kilo cada horas. tras - días de tratamiento sin respuesta positiva se recomienda nueva pcr del lcr, prolongando el tratamiento en caso de ser positivo. las itu constituyen una de las patologías infecciosas más frecuentes tanto en la comunidad como en el ámbito hospitalario, siendo responsables del - % de las sepsis graves que requieren ingreso en la uci. asimismo, son la causa más frecuente de bacteriemia secundaria de origen comunitario. los gérmenes que colonizan la uretra anterior o el introito vaginal ascienden hasta la vejiga dando lugar a la infección. la diseminación hematógena desde otros focos es rara, siendo en este caso stafilococcus aureus el germen más frecuente, seguido de cándida spp.. existen factores patogénicos que modulan el riesgo de itu que dependen del huésped y/o del germen responsable. los factores dependientes del huésped son: alteraciones del flujo urinario, alteraciones químicas u hormonales del epitelio uretral o genital, hábitos higiénicos, cateterismos y manipulación urinaria previas, embarazo y diabetes. en cuanto a los factores dependientes del germen, está la capacidad de adhesión de las bacterias al epitelio mediante los pili o fimbriae, la resistencia a los factores bactericidas del suero y la producción de hemolisinas, dando lugar a una gran virulencia. infección del tracto urinario inferior. aunque habitualmente son de curso benigno, la cistitis enfisematosa puede cursar con un cuadro de shock fulminante. el diagnóstico se realiza mediante prueba de imagen, observándose la presen-cia de gas en la pared vesical. los microorganismos responsables más frecuentes son e. coli y klebsiella, siendo rara la infección por anaerobios. urosepsis altas. suele darse en pacientes jóvenes y, en ocasiones, evoluciona de manera fulminante a shock séptico, con una mortalidad del %. en la pielonefritis aguda, infección del parénquima renal y/o sistema pielocalicial, suele caracterizarse clínicamente por la presencia de fiebre y escalofríos asociados a dolor en fosa renal, habitualmente acompañados o precedidos de síndrome miccional y, con menor frecuencia, náuseas y vómitos. no es infrecuente que en pacientes ancianos la única manifestación sea confusión o letargo. mediante prueba de imagen, ecografía de abdomen o tc se deberá descartar una causa obstructiva subyacente. los gérmenes habituales por orden de frecuencia son: e. coli, proteus mirabilis, klebsiella pneumoniae y enterococcus spp.. en los últimos años se ha producido un aumento en el número de aislamientos de bacterias productoras de betalactamasas de espectro extendido (blee) en las itu comunitarias, principalmente por k. pneumoniae y e. coli. los factores de riesgo para la presencia de blee son: sexo mujer, edad superior a años, diabetes mellitus, itu previas recurrentes, antecedentes de procedimiento invasivo urológico (cateterismo/sondaje) y el tratamiento previo con aminopenicilinas, cefalosporinas y fluorquinolonas . se realizará mediante el análisis del sedimento y la realización de tinción gram y cultivo de la orina. deberá realizarse de forma empírica en espera de los resultados del urocultivo. en infecciones no complicadas: ceftriaxona - g por vía intravenosa cada horas o tobramicina mg por vía intravenosa por kg cada horas. en caso de alergia, ciprofloxacino mg por vía intravenosa cada horas. la pielonefritis aguda deberá tratarse con un carbapenem (imipenen g por vía intravenosa cada horas) y en caso de alergia tigeciclina mg por vía intravenosa cada horas. si existen factores de riesgo para blee, el tratamiento de elección es un carbapenem (imipenen g intravenoso cada horas). las in son infecciones adquiridas durante la estancia en un hospital, no estando presentes ni en periodo de incubación al ingreso del paciente. las in constituyen un problema grave en los smi, conllevando una mayor morbimortalidad, junto con un aumento de los costes (debido a la prolongación de la estancia, gastos de tratamiento, pérdida de productividad del paciente por un retraso en su incorporación a la vida laboral, costes de desplazamiento de los familiares, etc.), junto con el riesgo de generación de gérmenes multirresistentes. el paciente crítico es especialmente vulnerable a la in, estimándose que entre el % y el % de los pacientes sufrirá una infección durante la estancia en la uci, frente a una frecuencia del % en los pacientes ingresados en planta de hospitalización [ ] [ ] [ ] . esta mayor susceptibilidad en los pacientes críticos a la in es debida a la convergencia de varios factores predisponentes: . factores de riesgo dependientes del paciente, como es la inmunosupresión, bien congénita o secundaria a fármacos (quimioterapia, corticoides, tratamiento inmunosupresor, etc.). . alteración de la inmunidad ligada a la patología aguda que motivó el ingreso en la uci. . la necesidad de uso de dispositivos invasivos (intubación, vía central, sonda vesical, etc.) que al romper la barrera natural de defensa facilitan la entrada de microorganismos. . la gran complejidad de estos pacientes, precisando numerosas cuidados y procedimientos, favoreciendo las oportunidades de transmisión cruzada. los gérmenes responsables más frecuentes de la in en la uci, aunque dependiendo del tipo de infección predominarán uno u otro germen, son los microorganismos gram negativos ( , %), seguidos de los gram positivos ( %), hongos ( , %) y otros microorganismos ( %). las infecciones más frecuentes adquiridas en la uci son las asociadas a dispositivos: la bacteriemia relacionada con catéter, la neumonía asociada a la ventilación mecánica (nav) y la infección relacionada con el sondaje uretral. estas tres entidades por su frecuencia y gravedad son motivo de seguimiento en el registro anual de estudio nacional de vigilancia de la infección nosocomial en servicios de medicina intensiva (envin-helics) en las uci españolas . la infección relacionada con dispositivos de derivación externa de líquido cefalorraquídeo es una complicación grave y frecuente en las uci neuroquirúrgicas, por lo que también se abordará en esta actualización. en la figura , se muestran los datos del estudio envin-helics de las infecciones asociadas a dispositivos en las uci españolas . se denomina bacteriemia a la presencia de microorganismos en la sangre. cuando la causa de una bacteriemia es un catéter (bien de origen central o periférico) hablamos de bacteriemia relacionada con catéter. la bacteriemia relacionada con catéter venoso central (brcvc) es la tercera in en frecuencia en el registro envin-helics. en el año supuso el , % de todas la in, con una densidad de incidencia (di) de , bacteriemias por . días de uso de catéter venoso central (cvc). la brcvc puede originarse a partir de tres vías patogénicas que enumeramos a continuación. vía exoluminal. los gérmenes que colonizan la piel penetran a partir del punto de inserción por la superficie exoluminal formando una biopelícula, con posterior diseminación hematógena. este mecanismo es más frecuente en la primera semana tras la inserción del catéter y está relacionada con la esterilidad en el proceso de inserción. la localización del cvc se relaciona con la frecuencia de infección, siendo mayor el riesgo para la localización femoral, seguida de yugular, subclavia y por último en las venas basílicas . los patógenos implicados suelen ser los saprófitos cutáneos, mayoritariamente gram positivos. vía endoluminal. la colonización del catéter se produce a través de las manipulaciones de las conexiones, migrando vía endoluminal formando una biopelícula. la bacteriemia es más tardía, generalmente a partir de las dos semanas de implantación del catéter y está relacionada con la higiene en la manipulación de las conexiones. en estas bacteriemias tardías o cuando la vía es de localización femoral suele haber mayor proporción de bacilos gram negativos y hongos. vía hematógena. es la menos frecuente, produciéndose la colonización del catéter a partir de un foco distal de infección vía hematógena. los patógenos más frecuentes en las brc son los gram positivos ( , %), seguidos de los bacilos gram negativos ( , %) y los hongos ( , %). dentro de los gram positivos los más frecuentes son el staphylococcus epidermidis ( %) y staphylococcus coagulasa negativo ( %). dentro de los bacilos gram negativos, klebsiella pneumoniae es el más frecuente ( , %), seguido de e. coli ( , %) y pseudomonas aeruginosa ( , %). las bacteriemias por hongos son fundamentalmente especies de candida (albicans y parapsilosis con la misma frecuencia y en menor proporción glabrata) . las manifestaciones clínicas pueden ser focales con induración, eritema y dolor en el punto de inserción o trayecto del catéter o sistémicas acompañándose de fiebre, escalofríos e hipotensión. no obstante, las manifestaciones locales son inespecíficas y pueden existir en ausencia de brcvc (sensibilidad menor al %). el diagnóstico de brcvc necesita la evidencia microbiológica de que el catéter es el origen de la bacteriemia y en ello reside la mayor dificultad diagnóstica. la retirada de un catéter implica la necesidad de una nueva canalización, técnica que no está exenta de riesgo. en los pacientes ingresados en la uci es frecuente la presencia de coagulopatía o trombopenia (riesgo elevado de hematoma y sangrado) o accesos vasculares limitados (quemados o accesos trombosados), por lo que en la práctica habitual con frecuencia es necesario mantener el catéter hasta confirmar que este es el foco de la bacteriemia. por tanto, la aproximación diagnóstica se divide en grupos . tras retirada del catéter venoso central. se define bacteriemia o fungemia relacionada con el catéter como el aislamiento del mismo microorganismo en los hemocultivos extraídos por punción en una vena periférica y en el cultivo cuantitativo o semicuantitativo de la punta del cvc retirado, en un paciente con clínica de sepsis, y sin otro foco de infección aparente. en caso de aislamiento de estafilococos coagulasa negativos se requerirá el aislamiento en al menos frascos de hemocultivos periféricos. se considera positivo el cultivo semicuantitativo (técnica de maki) de la punta del catéter si crecen más de unidades formadoras de colonia (ufc) por mililitro de sangre o en el cultivo cuantitativo de la punta (tras sonicación) crecen más de ufc. si no se ha retirado el catéter. se define bacteriemia o fungemia relacionada con el catéter como el aislamiento del mismo microorganismo en hemocultivos simultáneos cuantitativos en una proporción : en las muestras extraídas a través del catéter respecto a las obtenidas por venopunción. dado que este procedimiento es caro y costoso, en la práctica el método más usado es comparar el tiempo de diferencia hasta la positivación, considerándose significativo si la muestra extraída a través del cvc positiviza al menos horas antes que la extraída por venopunción. un % de las brcvc cursan con sepsis y un % con shock séptico. un estudio de casos-controles con datos extraídos del registro envin-uci demostró en pacientes críticos que la adquisición de una brc produce un significativo incremento de la mortalidad y la estancia, con una mortalidad iu-su bod-cvc atribuible del , % y una prolongación de la estancia de días . estos datos han sido corroborados por otros estudios, resaltando la mayor mortalidad y estancia en las bacteriemias por staphylococcus aureus resistente a meticilina . en , peter provonost publicó la experiencia de aplicar un paquete de medidas preventivas en el estado de michigan, consiguiendo una reducción de las brcvc de , por mil días de catéter a , durante los - meses de seguimiento. estos resultados impulsaron la puesta en marcha de un programa a nivel nacional denominado bacteriemia zero . el proyecto se implantó en enero de hasta junio en uci españolas, siendo el objetivo una reducción en la di a o menos episodios de bacteriemias por mil días de cvc (reducción del % respecto a la tasa media de los últimos años). el proyecto contaba con dos ramas: stop-brc y el plan de seguridad integral con el objetivo de crear una red de uci seguras en españa. la rama stop-brc incluía medidas de obligado cumplimiento relacionadas con la inserción y manejo de los cvc: . higiene de manos tanto durante la inserción como en la manipulación de los catéteres para evitar la colonización de la piel, conexiones o el propio catéter. . uso de máximas barreras durante la inserción con medidas de esterilidad máxima, incluido el uso de bata, gorro, guantes estériles y paño que cubra totalmente al paciente. . desinfección de la piel con clorhexidina. . preferencia a la hora de usar la localización en la subclavia. . retirada de catéteres innecesarios. la presencia de un catéter es el principal factor de riesgo para el desarrollo de bacteriemia, por lo que si se puede utilizar otra vía debe retirarse. no se recomienda la sustitución sistémica de los cvc ni el recambio con guía metálica. . manejo higiénico de los catéteres: antes de manipular un catéter (administrar medicación, cambiar sueros, etc.) debe realizarse la higiene de manos y la desinfección de las conexiones con clorhexidina. cambio de las líneas de infu-sión a los días, salvo en las perfusiones con lípidos o hemoderivados en las que se realizará diariamente. el proyecto fue un éxito, consiguiendo una reducción en la di a , bacteriemias por . días de cvc (se pretendía bajar a ), manteniéndose estos buenos resultados a lo largo en la actualidad. así, en el año la tasa de brcvc es de , bacteriemias por mil días de cvc, recomendándose que bz deba ser de obligado cumplimiento en las uci españolas. el tratamiento empírico dependerá de la situación del paciente y del tipo de catéter (fig. ) . los cvc de inserción periférica y los cvc no permanentes se han de retirar siempre que se sospeche que son el foco de origen de la bacteriemia. la existencia de signos locales de infección, aun sin bacteriemia relacionada, también es un criterio absoluto de su retirada. los cvc de uso permanente, sean o no tunelizados, se han de retirar en caso de: . infección persistente del punto de inserción. . signos de infección a nivel del túnel subcutáneo. . bacteriemia producida por ciertos microorganismos: s. aureus, bacilos gram negativos no fermentadores, hongos filamentosos, levaduras y micobacterias. . bacteriemia o candidemia persistente a pesar de haber transcurrido días desde el inicio de un tratamiento antimicrobiano adecuado. la terapéutica antimicrobiana tendrá que basarse en la identificación del agente causal, mediante hemocultivos y según antibiograma. en situación de shock séptico es necesario administrar un tratamiento empírico que ha de incluir antimicrobianos activos frente a los microorganismos gram positivos y gram negativos que más a menudo causan estas infecciones. la flora predominante en el hospital o en una determinada área del mismo y la existencia de patógenos multirresistentes pueden condicionar la elección de la terapia empírica. la cobertura empírica para bacteriemia por hongos deberá realizarse ante la existencia de shock séptico en un paciente crítico con colonización por cándida múltiple previa o en enfermos con procesos hematológicos y neutropenia asociada. la elección de fluconazol (en dosis de mg/día) o de una equinocandina (caspofungina, anidulafungina o micafungina en las dosis habituales) depende de la existencia o no de exposición previa a los azoles por parte del paciente (tabla ). una vez obtenido el germen, la antibioterapia irá dirigida según el antibiograma. en bacteriemia por estafilococo coagulasa negativa, se recomienda la retirada del catéter y valorar tratamiento - días. en las bacteriemias por s. aureus se recomienda la retirada del catéter, si esta opción no es posible, realizar un tratamiento local (sellado con vancomicina) y sistémico durante semanas, pero si persisten los síntomas a las horas se debe retirar el catéter y descartar la presencia de endocarditis o tromboflebitis séptica que, de estar presente, prolongará el tratamiento durante mes. en la bacteriemia por gram negativos se recomienda retirar el catéter y realizar tratamiento durante una semana. en la candidemia se debe retirar el catéter, y el tratamiento debe prolongarse hasta semanas tras el último hemocultivo negativo. debe realizarse un estudio del fondo de ojo para descartar la presencia de endoftalmitis (tratamiento durante semanas). la nav es la segunda infección más frecuente asociada a dispositivos, siendo responsable del % de las infecciones adquiridas en la uci. un % de los pacientes que precisan ventilación mecánica desarrollará una neumonía. en españa, en el año , según datos del registro envin-helics , la di fue de , neumonías por mil días de ventilación mecánica. se define como nav aquella que se desarrolla tras - horas de intubación endotraqueal. la patogenia de la nav h/ivtiene una estrecha relación con el tubo endotraqueal (tet). la nav es más frecuente en los primeros días tras la intubación y existen determinadas comorbilidades (epoc, paciente inmunodeprimido o enfermedad de base grave) o patologías agudas (traumatismo craneoencefálico, coma, sedación profunda o pacientes quemados con síndrome de inhalación) que favorecen el desarrollo de nav . existen tres vías en la patogenia de la nav que se enumeran a continuación. vía aspirativa. por macro-o microaspiración de secreciones procedentes de orofaringe y/o estómago. en el espacio subglótico se acumulan secreciones procedentes de la orofaringe o del tracto gastrointestinal, existiendo una colonización posterior de estas secreciones por gérmenes endógenos o exógenos. la colocación del tet mantiene las cuerdas vocales abiertas y permite el paso de las secreciones subglóticas. además, la pérdida de presión del neumotaponamiento permite el paso de dichas secreciones a la vía aérea inferior. a través del tet, durante la aspiración de secreciones, fibrobroncoscopias o nebulizaciones debido a una inadecuada higiene de manos, deficiente limpieza de los equipos o por condensación de agua en los circuitos del respirador. otras vías. por translocación bacteriana, al existir una disfunción de la mucosa intestinal que habitualmente actúa como barrera de protección entre los gérmenes de la luz intestinal y el torrente sanguíneo, se favorece el paso de bacterias y productos inflamatorios a la sangre. vía hematógena a partir de un foco infeccioso extrapulmonar. el momento del inicio de la neumonía va a influir en el tipo de patógeno y en el pronóstico. en las de inicio precoz (menos de días desde el inicio de la ventilación mecánica), el inóculo bacteriano se implanta en el momento de la intubación, suelen tener mejor pronóstico al ser producida por bacterias sensibles a los antibióticos. en las neumonías de inicio tardío (más de días desde el inicio de la ventilación mecánica) suele producirse por patógenos multirresistentes (bmr) y se asocia con un aumento de la morbimortalidad . no obstante, pacientes con neumonía precoz, pero que han recibido tratamiento antibiótico en los días previos o han estado hospitalizados, tienen un elevado riesgo de estar colonizados o infectados por bmr y deben ser considerados como si tuvieran neumonía tardía . los factores de riesgo para bmr se describen en la tabla . en general, en la nav los gérmenes más frecuentes son los gram negativos ( %), seguidos de gram positivos ( %), hongos ( %) y otros ( %), siendo pseudomonas aeruginosa el patógeno más frecuente ( %), seguido de staphylococcus aureus ( %) y klebsiella pneumoniae ( %). en la neumonía precoz, el patógeno más frecuente es staphylococcus aureus y en las tardías pseudomonas aeruginosa. la clínica consiste en un aumento de secreciones, de aspecto purulento, acompañado de fiebre y leucocitosis, junto con imagen de consolidación en la radiografía de tórax, dando lugar a un empeoramiento en la oxigenación. puede existir hipotensión si se acompaña de sepsis ( %), precisando un soporte hemodinámico si evoluciona a shock séptico ( % de las nav) ( fig. ) . la adquisición de una nav tiene un importante impacto sobre el pronóstico del paciente, asociándose a un aumento de la mortalidad y de la estancia: la mortalidad atribuible es de un %, prolongación de la ventilación mecánica entre y días y de la estancia media hospitalaria de días con un gasto total por cada episodio de nav de . dólares [ ] [ ] [ ] . el diagnóstico requiere la existencia de un diagnóstico clínico junto con la confirmación microbiológica. diagnóstico clínico. presencia de al menos signos (purulencia de las secreciones, temperatura > ºc y leucocitosis > . c/mm o leucopenia < . c/mm ), junto con la aparición de una opacidad en la radiografía de tórax. confirmación microbiológica. cultivo cuantitativo de en el lavado alveolar o de en una muestra de aspirado bronquial. la primera medida es retirar la intubación en cuanto sea posible junto con una estricta higiene de manos y asepsia durante la intubación y posteriores manipulaciones del tubo en ventilación mecánica. además, en la nav existen unas medidas preventivas específicas dirigidas a disminuir la producción, colonización o entrada en el árbol bronquial de las secreciones subglóticas: . cabecero de la cama a º para dificultar el reflujo de las secreciones gastrointestinales y su acumulación en el espacio subglótico. . aspiración de las secreciones del espacio subglótico mediante un tet con canal de aspiración a nivel subglótico, siendo la medida con mayor evidencia científica. . descontaminación de las secreciones subglóticas mediante el uso de antibióticos no absorbibles. su uso se asocia a una disminución de la incidencia de nav y de la mortalidad. el riesgo de seleccionar gérmenes multirresistentes ha dificultado su uso generalizado. . evitar su entrada en el árbol bronquial manteniendo una presión del neumotaponamiemto entre y cm h o. . disminuir el inóculo administrando en pacientes que se intuban en coma un tratamiento antibiótico profiláctico - días. en el año , se puso en marcha el proyecto neumonía zero (nz) , en las uci españolas, con el objetivo de disminuir la di de nav a episodios por mil días de ventilación mecánica (reducción del % respecto a la tasa media de los años anteriores). el proyecto contaba con unas medidas de obligado cumplimiento y otras altamente recomendables (tabla ). el proyecto nz, al igual que fue bz, ha sido un éxito, consiguiendo disminuir (y mantener la disminución en el tiempo) las tasas de nav. actualmente se considera un estándar de calidad smi con tasas por debajo de neumonías por mil días de ventilación mecánica. factores de riego de neumonía asociada a ventilación mecánica por patógenos multirresistentes tratamiento antibiótico en los últimos días nav que cursa con shock séptico sdra precediendo a la nav ingreso hospitalario de ≥ días previo a la nav técnicas de trr previo al inicio de la nav tratamiento antibiótico en los días previos tratamiento antibiótico en los días previos nav: neumonía asociada a ventilación mecánica; sdra: síndrome de distrés respiratorio del adulto; trr: técnica de reemplazo renal. el tratamiento empírico debe cubrir s. aureus, pseudomonas aeruginosa y otros bacilos gram negativos. el antibiótico inicial dependerá de si existe riesgo de infección por patógenos multirresistentes (tabla ): . sin factores de riesgo para patógenos multirresistentes: monoterapia con piperazilina-tazobactan ( g/ horas vía intravenosa) o cefepime o ceftazidima ( g/ horas vía intravenosa) o carbapenen (meropenem g cada horas o imipenen mg/ horas vía intravenosa) o levofloxacino ( mg/ horas vía intravenosa): si existe alergia a betalactámicos (aztreonan g/ horas vía intravenosa o levofloxacino). . si existe riesgo de samr el tratamiento debe incluir vancomicina mg/kg/ - horas o linezolid mg/ horas. . si existe riesgo de pseudomonas multirresistente asociar doble cobertura. . si hay riesgo de blee el tratamiento debe incluir un carbapenem. una vez obtenido el germen y el antibiograma, se desescalará y se realizará antibioterapia dirigida. en los pacientes ingresados en la uci, la itu relacionada con sonda uretral (su) es la infección asociada a dispositivos más frecuente, representando el % del total de las infecciones. su di es de , infecciones por mil días de sondaje . la permanencia de una su altera los mecanismos naturales de defensa del organismo. la su pone en contacto una zona intensamente colonizada como es el periné con una zona estéril como es la vejiga, proporcionando una puerta de entrada de los microorganismos a través de su superficie interna y externa. por otra parte, la su facilita el anidamiento de los microorganismos en forma de biopelícula, proporcionando un entorno favorable y de resistencia a los antibióticos. por tanto, la iu-su se puede adquirir por vías: . vía extraluminal. es la más frecuente ( %), produciéndose a partir de microorganismos endógenos que colonizan el periné o el tracto gastrointestinal. . vía intraluminal ( %). se adquiere por reflujo de microorganismos a través de desconexiones del sistema de drenaje o contaminación de la orina en la bolsa colectora. . la diseminación hematógena al tracto urinario es rara, siendo habitualmente producida por staphylococcus aureus. el factor de riesgo más importante para el desarrollo de iu-su es la permanencia de la sonda. este riesgo aumenta un - % por cada día de sondaje, estimándose que el % de los pacientes sondados desarrollan una iu-su a los días de sondaje. otros factores como sexo femenino, gravedad de la enfermedad de base, cirugía, edad mayor de años, diabetes mellitus, creatinina de m/dl y sondaje tras días de estancia en el hospital se asocian con un mayor riesgo para su desarrollo . cerca de un % de las iu-su se complican con bacteriemia secundaria, siendo factores de riesgo para su desarrollo la presencia de neutropenia, el tratamiento inmunosupresor, el sexo masculino, la infección por serratia y los días de hospitalización previa al desarrollo de la iu-su. los gérmenes más frecuentes son los gram negativos ( %), seguido de gram positivos ( %) y hongos ( %). escherichia coli es el patógeno más frecuente ( %), seguido de pseudomonas aeruginosa ( %), enterococcus faecalis ( %) y candida albicans ( %). los síntomas son variados e incluyen fiebre de nueva aparición, escalofríos, cambios en las características de la orina, hematuria, dolor en flanco o suprapúbico y alteración del estado mental en mayores de años. un % puede cursar con shock séptico y en un % existe bacteriemia secundaria. se basa en la presencia de signos o síntomas compatibles con infección urinaria, sin otro foco infeccioso, y el hallazgo de o más ufc/ml de una o más especies bacterianas en una muestra de orina obtenida a través de la sonda vesical o muestra de orina obtenida a mitad de la micción en un paciente al que se le retiró la sonda en un plazo de horas . entre un - % de las iu-su son evitables. si bien la tasa de mortalidad asociada es muy baja, los costes por episodio son de dólares, ascendiendo a . si se asocia bacteriemia. la mejor prevención es limitar su uso a los casos en los que exista verdadera indicación y su retirada lo antes posible. el tratamiento empírico dependerá del estado clínico del paciente y del riesgo de presentar bmr. en caso de shock séptico o riesgo de multirresistentes, el tratamiento es un carbapenem (meropenem g/ horas intravenoso) más ami-tabla formación y entrenamiento apropiado en el manejo de la vía aérea higiene estricta de manos en el manejo de la vía aérea control y mantenimiento de la presión del neumotaponamiento por encima de cm h o higiene bucal cada - horas utilizando clorhexidina ( , - , %) evitar, siempre que sea posible, la posición de decúbito supino a º favorecer todos los procedimientos que permitan disminuir de forma segura la intubación y/o su duración evitar los cambios programados de las tubuladuras, humidificadores y tubos traqueales descontaminación selectiva del tubo digestivo aspiración continua de secreciones subglóticas antibióticos sistémicos durante la intubación en pacientes con disminución del nivel de conciencia noglucósido (tobramicina mg/ horas intravenoso). en ausencia de las dos premisas anteriores estaría indicada la monoterapia con fosfomicina o un betalactámico antipseudomonas (cefepime o ceftacidima - g/ horas intravenoso-; piperacilina-tazobactan - g/ horas intravenoso-). se debe recambiar la sonda siempre en la infección por cándida y en el resto en caso de que la sonda lleve más de semanas de permanencia. la infección asociada a los sistemas de derivación de lcr externos (dve) es una grave complicación con una elevada morbimortalidad. su incidencia se sitúa entre el - %. la infección puede adquirirse por vía retrógrada intraluminal o por colonización tras la manipulación del catéter por vía extraluminal. son factores de riesgo para la infección las manipulaciones repetidas, los sistemas abiertos y los drenajes de más de días , . las bacterias gram positivas (s. aureus y estafilococos coagulasa negativos) suelen ser la etiología más frecuente, aunque los bacilos gram negativos (pseudomonas aeruginosa, klebsiella pneumoniae, enterobacter y acineobacter baumanii), incluidos los multirresistentes, van aumentando. cursa con un empeoramiento neurológico (alteración del nivel de conciencia o disfunción neurológica), junto con fiebre o febrícula. se basa en las manifestaciones clínicas y el estudio del lcr (citobioquímico y microbiológico). las características del lcr son pleocitosis moderada (con aumento de pmn), aumento de proteínas y lactato y disminución de la glucosa. deberá realizarse tinción de gram y cultivo tanto del lcr como de la punta del drenaje al retirarlo. el tratamiento implica la retirada del drenaje. si es preciso mantener un drenaje se colocará uno nuevo a ser posible por una vía de acceso diferente. la antibioterapia debe ser precoz debido a la elevada morbimortalidad, inicialmente guiada por el gram y posteriormente por el cultivo. si no se dispone de gram; la terapia empírica se realizará con vancomicina más meropenem ( g/ horas intravenoso) o ceftazidima ( g/ horas intravenoso) y en casos de alergia: linezolid ( mg/ horas intravenoso) más aztreonan ( g/ horas intravenoso). la adición al tratamiento sistémico de antimicrobianos por vía intratecal es controvertida, no estando de momento aprobado su uso por la food and drugs administration (fda). en principio se recomendaría su uso en: . infecciones producidas por gérmenes multirresistentes. . el antibiótico penetra mal o no alcanza una adecuada concentración en lcr. . cultivos de lcr persistentemente positivos. . cuando no es posible retirar el dve. . los antibióticos intratecales más utilizados son: vancomicina ( mg/día), aminoglucósidos (amikacina mg/día, gentamicina - mg/día) y colistina ( mg/día) disueltos en ml de suero fisiológico al , %, retirando previamente la misma cantidad de lcr, y manteniendo el drenaje cerrado - minutos. el tratamiento antibiótico deberá mantenerse - días o hasta la negativización de - cultivos de lcr. los autores declaran no tener ningún conflicto de intereses. muy importante ✔ metaanálisis ✔ artículo de revisión ✔ ensayo clínico controlado ✔ guía de práctica clínica ✔ epidemiología grupo de la guía multidisciplinar para el manejo de la neumonía adquirida en la comunidad. guía multidisciplinar para la valoración pronóstica, diagnóstico y tratamiento de la neumonía adquirida en la comunidad neumonía comunitaria grave. estudio descriptivo de años y utilidad de los criterios de la infectious diseases society of america y la american thoracic society en la identificación de los pacientes que requieren ingreso en una unidad de cuidados intensivos infecciones comunitarias que requieren ingreso en uci community-acquired pneumonia requiring hospitalization among u.s. adults advances in the causes and management of community acquired pneumonia in adults managing cap in the icu joint taskforce of the european respiratory society and european society for clinical microbiology and infectious diseases. guidelines for the management of adult lower respiratory tract infections infections in neurocritical care. neurocrit care escmid study group for infections of the brain (esgib). escmid guideline: diagnosis and treatment of acute bacterial meningitis febrile urinary tractinfection in the emergency room european centre for disease prevention and control. point prevalence survey of healthcare associated infections and antimicrobial use in european acute care hospitals estudio nacional de vigilancia de la infección nosocomial en servicios de medicina intensiva. envin helics. informe estudio de prevalencia de las infecciones nosocomiales en españa. informe prevention of intravascularcatheter-relatedinfections central venous accesssites for the prevention of venous thrombosis, stenosis and infection in patients requiring long-term intravenous therapy european centre for disease prevention and control. european surveillance of healthcare-associated infections and prevention indicators in intensive care units. hai-net icu protocol, versión . . stockholm: ecdc morbidity and mortality associated with primary and catheter-related bloodstream infections in critically ill patients the increased risks of death and extra lengths of hospital and icu stayf rom hospital-acquired bloodstream infections: a case-control study an intervention to decrease catheter-related bloodstream infections in the icu reducción de bacteriemias relacionadas con catéteres en los servicios de medicina intensiva de españa mediante una intervención multifactorial. proyecto bacteriemia zero. ministerio de sanidad y consumo impact of a national multimodal intervention to prevent catheter-related bloodstream infection in the icu: the spanish experience infecciones relacionadas con el uso de los catéteres vasculares neumonía asociada a la ventilación mecánica guidelines for the management of adults with hospitalacquired, ventilator-associated, and healthcare-associated pneumonia. the american thoracic society and the infectious diseases society of america management of adults with hospital-acquired and ventilator-associated pneumonia: clinical practice guidelines by the infectious diseases society of america and the attributable mortality of ventilator-associated pneumonia: a meta-analysis of individual patient data from randomised prevention studies mortality, attributable mortality, and clinical events as endpoints for clinical trials of ventilator-associated pneumonia and hospital-acquired pneumonia economic impact of ventilatorassociated pneumonia in a large matched cohort prevención de la neumonía asociada a ventilación mecánica en las ucis españolas preventing catheter-associated urinary tract infections in the intensive care unit infectious diseases society of america. diagnosis, prevention, and treatment of catheter-associated urinary tract infection in adults: international clinical practice guidelines from the infectious diseasessociety of america nosocomial infections in the neurointensive care unit key: cord- -fz ucwwm authors: freundt, eric c.; drappier, melissa; michiels, thomas title: innate immune detection of cardioviruses and viral disruption of interferon signaling date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: fz ucwwm cardioviruses are members of the picornaviridae family and infect a variety of mammals, from mice to humans. replication of cardioviruses produces double stranded rna that is detected by helicases in the rig-i-like receptor family and leads to a signaling cascade to produce type i interferon. like other viruses within picornaviridae, however, cardioviruses have evolved several mechanisms to inhibit interferon production. in this review, we summarize recent findings that have uncovered several proteins enabling efficient detection of cardiovirus dsrna and discuss which cell types may be most important for interferon production in vivo. additionally, we describe how cardiovirus proteins l, c and l(∗) disrupt interferon production and antagonize the antiviral activity of interferon effector molecules. picornaviridae is an important family of single-stranded, positive-polarity rna viruses that includes > genera with over species (zell et al., ) . within picornaviridae, the genus cardiovirus includes encephalomyocarditis virus (emcv), theiler's murine encephalomyelitis virus (tmev) and saffold viruses (safv). although emcv has been described as a potential zoonotic agent, safvs are the only cardioviruses known to regularly infect humans, with the vast majority of people showing evidence of infection (zoll et al., ; carocci and bakkali-kassimi, ) . emcv has been found to infect over host species and contains one serotype, mengo virus, which was isolated in in the mengo district of uganda (dick et al., ) . tmev was discovered in by max theiler and is found in wild mice and rats worldwide. tmev can cause different diseases, depending on the virus strain and host genetics, ranging from fatal encephalitis to a chronic demyelinating disease that has served as a model for multiple sclerosis (brahic et al., ) . the genome of cardioviruses is approximately . - . kb and contains and untranslated regions (figure ) . translation of the genome gives rise to a polyprotein that is cleaved by the c protease, leading to the production of proteins. two additional proteins, l * and b * , are expressed from alternate open reading frames. l * is only expressed by tmev and is important for infection of macrophages, persistence of the virus in mice and inhibiting rnase l (van eyll and michiels, ; sorgeloos et al., ) , b * results from a frameshifting mechanism conserved in cardioviruses that regulates the ratio of structural and non-structural proteins translated over time. protein b * itself is only thought to be important for replication of emcv, as mutants that abolish its expression had a small plaque phenotype. b * in tmev and safv is unlikely to act as a protein as it is predicted to encode a peptide of amino acids (loughran et al., ) . in this review, we focus on the ways in which cardioviruses trigger the innate immune response and the efficient mechanisms that they have evolved to suppress these signaling pathways. we consider which cell types may be most important for production of interferon (ifn) in vivo, and also describe how cardioviruses disrupt the functions of interferon effectors. double-stranded rna (dsrna) is a necessary product of picornavirus replication, as positive-stranded genome is copied to produce a negative-stranded, full-length template, which is in turn used to produce additional genomes. dsrna is recognized by several sensor proteins within the cell and triggers a signal transduction pathway that results in transcription of the type i ifn (ifn-α/β) genes, as well as ifn-λ. in the endosome, dsrna is detected by toll-like receptor (tlr ), which signals through the adaptor protein trif to activate ifn regulatory factor (irf- ) and nuclear factor kappa b (nf-κb) (yamamoto et al., ) . cytoplasmic dsrna is detected by the rig-i-like receptor (rlr) family of proteins, which includes rig-i (retinoic acid-induced gene i) and mda (melanoma differentiation-associated gene ). upon recognition of dsrna, these proteins undergo a conformational change that exposes n-terminal caspase activation and recruitment domains (cards). the rlrs are then capable of stimulating the mitochondrial antiviral signaling (mavs) protein, also known as ips- , cardif, and visa, which in turn activates tank-binding kinase- (tbk ), inducible i-κb kinase (ikk-ε) and irf- , which then translocates to the nucleus to facilitate transcription of ifn genes (reviewed in gebhardt et al., ) . although rig-i and mda both detect dsrna within the cytosol, their functions are non-redundant. rig-i recognizes relatively short dsrna species (< kb) with ppp or pp, which are produced in certain virus infections (hornung et al., ; pichlmair et al., ) . in contrast, mda recognizes long dsrna, which is present during picornavirus replication (kato et al., ; pichlmair et al., ) . thus, while rig-i is activated during infection with flaviviruses, paramyxoviruses, influenza, and others, mda is responsible for detection of picornaviruses (gitlin et al., ; kato et al., ; pichlmair et al., ; wang et al., ; feng et al., ) . the importance of mda for control of cardioviruses was demonstrated in mda -deficient mice, which failed to control emcv infection and did not efficiently produce ifn (gitlin et al., ; kato et al., ) . in addition to rig-like helicases described above, which activate the mavs pathway, another ifn-inducible rna helicase, moloney leukemia virus homolog (mov ) was reported to enhance ifn induction (cuevas et al., ) . mov expression in hek cells restricted emcv replication. interestingly, mov acts through irf- activation, in a rlr and mavs-independent way and signals require ikk-ε but not tbk . such mavs-independent pathways are likely not critical for global ifn production in emcv infected mice, given the major impact of mda or mavs deficiency in mice, but they may be important in specific cell types or in conditions where the other pathways may be less potent. laboratory of genetics and physiology (lgp ), also known as dhx , is also a member of the rlr family but lacks a card domain (yoneyama et al., ) . given its structural similarity and lack of a card domain, lgp was initially thought to negatively regulate dsrna recognition by rig-i, as its overexpression limited ifn induction by sendai virus and newcastle disease virus (rothenfusser et al., ) . however, although the negative effect of lgp on rig-i remained controversial, later studies have established that lgp acts as a co-activator of mda . mice deficient for lgp were impaired in responding to rna ligands for mda or to emcv infection (venkataraman et al., ; satoh et al., ) . lgp was also shown to increase the rate of mda interaction with rna and downstream signaling by facilitating the formation of numerous, shorter mda filaments (bruns et al., ) . thus, it appears that lgp can act as both a positive and negative regulator of rlr signaling, with the outcome likely dependent on the concentration of lgp (bruns and horvath, ) . however, recombinant mda was directly activated as measured by an atp hydrolysis assay by the replicative form of dsrna coxsackievirus b , showing that lgp is not essential for activation of mda in vitro by dsrna (feng et al., ) . both lgp and mda are important for detecting cardiovirus replication. mefs deficient for either protein produce lower amounts of ifn-β when infected with emcv (deddouche et al., ) . intriguingly, lgp may enhance activation of mda during emcv infection by binding to rna complementary to the leader (l) gene and forming a complex with mda . this rna sequence from l was also shown to be a potent activator of mda in the absence of virus infection (deddouche et al., ) . although dsrna can bind and activate recombinant mda in the absence of other proteins, it is likely that additional partners are required for efficient activation of mda in vivo. for example, mda is phosphorylated in resting cells, and requires dephosporylation by pp α/γ (wies et al., ; takashima et al., ) . additional proteins that participate in recognition of cardiovirus dsrna have recently been described, including a study showing that tar rna binding protein (trbp) interacts with lgp in a yeast two-hybrid screen (komuro et al., ) . lgp was found to interact with trbp in co-immunoprecipitation experiments and depletion of trbp by sirna reduced interferon production induced by tmev and emcv. moreover, trbp enhanced ifn induction by tmev and emcv when overexpressed. depletion of trbp did not affect induction of ifn by sendai virus, which is recognized by rig-i. this study establishes that trbp participates in detection of cardiovirus dsrna by lgp /mda but the mechanism and its importance in vivo remain to be elucidated. as trbp is a component of the rnai machinery (chendrimada et al., ; haase et al., ) , other molecules involved in rnai were assessed for their role in dsrna detection and protein activator of pkr (pact) was found to also participate in activation of ifn signaling by cardioviruses. when pact was depleted by sirna, interferon production was decreased during infection of both tmev and emcv. overexpression of pact also increased ifn activation when lgp was co-expressed with mda . intriguingly, single-stranded tmev genome enhanced the association of lgp and pact, which suggests that a secondary structure in the tmev genome might facilitate this interaction (miyamoto and komuro, ) . in a separate report, pact was shown to be required for induction of ifn by emcv but not sendai virus. this study also demonstrated that pact and mda were recruited to dsrna (poly(i:c)) but not single stranded rna, and that pact expression increased the amount of mda oligomerization (lui et al., ) . both trbp and pact have also been reported to bind to double stranded rna-dependent protein kinase (pkr), and pact can bind rig-i (park et al., ; patel and sen, ; kok et al., ) . at the present time, it is not clear if these interactions are important for mediating recognition of cardioviruses. yet another partner in detecting dsrna in cardiovirus infection was recently uncovered. a cdna screen to identify genes involved in regulating ifn signaling revealed that dhx expression increased transcription of an ifn-β reporter plasmid in response to high molecular weight (hmw) poly(i:c) (zhu et al., ) . depletion of dhx resulted in decreased phosphorylation of tbk and irf- in response to hmw poly(i:c) and emcv as well as decreased production of ifn-β. the authors also show that dhx binds to mda but not mavs or rig-i, and binding could only be detected when mda was activated by emcv or hmw poly(i:c). mechanistically, this study demonstrated that dhx mediated rna binding of mda by interacting with mda through its n-terminus and rna through its dexd and helicase domains, and that dhx promotes formation of mda filaments, which are required for activation (zhu et al., ) . dhx was also independently described to interact with rig-i (sugimoto et al., ) , although it may be of greater importance for activation of mda (zhu et al., ) . together, these studies show that multiple proteins facilitate recognition of dsrna by mda during cardiovirus infection (figure ). thus far, these proteins include lgp , dhx , pact and trbp. additional helicases are also likely to participate in rlr-dsrna complex formation but their activities remain to be clarified (oshiumi et al., ) . moreover, recent discoveries have identified a novel role for pkr in recognition of dsrna and activation of mda , which will be discussed below. as the number of proteins mediating mda activation grows, so does the number of questions about how this pathway functions. for example, what role does each of the proteins play and how do they work together mechanistically to activate mda ? do they play non-redundant roles, or do they function in the same way but in different cell types? resolving these questions will allow for deeper understanding of the first line of immune defense against rna viruses. upon recognition of dsrna, pkr controls virus infection by phosphorylating eukaryotic initiation factor (eif α), which inhibits translation (farrell et al., ) . in this way, an infected cell can suppress production of viral proteins. phosphorylation of eif α also leads to stress granule formation and induces autophagy, and both pathways are commonly observed during virus infection (paul and munz, ; poblete-duran et al., ) . not surprisingly, many viruses have evolved strategies to inhibit the activation or function of pkr (reviewed in garcia et al., ) , as evidenced by the fact that pkr-deficiency did not modify the survival time of emcv infected mice (yang et al., ) . in addition to its role in inhibiting translation, recent evidence has emerged to show that pkr participates in production of ifn. for example, pkr appears to be important for nuclear translocation of irf- following mda activation (pham et al., ) . pkr was found to bind to mda and this interaction was not disrupted by nuclease treatment, indicating that binding does not depend on the presence of rna. in pkr-deficient cells, emcv infection failed to induce irf- translocation to the nucleus. moreover, a constitutively active mutant of pkr induced ifn through mavs. the effect of pkr on induction of ifn required its catalytic activity but did not depend on phosphorylation of eif α (pham et al., ) . in a separate report, pkr was shown to be important for normal processing of ifn-β mrna, suggesting that pkr may function at multiple points in the ifn pathway (schulz et al., ) . additionally, activation of pkr by hmw poly(i:c) was shown to be inhibited in cells depleted of mda and mavs, suggesting that mavs influences activation of pkr. moreover, mavs and pkr were found to interact in co-ip experiments, and the interaction depended on the card domain in mavs and the dsrna binding domain of pkr (zhang et al., ) . together, these studies clearly indicate a role for pkr in mda -dependent ifn induction, although several mechanisms may be involved, which require clarification. the role of pkr in ifn production after cardiovirus infection remains to be resolved. in one study that examined mengo virus infection, knockdown of pkr led to decreased induction of ifn-β in hela cells, suggesting that pkr may play a role in the mda pathway during cardiovirus infection (langereis et al., ) . this observation fits with the model proposed by onomoto et al. ( ) , which was based on influenza virus infection and suggests that pkr triggers the formation of "antiviral stress granules" that serve as a recruitment platform for dsrna and rig-like helicases, thereby enhancing ifn production. pkr is, however, not essential for ifn production in cardiovirus-infected cells because mengo virus possessing a deletion in the zinc-finger of l, which abrogates its functions as an interferon antagonist, was found to induce interferon in the cells lacking pkr and rnase l (feng et al., ) . cardioviruses may also inhibit pkr through activity of the l protein, as stress granule formation was prevented by the l protein of mengo, tmev, and safv- during virus infection (borghese and michiels, ) . however, direct inhibition of pkr by l remains to be demonstrated. during infection, viral dsrna can also be released into the extracellular milieu, either non-specifically during lysis of infected cells or possibly intentionally by exocytosis to trigger innate immunity by uninfected cells. dsrna can then be endocytosed by neighboring cells and infiltrating immune cells and lead to ifn production. tlr recognizes dsrna in endosomes and signals through trif to activate irf- and nf-κb. the importance of tlr in context of cardiovirus infection may depend on the model of infection. for example, mice deficient for myd or tlr were not significantly more susceptible than wild-type mice to emcv infection . in a separate report, however, tlr -deficient mice had higher viral loads in the liver and heart and were more susceptible to infection (hardarson et al., ) . also, a study that evaluated the role of tlr in controlling a strain of emcv with tropism for β cells of the pancreas found that tlr protected mice from a fatal infection and that tlr -deficent mice produced less ifnβ early in infection ( and h post-infection). however, this deficiency was transient and mice lacking tlr produced levels of ifn-β equivalent to wild-type animals at h post-infection. in the same experiments, the authors demonstrated that mice lacking mda succumbed to the infection more rapidly than tlr -deficient mice and produced less ifn-β at h postinfection (mccartney et al., ) . finally, a recent report using intracerebral inoculation of the gdvii strain of tmev evaluated the importance of these molecules for control of virus replication and induction of ifn. trif-/-, myd -/-, and mice lacking both trif and myd showed wild-type levels of ifn induction, while mavs-deficient animals were slightly but significantly impaired. when trif, myd and mavs were all depleted, however, mice were unable to induce ifn. only mice lacking myd and trif, or mice lacking myd , trif and mavs showed increased titers of gdvii in the brain (pfefferkorn et al., ) . thus, both tlr and rlrs contribute to controlling virus replication in vivo, although the relative importance of these pathways may depend on the virus and route of inoculation. surprisingly, mda is also responsible for the vast majority of ifn produced from extracellular dsrna in vivo. mice deficient for mda produced substantially less ifn when administered polyi:c, whereas tlr -deficient mice responded like wild-type (gitlin et al., ) . these results raise the question of how dsrna taken up through endocytosis could gain access to the cytoplasm. the mechanism by which dsrna could be internalized and then access the cytoplasmic rlrs has been unresolved until a recent discovery identified sidt , the mammalian ortholog of the sid- dsrna transporter in caenorhabditis elegans, as a transporter of dsrna from the endosome to the cytoplasm. sidt was shown to be important for mediating detection of dsrna in the context of emcv infection in vivo. mice that were deficient for sidt failed to control replication, produced less ifn-β, and succumbed to infection (nguyen et al., ) . these data suggest that a crucial pathway for innate signaling in emcv infection is release of viral rna into the extracellular milieu, endocytosis, and subsequent transfer of viral rna to the cytoplasm to access mda . two proteins encoded by cardioviruses were shown to counteract ifn production in infected cells: protease c, which is responsible for the processing of the virus-encoded polyprotein, and the leader protein (l), which corresponds to the n-terminal peptide of the polyprotein. c is a cysteine proteinase with a trypsin-like serine protease fold, responsible for most cleavages occuring during the maturation of the viral polyprotein (pelham, ) . like c proteases of other picornaviruses that were shown to target critical factors involved in ifn induction such as rig-i (barral et al., ) , emcv c was reported to cleave traf family member-associated nf-kb activator (tank) in infected cells, thus disrupting the complex involving tbk , ikke and irf and limiting type i ifn production (huang et al., ) . likewise, emcv c was shown to target mov , an rna helicase that acts in a mavs-independent way, as a possible innate immune evasion mechanism (cuevas et al., ; figure ). l is a small, multifunctional protein of - amino acids expressed by all cardioviruses (figure ). l contains an n-terminal zinc finger motif (cys-his-cys-cys), an acidic domain, a serine/threonine rich domain, and a c-terminal theilo domain, which is present in safv and tmev but absent in emcv. l was shown to be dispensable for replication of tmev in cell culture but its loss inhibits spread in cells that have a functional interferon response, like l cells, and also impairs the viral persistence in vivo (van pesch et al., ) . l deletions in figure | interferon antagonism by the cardiovirus c protease. the c protease is responsible for cleavage of the cardiovirus polyprotein produced from translation of the genome. in addition, the cardiovirus c protease cleaves host proteins, such as tank and mov , to prevent the cell from producing ifn. frontiers in microbiology | www.frontiersin.org emcv prevent the virus from shutting off host protein synthesis and enable interferon production (zoll et al., (zoll et al., , . mengo virus containing a mutation in the zinc finger of l failed to inhibit ifn synthesis and its replication was inhibited during low moi infections in vitro. in mice lacking the ifn α/β receptor, the mutant virus behaved as wild-type, but in wild-type mice, replication of the l mutant virus was impaired and it failed to cause disease, demonstrating that activity of l is important for pathogenesis in vivo (hato et al., ) . mutations in the zinc finger domain or the theilo domain of tmev or safv l inhibit its ability to antagonize interferon signaling (ricour et al., ) . a critical step in production of ifn following detection of viral replication by mda and other molecules is nuclear translocation of irf- and nf-κb. these proteins enable transcription of the ifn genes to produce mrna, which must then be exported from the nucleus for translation. since picornaviruses do not replicate within the nucleus, many viruses within this family disrupt nucleocytoplasmic trafficking, which results in inhibition of ifn production and translocation of nuclear proteins to the cytosol to benefit viral replication (reviewed in yarbrough et al., ; flather and semler, ) . the mechanism of how l interferes with ifn production may be due to its abilities to disrupt nucleocytoplasmic trafficking, activation of irf- , and assembly of stress granules in infected cells (figure ) . each of these activities will be explored below. the l protein of cardioviruses perturbs the function of the nuclear pore complex (npc) (porter et al., ) . in mammals, the npc consists of approximately different proteins, called nucleoporins (nups) and enables transit across the nuclear membrane (gorlich and kutay, ; wente and rout, ) . while small molecules and ions are able to diffuse through the npc, molecules larger than approximately - kda require active transport, which is regulated by transport receptors called karyopherins (yarbrough et al., ) . transport into the nucleus requires a short amino acid motif, called a nuclear localization sequence (nls) that can interact with either the α or β subtypes of karyopherins, depending on the sequence of the protein's nls. binding and dissociation of nls-containing proteins by karyopherins is also regulated by small gtpase ran. in the cytosol, ran is bound to gdp and can bind cargo proteins. once in the nucleus, however, ran is converted to the gtp bound form by the ran guanine nucleotide exchange factor (rangef) and dissociates from cargo. export then requires a nuclear export sequence (nes) that binds to karyopherins bound to rangtp, and dissociation of this complex occurs in the cytoplasm when a ran gtpase-activating protein (rangap) hydrolyzes gtp to gdp. in this way, the rangdp/gtp gradient regulates directional transport into and out of the nucleus. localization of l to the nucleus depends on expression of a, which contains a nls in its c-terminus (groppo et al., ) . upon nuclear localization, l interacts with ran with high affinity and a is displaced as the binding sites for a and ran partially overlap (petty et al., ) . l from emcv, tmev, and safv induce hyper-phosphorylation of nups including nup and nup (ricour et al., ; ciomperlik et al., ) , likely by recruiting and activating a kinase, which may be facilitated by l binding of exportins crm and cas (ciomperlik et al., ) . chemical inhibition of erk and p was able to block l-mediated hyper-phosphorylation of nups (porter et al., ) . additionally, l of emcv is phosphorylated by casein kinase (ck ) and this phosphorylation is required for nup phosphorylation, although ck did not phosphorylate l of safv or tmev . it is possible, although it remains to be shown, that these kinases also play a role in inhibition of nucleocytoplasmic trafficking by l of tmev and safv. in addition to its role in disrupting nucleocytoplasmic trafficking, tmev and mengo l prevent production of type i ifn in infected cells by interfering with irf- dimerization and tmev l also prevents export of mrna from the nucleus (delhaye et al., ; ricour et al., ) . for both tmev and mengo virus, dimerization of irf- was impaired despite the protein having been phosphorylated. inactivation of irf- occurs despite reports that it accumulates in the nucleus of infected cells (delhaye et al., ) . these data suggest that dsrna is detected in cardiovirus infected cells leading to activation of mavs and downstream kinases, but that irf- is unable to induce ifn transcription. stress granules can form in cells during virus infection and often result from inhibition of translation following phosphorylation of eif α by pkr (white and lloyd, ) . the l protein of mengo, tmev, and safv- inhibits stress granule formation during infection and ectopic expression of l was able to prevent thapsigargin-and arsenite-induced stress granules (borghese and michiels, ) . stress granules formed during infection with viruses containing deletions in the zinc-finger domain or a mutation in the theilo domain of l, indicating that these motifs are also important for inhibition of stress granules (borghese and michiels, ) . while l inhibits nucleocytoplasmic trafficking, stress granule formation, and possibly pkr activation, it has not been possible to uncouple these events using l mutants. when one function of l is disrupted, all functions are simultaneously impaired. therefore, it remains possible that l inhibits ifn production by blocking pkr activation, by interfering with irf- dimerization or nucleocytoplasmic trafficking, or through a combination of these mechanisms (figure ) . the importance of these antiviral pathways in controlling infection is underscored by the variety of mechanisms that viruses have evolved to prevent their activity. for example, the l protein of foot-and-mouth disease virus (fmdv), a picornavirus in the genus aphthovirus, has proteolytic activity whereas the l protein of cardioviruses does not. despite the major differences in these proteins, they all still function to inhibit induction of ifn. fmdv l pro can cleave eif g (devaney et al., ; kirchweger et al., ; guarne et al., ) and therefore reduce translation of cellular mrnas, and can also perturb ifn transcription by cleaving nf-κb (de los santos et al., . however, in the once in the nucleus, l interacts with high affinity with ran gtpase, thus displacing a. the l-ran complex would activate a kinase and trigger nucleoporin hyper-phosphorylation, thereby leading to nuclear pore complex dismantling and to nucleocytoplasmic trafficking perturbation. on the other hand, l may inhibit pkr, thus preventing translation arrest through eif α phosphorylation and therefore block assembly of stress granules. inhibition of ifn gene transcription by l may result from irf- trafficking perturbation and/or from the absence of pkr-enhanced dsrna detection by mda . context of a chimeric mengo virus infection, fmdv l pro was less effective at inhibiting ifn induction in vitro and in vivo (hato et al., ) . similar convergent evolution is apparent when considering the a protein of picornaviruses. whereas a functions as a protease for most picornaviruses and cleaves mediators of type i interferon signaling, this activity is not present in cardioviruses. nevertheless, l still targets some of these same molecules for inactivation (agol and gmyl, ) . additionally, both a of enteroviruses and l of cardioviruses inhibit stress granule assembly (yang et al., ) . the functions of l appear to be sufficiently important to the virus so that it maintains high levels of l expression throughout infection. emcv and tmev undergo a frameshift during translation later in infection by a binding to a stem-loop structure in the genome (napthine et al., ) . this frameshift decreases expression of non-structural proteins bc- abcd by - % (finch et al., ) . a follow up study using metabolic labeling estimated the frameshifting to be - % efficient (ling and firth, ) . this mechanism may allow for cardioviruses, and perhaps other picornaviruses, to increase the translation of structural proteins later in infection. due to its position in the genome, however, l expression would remain high throughout infection despite it not having a structural role for virus assembly. thus, it may be important for cardioviruses to express sufficient levels of l to counteract the immune response throughout the replication cycle. with effective ways to inhibit the production of interferon during infection, control of cardioviruses likely depends on nearby uninfected cells to produce interferon. intriguingly, these pathways seem to also depend on mda , although tlr may also be important in certain cell types such as plasmacytoid dendritic cells (hornung et al., ) . as discussed, recent data suggest a model where viral dsrna is released, endocytosed, and then the rna is translocated to the cytosol where it is detected by mda . in the cns, astrocytes appear to be the primary producers of ifn-β for several neurotropic viruses that preferentially infect neurons, such as tmev and la crosse virus (kallfass et al., ; pfefferkorn et al., ) . using transgenic mice that expressed firefly luciferase under the control of the ifn-β promoter restricted to different cell types, the authors were able to determine that % of ifn-β production during a neurotropic tmev infection was from astrocytes, whereas only % was from neurons, which are the primary target of infection. in mice lacking mavs, ifn-β production was slightly but significantly reduced, suggesting that the rlr pathway is active during infection but may not be the only pathway activated by tmev in astrocytes. intriguingly, mice deficient for myd and trif did not show a significant decrease in ifn-β induction, although induction of ifn-β was completely abrogated in mice deficient for mavs, myd and trif. therefore, it appears that both rlr and tlr signaling are important for ifn-β production after tmev infection of the cns. astrocytes were also shown to be primary producers of ifn during infection with rabies virus and vesicular stomatitis virus. in the case of rabies virus, astrocytes are stimulated to produce ifn by an abortive infection. that the virus is unable to replicate fully may prevent expression of viral interferon antagonists and allow for robust production of ifn. how viral replication is prevented in these cells will be important to uncover and may lead to novel insights about viral control in vivo. it is likely that abortive infection of astrocytes occurs during infection by tmev. however, this remains to be demonstrated and viral rna may well be encountered by other means. mda is critical for induction of ifn against cardioviruses in the periphery as well. ex vivo, cells such as macrophages, conventional dendritic cells and fibroblasts depend on mavs for production of ifn in response to dsrna (sun et al., ) . similarly, mda was shown to be essential in these cells for type i ifn production after emcv infection, in contrast to pdcs which induce ifn production in a tlr-dependent fashion (gitlin et al., ; kato et al., ) . after emcv infection of mice, some ifn is produced through tlrs, likely by pdcs, but most ifn was produced by mda activation (gitlin et al., ; kato et al., ) . levels of ifn-i were strongly decreased in the serum of mda -deficient mice infected by emcv. whereas mda expression strongly influenced survival in response to infection, the effect of myd depletion had a modest effect and loss of trif or rig-i did not affect survival . thus, mda is essential for controlling emcv infection in the periphery. interferon secreted by infected cells binds to its receptor on surrounding cells, activating a signaling cascade that leads to expression of hundreds of interferon-stimulated genes (isgs). two of these isgs, pkr and oligoadenylate synthetases (oas) are part of the best-characterized interferon effector pathways. as described earlier, pkr is likely antagonized by the l protein, as l inhibits pkr-induced stress granule assembly. moreover, a recent study reported increased sumo conjugation figure | inhibition of rnase l activation by l * tmev l * binds rnase l ankyrin repeats and (numbered) through a direct protein-protein interaction, thereby preventing association of - a with rnase l monomers and the consequent dimerization and activation of the enzyme. of pkr in emcv-infected cells, which dampens pkr activation and promotes caspase-dependent pkr degradation (maarifi et al., ) . oligoadenylate synthetases are enzymes responsible for rnase l activation. cardioviruses have evolved two strategies to interfere with the oas-rnase l pathway. in an infected cell, oas are activated by dsrna and produce - oligoadenylates ( - a). binding of two - a molecules to the ankyrin domain of the latent endoribonuclease rnase l triggers its dimerization and activation (figure ) . active rnase l then cleaves viral and cellular ssrna leading to decreased viral replication and ultimately to apoptosis of the cell. interestingly, rna fragments generated by rnase l can amplify ifn production in a rig-i, mda and mavs-dependent way (malathi et al., ) . rnase l targets both viral and cellular mrna but is also predicted to cleave the genome of ssrna viruses, as reported for emcv (li et al., ) . in addition to -phosphodiesterases and phosphatases that tightly regulate the system by degrading - a within minutes of their synthesis, rnase l activity can be negatively regulated by the rnase l inhibitor (rli/abce) (bisbal et al., ) . rli/abce expression is induced by emcv and correlates with rnase l inhibition (martinand et al., ) . accordingly, overexpression of rli/abce inhibited the action of ifn against emcv (bisbal et al., ) . rnase l inhibition by emcv-induced rli is, however, partial as rnase l antiviral activity against emcv was demonstrated in vitro using dominant negative rnase l and oas overexpression (chebath et al., ; zhou et al., ) and in vivo, in rnase l-deficient mice, which presented increased emcv infection and mortality compared to wild-type mice (zhou et al., ) . the l * protein of tmev was found to potently inhibit rnase l through a direct protein-protein interaction (sorgeloos et al., ) . mechanistically, l * binds to rnase l ankyrin repeats and , thereby preventing - a binding to the enzyme and further activation steps (drappier et al., ; figure ). in wild-type macrophages, replication of l * -mutant was significantly impaired as compared to that of the wild-type virus (sorgeloos et al., ) . in contrast, l * -mutant and wildtype viruses replicated to the same level in rnase l-deficient primary peritoneal macrophages. moreover, l * was shown to be active in vivo in the context of mhv chimeric viruses; l * could substitute for another viral rnase l inhibitor, namely the ns phosphodiesterase of mhv, in the liver of infected mice (drappier et al., ) . the fact that the virus devotes one of its proteins to rnase l antagonism highlights the importance of this antiviral pathway against tmev. interestingly rnase l inhibition by l * is highly species-specific; l * of a mouse tmev strain inhibits mouse rnase l but not its orthologs from other species including rat (sorgeloos et al., ; drappier et al., ) . accordingly, l * of a rat tmev strain inhibits rat but not mouse rnase l. theiler's murine encephalomyelitis virus is the only cardiovirus expressing l * , and thus the only cardiovirus known to directly inhibit rnase l. this could stem from its tropism for macrophages, which are the main tmev target during the chronic phase of infection and in which the oas-rnase l system is particularly active (zhao et al., ) . however, macrophages were reported to play important roles in emcv pathogenesis, including for viral replication and dissemination in piglets (papaioannou et al., ) and as reservoir cells for emcv persistence in rats (psalla et al., ) . since emcv is sensitive to rnase l activity, it is possible that another emcv protein will have developed some rnase l antagonistic activity, which might be identified using the appropriate host-pathogen context. interactions between safvs and rnase l have yet to be described, but it is also likely that these viruses have evolved ways of inhibiting this pathway. given the many ways that picornaviruses inhibit interferon production and signaling in infected cells, it is not surprising that the most important producers of ifn would be uninfected or abortively infected cells. indeed, cardioviruses efficiently block ifn in infected cells but loss of mda in mice causes them to be more susceptible to virus infection. these data indicate that detection of cytoplasmic dsrna by mda occurs in cells that are not productively infected (gitlin et al., ) and the recent finding that sidt mediates this process opens many new exciting areas of research (nguyen et al., ) . which molecules might be important for release of viral rna? is there a role for exosomes in this process? how might these pathways be stimulated pharmacologically? preventing release of viral rna and subsequent detection by uninfected cells may represent selective pressure favoring non-lytic release. given the exquisite genetic malleability in response to natural selection displayed by viruses, it is likely that viruses will have evolved mechanisms of inhibiting detection of viral rna by uninfected cells, perhaps by restricting dsrna release or by secreting proteins that inhibit rna transport into uninfected cells. it will be exciting to see how discoveries unfold in this area of research. several recent studies involving cardioviruses have revealed a more complicated picture regarding initial detection of replicating rna and induction of ifn. while it is clear that the helicases lgp , dhx , pact and trbp work in concert with mda for detection of dsrna, it remains to be determined how these molecules coordinate and interact and whether they function in a cell-type specific manner. it will also be important to resolve the way in which pkr functions to activate ifn signaling. future studies in this area will likely have broad relevance for innate detection of viruses. finally, the mechanisms by which l disrupts nucleocytoplasmic trafficking, stress granule formation, and interferon production clearly require further clarification. mutational analysis of l has revealed that these activities are tightly coupled, suggesting that the l interacts with protein(s) that can serve as a common node in each of these pathways. as ifn signaling and stress granules are important for a variety of viral pathogens, answers to these questions may provide broadly relevant insight into host-pathogen interactions. ef, md, and tm wrote the manuscript and approved its final version. ef was supported by a david delo research grant. md was supported by action de recherches concertée (arc). research in the tm lab was supported by the belgian fund for medical research (frsm, pdr # t. . ) and by eos joint program of fonds de la recherche scientifique -fnrs and fonds wetenschapelijk onderzoek -vlaanderen -fwo (eos id: ). viral security proteins: counteracting host defences rig-i is cleaved during picornavirus infection encephalomyocarditis virus leader is phosphorylated by ck and syk as a requirement for subsequent phosphorylation of cellular nucleoporins cloning and characterization of a rnase l inhibitor. a new component of the interferon-regulated - a pathway the leader protein of cardioviruses inhibits stress granule assembly the genetics of the persistent infection and demyelinating disease caused by theiler's virus lgp synergy with mda in rlrmediated rna recognition and antiviral signaling the innate immune sensor lgp activates antiviral signaling by regulating mda -rna interaction and filament assembly the encephalomyocarditis virus constitutive expression of ( '- ') oligo a synthetase confers resistance to picornavirus infection trbp recruits the dicer complex to ago for microrna processing and gene silencing three cardiovirus leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways cardiovirus leader proteins bind exportins: implications for virus replication and nucleocytoplasmic trafficking inhibition mov provides antiviral activity against rna viruses by enhancing rig-i-mavs-independent ifn induction the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response degradation of nuclear factor kappa b during foot-and-mouth disease virus infection a conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function identification of an lgp -associated mda agonist in picornavirus-infected cells the leader protein of theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins leader protein of foot-and-mouth disease virus is required for cleavage of the p component of the cap-binding protein complex mengo encephalomyelitis; a hitherto unknown virus affecting man a novel mechanism of rnase l inhibition: theiler's virus l * protein prevents - a from binding to rnase l phosphorylation of initiation factor elf- and the control of reticulocyte protein synthesis mda detects the double-stranded rna replicative form in picornavirus-infected cells characterization of ribosomal frameshifting in theiler's murine encephalomyelitis virus picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positivestrand rna virus the dsrna protein kinase pkr: virus and cell control discrimination of self and non-self ribonucleic acids essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus transport between the cell nucleus and the cytoplasm mutational analysis of the emcv a protein identifies a nuclear localization signal and an eif e binding site structure of the foot-and-mouth disease virus leader protease: a papainlike fold adapted for self-processing and eif g recognition trbp, a regulator of cellular pkr and hiv- virus expression, interacts with dicer and functions in rna silencing toll-like receptor is an essential component of the innate stress response in virus-induced cardiac injury the mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor differential ifn-alpha/beta production suppressing capacities of the leader proteins of mengovirus and foot-and-mouth disease virus '-triphosphate rna is the ligand for rig-i encephalomyocarditis virus c protease attenuates type i interferon production through disrupting the tank-tbk -ikkepsilon-irf complex visualizing production of beta interferon by astrocytes and microglia in brain of la crosse virus-infected mice length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene differential roles of mda and rig-i helicases in the recognition of rna viruses foot-and-mouth disease virus leader proteinase: purification of the lb form and determination of its cleavage site on eif- gamma the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response the tar-rna binding protein is required for immunoresponses triggered by cardiovirus infection mda localizes to stress granules, but this localization is not required for the induction of type i interferon rnase l mediates the antiviral effect of interferon through a selective reduction in viral rna during encephalomyocarditis virus infection an analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators ribosomal frameshifting into an overlapping gene in the b-encoding region of the cardiovirus genome pact facilitates rna-induced activation of mda by promoting mda oligomerization differential effects of sumo and sumo on pkr activation and stability small self-rna generated by rnase l amplifies antiviral innate immunity rnase l inhibitor is induced during human immunodeficiency virus type infection and down regulates the - a/rnase l pathway in human t cells rna sensor-induced type i ifn prevents diabetes caused by a beta cell-tropic virus in mice pact is required for mda -mediated immunoresponses triggered by cardiovirus infection via interaction with lgp protein-directed ribosomal frameshifting temporally regulates gene expression sidt transports extracellular dsrna into the cytoplasm for innate immune recognition critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity accessory factors of cytoplasmic viral rna sensors required for antiviral innate immune response pathogenesis of encephalomyocarditis virus (emcv) infection in piglets during the viraemia phase: a histopathological, immunohistochemical and virological study tar rna-binding protein is an inhibitor of the interferon-induced protein kinase pkr pact, a protein activator of the interferoninduced protein kinase, pkr autophagy and mammalian viruses: roles in immune response, viral replication, and beyond translation of encephalomyocarditis virus rna in vitro yields an active proteolytic processing enzyme binding interactions between the encephalomyocarditis virus leader and protein a abortively infected astrocytes appear to represent the main source of interferon beta in the virus-infected brain pkr transduces mda -dependent signals for type i ifn induction rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates activation of mda requires higher-order rna structures generated during virus infection who regulates whom? an overview of rna granules and viral infections a picornavirus protein interacts with ran-gtpase and disrupts nucleocytoplasmic transport nucleoporin phosphorylation triggered by the encephalomyocarditis virus leader protein is mediated by mitogen-activated protein kinases pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in rats inhibition of mrna export and dimerization of interferon regulatory factor by theiler's virus leader protein the rna helicase lgp inhibits tlrindependent sensing of viral replication by retinoic acid-inducible gene-i lgp is a positive regulator of rig-i-and mda -mediated antiviral responses protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity evasion of antiviral innate immunity by theiler's virus l * protein through direct inhibition of rnase l helicase proteins dhx and rig-i cosense cytosolic nucleic acids in the human airway system the specific and essential role of mavs in antiviral innate immune responses riok -mediated phosphorylation of mda interferes with its assembly and attenuates the innate immune response influence of the theiler's virus l * protein on macrophage infection, viral persistence, and neurovirulence the leader protein of theiler's virus inhibits immediate-early alpha/beta interferon production loss of dexd/h box rna helicase lgp manifests disparate antiviral responses mda and mavs mediate type i interferon responses to coxsackie b virus the nuclear pore complex and nuclear transport regulation of stress granules in virus systems dephosphorylation of the rna sensors rig-i and mda by the phosphatase pp is essential for innate immune signaling role of adaptor trif in the myd -independent toll-like receptor signaling pathway picornavirus a protease regulates stress granule formation to facilitate viral translation deficient signaling in mice devoid of double-stranded rna-dependent protein kinase viral subversion of nucleocytoplasmic trafficking shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity ictv virus taxonomy profile: picornaviridae ips- plays an essential role in dsrna-induced stress granule formation by interacting with pkr and promoting its activation antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology interferon action and apoptosis are defective in mice devoid of ' , '-oligoadenylate-dependent rnase l impact of rnase l overexpression on viral and cellular growth and death dhx functions as an rna co-sensor for mda -mediated emcv-specific antiviral immunity saffold virus, a human theiler'slike cardiovirus, is ubiquitous and causes infection early in life mengovirus leader is involved in the inhibition of host cell protein synthesis the mengovirus leader protein suppresses alpha/beta interferon production by inhibition of the iron/ferritin-mediated activation of nf-kappa b key: cord- -mzzvmyea authors: shumilak, geoffrey; sligl, wendy i. title: moving past the routine use of macrolides—reviewing the role of combination therapy in community-acquired pneumonia date: - - journal: curr infect dis rep doi: . /s - - - sha: doc_id: cord_uid: mzzvmyea purpose of review: despite advances in diagnostic microbiology and sepsis management, community-acquired pneumonia (cap) remains a significant cause of morbidity and mortality. current recommendations regarding the use of beta-lactams in combination with macrolides published in clinical practice guidelines are variable and based on low-quality evidence that is frequently retrospective, observational, and heterogeneous in nature. while population-based studies have historically suggested improved clinical outcomes with the routine use of macrolide combination therapy in hospitalized patients with cap, emerging evidence from recent randomized controlled trials has challenged this practice. in this article, we discuss the historical rationale and current evidence for combination macrolide therapy in the management of cap. recent findings: recent randomized controlled trials have assessed the non-inferiority of beta-lactam monotherapy compared to beta-lactam/macrolide combination therapy in adult patients hospitalized with cap. beta-lactam monotherapy was associated with equivalent clinical outcomes in patients with mild to moderate cap. patients with severe cap managed with beta-lactam monotherapy have demonstrated worse clinical outcomes when compared to patients treated with combination therapy. in addition, previous beta-lactam exposure prior to hospitalization has not been shown to negatively impact outcomes in patients managed with beta-lactam monotherapy in the hospital. summary: current evidence supports the use of beta-lactam monotherapy in adult patients hospitalized with mild to moderate cap. while existing evidence supports the use of combination therapy in patients with severe pneumonia, further large-scale randomized controlled trials are urgently needed to clarify the role of combination therapy in this population. community-acquired pneumonia (cap) remains a significant cause of morbidity and mortality, resulting in an estimated . million adult hospitalizations annually in the united states [ •, ] . despite advances in sepsis recognition and management and being a core measure by the centers for medicare & medicaid services, the in-hospital mortality of patients admitted with cap remains high at . % with all-cause mortality at days, months, and year estimated to be %, %, and %, respectively [ •, ] . the broad spectrum of pathogens causing cap remains unchanged and includes typical bacteria (streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis), atypical bacteria (mycoplasma pneumoniae, chlamydia pneumoniae, legionella spp.), viruses (such as rhinovirus, influenza, parainfluenza, respiratory syncytial virus, coronaviruses, human metapneumovirus) and less frequently staphylococcus aureus, gram-negative bacilli, fungi, and mycobacteria [ ] . while diagnostic techniques to facilitate the accurate identification of causative agents have improved, including the rapid evolution of commercial multiplex pcr assays, an etiologic agent is identified in fewer than half of cap cases and frequently leaves clinicians to rely on empiric antimicrobials for management [ , •] . macrolide antimicrobials are commonly prescribed agents for the management of respiratory tract infections [ ] . as a class, macrolides are bacteriostatic and function by inhibiting bacterial protein synthesis through reversible binding to s ribosomal rna of the s ribosomal subunit, resulting in inhibition of rna-dependent protein synthesis [ ] . however, widespread resistance to macrolides due to point mutations in the s rrna binding site has significantly limited their utility [ , ] . s. pneumoniae resistance to macrolides has demonstrated regional variability with rates ranging from % in north america to over % in vietnam while m. pneumoniae demonstrates similar resistance ranging from % in the united states to over % in regions of japan [ ] [ ] [ ] [ ] . aside from antimicrobial properties, macrolides are also prescribed for their immune modulating effects, particularly in the setting of chronic inflammatory lung diseases. m a c r o l i d e s h a v e b e e n s h o w n t o s u p p r e s s p r oinflammatory cytokines [ ] , decrease polymorphonuclear cell recruitment [ ] , attenuate reactive oxygen species [ ] , and alter key transcription factors [ ] . while several small studies have shown that immune modulation from macrolides may also be beneficial in acute cap by reducing inflammatory mediators, supportive research linked to clinical outcomes remains limited and a clear benefit has not been established [ , ] . a recent effort to characterize causative cap pathogens through the use of modern diagnostic tests has been published in the center for disease control and prevention (cdc) etiology of pneumonia in the community (epic) study [ • ]. the cdc epic study was a prospective, multicenter, population-based active surveillance study of patients with radiographically confirmed pneumonia at five us hospitals between and . in addition to conventional diagnostic tests, extensive molecular and serologic testing was employed to characterize causative pathogens. in this cohort, human rhinovirus, influenza, and s. pneumoniae were the most common etiologic agents. atypical pathogens were infrequently identifiedwith the incidence of m. pneumoniae, c. pneumoniae, and l. pneumophila each representing less than . cases per , adults in the community. this study provides contemporary, comprehensive microbiologic cap data that is essential in informing guidelines and choosing empiric therapies wisely. the low incidence of atypical pathogens is of particular interest. published recommendations regarding the use of beta-lactams in combination with macrolides in current clinical practice guidelines are variable and based on relatively low quality evidence that is frequently retrospective, observational, and heterogeneous in nature. last updated in , the joint infectious disease society of america (idsa)/american thoracic society (ats) guidelines for cap recommend empiric combination therapy with a beta-lactam plus macrolide or monotherapy with a respiratory fluoroquinolone (e.g., moxifloxacin) for adult patients hospitalized with cap in a non-icu setting [ ] . combination therapy is recommended for all patients with severe cap (inpatient icu). in contrast, guidelines published by the british thoracic society (bts, ) and national institute for health and care excellence (nice, ) recommend beta-lactam monotherapy for lowseverity cap with consideration of combination therapy only in moderate to severe cap [ , ] . the body of evidence used to support current idsa/ats guideline recommendations that advocate for combination therapy with a beta-lactam plus macrolide in the management of hospitalized adult patients with cap originates from a series of large, retrospective cohort studies that showed improved clinical outcomes in patients treated with combination therapy. gleason et al. evaluated the relationship between initial antimicrobial therapy and clinical outcomes in a retrospective cohort of , medicare patients hospitalized with pneumonia [ ] . thirty-day mortality was lower when a macrolide was combined with a second-generation cephalosporin (hr, . ; % ci, . - . ) or third-generation cephalosporin (hr, . ; % ci, . - . ) compared to conventional beta-lactam monotherapy. houck et al. similarly evaluated a retrospective cohort of medicare patients hospitalized with pneumonia in western states during the years , , and [ ] . therapy with a macrolide in combination with a beta-lactam was associated with a significant reduction in mortality (aor, . ; % ci, . - . ) in but this effect was not observed during the other years of study. dudas et al. subsequently performed a prospective, observational cohort study at community hospitals to evaluate compliance with ats guidelines in patients with non-severe community-acquired pneumonia and compared outcomes between patients administered varying combinations of antimicrobials [ ] . compliance with ats guidelines was found to be % and the addition of a macrolide to a beta-lactam was associated with decreased mortality and reduced length of hospital stay. brown et al. further reinforced these findings by evaluating a retrospective cohort of , patients in a hospital claims database [ ] . in this cohort, macrolide combination therapy was associated with decreased mortality, length of hospital stay, and healthcare-associated costs overall. based on the findings of these large observational studies, many clinical practice guidelines recommend combination therapy with a beta-lactam plus macrolide or monotherapy with a respiratory fluoroquinolone as first-line therapy for hospitalized adult patients with cap. however, this recommendation for routine combination therapy for the management of cap has remained a contentious issue. despite the body of (predominantly retrospective, observational) evidence that demonstrates improved clinical outcomes, many clinicians regard this evidence (which grounds guideline recommendations) to be low quality and have called for further prospective, randomized-controlled trials to address this question. the routine use of macrolides in hospitalized adults to augment the spectrum of antimicrobial coverage in mild to moderate cap patients to include atypical pathogens has been questioned owing to the infrequent identification of these pathogens. m. pneumoniae and c. pneumoniae are also specifically known for producing relatively mild illness with minimal existing literature that targeted antibiotic therapy impacts patient mortality [ ] . additionally, any adjunctive benefit from the postulated anti-inflammatory effect of macrolides in the treatment of cap remains unproven. given the increasing resistance of s. pneumoniae and m. pneumoniae isolates to macrolide antibiotics, unnecessary exposure to potential harms associated with macrolide use (potential cardiotoxicity, clostridium difficile infection, etc.), and the economic costs of unnecessary combination therapy, many have called the routine use of combination beta-lactams plus macrolides into question [ ] [ ] [ ] [ ] [ ] . additional studies have attempted to bring clarity to this controversy. a systematic review and meta-analysis performed by the cochrane acute respiratory infections group explored if the addition of atypical coverage to an antimicrobial regimen would improve patient outcomes [ ] . in trials, including patients hospitalized with cap, there was no overall difference in mortality (rr . , % ci: . - . ) between the atypical and the typical coverage arms. a trend towards fewer clinical failures was observed in the atypical therapy arm, but this did not achieve statistical significance (rr . ; % ci: . - . ). of note, the cochrane review only included one study that specifically focused on the comparison of beta-lactam monotherapy to beta-lactam/macrolide combination therapy. asadi et al. also compared macrolides to other treatment regimens in adults hospitalized with cap in a systematic review and metaanalysis including studies and , patients [ ] . while overall macrolide use was associated with a statistically significant reduction in mortality when compared to nonmacrolide use, the survival advantage disappeared ( . vs . %; rr . ; % ci: . - . ) when analyses were restricted to rcts. in recent years, researchers have attempted to address the paucity of high-quality evidence with several prospective, randomized-controlled trials aimed at evaluating the utility of combination antimicrobial therapy in hospitalized adults with cap. garin et al. tested the non-inferiority of beta-lactam monotherapy compared with beta-lactam/macrolide combination therapy in moderately severe patients with cap in an openlabel, multicenter, non-inferiority randomized control trial [ ••] . the study was conducted in six acute care hospitals in switzerland from to . the primary endpoint in this trial was the proportion of patients not achieving clinical stability at day . after days of therapy, % in the monotherapy arm and % in the combination arm had not reached clinical stability ( % difference, p = . ), exceeding the predefined non-inferiority boundary. patients identified to have atypical pathogens or severe pneumonia (psi ≥ ) were less likely to reach clinical stability with monotherapy while patients infected with typical pathogens or mild to moderate cap severity (psi to ) had equivalent outcomes. secondary endpoints that included overall mortality, need for icu admission, complications, length of stay, and recurrence of pneumonia within days did not differ significantly between the two groups. postma et al. designed the cap-start trial, a multicenter cluster-randomized, crossover, non-inferiority trial conducted at hospitals in the netherlands to determine if empiric therapy for cap with beta-lactam monotherapy was noninferior to combination beta-lactam/macrolide therapy or a respiratory fluoroquinolone [ ••] . adult patients were included if they demonstrated radiographic evidence of pneumonia and compatible clinical findings. patients were excluded if they had cystic fibrosis, positive legionella antigen, recent hospitalization, or were from a long-term care facility. the unadjusted -day all-cause mortality was . % (betalactam monotherapy), . % (fq monotherapy), and . % (beta-lactam/macrolide combination). adjusted comparisons for -day all-cause mortality favored beta-lactam monotherapy with a . % lower risk of death when compared to macrolide combination therapy ( % ci: − . to . ). time to oral antibiotic de-escalation, length of hospital stay, and occurrence of complications during hospital stay were not significantly different between the groups. while criticisms of this study included a high rate of protocol deviation in the beta-lactam monotherapy group to include atypical coverage, subgroup analyses of strategy-adherent and antibiotic-adherent groups revealed no significant difference in outcomes. van werkhoven et al. further evaluated outcomes in a subset of patients from the cap-start trial that had previous beta-lactam exposure prior to hospitalization [ ••] . thirty-day mortality, hospital length of stay, and frequency of treatment escalations were compared between patients that were continued on beta-lactam monotherapy and patients that received beta-lactams and atypical coverage. despite the betalactam monotherapy group being older, having more medical comorbidities, and longer duration of symptoms prior to admission, the authors found no significant differences between groups with respect to -day mortality and hospital length of stay. while these recent trials performed by garin, postma, and van werkhoven provide evidence to support a strategy of beta-lactam monotherapy for patients presenting with mild to moderate cap, the role of combination beta-lactam/ macrolide therapy in critically ill patients with severe cap remains unclear. the use of macrolide combination therapy in severe cap is endorsed in various clinical guidelines (table ) . again, however, the data to support macrolide use is of relatively low quality and does not clearly explain whether improved outcomes in this population are derived from the antimicrobial or immune-modulatory properties of these agents. baddour et al. previously evaluated the impact of combination antibiotic therapy on adult patients with severe bacteremic pneumococcal pneumonia in a prospective, observational multicenter study [ ] . while overall -day mortality did not differ significantly in this cohort, combination therapy was associated with significantly reduced mortality among a subset of critically ill patients ( . vs . %, p < . ) regardless of nationality, level of icu support, class of antibiotics, and in vitro activity of the prescribed antibiotics. while this study was not limited to combination macrolide therapy, beta-lactam/macrolide therapy remained the most common regimen in the study population. multiple retrospective cohort studies have shown a similar effect in critically ill patients with severe pneumococcal pneumonia complicated by bacteremia [ ] . in addition, sligl et al. explored the impact of beta-lactam/macrolide combination therapy in critically ill patients in a systematic review and meta-analysis involving nearly , critically ill patients from observational studies and found that combination therapy including macrolides was associated with a significant reduction in mortality [ ] . a subgroup analysis of the cohort evaluated by garin et al. also supports this literature-demonstrating that patients with severe pneumonia were less likely to achieve clinical stability with beta-lactam monotherapy. despite the lack of randomized trials in severe cap, the available evidence is consistent with current recommendations published in the joint idsa/ats guidelines for management of cap in patients requiring icu admission. further research to characterize the potential immune-modulatory effects of macrolides in patients with severe cap is warranted, specifically because this population is more likely to have concomitant sepsis and deleterious systemic inflammation. the utility of macrolide combination therapy in the management of cap has been a source of significant controversy with a large body of conflicting evidence. considering the increasing resistance of typical bacterial isolates (specifically s. pneumoniae) to macrolides and the infrequent identification of atypical pathogens as causative agents of pneumonia, clinicians need to carefully consider the utility of macrolides in the treatment of cap. in addition, clinicians need to balance any potential incremental benefit from the anti-inflammatory effect that macrolides may possess against the urgent need for antimicrobial stewardship and responsible antibiotic use. while population-based epidemiologic studies have historically suggested a benefit of adjunctive macrolides in the management of cap, most of these studies are of relatively low quality. emerging evidence from several prospective studies has challenged this practice by failing to show a significant improvement in relevant clinical outcomes in patients hospitalized with mild to moderate cap. while existing evidence still supports the use of combination therapy in critically ill patients with severe cap, further randomized-controlled trials are urgently needed to clarify the role of combination therapy in this population. in particular, the potential immune modulatory effect of macrolides in sepsis remains an area of uncertainty. based on the current literature, we suggest a pragmatic approach pending further high quality evidence to guide therapy. for patients with mild-moderate cap requiring hospitalization, we suggest treatment with beta-lactam monotherapy (e.g., ceftriaxone) or a respiratory fluoroquinolone (e.g., moxifloxacin). for patients with severe pneumonia or those requiring icu-level care, we suggest beta-lactam/macrolide (e.g., ceftriaxone/azithromycin) combination therapy. empiric therapies, however, may differ based on local epidemiology and antimicrobial susceptibility patterns in each individual health region or institution. conflict of interest geoffrey shumilak and wendy sligl declare that they have no conflicts of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. prospective population-based cohort study evaluating incidence of cap in consecutive hospitalized patients at all adult hospitals in louisville, kentucky. calculation of annual population-based incidence of cap, mortality at , , and months following hospitalization national hospital ambulatory medical care survey: emergency department summary tables deaths: final for douglas and bennett's principles and practice of infectious diseases. chapter : acute pneumonia the canadian cap working group. summary of canadian guidelines for the initial management of community-acquired pneumonia: an evidence-based update by the canadian infectious disease society and the canadian thoracic society active population-based surveillance study of immunocompetent adults hospitalized with community-acquired pneumonia. in addition to conventional diagnostic testing, extensive molecular and serologic testing was employed to characterize causative pathogens and provide contemporary data on the causative agents of cap. despite extensive testing the value of macrolide-based regimens for community-acquired pneumonia structural basis for the interaction of antibiotics with the peptidyl-transferase centre in eubacteria the mechanism of action of macrolides, lincosamides and streptogramin b reveals the nascent peptide exit path in the ribosome antimicrobial resistance among streptococcus pneumoniae in the united states: have we begun to turn the corner on resistance to certain antimicrobial classes? antibiotic resistance threats in the united states high prevalence of antimicrobial resistance among clinical streptococcus pneumoniae isolates in asia (an ansorp study) macrolide-resistant mycoplasma pneumoniae, united states nationwide surveillance of macrolide-resistant mycoplasma pneumoniae infection in pediatric patients macrolide antibiotics as biological response modifiers effect of erythromycin on haemophilus influenzae endotoxininduced release of il- , il- and sicam- by cultured human bronchial epithelial cells membrane-stabilizing, antiinflammatory interactions of macrolides with human neutrophils molecular mechanisms of anti-inflammatory action of erythromycin in human bronchial epithelial cells: possible role in the signaling pathway that regulates nuclear factor-kappa b activation antiinflammatory effects of adjunctive macrolide treatment in adults hospitalized with influenza: a randomized controlled trial immunomodulatory effects of macrolides during community-acquired pneumonia: a literature review infectious diseases society of america/ american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults bts guidelines for the management of community acquired pneumonia in adults: update diagnosis and management of community and hospital acquired pneumonia in adults: summary of nice guidance associations between initial antimicrobial therapy and medical outcomes for hospitalized elderly patients with pneumonia empiric antibiotic therapy and mortality among medicare pneumonia inpatients in western states antimicrobial selection for hospitalized patients with presumed community-acquired pneumonia: a survey of nonteaching us community hospitals impact of initial antibiotic choice on clinical outcomes in community-acquired pneumonia: analysis of a hospital claims-made database antibiotics for communityacquired lower respiratory tract infections secondary to in children antibiotic treatment of moderate-severe community-acquired pneumonia: design and rationale of a multicenter cluster-randomized cross-over trial the controversy of combination vs monotherapy in the treatment of hospitalized community-acquired pneumonia does empiric therapy for atypical pathogens improve outcomes for patients with cap? azithromycin and the risk of cardiovascular death fluoroquinolone and macrolide exposure predict clostridium difficile infection with the highly fluorquinolone and macrolide-resistant epidemic c. difficile strain bi/nap / empiric antibiotic coverage of atypical pathogens for community-acquired pneumonia in hospitalized adults macrolide-based regimens and mortality in hospitalized patients with community-acquired pneumonia: a systematic review and meta-analysis evaluated the non-inferiority of beta-lactam monotherapy compared with beta-lactam/macrolide combination therapy in moderately severe cap in an open-label, multicenter, noninferiority randomized control trial. failed to meet primary outcome and demonstrate non-inferiority of beta-lactam monotherapy in moderately severe community-acquired pneumonia. subgroup analyses revealed equivalent outcomes in patients with mild to moderate cap (psi - ) but worse outcomes in patients managed with beta-lactam monotherapy and severe cap multi-center cluster-randomized, crossover, non-inferiority trial conducted at hospitals in the netherlands to determine if empiric therapy for cap with beta-lactam monotherapy was non-inferior to combination beta-lactam/macrolide therapy or a respiratory fluoroquinolone. -day all-cause mortality was lowest in the betalactam monotherapy arm. adjusted comparisons for -day all-cause mortality favored beta-lactam monotherapy with a . % lower risk of death when compared to macrolide combination therapy. time to oral antibiotic de-escalation evaluation of outcomes in a subset of patients from the cap-start trial that had previous beta-lactam exposure prior to hospitalization. compared thirty-day mortality, hospital length of stay, and frequency of treatment escalations between patients that were continued on beta-lactam monotherapy and patients that received beta-lactams and atypical coverage combination antibiotic therapy lowers mortality among severely ill patients with pneumococcal bacteremia macrolide therapy is associated with lower mortality in community-acquired bacteremic pneumonia macrolides and mortality in critically ill patients with community-acquired pneumonia: a systematic review and metaanalysis key: cord- -yd idp authors: zhang, chengfei; yan, yan; he, hongwang; wang, li; zhang, na; zhang, jie; huang, hongjun; wu, nannan; ren, hua; qian, min; liu, mingyao; du, bing title: ifn-stimulated p y( ) protects mice from viral infection by suppressing the camp/epac signaling pathway date: - - journal: j mol cell biol doi: . /jmcb/mjy sha: doc_id: cord_uid: yd idp among the most important sensors of extracellular danger signals, purinergic receptors have been demonstrated to play crucial roles in host defense against infection. however, the function of p receptors in viral infection has been little explored. here we demonstrated that p y( ) and its ligand adp play an important role in protecting hosts from viral infections. first, we demonstrate that p y( ), as a typical interferon-stimulated gene, is induced together with extracellular adp during viral infection. most importantly, extracellular adp restricts the replication of different kinds of viruses, including vesicular stomatitis virus, newcastle disease virus, herpes simplex virus , and murine leukemia virus. this kind of protection is dependent on p y( ) but not p y( ) or p y( ), which are also considered as receptors for adp. furthermore, cyclic adenosine monophosphate and epac are downregulated by extracellular adp through the p y( )-coupled gi alpha subunit. accordingly, inhibition or deletion of epac significantly eliminates adp/p y( )-mediated antiviral activities. taken together, our results show that p y( ) and adp play pivotal roles in the clearance of invaded virus and have the potential as antiviral targets. upon viral infection, the host can sense the virus through the recognition of pathogen-associated molecular patterns by pattern recognition receptors, especially toll-like receptors (tlrs) and rig-i-like receptors (rlrs) (kawai and akira, ; chen et al., ) . when activated by viral nucleic acids, tlrs and rlrs will activate the tbk -irf axis, leading to type i interferon (ifn) expression and secretion (garcia-sastre and biron, ; he et al., ) . then, the released ifns induce hundreds of interferon-stimulated genes (isgs) via the janus kinase-signal transducer and activator of transcription (jak-stat) signaling pathway to clear the invaded virus (platanias, ; garcia-sastre and biron, ; shu et al., ) . among them, the double-stranded rna-dependent protein kinase pkr, the myxovirus resistance gene mx, the ′- ′ oligoadenylate synthetases, and interferon induced transmembrane protein (ifitm ) have been shown to disrupt viral infection in various manners rafique et al., ; liu et al., ) . more than genes have been characterized as isgs. however, their roles and mechanisms in fighting against multiple distinct classes of viruses remain largely unexplored. purinergic signaling and purinergic receptors are important in both innate and adaptive immune responses. purinergic signaling was first reported in and, after several decades of study, many of the biologic effects of atp, utp, adp, udp, and adenosine signaling were discovered (eltzschig et al., ; idzko et al., ) . in tissue injury or pathogen infection, nucleotides can be released from stressed cells as a danger signal, alerting the cells by interaction with purinergic receptors in an autocrine or paracrine manner . nineteen different purinergic receptor subtypes have been identified, which can be classified into p and p receptors. four p receptor subtypes can be activated by adenosine. p receptors can be further divided into p y receptors (p yrs) and p x receptors (p xrs) based on their structures and distinct signaltransduction mechanisms. p yrs are g-protein-coupled receptors for atp, adp, utp, and udp, while p xrs are nucleotide-gated ion channels and can be activated by atp (ralevic and burnstock, ; junger, ; idzko et al., ) . many studies revealed crucial roles for p receptors during inflammatory and infectious diseases. for example, atp-triggered p x activation is crucial for the activation of nlrp inflammasome and the subsequent release of interleukin- β (di virgilio et al., ) . atp/utp receptor p y signaling has been identified as a 'find-me' signal to recruit motile phagocytes, thereby promoting clearance of apoptotic cells or bacteria (elliott et al., ; chen et al., ) . our previous study demonstrated that the adp-mediated p y /p y signal pathway protects the host defense against bacterial infection via erk signaling (zhang et al., ) . however, whether adp-(or its receptors-) mediated immune regulation is also involved in viral infection remains unclear. p y , p y , and p y are all receptors for adp. p y binds to gtp-binding protein subunit alpha q (gq), which can evoke calcium signaling. while p y and p y interact with gtpbinding protein subunit alpha i (gi), which can suppress the activity of adenylate cyclase (adcy) and the production of cyclic adenosine monophosphate (camp) (zhang et al., ; shinozaki et al., ) . in this study, we demonstrate that p y , as an isg, and its ligand, adp, is released from infected cells as a danger signal during viral infection, which restricts the replication of both dna and rna viruses. adp/p y -mediated protection against viral infection operates by suppressing the expression of exchange protein activated by camp (epac ), which is an alternative key intracellular sensor for camp. therefore, we demonstrated that adp/p y restricts viral infection through suppressing the camp/epac signaling pathway, which has potential therapeutic significance in the prevention and control of viral diseases. the expression of p y is increased by ifn through jak/stat pathway to identify p y family members potentially involved in antiviral immune responses, we treated mouse bone marrowderived macrophages (bmdms) with ifn-α, ifn-β, and ifn-γ to appraise the rna expression of all p y family members by quantitative real-time pcr (q-pcr). as shown in figure a , the rna expression of p y was upregulated obviously by all three ifns. the protein level of p y also upregulated by ifn-β ( figure b) . besides, when blocked ifn receptor (ifnar) with the anti-ifnar antibody, the upregulated expression of p y was decreased ( figure c ), suggesting that p y upregulation is dependent on signals downstream of ifn. the jak/stat pathway is crucial for ifn-induced cellular antiviral signaling. to determine whether ifn-induced p y upregulation via the jak/stat pathway, we pretreated mouse peritoneal macrophages (pems) with ruxolitinib (a jak inhibitor) and fludarabine (a stat inhibitor) and found that ifnβ-induced p y expression was blocked by both inhibitors ( figure d and e). inhibition of stat also reduced vsv-induced p y expression ( figure f ). in addition, knockout of tlr to block tlr -mediated activation of ifn also suppressed poly(i:c)-induced p y expression ( figure g ). taken together, these data demonstrated that p y has the typical characteristics of isgs. as an endogenous ligand to purinergic receptors, adp can be released from injured cells to activate p y , p y , and p y receptors. to investigate the potential role of adp as a danger signal in viral infection, the release of endogenous adp in viralinfected cells was measured. as shown in figure a and b, extracellular adp in vsv-infected cell supernatant was clearly increased in a time-dependent manner. besides, ndv-infected bmdms and hsv- -infected hek t cells also release large amounts of adp ( figure c and supplementary figure s a ), suggesting adp can be released as a danger signal during viral infection. to investigate the biological significance of adp, we pretreated the cells with adp before vsv infection. to our surprise, the rna replication of vsv in adp-treated raw . cells was reduced significantly in a time-( figure d ) and concentration-( figure e ) dependent manner. accordingly, the viability of vsv-infected raw . cells was increased dramatically by adp in a concentration-dependent manner while have little influence on uninfected cells ( figure f and supplementary figure s b ), indicating that extracellular adp protects cells from vsv infection. to confirm whether adp has broad-spectrum antiviral activities, raw . cells and bmdms were pretreated with adp before vsv, ndv, and hsv- infection. as shown in figure g -j and supplementary figure s c , the rna expression of vsv, ndv, hsv- and the titers of vsv were all strikingly decreased by adp. similar results were observed in hek t cells, where cell cytopathic effects were induced by vsv and hsv- , as determined using a crystal violet staining assay (supplementary figure s d and e). we further infected hek t and hela cells with vsv-gfp and mlv-gfp and found that the number of gfppositive cells was decreased in adp-treated cells, indicating that vsv-gfp and mlv-gfp were restricted by adp (supplementary figure s f-i). to rule out the possibility that adp degradation products mediate the suppression of viral replication, we treated cells with non-hydrolyzable adp analog adp-β-s. as shown in supplementary figure s j , adp-β-s has a similar role as adp in restricting vsv replication. taken together, our data suggested that extracellular adp has broad-spectrum antiviral activities, which have great potential in protecting hosts from a viral infection. to explore the key receptors involved in adp-mediated antiviral activities, we detected the expression of p y , p y , and p y after vsv infection. as shown in figure a , only the expression of p y was increased significantly, whereas the expression of p y and p y was little changed in vsv-infected raw . cells. similar data were observed in poly(i:c)-treated bmdms ( figure b ). we then used mrs and mrs to inhibit p y and p y , respectively, to verify whether p y and p y contributed to adp-mediated antiviral activities. we found that adp-mediated antiviral activities were little influenced by mrs but significantly decreased by mrs as shown in figure c -f, and both of mrs and mrs have little influence on cell viability (supplementary figure s a and b). consistently, when infected wild-type, p y -, p y -, and p y deficient bmdms with vsv, the vsv rna replication was increased only in p y -deficient bmdms, not in p y -or p y -knockout bmdms ( figure g ). meanwhile, adp-reduced vsv rna replication was compensated in p y -deficient pems ( figure h) . furthermore, the protein expression of vesicular stomatitis virus glycoprotein (vsv-g) was significantly increased in p y -deficient pems and overexpression of p y in hela cells significantly repressed vsv replication ( figure i ; supplementary figure ifn signal pathway increases p y expression. (a) mouse bmdms were treated with ifn-α, ifn-β, or ifn-γ ( ng/ml) for h, and mrna expression of p y family was detected by q-pcr. (b) mouse pems were treated with ifn-β ( ng/ml) for or h, and the expression of p y protein was detected by western blotting. (c) mouse bmdms were pretreated or not with anti-ifn-α/β receptor igg ( μg/ml) for h before being treated with ifn-β ( ng/ml) for h, and the expression of p y mrna was detected by q-pcr. (d) mouse pems were pretreated or not with ruxolitinib ( μg/ml) for h and then stimulated with ifn-β ( ng/ml) for h. the expression of isg and p y mrna was detected by q-pcr. (e) mouse pems were pretreated with or without fludarabine ( μg/ml) for min and then stimulated with ifn-β ( ng/ml) for h. the expression of isg and p y mrna was detected by q-pcr. (f) mouse pems were pretreated or not with fludarabine ( μg/ml) for min and then infected with vsv (moi = ) for h. the expression of p y mrna was detected by q-pcr. (g) tlr +/+ and tlr −/− mouse pems were treated with poly(i:c) ( μg/ml) for h. the expression of ifn-β, isg , and p y mrna was detected by q-pcr. data are shown as mean ± sd. **p < . ; ***p < . . figure s c and d). taken together, our data suggested that p y deficiency facilitates vsv infection and adp-mediated antiviral activities primarily through the p y receptor. to further confirm the in vivo antiviral activities of adp/p y , we challenged wild-type and p y -deficient mice with a lethal and then infected with vsv (moi = ) for h. vsv titers in supernatants were measured by plaque assay. (i and j) mouse bmdms were treated with adp ( μm) for h and then infected with ndv (moi = . ) and hsv- (moi = . ) for h virus rna replicates were detected by q-pcr. data are shown as mean ± sd. *p < . ; **p < . ; ***p < . . dose of vsv. as shown in figure a , the survival of adp-treated wild-type mice was increased obviously, but there was little change in that of p y -deficient mice. p y -deficient mice were more susceptible to vsv infection. on the other hand, if we intraperitoneally injected mice with adp to activate p y , vsv distributions in the liver, spleen, and lung were all significantly decreased ( figure b ). similar data were observed during hsv- infection ( figure c ). besides, more vsv rna replicates were found in p y -deficient mice liver ( figure d ). as a neurotropic virus, vsv can invade the nervous system and is transmitted directly via the transcutaneous or transmucosal route. thus, to mimic the natural infection of vsv, we treated mice with adp through nasal dripping and then intranasally infected with vsv. as shown in figure e and f, vsv rna replication and vsv-g in olfactory bulbs were both significantly reduced by adp. taken together, these data demonstrated that adp/p y plays an important role in protecting mice from vsv infection, suggesting that adp/p y has potential as a novel antiviral drug target. adp/p y restricts viral replication by inhibiting camp signaling type i ifn plays pivotal roles in fighting against the invaded virus, so we tested whether it was involved in adp/p y mediated antiviral activities. however, when we treated bmdms with adp, the rna expression of ifn-α and ifn-β was little changed (supplementary figure s a and b) . p y signaling can suppress the activity of adcy and the production of camp. to confirm that, raw . cells were pretreated or not with mrs before being treated with adp. as shown in figure a , adp significantly suppressed the production of camp, while p y antagonist mrs rescued this phenomenon. adp ( mg/kg) for h and then intraperitoneally injected with vsv ( × pfu/g). mice were executed for days and mouse survival was recorded for days. (b) six-week-old mice were intraperitoneally treated with adp ( mg/kg) for h and then intraperitoneally injected with vsv ( × pfu/g) for h. vsv rna replicates in organs were detected by q-pcr. (c) six-week-old mice were intraperitoneally treated with adp ( mg/kg) for h and then intraperitoneally injected with hsv- ( × pfu/g) for h. hsv- rna replicates in livers were detected by q-pcr. (d) eight-week-old p y +/+ or p y −/− mice were intraperitoneally injected with vsv ( × pfu/g) for h and vsv rna replicates in livers were detected by q-pcr. (e) six-week-old mice were treated with adp ( mg/kg) through nasal dropping for h and then intranasally infected with vsv ( × pfu/g) for h. vsv rna replicates in olfactory tissues were detected by q-pcr. (f) six-week-old mice were treated as in e, and vsv invaded in olfactory tissues were detected by immunofluorescence. scale bar, μm. data are shown as mean ± sd. **p < . ; ***p < . . however, to our surprise, vsv infection caused an obvious increase in cellular camp levels ( figure b) . besides, p y -deficient pems showed higher cellular camp levels than wild-type pems during vsv infection ( figure c ). so, we examined the role of camp in adp/p y -mediated antiviral activities. we first found that adcy activator forskolin blocked adp-mediated the cells were then infected with vsv (moi = . ) for h. vsv rna replicates were detected by q-pcr and vsv titers in supernatants were measured by plaque assay. (i) raw . cells were pretreated or not with kh ( μm) for h before being treated with adp ( μm) for h. the cells were then infected with hsv- (moi = . ) for h, and hsv- rna replicates were detected by q-pcr. data are shown as mean ± sd. *p < . ; **p < . ; ***p < . ; ns, not significant. antiviral activity completely ( figure d and e). furthermore, camp treatment promoted viral replication obviously and also rescued the adp-reduced cell cytopathic effects and vsv replication ( figure f and g; supplementary figure s c and d). consistent with this, kh (a selective inhibitor of adcy) treatment significantly inhibited cell cytopathic effects and vsv replication (supplementary figure s e and f). adp-mediated antiviral activities were also significantly blocked by kh ( figure h and i). the cell viability assays revealed that these phenomena were not caused by the toxicity of forskolin, camp, and kh (supplementary figure s g-i) . taken together, these results suggested that camp plays a key role in adp/p y -mediated antiviral activities. adp/p y restricts viral replication in an epac -dependent manner when intracellular camp is increased by upstream signaling, epac and protein kinase a (pka) could be activated as classical sensors for camp. thus, we treated hela cells with esi- (epac / inhibitor), hjc (epac selective inhibitor), and h (pka inhibitor) at a safety concentration (supplementary figure s a -c). as shown in supplementary figure s d , the replication of vsv is dramatically suppressed by both esi- and h , but not hjc , suggesting that epac and pka may be involved in adp/p y -mediated restriction to vsv. however, when we inhibited pka with h , the replication of vsv ( figure a ) and hsv- ( figure b ) in hela cells are still suppressed by adp, whereas adp-reduced vsv, hsv- , and ndv replication was significantly blocked by esi- , but not hjc ( figure c -f and supplementary figure s e) . besides, the adpreduced vsv-g expression was also blocked by esi- ( figure g) . these data show that epac is crucial in adp/p y mediated antiviral activities. in addition, we generated an epac knockout cell line using the crispr/cas system ( figure h ). as shown in figure i and j, when epac is deficient, adp-mediated suppression of vsv rna replication and vsv-g expression were eliminated. taken together, these results demonstrated that adp/p y -mediated antiviral activities are dependent on epac . inhibiting epac with esi- in raw . cells significantly suppressed vsv and hsv- replication, and epac knockout also inhibited hsv- replication, demonstrating that either inhibition or deletion of epac has broad-spectrum antiviral activities (supplementary figure s f-h) . to further confirm the key role of epac in viral infection, we detected the expression of epac in viral-infected cells. as shown in figure a , when infected raw . cells with vsv, ndv, and hsv- , rna expression of epac was increased significantly. deficiency in p y maintained higher expression of epac in pems after infection with vsv ( figure b ). in addition, the expression of epac protein also upregulated after virus infection as shown in figure c and supplementary figure s a , b. however, to our surprise, the increased expression of epac induced by vsv was reduced by adp in a dose-dependent manner (supplementary figure s c and d) . to exclude that the downregulation of epac may be due to the attenuation of vsv replication by adp, cell lines were only treated with adp. as shown in figure d -f and supplementary figure s e , the endogenous rna expression of epac was directly inhibited by adp. the protein expression of epac was also inhibited by adp ( figure g and h). taken together, these data demonstrated that the virusincreased epac expression is suppressed by adp, which restricts the viral infection in host cells. p y and p y , as membrane receptors for adp, are both gi-coupled receptors that inhibit the activity of adcy and reduce the production of camp (vitiello et al., ) . p y is highly expressed in platelets and plays a central role in platelet activation, which has become a drug target for atherothrombotic diseases, whereas p y is mainly expressed in the bone marrow, spleen, liver, and brain (savi et al., ; vitiello et al., ) . previous studies have shown that p y and p y are both involved in the development of pain behavior during nerve injury and the expression of pro-inflammatory cytokines (kobayashi et al., ; liu et al., ) . however, the role of p y and p y receptors in viral infection remains unclear. here we demonstrate that p y is upregulated by type i ifn (p y is little changed) and its endogenous ligand, adp, is released as danger signals from virusinfected cells. consequently, extracellular adp-activated p y protects the host against viral infection in a camp-dependent manner. thus, our study reveals an important role of adp/p y in fighting against viral infection, which demonstrates the potential of purinergic receptors as novel antiviral targets. upon viral infection, the host initiates a series of signaling cascades to induce the production of type i ifn, which results in the induction of isgs to exclude invaded virus (ooi et al., ; zhan et al., ) . previous studies reported that isgs could further promote the production of type i ifn to eliminate the virus, such as isg and ifit (lu et al., ; liu et al., ) . therefore, we tested whether adp/p y influences the expression of type i ifn. our results show that adp enhanced ifn expression by only a little. because adp clearly inhibited virus replication, we hypothesized that there must be another way for adp/p y to restrict viral infection. thus, the classic gi-coupled signaling pathway came to mind. interestingly, we found that adp-inhibited vsv replication and cell cytopathic effects were rescued by both adcy activator forskolin and camp. moreover, similar to adp, the adcy inhibitor kh also inhibited vsv replication, suggesting that camp plays a key role in adp/p y mediated antiviral activities. as the first identified second messenger, camp plays a crucial role in microbial pathogenesis (mcdonough and rodriguez, ) . pka and epac are the two classical sensors for intracellular camp, which are essential for many biological processes (gloerich and bos, ) . pka plays important roles in metabolism, cell cycle, cell migration, differentiation, and apoptosis (yan et al., ) . previous studies have reported that pka can inhibit ifn induction of the jak/stat pathway (david et al., ) . recently, a new study reported that pka catalytic (pkac) figure adp-mediated antiviral activities are dependent on epac . (a) hela cells were treated or not with h ( μm) for h before being treated with adp ( μm) for h and then infected with vsv (moi = . ) for h. vsv rna replicates were detected by q-pcr. (b) hela cells were treated or not with h ( μm) for h before being treated with adp ( μm) for h and then infected with hsv- (moi = . ) for h. hsv- rna replicates were detected by q-pcr. (c) hela cells were pretreated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with vsv (moi = . ) for h and vsv rna replicates were detected by q-pcr. (d and e) hela cells were pretreated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with hsv- (moi = . ) or ndv (moi = . ) for h and viral rna replicates were detected by q-pcr. (f) raw . cells were treated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with hsv- (moi = . ) for h, and hsv- rna replicates were detected by q-pcr. (g) hela cells were pretreated or not with esi- ( μm) for h before being exposed to adp ( μm) for h. the cells were then infected with vsv (moi = . ) for h and vsv-g protein levels were detected by western blotting. (h) epac +/+ and epac −/− hela cells were exposed to vsv (moi = . ) for h, and the expression of epac was detected by western blotting. (i) epac +/+ and epac −/− hela cells were treated with adp ( μm) for h and then infected with vsv (moi = . ) for h. vsv rna replicates were detected by q-pcr. (j) epac +/+ and epac −/− hela cells were treated with adp ( μm) for h and infected with vsv (moi = . ) for h. vsv-g protein levels were detected by western blotting. data are shown as mean ± sd. *p < . ; **p < . ; ***p < . ; ns, not significant. subunits α and β could negatively regulate rna virus-triggered signaling and ultimately attenuate the innate antiviral response (yan et al., ) . epac has two isoforms, epac and epac . the camp/epac signal pathway is important for controlling various cellular functions, including cell adhesion, exocytosis, and microtubule dynamics. previous studies have reported that epac is a potential target for the treatment of fatal rickettsioses and that the silencing of epac gene expression renders cells resistant to mers-cov infection (gong et al., ; tao et al., ) . in our study, we found that both pka and epac inhibitors suppress viral replication. however, only epac inhibition could block adp/p y -mediated antiviral activities. also, we found that the epac inhibitor could not block adp/p y -mediated antiviral activities. these findings suggest that adp/p y -mediated antiviral activities are mainly dependent on epac . knockout of epac in hela cells showed similar results and further proved this point. taken together, our results showed that, upon viral infection, the virus can trigger an upregulation of intracellular camp levels to activate epac and thereby facilitating virus replication. however, the virus also triggers adp released from these cells, which will activate p y and suppress the camp/ epac signal pathway to restrain virus replication. this phenomenon revealed a negative feedback regulation in animals during viral infection, and that activation of p y and inhibition of the camp/epac signal pathway are potential antiviral strategies. p y , p y , and p y knockout mice were described previously (zhang et al., ) . tlr knockout mice were a gift from professor yuping lai (east china normal university) and were described previously (wu et al., ) . c bl/ wild-type mice were purchased from the shanghai laboratory animal company. all of the mice were maintained in specific pathogen-free (spf) facilities at the animal center of east china normal university. all animal experiments were done following institutional guidelines. reagents adp, adp-β-s, mrs , forskolin, camp, kh , esi- , h , anti-β-actin antibody, and anti-vsv-g antibody were purchased from sigma-aldrich. ruxolitinib was from targetmol. fludarabine was obtained from calbiochem. polyinosinic-polycytidylic acid [poly(i:c)] was purchased from invivogen. adp-glo™ kinase assay kit was purchased from promega. mrs and anti-gpr (p y ) antibodies were purchased from abcam. hjc was purchased from selleck. the anti-epac antibody was from bioss. primescript rt master mix and sybr premix ex taq were from takara. ifn-α, ifn-β, and ifn-γ were purchased from sino biological inc. the camp elisa kit, mouse ifn-α/β receptor block/neutralize polyclonal goat igg (af ), and normal goat igg control were from r&d systems. p y plasmid was obtained from biogot technology, co., ltd. the bmdms were prepared as follows: bone marrow cells were collected from tibias and femurs by flushing with dmem, and the cell suspension was filtered through a -μm cell strainer to remove any cell clumps. bone marrow cells were then cultured in dmem containing % fbs, % penicillin/streptomycin, and % l culture supernatants. the pems were prepared as follows: mice were intraperitoneally injected with ml of % sterile thioglycollate medium and, days later, mice were sacrificed and the peritoneal cavities washed with ml pbs. the cell suspension was filtered through a -μm cell strainer to remove any cell clumps. pems were then cultured in dmem containing % fbs and % penicillin/streptomycin. raw . , hek t, and hela cells were obtained from the american type culture collection and cultured in dmem containing % fbs and % penicillin/streptomycin. after stimulation, total rna was extracted with rnaiso plus (takara) from the cells. cdna was synthesized from extracted total rna using the primescript rt master mix perfect real time kit (takara) according to the manufacturer's protocol. quantitative real-time pcr was performed using a sybr-green premix (takara). the sequence-specific primers are listed in supplementary table s . adp release assay raw . cells, mouse bmdms, and hek t cells were plated in -well plates ( × cells per well) overnight and then infected with viruses for various periods. adp release into the cell culture supernatants was detected using adp-glo™ kinase assay kit according to the manufacturer's protocol. raw . cells were plated in -well plates ( × cells per well) overnight. the cells were treated with adp for h and then infected with vsv for h. mts ( μl, promega) was added to each well. after incubated for h at °c, the absorption was measured at nm. cell viability of the untreated group was normalized to %. hela and hek t cells were seeded into -well plates ( . × cells per well) overnight. the cells were treated with adp for h and then infected with vsv-gfp or mlv-gfp for h. cells containing virus were determined after gating on gfp-positive cells. hek t cells were plated in -well plates ( × cells per well) overnight. twelve hours after vsv (moi = . ) or hsv- (moi = . ) infection, cells were fixed with % paraformaldehyde in pbs for min at room temperature. after incubation with crystal violet for min, the cells were then washed with pbs five times before the photographs were taken. cells were grown in -well plates. after stimulation, cells were lysed with radio immunoprecipitation assay (ripa) buffer supplemented with protease and phosphatase inhibitors. whole-cell lysates were separated by % sds-page, transferred to nitrocellulose membranes, and blocked with % bsa. immunoblots were hybridized with the respective primary antibodies. protein bands were visualized with the appropriate fluorescent secondary antibodies and the odyssey laser digital imaging system (gene company). cells were infected with viruses for the indicated time and moi. for mouse survival assay, -week-old p y +/+ or p y −/− mice were intraperitoneally treated with adp ( mg/kg) or not treated for h and then intraperitoneally injected with vsv ( × pfu/g). executed these for days and mouse survival was recorded for days. for in vivo studies of viral replication, to -week-old mice were intraperitoneally treated with adp ( mg/kg) or not treated for h and then intraperitoneally injected with vsv ( × pfu/g) or hsv- ( × pfu/g) for the indicated time and viral rna replicates in organs were detected by q-pcr. vero cells were plated in -well plates ( × cells per well) overnight. the supernatants from vsv-infected cells were serially diluted and infected vero cells for h. then the supernatant was removed and the cells were covered with dmem containing % low-melting point agarose. plaques were counted after h. for immunofluorescence assay, -week-old mice were anesthetized with ketamine/rompun and treated with mg/kg adp through nasal dropping for h before being intranasally infected with vsv ( × pfu/g). after h of infection, mice were sacrificed, and the olfactories were fixed with % paraformaldehyde in pbs overnight. slices ( -μm) from olfactories were permeabilized with . % triton x- and blocked with % bsa. tissues were then incubated with anti-vsv-g antibody overnight and alexa-fluor- -conjugated secondary antibody for h. finally, the tissues were stained with dapi and visualized with a fluorescence microscope. for camp detection, raw . cells or pems were plated in -well plates ( × or × cells per well, respectively) overnight. after stimulation, the cells were lysed with μl cell lysis buffer and the amounts of camp were measured by elisa (r&d systems) according to the manufacturer's instructions. to generate an epac -knockout cell line, a crispr/cas system was used. the epac -knockout target sequence used was ′-aaatgcactccggaccacg- ′. a lenticrisprv plasmid containing the epac -knockout target sequence was transduced into hela cells. three days later, puromycin-resistant single clones were selected, and the epac -knockout clones were identified by western blotting with the anti-epac antibody. all data were analyzed using the graphpad prism software and shown as mean ± sd. the student t-test (two-tailed) was used to analyze the significance of data and p-value < . was considered statistically significant. supplementary material is available at journal of molecular cell biology online. induction of siglec-g by rna viruses inhibits the innate immune response by promoting rig-i degradation purinergic signaling: a fundamental mechanism in neutrophil activation purinergic p y receptor modulates stress-induced hematopoietic stem/progenitor cell senescence activation of protein kinase a inhibits interferon induction of the jak/stat pathway in u cells the p x receptor in infection and inflammation nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance purinergic signaling during inflammation impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action. microbiol type interferons and the virushost relationship: a lesson in detente epac: defining a new mechanism for camp action exchange protein directly activated by camp plays a critical role in bacterial invasion during fatal rickettsioses errα negatively regulates type i interferon induction by inhibiting tbk -irf interaction nucleotide signalling during inflammation immune cell regulation by autocrine purinergic signalling the role of pattern-recognition receptors in innate immunity: update on toll-like receptors multiple p y subtypes in spinal microglia are involved in neuropathic pain after peripheral nerve injury interferon-inducible cholesterol- -hydroxylase broadly inhibits viral entry by production of -hydroxycholesterol ifn-induced tpr protein ifit potentiates antiviral signaling by bridging mavs and tbk p y and p y receptors involved in adpβs induced the release of il- β, il- and tnf-α from cultured dorsal horn microglia isg enhances the innate antiviral response by inhibition of irf- degradation the myriad roles of cyclic amp in microbial pathogens: from signal to sword novel antiviral host factor, tnk , regulates ifn signaling through serine phosphorylation of stat mechanisms of type-i-and type-ii-interferon-mediated signalling positional effect of phosphorylation sites and in the cytoplasmic domain of the e protein of hepatitis c virus a genotype: interferon resistance analysis via sequence alignment receptors for purines and pyrimidines the active metabolite of clopidogrel disrupts p y receptor oligomers and partitions them out of lipid rafts transformation of astrocytes to a neuroprotective phenotype by microglia via p y receptor downregulation adap is an interferon stimulated gene that restricts rna virus entry blocking of exchange proteins directly activated by camp leads to reduced replication of middle east respiratory syndrome coronavirus immunoregulation through extracellular nucleotides hyperglycaemia inhibits reg a expression to exacerbate tlr -mediated skin inflammation in diabetes pkacs attenuate innate antiviral response by phosphorylating visa and priming it for march -mediated degradation phosphatase pp negatively regulates type i ifn production and antiviral innate immunity by dephosphorylating and deactivating tbk two disparate ligand-binding sites in the human p y receptor extracellular adp facilitates monocyte recruitment in bacterial infection via erk signaling this work was supported by the national key r&d program of china ( yfa to b.d.), the national natural science conflict of interest: none declared. key: cord- - um jmhk authors: imperiale, michael j.; casadevall, arturo title: a new approach to evaluating the risk–benefit equation for dual-use and gain-of-function research of concern date: - - journal: front bioeng biotechnol doi: . /fbioe. . sha: doc_id: cord_uid: um jmhk in the twenty-first century, biology faces a problem that has previously vexed other disciplines such as physics, namely the prospect that its knowledge domain could be used to generate biological agents with altered properties that enhanced their weapon potential. biological weapons bring the additional dimension that these could be self-replicating, easy to manufacture and synthesized with commonly available expertise. this resulted in increasing concern about the type of research done and communicated, despite the fact that such research often has direct societal benefits, bringing the dual-use dilemma to biology. the conundrum of dual use research of concern was crystallized by the so-called “gain-of-function” type of experiments in which avian influenza viruses were endowed with new properties in the laboratory such as increased virulence and the capacity for mammalian transmission. after more than a decade of intensive discussion and controversy involving biological experiments with dual-use potential, there is no consensus on the issue except for the need to carry out such experiments in the safest conditions possible. in this essay, we review the topic with the hindsight of several years and suggest that instead of prescribing prohibitions and experimental limitations the focus should be on the importance of scientific questions at hand. we posit that the importance of a scientific question for medical and scientific progress provides a benchmark to determine the acceptable level of risk in biological experimentation. revolution in the late twentieth century that the potential of biology, and in particular microbiology, in biological warfare and terrorism came into focus. these fears came true with a single act of terrorism involving the mailing of anthrax spores in the u.s. in , which caused several deaths and considerable disruption to government facilities such as congressional offices and the postal system (bush and perez, ) . this act of bioterrorism catalyzed a series of events that led to greater awareness of the potential of using biological knowledge in nefarious ways, new regulations in biological research in the u.s. such as the select agents and toxins list, and heighted concerns about some types of microbiology research. one of the immediate effects of the post- environment was increased scrutiny of published findings, and several papers were particularly noteworthy for eliciting discussion and concern. in , researchers in australia showed that insertion of the gene for il- into the ectromelia virus genome defeated vaccine immunity (jackson et al., ) , a report that drew immediate concerns because of its possible implication for similar effects with variola major virus. similarly, the finding that complement inhibition potentiated variola virus virulence suggested a mechanism for enhancing its pathogenicity (rosengard et al., ) . an analysis of the u.s. milk distribution system revealed vulnerable nodes where the introduction of botulinum toxin could lead to mass casualties (wein and liu, ) , leading to an outcry about journals publishing research that could be exploited by terrorists. advances in molecular biology led to the chemical synthesis of poliovirus (cello et al., ) and the reconstruction of the pandemic influenza virus (tumpey et al., ) , which greatly heightened concern about the re-introduction of extinct, controlled, and new viruses to naïve human populations. in this environment, the u.s. government created the national science advisory board for biosecurity (nsabb) in to provide guidance on issues relating to biological risk and security. both authors of this article were inaugural members of the nsabb and served on that board for years. when the nsabb first began their deliberations, one of the major early topics was that of dual-use research in the biological sciences. this issue had previously vexed the physics community in the twentieth century as advances in physics and associated technologies had led to radar, nuclear weapons, ballistic missiles, and other military hardware. however, there was little or no precedent for similar concerns in the biological research community, especially since biological weapons were outlawed by the biological weapons convention in . moreover, the term dual use, which had meant civilian and military use in the physics arena, was redefined in its application to biology by an nrc report in to mean technologies and information that had both beneficial and maleficent use (nr council, ) . nevertheless, among the early accomplishments of the nsabb was formulating a definition for dual use research of concern (durc), which limited the scope of worrisome work to a small subset of biological research. the nsabb also generated a set of recommendations for communicating durc in publications and other venues. the work of the nsabb proceeded quietly and in relative obscurity until when the u.s. government learned about two submitted manuscripts reporting that highly pathogenic avian influenza virus (hpaiv) could be made mammalian transmissible by laboratory passage in ferrets, and sought an opinion from the board as to whether publication should be permitted. before considering the gof controversy involving influenza virus, it is worthwhile reviewing what is meant by "gof" and the limitations of the lexicon in the controversy. at the most basic level, a gof experiment, as the name implies, is one that gives a biological entity any new property. gof experiments can be highly beneficial and can generate pest resistant crops, microbes expressing proteins for medical therapy such as recombinant human insulin, and new cancer therapies by enhancing lymphocyte function. the controversial aspects of gof experiments in microbiology arise when microbes are modified to have new properties that can enhance their virulence and transmissibility because such experiments raise worries about new diseases, outbreaks, pandemics, biosafety, and biosecurity. in the experience of the authors, the nsabb probably did not anticipate what a problematic dual-use paper would look like until they had the opportunity to consider the two papers that reported the gain of mammalian transmissibility for hpaiv (herfst et al., ; imai et al., ) . what made the two papers highly controversial was that they reported that only a few amino acid changes were sufficient to confer mammalian transmissibility to the h n hpaiv, and there was concern that this information could be used to generate a virus capable of pandemic potential with high mortality by anyone with basic molecular biology and virology skills. the nsabb initially recommended redacting the sequence information, but this was considered not feasible given the legal framework in the u.s., and the two papers (herfst et al., ; imai et al., ) were eventually published in after some modifications to the text. the arguments, counterarguments, and events surrounding that episode are described in various publications (berns et al., a,b; fouchier et al., a) , often by the participants themselves, and will not be repeated here. the major outcome of the great gof controversy of is that it defined and crystallized some of the issues of dual-use research in biology by providing clear examples of experiments that were of great scientific value while also raising biosecurity and biosafety concerns. although both papers were eventually published with full information, the debate and controversy did not solve the central questions of what work should be performed moving forward and how it should be reported. in fact, the controversy erupted again in the summer of following the publication of additional papers describing other gof experiments (e.g., watanabe et al., ) as well as a series of biosafety lapses at u.s. government laboratories, which led the government to impose a pause on these types of experiments with certain viruses until the nsabb could formulate new recommendations and guidelines. the pause was lifted in mid-december, , alongside policy guidance about how gof funding decisions should be made going forward. one of the most vexing questions of the problem of dual-use research is publication of scientific findings. in the u.s., the research community is guided by national security decision directive (nsdd) , formulated in by the reagan admini stration, which states that all fundamental research results could be published freely. in other words, there was either open publication or classification, and nothing in between. viewed from the context of nsdd- the papers involved in the gof controversy were either to be published in their entirety or classified, with the latter alternative being impractical and unfeasible since the work had been carried out in an unclassified environment. with this binary policy, redacting papers because they might contain information useful for nefarious purposes is not an option unless export control regulations are invoked [for a full discussion of the legalities involved, see nas em durc report (national academies of sciences engineering and medicine, )]. consequently, when faced with gof papers containing information that could conceivably be used to enhance the pathogenicity or transmissibility of a virus, editors and journals have almost always opted for full publication, usually requiring more details from the authors about biosafety and biosecurity methods, and often publishing an accompanying editorial emphasizing the scientifically useful aspects of the research [for examples, see dermody et al. ( dermody et al. ( , a ]. such decisions have sometimes elicited strong criticism from members of the virology community (dermody et al., b; wain-hobson, a) . a more recent situation illustrates the difficult issues faced by authors, editors, and journals when publishing papers that contain durc. in , the journal of infectious disease faced the quandary of receiving a paper reporting a new botulinum toxin that was resistant to neutralization by the then-available antisera. in response, the journal took the remarkable decision of publishing the paper without the toxin sequence and vowed to keep the data confidential until an antidote was available (casadevall et al., ; dover et al., ; relman, ; hooper and hirsch, ) . a major factor in withholding the sequence data was the realization that this toxin could be easily produced using standard techniques if the toxin sequence was available and that the availability of such a toxin might pose a major public health risk. this decision was highly controversial since without the sequence information other investigators could not verify the findings reported in the publication or begin to develop medical countermeasures. in fact, subsequent work revealed that the new toxin was indeed neutralized by available sera, thus diminishing the initial concerns (enserink, ) . on one hand, withholding the sequence information from public scrutiny was the responsible action given the potential threat, while on the other hand, such an action delayed verification of the findings and the generation of countermeasures had the threat been real. clearly, manuscripts containing durc pose vexing problems for journals that often must make such decisions alone. at american society for microbiology journals, there are protocols in place for reviewing such manuscripts and the journals have available many individuals with microbiological and safety expertise who can provide input (casadevall et al., ) . other situations are likely to pose different challenges, but it is clear that journals are often poorly equipped to handle the difficult problems involved in publishing durc content. in this regard, we have argued for the need of a national board that can help journals make publication decisions (casadevall et al., ) , although to date no such body exists. another major challenge to the control of durc information is the emergence of preprint servers in biology that allow the posting of research findings before peer review and the proliferation of predatory open access journals that will publish essentially any paper for a fee. consequently, there now exist alternative systems for publication even if standard journals decline to publish a particular article over durc concerns. bypassing traditional publication methods could also allow authors to avoid government scrutiny. it is not an exaggeration that the situation today regarding durc and gof experiments remains highly unsatisfactory. experimental work involving gof experiments on pathogens with pandemic potential is highly regulated with proponents of such work arguing that restrictions on experimental design are unwarranted (fouchier et al., b) while opponents of such experiments argue that such work cannot be justified (wain-hobson, a,b). proponents of gof experiments have noted that data generated in such experiments is useful in epidemiological surveillance (schultz-cherry et al., ), whereas opponents have argued that such work cannot be morally justified because of its inherent risk (lipsitch and galvani, ) . in the u.s., government-mandated pauses on certain types of experiments remain in effect for federally funded research but such prohibitions do not extend to other countries, or to work funded by other sources. although the nsabb continues to analyze and debate the issues involved with durc and gof experiments in the u.s., there is no comparable body on the international scene, where much of this type of research is done. there is no consensus in the scientific community on whether the value of some experiments justifies the risks involved. the central problem in finding a consensus is that the value of the research cannot be measured in real time, while assessments of risk involve assumptions that can lead to widely divergent estimates. for example, some risk-benefit calculations have suggested that a high consequence accident is likely to occur in the near future (lipsitch and bloom, ; lipsitch and inglesby, ) , while others have estimated such probabilities to be near zero (fouchier, ) . however, there is some evidence that the incessant debate, which shows no sign of resolution, is taking its toll on the virology community as shown by surveys suggesting that scientists in training may avoid these areas of research (pfeiffer, ) . this is of particular concern given that recent years have seen new viral threats in the forms of the west africa ebola outbreak, the emergence of zika virus in the americas, and middle east respiratory syndrome coronavirus in the arabian peninsula, highlighting the importance of virology in human preparedness against pandemic threats casadevall, a, ) . one of the most important developments in the past few decades has been a change in the zeitgeist of the field that concerns itself with durc research and gof experiments. in the early years of the twenty-first century, as the u.s. reeled from the / terrorist attacks, which were shortly followed by the anthrax spore attacks, the focus was primarily on biosecurity. at that time, the concern was that terrorists and state actors would use the tools of the molecular biology revolution to create new and more devastating biological weapons. however, as the years passed and no new attacks developed, combined with the attribution of the anthrax spores in mail attacks to a lone insider in a u.s. government laboratory (bhattacharjee and enserink, ) , this security threat appeared to recede. at the same time, a series of unfortunate biosafety lapses in government laboratories heightened concerns about biosafety. hence, today people (at least those outside the security community) appear to be more worried about an unintended release of a pathogen with pandemic potential than a deliberate biological attack. this change in emphasis from biosecurity to biosafety has developed slowly and could easily change if there is another deliberate biological attack or if becomes apparent that adversaries are developing biological weapons. a major problem contributing to the unsatisfactory nature of the current situation is that the nsabb definition of durc does not work well in day-to-day practice. the nsabb defined durc as "life sciences research that, based on current understanding, can be reasonably anticipated to provide knowledge, information, products, or technologies that could be directly misapplied to pose a significant threat with broad potential consequences to public health and safety, agricultural crops and other plants, animals, the environment, materiel, or national security. " although the crafting of this definition was a major achievement at the time in being able to limit the scope of durc to a relatively small portion of biomedical research, thus leaving most biological research to proceed unfettered, subsequent experience showed that the clause "reasonably anticipated" is so subjective in nature that it requires a risk assessment beyond the capabilities of most scientists, reviewers, and editors (casadevall et al., ) . in essence, the definition was helpful in keeping whole swaths of biological research outside the durc category, such as cancer and immunology research, but it difficult to apply in determining what is included in the durc definition. also contributing to an unsatisfactory situation are concerns whether such regulations as the select agents and toxins list help or hinder societal security. on one hand, placing great restrictions on the accessibility to a number of agents and toxins does increase security by making them more difficult to obtain, with the caveat that these are naturally occurring agents that could be obtained from nature by a determined actor. on the other hand, there is the concern that focusing on lists and a relatively short list of agents means that the overwhelming majority of microbial threats are not on the durc radar screen. for example, zika virus emerged as a pathogen of pandemic potential with little or no anticipation from experts. hence, making lists and focusing attention on those agents in the list has the paradoxical potential to reduce biosecurity since regulations are tightened for listed agents, possibly hindering research, while other dangerous agents are neglected (casadevall and relman, ) . a similar argument applies to the current durc oversight policy of the u.s. government, which is focused on a specific list of agents. this process, which is akin to searching under the lamppost, carries great danger because it ignores what could be significant threats. in this regard, it is noteworthy that fungal pathogens are seldom considered as biological threats despite the fact that members of this kingdom have significant weapon potential (casadevall and pirofski, ; casadevall, ) . against this backdrop of dissatisfaction is the fact that science continues to progress very rapidly, introducing new technologies such crispr/cas , gene drives, and more efficient synthetic biology, each of which brings with it new possibilities for research that improves the human condition as well as new tools for nefarious purposes. furthermore, as the technologies improve they are increasingly accessible to more individuals and countries for whom this type of research was previously beyond reach. hence, the problem of durc is likely to become significantly more urgent in the near future. in a world where new infectious agents that can rapidly spread among vulnerable populations are described on a regular basis, humanity needs a healthy research establishment focused on microbial threats. it is estimated that there are a minimum of , mammalian viruses (anthony et al., ) : some fraction of these are probably zoonotic events waiting to happen. as ian malcolm, the fictional character created by michael crichton in the novel jurassic park, stated, "life finds a way. " information and knowledge are the best defenses against these threats. in addition to terror from nature, a new crisis will almost certainly occur in the future arising from a deliberate attack, a new provocative paper, or another biosafety lapse. gof experiments and durc research are essential for preparedness and the question is not whether this research should be pursued but rather how to do it safely and mitigate risk. to date, each of the controversies has been reactive, with proponents and opponents of such experiments responding to a specific finding or study. after more than a decade of discussion on what constitutes durc, benefits and risks of gof experiments, regulations, pauses, and moratoriums, it is increasingly apparent that current approaches are inadequate for the challenges at hand. given these limitations, we have proposed a new framework for durc research that focuses on answering specific questions (imperiale and casadevall, b) . hence, instead of prohibiting certain types of experimentation, we suggest that the way forward is to focus on specific scientific questions and the problems that need answers. for example, if there is a need to determine whether a particular feral virus pathogen has the capacity for mammalian infection and transmission, then gof experiments performed in safe and controlled conditions can be justified. on the other hand, endowing hiv with new transmission properties is not a medically important gof experiment (national academies of sciences engineering and medicine, ). all human activities that involve probing the unknown, ranging from space exploration to tissue culture procedures, carry some degree of risk, and it is nature of humanity to accept risk to attain progress. opponents and proponents of this type of experimentation need to maintain open channels for continued discussion because the dialectic of ideas is likely to result in better decisions. institutional bodies such as the nsabb need to be supported for they constitute important venues for such discussion and recommendations that mitigate risk. in fact, it is important to create similar institutions that can work at the international level since u.s.-based research is a small portion of all microbiological work done worldwide. most importantly, we should remain optimistic that the research community can do the research necessary to obtain information critical to protect our species while minimizing the risks of such work. a strategy to estimate unknown viral diversity in mammals public health and biosecurity. adaptations of avian flu virus are a cause for concern policy: adaptations of avian flu virus are a cause for concern anthrax investigation. fbi discusses microbial forensics -but key questions remain unanswered the anthrax attacks years later don't forget the fungi when considering global catastrophic biorisks on the need for a national board to assess dual use research of concern dual-use research of concern (durc) review at american society for microbiology journals redaction of sensitive data in the publication of dual use research of concern the weapon potential of human pathogenic fungi microbial threat lists: obstacles in the quest for biosecurity? chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template a new determinant of h n influenza virus pathogenesis in mammals sequence changes associated with respiratory transmission of h n influenza virus in mammals the decision to publish an avian h n influenza virus gain-offunction experiment molecular characterization of a novel botulinum neurotoxin type h gene smallpox and the indians in the american colonies biosecurity. as new botulism threat implodes, more questions studies on influenza virus transmission between ferrets: the public health risks revisited pause on avian flu transmission research the pause on avian h n influenza virus transmission research should be ended airborne transmission of influenza a/h n virus between ferrets novel clostridium botulinum toxin and dual use research of concern issues experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets the importance of virology at a time of great need and great jeopardy a new synthesis for dual use research of concern zika virus focuses the gain-of-function debate expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox rethinking biosafety in research on potential pandemic pathogens ethical alternatives to experiments with novel potential pandemic pathogens moratorium on research intended to create novel potential pandemic pathogens dual use research of concern in the life sciences: current issues and controversies biotechnology research in an age of terrorism is the debate and "pause" on experiments that alter pathogens with pandemic potential influencing future plans of graduate students and postdoctoral fellows? inconvenient truths" in the pursuit of scientific knowledge and public health variola virus immune evasion design: expression of a highly efficient inhibitor of human complement influenza gain-of-function experiments: their role in vaccine virus recommendation and pandemic preparedness characterization of the reconstructed spanish influenza pandemic virus an avian h n gain-of-function experiment of great concern the irrationality of gof avian influenza virus research circulating avian influenza viruses closely related to the virus have pandemic potential analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk biological warfare at the siege of caffa both authors contributed to the writing of this manuscript. references conflict of interest statement: 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. provided the original author(s) and the copyright owner 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- -x hq b authors: versluys, anne birgitta; boelens, jaap jan title: morbidity and mortality associated with respiratory virus infections in allogeneic hematopoietic cell transplant: too little defense or harmful immunity? date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: x hq b the impact on morbidity and mortality of community acquired respiratory virus (carv) infections in patients undergoing allogeneic hematopoietic cell transplant (hct) is widely studied. here we give an overview of the current literature on the incidence and chance of progression to severe disease in this highly immune compromised population. we discuss the issue whether it is predominantly direct viral damage that causes clinical deterioration, or that it is in fact the allogeneic immuneresponse to the virus that is most important. this is an important question as it will guide therapeutic decision making. it asks for further collaborative studies focusing on sensitive surveillance with pcr techniques and relating clinical data with parameters of immune reconstitution. community acquired respiratory virus infections (carv) include a variety of viruses such as rhinovirus, coronavirus, respiratory syncytial virus (rsv), influenza virus, para influenza virus, and metapneumo virus. carv infections range from asymptomatic carriership to significant respiratory disease. prevalence of carv largely depends on season, detection mode, age of patient and immune status (shah et al., ; hirsch et al., ; green, ) . influenza and rsv have significant seasonal variation, whereas para influenza or rhinovirus cause disease year round (green, ) . highly sensitive diagnostic techniques like polymerase chain reaction (pcr) for the detection of viral dna and rna reveal a high prevalence of carv in the normal population. in healthy children attending day care prevalence is as high as % (moe et al., ) . in children admitted to hospital for respiratory disease - % are tested pcr positive, almost twice as high as in adults admitted to hospital for respiratory disease (ching et al., ) . in the immune compromised pediatric population the prevalence of carv is around - % (fazekas et al., ) with mostly mild symptoms at time of detection. there are many reports on carv prior to, or early after, hematopoietic cell transplantation (hct). recently a large multicenter retrospective analysis in , pediatric hct recipients showed an incidence of . % symptomatic carv infections within year after transplant (fisher et al., ) . surveillance studies in the same population on nasopharyngeal aspirates (npa) routinely performed prior to transplant showed an incidence of % (versluys et al., ) . most studies discuss the risk of progression to viral pneumonia, for various types of carv. only few groups looked at long term outcome. results are conflicting, reported risk of progression is - % (bredius et al., ; hirsch et al., ; chemaly et al., b; fisher et al., ; green, ) . hct is a curative treatment for several malignant and non-malignant childhood diseases. its success is limited by infections, alloimmunity and toxic events. respiratory viruses contribute to post-transplantation morbidity and mortality in different ways. here we provide an overview of recent literature on carv in the hct setting, focusing on the risk of progressive viral lung disease, the role of viruses in the lung microbiome and the potential viral trigger for allo-immunity after hct. reported incidence of rsv in hct-recipients varies between and % (shah et al., ; robinson et al., ; fisher et al., ) , depending largely on season, patients' age and detection methods. progression to lrti occurs in - % (shah et al., ; kim et al., ; fisher et al., ) , with risk factors for progression related to age, donor source, use of steroids, immune status, and concomitant infections (renaud et al., ; chemaly et al., b; kim et al., ; shah et al., ) . mortality rates are around % (chemaly et al., a; robinson et al., ; fisher et al., ) it is important to notice the in most pediatric studies, rsv-positive patients were treated with the antiviral drug ribavirin (molinos-quintana et al., ) , with anti-rsv monoclonal antibodies (palivizumab) or with nonspecific intravenous immunoglobulins (ivig) (el-bietar et al., ) , or with a combination (chávez-bueno et al., ) . aerosolized ribavirin and ivig is recommended for adult hct recipients with rsv lrti (dignan et al., ; waghmare et al., ) . para influenza virus (piv) in hct recipients is systematically reviewed by shah et al. ( a) . the incidence of piv in hct recipients is % (range . - %), % progressing to lrti. significant predictors of lrti progression were infection within days after hct, lymphocytopenia/neutropenia at the onset of infection, use of corticosteroids, younger age, and respiratory co-infections. reported overall mortality is % ( - %), in piv-lrti %. there is currently no licensed therapy for piv pneumonia. influenza is diagnosed in approximately - % of hct recipients, and in up to % of patients with respiratory symptoms in the flu-season. progression to viral pneumonia occurs in - % of patients, and is associated with mortality in - % (fisher et al., ; green, ) . these numbers are strongly influenced by seasonal outbreaks and the subtype of the influenza virus. risk factors for progressive influenza disease are lymphocytpenia/neutropenia and steroid use (kmeid et al., ) . in contrast to many other respiratory viruses, influenza can be treated with neuraminidase inhibitors (waghmare et al., ; green, ) . human rhino virus (hrv)is the most common cause of respiratory virus infections in both immunocompetent and immunocompromised individuals. reported incidence in hct recipients is - % (versluys et al., ; shah et al., ; campbell et al., ; fisher et al., ) . for a long time there was uncertainty about the ability of hrv to cause lower respiratory tract disease. recent studies however, suggest that hrv may be a clinically significant pathogen with the potential to cause serious pulmonary disease in hct recipients (campbell et al., ; seo et al., seo et al., , versluys et al., ) with risk of progression to lrti of - % (shah et al., ; campbell et al., ; fisher et al., ) and hrv related mortality of - % (shah et al., ; campbell et al., ; fisher et al., ) . there is increasing interest in the pathogenecity of human metapneumo virus (hmpv). in a systematic review (shah et al., b) shah et al. summarized all published data on hmpv in patients with hematologic malignancies or undergoing hct. about one third of described cases were children. they report an overall incidence of % (range - %), with a risk for progression to lrti of % (range - %) and a mortality rate of % ( - %) in the total hmpv positive group, and % in the hmpv-lrti group. other respiratory viruses like bocavirus (bov) and coronavirus (cov) in hct setting, are scarcely studied. prevalence of either bov or cov is % in the adult hct population with respiratory symptoms. a significant proportion of the cov infected patients required hospitalization, and some progressed to lrti. in contrast, bov detection was rare and almost always related to co-pathogens (pinana et al., ) . a surveillance study on bov in children found % of children with rti to be pcr positive, as well as % of the healthy controls; thus showing a high prevalence of the virus without necessarily causing disease. progression to lower infection however was associated with higher bov load and viremia, suggesting a pathogenic role in a subgroup of patients (christensen et al., ) . figure shows the incidence of carv in mainly adult hct population, and the impact of carv on lrti and mortality. profound immunosuppression in patients undergoing hct obviously leads to a greater risk of infection, with prolonged shedding of the virus, a higher chance of transmission of disease and a greater risk of progression to severe lower respiratory tract disease. as antiviral therapy is only effective in the minority of the respiratory viruses, and vaccines are not widely available, prevention is very important. the working group of the fourth european conference on infections in leukemia (ecil- ) reviewed the literature on carv in leukemic patients and patients after hct (hirsch et al., ) . for prevention they recommend infection control measures like good personal hygiene, avoiding contact with individuals with a respiratory tract infection and restricting young children from visiting patients. administration if ivig preparations in patients with hypogammaglobulinemia (igg < g/l), and the use of intravenous monoclonal antibody specific for the rsf-f protein (palivizumab) during rsv outbreaks may be considered. they do not advocate routine screening for carv. deferral of chemotherapy or conditioning should be considered, figure | incidence of common respiratory viral infections (crv) and associated progression to lower respiratory tract infections (lrti) and mortality in hct recipients. updated from shah et al. ( ) , based on (versluys et al., ; shah et al., ; chemaly et al., b; campbell et al., ; robinson et al., ; fisher et al., ; green, ) . as well as treatment for rsv and hpiv only in the hct setting. in a joint working group in the uk (dignan et al., ) has reviewed the available literature and made recommendations for the diagnosis and management of respiratory viral infections in patients with hematological malignancies or those undergoing hematopoietic stem cell transplantation. as far as prevention is concerned they come to the same conclusions as ecil- , and add the recommendation for influenza vaccinations in household contacts and medical staff, and post-exposure prophylaxis with oseltamivir in hct patients who have been in contact with influenza. in a recent large prospective study, including adults and children undergoing allogeneic hct, clinical outcomes associated with respiratory viruses (rv) detected prior to hct were analyzed (campbell et al., ) . multiplex pcr testing for rv was done on nasal washes or nasopharyngeal swabs. in % of patients a rv was detected, % of them being asymptomatic. in the pediatric subgroup, defined as aged < years, the prevalence of rv was higher ( %), with a larger proportion being without symptoms ( %). the rv positive patients were significantly younger and had higher risk underlying disease with lower lymphocyte count. overall mortality at day was significantly higher in rv-patients than in non-rv patients ( . vs. . %; hr . ; % ci . - . ; p = . ). in % of the deceased patients the cause of death was thought to be directly related to the pre-hct rv, no data are given about the cause of death in the other patients. patients with rhinovirus performed worse compared to the other rv. this may be partly explained by the fact that most patients with rsv or influenza where treated with antiviral therapy or had their transplant delayed. data on longer follow up are lacking (campbell et al., ) . hutspardol et al. retrospectively studied treatment related mortality (trm) and long-term pulmonary complications in children who had respiratory symptoms and a rv detected within days after allogeneic hct. the overall frequency of documented rv infections was . %, half of the patients presented with signs of a lrti and mortality rate at day was %. cause of death was pneumonitis/ards in all, with symptoms occurring on day - after hct. during follow up ( . years, range . - . ) no chronic pulmonary complications nor allo-immune lung syndrome was observed (hutspardol et al., ) . with regard to long term pulmonary function chien et al. studied , adult hct recipients by performing routine pulmonary function tests years after hct. airflow obstruction, defined as an annualized decline in fev of more than %, occurred in % of patients and had impact on overall mortality. higher age at transplant, gvhd category, pulmonary function pre-transplant and the occurrence of a respiratory virus infection within the first days after hct were significant risk factors for airflow obstruction (chien et al., ) . erard et al. further studied the association of rv and airflow decline, and found that this was particularly true in patients after lrti caused by parainfluenza virus or respiratory syncytial virus (erard et al., ) . in a retrospective study among , pediatric hct recipients in us centers . % acquired symptomatic rv within the first year after hct (fisher et al., ) . in line with others, rhinovirus was the most common virus, followed by rsv and piv. rv was detected after a median of ( - ) days after hct. most children had urti only, in patients with hmpv there was significantly more lrti. during months follow up % required mechanical ventilation and % had significant pulmonary sequelae like bronchiolitis obliterans, subacute pulmonary problems and other not specified pulmonary complications. all cause mortality among rv positive patients was %, compared to % in the non-carv group. recent steroid exposure and rv detection within days after hct were poor prognostic factors for morbidity and death. at least % of death were not attributable to carv infection. the timing of the events is also remarkable, as % of deaths occurred more than days after diagnosing carv infection, which is at least months after hct for most. the widespread use of pcr diagnostics has led to an increase in the detection of carv in patients undergoing hct. many of these patients become symptomatic and a significant proportion develops lrti. there is a clear increased risk for mortality in carv positive patients. hence, prevention and development of anti-viral drugs are of great importance. however, one could debate about the reason for severe morbidity and mortality in carv positive patients. how do you diagnose progressive viral infection? the carv will not be cleared for months because of the immunocompromised state of the host after hct, so finding positive pcrs is not convincing enough. timing of (progression of) symptoms in relation to immune reconstitution might be helpful in answering the question if it is progression of viral damage or if the donor derived immunity actually is targeting the lung. in last decade more and more evidence has emerged that "triggered" alloreactivity may play a crucial role in toxicity and mortality. this holds true for hct, but is also recognized in solid organ transplantation. in the context of lung transplantation several studies have examined the role of rv in the development of chronic lung allograft dysfunction (clad), a form of chronic rejection of the lung (kumar et al., (kumar et al., , fisher et al., ) . many, but not all, reported an association between rv and clad. pooled analyses of studies on rv and clad (vu et al., ) did not confirm the association, mainly due to the heterogeneity of studies and limitations in design, diagnostic techniques and definitions. fisher et al. tried to overcome these limitations by studying a more homogenous cohort of lung transplant recipients, using modern molecular assays to detect rv and applying consensus definitions of clad (fisher et al., ) . in patients, ( %) developed clad at a median of weeks (interquartile range (iqr): - weeks). in patients ( . %) a respiratory viral episode was seen, after a median of weeks post lung transplantation (iqr: - weeks). in multivariate analysis rv was associated with clad (hr . , % ci . - . ; p = . ). this association was stronger the more proximate the rv occurred after lung transplantation. our group studied the role of respiratory viruses (rv) in immune mediated lung disease after hct, analogous to this phenomenon as described after lung transplantation (versluys et al., (versluys et al., , . the host-vs.-graft chronic allograft rejection in lung transplantation is in many ways comparable to the graft-vs.-host inflammation in hematopoietic cell transplantation. in a cohort of children undergoing allogeneic hct routine npa and bal sampling for the presence of rv was done prior to transplant. rv was found in % ( % in bal/npa, % in npa-only). rhinovirus was the most frequently detected rv ( %). allo-immune lung syndrome (allo-ls), defined as bronchiolitis obliterans syndrome (bos) or idiopathic pneumonia syndrome (ips), occurred in %, after a median of . weeks ( - weeks). rv-positivity in bal was a predictor for allo-ls (hr . , % ci . - . ; p = . ). no other predictors were found. the hypothesis is that rv causes epithelial damage and triggers an allogeneic immune response leading to severe lung disease. so the lung disease does not occur primarily from progressive viral infection during the period of low immunity, but from allo-immune mediated damage weeks after hct. this inflammatory aspect of disease might explain the reported benefit of steroid use on the risk of mechanical ventilation among hct recipients with influenza (choi et al., ) , and the controversy on risks of steroid use in case of carv after hct (waghmare et al., ) . more and more is known about the role of microbiota in human health. so far research has largely focused on the gut microbiome, also in the context of gvhd. but the microbial ecosystem at other body sites, including the respiratory tract is attracting growing attention. host and environmental factors influencing the respiratory microbiota include genetics, microbial exposure (birth mode, feeding type, day care), vaccination, infections and antibiotics (man et al., ) . viral infection interacts with the microbiome by disrupting the airway epithelial barrier facilitating bacterial adhesion, liberating host derived nutrients and decreasing muco-ciliary clearance. in addition respiratory viruses can modulate innate and adaptive immune responses promoting bacterial colonization (man et al., ) . moreover, it is becoming clear that the virome should be seen as a part of the microbiome, that affects the function of the host immune system (cadwell, ) . the role of a disturbed respiratory microbiome/virome in lung disease is postulated for asthma and chronic obstructive pulmonary disease (copd) (zou et al., ) . impact of carv on outcomes after hct is an intriguing topic where pathogenesis is not completely understood. is the poor immune system associated with progressive infection? does alloimmunity play a crucial role in lung toxicity? most studies describe data on symptomatic patients where carv is detected at time of symptoms. only few studies report on pre-hct sampling, although we know a large proportion of our patients is carv positive with only mild symptoms. time of (worsening) of symptoms, warranting viral diagnostics and thus detecting the carv, is often weeks after hct. are these nosocomial acquired viruses, or were these virus already present and giving symptoms after a certain period of time? are the viruses acquired after discharge? but then immunity is usually restored to a certain degree. from various infectious diseases, like rsv bronchiolitis (fonseca et al., ) , immune reconstitution inflammatory syndrome (iris) in hiv patients (with cryptococcal meningitis, cmv retinitis or bcg-itis) (walker et al., ) or iris in non-hiv immunesuppressed patients with immune recovery (followed by worsening of treated tuberculosis, idiopatic pneumonia or hepatitis) (sueki et al., ) , we know the harmful effect of immune response on the patient. the debate about inappropriate immune response on infectious triggers is especially intriguing in the allogeneic setting. the definition criteria for alloimmune mediated lung syndromes (panoskaltsis-mortari et al., ; jagasia et al., ) describe the clinical, radiologic and functional aspects of lung pathology, with exclusion of other evident causes of this phenotype, like heart failure and infection, including respiratory virus infection. one can argue if this holds true for respiratory viruses detected by pcr. the detection modes have become much more sensitive over time, so the impact of positive findings on the disease criteria should be reevaluated. in the hct population with its high prevalence of rv, these viruses will have a long persistence making them detectable for weeks after initial infection, with uncertain meaning for their role in pathology. an interesting paper in this matter was recently published by seo et al. ( ) . in patients with ips, they went back to bal samples at time of diagnosis, and applied more sensitive diagnostics for microbial pathogens. in % of patients an occult pathogen was found, % being a respiratory virus. all patients were treated with steroids because of ips. overall mortality was higher in the group of patients with an occult pathogen, than in the group without. the authors conclude that these patients had had to be excluded as ips patients, that they had infectious pneumonia and that steroid treatment had adversely influenced their outcome. however, as we are not informed about rv status pre-hct, this could also be persisting rv after hct, triggering immune-mediated lung disease (ips). in that situation steroids are beneficial in the treatment at the moment of clinical deterioration. in conclusion, despite the growing awareness of carv infections in hct patients, well-designed studies are lacking that systematically evaluate diagnostic and therapeutic strategies of carv. only then we will be able to better understand the direct viral impact and the indirect alloimmune pathology, both largely influencing clinical outcome of patients. detailed longitudinal studies, combining data from microbioma/virioma surveillance with data on immunerecovery after hct and clinical outcome are needed to better understand pathogeneic mechanisms involved in lung disease and carv after hct. this insight should largely influence the therapeutic decision of delaying transplant, treating rv and most important increasing or decreasing immune suppression after transplant. prospective study of respiratory viral infections in pediatric hemopoietic stem cell transplantation patients the virome in host health and disease clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant intravenous palivizumab and ribavirin combination for respiratory syncytial virus disease in high-risk pediatric patients respiratory syncytial virus infections in children with cancer management of respiratory viral infections in hematopoietic cell transplant recipients and patients with hematologic malignancies airflow obstruction after myeloablative allogeneic hematopoietic stem cell transplantation respiratory virus detection and co-infection in children and adults in a large australian hospital in - differences in clinical outcomes after influenza a/h n and seasonal influenza among hematopoietic cell transplant recipients human bocavirus in children: mono-detection, high viral load and viraemia are associated with respiratory tract infection the, bcsh/bsbmt/uk clinical virology network guideline: diagnosis and management of common respiratory viral infections in patients undergoing treatment for haematological malignancies or stem cell transplantation rsv infection without ribavirin treatment in pediatric hematopoietic stem cell transplantation airflow decline after myeloablative allogeneic hematopoietic cell transplantation: the role of community respiratory viruses prevalence and clinical course of viral upper respiratory tract infections in immunocompromised pediatric patients with malignancies or after hematopoietic stem cell transplantation a multicenter consortium to define the epidemiology and outcomes of inpatient respiratory viral infections in pediatric hematopoietic stem cell transplant recipients symptomatic respiratory virus infection and chronic lung allograft dysfunction factors affecting the immunity to respiratory syncytial virus: from epigenetics to microbiome viral pneumonia in patients with hematopoietic cell transplantation and hematologic malignancies ecil- ): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus significant transplantation-related mortality from respiratory virus infections within the first one hundred days in children after hematopoietic stem cell transplantation national institutes of health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: i. the diagnosis and staging working group report respiratory syncytial virus in hematopoietic cell transplant recipients: factors determining progression to lower respiratory tract disease outcomes of influenza infections in hematopoietic cell transplant recipients: application of an immunodeficiency scoring index clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant a prospective molecular surveillance study evaluating the clinical impact of community-acquired respiratory viruses in lung transplant recipients the microbiota of the respiratory tract: gatekeeper to respiratory health respiratory virus detection and clinical diagnosis in children attending day care intravenous ribavirin for respiratory syncytial viral infections in pediatric hematopoietic sct recipients an official american thoracic society research statement: noninfectious lung injury after hematopoietic stem cell transplantation: idiopathic pneumonia syndrome epidemiologic and clinical characteristics of coronavirus and bocavirus respiratory infections after allogeneic stem cell transplantation: a prospective single-center study mortality rates of human metapneumovirus and respiratory syncytial virus lower respiratory tract infections in hematopoietic cell transplantation recipients respiratory syncytial virus infections in pediatric transplant recipients: a canadian paediatric surveillance program study idiopathic pneumonia syndrome after hematopoietic cell transplantation: evidence of occult infectious etiologies human rhinovirus detection in the lower respiratory tract of hematopoietic cell transplant recipients: association with mortality immunodeficiency scoring index to predict poor outcomes in hematopoietic cell transplant recipients with rsv infections management of respiratory viral infections in hematopoietic cell transplant recipients parainfluenza virus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review human metapneumovirus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review immune reconstitution inflammatory syndrome in non-hiv immunosuppressed patients strong association between respiratory viral infection early after hematopoietic stem cell transplantation and the development of life-threatening acute and chronic alloimmune lung syndromes infection with a respiratory virus before hematopoietic cell transplantation is associated with alloimmune-mediated lung syndromes respiratory viruses in lung transplant recipients: a critical review and pooled analysis of clinical studies how i treat respiratory viral infections in the setting of intensive chemotherapy or hematopoietic cell transplantation immune reconstitution inflammatory syndrome in hiv-infected patients research on the human virome: where are we and what is next all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the reviewer pv declared a past co-authorship with one of the authors jb to the handling editor.the remaining author declares 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 © versluys and boelens. 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- -oecpqf j authors: nan title: aspho abstracts date: - - journal: pediatr blood cancer doi: . /pbc. sha: doc_id: cord_uid: oecpqf j nan myelodysplastic syndrome (mds) and frequently arise in the context of inherited bone marrow failure (bmf) syndromes, such as shwachman diamond syndrome (sds). monosomy /del( q) is associated with high grade mds and propensity to progress to acute myelogenous leukemia, a major cause of morbidity and mortality for patients with inherited bmf. development of non-transplant strategies to treat bone marrow failure without simultaneously stimulating outgrowth of malignant clones remains a major challenge. objectives: the aim of this study is to investigate the molecular consequences of del( q) in the context of bmf with the goal of developing more effective treatments. design/method: to study the biological and molecular consequences of monosomy/del( q) in bmf, induced pluripotent stem cells were generated from sds patients (sds-ipsc) . a deletion of the mds-associated region of the long arm of chromosome was then introduced using a previously published modified cre-lox approach. results: the sds ipsc phenocopied bone marrow failure with slow proliferation and impaired hematopoietic differentiation. we next explored whether deletion of q conferred a relative fitness advantage within the context of bone marrow failure. proliferation of the sds-del( q) ipscs was reduced below that of both the isogenic sds ipscs and normal controls without an increase in cell death. sds-del( q) demonstrated reduced hematopoietic differentiation compared with isogenic sds cells. these data demonstrate that deletion of q fails to confer a relative growth advantage relative to isogenic sds ipscs and results in further impairment of hematopoiesis. to gain insight into the mechanisms of del q-associated clonal evolution in sds, we performed rna sequencing (rnaseq) of sds+/-del( q) ipsc. expression of tgf pathways and their downstream targets were reduced in sds-del( q) ipscs compared to isogenic sds ipsc. single cell rnaseq analysis of primary sds bone marrow cells confirmed that the tgf pathway is hyperactivated in sds. western blot analysis showed increased phospho-smad levels in sds ipscs compared to sds-del( q) and normal controls, while total levels of smad were unchanged. pharmacological targeting of tfg with small molecule inhibitors resulted in selective improvement of sds hematopoietic colony formation and myeloid differentiation without stimulating outgrowth of the isogenic sds-del( q) cells or normal controls. these results demonstrate that del( q) reverses the tgf pathway hyperactivation of sds. furthermore, inhibition of tgf selectively rescues hematopoiesis in sds but not in isogenic del q cells, suggesting a potential strategy to treat bone marrow failure without stimulating del q clonal outgrowth. background: standard therapy of medulloblastoma consists of treatment with alkylating agents and radiation after surgical resection. although a statistically significant increase in survival is reported with this regimen, / rd recur and become resistant this class of agents ultimately leading to mortality. large numbers of somatic mutations were observed in recurrent medulloblastoma (rm) after alkylating agent and radiation treatment. high mutation rates in tumors can have twofold effect; ) a large number of non-synonymous mutations that have no role as drivers can still cause functional tumor antigens increasing the neoantigen burden and immunogenicity. moreover, ) such tumors can gain mutations in canonical or non-canonical dna repair pathways leading to a gain in the number of mutations as seen in case of glioblastoma, this can lead to even higher accelerated mutational rate. evidences suggest that high mutational load can cause higher neoantigen burden thereby making the tumor more susceptible to immune checkpoint inhibition. we propose that post therapy recurrent medulloblastoma gain mutational signature and immunophenotype of malignancies demonstrating clinical response to immune checkpoint therapy. objectives: ) rm has molecular signatures identical to tumors with high immunogenicity and clinical response to immune check point inhibition. ) rm has the immune inflammatory phenotype; harboring high percentage of tumor infiltrating lymphocytes (tils), macrophages and monocytes. design/method: to test our hypothesis, we downloaded the raw bam files of previously published data from international cancer genome consortium (icgc) . this set of about matched primaries and recurrent medulloblastoma cases forms our discovery cohort. we have called somatic variants using the gatk pipeline by the broad institute. to validate our key findings, we have procured human medulloblastoma specimens and are conducting whole exome sequencing. the primary assays utilized to assess immunogenicity are immunohistochemical (ihc) staining of formalin fixed and embedded recurrent medulloblastoma tissue to identify tils, tumor associated macrophages and other markers. mg/m had dlts of dyspnea (grade )/hypoxia (grade ) but no dlts were observed in any other cohort. adverse events were generally mild to moderate, consistent with the safety profile observed in adults. across the desc cohorts, plasma concentrations were dose-proportional and steady state concentrations were lower on day vs. day . mean systemic exposure in the mg/m cohort was ∼ -fold greater compared with the adult rp d of mg bid. a pk:pd relationship between tazemetostat exposure and h k me levels in peripheral blood monocytes and granulocytes was observed in the desc phase. consistent and significant post-dose reductions in h k me occurred at doses ≥ mg/m . further analysis of twelve patients treated at the rp d confirmed that h k me inhibition was maximally inhibited. doses - mg/m showed confirmed objective responses (cr/pr) per recist/rano in patients with es (n = ), chordoma (n = ), and atrt (n = ). background: previous studies established that the platelet/ fibrin(ogen) axis promotes metastatic potential by impeding the clearance of newly formed micrometastases by natural killer (nk) cells. however, multiple important questions remain, including the potential of fibrin(ogen) to promote metastasis through interactions with cells other than platelets (e.g., inflammatory cells), and the fundamental question of whether fibrin polymerization is required for metastasis. objectives: determine the role of fibrin polymerization and fibrin(ogen) engagement of integrins iib and m in metastasis. design/method: we performed experimental and spontaneous metastasis assays in immunocompetent mice carrying specific fibrinogen structure/function alterations. results: expression of a mutant fibrinogen lacking the binding motif for the leukocyte integrin m (fib - a) significantly decreased metastatic potential relative to wildtype fibrinogen, suggesting a role for fibrin(ogen)inflammatory cell interactions mediated by m in metastasis. to directly determine the importance of thrombinmediated fibrin polymerization in metastasis, we analyzed metastatic potential in fibaek mice, which carry a form of fibrinogen essentially "locked" in the soluble state due to a mutation in the a chain thrombin cleavage site. metastatic potential in fibaek mice was diminished relative to control mice, speaking to the importance of thrombin-mediated fibrin polymerization in the metastatic process. however, the fibaek mice retained significant metastatic potential relative to complete fibrinogen deficiency, indicating that fibrinogen monomer retains significant prometastatic properties. in order to better define the role of fibrin(ogen)-platelet interactions in metastasis, we compared metastatic potential in control and fib Δ mice, carrying a form of fibrinogen lacking the chain binding motif for the platelet integrin iib . surprisingly, this mutation had no impact on metastatic potential. together, these studies suggest fibrinogen plays a multifaceted role in metastasis. fibrin(ogen)-leukocyte interactions mediated by m appear to have a role in metastasis. previous studies showed that macrophages promote the metastatic potential of circulating tumor cells, which may represent at least one important m expressing cell type whose prometastatic behavior is influenced by fibrin(ogen) interactions. these studies show that thrombin-mediated fibrin polymerization promotes metastasis, but soluble fibrinogen retains some significant prometastatic capacity. surprisingly, loss of the fibrinogen chain iib binding motif had no impact on metastasis. given the established importance of platelets in metastasis, these findings suggest that fibrin (ogen) is capable of platelet stabilization through mechanism(s) independent of this iib binding motif. platelets may bind polymerized fibrin at other sites, and/or fibrin interactions with other matrix proteins capable of binding iib are sufficient to support platelet functions required for metastasis. the role of platelets in hemostasis and thrombosis is well defined, but it is becoming increasingly evident that platelets also assist in host defense and inflammation. platelets participate in the innate immune system through direct antimicrobial activity and interactions with effector cells (chapman , garlanda , kapur ). in the adaptive immune system, platelets recruit and costimulate t-cells, and promote b-cell differentiation and antibody class switching (kapur , morrell ). the question remains: which mechanisms influence platelet immune function and are they developmentally regulated? preliminary studies in the palis lab have revealed significant dif-ferences in embryonic versus adult platelet gene expression, including regulators of immune and inflammatory responses such as beta -microglobulin (b m) and major histocompatibility complex class i (mhc ). mhc is expressed on all cell surfaces except red blood cells and its molecular chaperone b m is a marker of inflammation highly expressed in platelet alpha granules (zufferey ). preliminary data from the morrell lab reveals a mass release of b m during platelet activation, which drives monocyte differentiation to an inflammatory phenotype through tgfb receptor signaling. we therefore sought to determine whether developmental changes in platelet b m expression mediate differences in platelet-mediated monocyte activation. with trilineage hematopoiesis with a predominance of early myeloid precursors, with full maturation. microarray, elane and sbds sequencing and deletion/duplication analyses were negative. immunologic evaluation was significant for agammaglobulinemia and an absence of memory (cd +cd +) b cells. a gene primary immunodeficiency panel revealed two variants of unknown significance-c. g>a and c. g>t in dnmt b; one previously reported in association with icf . parental testing demonstrated parental heterozygosity. centromeric instability was confirmed in mitogen stimulated lymphocytes showing characteristic, multibranched chromosomes containing at least arms of chromosome and joined near the centromere. decondensation of the qh and qh regions and triradial configuration of chromosome was noted, and a diagnosis of icf syndrome was made. the patient was started on monthly intravenous immunoglobulin (ivig). prophylaxis for pneumocystis jiroveci pneumonia and respiratory syncytial virus was initiated. a / matched sibling hsct is being planned. demonstrated the diagnosis of high grade osteosarcoma. the patient was started on multi-agent chemotherapy with planned a whole femur prosthesis at time of local control. cases of osteosarcoma have been described in the literature in patients with nf (median age; years, range - years) with slightly male predominance ( cases). the femur was the most common site of involvement ( cases). four patients died of metastatic disease despite surgery and multi-agent chemotherapy. conclusion: nf represents a major risk factor for development of malignancy and uncommonly osteosarcoma in adolescents and adults. we report a rare case of an extensive involvement of osteosarcoma of the left femur in a child with known diagnosis nf . this presentation should alert the pediatric oncologists to monitor for bone tumors in patients with nf by physical exam and detailed medical history. hasbro children's hospital, providence, rhode island, united states background: dysautonomia is a paraneoplastic syndrome most commonly described in adult malignancies. despite current therapies aimed at symptoms management, it is often debilitating. we present a case of a -year-old girl who initially presented with autonomic dysfunction and was subsequently found to have hodgkin lymphoma. objectives: describe hodgkin lymphoma presenting with dysautonomia and discuss symptom management with rituximab design/method: case report a year-old-girl presented with severe symptoms of orthostatic hypotension necessitating prone positioning to prevent syncopal episodes. additionally, she reported anhidrosis, xerostomia, urinary retention, and constipation. she had unmanageable peripheral neuropathic pain despite multiple analgesia medications. initially, it was suspected that her symptoms were caused by an atypical presentation of guillain-barre syndrome. she was treated with intravenous immunoglobulin g, without response. due to a suspicion of a paraneoplastic syndrome a positron emission test/cat scan (pet/ct) was performed and revealed widespread fdg-avid nodal and splenic disease. pathology from a thoracoscopic biopsy of a mediastinal lymph node demonstrated classical hodgkin lymphoma. she was classified as stage ivb. a paraneoplastic panel obtained during the first cycle of chemotherapy revealed elevated anti-amphiphysin antibodies and glutamic acid decarboxylase (gad) antibodies. therapy was initiated with abe-pc (doxorubicin, bleomycin, etoposide, prednisone, cyclophosphamide) ; vincristine was held given her significant neuropathy. due to persistence of autonomic symptoms following her first cycle and presence of antiamphiphysin and gad antibodies, rituximab was incorporated into her treatment. following two cycles abe-pc, she had a rapid early response by fdg-pet/ct. she completed an additional three cycles of abd-pc. end of therapy imaging demonstrated complete response with a single persistent mildly fdg-pet avid lymph node (deauville ) and her antibodies were negative. she continues treatment of maintenance rituximab with significant improvement, but not resolution, of her orthostatic hypotension. at this time, the patient can ambulate with assistance. constipation and urinary retention have fully resolved and, her peripheral neuropathy, xerostomia, anhidrosis have improved. conclusion: this is rare case of a pediatric hodgkin lymphoma patient developing dysautonomia associated with antiamphiphysin and glutamic acid decarboxylase antibodies and subsequently managed with chemotherapy and rituximab. clinicians should be suspicious of a paraneoplastic syndrome when a neurologic disorder fails to improve with standard treatment. results: labs obtained at an outside hospital one month prior to presentation showed absolute neutrophil count (anc) and hemoglobin . g/dl. she presented to our institution with days of fever, hepatomegaly cm below costal margin, a white plaque on her tongue, and circumferential perianal ulceration. labs were significant for anc and hemoglobin . g/dl. anti-granulocyte antibody testing was positive. bone marrow biopsy showed arrest of neutrophil maturation. after initiation of filgrastim ( . mcg/kg/day), her anc increased to > and repeat bone marrow biopsy demonstrated left shifted myelopoiesis. biopsy of her oral lesion demonstrated invasive actinomyces prompting a prolonged course of antibiotics. biopsies of her oral and anal lesions were reported as myeloid sarcoma without mll rearrangement. chemotherapy was not initiated due to complete resolution of both lesions within weeks of initiating filgrastim and appropriate antibiotic coverage. she has not developed any further lesions concerning for malignancy. testing for common genes associated with severe congenital neutropenia and autoimmune lymphoproliferative syndrome was negative. her immunoglobulin levels and the measurement of age-appropriate vaccine responses were normal. after her lymphocyte subpopulation analysis indicated a selective deficiency in cd positive t-lymphocytes (absolute cd cell count ), the severe combined immunodeficiency panel from genedx showed compound heterozygous mutations in results: a male infant was born with a large thigh mass. the child was clinically well aside from restricted movement of affected leg. mri showed mass expanding into pelvis without other lesions. an interventional-radiology guided core biopsy of the mass was reported as high-grade spindle cell sarcoma without etv rearrangement. surgery was deferred because of concern that it would result in excessive morbidity. the mass was treated with vincristine and dactinomycin per infantile fibrosarcoma protocols. after months of therapy, no significant change in size of the mass was noted on physical exam or imaging. repeat biopsy was obtained to confirm diagnosis and allow for expanded tumor testing. this biopsy showed triphasic distribution of adipose, fibrous and mesenchymal tissue consistent with fhi with rare sarcomatous foci. additional chemotherapy was deferred and the child was followed clinically. his tumor has remained approximately the same size and still unresectable. next generation sequencing of tumor utilizing panel based technology revealed braf-erc fusion consistent with braf activating mutation. this mutation was confirmed by fluorescent in situ hybridization (fish) probe for braf. braf and mek inhibitors have been pursued as treatments to decrease size of tumor and allow for resection. conclusion: braf mutations have been characterized in a variety of malignancies. inhibition of braf and downstream signaling components has produced promising results in a variety of patients. this is the first case report of a braf mutation in a fhi. although management of fhi is typically surgical, this does suggest a potential therapeutic target and may allow for improved surgical outcomes especially in cases where up-front surgery would result in unacceptable morbidity. genetic sequencing of fhi and other rare tumors is an important tool and has the potential to identify mutations amenable to targeted therapies. background: icf is a rare autosomal recessive disorder characterized by hypo-or agammaglobulinemia and often opportunistic infections suggesting t-cell dysfunction. it is further categorized into subtypes - based on mutations in dna methylation. mutations in the helicase-lymphoid specific (hells) gene, which is required for t-cell proliferation and participates in de novo dna methylation, are characteristic of icf type (icf ). of approximately reported cases of icf, less than percent are characterized as icf . while malignancy has been reported in icf (angiosarcoma, acute lymphoblastic leukemia), and icf (hodgkin lymphoma), here we describe the diagnosis and management of an icf patient with neuroblastoma and neutropenia, which has not been previously described. objectives: describe a novel phenotype and mutation of icf and its management to further expand our understanding of this disease. results: a month ex- week premature male with bronchopulmonary disease and failure to thrive presented with acute respiratory failure in the setting of recent viral bronchiolitis with associated chronic diarrhea. he was subsequently diagnosed with multiple infections including pjp pneumonia, norovirus, parainfluenza, rhinovirus, and pseudemonal cellulitis. he presented with profound neutropenia and agammaglobulinemia with presence of b and t cells on lymphocyte phenotyping. ct revealed a paraspinal mass that was mibg-avid on further study, strongly suggesting neuroblastoma. bone marrow was normocellular and negative for malignancy, however revealed marked granulocytic hypoplasia and maturation arrest concerning for severe congenital or, less likely, immune-mediated neutropenia. metastatic workup was negative. whole exome sequencing revealed a homozygous variant of unknown significance (c. t>c) in the hells gene, portending a working diagnosis of icf syndrome. immunoglobulin supplementation, pentamidine prophylaxis, and g-csf were initiated. he was able discontinue g-csf after months of treatment. his neuroblastoma, initially categorized as l , met criteria for observation. however, followup mri revealed interval growth nearing the spinal canal. he underwent tumor resection, confirming mycn non-amplified, favorable histology neuroblastoma. after infectious prophylaxis and immunologic support were initiated, he incurred two other hospitalizations, the first for g-tube cellulitis and the second for parainfluenza respiratory illness. he now has stable neutrophil counts off g-csf and remains in remission from neuroblastoma. current plan is to proceed with bone marrow transplantation for immunodeficiency. conclusion: icf has not previously been described with neutropenia or neuroblastoma. this report not only describes a novel mutation and phenotype of icf and the management thereof, but also reveals the potential curative role of bone marrow transplantation in such disease. staten island university hospital -northwell health, staten island, new york, united states background: desmoid tumors are rare tumors that arise from highly differentiated fibroblasts. they occur in isolation or as part of the disease spectrum of familial adenomatous polyposis (fap) . fap mutations between codons - typically correlate with increased extraintestinal disease such as desmoid tumors and upper gastrointestinal polyps. we describe a patient with a large intra-abdominal desmoid tumor who is heterozygous for a c. c>t (p.arg cys) apc gene mutation. we are not aware of any other patients reported with this germline apc mutation presenting with a desmoid tumor. objectives: to discuss a novel apc mutation and the presentation of a rare case. design/method: review of clinical presentation, genetic analysis and management of a rare tumor. a -year-old female with no significant medical history presented with abdominal asymmetry and intermittent pain. she reported urinary urgency, shortness of breath, early satiety, decreased appetite and a -pound weight loss over the course of months. ct scan of the abdomen demonstrated a × cm abdominal tumor abutting the local organs but no presence of bowel obstruction. a biopsy revealed a spindle cell neoplasm favoring fibromatosis. there was no known family history of fap, colon cancer, or desmoid tumors. apc gene mutation analysis demonstrated a c. c>t (p.arg cys) heterozygous gene variant. due to size and location of the tumor, it was initially deemed unresectable. the patient was started on a course of monthly liposomal doxorubicin. she tolerated the initial cycles well and interval ct after cycles of chemotherapy revealed a % decrease in tumor volume. variability exists in phenotypic presentation with regards to the location of the afp mutation locus. while fap mutations associated with desmoid tumors typically have changes in the - codon region, our patient presented with a heterozygous mutation resulting in a missense mutation at codon . due to the change in polarity and size, the mutation is not considered to be of conservative nature. we are only aware of one other report of this mutation, which occurred in an individual with a personal and family history of colon cancer. we are not aware of any patients with desmoid tumors who also have this germline apc gene mutation. our case report highlights an apc gene mutation that is not well-described; we are not aware of any other cases of this mutation reported in patients with desmoid tumors. future evaluation and tracking of this mutation may lead to the determination of further clinical significance. background: over time, advanced care planning for location of death has been associated with increased deaths at home rather than in the hospital. in some cases, however, complex management and symptom control can prevent families from achieving their goal of keeping their child out of the hospital and at home at the end of life. ascites is a sequelae of many conditions including malignancy that might lead to significant morbidity. increasingly, interventional procedures are being utilized. peritoneovenous "denver" shunts are placed internally with one end in the peritoneal space and the other buried within a major vessel such as the svc. a one-way valve and pump buried under the skin allows the patient to pump fluid from the peritoneal to the vascular space. the shunt is used frequently in adults, but has not seen much use in pediatric oncology patients. objectives: to describe a case of a terminally ill patient with refractory wilms tumor with ivc involvement who received symptomatic relief with denver shunt placement. results: an -year-old female was diagnosed with relapsed, refractory, metastatic wilms tumor with pulmonary and hepatic involvement, with tumor extension to the hepatic veins and ivc. multiple chemotherapeutic regimens and palliative radiation to the ivc were administered, but her disease continued to progress, leading to pressure on the portal vein and portal hypertension. the resulting ascites was causing the patient significant pain and was difficult to manage. the patient's code status was changed to dnr/dni after discussion with her mother, who identified a desire to have the child die at home as comfortably as possible. a peritoneovenous shunt was placed in order to control the patient's pain and avoid frequent medical procedures and therapies. despite initial anxiety, the patient was able to utilize the pump and achieve significant improvement in her ascites and pain. she was able to spend the remaining six weeks of her life at home. ascites is a common phenomenon of end stage disease. peritoneovenous shunts are a treatment modality that may be considered to allow for pain control at the end of life for pediatric oncology patients with ascites. the procedure is relatively low risk, allows for self-control of the pump to maintain comfort, and is easy enough to use by the patient or family. background: extraneural metastases (enm) from pediatric glioblastoma multiforme (gbm) are rare, with an estimated frequency of . %. etiologic factors include multiple neurosurgical procedures and sarcomatous dedifferentiation. their occurrence can seriously affect the patient's quality of life and survival. while enms have been well documented in adults, pediatric cases have not been previously summarized. a year old male with a cerebral gbm developed extension of disease outside of the neuraxis approximately months post initial presentation and at the time of disease progression. metastases included exracranial temporal lesions, cervical and mediastinal lymph nodes and s of s bilateral lung nodules. a large pleural-based soft tissue metastatic focus was identified on imaging when the patient presented with respiratory distress secondary to a right tension pneumothorax, which was recognized and managed promptly. we summarize the main reported cases in literature to better define risk factors for and evaluate the proposed mechanisms underlying these systemic metastases. design/method: we performed a literature review on the pubmed database using the terms gbm and enm. patients under years of age who met the weiss criteria for the diagnosis of enm from primary cns tumors were included. results: our patient fulfilled two of the three weiss criteria with confirmed gbm at the primary site with all enm in the temporal soft tissue and cervical lymph nodes displaying histopathologic features similar to the primary cns tumor. the intrathoracic adenopathy and lung nodules detected upon chest imaging during workup for respiratory distress were assumed to represent additional metastatic foci. our literature review identified pediatric patients with enm from gbm with a median age of years (range . - years) and a slight female predominance ( % females vs. % males). the most common sites of metastases reported were pleura/lungs, bones, lymph nodes and liver. in of patients, metastases were associated with csf shunting. conclusion: pediatric oncologists should have an increased index of suspicion when caring for patients with gbm, particularly those who have undergone shunting procedures and present with systemic symptoms including bony pain, respiratory changes, transaminitis or cytopenias which should prompt timely investigation for enm. although enm of cns tumors carry very poor prognosis, their diagnosis has potential therapeutic importance because treatment of metastatic lesions may alleviate symptoms and improve the quality of life. additional studies may be warranted to evaluate the incidence of enm that can provide valuable insight into the pathogenesis and biology of high-grade gliomas. nicklaus children's hospital, miami, florida, united states background: sinusoidal obstruction syndrome (sos) has been reported in patients undergoing intensive chemotherapy and as a complication post-hematopoietic stem cell transplan-tation. sos may be complicated by portal hypertension, hepatorenal disease or multi-organ failure. however, despite treatment, there may be further potential complications that can be anticipated in patients with history sos. we report two patients with history of sos presented with post-procedural bleeding after gastric tube placement. we believe that their presentations may be associated to their previous diagnosis of sos. design/method: pubmed search was done with search for terminology including "sinusoidal obstruction syndrome" "defibrotide", and "bleeding". papers relevant to our cases were selected for literature review. results: case : a year-old female with history of desmosplastic medulloblastoma status-post resection and intensive chemotherapy was diagnosed with sos one month after her second part of planned tandem transplant. she was managed with paracentesis and defibrotide. due to malnourishment, patient had a gastric tube placement months after she completed therapy and had an episode of upper gastrointestinal bleeding postoperatively from the g tube site. case : similarly, a year-old male diagnosed with anaplastic medulloblastoma status post resection and adjuvant multiagent chemotherapy. his treatment course was complicated with sos after the second cycle of induction chemotherapy which responded to -day course of defibrotide. likewise, the patient had a major bleeding event from the g-tube site approximately two months after sos diagnosis. defibrotide was discontinued in both cases before g-tube placement. both patients had no previous history of bleeding disorders or relevant family history. in addition, comprehensive laboratory evaluations were within normal limits before both procedures. in sos, there is blockage of fluid out of the liver that leads to congestion, ascites, ischemia of the liver, and post-sinusoidal portal hypertension. two related causes of sos should be considered as an explanation for g-tube bleeding. similar patients should have close monitoring postoperatively or if possible surgical intervention should be delayed until the sos process has been evolved. nicklaus children's hospital, miami, florida, united states background: the development of treatment related acute myeloid leukemia (t-aml) and myelodysplastic syndromes (t-mds) is a potential complication after cytotoxic chemotherapy or radiation therapy. the incidence of development of t-aml/t-mds varies from - % depending on the treatment regimen used. cutaneous myeloid sarcoma (ms) is a common presentation of extramedullary leukemia and usually occurs in the setting of aml. we report a rare case of cutaneous ms in an adolescent female after successful treatment for ovarian yolk sac tumor (yst) stage i with bep (bleomycin, etoposide and cisplatin) therapy. the ms was managed only with biopsy and close observation. design/method: a pubmed search was conducted for queries including t-aml/t-mds, cytotoxic agents, cutaneous myeloid sarcoma, regression. relevant papers were selected for literature review. a year-old female was diagnosed with a left ovarian yolk sac tumor, for which she underwent left salpingooophorectomy and successfully completed cycles of bep over months. during routine follow-up months after initiation of treatment for ovarian yst, she was noted to have a small, non-tender, indurated nodule on the left side of her upper back approximately cm in diameter. punch biopsy of the skin nodule was performed and pathology was positive for cutaneous myeloid sarcoma. at the time of next follow-up less than one month later, the skin lesion had resolved. two subsequent bone marrow aspirates were performed one month apart and were negative for leukemic involvement or mds. examinations and work-up including whole body pet with ct scan were negative for evidence of disease. although cutaneous ms can be regarded as the herald of systemic myeloid disease rather than a localized process, our patient was monitored periodically with physical exam and laboratory evaluations. she remains free of disease more than four years after the presentation of cutaneous ms without any further treatment. spontaneous regression ms has been previously reported. the authors would like to stress that a conservative approach with close observation could be an option in cutaneous ms even with history of chemotherapy exposure. nesreen ali, iman sidhom, sonia soliman, sherine salem national cancer institute, cairouniversity, egypt children cancer hospital egypt, egypt background: acute leukemia is the commonest malignancy in childhood. the coincidental occurrence of leukemia with hemophilia is extremely rare. hemophilia is a congenital rare x linked bleeding disorder. the main complication of the two diseases is bleeding diathesis which may be lifethreatening due to many factors, deficiency of coagulation factors in hemophilic patients, thrombocytopenia from disease and chemotherapy in leukemic patients, certain cytotoxic drugs such as asparaginase which may result in coagulation disorders and infection which may lead to disseminated intravascular coagulation. objectives: reporting such a case is imperative to set up treatment guidelines for prevention of bleeding and to optimize the therapeutic approach for these patients. design/method: seventeen years old boy, presented to children cancer hospital egypt in june with pallor and multiple ecchymoses.he was diagnosed with precursor b acute lymphoblastic leukemia, cerebrospinal fluid (csf) was free, the chromosomal analysis revealed hypodiploidy , xy. he had moderate type of hemophilia a since birth, factor viii level was . % at time of diagnosis, coagulation profile revealed prolonged partial thromboplastin time (normal - ), factor viii was low %, prothrombin concentration and prothrombin time were normal % and seconds, virology screening for hepatitis b core igg/igm, hbs ag, hiv and hc igg /igm were negative.the patient started induction total xv sjcrh protocol, factor viii unit/kg was given at presentation before doing bone marrow aspiration(bma), csf and as a prophylactic before intramuscular asparaginase injection, intrathecal and bma. it was given immediately within hours before the procedures and platelets transfusion was given regularly to maintain platelets count about , . the minimal residual disease by flow cytometry was . % and . % at d and d induction. results: our patient received his induction and reintensification chemotherapy without any major bleeding event which reveals the success of our guidelines for the prevention of bleeding. he developed very early relapse at w maintenance by the same clone. he received salvage chemotherapy but didn't achieve remission and died out of disease and resistant clone. the development of leukemia on top of hemophilia is a major problem. bleeding complication during chemotherapy can be prevented by regular prophylactic factor viii and platelets concentrate transfusion with good supportive care. life threating bleeding complication may be correlated with the severity of hemophilia. we need to collect data about the biology of leukemic cells, complications, and cause of death to optimize care for these patients. background: mucoepidermoid carcinoma (mec) is a rare malignancy that arises from exocrine glands in the upper aerodigestive tract and tracheobronchial tree. conventionally, mec diagnosis is based on histology, with prognosis based on the extent of resection and detection of metastases. mec is characterized by a translocation of chromosomes q and p resulting in a fusion between the mect and maml genes, that occurs in - % of cases. this fusion transcript has been recognized to have a favorable impact on disease features and prognosis of mec. however, recent studies indicate that high grade mec can have mect -maml fusion positivity and multiple other genomic imbalances that have not been studied in much detail. owing to the rarity of mec tumors, more definitive data related to the clinical and prognostic significance of these molecular markers are limited. objectives: . identify the presence or absence of mect -maml fusion in the tissue of our patient. . analyze the incidence of the fusion in mec cases in children and young adults retrieved from the iowa cancer registry. . determine if fusion status correlates with clinical, pathological and outcome data in our cohort. design/method: we describe the case of a year-old caucasian male who presented with recurrent pneumonia, persistent cough and radiographic evidence of right lobar collapse. bronchoscopy revealed an endobronchial lesion and the patient underwent right upper lobe sleeve resection. pathology report was consistent with low grade muco-epidermoid carcinoma. we retrieved archived formalin-fixed paraffinembedded (ffpe) specimens of pediatric and young adult mec cases (ages - ) reported in iowa from - using the iowa cancer registry. testing for the mect -maml fusion in the index case and ffpe specimens will be done using a custom-designed laboratory validated next generation sequencing (ngs) assay with the ability to detect novel fusion partners. clinical, pathological and outcome data (age, sex, tumor site, tumor size, nodal metastases, clinical stage, histologic grade, treatment and follow up) will be analyzed to correlate with fusion status. the mect -maml fusion tested positive in our index patient. we will obtain irb approval to test for the fusion in the archived ffpe specimens and correlate clinical, pathological and outcome data. conclusion: mect -maml fusion is a frequent event in mec that has prognostic and potential therapeutic applications in adults. the results of this study may enlighten the clinical management of mec in children and young adults. children 's mercy hospital, kansas city, missouri, united states background: mutations in the samd gene are associated with a rare syndrome comprising of myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (mirage syndrome). diagnosis is made through exome sequencing. in the largest reported case series, of eleven patients diagnosed with mirage syndrome, two developed loss of chromosome . given the potent growth restricting activity of samd mutants, the loss of chromosome is considered the first documentation of adaptation by aneuploidy mechanisms in humans and led to myelodysplastic syndrome (mds), with deaths occurring from related complications at and years of age. objectives: to report a case of mirage syndrome with congenital thrombocytopenia progressing to bone marrow failure, managed uniquely with bone marrow transplantation. results: male born at weeks gestation with prenatal diagnosis of iugr, two vessel cord, oligohydramnios was found to have ambiguous genitalia, adrenal insufficiency, partial panhypopituitarism and congenital thrombocytopenia with bone marrow showing absence of megakaryocytic precursors. severe thrombocytopenia was present from birth. bone marrow evaluation demonstrated a hypocellular marrow with markedly reduced megakaryocytic and myeloid precursors and no evidence of myelodysplasia. he required gastric tube placement for failure to thrive, had a laryngeal cleft repaired and developed focal segmental glomerulosclerosis. mpl gene testing for congenital amegakaryocytic thrombocytopenia was negative. testing for fanconi anemia, shwachman-diamond syndrome and dyskeratosis congenita was also negative. approximately % of cells had loss of heterozygosity on chromosome q. exome sequencing showed that he is heterozygous for a de novo gain of function variant, c. g>a (p.arg gln), identified in the samd gene, confirmed by sanger sequencing and consistent with a diagnosis of mirage syndrome. at years of age, he developed pancytopenia requiring frequent transfusions with platelets and packed red blood cells. he underwent a successful bone marrow transplant at years of age without significant complications, and remains transfusion independent without cytopenias greater than months from bone marrow transplantation. conclusion: it is imperative to pursue work up for persistent congenital thrombocytopenia in a stepwise multidisciplinary manner. to the best of our knowledge, this is the first case of mirage syndrome associated bone marrow failure treated with bone marrow transplant. due to the individual rarity of mirage syndrome and pediatric myelodysplastic syndrome, it is important to maintain an index of suspicion given their association and explore bone marrow transplant as a therapeutic option. results: the patient demonstrated disease regression, initially, and continued without disease progression for months. the regimen has been well tolerated with only minimal side effects of dry skin (ctcae grade ) and a transient episode of brief erythrodysesthesia (ctacae grade ) that resolved spontaneously. the combination of sorafenib and capecitabine was effective and well tolerated in this adolescent patient with fl-hcc. our observations, although in a single patient, lend support for further testing of this novel oral chemotherapy regimen in patients with fl-hcc, a disease for which there is no effective standard chemotherapy approach. background: epstein-barr virus (ebv) is a ubiquitous virus associated with a broad range of malignancies due to its oncogenic potential. history of organ or bone marrow transplantation, immunosuppressive therapy, and primary or acquired immunodeficiency syndromes increases the risk of ebvassociated tumors. epstein-barr virus associated smooth muscle tumors (ebv-smt) are unique and rare neoplasms typically discovered in immunocompromised patients. most information related to pathogenesis and therapeutic options is limited to case reports and case series of adult patients. there are several gene expression pathways that ebv utilizes, the most notable of which is the mammalian target of rapamycin (mtor) pathway. the mtor pathway performs a key role through integrating various cell growth signals and factors to regulate protein synthesis and metabolism related to smooth muscle proliferation. sirolimus is an immune modulating therapy that targets the mtor pathway to block activation of lymphocytes. objectives: several case reports have demonstrated shortterm clinical remission of ebv-smt in adult patients with the use of sirolimus. we report the first case of long-term background: bilateral neuroblastoma is characterized as neuroblastoma arising in both adrenal glands, a rare presentation with little data on its genetic make-up. a two-monthold patient was diagnosed with bilateral neuroblastoma in our clinic. her risk assignment was based on biopsy of the left adrenal lesion, which showed mycn amplification, an unfavorable genetic marker. treatment regimen was intensified accordingly and after courses of chemotherapy tumors were excised. patient went on to receive a stem cell transplant and immunotherapy. with no knowledge of genetic similarity between the two tumors it is unclear whether biopsy of the right lesion would have yielded similar results or whether bilateral biopsies are needed for risk assessment of bilateral neuroblastoma. objectives: utilize whole exome sequencing (wes) to characterize the genomic signature of bilateral adrenal neuroblastomas excised following chemotherapy treatment. design/method: paraffin-embedded samples from left (l) and right (r) tumors underwent wes at the broad institute. we analyzed resulting data including somatic variant calls, indel mutations, and copy number variants (cnvs) using ingenuity software to evaluate and compare differences between the two tumor samples. preliminary analysis of the data shows important descriptive information on the two tumor samples. out of somatic mutations in the r tumor cells and mutations in the l tumor cells, only two common somatic mutations were present. out of cnv calls in the r tumor and in the l tumor, cnvs were common between the two tumors, or % of each tumor's cnv calls. there was a fold higher frequency in gains versus losses. the median size of the common cnvs was , (range to , , bp). cancerrelated genes with increased copy numbers included transcription factors, receptors for signal transduction pathways, and histone methylation proteins. conclusion: preliminary analysis of the wes results of the two adrenal tumors show some genomic divergence. because the tumor tissue was exposed to chemotherapy prior to excision it is difficult to determine whether genomic divergence is a result of independently originated tumors or subsequent adaptation to chemotherapy of a clonal cell population. the high number of common cnvs in the two tumors points to a common cell of origin, however the low number of common somatic mutations does not fit that picture. a future study to help elucidate the question will be wes of the original biopsy tissue to provide information on tumor mutations prior to the effects of chemotherapy. baylor college of medicine, houston, texas, united states background: although there has been significant improvement in the overall survival rates of children with cancer many children will still die from their illness or complications secondary to treatment. research surrounding the deaths of children who succumb to their disease is warranted to ensure we are providing the best care possible for these patients. objectives: this case series aims to explore pediatric cancer deaths by focusing on perhaps the most extreme cases of high intensity end of life care. we explore those patients whom we know are dying or our very likely to die as evidence by their do not resuscitate (dnr) orders. in all of these cases despite the patients very grim prognosis, their great likelihood of death and limitations placed of resuscitation methods all patients continued end of life care in the pediatric intensive care unit (picu). the primary medical records of all children with a cancer diagnosis who died between february , and january , in the picu with a dnr order seven days or earlier prior to death. each medical history included disease-directed treatment history and response with particular attention to the events surrounding the terminal admission. results: eight patients met criteria for this study representing . % of all cancer patients who died during this time period and . % of those who died in the icu. the average time between dnr and death is . days ( days - days). the average length of terminal admission was . days ( day - days). the average time between diagnosis and dnr is . months ( months - months). the average time between diagnosis and death is . months ( months - months). conclusion: these cases highlight the journey that patients, families and providers endure leading up to death. medical care is complex, there are very few absolutes that are encountered when caring for patients and decisions around limiting or withdrawing medical care are made in a context of the prior journey. . these cases help to understand the complexity of death and how two seemingly opposite ideals can be congruent in the event of an anticipated death. most of these cases show the need for improved anticipatory guidance surrounding death and greater consideration for de-escalation of care when death is expected. the hospital for sick children, toronto, ontario, canada background: rhabdomyosarcoma (rms) is the most common soft tissue sarcoma in children, with embryonal (erms) and alveolar (arms) representing the most common subtypes. arms tumors are associated with inferior outcome when compared to erms, and they are characterized in about % of the cases by a t ( ; ) or t( ; ) chromosomal translocation with creation of a pax -foxo or pax -foxo fusion gene, respectively. it is increasingly clear that the pax-foxo fusion status is an important poor prognostic factor, thus the histological classification tends to be replaced by the fusion status, particularly in terms of risk stratifica-tion in contrast to arms, there are no recurrent chromosome alterations in erms; however, there are multiple numerical chromosome changes that are frequent in these tumours: gain of chromosome , , and have been found in to % of emrs karyotypes. moreover, erms tumors show frequently allelic loss, the .p . chromosomal region being the most frequently involved. recently, novel gene fusions have been described also in erms tumours. these fusions involved mainly the ncoa and or the vggl genes. the rearrangement partners are variable, and include, i.e. pax ( q ), srf ( q ) and tead ( p ). objectives: to present a patient who died as a consequence of brain metastases while on therapy in the setting of an foxo negative rms and the identification of a new translocation t( ; )(q ;q ). design/method: case report and retrospective review of the literature. we report a case of pelvic embryonal rhabdomyosarcoma in a -month old boy. he was treated as per cog arst intermediate risk group, but unfortunately was found to have a large cerebellar tumour during the course of his chemotherapy treatment and he subsequently passed away. a novel translocation between chromosomes and was observed in of metaphase cells by g-band analysis in the autopsy sample of the brain lesion. breakpoints of the translocation were estimated to be at q and q . there were no additional clonal chromosome abnormalities in the tumour cells. conclusion: erms tumors with fusion genes involved have been exclusively described in patients less than months of age; they seem to be associated with spindle cell histology and, a favorable outcome. in our patient, a novel ( ; ) translocation was found and clinically, the patient had a dismal outcome. further studies are indicated to inquire whether this finding is of significance in term of prognosis for these patients. children 's national medical center, washington, district of columbia, united states background: iatrogenic immunodeficiency-associated lymphoproliferative disorders (lpds) are a group of lymphoid s of s proliferations or lymphomas that are well known to be associated with an immunosuppressed state. these disorders most commonly occur following hematopoietic or solid organ transplantation (called post-transplant lymphoproliferative disorders or ptld), but cases have also been described during the treatment of autoimmune and rheumatologic disorders by immunosuppressive and immunomodulatory medications. these disorders are strongly associated with infection by the epstein-barr virus (ebv) as a result of impaired immune function in the immunosuppressed state. while this phenomenon has been well documented in autoimmune conditions, cases affecting pediatric patients while on antileukemia chemotherapy are lacking. background: atypical teratoid/rhabdoid tumor (at/rt) of the central nervous system (cns) in children younger than years old has a prevalence of % to % and accounts for . % of all pediatric cns tumors. only - % of patients have leptomeningeal dissemination. rhabdomyosarcoma is the most common soft tissue tumor in childhood, but represent only - % of all pediatric cancers. rarely, it can metastasize or even directly extend into the cns, but typically, cases of cns involvement arise either from parameningeal areas or other primary sites. primary spinal or meningeal rhabdomyosarcoma is extremely rare. objectives: our objective is to describe two unique cns malignancies presenting as rare, primary leptomeningeal disease. design/method: case a -month-old female presented with vomiting, fatigue and listlessness, despite a normal head ct and brain mri. csf showed hypoglycorrhachia and mild pleocytosis. ceftriaxone was started, but she developed nuchal rigidity and cranial nerve vii palsy. repeat brain mri showed evolving leptomeningeal enhancement concerning for meningitis. she gradually developed worsening opisthotonus and ultimately a brain biopsy of the temporal lobe was consistent with at/rt. case a -year-old male presented with new generalized tonic-clonic seizure activity and intermittent headaches with photophobia, phonophobia, and vomiting. brain mri was significant for enhancement of interpenducular and suprasellar cisterns extending to the optic nerves and chiasm most consistent with meningitis. neurosurgery ultimately placed a lumbar drain for hydrocephalus, and a tissue biopsy demonstrated primary meningeal rhabdomyosacroma. results: in case , our patient's temporal lobe biopsy demonstrated grade iv malignant tumor cells consistent with atypical teratoid/rhabdoid tumor. fish demonstrated a homozygous deletion of smarcb ( q . ). she was started on chemotherapy per the dana farber at/rt protocol but ultimately was discharged home on hospice. in case , our patient's lumbar arachnoid biopsy demonstrated cellular tumor consistent with group iiia embryonal rhabdomyosarcoma. immunostaining was positive for cd , desmin, myogenin, and myo-d with neural markers ema and gfap highlighting the meninges but without a neural component to the tumor. he completed craniospinal radiation to gy total with lumbar boost to . gy total. he is currently receiving chemotherapy per arst protocol. conclusion: these two cases are particularly instructive because of their similar initial presentations and neuroimaging, but with very different and unique diagnoses. university of iowa, iowa city, iowa, united states background: ebf -pdgfrb fusion causes ph-like b-cell acute lymphoblastic leukemia (b-all), which has a philadelphia positive phenotype without the bcr-abl translocation. this is one of several mutations associated with ph-like b-all and leads to downstream overexpression of tyrosine kinase. ebf -pdgfrb fusion accounts for about % of children with ph-like b-all. patients with ph-like b-all previously had poorer outcomes with conventional chemotherapy. the addition of tyrosine kinase inhibitors (tki), like imatinib, has improved the outcome for many patients predicted to have tki sensitive mutations. objectives: to review clinical characteristics and outcomes of two cases of ph-like b-all at the university of iowa stead family children's hospital and to compare these outcomes to similar cases reported in the literature. design/method: a retrospective chart review was performed for two cases of ph-like b-all diagnosed and treated at the university of iowa stead family children's hospital. results: both patients were males diagnosed at years of age with high wbc count ( , and , ) and positive for ebf -pdgfrb gene fusion. patient (pt ) was cns b at presentation while patient (pt ) was cns negative; neither had testicular involvement. both started treatment according to cog protocol aall . peripheral blasts cleared by induction day for pt and induction day for pt . at end of induction, pt had m bone marrow and pt had m bone marrow but mrd %. dasatinib was started induction day for pt and induction day for pt . pt was still not in remission at end of consolidation; bone marrow cell culture for tki resistance showed best response to dasatinib. pt proceeded to anti-cd car t-cell therapy followed by tbi-based matched unrelated donor bone marrow transplant. pt had negative mrd at the end of consolidation and continues chemotherapy according to aall , dasatinib arm. both patients are currently clinically well. our patients had the same tyrosine kinase gene fusion and similar initial clinical courses. while both patients had persistent disease at end of induction, pt had almost % blasts while pt had significant reduction of disease burden before starting tki. pt showed good response with the addition of dasatinib while pt did not. these findings suggest that response to conventional chemotherapy may potentiate the effect of tki and may predict overall outcome. there are likely additional factors which must be taken into account when determining response to tki for patients with ph-like b-all which have not yet been identified. background: medulloblastoma is the most common malignant brain tumor of childhood. classically, medulloblastoma presents as a well-defined mass lesion in the cerebellum, with a high rate of metastatic dissemination. primary leptomeningeal medulloblastoma (plmb) is an exceedingly rare type of medulloblastoma presentation with a dismal prognosis in which patients present with isolated leptomeningeal disease without an associated mass. to our knowledge, only three pediatric and three adult cases of plmb (ages - years) have been reported, all of which died within months of diagnosis. this is the first case of plmb to report a molecular classification. objectives: to report the case of a pediatric patient with plmb in which histopathologic and molecular characterization was performed and to describe the patient's treatment and clinical course. design/method: retrospective review of the patient's electronic medical record and review of the literature. a -year-old boy presented with headache, vomiting, diplopia, and fatigue. physical examination revealed upward gaze palsy, left-sided extremity and facial weakness, and ataxia. magnetic resonance imaging (mri) of the brain revealed diffuse cerebellar leptomeningeal enhancement and edema without an identifiable mass and moderate hydrocephalus. mri of the spine and cerebral spinal fluid analysis were normal. a diagnosis of cerebellitis was rendered, and the patient underwent placement of a ventriculoperitoneal shunt. an extensive infectious, neurologic, rheumatologic, and oncologic workup did not identify an etiology. empiric antibiotics, high-dose steroids, and intravenous immunoglobulin therapy yielded minimal improvement. two months later, repeat mri of the brain performed for declining mental status demonstrated progressive thickening of cerebellar leptomeningeal disease. a suboccipital craniectomy with decompression and cerebellar biopsy were performed. pathologic examination revealed a diagnosis of plmb, classic histology, non-wnt/non-shh, without gain/amplification of myc/mycn, and p wild type pattern. craniospinal radiation to cgy with a cgy boost to the posterior fossa was delivered with concurrent carboplatin/vincristine over six weeks. two months following chemoradiation, mri of s of s the brain demonstrates significantly reduced pathological leptomeningeal enhancement of the cerebellum, and the patient is awaiting initiation of systemic chemotherapy while recovering from a surgical wound infection. conclusion: plmb is extremely rare but should be considered in patients with cerebellitis and diffuse leptomeningeal involvement who are refractory to medical management or in whom an etiology has not been identified. cerebellar biopsy is recommended early to enable timely treatment and improved outcomes. molecular classification should be performed in cases of plmb to further characterize this disease, inform treatment decisions, and improve clinical outcomes. background: primary intracerebral osteosarcoma is extremely rare and limited to case reports. ptpn gain of function is associated with noonan syndrome, which has increased risk of multiple cancer types including brain tumors, but osteosarcoma has never been described. ptpn mutations have been reported in many cancers as both oncogenes and tumor suppressors, however no ptpn mutations have been described in osteosarcoma. pdgfr-a is a growth factor receptor whose activation is implicated in several malignancies. pdgfr-a and ptpn concurrent mutations are described in glioblastoma. there is no known link between holoprosencephaly, noonan syndrome, and osteosarcoma. we report a case of multifocal intracerebral osteosarcoma in a child with lobar holoprosencephaly and chronic subdural hemorrhage and discuss the genetic changes found in the tumor. design/method: a seven-year-old caucasian female, with a known diagnosis of lobar holoprosencephaly, chronic subdural hemorrhage and well controlled seizure disorder presented with status epilepticus shortly after completing antibiotic therapy for infection of subdural hematoma. mri showed diffuse dural thickening with mass lesions in the frontal lobe, temporal lobe, and the parasagittal region, the largest of which was contiguous with the subdural space but none of the lesions were associated with bone on mri or by direct neurosurgical visualization. tissue obtained for concern for recurrent infec-tion resulted in a diagnosis of high grade osteosarcoma. dna analysis was performed to help guide treatment choice. results: standard metastatic work-up was negative for skeletal primary tumor or metastatic lesions outside of the brain. she was treated with high dose methotrexate for two cycles per modified aost . despite maximal supportive care, she quickly developed rapid tumor growth as well as intratumoral hemorrhage with resultant herniation and death from respiratory failure just three months after diagnosis. tumor gene sequencing discovered three mutations with described roles in cancer: pdgfra d >vr, kdm a loss of exons - , and ptpn a v. conclusion: to our knowledge, primary multifocal extraosseus intracerebral osteosarcoma has not been previously described. despite known cns penetration of high dose methotrexate, this tumor proved resistant and aggressive. holoprosencephaly is associated with a multitude of known genetic drivers, but none are found in this case. furthermore, the genetic changes in this tumor are not typical for osteosarcoma. pdgfr-a over-expression is described in osteosarcoma, but is not clearly correlated with worse overall survival. further research is required to determine the role of ptpn in osteosarcoma. background: anaplastic lymphoma kinase (alk) encodes a receptor tyrosine kinase whose activation induces pathways associated with cell proliferation, angiogenesis, and cell survival. alk rearrangements are rare in neuroblastoma, while alk mutations and gene amplification occur more frequently. alk mutations have been found to be associated with increased alk protein expression that is associated with a worse prognosis. alk is commonly mutated in neuroblastoma at three hotspots (f , r , and f ). the eml -alk rearrangement has mostly been associated with lung adenocarcinomas, with only a few cases of non-lung cancers found. it has never been reported in neuroblastoma. multimodal therapy and to report the successful management of treatment related iron overload. results: a -year old male presented with abdominal swelling and ct showed a right kidney mass and bilateral lung nodules. he underwent right radical nephrectomy with lymph node sampling. pathology was reviewed centrally and revealed wilms tumor with diffuse anaplasia with rhabdomyosarcoma arising within the stromal component and of nodes positive. he received adjuvant intensive chemotherapy and radiation to the hemiabdomen and whole lungs. the -week chemotherapy regimen was vincristine, doxorubicin, cyclophosphamide (per cog arst ) alternating with carboplatin and etoposide (per cog aren revised uh- ). treatment was complicated by multiple episodes of fever and neutropenia and anorexia requiring g-tube placement. post-therapy, he had persistent neutropenia and thrombocytopenia without related complications. every months for evaluations he underwent a bone marrow which revealed normocellular marrow with maturing trilineage hematopoiesis. evaluation for a bone marrow failure syndrome was unrevealing. starting at months into therapy and all posttherapy imaging showed splenomegaly. he received units of packed red blood cells through the duration of therapy. he was diagnosed with iron overload based on serum ferritin and imaging, including t *mri. he received therapeutic phlebotomy for years with normalization of serum iron studies, t * of the heart, and liver iron concentration. he is more than years from completing therapy with no evidence of recurrent disease. asymptomatic cytopenias persist and he has no evidence of iron overload. conclusion: though a rare development, clonal sarcomatous transformation can occur in wilms tumor. our patient's tumor was successfully treated with intensive multimodal therapy targeting the diffusely anaplastic wilms and the rhabdomyosarcomatous component. treatment-related iron overload in a pediatric patient with a solid tumor was successfully treated with phlebotomy. consideration should be given to screen patients with solid tumors who receive multiple packed red cell transfusions for iron overload at the completion of cancer therapy. primary children's hospital, university of utah, salt lake city, utah, united states background: malignant solid tumors are less frequently encountered in infants. primitive myxoid mesenchymal tumors of infancy (pmmti) are a myofibroblastic malignancy and cases are rarely reported in the literature. cure is achieved in the majority of cases with surgical resection, however treatment for unresectable cases remains an enigma. recently published literature postulates that the newly discovered bcor duplication found in pmmti is tumorigenic via an epigenetic pathway. this molecular signature resembles that of clear cell sarcoma of the kidney (ccsk) and the growing number of bcor mutated sarcomas. a similar chemotherapeutic backbone and local control used for ccsk, has been proposed for the unresectable subset of pmmti. utilizing this approach a month-old with relapsed disease has remained disease free for months. however, given the rarity of this disease and the lack of published literature, there is no known standard of care treatment for unresectable and/or recurrent ppmti. we report a case of unresectable recurrent pmmti, a rare infant tumor, with less than cases reported. design/method: medical record, radiological studies, pathology and literature was reviewed. results: our patient is a now month-old female who presented with constipation and lower extremity weakness in the first weeks of life. an mri demonstrated a large lumbar epidural mass with spinal cord impingement. given prolonged (> days) neurological symptoms and location, emergent chemotherapy was initiated. biopsy showed a bcor positive, primitive myxoid mesenchymal tumor of infancy (pmmti). she was treated with ifosfamide, carboplatin and etoposide, and demonstrated clinical and radiographic response. we gave two additional cycles of cyclophosphamide, carboplatin and etoposide until surgical resection was feasible followed by two post-surgical cycles of chemotherapy. unfortunately, four month post-therapy mri demonstrated two new lesions; an unresectable paraspinal soft tissue mass and a left iliopsoas groove mass. given bcor association and reported successful therapy with vincristine, doxorubicin, cyclophosphamide alternating with ifosfamide and etoposide, we elected to incorporate vinca-alkaloid and anthracycline into her regimen. she is being treated with vdc/ie with plan for radiation consolidation. conclusion: pmmti is a locally aggressive tumor, for which surgical resection is curative. for those not amendable to resection, best care practices are still being determined. we report a case of pmmti initially responsive to chemotherapy, but not curative. this is the second case to conclusively demonstrate chemo-responsiveness. bcor mutation seems to be a common feature of this cancer; its role in the pathogenesis and as a target is an area of investigation. medical college of wisconsin, milwaukee, wisconsin, united states background: atypical teratoid/rhabdoid tumors (atrt) are central nervous system (cns) tumors that most commonly occur in very young children. there is no widely accepted standard of care for atrt patients, and while survival rates are improving they are historically poor. patients with metastatic disease to the spine at diagnosis have a worse prognosis, and for patients > years old, the presence of metastatic disease often results in the use of craniospinal radiation. the importance of correctly identifying metastatic disease at diagnosis aids in decision making and can have both prognostic and therapeutic implications. mr imaging at diagnosis is used to identify metastatic disease; however, here we present a case of diffuse leptomeningeal enhancement that spontaneously resolved after resection of a primary supratentorial atrt. objectives: to describe the resolution of diffuse leptomeningeal enhancement after resection of a primary atrt tumor in a -month-old prior to any adjuvant therapy. results: a -month-old male presented with a month history of vomiting and weight loss, regression of gross motor developmental milestones, and left hemiparesis. a brain mri demonstrated a × . × . cm solid and cystic right atrial mass with diffusion restriction and post-contrast enhancement. smooth diffuse enhancement was noted along the surface of the brainstem and within the interpeduncular fossa. a spine mri demonstrated diffuse circumferential post-contrast enhancement along the surface of the entire spinal cord. the patient underwent a successful near total surgical resection of the primary mass. pathology confirmed the loss of ini- staining in tumor cells, consistent with a diagnosis of atrt. no immediate adjuvant radiation or chemotherapy was given. repeat imaging was completed days after resection. brain mr demonstrated expected post-operative changes within the surgical cavity without definitive residual mass or leptomeningeal enhancement. spine mr demonstrated complete resolution of the previously seen circumferential enhance-ment along the entire spinal cord. csf evaluation at that time was negative for tumor cells. after recovery from surgery, chemotherapy treatment was initiated. conclusion: leptomeningeal enhancement at the time of diagnosis of atrt has historically been considered clear evidence of metastatic disease. this case raises questions about the previously accepted etiology of these imaging changes and suggests that widespread leptomeningeal enhancement should be carefully interpreted in future patients with similar imaging findings. in this setting, clinicians should consider repeat imaging following primary surgical resection in order to provide appropriate prognostic information and inform therapeutic decisions. poster # primary ewings sarcoma of cervical cord mimicking cauda equina syndrome sucharita bhaumik, joshua chan nyu winthrop hospital, mineola, new york, united states background: ewing's sarcoma (es) is a malignant primary bone tumor usually involving long bones. primary es of spine is quite uncommon ( . %) and its location in the cervical spine is even more rare. cauda equina syndrome (ces) is symptoms due to damage to the bundle of nerves below the end of the spinal cord known as the cauda equina (low back pain, radiating shooting pain down the legs, paraplegia, and loss of bowel or bladder control). it often occurs with lesions of lumbosacral spine. treatment with high-dose steroids may provide pain relief and improved neurologic function (by reducing edema) while awaiting diagnostic studies objectives: to demonstrate an unusual clinical presentation and emergent management of cervical es presenting with ces like symptoms. : year old male presented with a left sided posterior neck mass. soon after, he developed weakness of left arm, urinary and stool retention and inability to walk or bear weight in both legs. on physical exam a left tempero-occipital × cm fixed, non-tender, non-fluctuant mass was noted as well as motor and sensory impairment of left upper extremity, bilateral spastic paraplegia and loss of sphincter control. mri cervical spine showed a left cervical tumor with moth eaten appearance involving the vertebral bodies of c -c , adjacent muscles, displacing vital structures of the neck and compressing the cervical spinal cord. the thoracic and lumbosacral spine had no disease involvement. due to rapidly worsening spinal cord compression he was emergently treated with high dose steroids. he gained back all function in his extremities and regained bowel and bladder control. this eliminated need for urgent neurosurgical intervention. results: biopsy of the neck mass showed small blue round cells consistent with es with ewsr gene rearrangement. staging work up revealed no additional metastatic involvement. he then initiated treatment for localized es with systemic chemotherapy and radiotherapy and has had excellent response to treatment so far. conclusion: this is the first known case of non metastatic primary cervical es mimicking ces where an acutely enlarging mass presented with rapidly progressive neurologic deficits due to compression of anterior spinothalamic tract. in these unusual presentations of ces without lumbodorsal involvement it is important to consider cervical lesions. early rapid steroid initiation should be considered while awaiting biopsy results to prevent worsening cord compression followed by es focused treatments. this increases the chance of a successful outcome. the initial improvement with steroids may confuse the tumor with being a lymphoma children 's mercy hospital, kansas city, missouri, united states background: von willebrand disease (vwd) is a relatively common bleeding disorder with a high degree of genotypic and phenotypic variation. bleeding is usually mucocutaneous but can be severe and include muscle and joint bleeds especially in type vwd patients. most common bleeding management consists of desmopressin, anti-fibrinolytics, and/or plasma-derived antihemophilic factor/von willebrand factor (ahf/vwf) complex. a recombinant vwf has become available in the last few years. anaphylaxis and inhibitor development in vwd are rare. objectives: to describe the rare clinical manifestation of anaphylaxis to factor concentrate in a patient with severe type vwd. results: a -year-old female with severe type vwd [baseline vwag %, activity < %, factor viii (fviii) %] originally presented with heavy menstrual bleeding (hmb) leading to anemia requiring blood transfusion. she underwent placement of a levonorgestrel-releasing intrauterine device (lngiud) and began norethindrone. her hmb continued despite the lngiud and an increase in norethindrone dosing. plasma derived ahf/vwf complex was administered, which she had previously received. following the infusion, the patient developed anaphylaxis with hives, wheezing, tachycardia, and itching requiring doses of diphenhydramine and dose of hydrocortisone with resolution of symptoms. subsequently, she received recombinant vwf without incident. however, due to her low fviii level, she also required treatment with a full length recombinant fviii product. she again developed hives and itching after this infusion. she has since received recombinant vwf with recombinant fviii/fc fusion protein without further allergic reaction. there was no evidence of an inhibitor with her most recent post-infusion vwf level was %, factor viii %. conclusion: anaphylaxis to plasma derived factor products has been documented far less frequently within the vwd population compared to those with hemophilia and is typically seen in those with large gene deletions, usually with type disease. therefore, similar type vwd patients with severe disease may benefit from gene sequencing. it is unclear in this patient's case to which aspect of her treatment she is allergic, as she reacted to plasma-derived ahf/vwf and full length recombinant fviii, but not recombinant vwf or recombinant fviii/fc fusion protein. we hypothesize that she may be allergic to an epitope in the fviii b domain, or that the presence of fc fusion may have had a protective effect. further investigation including genetic analysis is planned. nodules. biopsies were consistent with neuroendocrine carcinoma, large cell type (g ). next generation sequencing revealed a khdrbs -braf fusion. he received conventional cytotoxic chemotherapy regimens both with cisplatin/doxorubicin, capecitabine/temozolomide, and doxorubicin/etoposide, but achieved a minimal response followed by rapid disease progression, massive ascites, and renal failure secondary to bilateral ureteral obstruction. results: based on his prior genomic testing, therapy with single agent mek inhibitor (trametinib) was initiated. this produced a rapid, dramatic response with greatly reduced disease burden at all sites, resolution of ascites and return to completely normal activity within months. this response lasted for approximately months before the tumor again progressed. further therapy with an erk inhibitor was ineffective, and the patient expired from progressive disease. located on the chromosome q , the braf oncogene, as part of the ras/mapk pathway, is involved in cellular proliferation, differentiation, migration, and apoptosis. braf mutations are recognized in a wide range of adult malignancies: thyroid cancers, non-small cell lung cancer, cholangiocarcinoma, ovarian cancers, and multiple myeloma. braf mutations have also been described in adult neuroendocrine carcinoma of the colon. trametinib is a highly specific inhibitor of mek /mek , a downstream mediator in the braf pathway. it has demonstrated activity in a number of tumors including advanced melanoma and gliomas. trametinib was chosen for this patient based on his atypical braf fusion. we believe this is the first documented case of its successful use in neuroendocrine carcinoma in the pediatric population. conclusion: this case demonstrates the presence of braf fusion in a case of pediatric neuroendocrine carcinoma and significant response to single agent mek inhibition in this context. this cases raises the question as to whether the combination of a targeted inhibitor, in addition to either conventional chemotherapy or other braf inhibitors, might offer a better approach to therapy than current treatment options. albany medical center, albany, new york, united states background: warm autoimmune hemolytic anemia (waiha) is characterized by autoantibody, and occasional complement binding of protein antigens, on the surface of red blood cells at temperatures ≥ oc resulting in targeted destruction. we describe the case of a year old male with a history of evan's syndrome, poor immune response to vaccines and lymphoid hyperplasia, presenting with altered mental status and severe anemia, found to have a warm igg pan agglutinin with evidence of both intra and extravascular hemolysis. his course was complicated by respiratory failure requiring intubation, pulmonary emboli, enterococcus bacteremia and hypertension. he received multiple transfusions with only transient increases in hemoglobin. the aiha was refractory to multiple rounds of treatment with high dose steroids, ivig, rituximab, cyclophosphamide, bortezomib, plasma exchange and mycophenolate mofetil (mmf). objectives: given the refractory nature of our patient's aiha the decision was made to trial eculizumab, a monoclonal antibody targeting c complement, preventing its cleavage and activation, and shown to be effective in treatment of atypical hemolytic uremic syndrome and hemolysis due to an igm cold agglutinin. prior to eculizumab infusion, ch and sc b- assays were significantly elevated. design/method: the patient was given two doses of eculizimab days apart. results: his hemoglobin steadily rose independent of red cell transfusions with a corresponding decrease in reticulocyte count, ldh and ch levels. the patient has remained stable with a normal hemoglobin ( - g/dl) on maintenance steroids and mmf. although we cannot definitively conclude that eculizumab directly caused his recovery, the clinical course post-eculizumab suggests this may be an efficacious treatment for aiha. genetic testing showed monoallelic frameshift mutation of the nfkb gene and monoallelic missense mutation of the dock gene. given the role of nfkb in both immunodeficiency and autoimmunity, it is thought that the patient's phenotype is due to nfkb haploinsufficiency and he is currently considering hematopoietic stem cell transplant. st. joseph's regional medical center, paterson, new jersey, united states background: heterozygous -thalassemia typically manifests as thalassemia minor, characterized by mild microcytic hypochromic anemia with minimal clinical ramifications. coinheritance of -globin gene triplication has been reported to exacerbate the clinical and hematological phenotype ofthalassemia trait, due to increase in the alpha/non-alpha-chain imbalance. reported phenotypes range from asymptomatic thalassemia minor to moderate thalassemia intermedia, usually diagnosed in adulthood without transfusion dependence. this combination has been described in mediterranean, european and asian populations, but rarely reported in hispanics. objectives: to report two cases of unusually severethalassemia intermedia in hispanic patients with heterozygosity for triplicated -globin gene and a ( )-thalassemia allele. results: case : sixteen-month-old male of mexican descent presented with persistent microcytic anemia and jaundice. peripheral smear showed nucleated rbcs with basophilic stippling and target cells. hemoglobin electrophoresis revealed: hba- %, hbf- %, hba - . %. -globin gene testing revealed heterozygosity for ( ) mutation (ivsi-i, g→a). given the unusually severe anemia, -gene testing was performed which showed -globin gene(anti . ) triplication ( / ). at four years, he had splenomegaly and bilateral maxillary prominence. head ct showed irregular contour of the parieto-occipital region due to medullary expansion. due to significant persistent anemia ( - g/dl) and progressive bony deformities of the skull, patient began chronic transfusions at age eight after family declined splenectomy.case : fifteen-year-old female, of peruvian and honduran descent, presented for evaluation prior to cholecystectomy for gallstones and recurrent ruq pain. father had known thalassemia trait. her hb was . g/dl with hypochromia, microcytosis, and target cells. electrophoresis indicated -thalassemia trait (hba- %, hba - . %, hbf- . %), confirmed by gene testing (heterozygous for a ( ) mutation in codon c>t). given jaundice and gallstones, -globin gene analysis was ordered showing triplication ( / ). ruq pain resolved post-cholecystectomy, but she developed persistent painful splenomegaly. she began hydroxyurea to increase gamma-globin production and decrease excess alpha chains, but it was discontinued due to hematological toxicity. due to recurrent luq pain and progressive splenomegaly, she underwent laparoscopic splenectomy at age with resolution of symptoms and improved hemoglobin. conclusion: -globin gene testing should be considered in -thalassemia carriers with an atypical clinical presentation including hispanic patients. the wide variability in the phenotypic expression of (anti . ) mutation andthalassemia trait suggest interplay of other genetic factors which remain undefined. the clinically significant presentation amongst certain subjects, as in our two cases, makes it imperative to identify these factors to aid in phenotype prediction and genetic counseling. ashley bonheur, shivakumar subramaniyam, jogarao vedula, sucharita bhaumik nyu winthrop hospital, mineola, new york, united states background: wilms tumor (wt) is one of the most common solid malignant neoplasms in children. a diverse range of genes and mechanisms are implicated in wt pathogenesis. predisposing syndromes result from a disruption of wt gene, crucial for renal and gonadal embryogenesis. another gene is wt gene locus at p , an area of imprinting. the p tumor suppressor gene on chromosome p . is seen in patients with anaplastic histology. in addition to these genes, whole and partial chromosome gains of q, , q, , , & and losses of p, p, q, q, as well as loss of heterozygosity (loh) are commonly seen. some genetic markers appear to be predictive of outcome and are now incorporated into the assigning of risk-directed therapy. patients with loh at chromosome p and q are treated with more intensive chemotherapy, as they have been associated with increased risk of relapse and mortality. objectives: to describe a new complex translocation involving chromosome , , and in a case of pediatric wt. design/method: a four-year old female presented with abdominal pain and emesis. on exam, patient had a firm and large abdominal mass. radiologic studies revealed a complex lobulated right renal mass. right radical nephrectomy was performed. histopathologic studies showed wt with triphasic histologic features with blastema predominance, invasion of the lymphovascular and perinephric adipose tissues, perinephric lymph node involvement and no anaplasia. chest ct scan showed bilateral lung metastases. tumor cytogenetics showed an abnormal karyotype, a complex translocation of , , and . the rearrangement occurred due to translocation between chromosomal bands q and q , with an insertion of q - on the q region. pcr based genotyping using microsatellite markers additionally identified loh for chromosome p and q . the patient was treated for high risk stage iv wilms tumor with favorable histology and received intensive chemotherapy and radiation therapy to the flank and the lungs. she is now in remission months after, with no evidence of recurrence on surveillance scans. complex translocations associated with wt have not been rigorously studied. a question for further study is whether there is any relationship between recurrence potential with a complex translocation compared to common chromosomal abnormalities. further knowledge of the molecular pathology and genetic changes in wt will help the development of new targeted therapies, as well as new biomarkers to aid diagnosis, risk stratification, and monitoring of treatment and relapse. results: a week-old girl was referred for evaluation of an abnormal newborn screen. mother was a known carrier of hb khartoum trait while father was a known carrier of thalassemia trait. patient's hemoglobin quantification performed by capillary zone electrophoresis showed hbf %, hb variant %, and no detectable hba. the hb variant ran in the d zone, a pattern consistent with mother's hb. alkaline agarose gel electrophoresis banding pattern showed f/s. acid agarose gel electrophoresis pattern showed v/f. later testing revealed abnormal isopropanol stability with + precipitation at minutes. this electrophoresis pattern is consistent with the pattern previously reported of hb khartoum. clinically, the patient is a healthy, active child whom we have followed for two years. she has not had any significant anemia outside of her physiologic nadir. she has not had any hemolytic episodes, and her bilirubin levels have always been within the normal range conclusion: to the best of our knowledge, this is the only reported case of hb khartoum/ thalassemia. the proline to arginine substitution of hb khartoum introduces a charged group on the chain at the site of contact. the resulting unstable chains can dissociate into monomers and favor the formation of methemoglobin, leading to hemoglobin instability. we had wondered if this unstable hemoglobin might result in clinical hemolysis when challenged with oxidative stress, such as in periods of infection. however, in the two years we have followed this patient, she has never had a hemolytic episode. at two years of age, she has hbf . %, hb khartoum . %, and hba . %. whether hbf elevation is protective from oxidative stress remains to be determined as we continue to follow this child. university of puerto rico -medical science campus, san juan, puerto rico, united states background: gm gangliosidosis is a lysosomal disorder caused by -galactosidase deficiency due to mutations in the glb gene. it is a rare autosomal recessive neurodegenerative disorder with an incidence of about : , - : , live births worldwide. this neurological disorder has three clinical forms. gm type , or infantile form is characterized by psychomotor regression by the age of months, visceromegaly (hepatosplenomegaly), macular cherry red spot, facial and skeletal abnormalities, seizures, and profound intellectual disability. we present a -year-old female with gm type and acute lymphocytic leukemia (all). design/method: she was diagnosed with gm type at the st months of age and family history was remarkable for an older sister with gm type . diagnostic studies reveal homozygous exon of the glb gene for a sequence variant defined as c. c>t, predicted to an amino acid substitution p.aarg cs. results: patient presented to our hospital with petechiae in lower extremities, pallor and intermittent tracheal bleeding. physical examination shows a hemodynamically stable girl that is chronically ill dependent of mechanical ventilation, severe mental retardation and scatter petechiae at upper and lower extremities. laboratory workup revealed severe normocytic anemia (hgb: . g/dl) with immature peripheral cells and thrombocytopenia ( × /l). serum chemistry revealed increase ldh ( u/l), increase hepatic enzymes (ast: u/l), normal uric acid level. there was no evidence coagulopathy. chest x ray was unremarkable except for evidence of chronic pulmonary illness. abdominal sonogram hepatosplenomegaly. during hospitalization, bone marrow aspirate and biopsy was performed which was diagnostic of b cell acute lymphoblastic leukemia (all) with . % lymphoblast and orderly myeloid/erythroid maturation. flow cytometry: % b lymphoblast with aberrant phenotype c/w b-acute lymphoblastic leukemia. karyotype revealed hyperdiploid female of favorable prognosis. cytogenetic by fish: hyperdiploid all with extra copies of runx and igh (no bcr-abl translocation). family was oriented about the new diagnosis and the dismal prognosis in conjunction to her primary condition. parents agree on no chemotherapy treatment for all with only supportive treatment. to this date, there is no evidence in literature that has previously described association of gm and leukemia. life expectancy of patient's primary condition is null therefore, correlation with leukemia might not be a coincidental finding. this patient opens the possibility of malignancy as part of gm type thus, malignancy diagnosis should be considered as part of their medical lifetime course. university of south florida, tampa, florida, united states background: hematological manifestations related to hiv infection are not uncommon, with thrombocytopenia having an estimated prevalence of - %. the pathophysiology is likely multifactorial. studies suggest that the primary mechanism may be immunologic resulting in accelerated platelet destruction. additional theories suggest that infection of megakaryocytes may also play a role causing inadequate platelet production. treatment of hiv-related thrombocytopenia is challenging. first-line treatments include initiation and optimization of antiretroviral therapies, immunoglobulin (ivig), and glucocorticoids. however, this approach is not effective in all patients and second line treatment options are less well studied, particularly in the pediatric population. objectives: we aim to present and discuss the case of a year old patient with perinatally acquired hiv- infection and persistent thrombocytopenia who, after failing first line therapies, showed normalization of platelet count on the novel thrombopoietin receptor agonist, eltrombopag. design/method: a retrospective chart review of the case patient's medical record was conducted. additionally, a thorough literature review was performed on this topic including the pathophysiology of hiv related thrombocytopenia and its treatment modalities. the patient required monthly ivig infusions for about year, but did not show a sustained response, often with platelet count dropping to less than , in between infusions. after initiation of mg eltrombopag daily the patient showed a sustained increase in platelet count (range , - , ). during a brief week lapse in eltrombopag treatment his platelet count dropped to , . upon re-initiation of therapy his count increased to , . the patient has remained asymptomatic, off of ivig for over one year, with undetectable hiv viral load and greater than cd t cell counts. no side effects or grade laboratory abnormalities were reported. conclusion: treatment of hiv-related thrombocytopenia can be challenging. first line therapies, including ivig and glucocorticoids, are not effective in all patients. several other treatment modalities have been utilized, including anti-d immunoglobulin, dapsone, danazol, interferon alfa, vincristine, thrombopoetic growth factors including romiplostim and eltrombopag, or splenectomy, but these are less well studied. this represents the first reported case of a pediatric patient with hiv who showed a positive response to eltrombopag with a sustained improvement in platelet count and no adverse effects from treatment. eltrombopag may be a safe alternative to first line therapies in those patients with hiv and refractory thrombocytopenia, however additional studies are needed. university of illinois college of medicine at peoria, peoria, illinois, united states background: achromobacter xylosoxidans is a gram negative rod with peritrichous flagella which causes rare opportunistic infections most commonly encountered by immunocompromised patients. it is primarily associated with uncomplicated bacteremia, cather-associated infections, and pneumonia. most reports of bacteremia associated with a. xylosoxidans are nosocomial, associated with neoplasm, and occurring mainly in adults. most reported infections with a. xylosoxidans in children are associated with cystic fibrosis. there are very few reported cases of septic shock from a. xylosoxidans bacteremia and pneumonia in the pediatric oncology population. objectives: to describe a rare case of a. xylosoxidans septic shock in a pediatric patient with relapsed neuroblastoma results: a -year old boy with history of stage iv highrisk neuroblastoma underwent standard frontline therapy with chemotherapy, hematopoietic stem cell transplant, radiation therapy, and immunotherapy, followed by a dfmo trial for maintenance. his -month follow-up scans demonstrated relapse and he was subsequently treated with additional chemotherapy, surgical resection, and mibg therapy, crizotinib for an eml -alk fusion and finally ifosfamide, carboplatin and etoposide (ice). he developed neutropenic fevers and was started on cefepime, vancomycin and fluconazole. blood cultures were initially negative. on the th day of fever, his previously scheduled pet scan was performed during hospitalization and showed new pulmonary opacities. he did not have respiratory symptoms, but therapy was escalated to meropenem, vancomycin and amphotericin. emergent bronchoscopy was performed the same day, with all bacterial and fungal cultures remaining negative. overnight, he developed tachypnea and saturations in the upper s, requiring nasal cannula. ir-guided lung biopsy was performed the next day, a flexible bronchoscopy was done to remove blood clots in the airway, the patient was placed on a ventilator, femoral lines were placed, granulocytes ordered and pressors were started for deterioration to presumed septic shock. arterial and femoral lines were placed but patient continued to have hemodynamic instability on multiple pressors. the following day, blood and respiratory cultures returned positive for results: at days after the start of iti, the inhibitor was < . bu and continued undetectable months after initiation of iti therapy. in this patient, iti with high-dose plasma-derived factor viii and von willebrand factor (vwf) complex was well tolerated and effective. genetic analysis confirmed a large factor viii gene duplication of exons to . we believe our patient developed inhibitor so quickly ( exposure days) due to the possibility of this mutation causing a frameshift that introduces a premature termination codon. this might be functionally similar to a deletion in the factor viii gene which poses the highest risk for inhibitor development in patients with severe hemophilia a. this variant has only been identified previously in two unrelated patients diagnosed with severe hemophilia a. this duplication is not listed in dbsnp variant database, nor observed in the general population database. our case proves the effectiveness of this method for patients with severe hemophilia a and an inhibitor. it also shows that more research is needed to identify patients at risk for inhibitor development. background: mercaptopurine ( -mp) is a prodrug that is a core component of maintenance chemotherapy for patients with a diagnosis of acute lymphoblastic leukemia (all). suppression of the neutrophil count is used to demonstrate adequate dosing of -mp during this phase of therapy. bone marrow suppression is mediated by the active metabolite -thioguanine ( -tgn), whereas the metabolite -methylmercaptopurine nucleotides ( -mmpn) has been shown to cause hepatotoxicity. allopurinol has been used infrequently in all maintenance therapy in the setting of skewed metabolism when adequate myelosuppression is difficult to achieve due to excessive hepatic toxicity. when given in combination with allopurinol a reduced dose of -mp may result in both increased -tgn levels and decreased -mmpn levels. objectives: describe the characteristics and clinical course of patients treated with allopurinol and reduced dose -mp during maintenance chemotherapy for all. we performed a retrospective chart review of patients at aflac cancer and blood disorders center of children's healthcare of atlanta with new diagnoses of b or t-cell all who received allopurinol during maintenance chemotherapy. we identified eleven patients with b-cell or tcell all who received allopurinol adjunctive therapy during maintenance chemotherapy at a single institution between - . these patients received adjunctive allopurinol for - weeks (median weeks) with reduced -mp ( - % of full dose). all ten patients with genetic testing for thiopurine s-methyltransferase (tpmt) had wildtype genotype associated with normal enzyme levels. indications for allopurinol use were most commonly unfavorable -mp metabolite levels, transaminitis (n = ), pancreatitis (n = ) and hyperbilirubinemia (n = ). favorable metabolite shift was achieved in all patients. liver enzymes improved in of patients with transaminitis after initiation of allopurinol/reduced -mp. three patients who experienced pancreatitis during maintenance did not have recurrence after initiation of allopurinol ( of these patients previously reported). six patients developed pancytopenia while on allopurinol, and two of those patients developed pancytopenia severe enough to require allopurinol cessation. four patients developed isolated anemia (hgb < . g/dl) without thrombocytopenia or severe neutropenia. no patient has experienced a recurrence of leukemia. overall, treatment with allopurinol and reduced dose -mp was successful in producing a favorable -mp metabolite distribution and reducing toxicity. therapy was generally tolerated; however a major and notable side effect was pancytopenia, in two cases severe enough to stop allopurinol treatment. anemia may be more prominent with allopurinol usage. allopurinol effect is variable among individual patients despite normal tpmt genotypes. baylor college of medicine, houston, texas, united states background: congenital sideroblastic anemia, b-cell immunodeficiency, periodic fevers and developmental delay syndrome (sifd) is a rare inherited sideroblastic anemia syndrome, first described in with clinically similar cases. genetic variations of trnt were identified as causative. objectives: to present an unusual presentation of a patient with sifd complicated by diagnosis of concomitant alpha thalassemia trait. design/method: retrospective chart review. a five month old male infant was referred to our hematology center for evaluation of elevated hemoglobin barts identified on newborn screen. despite numerous attempts, blood work was unable to be collected. at seven months of age he had microcytic anemia (hemoglobin . g/dl, mean corpuscular volume fl) more severe than what would be expected with alpha thalassemia trait. no variant hemoglobin was identified with isoelectric focusing or high performance liquid chromatography. by nine months of age he developed growth failure, intermittent emesis with fevers, developmental delays (predominantly gross motor), hearing loss, a disproportionally large head and coarse, thinning hair. over the next ten months, he was seen by numerous specialists for seemingly unconnected problems including sensorineural hearing loss, elevated liver enzymes and growth hormone deficiency. alpha globin analysis revealed deletion of two alpha globin genes. at months of age, he was admitted with one week of fevers, jaundice, and emesis. peripheral blood smear showed microcytic hypochromic anemia with marked anisopoikilocytosis including target cells, elliptocytes, tear drops, spherocytes, poikilocytes, marked polychromasia, and coarse basophilic stippling. given the inconsistency of his laboratory findings with the diagnosis of alpha thalassemia trait and clinical syndromic findings, bone marrow biopsy was performed which revealed rare ringed sideroblasts. one month later whole exome sequencing revealed trnt splicing variant c. - c>g and novel missense variant c. a>t consistent with sifd. hemoglobin barts on newborn screen with moderate to severe microcytic anemia directed initial diagnostic work-up towards variant alpha thalassemia. as additional medical conditions developed the focus shifted to a unifying syndrome. compared to previously described cases, our patient was diagnosed at an older age, presented with anemia rather than episodes of febrile illnesses, and had rare sideroblasts on bone marrow examination. diagnosis in this case led to identification of the novel c. a>t variant in his sister who had similar, but milder, features. sifd is a rare disease with variable phenotypic severity making diagnosis challenging without high index of suspicion which is crucial for appropriate management. wiseman, blood, . chakraborty, blood, background: cholelithiasis is uncommon in childhood. cholelithiasis is known to occur more frequently in children with predispositions, including female sex, obesity, parenteral nutrition, previous abdominal surgery, use of oral contraceptives, family history of gallstones, chronic hemolytic anemias, hepatobiliary disease, or exposure to specific drugs. although there have been occasional case reports linking cholelithiasis to childhood leukemia or leukemia therapy, the prevalence and risk factors of cholelithiasis in patients with childhood leukemia remain unclear. objectives: to estimate the prevalence of cholelithiasis in patients diagnosed with childhood acute lymphoblastic leukemia (all), and to evaluate possible risk factors for the development of cholelithiasis in patients with childhood all. we performed a computer-assisted review of the electronic medical records of patients diagnosed for b or t-cell all at children's healthcare of atlanta in the period from to . patients with diagnoses of cholelithiasis, cholecystitis or who had a cholecystectomy were identified. possible risk factors of age, sex, bmi, history of abdominal surgery and parenteral nutrition use were abstracted. patients with underlying chronic hemolytic anemia or pre-existing gallbladder disease were excluded. results: seventeen cases of cholelithiasis and cases of cholecystitis without documented cholelithiasis were identified. among patients with cholelithiasis, were female. median age at diagnosis of cholelithiasis was . (range . - . ) years. seven patients had no symptoms referable to cholelithiasis at the time of diagnosis. the median age of leukemia diagnosis among these patients was . (range . - . ) years. the median interval from diagnosis of leukemia to gallbladder disease was . years. four patients had bmi over the th percentile for age. two patients had a prior history of intraabdominal surgery. no patient received oral contraceptive pills. six patients received parenteral nutrition for more than days. there was no documented family history of cholelithiasis. seven patients did not receive any cholelithiasis directed therapy. two patients were managed with medical management only, with endoscopic retrograde cholangiopancreatogram with stone extraction, and with cholecystectomy. our study estimates the prevalence of cholelithiasis in childhood lymphoblastic leukemia to be . %, higher than the reported prevalence in the general pediatric population of . - . %. although our cohort size is small, it appears that all therapy and supportive care modalities associated with all are likely to play a larger role in the development of cholelithiasis than known predisposing factors in the general population. further studies are warranted. background: an uncommon side effect of intravenous immunoglobulin (ivig) administration is clinically apparent, sometimes severe hemolysis. we describe a severe case of coombs-positive hemolytic anemia secondary to ivig administration. ivig is a blood derivative manufactured from pools of , to , individual plasma donations. ivig is not abo-type restricted, so anti-a, anti-b and anti-a,b isoagglutinins are detectable. objectives: to describe a rare but serious type of transfusion reaction leading to gross hemolysis after ivig administration. results: a -year-old male with a past medical history of obstructive sleep apnea and obesity was admitted to the pediatric intensive care unit for adenoviral pneumonia and subsequent respiratory failure requiring mechanical ventilation. he had a complex hospital course with many complications including acute respiratory distress syndrome (ards), septic-shock, and coombs-positive hemolytic anemia. the patient was treated with commercial ivig (baxter/baxalta) -mg/kg daily for five days. he had two isolated episodes of severe hemolysis in relation to ivig administration requiring multiple transfusions of packed red blood cells (prbc). examination of pre-transfusion peripheral blood smear showed spherocytosis with rouleaux formation and large clumped rbc aggregates. the patient's blood type was classified as blood group a, rh-negative and his initial prbc transfusions were of this type. subsequently, the patient's coombs test was found to be positive using polyspecific and anti-igg typing sera. the patient's antibody screen against reagent group o screening cells was negative ruling out autoimmune hemolytic anemia. however, type specific anti-a antibodies were detected in his plasma as well as the acid eludate prepared from the coombs-positive red blood cells. it was concluded that the patient's hemolysis was due to anti-a antibodies presumed to arise from ivig. the patient's rbc transfusions were changed to o-negative blood and the hemolytic process resolved. the patient ultimately died due to complications of ards. although hemolysis is a known side effect of ivig, it is rarely considered when deciding to administer ivig. in addition, it has rarely been described in the pediatric population. ivig is used in the treatment of a growing number of medical conditions. due to the critical nature of many of these patients, hemolysis secondary to ivig may not be considered and continued blood transfusions with the patient's specific blood type may be used. it is crucial to remember that severe hemolysis can occur from ivig, and the importance of transfusing with blood group o, rh-negative blood when applicable. university of maryland medical center, children's hospital, baltimore, maryland, united states background: coagulopathy is a well-described complication of acute promyelocytic leukemia (apml), and remains a leading cause in induction failure. with treatment, coagulopathy associated with apml has been shown to rapidly improve. multiple organ dysfunction syndrome (mods) in apml, including acute respiratory distress syndrome (ards), has been associated with infection, traumatic injury, malignant infiltration, and cytokine release syndrome. when mechanical ventilation is no longer sufficient, extracorporeal membrane oxygenation (ecmo) can be considered; however, coagulopathy, severe end-organ damage, and malignancy are all relative contraindications to initiation of treatment. we report the case of a -year-old female presenting in respiratory failure, disseminated intravascular coagulopathy (dic), with intracranial hemorrhage, and mods, diagnosed with apml, successfully treated with ecmo therapy. design/method: retrospective case analysis and literature review. our patient, a -year-old female was admitted in respiratory failure and altered mental status, following a fall shortly prior to presentation. initial laboratory values were notable for pancytopenia, dic, and acute renal failure. a non-contrast head ct showed left temporal lobe intraparenchymal hemorrhage. she was diagnosed with apml by peripheral smear, later confirmed by fish for t( : ), and was started immediately on high-risk induction chemotherapy as per cog protocol aaml , including all-trans retinoic acid, arsenic trioxide, idarubicin, and dexamethasone. cvvhd was required for acute renal failure. despite maximal respiratory support, she remained hypoxemic, with oxygenation index of , pao /fio ratio of . ecmo was initiated hours after start of induction, hours after admission. coagulopathy resolved on day of induction, ecmo was discontinued after days, mechanical ventilation and cvvhd were stopped after days and she continued to improve, eventually achieving remission with few neurologic side effects. despite relative contraindications to ecmo, this patient was successfully treated with ecmo without significant neurologic side effects. the correction of her coagulopathy was multifactorial: ) restoration of adequate oxygen delivery via ecmo improving endothelial function; ) successful organ support to allow sufficient response to induction chemotherapy with atra leading to the terminal differentiation of leukemic blasts; ) complement and contact system activation through contact with ecmo circuitry. this case illustrates that ecmo can still be considered in patients despite coagulopathy and end organ damage. sinai hospital of baltimore, baltimore, maryland, united states background: primary polycythemia vera is an extremely rare diagnosis in the pediatric patient and is defined by a marked elevation of red blood cells due to erythropoietin-independent mechanisms. presentations of this disorder range from the asymptomatic person to severe thrombotic events, such as budd-chiari syndrome or cerebrovascular stroke. mutations in the jak gene are found in adult and pediatric patients with polycythemia vera; however, the jak v f mutation is less commonly identified in pediatric patients. we describe an otherwise healthy -year-old female who presented with a significantly elevated total erythrocyte count, hemoglobin, and platelets, incidentally discovered upon routine annual blood work obtained by her pediatrician. design/method: this is a report and discussion of a rare case. demonstrated cellular marrow with trilineage hematopoiesis and no dysplasia. cytogenetics were not assessed. his hemoglobin and platelet count recovered but leukopenia and neutropenia persisted. follow-up evaluation at three months revealed fevers, ongoing cytopenias, a one-month of a nodular skin rash on the trunk and extremities resembling erythema nodosum, and hepatitis (peak alt and ast of , and , , respectively). following clinical evaluation, a skin biopsy was performed and was remarkable for atypical lymphocytes within the subcutis with t-cell markers, a high ki- , and positive tia- , perforin, and -f immunoperoxidase stains. negative stains for cd , cd , and ebv were noted. these results are consistent with sptcl. additional evaluation did not support a diagnosis of hlh. a staging evaluation was performed. pet-ct showed widespread hypermetabolic subcutaneous activity in the legs, trunk and skull and diffuse marrow hyperplasia. bone marrow demonstrated involvement with precursor b-cell acute lymphoblastic leukemia, with a mll gene rearranagement. his skin biopsy was retrospectively stained with tdt, cd , pax- , cd a, and cd with negative results, and a blood smear taken at the time of the skin biopsy did not demonstrate leukemic cells. conclusion: this is the first report of a patient with sptcl having a synchronous malignancy. the patient is doing well, currently in the maintenance phase of treatment for his all, and his skin disease has resolved on pet-ct. while it is possible that his presentation was a function of chance, the possibility of an underlying immune dysfunction or cancer predisposition warrants further investigation. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hereditary xerocytosis (hx) is a rare red blood cell (rbc) dehydration disorder, characterized by variable hemolysis and propensity to iron overload. hx is often misdiagnosed as hereditary spherocytosis (hs). while splenectomy is curative for hs, it is relatively contraindicated in hx due to a substantial thromboembolism risk, signifying the importance of delineating these diseases. blood smear abnormalities are variable and often insufficient to make an accurate diagnosis. osmotic-gradient ektacytometry and genetic confirmation are critical in distinguishing these overlapping disorders. objectives: describe a family with hx, initially misdiagnosed as hs. discuss the importance of distinguishing these disorders and the utility of ektacytometry in making this distinction. design/method: a -year-old caucasian male was diagnosed with hs after presenting with prolonged neonatal jaundice starting on the first day of life. he described mild scleral icterus and history of intermittent jaundice and dark urine, without need for transfusions. his father, paternal uncle and paternal grandmother were all diagnosed with hs during childhood and underwent cholecystectomy. additionally, his father underwent splenectomy for abdominal pain. the child's blood counts revealed compensated anemia (hb . gm/dl) and reticulocytosis (arc × /mcl) with increased mcv ( . fl) and mchc ( . gm/dl). blood smear showed increased polychromasia and poikilocytosis with rare spherocytes and few stomatocytes. while the child had normal ferritin, his father had iron overload (ferritin ng/ml) despite no prior transfusions. osmotic-gradient ektacytometry profile of the child and father's rbcs showed a characteristic left-shifted, bell-shaped curve with decreased omin and ohyp, diagnostic of hx. the family is currently undergoing genetic studies. despite clinical similarities between hs and hx, distinguishing these diseases has significant management implications. hx is a disorder of rbc permeability, causing shortened rbc survival. stomatocytes on blood smear can raise suspicion for hx, but are insufficient to make an accurate diagnosis. identifying characteristic biomechanical membrane properties using osmotic-gradient ektacytometry is the gold standard for clinical diagnosis, which can then be confirmed by molecular studies. hs and hx can be easily and reliably distinguished using ektacytometry, as both disorders have very distinctive curves representing different rbc deformability patterns. after hx diagnosis was made, we counseled the family against splenectomy, as the risk of thromboembolism is significantly increased in hx compared to hs, and the father was diagnosed with iron overload. conclusion: hx is commonly misdiagnosed as hs. this case highlights the importance of making this distinction, and the utility of osmotic-gradient ektacytometry in reliably distinguishing these conditions. penn state health children's hospital, hershey, pennsylvania, united states background: relapsed acute myeloid leukemia (aml) presenting as an isolated central nervous system myeloid sarcoma (cns ms) is very rare and its treatment is not well-defined. thiotepa, vinorelbine, topotecan and clofarabine (tvtc) has been successful for re-induction therapy to induce remission prior to hematopoietic stem cell transplant (hsct). objectives: to describe our experience in utilizing tvtc therapy in two children with no extramedullary disease at initial diagnosis who presented with relapsed aml as intracranial myeloid sarcomas. results: case : month-old female was diagnosed with flt negative aml and completed treatment per the children's oncology group (cog) aaml study on the low risk arm without bortezomib. cerebral spinal fluid (csf) negative at diagnosis. fish testing positive for tcf gene deletion of unknown significance. mrd was undetectable after induction i and remained undetectable after each cycle. nine months off therapy, recurrent headaches prompted mri imaging which revealed two posterior fossa masses. csf and bone marrow testing were negative. stereotactic biopsy of the larger mass confirmed recurrence of aml. patient underwent two cycles of tvtc with a total of seven doses of intrathecal cytarabine with almost near resolution of the cns ms. completed cranial radiation and proceeded to allogeneic stem cell transplant with unrelated cord marrow donor and is disease free at approximately day + .case : year-old female diagnosed with flt and mll negative aml and completed treatment per cog aaml study on the low risk arm without bortezomib. csf negative at diagnosis. mrd was undetectable after induction i and completed therapy without complications. two months off therapy, a retrospective analysis of her diagnostic bone marrow by the cytogenetic laboratory to test a new panel identifying novel q partners revealed a cryptic insertional : (mllt /mll(kmt a) translocation. at four months off therapy, acute mental status changes prompted mri imaging which revealed two intracranial ms and lumbar spine involvement. resection of the larger lesion for symptomatic relief confirmed the mllt /mll(kmt a) fusion. csf positive for blasts and marrow negative for relapsed disease. patient completed two cycles of tvtc with a total of seven doses of it cytarabine with near resolution of cns disease (only mm contrast enhancement in the medulla). she received craniospinal radiation and is awaiting improvement in her cardiac function before proceeding to hsct. conclusion: tvtc is a successful reinduction regimen for relapsed aml with cns ms prior to hsct. background: acute severe anemia can be a life-threatening medical condition. the differential is quite broad for possible etiologies of acute severe anemia, including autoimmune hemolytic anemia (aiha) and atypical hemolytic uremic syndrome (ahus). autoimmune hemolytic anemia is an antibody-mediated process that targets the protein antigens located on the surface of red blood cells. treatment options for aiha include corticosteroids, with up to % of patients being responsive, with some requiring splenectomy. atypical hemolytic uremic syndrome is a medical urgency, defined as the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. the etiology is usually due to genetic causes, or less commonly, due to autoantibodies or idiopathic reasons. prognosis is very poor. objectives: differentiating between autoimmune hemolytic anemia and atypical hemolytic uremic syndrome can be a time-sensitive diagnostic dilemma while the patient is in critical condition, but this important delineation can vastly alter therapeutic options. design/method: here we discuss two cases highlighting the diagnostic workup involved in differentiating between atypical hemolytic uremic syndrome and autoimmune hemolytic anemia. patient a is a -year-old male who presented in extremis with severe anemia, uremic encephalopathy, and severe acute renal injury requiring hemodialysis and multiple blood transfusions. patient b is a -month-old male, who also presented in extremis with respiratory failure secondary to adenovirus/rhinovirus/enterovirus, with acute progressive renal failure and microangiopathic hemolytic anemia, requiring hemodialysis and cardiorespiratory support. : patient a underwent a full hematologic and infectious disease workup. subsequent laboratory studies confirmed enteropathogenic e.coli (epec) in the patient's stool; blood cultures remained negative. renal biopsy results were consistent pigment nephropathy. bloodwork indicated positive direct coombs. patient a was ultimately treated with steroids mg/kg/day, with significant improvement. patient b also included a full hematologic work-up, including adamts activity and ahus genetic panel, as well as full infectious disease work-up. subsequent laboratory test-ing revealed blood cultures growing streptococcus pneumoniae, with adamts activity at % (adult ref range: >/ = %), and normal complement levels. imaging findings also supported diagnosis of ahus. the management of a critically ill patient with acute severe anemia requires a thorough hematologic and infectious disease work-up. while molecular and genetic are helpful in definitive diagnosis of ahus, the utility of such results is limited by time. overlapping clinical presentation of a patient in extremis due to acute severe hemolytic anemia with progressive renal failure presents a rather broad differential, with time-sensitive treatment and prognostic implications. the favorable response to steroids delineates aiha from hus. background: d- -hydroxyglutaric aciduria (d- -hga) is a rare metabolic disorder characterized by developmental delay, hypotonia, and bi-allelic mutations in d- hydroxyglutarate dehydrogenase (d hgdh) or isocitrate dehydrogenase (idh ). metaphyseal chondromatosis with d- -hydroxyglutaric aciduria (mc-hga) is a type of d- -hga that has been previously reported in seven patients (omim ; pmid ), three of whom had somatic mosaicism for r variants in isocitrate dehydrogenase (idh ). we describe a -year-old boy with mc-hga who subsequently developed acute myeloid leukemia (aml) and was found to have a r variant in idh in a leukemic bone marrow sample. we report the first case of aml with this metabolic disorder. design/method: a -year-old hispanic boy presented with short stature, developmental delay, abnormal skin pigmentation, and unilateral congenital cataract. workup revealed multiple skeletal enchondromatosis and elevated urine d- -hydroxyglutaric acid levels. he was diagnosed with mc-hga. no pathogenic variants in d hgdh, idh and idh were identified in peripheral blood. germline testing with biopsies of skin lesions was declined by the family. two years later, he presented with streptococcal sepsis and pancytopenia. blasts were noted on peripheral smear. bone marrow morphology was consistent with acute myelomonocytic leukemia (∼ % blasts). chromosome analysis showed normal xy, and molecular testing by pyrosequencing idh and idh revealed a r c variant in idh ( % mosaicism). the patient is being treated as per the cog study aaml . end of induction i bone marrow aspirate was hemodiluted, but there was no obvious residual disease by flow cytometry ( . - . % sensitivity) or morphology. the previously identified idh variant was no longer detectable (limit of detection < %). although targeted therapy for aml with idh mutation is currently in phase i clinical trials in adults, there is no safety or efficacy data for using idh inhibitors in children. treatment with ivosidenib is therefore not currently an option for our patient. conclusion: this is the first case of aml reported with this rare metabolic disorder. somatic r variants in idh have been identified in three other mc-hga cases. this same mutation leads to the accumulation of d- -hydroxyglutarate in gliomas and aml. without any confirmed germline mutation or somatic mosaicism testing of multiple specimen sources, we can only speculate that the patient has an underlying somatic idh mutation associated with mc-hga which subsequently led to leukemogenesis. we present the first case of this association, to increase index of suspicion for development of aml in children with metabolic disorders associated with variants in idh . background: congenital combined deficiency of the vitamin k-dependent coagulating factors (vkcfd) is a rare heterogeneous autosomal recessive bleeding disorder. vkcfd is caused by mutations in the genes of either gamma-glutamyl carboxylase (ggcx) or vitamin k epoxide reductase complex (vkorc), which are responsible for the gammacarboxylation of vitamin k dependent proteins (vkdps) allowing for their activation. the clinical presentation ranges from no bleeding to intracranial hemorrhage. to date, vkcfd has been reported in few patients worldwide. objectives: we report a case of a girl with novel homozygous mutation of the ggcx gene, highlighting her clinical and biochemical characteristics with a review of the literature. a -month-old girl of consanguineous emirati parents, presented to our hospital with a history of bleeding from puncture site after receiving her second-month vaccine. that was associated with episodes of mild mucosal bleeding. review of systems was negative for jaundice, steatorrhea and failure to thrive and physical exam was unremarkable. investigations revealed markedly prolonged pt and aptt with high inr. fibrinogen, hemoglobin and platelets were always normal. activities of vitamin k-dependent factors including fii, fvii, fix, fx, protein c and s were all low. a measurement of proteins induced by vitamin k absence (pivka-ii) was done and came very high. this was associated with a mild elevation in liver enzymes but normal liver function test. the picture was supporting vitamin k deficiency, and as a result, she was started on oral vitamin k supplements of mg/day. she responded partially to vitamin k and required higher doses to stabilize her inr. after excluding acquired causes and due to her requirement of high doses of vitamin k, a mutation in either ggcx or vkorc genes was suspected. genetic analysis was conducted for her which revealed a novel missense homozygous mutation in the ggcx gene (c. a>t) confirming the diagnosis of combined deficiency of vitamin k-dependent clotting factors type . the asymptomatic parents were both heterozygous for the same mutation. results: she is currently stable on mg/day of vitamin k supplements. conclusion: vkcfd is a rare bleeding disorder with an overall good prognosis due to the availability of several effective therapeutic options. the function of the mutated gene is unknown. our patient demonstrated a partial response to vitamin k supplements suggesting presence of a residual carboxylation capacity and a possible role of this gene in the enzymesubstrate interactions. university of alabama at birmingham, birmingham, alabama, united states s of s background: gata is a zinc finger transcription factor that plays a critical role in the regulation of hematopoiesis and lymphatic angiogenesis. mutations leading to gata deficiency (gd) have been linked to a variety of clinical conditions. patients with gd have a striking predisposition to develop myelodysplastic syndrome (mds), acute myeloid leukemia (aml), or chronic myelomonocytic leukemia (cmml). acute lymphoblastic leukemia (all) has not been associated with gd, although the association of bcell all and gd has been previously reported. objectives: to describe a unique association of gata deficiency and t-cell all in a young child. results: an -year-old female presented with a one-week history of fever and malaise. she had a significant past medical history of verruca plantaris and self-resolving leukopenia associated with febrile illnesses. significant family history included sister with neutropenia and human papilloma virus (hpv) infection, and mother with neutropenia, monocytopenia, atypical mycobacterial infections, and hpv infection. peripheral blood revealed hemoglobin . g/dl, hematocrit . %, platelets , /ul, and white blood cell , /ul (neutrophils /ul, lymphocytes /ul, monocytes /ul). patient underwent a bone marrow biopsy demonstrating lymphoblast infiltration. flow cytometry analysis demonstrated monoclonal lymphoid blast population that co-expressed cd , cd , cd , nuclear tdt, cd , however, lacked expression of cd , cd , cd , cd , hla-dr, or myeloperoxidase. findings were consistent with tcell all with aberrant myeloid markers. cytogenetics analysis revealed ,xx,dic( ; )(p . ;p . ). patient began treatment as per children's oncology group aall and achieved remission at the end of induction. course of therapy was complicated by episodes of fever, reciprocating junctional tachycardia, asparaginase-associated thrombosis, viral meningitis, recurrent episodes of verruca plantaris, and resistant streptococcus pneumoniae or haemophilus parainfluenza infections causing chronic cough. later, she was also found to have low igm levels; after completion of therapy, she developed monocytopenia. lymphocyte subset panel revealed absent b cells, decreased number of natural killer (nk) cells, and cd /cd inversion. further work-up included gata sequence analysis that showed heterozygous nonsense mutation (c. c > t/c; reference nm_ ) likely resulting in gata haploinsufficiency. patient continues to be in remission, is receiving monthly immunoglobulin replacement and is on azithromycin for atypical mycobacterial prophylaxis. surveillance bone marrow biopsies have shown no evidence of mds or leukemia, however, have demonstrated persistent hypocellularity. the possibility of undergoing an allogeneic bone marrow transplant is actively being discussed given its curative potential. clinicians should be aware that t-cell all may be associated with gata deficiency. cincinnati children's hospital medical center, cincinnati, ohio, united states background: treatment for severe hemophilia a is centered on factor viii (fviii) replacement therapy. development of an alloantibody (inhibitor) against fviii is a significant treatment complication occurring in as many as - % of patients. high titer inhibitors render treatment with factor viii ineffective, necessitating the use of bypass agents that may not achieve hemostasis with the same efficacy. considering the substantial ramifications of inhibitor development on treatment, eradication of inhibitors is of great importance to achieve adequate hemostasis in this patient population. desensitization by immune tolerance induction (iti) is the primary method of inhibitor elimination. however, not all patients respond to iti. immunomodulation may be considered as the next line of therapy, although controversy remains in regards to agent selection and use. objectives: there is incomplete data on the use of immunomodulation therapy for inhibitor eradication in severe hemophilia a. we present a case of a pediatric patient with severe hemophilia a and high titer inhibitor who failed initial iti therapy to better illustrate potential treatment options for the future. design/method: a retrospective chart review was performed on a patient with severe hemophilia a at cincinnati children's hospital medical center. results: an -year-old caucasian male with severe hemophilia a secondary to intron inversion, was initially diagnosed following extensive bleeding after circumcision at birth. he was identified as having an inhibitor ( bethesda units (bu)) at months of age after exposure days of treatment. he failed multiple attempts of iti, with recombinant and plasma-derived (pd) fviii. he was advanced to immunomodulation therapy in combination with pdfviii, however demonstrated anaphylaxis to rituximab and ofatumumab. he underwent tolerization to rituximab, and received a six month course with a partial response (nadir of . bu). months following last dose of rituximab, a rising inhibitor titer ( . bu) was found. mycophenolate mofetil (mmf) was initiated with subsequent inhibitor stabilization and a decreasing titer ( . bu) over the course of the following year. mmf has been well tolerated without major side effects or infection throughout therapy. conclusion: development of an inhibitor against fviii is a considerable complication in patients with severe hemophilia a. use of immunomodulatory therapies following iti failure remains controversial. mmf has not been well studied in this patient population. we report a case of a patient who is being successfully treated with mmf with minimal side effects. further prospective studies should be considered to further define the role of mmf immunomodulation therapy. background: down syndrome (ds) children with aml (ds aml) have higher cure rates than their non-ds counterparts. outcomes for refractory/relapsed cases, however, remain dismal. somatic mutations of the gene encoding the transcription factor gata in ds aml patients are responsible for the observed hypersensitivity of ds aml blasts to cytosine arabinoside (ara-c). in view of excellent survival rates (approaching %) of ds aml patients, the ongoing children's oncology group (cog) aaml study seeks to determine the feasibility of treating standard risk (minimal residual disease/mrd negative) ds aml patients using a reduced dose ( -fold decrease) ara-c backbone. although results from japanese trials with this approach are promising, north american and european data are conflicting. although chromosome rearrangements in ds aml do not appear to carry the same adverse prognostic significance as in non-ds aml, monosomy in ds aml patients has been associated with a moderately worse outcome. isochromosome q, however, is rare and has only been reported in previous cases of ds aml. objectives: to report our institutional experience of very early relapse involving cases of ds aml patients treated per the reduced dose ara-c arm ( . g/m ) of the aaml study. design/method: we hereby report the disease course and cytogenetics of the above ds aml patients. : patient is a month old caucasian female who had gata mutation negative aml. patient is a -year old caucasian male whose chromosomal analysis revealed isochromosome q ( copies of the long arm of chromosome ). both patients achieved negative mrd (< . %) after induction i chemotherapy with thioguanine, low-dose ara-c and daunorubicin and proceeded per the reduced dose ara-c arm of aaml . patient relapsed immediately after completion of chemotherapy. salvage chemotherapy with mitoxantrone/high dose ara-c (hidac) failed to induce a second remission and the patient subsequently died of disease. patient relapsed within months from end of therapy. the patient underwent salvage chemotherapy utilizing a hidac backbone and remains in disease remission. the noted very early relapse following a reduced dose ara-c regimen in our above ds aml children suggests that testing for gata mutation and chromosome rearrangements may play a useful role in the development of future risk-stratified treatment strategies for ds aml. university of rochester, rochester, new york, united states background: in developed countries in the st century, severe nutritional deficiency is not an often considered differential diagnosis of unexplained childhood anemia. aside from iron deficiency anemia, vitamin deficiency severe enough to impact hematopoiesis is uncommon in the general pediatric population. here we present the unique case of a -monthold infant who presented with intermittent emesis, failure to thrive (ftt), developmental delay, macrocytic anemia, and neutropenia which was initially concerning for a congenital bone marrow failure syndrome. instead, she was discovered to have an underlying, potentially familial deficiency of b . objectives: . to describe the unique case of an infant with b deficiency. . to outline the importance of including b deficiency in the differential diagnosis of unexplained megaloblastic anemia in children. a -month-old exclusively breastfed infant presented for gastroenterology evaluation due to persistent emesis and poor weight gain over the course of months. her history was notable for delayed developmental s of s milestones and hypoactivity. marked pallor prompted hematologic evaluation, which revealed concern for macrocytic anemia (hemoglobin . g/dl, mcv ), reticulocytopenia ( . × ^ / l), and neutropenia (anc . × ^ /l). an otherwise reassuring physical examination and laboratory evaluation was notable only for the discovery of an undetectable b level and marked hyperhomocysteinemia ( mol/l). her hemoglobin (hgb) continued to decline (to . g/dl) over the first few days after presentation, and she required red blood cell (rbc) transfusion. within only a few days of initiation, daily cyanocobalamin injections resulted in a robust reticulocytosis response, improved hgb, immediate normalization in the neutrophil count, and resolution of hyperhomocysteinemia. additional history and laboratory evaluation from the patient's mother revealed a concurrent, asymptomatic maternal b deficiency as well as a history of a need for b supplementation in the maternal grandfather, raising concern for an inherited etiology. despite the rarity of vitamin-deficient hematologic abnormalities in the general pediatric population, b deficiency should be considered as a potential cause of an otherwise unexplained megaloblastic anemia, especially in the setting of concurrent ftt and neurodevelopmental delay. a detailed family history should be obtained in such cases and may have helped to prevent this patient's clinical sequelae had the deficiency been discovered sooner. our patient has experienced a favorable clinical response to b supplementation, attesting to the importance of vitamin b in early childhood growth and development. background: peg-asparaginase is universally utilized in the treatment of pediatric acute lymphoblastic leukemia (all). despite its high efficacy in this disease, it is associated with hypersensitivity and allergy in - % of patients. protracted anaphylaxis has been described in circumstances such as severe food allergy with ongoing allergen exposure; however, it has not yet been described in relation to peg-asparaginase. we describe the first reported case of protracted anaphylaxis after peg-asparaginase administration, provide guidance as to time course and management of protracted anaphylaxis, as well as evidence that erwinia asparaginase may be safely administered even in this high risk population. objectives: to provide guidance regarding the duration, course and management of protracted, severe anaphylaxis after peg-asparaginase therapy. a year old male with very high risk all presented for consolidation therapy with peg-asparaginase (intramuscular) and vincristine. one hour after administration, he developed generalized hives and angioedema, for which he was given diphenhydramine. he then quickly developed progressive hives, angioedema, subjective throat and chest tightness, and wheezing. he was treated with diphenhydramine, epinephrine, albuterol, and methylprednisolone with resolution of symptoms. one hour later, symptoms recurred and the patient became hypotensive; he was retreated with methylprednisolone and epinephrine, and was transferred to the pediatric intensive care unit (picu). in the picu, he was placed on an epinephrine drip, and continued on methylprednisolone, diphenhydramine, cetirizine, albuterol, and ranitidine. the epinephrine drip was successfully discontinued after hours, and his other medications were gradually weaned over the course of two weeks. of note, the patient did have st segment changes in his electrocardiogram during the first hours of anaphylaxis. these were associated with normal ventricular function as per echocardiogram, and resolved within one week. this patient has subsequently tolerated multiple doses of erwinia asparaginase (intramuscular) without premedication. this patient was acutely managed in the pediatric intensive care unit with steroids, anti-histamines, and continuous infusion epinephrine. symptoms consistent with severe anaphylaxis including hives, angioedema, throat and chest tightness, wheezing, and hypotension persisted for a total of four days before finally resolving. he has thus far tolerated multiple doses of erwinia asparaginase without any symptoms of allergy, hypersensitivity, or anaphylaxis. protracted severe anaphylaxis after peg-asparaginase therapy can be successfully managed with multi-agent therapy, including antihistamines, steroids, and continuous infusion epinephrine. re-challenge with an alternate form of asparaginase may be tolerated, even in a patient with protracted anaphylaxis to peg-asparaginase. ucsf benioff children's hospital oakland, oakland, california, united states background: vincristine (vcr) is widely used in pediatric cancers. unlike most cytotoxic agents, hematopoietic toxicity is uncommon. vcr-induced anemia has been observed but its mechanism has not been well studied. vinca alkaloid-induced membrane changes were seen in early studies of hereditary spherocytosis (hs) and anecdotal cases suggest vcr may increase hemolysis in such patients. here we describe a case involving severe vcr-induced anemia in a patient with hs and an explanation as to the mechanism. objectives: to describe the mechanism of vcr-induced anemia in hs. design/method: case report. a year-old female with hs was diagnosed with t-lymphoblastic lymphoma. she had required packed red blood cell (prbc) transfusions as a neonate and thereafter had done well without episodes of acute hemolysis or aplasia. complete blood counts (cbc's) demonstrated a compensated hemolysis, and she did not require further transfusions until she commenced chemotherapy. by the start of maintenance she had received many more prbc transfusions than the average patient. intermittent drops in hemoglobin (hb) did not correlate with any particular agent, and she had stable, mild splenomegaly. a clear pattern emerged during maintenance. her hb was - g/dl at monthly clinic visits, when she received vcr, intermittent intrathecal methotrexate, and corticosteroids. within - days, her hb dropped to . ± . g/dl, and reticulocyte count decreased from . to . ± . %. transfusion at day corrected hb, and the reticulocytes and hb returned to baseline. white blood cell and platelet counts did not change after vcr. blood samples from pre, immediately post, and days post vcr were analyzed and rbc characteristics and markers of hemolysis were not significantly different. ektacytometry showed identical curves, indicating no change in rbc deformability. in vitro incubation of patient blood samples with vcr also did not affect the osmotic deformability, confirming that a change in rbc rigidity was unlikely the reason for the drop in hb. these data indicate that a dysregulation of erythropoiesis was responsible for the anemia after vcr, rather than damage of peripheral rbc's. in most patients, maintenance therapy for lymphoblastic lymphoma does not cause severe anemia, likely because a temporary reduction in erythropoiesis in patients with a normal rbc survival and low reticulocyte count is not noticed. however, in a patient with decreased rbc survival and a brisk reticulocytosis, a disruption in rbc generation is more apparent. in conclusion, vcr administration to patients with an rbc disorder warrants close observation for potentially severe vcr-induced anemia. background: the addition of tyrosine kinase inhibitors (tki) to conventional chemotherapy has improved outcomes for pediatric patients with philadelphia chromosome-positive (ph+) acute lymphoblastic leukemia (all), however there remains an increased risk of relapse compared to other types of childhood all. typically, in relapsed disease the philadelphia chromosome persists and several mechanisms of resistance involving acquired mutations of the bcr-abl chimeric oncoprotein have been reported. objectives: describe a unique case of a pediatric patient with ph+ b-precursor all relapsing with b-precursor all without the philadelphia chromosome. results: an -year-old boy was diagnosed with ph+ bprecursor all with the presence of the t( ; )/bcr-abl translocation by cytogenetics and fluorescence in situ hybridization (fish), respectively. additional abnormalities included gains of runx and loss of one copy of etv . a remission bone marrow with negative minimal residual disease (mrd) was achieved at the end of induction with dasatinib and the esphall chemotherapy backbone. duration of tki therapy was two years post diagnosis. nearly one year after the completion of therapy, cytopenias prompted a bone marrow investigation. relapsed b-precursor all was established by immunophenotyping, however fish analysis did not identify the bcr-abl rearrangement. moreover, quantitative reverse transcriptase pcr was negative for the bcr-abl fusion transcript. again fish analysis of the bone marrow revealed multiple additional copies of runx and mono-allelic loss of etv , similar to the initial diagnostic sample. the patient was re-induced per aall anticipating a ph+ all relapse. however, with confirmation of the loss of the ph+ clone, tki therapy was not re-initiated. due to positive mrd of . % at the end of re-induction therapy, the patient was salvaged with blinatumomab therapy and subsequently underwent an allogenic stem cell transplant with a sibling donor. conclusion: this is the first known report of a pediatric patient with ph+ b-precursor all who developed recurrent b-precursor all without the philadelphia chromosome. the persistent findings of gain of runx and loss of etv makes it unlikely that a second unrelated b-precursor all developed following successful treatment of the original disease. this case highlights the possibility of a genetically distinct subclone present at the onset of disease that shared abnormalities of runx and etv but did not contain the philadelphia chromosome. nevertheless, the subclone harbored leukemogenic potential in the absence bcr-abl expression. it is plausible that the predominant clone present at diagnosis was effectively treated with dasatinib and extinguished, but the bcr-abl -negative clone persisted in the face of tki therapy. background: ligneous conjunctivitis is a rare form of pseudomembranous conjunctivitis that develops specifically in patients with type plasminogen deficiency. lack of plasmin activity in those patients result in defective fibrinolysis and formation of fibrin-rich membranous material/ masses that develops on the palpebral conjunctiva as well as other sites in the body.current management involve surgical excision of the masses that is usually complicated by multiple recurrences. recently, use of topical plasminogen concentrates helped delaying recurrence, but currently, those concentrates are not commercially available. we report on a -year-old omani girl, with hypoplasminogenemia who required optimization of plasminogen level at the time of surgery to delay/ prevent recurrence. objectives: case report on the peri-operative use of ffp versus cryopricipitate transfusion as an alternative replacement of plasminogen during surgical excision of ligneous conjunctivitis. design/method: pharmacokinetic study was performed to assess plasminogen recovery after ffp ( ml/kg) and precipitate ( bag/ kg) transfusion results: plasminogen levels remained subnormal after either ffp or cryoprecipitate administration. with ffp, the maximum concentration reached was almost % of normal. although half-life of plasminogen is known to be - . days, the patient seemed to have a high catabolic rate after receiv-ing cryoprecipitate, with plasminogen levels reaching basal levels within hours. because of the better recovery profile with ffp, we opted to give ffp before and after surgery. peri-operative management included ffp transfusion at ml/kg/ hours one day before and for days post operatively, followed by ml/kg once daily from day - , then ml/kg on th post-operative day. topical treatment was initiated using antibiotic and steroids ed on the day of surgery, followed by heparin ed on the second day. on follow up, she used topical heparin, cyclosporine, prednisolone, and topical lubricant eye drops for variable duration. clinical picture remained stable for almost year post operatively, when she started to develop recurrence of ligneous lesions again. background: ponatinib (inclusig®, ariad pharmaceutical) is a rd generation multi-targeted tyrosine kinase inhibitor (tki) approved for treatment of adults with chronic myeloid leukemia (cml) and philadelphia chromosomepositive acute lymphoblastic leukemia (ph+ all) resistant to or intolerant of other tkis. ponatinib has numerous drug-drug interactions and a black box warning for associated serious adverse vascular events and hepatotoxicity. for this reason, ponatinib use has been confined to specific high-risk populations. however, in patients who prove refractory to other therapies, the potential benefits of ponatinib may outweigh risks. to date, ponatinib has not been studied in the pediatric/adolescent and young adult (aya) population. furthermore, literature describing the use of ponatinib alone or in combination with other agents in pediatric oncology patients is scarce. objectives: to describe a single institutional experience using ponatinib in the pediatric patients with ph+ all. design/method: two cases of ponatinib use in pediatric ph+ patients resistant to other tkis were identified at our institution and are described. peripheral blood samples obtained from both patients identified bcr-abl p fusion transcripts and sanger sequencing was used to identify resistant mutations. results: our first case is a -year-old female who received upfront multi-agent chemotherapy plus dasatinib for ph+ all. relapse was confirmed on end-of-therapy bone marrow evaluation, thus bcr-abl mutation testing was performed and revealed a t i mutation. ponatinib was initiated then discontinued after one week due to clinically significant fluid retention with peripheral edema and bilateral pleural/pericardial effusions. the second case is a lateadolescent female with ph+ all who relapsed -years after stem cell transplant (sct). following relapse, tki therapy included both imatinib and dasatinib. due to persistence of bcr-abl fusion transcript despite tki therapy she was switched to ponatinib. shortly following initiation of ponatinib she developed a diffuse, maculopapular rash, which persisted despite dose reduction, resulting in ultimate discontinuation of the drug. bcr-abl mutation testing identified f l and f v resistance-conferring mutations. to date, there is scant existing literature detailing the use of ponatinib in pediatric patients. appropriate dosing is undefined and side effect profile not well described, particularly when used concurrently with other chemotherapeutic agents. thus, this case series reporting the response to and toxicity of ponatinib in pediatric ph+ all patients has important clinical implications. additionally, this is the first report of a pediatric ph+ all patient with documented t i mutation underscoring the importance of bcr-abl mutational testing, particularly at the time of relapse. cooper university hospital, camden, new jersey, united states background: myh -related disorder is a rare autosomal dominant disease, encompassing several subtypes: may hegglin anomaly, epstein syndrome, fechtner's syndrome, and sebastian syndrome. heterozygous mutations are seen in the gene encoding non-muscle myosin heavy chain iia (nmmhc-iia) which is involved in cell motility as well as functions to maintain cellular shape and integrity. the presentation of myh -rd is mainly characterized by macrothrombocytopenia, but various related expressions exist: nephritis often leading to renal failure, cataracts and sensorineural deafness ( ). a -year-old girl with history of extensive dental caries, hyperactivity, and speech delay due to suspected hearing loss was incidentally found to have thrombocytopenia at the time of genetic evaluation. she did not have any bruising or excessive bleeding. she did not respond to observation, immunoglobulins, or steroid therapy. her platelet count remained persistently low ( - k/ul). she underwent extensive evaluation to rule out platelet disorder vs. coagulation defect. her peripheral smear showed enlarged platelets by giemsa stain but no inclusion bodies were noted in granulocytes. her platelet aggregation and platelet surface glycoprotein by flow cytometry were negative. her coagulation profile was also normal. objectives: this case report summarizes the complexity in diagnosing myh -rd in a pediatric patient. design/method: since a unifying diagnosis for her clinical presentation was not apparent, whole exome sequencing (wes) was undertaken. results: wes revealed the r c heterozygous pathogenic variant, located in exon in the myh gene. myh gene alteration explained the patient's clinical features of macrothrombocytopenia and hearing loss. this mutation was paternally inherited, and her father demonstrates mosaicism. he was asymptomatic with normal platelet count but his morphology showed enlarged platelets with no inclusion bodies in granulocytes. when dealing with patients who have mild or no symptoms of bleeding diathesis but evidence of persistent macrothrombocytopenia, considering a platelet disorder belonging to myh -rd can help delineate certain predisposing syndromes and guide clinical management. patients are likely to benefit from early genetic testing while receiving supportive therapy. wes can highlight syndromes and provide information on recurrence risk for families. the renal and hearing abnormalities are indistinguishable between epstein and fechtner's syndromes, but the pathogenic variants differ ( ). the genotype-phenotype correlation implies that our patient may have either syndrome, although clinical features compatible with nephritis have yet to manifest. patients should be monitored closely for long-term progression of myh disease, and treatments should be initiated accordingly. we present an -year old female evaluated by genetics at birth due to prenatal microcephaly. chromosomes and microarray were normal. at age she developed standard risk pre-b-cell acute lymphoblastic leukemia (all). she completed treatment in and has been doing well in the interim, remaining in complete clinical remission. during and after treatment she exhibited developmental delay and neurocognitive deficits. at age her height and weight were at or below the th centile and head circumference was below the nd centile (approximately standard deviations below the mean and corresponding to the th centile for a -month-old girl). bone age was appropriate. she had a distinctive triangular face with micrognathia and a pointed nose resembling a seckel-like syndrome. the patient also had clinodactyly of the th toes, zygodactylous triradius involving the nd and rd left toes, tendency to sydney line in the right palm and a radial loop in the left middle finger. the patient's unique clinical presentation prompted a more thorough genetic evaluation, which led to a novel finding we feel is clinically significant with regard to the development of malignancy. design/method: whole exome sequencing (wes) was performed on the patient as well as her biological parents (trio). a de novo heterozygous mutation in the gene pcdh with potential relation to the phenotype was discovered. this c. dupa variant causes a frameshift starting with codon asparagine , changing this amino acid to a lysine residue and creating a premature stop codon at position of the new reading frame denoted p.asn lysfsx . this variant is predicted to cause loss of normal protein function via protein truncation or nonsense-mediated mrna decay. conclusion: pcdh is a member of the protocadherins family which is important in cell-to-cell adhesion and synaptic function in the central nervous system and is highly expressed in areas of the brain involved in higher cortical function and speech. aberrant expression of protocadherins has been associated with the development of malignancies in many organ systems. with regards to leukemia, the methylation status of this gene at diagnosis has been implicated in the prognosis of all and could be used as a biomarker to predict relapse. this patient's de novo mutation and clinical presentation are unique to what has been previously presented in the literature. we feel that this mutation is a clinically significant finding that may shed light on the role of this gene in the development of hematopoeitic malignancies. background: acquired hemophilia a (aha) is an uncommon and potentially life-threatening hemorrhagic disease characterized by sudden onset of bleeding in patients with neither personal nor family history of bleeding dyscrasia. it is usually seen in adults with autoimmune diseases, solid tumors, lymphoproliferative diseases, pregnancy or during the postpartum period; occurrence in the pediatric population has rarely been reported. we report a case of an otherwise healthy teenager who was found to have aha when he presented with acute onset of atraumatic soft tissue hematoma. results: a -year old male of middle eastern descent with history of congenital absence of the right external ear, but otherwise in good general health, presented to our emergency department with a three day history of progressive worsening of right lower leg pain, swelling, and paresthesia, without preceding history of trauma. evaluation by the pediatric orthopedics service documented significantly elevated compartment pressures, necessitating immediate four-compartment fasciotomy. pre-operative labs were significant for prolonged activated partial thromboplastin time (aptt) of . ( . - . ) seconds with normal prothrombin time (pt) and international normalized ratio (inr). ptt did not correct on mixing studies, suggesting the presence of a circulating anticoagulant. factors xii and xi were in the normal range; factor ix was elevated, ( - ). factor viii level was % and fviii inhibitor level was . bethesda units (< . ), confirming the diagnosis of aha. work up for autoimmune disease was negative. his bleeding and surgical hemostasis were managed with recombinant factor vii (novoseven) mcg/kg every hours for hours post operatively, with gradual interval prolongation. factor viii antibody eradication was managed with prednisone mg/kg/day. factor viii and inhibitor levels normalized by day of hospitalization. recombinant factor vii was discontinued; steroids were gradually tapered and discontinued at discharge (hospital day ). conclusion: acquired hemophilia is likely an underdiagnosed condition in pediatrics. while it is typically seen in adults with underlying autoimmune disease, solid tumors, lymphoproliferative disease, or during pregnancy or the postpartum period, pediatric cases may have no identifiable etiology. this case highlights the importance of considering this diagnosis in any patient with unexplained bleeding regardless of their age, so as to intervene early and prevent adverse consequences. university of oklahoma, oklahoma city, oklahoma, united states background: myeloid neoplasms associated with eosinophilia is a rare subtype of chronic leukemia characterized by clonal eosinophilia. the true incidence is unknown due to its rarity and possible classification as idiopathic hypereosinophilia syndrome. the most common chromosomal aberrations involve platelet-derived growth factor receptors (pdgfrs). we report one such rare case in a pediatric patient. most of the pediatric management of this entity is derived from adult case reports and case series. objectives: to describe a case of chronic leukemia presenting as eosinophilia results: a previously healthy year old caucasian male presented with a several week history of migrating joint pain, splenomegaly, and abnormal blood counts with leukocytosis, thrombocytopenia and absolute eosinophilia. white blood cell differential showed myeloid precursors suggestive of chronic myeloid leukemia. bone marrow evaluation showed % blasts and % eosinophils. bcr-abl testing was negative, ruling out cml. fish analysis for eosinophilic clonality revealed deletion of chic gene, resulting in fip l /pdgfra fusion gene, diagnostic for myeloid neoplasm with eosinophilia associated with pdgfr abnormalities. treatment was started with tyrosine kinase inhibitor (tki), imatinib mg daily. within months, fish analysis for fusion gene was negative. after approximately months of daily imatinib, he was switched to maintenance dose of mg weekly. he is approximately months since diagnosis and doing well on maintenance imatinib. in , the who revised its classification of some chronic eosinophilic leukemias to myeloid and lymphoid neoplasms associated with eosinophilia and rearrangement of pdgfra, pdgfrb, fgfr . the most common abnormality is the fip l /pdgfra fusion gene. other less common abnormalities include fusion genes kif b-pdgfra and etv -pdgfrb and point mutations in pdgfra . some features of chronic eosinophilic leukemia include absolute eosinophilia, splenomegaly, elevated vitamin b and tryptase levels, and organ damage from eosinophil infiltrates and cytokine release. patients with rearrangements or mutations involving pdgfra are usually very responsive to imatinib. starting doses have not been well studied or established. experts recommend co-administration of corticosteroids during the first few days of imatinib therapy in patients with a history of cardiac involvement and/or elevated serum troponin levels to prevent myocardial necrosis, a rare complication of imatinib therapy in eosinophilic patients. fortunately our patient did not have cardiac involvement and to date has not exhibited signs of chronic tki toxicity. conclusion: myeloid neoplasms with eosinophilia constitute a rare form of chronic leukemias. they are often associated with pdgfr abnormalities and are usually very responsive to tyrosine kinase inhibitor therapy. walter reed national military medical center, bethesda, maryland, united states background: germline samd l mutation is a rare cause of constitutional bone marrow failure with a unique propensity for clonal evolution to monosomy and mds. objectives: previous case series have demonstrated diverse clinical outcomes in patients with a germline samd l mutation. our case presents a novel samd l mutation (p.val leu). additionally, the case highlights the challenges in clinical decision making for a patient with a gene mutation that is known for clonal evolution towards monosomy with risk of progression to myeloid malignancy, but also known for self-correction through uniparental disomy or inactivating mutations which results in disease remission. design/method: a retrospective chart review and review of the literature was performed. dna was isolated from peripheral blood and used for whole exome sequencing. a peripheral blood sample from the patient's mother and father showed no samd l mutation. skin biopsies of the patient and parents were evaluated for uniparental disomy or new mutations. to determine the pathogenicity of this novel mutation, the specific samd l mutant dna was transfected into the human embryonic kidney cell line to assess its role in inhibiting cell proliferation. our patient presented at months of age with pancytopenia and hypocellular bone marrow in the setting of s of s sepsis. he had evidence of dysfunctional immune activation with hemophagocytosis and elevated soluble il with simultaneous severe hypogammaglobulinemia. analysis of the peripheral blood showed no increase in chromosomal breakage, normal telomere length, and normal flow cytometry. gene testing for primary hemophagocytic lymphohistiocytosis and inherited bone marrow failure were negative. after the patient recovered from his presenting illness, a repeat bone marrow biopsy demonstrated improved cellularity with myelodysplasia and cytogenetics significant for monsomy .whole exome testing demonstrated a novel samd l mutation. the patient continued to require intermittent ivig and failed to demonstrate appropriate leukocytosis with intermittent infections. on repeat bone marrow evaluation over the course of months, the patient demonstrated no evidence of evolution towards self-correction and had a persistent monosomy clone. the patient is scheduled to undergo a matched unrelated donor bone marrow transplant. our case highlights the unique clinical picture associated with constitutional marrow failure and clonal evolution secondary to a novel samd l mutation which is thought to cause pancytopenia by inhibiting cellular proliferation and often results in the development of monosomy which rescues hematopoiesis but with a risk for malignancy. background: notable labs developed a flow cytometricbased assay with a custom robotic platform to test fdaapproved drugs for anti-cancer activity against individual patient's tumor cells. this personalized assay is a potential method for identifying novel agents and drug combinations to treat aml patients who have failed standard therapies. objectives: to present the case of a teen who underwent successful treatment of relapsed aml post-sct with bortezomib, panobinostat, and dexamethasone-a regimen selected based upon results of notable lab testing. results: a -year-old male with m -aml had an isolated bone marrow relapse months after completion of scheduled therapy. at relapse, his aml was flt -itd positive. he achieved a second remission with negative mrd and underwent matched sibling donor bmt after busulfan/cyclophosphamide conditioning. bma performed on day + was mrd positive ( . %). repeat bma done on day + showed . % mrd. he started sorafenib on day + . he received donor lymphocyte infusion (dli) on day + , then received cycles of azacitadine (aza) followed by dli. marrow mrd by flow after sorafenib alone, sorafenib with dli, and sorafenib with aza/dli were %, . %, and . %, respectively. treatment was complicated by varicella meningitis, grade i skin agvhd, febrile neutropenia and c. difficile colitis, and metapneumovirus pneumonia. despite extremely low levels of leukemia (marrow mrd . %), notable lab testing performed on the patient's leukemia cells from marrow collected after aza/dli/sorafenib revealed sensitivity of his leukemic blasts to a combination of bortezomib, panobinostat, and dexamethasone. because of prolonged cytopenias, multiple infectious complications, and persistently positive mrd, he discontinued aza/dli/sorafenib and on day + started bortezomib . mg/m iv on days , , , and ; panobinostat mg po on days , , , , , ; and dexamethasone mg po on days , , , , , , , and . chemotherapy cycle started days later. he tolerated treatment without side effects and with resolution of rash and cytopenias. he achieved full donor chimerism, negative flt -itd, and complete remission by morphology and flow after two cycles. notable lab testing is a powerful tool for evaluating the sensitivity of small populations of leukemic blasts to novel drug therapy. results from notable lab testing may serve as a useful guide for treatment selection after failure of standard aml therapy. this patient achieved morphologic and mrd remission post-sct with bortezomib, panobinostat, and dexamethasone-a regimen predicted to be efficacious based upon notable lab results. maria ahmad-nabi, christine knoll, sanjay shah, esteban gomez, lori wagner phoenix children's hospital, phoenix, arizona, united states background: development of inhibitors in patients with factor ix deficiency (fixd) is a well-recognized complication occurring in - % of patients. within this subset a small percentage can develop anaphylaxis to factor. desensitization with cyclophosphamide, an alkylating agent used in the management of various oncologic malignancies, and reported for use in factor viii desensitization has been previously unreported for use in desensitization in patients with fixd. rituximab, an anti-cd antibody, however has been used. objectives: to induce immune tolerance (it) in patients with inhibitors to factor ix with either novel or under reported methods using cyclophosphamide and/or rituximab. we report a case series of patients at phoenix children's hospital with fixd who achieved it with cyclophosphamide and/or rituximab. results: patient one was a year old male with severe fixd, who at the time of desensitization had inhibitor levels of bu. he was desensitized with cyclophosphamide, then admitted for infusion of recombinant factor ix. he experienced a few minor symptoms of intolerance including an urticarial rash which was self-limited, and hemarthrosis of the right elbow on day which responded to novo . he tolerated the remainder of his infusion without issues. he continued recombinant factor ix daily, and returned to clinic for monthly cyclophosphamide for months. he did develop urticaria with hemarthrosis and spontaneous muscle bleeds which were tempered with zantac, zyrtec, solumedrol, and benadryl. he remained without a recurrence of inhibitors, however did have intermittent hemarthrosis of his ankles thereafter requiring prophylactic twice daily dosing recombinant factor ix. patient two was a year old male with severe fixd and a family history of anaphylaxis to factor causing early death in all male relatives with the disease. he had never received factor ix and did not have a detectable inhibitor prior to desensitization. he successfully underwent desensitization to recombinant factor ix with rituximab in the icu, and returned to clinic for weekly infusions x . he experienced no adverse reactions concerning for anaphylaxis. he continued to tolerate factor ix products without evidence of intolerance, development of inhibitors, and continues on as prophylactic dosing of recombinant factor ix every other day. our experience at a single institution proves cyclophosphamide as a novel agent for inducing it in those with fixd and anaphylaxis. it also provides further evidence that rituximab can desensitize patients with severe fixd. differences include longer duration for cyclophosphamide therapy ( months vs month). background: cartilage-hair hypoplasia (chh) is an autosomal recessive chondrodysplasia associated with defective cell-mediated immunity caused by mutations in the ribonuclease mitochondrial rna processing (rmrp) gene. cancer incidence is -fold higher in patients with chh than in the general population, especially non-hodgkin lymphoma. the use of rituximab, an anti cd antibody, results in decreased host b-cell number and impaired humoral function for - months. the safety of rituximab in pediatric patients with cancer and immunodeficiency is not well documented. a diagnosis of underlying immunodeficiency may discourage physicians from using rituximab due to the risk of severe bacterial infection or viral re-activation. objectives: to report a case of burkitt lymphoma in a young adult female with chh and defective cellular immunity successfully treated with rituximab. results: an -year old amish female with disproportionate short stature presented to our center for management of stage iv biopsy proven burkitt lymphoma with myc rearrangement. she had presented a week earlier with cervical, occipital, and submandibular lymphadenopathy, splenomegaly; fevers, night sweats, and weight loss for - weeks. on exam, her height was three feet associated with brachydactyly, mild bowing of the legs, normal size head without frontal bossing, fine and sparse hair. she had normal intelligence. her pattern of dysmorphisms was suggestive of chh (genetic testing not performed at time of diagnosis). pet-ct scan showed stage iv disease with involvement of cervical lymph nodes, spleen, iliac bone and bone marrow. treatment with standardintensity fab/lmb therapy (group c) with the addition of rituximab was initiated. she had an incomplete response to cop (∼ % reduction of tumoral masses) but achieved complete remission after copadam . her course was complicated with severe varicella zoster but she completed therapy and remains in complete disease remission for months after treatment completion. genetic testing subsequently performed proved homozygosity for chh with a n. a>g variant. she had no other opportunistic infections during or after therapy. conclusion: the use of rituximab was both safe and beneficial in our patient despite defective cell mediated immunity secondary to chh suggesting that rituximab may be safe to use in patients with cellular immune deficiencies. background: hemophilia a and b are bleeding disorders characterized by deficiency in factor viii or ix, respectively. spontaneous or provoked hemarthrosis is a known complication of hemophilia. repetitive episodes of hemarthrosis can lead to debilitating hemophilic arthropathy. lyme disease is a tick-born infection which is endemic to increasing parts of the united states. chronic lyme disease, the phase in which lyme arthritis typically develops, occurs months to years after initial infection and is characterized by swelling of one or more large joints generally in the absence of systemic symptoms. objectives: review cases of hemophilia a and b patients with episodes of provoked hemarthrosis refractory to intensive recombinant factor replacement therapy found to have concurrent lyme arthritis. design/method: we report two clinical cases and review relevant literature. results: first, we report a year-old male with moderate hemophilia a with a provoked knee hemarthrosis which failed to improve despite months of intense factor replacement therapy requiring multiple hospitalizations. factor replacement regimens included twice daily standard half-life recombinant factor viii products or daily to every other day extended half-life recombinant factor viii products with trough levels aimed as high as - %. factor viii pk studies were obtained for dosing, to confirm adherence, and to evaluate for subclinical inhibitors (inhibitor testing was negative). given protracted symptoms additional workup for hemarthrosis was pursed. lyme titers were positive for ( )igg, though negative for igm. he was treated with days of doxycycline during which time hemarthrosis greatly improved on examination and imaging, and he was able to recover function through physical therapy. second, we report a year-old male with moderate hemophilia b who required multiple hospital admissions for a provoked knee hemarthro-sis with no improvement in symptoms despite weeks of daily or twice daily factor replacement with standard halflife recombinant factor ix products aiming for % correction. we performed inhibitor testing (which was negative) and pk studies to assess for non-detectable inhibitors, dosing and adherence. lyme testing was positive for ( )igg, though negative for igm. he was treated with amoxicillin for days during which time hemarthrosis significantly improved on examination and imaging. diagnosis and follow-up imaging studies for both patients included mri and serial bedside ultrasounds performed as per uc san diego school of medicine mskus guidelines. background: relapse/refractory aml following allogeneic hematopoietic stem cell transplant (hsct) holds a high mortality rate. current relapse/refractory therapy modalities for younger patients may include re-induction with a clofarabinebased regimen followed by second allogeneic hsct. even for patients who undergo second hsct, the five-year survival rate is dismal. new therapies, including small molecule inhibitors, are being studied in the post-hsct relapse setting or those unfit for hsct with promising results. venetoclax is a small molecule inhibitor that has received breakthrough designation for aml treatment in elderly patients objectives: to report a young adult aml patient with relapse post hsct who was successfully re-induced with topotecan, vinorelbine, thiotepa, clofarabine (tvtc) and has sustained remission with venetoclax maintenance therapy. this approach appears to be unique in terms of reported literature. results: our patient is now a -year-old female noted to have mll rearranged aml at initial diagnosis when she was years old. she underwent chemotherapy consisting of cytarabine/daunorubicin according to standard + . due to persistent disease, she was re-induced with g-csf, clofarabine, and high-dose cytarabine (gclac) which put her in cr. her course was complicated by sepsis, colitis, gastrointestinal bleed, deep venous thrombosis, and transfusionassociated circulatory overload. given her co-morbidities, she received another cycle of clofarabine/cytarabine, and then proceeded to reduced intensity allogeneic hsct, according to bmt ctn . the patient tolerated hsct well and experienced no transplant-related complications, including no acute or chronic gvhd. unfortunately, she relapsed about month's post-hsct. initial salvage therapy consisted of another course of g-clac, but due to persistent disease the decision was made to re-induce her with topotecan, vinorelbine, thiotepa, and clofarabine (tvtc). during this time however, she was found to have extensive infection with a fusarium species requiring a course of anti-fungal therapy. bone marrow evaluation showed no residual disease with an mrd of < . %. once the absolute neutrophil count recovered, the patient was started on single-agent venetoclax for maintenance therapy, which has been well-tolerated. she remains in morphologic remission for over months. we describe herein a young adult with multiply relapsed aml wherein tvtc re-induction, followed by maintenance with venetoclax were safely used in the post-hsct setting. venetoclax therapy in the relapsed aml setting warrants further study. background: vitamin b deficiency is uncommon in children in developed countries, especially in the absence of risk factors like malabsorption or inadequate dietary intake. it often presents with non-specific symptoms and signs and can elude diagnosis. the recognition and treatment of vitamin b deficiency is critical as it can lead to bone marrow failure as well as severe neurological and developmental problems in children. to increase index of suspicion of vitamin b deficiency anemia in children. we report a rare case of vita-min b deficiency anemia in a child who presented with a severe macrocytic anemia, with signs of hemolysis and concern of malignancy. design/method: an almost three-year-old previously healthy girl presented with a few day history of fever, emesis, fatigue and pallor. she had no dysmorphic features, hepatosplenomegaly or lymphadenopathy on exam, growth and development were normal. laboratory findings showed severe macrocytic anemia (hemoglobin . grams/dl; mcv . fl) with reticulocytopenia. signs of intravascular hemolysis were present with elevated lactate dehydrogenase ( , units/l) and haptoglobin below assay limit. immune-mediated hemolysis was ruled out. initial picture of a hemolytic anemia was compounded by other findings of moderate neutropenia, mild thrombocytopenia and peripheral smear showing occasional blasts. further workup was done with a broad differential diagnosis that included leukemias, hemolytic anemias, bone marrow failure syndromes, and specific deficiencies. results: workup revealed abnormally low vitamin b levels along with significantly elevated homocysteine and methylmalonic acid levels indicating functional vitamin b deficiency. bone marrow evaluation showed megaloblastic anemia and dyserythropoiesis consistent with vitamin b deficiency, and ruled out leukemia. vitamin b deficiency can cause a hemolytic anemia like picture secondary to intramedullary hemolysis due to ineffective erythropoiesis. myeloid precursors are also affected which can lead to neutropenia, thrombocytopenia, and abnormal peripheral blood cells. in our patient, initial symptomatic anemia was treated with blood transfusion, followed by intramuscular vitamin b injections with normalizing lab values. so far, workup for an etiology for vitamin b deficiency is negative except for an equivocal range of anti-parietal cell antibodies raising concerns for pernicious anemia; however it is rare in this age group. another rare condition is an inborn error of the cobalamin transporter. she is currently on oral vitamin b supplementation and further workup will be planned based on response. conclusion: this case highlights the importance of early consideration and thorough evaluation of vitamin b deficiency in children with unclear etiology of anemia, so that prompt treatment can be initiated. memorial hospital/ university of miami, miami, florida, united states background: despite great success in the treatment of acute lymphoblastic leukemia (all), the outcomes for patients with relapsed all remain poor. prognostic indicators include timing and site of relapse. blinatumomab, is the first agent in its class that simultaneously binds cd -positive cytotoxic t cells to cd -positive b cells resulting in lysis of malignant cells. however, mechanisms of leukemia resistance to blinatumomab are unclear. objectives: to describe a case with multiple sites of extramedullary (em) relapse during blinatumomab therapy. results: a -year-old hispanic male with philadelphia positive, cd -positive b-precursor cell all refractory to chemotherapy, had failed a bone marrow (bm) and was placed on blinatumomab and imatinib. he achieved minimal residual disease (mrd)-negative systemic remission, but during his fifth cycle developed bilateral periorbital masses. biopsies confirmed cd -negative isolated em relapsed disease, which was treated with radiation therapy (rt). there was notable resolution of em disease and he continued systemic therapy. subsequently, he presented with a painful left scapular swelling. imaging showed muscle and lung parenchymal em relapse with cd -positivity confirmed on histology. he continued on blinatumomab with localized rt while awaiting car-t cell therapy. his bm mrd remained negative until he developed systemic mrd-positivity with cd -positive blasts following the sixth cycle. primary resistance to blinatumomab is poorly understood. it is proposed that expansion of cd -negative clones or downregulation of cd following blinatumomab may play a role. this was observed in our patient's periorbital relapse; but subsequent em and systemic relapses were cd -positive, consistent with the co-existence of multiple clones in relapsed all. it has also been postulated that em relapse could be linked to the failure of blinatumomab or t cells to migrate to em sites of disease or drug inactivation by the microenvironment. the second em relapse in our patient, with cd -positive disease suggests this as a possible mechanism of relapse. this was reported in patients with cd positive non-hodgkin lymphoma (nhl), and higher doses of blinatumomab however, have shown promising results in this population. despite blinatumomab's effectiveness in inducing remissions in patients with refractory/relapsed all, it appears to have limitations in patients with em disease. these may arise either from the multiclonality associated with relapsed all or due to the emergence of resistance to blinatumomab, including failure to migrate to em sites. background: cyclic neutropenia is a rare hereditary disorder, characterized by recurrent neutropenia, cycling at about week intervals, with variable associated symptoms including oral ulcers and fever. there are reported cases of cyclic neutropenia associated with chronic inflammation leading to development of reactive aa amyloidosis. one patient also presented with amyloid goiter. we report a new case of cyclic neutropenia with associated renal and thyroid amyloid. design/method: a -year-old female presented with a month history of thyromegaly, and recurrent aphthous ulcers associated with fevers. laboratory workup showed severe neutropenia, anemia, azotemia, and abnormal thyroid function, with an absolute neutrophil count - / l, hemoglobin - . g/dl, serum creatinine - . mg/dl, and uric acid - . mg/dl. thyroid stimulating hormone was elevated - . iu/ml, and normal free t . urinalysis showed + protein, + blood, and - urine red blood cells/hpf. chest radiograph showed mild narrowing of the trachea from thyroid compression. bone marrow biopsy showed a hypocellular marrow, with tri-lineage hematopoiesis, left shifted myeloid maturation with very rare mature neutrophils. both renal biopsy and thyroid fine needle aspiration revealed abundant amyloid. of note, her father had aa amyloidosis, resulting in end-stage renal disease (esrd) requiring hemodialysis, and recurrent aphthous ulcers. the family history suggested a familial predisposition. genetic testing revealed a pathogenic elane c. a>t gene mutation with autosomal dominant inheritance confirming the diagnosis of cyclic neutropenia. we treated our patient with daily granulocyte colony stimulating factor to reduce the burden of chronic inflammation induced by cyclic neutropenia, and to preserve renal and other end organ function affected by further amyloid deposition. results: proband with elane gene mutation positive cyclic neutropenia, amyloidosis of thyroid and kidney, with a positive paternal history of aa amyloidosis resulting in esrd. cyclic neutropenia may result in chronic inflammatory states leading to secondary amyloidosis. university of kentucky, lexington, kentucky, united states background: overall survival of burkitt lymphoma (bl), regardless of stage, is greater than % in the pediatric population when treated with multi-agent chemotherapy. adenovirus is a common, usually self-limited infection within the pediatric population; however, findings can vary within an immunocompromised host. hepatitis is a rare complication, with very few reports of radiologic findings in this patient population. we discuss a three year old male with history of bl who presented with clinical and radiographic evidence of relapse but was found to have adenovirus hepatitis. design/method: a case report of a patient with bl in complete remission after completion of standard of care chemotherapy, who presented with return of high fever, elevated ldh, transaminitis and hepatic lesions. we describe the hepatic imaging and pathology consistent with adenovirus hepatitis in this immunocompromised host. our patient presented at three years old with a six week history of worsening abdominal pain and fevers. he was found to have a right sided pleural effusion, multiple lesions of the liver, and diffuse abdominal lymphadenopathy; biopsy of lymph tissue was consistent with bl. he completed therapy per anhl arm b and was in a complete remission at the end of planned therapy. one month after completion of therapy, he returned with high fever, abdominal pain and transaminitis, similar to his initial presentation. ct scan showed multiple hypodense discrete lesions throughout the liver and re-accumulation of right sided pleural effusion. ldh peaked at u/l (uln u/l). uric acid remained within normal limits. bilirubin peaked at . mg/dl, conjugated . mg/dl. liver biopsy was performed, showing smudgy nuclei with immunohistochemical staining positive for adenovirus. there was no evidence of lymphomatous involvement. resolution of hepatic lesions and transaminitis, with normalization of ldh and fever, occurred with symptomatic treatment alone. adenovirus is known to cause systemic disease in immunocompromised patients and rarely hepatitis. no pediatric patients with discrete hepatic lesions secondary to adenovirus have been reported in the literature. three cases of discrete hepatic lesions have been reported in adult immunocompromised patients, two with fatal fulminant liver failure and one who required cidofovir. this case demonstrates that a common pediatric viral infection can present with lesions concerning for metastatic disease in a pediatric lymphoma patient. prompt diagnosis is vital in the management of these patients when recurrent lymphoma is in the differential. background: heparin induced thrombocytopenia (hit) is an immunologic process in which antibodies bind a heparin complex and cause a paradoxical hypercoagulable state. ramifications of this process may include a multitude of thrombotic events and bleeding complications secondary to platelet consumption. in our patient, hit manifested as increased bruising, an acute decrease in platelet count, and continual clotting of her crrt circuit. hit, although rare in pediatrics, should be included in the differential for children with thrombocytopenia who have received heparin products. to present a unique case report of a critically ill pediatric patient who developed hit in the presence of multiorgan system failure and to discuss the challenges encountered with identification of an alternative anti-coagulant. results: a yo obese, caucasian female child presented to our facility with bilateral pulmonary emboli (of unclear etiology). initially, she was started on a continuous heparin infusion, but was transitioned to enoxaparin within days without issue. five days after enoxaparin was initiated, the patient developed acute kidney injury (evidenced by increasing creatinine) attributable to her biventricular heart failure. due to her need for continuous renal replacement therapy (crrt), she was transitioned back to a continuous heparin infusion. whereas her initial platelet count on transition was normal, she developed severe thrombocytopenia ( , ul) within hours. due to intermediate risk but low suspicion for hit, pf antibodies were sent which were positive. after much discussion, she was transitioned to an argatroban infusion which was titrated according to ptt levels. within hours, her platelet count normalized. at discharge, she was prescribed apixaban for anti-coagulant management. conclusion: hit is an uncommon presentation in the pediatric population. given its rarity, there is often a delay in diagnosis which increases risk of complications such as bleeding, stroke, and limb ischemia. even if the diagnosis is suspected or proven, there may be challenges in initiating alternative agents as limited data exists on pediatric options. as argatroban remains the treatment of choice for patients with hit, experience in pediatric patients is limited, and dosing recommendations have been extrapolated from adult studies. anecdotal data exists for use of bivalirudin in children, although studies, primarily, focus on use in specific cardiac cases. in our patient's case, choice was further complicated by renal failure. this case study highlights the need for further research regarding the identification of a secondary anti-coagulant agent for use in pediatric patients with hit. background: subcutaneous panniculitis-like t-cell lymphoma (sptl) is a rare form of non-hodgkin's lymphoma characterized by infiltration of cytotoxic t-cells into subcutaneous tissue. sptl occurs in both adults and children and can present in both patient populations as either alpha/beta or gamma/delta subtypes. patients with the gamma-delta phenotype have an overall poorer survival, although the exact etiology is unclear. interestingly, both subtypes of sptl can present with secondary hemophagocytic lymphohistiocytosis (hlh), and this is associated with a worse prognosis. currently, there are no standardized treatment protocols for sptl, and clinical management includes watchful waiting, corticosteroids/immunosuppression, chemotherapy, and stem cell transplant. the primary objective was to compare how two patients with the same diagnosis responded acutely to therapy. we performed a retrospective chart review of two pediatric patients at our institution who were diagnosed with alpha/beta sptl and secondary hlh. we examined each presentation, treatment course, and outcome. we then completed a brief review of the current literature describing treatment of and outcomes for sptl with secondary hlh. results: these two patients presented in a similar manner with signs and symptoms of hlh. each was then subse-quently diagnosed with alpha/beta sptl after biopsy of cutaneous nodules and each had diffuse disease, as measured by pet. however, they demonstrated vastly different acute responses to therapy. one patient was pre-treated with systemic glucocorticoids before receiving definitive chemotherapy and tolerated therapy well as an outpatient. the other patient started systemic chemotherapy without steroid pretreatment and developed severe cytokine storm characterized by hypotension, cardiac dysfunction, multi-organ failure and cytokine elevation. both patients achieved complete remission (cr) after treatment with chop chemotherapy and remain disease-free - months off therapy. in patients presenting with sptl and secondary hlh, we propose that initial treatment with antiinflammatory or anti-cytokine therapy can decrease, or even prevent, the possibility of life threatening cytokine release as a result of cytotoxic chemotherapy. background: congenital dyserythropoietic anemia type ii (cda ii) is a rare autosomal recessive disorder, rarely presenting in the neonatal period. iron overload often occurs as a late sequela of ineffective erythropoiesis and intramedullary hemolysis. objectives: to report the novel use of iron chelation in an infant with cda ii associated with severe iron overload. the patient is a -month-old, former -week infant with prenatal non-immune hydrops and transfusion-dependent fetal anemia who presented with persistent anemia, reticulocytopenia, hyperbilirubinemia, liver dysfunction, and hyperferritinemia. his initial ferritin was . ng/ml, tibc ug/dl, and transferrin mg/dl. his bone marrow biopsy showed trilineage hematopoiesis and erythroid dyspoiesis characterized by binucleation of late-stage precursors. genetic testing revealed a compound heterozygous missense mutation and splice site mutation in the sec b gene, confirming the diagnosis of cda ii. initial liver biopsy revealed mild portal fibrous expansion, and abundant hepatic iron deposition. his ferritin continued to increase, peaking at , ng/ml, along with liver enzymes peaking at an alanine aminotransferase (alt) of u/l and aspartate aminotransferase (ast) of u/l. ferriscan showed an elevated estimated liver concentration of . mg/g dry tissue. repeat liver biopsy months later showed giant cell hepatitis with worsening mild portal fibrosis and hemosiderosis. additionally, tissue liver iron concentration was mcg/g dry weight. cardiac t * mri revealed mild cardiac iron deposition. given his significant degree of iron overload, deferoxamine was used to reduce hemosiderosis and liver morbidity in preparation for bone marrow transplantation. the patient received deferoxamine mg/kg/day iv x days/week for three months, without any clinically significant adverse events. blood counts and hepatic and renal function were monitored weekly without any abnormalities. growth parameters and liver enzymes significantly improved while receiving chelation therapy. as a noninvasive, cost-effective method, serum ferritin levels were monitored monthly to gauge response to treatment. despite receiving blood transfusions every - weeks, serum ferritin decreased to ng/ml and liver enzymes decreased to alt u/l and ast u/l prior to bone marrow transplantation. we report the use of deferoxamine in a patient with cda ii less than years of age, for treatment of iron overload. our patient tolerated deferoxamine well without significant adverse events or organ toxicity. deferoxamine may be a well-tolerated method of reducing iron burden in young patients with iron-loading pathologies. background: low grade gliomas with kiaa- -braf fusions typically have a favorable prognosis with infrequent rates of high grade transformation, low rates of metastasis and even lower rates of extra cns metastasis. while highgrade transformation has been reported for tumors with braf v e mutations and cdkn a deletions, it has not been pre-viously reported in gliomas with kiaa- -braf fusions. while there are case reports of high-grade cns malignancies metastasizing through a ventriculo-peritoneal (vp) shunt, low-grade gliomas metastasizing in this manner are extremely rare. objectives: to describe a unique case of peritoneal tumor dissemination of a braf fusion positive high grade neuroepithelial tumor in a child with a vp shunt placed for multifocal braf fusion positive low grade astrocytomas results: an eight-year-old male was initially diagnosed with multifocal low-grade astrocytomas of the hypothalamus and c -c spinal cord. initial testing revealed the kiaa- -braf fusion, but no cdkn a or braf v e mutation. initial surgical management included a vp shunt and resection of the cervical spinal lesion. he received vincristine and carboplatin, followed by transition to vinblastine given new thoracic metastatic lesions after months of therapy. at months after diagnosis, scans were concerning for diffuse leptomeningeal progressive disease and new intracranial lesions, necessitating craniospinal radiation. following a near cr, he presented months later with acute onset of abdominal pain. a ct scan revealed peri-renal and perirectal soft tissue masses, confirmed by exploratory laparotomy to be peritoneal tumor dissemination of high grade neuroepithelial tumor. a kiaa -braf fusion was noted and confirmed by rt-pcr, identical to that seen in the original cns tumors. additional findings included deletion of chromosome p (without q loss) and heterozygous and homozygous deletion of cdkn a found by fish. brisk mitotic activity justified a high-grade designation. salvage chemotherapy consisted of cycles of ice with subsequent resolution of pet-avid disease and only minimal peri-nephric tissue remaining. given the favorable response, surgical resection and multiple tissue biopsies were performed which documented no residual active disease. the shunt was revised and he started trametinib for maintenance. we present a unique case of peritoneal dissemination of high grade neuroepitheial tumors with the same kiaa- -braf fusion as multifocal low grade astrocytomas in a child with a vp shunt. this raises suspicion for tumor metastasis and transformation to a higher grade malignancy versus two distinct diseases, which may be indicative of an underlying cancer predisposition. texas children's hospital, houston, texas, united states background: polycythemia is a common referral to hematology. it is important to evaluate for a high oxygen affinity hemoglobinopathy, ensuring appropriate testing is performed for early diagnosis and avoidance of additional tests and procedures. a year old mexican female presented with an elevated hemoglobin and hematocrit, symptoms of plethora of her hands and feet, chest pain, palpitations, and fatigue. further confounding the picture, she also had significant menorrhagia and iron deficiency. she was diagnosed with the rare high oxygen affinity hemoglobin new mexico variant, only previously described once in the literature in a year old black boy. objectives: the patient initially presented at age with a hemoglobin of . g/dl and a hematocrit of . %. initial work up consisted of a hemoglobin electrophoresis which diagnosed sickle cell trait, a co-oximetry panel which was normal, and erythropoietin level of mu/ml, also normal. she was then lost to follow up and re-referred at age . she is a competitive basketball athlete, and at that time, she presented with a hemoglobin of . g/dl, and hematocrit of %. erythropoietin level continued to be normal at mu/ml. design/method: cardiology was consulted regarding chest pain and palpitations with a normal evaluation. chest x-ray was also normal. a bone marrow aspirate and biopsy was performed with results significant for mild erythroid hyperplasia and mild reticulin fibrosis. jak mutation, von hippel lindau, bpgm, and hereditary erythrocytosis mutations including phd , hif a, and epor mutation analysis were sent, all of which were normal. testing to mayo clinic for p rbc oxygen dissociation returned low at mmhg ( - mmhg normal range) and subsequently a hemoglobin electrophoresis identified a hemoglobin variant leading to beta globin gene sequencing. results: patient found to be heterozygous for hemoglobin new mexico, with . % hb new mexico and . % hba, and . % hba . there was no evidence of hbs. when evaluating patients with polycythemia, maintaining a high index of suspicion for high affinity hemoglobinopathies may eliminate further unnecessary and invasive testing for patients. caution should be used when using hemoglobin electrophoresis testing since hb new mexico is known to migrate similarly to hbs on hplc with minimal change that may not be detected in regular laboratories. most high affinity hemoglobinopathies are reported to not have significant symptoms. in this case, our patient complains of fatigue, occasional palpitations and plethora of hands and feet. we will need to further follow this patient for possible attributable symptomatology. divya keerthy, simone chang, warren alperstein, patricia delgado, claudia rojas, ofelia alvarez, matteo trucco university of miami jackson memorial hospital, miami, florida, united states background: improved technology is enabling detection of previously unidentified translocations and mutations in otherwise unclassified sarcomas. one such mutation is the bcl- co-repressor -internal tandem duplication (bcor-itd) allowing for the new classification of bcor positive undifferentiated round cell sarcomas (urcs). this sarcoma has a similar appearance to clear cell sarcoma of the kidney (ccsk), potentially representing an extra-renal manifestation of this tumor, but their clinical pathologic features are not identical. objectives: this case highlights how recombinant polymerase chain reaction (rt-pcr) and bcor immunohistochemical staining can ease the diagnosis of this rare sarcoma. results: a month-old female presented for right sided pre-septal cellulitis and a temporal subcutaneous mass. the detection of multiple other subcutaneous nodules on exam raised the concern for malignancy and she was admitted for evaluation. she had two subcutaneous masses on her abdomen, with more cutaneous masses on her legs, back, shoulder, cheek and submandibular areas. she lacked spontaneous lower limb movement and had bilateral clonus. imaging confirmed multiple masses throughout the body including paravertebral area from t to l , bilateral adrenal glands, left kidney and muscles of upper and lower extremities. initial differential included neuroblastoma, infantile myofibromatosis, rhabdomyosarcoma or atypical presentation of a renal tumor. however, synaptophysin and chromogranin stains were negative. with standard immunohistochemistry, the tumor could be only broadly classified as "undifferentiated sarcoma" maintaining the diagnostic challenge. using rt-pcr in the setting of a morphologically primitive round cell neoplasm with strong bcor expression, two external institutes simultaneously diagnosed the tumor as bcor-urcs. the primary lesion is unknown but potentially may have arose from the kidney. bcor-urcs has a heterogeneous histology with tumor cells appearing monomorphic in nests of - cells separated by septa with uniform nuclei. there is frequently an "orphan annie eye" appearance and sparse cytoplasm to the cells. diagnosis cannot be made solely on evaluation of this nonspecific histology. rt-pcr uses the genetic abnormality in undifferentiated sarcomas to narrow the differential and bcor immunohistochemical staining provides further context. bcor has significant diagnostic value given its sensitivity and specificity in urcs. another potential marker includes ywhae-nutm b fusions, which occur in smaller subset of cases, but requires further study. rt-pcr has helped further classify tumors leading to the diagnosis of a rare undifferentiated sarcoma with bcor overexpression. while this technology is beneficial, its availability is limited. if accessibility improves, earlier identification and treatment may be possible maximizing the chance for a positive outcome. background: hematohidrosis is a rare condition that mimics bleeding disorders. cases present with oozing blood tinged fluid from various sites like eyes, ears, nose, skin, etc. reported causes of this condition were stress or fear, physical activity, psychological disorders. the condition is self-limited and don't affect the general condition of the patients, but it may contributes to psychosocial problems and may increases their stress and anxiety. so this condition needs to be promptly treated. to test the response of this disease and the associated headache to propranolol treatment. design/method: our case female patient years old st offspring of non consanguineous marriage, was admitted with recurrent episodes of oozing blood tinged fluid from eyes, ears and nose months before admission, about . - ml from each orifice, lasted - minutes and subsided spontaneously. it could involve the sites simultaneously or - sites. the number of attacks was - times per day then gradually increased to - times per day. later on the patient developed a bleeding attack from umbilicus. these attacks were aggravated by stress and physical activity and decreased with rest and sleep. the condition was associated with severe headache involving the whole head, throbbing in nature of gradual onset, increased by physical activity and relieved by analgesics. the condition was not associated with vomiting, blurring or diminution of vision, ocular pain, eye discoloration. no earache, tinnitus or diminution of hearing. there was no other form of discharge from eyes, ears or nose. no history of ecchymotic patches, bleeding from other orifices or blood product transfusion. no history of trauma, drug intake, fever or rash. no symptoms of other system affection. past history of recurrent attacks of epistaxis and two operations were done that passed without remarkable bleeding. no similar condition in the family physical examination was free, no evidence of psychological problems. complete blood count, coagulation profile, platelets function, factor and c.t brain were normal. oozing fluid from the patient was analyzed showed the same components as blood. results: our case started oral propranolol . mg/kg/day based on its use in similar cases in literature. the frequency of attacks and headache reduced then stopped after months of treatment and didn't recur after stoppage of propranolol. propranolol can treat this condition successfully. further investigations are needed to determine the link between this condition and severe headache our case was suffering from. background: wilms tumor is the most common renal solid tumors of childhood and is derived from primitive metanephric cells located in the kidney. primary extra-renal wilms tumors (erwt) are extremely rare, estimated to comprise . - % of all wilms tumors. despite similar histologic appearance intrarenal and erwts differ in embryologic tissues of origin. erwts arise from the more primitive mesonephric or pronephric origin and, therefore, can develop anywhere along the craniocaudal migration pathway of these primitive tissues, most often retroperitoneal, inguinal/genital, lumbosacral/pelvic and mediastinal. these tumors are typically staged and treated per national wilms tumor study (nwts) guidelines, and, by definition, are stage ii or greater due to location beyond the kidney borders. based on the cases reported in the literature, outcomes for erwt are comparable to renal wilms tumors with an % local recurrence rate and an % two-year event-free survival. we report the first case of a stage iii testicular extrarenal wilms tumor in an -month-old male with an intrabdominal undescended testis who underwent complete surgical excision followed by chemotherapy and inguinal radiation. results: a full term -month old male underwent orchipexy for an undescended left testicle. the testicle was noted to be grossly abnormal with a pea-sized thickened tissue adherent to the upper pole and a separate mass outside of the scrotum on the superior epididymis. both masses were removed, and s of s pathology demonstrated wilms tumor with favorable histology and negative margins. ct imaging of the chest, abdomen and pelvis were negative for a primary renal tumor, local residual disease, pathologic lymph node enlargement or distant metastases. the tumor was classified per nwts as stage iii due to tumor removal in multiple pieces. the patient completed dd- a treatment with vincristine, doxorubicin and dactinomycin per aren with cgy left inguinal radiation. he is currently months off therapy without clinical or radiographic evidence of recurrent disease. primary erwt is an extremely rare malignant neoplasm associated with challenges in diagnosis, staging and treatment. based on the cases reported in the literature, outcomes are similar to that of intrarenal wilms tumor. there are four pediatric paratesticular wilms tumors reported in the literature and, to the best of our knowledge, this is the first case of stage iii testicular wilms tumor successfully treated with dd- a chemotherapy and radiation. in erwt, nwts guidelines for staging and treatment should be applied with evaluation of both kidneys to exclude an intrarenal primary tumor. background: patient is a yo f, with esrd secondary to atypical hus versus ttp, who presented with thrombotic microangiopathy, aki, thrombocytopenia and anemia after a living unrelated donor kidney transplant. patient initially had downtrending creatinine. on post-op day , hematology was consulted for an increasing ldh and drop in platelets. peripheral smear was notable for an absence of schistocytes. yet, biopsy of the kidney revealed microthrombi. the patient was diagnosed with a thrombotic microangiopathy. plasmapharesis was initiated on day # , at which time ms r was noted to have significantly elevated creatinine. plasmapharesis did not yield any correction in labs and significant bruising developed. patient was started on eculizimab; plasmapharesis was stopped. shortly after, creatinine, anemia and thrombocytopenia corrected to levels at which she was discharged. overall, patient was found to have progressive anemia, thrombocytopenia, an increasing creatinine and ldh ( s) concerning for atypical hus, despite absence of schistocytes on peripheral smear. she responded well to eculizimab, with correction of hematologic changes during induction. she was discharged on eculizimab and continued to respond with normalizing platelet counts and hemoglobin. the differential in light of patient's thrombotic microangiopathy and thrombocytope-nia also included ttp. yet, adamts remained normal. dic was unlikely given normal fibrinogen level and d-dimer. objectives: presentations of atypical hus vs ttp. discuss eculizumab as a treatment of atypical hus. highlight atypical presentations of illness in transplant patients. results: despite absence of schistocytes by smear, pt was diagnosed with atypical hus based on presentation and after failing plasmapharesis, she responded well to eculizumab. though her presentation was abnormal, her response to this antibody that blocks the complement cascade suggests that she was experiencing a complement-mediated process. there are rare documented cases in the literature of atypical hus without schistocytes. hemolytic uremic syndrome (hus) is characterized by hemolytic anemia, thrombocytopenia and acute kidney injury. atypical hus is a diagnosis of exclusion, not due common etiologies such as shiga toxin. among atypical causes are complement-mediated forms, caused by an antibody to complement factor. in addition to plasmapharesis, renal transplant and supportive care, the mainstay of treatment for atypical hus is eculizumab (an antibody that blocks the complement terminal cascade). this case describes a patient unique in that, she was diagnosed with atypical hus without any schistocytes by smear. secondly, she responded to eculizumab, with unremarkable gene studies. finally, this case highlights that transplant patients often have unique presentations. nicklaus children's hospital, miami, florida, united states background: synovial sarcoma is a spindle cell tumor categorized as a soft tissue sarcoma. the chromosomal translocation t(x; ) leading to the ss -ssx fusion protein is unique to this sarcoma. it is a slow growing tumor with common recurrences and often, at presentation, with evidence of metastatic disease. if resection is not feasible, then neoadjuvant with adjuvant chemotherapy is recommended. metastasis carries an unfavorable prognosis given synovial sarcoma historically does not respond well to chemotherapy. trabectedin is a well-tolerated alkylating agent currently indicated for the treatment of liposarcoma and leiomyosarcoma. we present a -year-old male with metastatic synovial sarcoma to the lungs that progressed and was refractory to chemotherapy. he was administered trabectedin as a form of palliative chemotherapy, with significant clinical and radiographic response. design/method: pubmed search was done with search for terminology including "synovial sarcoma" and "trabectedin". papers relevant to our case were selected for literature review. a -year-old male patient presented with a large right axillary mass. initial imaging showed a heterogeneous multiseptated mass invading the subscapularis and teres major muscles along with innumerable lung nodules. biopsy confirmed diagnosis of monophasic synovial sarcoma. the patient was started on protocol arst with ifosfomide, mesna, doxorubicin. he completed cycles followed by radical resection and sessions of radiation. due to progression of disease multiple chemotherapy regimens were tried including topotecan and cyclophosphamide, protocol advl with lorvotuzumab, and pazopanib. imaging of the chest continued to show significant progression of metastasis. the patient's clinical status deteriorated with worsening respiratory status, requiring l of oxygen therapy, and inability to ambulate. he was started on trabectedin . mg/m for palliative care. after cycles of treatment patient was no longer requiring oxygen and was ambulating without assistance. radiological imaging showed significant reduction in number and size of lung nodules. trabectedin is a recently approved alkylating agent for the management of sarcomas resistant to first line treatment. response in synovial sarcoma is scarcely documented in the pediatric population. epidemiology places the most common age group in the young adults and children. our case opens the doors to further consideration of the use of trabectedin in the pediatric patient with metastatic synovial sarcoma. background: gata is an x-linked gene that plays critical role in hematopoiesis. mutations of gata gene can be associated to various blood disorders including diamond blackfan anemia, cytopenia, congenital dyserythropoietc anemia and acute megakaryoblastic leukemia. we report a patient with macrocytic anemia and platelet dysfunction who carries a novel gata mutation that has not been reported. results: a now -month-old male with complex medical history including prematurity at weeks, dysmorphic features, global developmental delay, hyperinsulinism, hypogonadotropic hypogonadism, growth hormone deficiency, micropenis, failure to thrive, patent ductus arteriosus status post ligation, and severe hypotonia, was referred to hematology at months old for resolved, transient thrombocytopenia and macrocytic anemia since month of age. chromosomal microarray showed chromosome deletion of q . , which is the rps gene. he doesn't have a family history of diamond blackfan anemia (dba), despite mom having the same rps mutation. he was then diagnosed with dba. his lab workup showed mild macrocytic anemia (hgb . g/dl, mcv fl), normal to inappropriately low reticulocyte count, normal white blood cell and platelet counts, hgf %, erythroid ada . eu/gm hgb (elevated). he has abnormal pfa- , with prolonged closure time of both adp and epinephrine. he had low von willebrand antigen and ristocetin cofactor activity. he has severe pancreatic insufficiency. bone marrow biopsy showed normocellular marrow with trilineage hematopoietic maturation, without ringed sideroblasts. since mother has the same rps gene mutation, maternal labs were done and showed no evidence of macroytosis or anemia. the diagnosis of dba was questioned. whole exome sequencing did not identify any pathogenic sequence changes in the coding regions of rps gene, but detected a gata mutation r w, which was reported variant of uncertain significance. his mother shares the same mutation and is asymptomatic, but she may not be affected since gata iis xlinked. his father doesn't harbor the gata mutation. conclusion: gata gene encodes zinc finger dna binding hematopoietic transcription factor, which is important during erythroid differentiation. gata mutation r w has not been reported in literature and is a novel variant of gata mutation, which might be contributing to this patient's clinical picture. further studies are warranted to confirm gata mutation r w to be a pathogenic sequence change. alexander boucher, tomoyuki mizuno, alexander vinks, greg tiao, stuart goldstein, james geller cincinnati children's hospital medical center, cincinnati, ohio, united states background: hepatoblastoma (hb), the most common pediatric primary hepatic malignancy, can be associated with specific congenital syndromes. recently, chronic kidney disease and genitourinary anomalies have been linked to hb. cisplatin is a key chemotherapeutic agent in treating hb but its renal clearance and toxicity profile can limit its use for those with end-stage renal disease (esrd). objectives: using an institutional case series, we present data using cisplatin for hb in dialysis-dependent esrd and define recommended dosing for future use. design/method: a chart review of patients with concurrent hb and esrd on dialysis treated with cisplatin at our institution was undertaken. demographic data, diagnostic history, tumor pathology, alpha fetoprotein (afp), hearing assessments, dosing schema, treatment outcomes, and therapyrelated toxicities were reviewed. total cisplatin levels were collected at time points within days after each infusion. free cisplatin levels were also collected for infusions, as were dialysate cisplatin levels. pk parameters were generated using bayesian estimation with a published population pk model as a priori information. results: three patients meeting these criteria were identified. each had "low risk" (non-metastatic resectable) disease at presentation and underwent upfront resections. all had congenital renal anomalies with esrd prior to their hb diagnosis. all cisplatin infusions were given over hours, followed hours later by hemodialysis. patients and received cisplatin at % of children's oncology group's ahep weight-based dosing ( . mg/kg). patient received % of ahep body surface area-based dosing ( mg/m ) during cycle but required a second dose reduction ( mg/m ) for cycle due to prolonged cisplatin exposure (total area under the curve mg⋅h/l; average for all seven evaluable cycles mg⋅h/l) and early sensorineural hearing loss at - hz. no other hearing loss in any patient was identified; mild toxicities also included grade - emesis and grade neutropenia and thrombocytopenia. the median (range) of clearance, volume of distribution at steady-state, and elimination half-life at terminal phase for total platinum were . ( . - . ) l/hour/ kg, . ( . - . ) l/ kg and ( - ) hours, respectively. patients and received cycles with rapid afp normalization. patient required an additional cycles, for a likely second primary hb year after initial therapy. cisplatin can be used successfully in pediatric patients with esrd on hemodialysis to treat hb with minimal morbidity using % standard mg/kg-based dosing ( . mg/kg), achieving pharmacologically appropriate cisplatin exposures. background: treatment for immune thrombocytopenia (itp) has been grouped into rescue and maintenance therapy and often is reserved for patients with bleeding, severe thrombocytopenia, or for improvement in quality of life. splenectomy is considered one of the more invasive but definitive treatments with success rates of - %. treatment of itp can be more difficult in the setting of previous treatment with immune modulation or when the patient is immunocompromised and not a candidate for splenectomy. objectives: present an interesting case of a patient with an autoimmune disease that presented with severe thrombocytopenia, un-responsive to rescue therapy, and requiring emergent splenectomy in the setting of acute intracranial hemorrhage (ich). a year old female with a history of juvenile dermatomyositis presented with a fine purpuric rash on her extremities, wet purpura, and a platelet count of k/ l. bone marrow evaluation at that time was consistent with itp. she was on cyclosporine and plaquenil for dermatomyositis. platelets failed to increase after three doses of intravenous immunoglobulin and high dose steroids. following a two week course of oral prednisone and eltrombopag, she presented with persistent severe thrombocytopenia of k/ l, anemia of . g/dl, and a lower gi bleed. she was started on amicar, novo-seven, rituximab, and given platelet transfusions with no improvement in bleeding. subsequently, she developed a subdural hematoma with midline shift. surgery performed an emergent open splenectomy with concurrent continuous platelet transfusion. results: she was monitored closely post operatively and, due to ich, transfused to maintain platelets greater than k/ l. by week post-op she had normal platelet counts off transfusions. all medications were stopped within three days of discharge. she represented eight days later with abdominal pain and thrombocytosis and was found to have a portal vein, splenic vein and mesenteric vein thrombosis. she was started on lovenox therapy and admitted for monitoring due to her history of ich. it is unknown whether our patient's underlying immune dysregulation and history of treatment with immunosuppressive medications may have contributed to her unresponsiveness to multiple therapeutic agents. in addition, her significant bleeding did not allow us to fully evaluate her response to second tier therapy. this adds to the scarcity of literature of itp response in pediatric patients with autoimmune disease, and may support more aggressive therapy upfront in these patients. background: multivisceral organ transplantation involves concurrent transplantation of the stomach, pancreas, liver, and intestine with splenectomy, and has been classically used in the pediatric population for infants with intestinal failure from disorders affecting foregut integrity. while there is some data demonstrating its efficacy in adults with low-grade abdominal malignancies, it has not been traditionally used for hepatocellular carcinoma treatment. to describe a unique pediatric case of multivisceral organ transplantation as definitive therapy for refractory fibrolamellar hepatocellular carcinoma in an adolescent male. a year old male presents with a history of fibrolamellar hepatocellular carcinoma, tumor invasion of the portal vein, severe portal hypertension complicated by bleeding esophageal varices and hypersplenism. he had two treatments with yttrium- radioembolization, without significant response. he completed six cycles of traditional chemotherapy in combination with sorafenib with resolution of petavidity, but minimal decrease in tumor size and continued portal hypertension. since his disease remained relatively stable for over years, he was evaluated and listed for multivisceral organ transplantation. at approximately years and months after diagnosis, he underwent en bloc liver, pancreas, stomach, small bowel, and colon transplant with splenectomy. a single lymph node was positive for malignancy at the time of resection. in addition to expected post-transplant complications, he also developed skin only acute graft versus host disease at weeks after transplant, treated successfully with a thymoglobulin course. he clinically improved and was back to his baseline activity level, on full oral feedings within months post-transplantation. at three and six month post-transplantation, there is no concern for relapsed hepatocellular carcinoma on comprehensive imaging and evaluation. he is maintained on protocol immunosuppression and posttransplant support. we present the first known case of successful multivisceral organ transplantation in the treatment of refractory pediatric fibrolamellar hepatocellular carcinoma. background: hematohidrosis is a rare disorder that presents with spontaneous excretion of whole blood from intact skin or mucosa. diagnosis is based on clinical observation of the occurrence with the proven presence of erythrocytes and other blood components, without other abnormalities to account for the phenomenon. the existing literature is scarce and consists of primarily case studies. most reports describe bleeding from facial sites around the eyes, ears, and nose. the available literature suggests anxiety and physical or emotional stress reactions as the most common inciting events. little evidence exists regarding the ideal therapeutic approach, however propranolol has been used successfully to reduce bleeding frequency and severity in multiple case reports. a specific genetic etiology has not been elucidated, and no familial cases have previously been reported. we present a pair of half-siblings, both of whom presented with spontaneous cutaneous and mucosal bleeding before two years of age, and report on preliminary results of propranolol therapy. tanzania. at months of age, he became ill and developed spontaneous bleeding from his ears, nose, and scalp. he continued to have frequent bleeding episodes, usually related to illness or physical distress. a bleeding diathesis work-up was unremarkable, however some episodes were severe enough to require transfusions. the patient was subsequently diagnosed with hiv and hepatitis b, presumably acquired via unscreened blood product transfusions. patient b is an infant female born to the same mother as patient a, with a different father. she was healthy until two months of age when she developed spontaneous bleeding from the hairline, eyelids, ears and genital/rectal area. bleeding episodes were nearly always associated with irritability and crying. extensive coagulation workup was unremarkable. results: propranolol therapy was started in both patients, titrated to a goal of mg/kg/day. in both patients, the frequency and duration of bleeding episodes significantly improved. patient b continues to have milder occasional bleeding episodes from her eyes, ears and scalp but has significantly less discomfort and irritability during the episodes. conclusion: to our knowledge, there are no prior reports involving two related patients with hematohidrosis. this case series suggests that there may be a genetic predisposition which has yet to be identified. propranolol has shown effectiveness in reducing symptom frequency and severity. background: gliomas are the most common central nervous system tumors in children. they are classified into different grades based on genotype (idh, braf, tsc, etc.). lowgrade gliomas such as oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas are classified as grades i and ii. of the molecular level alterations this case report focuses on the braf v e mutation. braf is a member of the raf family of serine/threonine protein kinases and it plays an important role in cell survival, proliferation and terminal differentiation. objectives: here we discuss two cases where dabrafenib, a braf kinase inhibitor, was utilized in the management of gliomas. the cases focus on the use of dabrafenib late versus early in disease course. design/method: patient jl is a year old female who was diagnosed with a low-grade glioneuronal tumor (c -t with a metastatic lesion to the brain) in . jl was treated with chemotherapy, radiation, and surgical resection. despite treatment, the patient's disease progressed. she developed lower extremity dysfunction, urinary incontinence, poor truncal control, and hydrocephalus. dabrafenib was started after the braf v e mutation was confirmed. patient lg is a year old female who presented in november with left facial and upper extremity weakness. ct and mri scans demonstrated a mixed solid and cystic lesion extending from the optic chiasm and hypothalamus to the right thalamus and posterior basal ganglia with additional involvement of the right cerebral peduncle. neurosurgical intervention was undertaken and dabrafenib was started after the braf v e mutation was confirmed. results: patient jl's mri scans have demonstrated improvement of the spine with diminished areas of enhancement along thecal margins, decreased volume and enhancement within the trigeminal plate cistern and resolution of ependymal enhancement within the right ventricle. the patient's most recent mri exhibits no disease progression in head or spine. jl has shown improvement clinically since starting dabrafenib. patient lg has shown improvement in strength and recent mri of the brain has shown resolution of enhancement along surgical resection margins, decreased hyperintensity along the inferomedial aspect of the right basal ganglia and no new enhancements. conclusion: low grade gliomas can alter a person's quality of life and even lead to life threatening complications. often the standard chemotherapy, radiation and surgery don't prevent these complications. genetic analysis can help clinicians target therapy towards certain mutations such as braf v e. dabrafenib has shown to decrease tumor burden, early utilization as therapy can help prevent morbidity and mortality. children's hospital of pittsburgh of upmc, pittsburgh, pennsylvania, united states background: copper is an essential cofactor in enzymatic reactions essential to proper hematologic, skeletal, neurologic and vascular function. copper requirements in children over the age of are mg/day, which is readily acquired in a typical diet. copper deficiency is known to occur in patients with the rare x-linked mutation and in older individuals with gastrointestinal bypass surgery; however, it is rarely reported in other conditions. objectives: to highlight individuals with autism spectrum disorders or developmental delay with a limited dietary repertoire are at risk for copper deficiency, thus a high index of suspicion must exist in order to diagnose the disorder. design/method: a y/o boy with a prior diagnosis of global developmental delay and oral aversion presented with slowly progressive fatigue, weakness, gait instability, and weight loss. his longstanding feeding difficulties were refractory to intensive feeding programs. his daily diet consisted of - oz of milk and - individual servings of butterscotch pudding ( - calories/day, . mg iron/day). initial complete blood count demonstrated white blood cell count of . , absolute neutrophil count of , hemoglobin of . , mean corpuscular volume of < , reticulocyte count of . , platelet count of . review of his peripheral blood smear revealed microcytic, hypochromic red cells without marked fragmentation, anisopoikilocytosis and ringed sideroblasts; there were no morphologic abnormalities of his leukocytes or platelets. iron studies demonstrated ferritin of , total iron binding capacity of , and % iron saturation. he had no evidence of b , folate deficiency or blood loss. additional evaluation revealed a serum copper level of (range - ), and cerulosplasmin of . (range - ). results: once a diagnosis of copper deficiency was made, the patient promptly began a course of parenteral copper repletion. he received iv copper mcg/kg/day x days then weekly intravenous infusions. given his malnutrition, a gtube was placed to begin oral copper repletion and enteral nutrition. within weeks his copper level improved as well as his blood counts. unfortunately, although his blood counts and copper levels normalized, his neurologic status remains below his old baseline although, he has made gains in his gross and fine motor abilities. conclusion: acquired copper deficiency in the pediatric population is a rare event but given the hematologic and neurologic consequences, prompt recognition and treatment is important. this patient's clinical course demonstrates the need to have a high index of suspicion of concomitant nutritional deficiencies other than those routinely evaluated such as iron, b and folate. background: lymphoepithelioma-like thymic carcinoma (lelc) is a rare, aggressive neoplasm with a high rate of invasion, metastasis and recurrence. there are no known curative therapies for metastatic lelc. we report the case of a -year-old male who presented with metastatic ebv positive lelc. sites of disease included a large primary anterior mediastinal mass and metastases to hilar lymph nodes, lungs and liver. he was initially treated with cisplatin and fluorouracil followed by mediastinal radiation. he had a partial response to therapy but his end of therapy scans showed disease progression in lungs, liver, and hilar, supraclavicular and axillary lymph nodes. objectives: molecularly targeted therapies tailored to the patient's genetic profile offer a novel approach to obtain improved survival outcomes. design/method: the patient enrolled on a precision medicine trial, nmtrc : molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancer (nct ). in this study, tumor/normal whole exome sequencing and tumor rna sequencing were performed and a molecular report detailing the results of genomic and gene expression analysis was generated. a treatment plan was designed within a molecular tumor board comprising oncologists, pharmacists, genomicists, and molecular biologists with domain expertise. results: exome sequencing revealed somatic coding point mutations and no structural mutations (focal copy number changes or translocations). candidate somatic driver mutations included tp s x and r w as well as kit n k. both genes have been previously implicated in thymic carcinoma. rna expression analysis demonstrated aberrant activation of biological pathways, including overexpression of kit, hdac , and , tyms, and dhfr. the molecular tumor board selected the combination of pemetrexed ( mg/m ) on day of a day cycle, imatinib ( mg daily), and vorinostat ( mg days - , - , and - ) . on day of cycle , he was admitted with a herpes zoster infection and imatinib was discontinued in order to reduce risk of herpes zoster recurrence. imaging after cycles showed a complete metabolic response on f- fdg pet and a partial response by ct size criteria. as of december , the patient had received cycles of pemetrexed and vorinostat. scans in december showed an increase in the size and metabolic activity of two right lower lobe pulmonary nodules. there were no new sites of disease and imatinib was re-started. background: systemic lupus erythematosus (sle) is a chronic autoimmune disease that affects multiple organ systems and is associated with many different autoantibodies. patients can present with vague constitutional symptoms including fever, rash, fatigue, and weight loss. some of the various hematologic manifestations of sle include anemia of chronic disease, leukopenia, autoimmune hemolytic anemia (aiha), and idiopathic thrombocytopenic purpura (itp). these can be the presenting signs of sle. evans syndrome (es), a disease characterized by itp and aiha, is a rare hematologic manifestation of sle. neurofibromatosis (nf ) is a relatively common neurocutaneous disorder. these patients are at risk of developing benign and malignant tumors. its association with autoimmune disorders, including sle, remains rare. objectives: there are few cases in the literature that have patients with the combination of sle and nf . this is the only case that has a patient with sle, nf , and es. results: a -year-old caucasian female presents with two months of vaginal bleeding, weight loss and petechiae. her exam is remarkable for petechiae and café au lait macules. laboratory findings show severe anemia and thrombocytopenia. she receives blood and platelet transfusions during stabilization, and a bone marrow aspirate is performed to rule out a malignancy which is negative. based on the presence of thrombocytopenia and a positive coombs test, an autoimmune process such as es is considered. screening tests for sle reveal positive antinuclear and anti-double stranded dna antibodies as well as low complement. she receives intravenous immunoglobulin and methylprednisolone and eventually her vaginal bleeding slows and her counts recover. she begins sle therapy with hydroxychloroquine and azathioprine. due to the presence of café au lait macules on her exam, a genetics evaluation is performed and the patient is also diagnosed with nf . to date, there are seven cases of sle with nf reported in the literature, only two of which are pediatric cases. there are no reports of the combination of sle, nf , and es. conclusion: es is a rare hematologic manifestation of sle but can be the initial presentation of this disease. one large study estimates % of childhood-onset sle cases are observed to have es. screening for sle should be considered in all es patients even in the absence of typical clinical findings. association of nf and sle has been rarely described. whether this association reflects a causal relationship or is coincidental needs more investigation. (lube, ped blood & cancer, ) . university of california san diego, la jolla, california, united states background: high grade glioma (hgg) has poor outcomes in adults and children. extraneural metastases are very rare in hgg, and poorly characterized with only a few small case series in adults and only isolated case reports in pediatrics. no genomic data has previously been published for any children with hgg who develop extraneural metastases. objectives: our objective is to describe the natural history of two children with hgg and bony extraneural metastases, comparing their clinical characteristics as well as whole exome sequencing data for both tumors. this information would suggest similar patients should be monitored closely for extraneural metastasis and may benefit from more systemic therapy. design/method: we present a case series of two patients who presented with hgg and had development of bony metastases less than six months after initial diagnosis. both patients had molecular profiling with whole exome sequencing (wes). the first patient was an -year-old male with a tumor found in the left lateral ventricle invading into the fornices, hypothalamus, and left midbrain, who had subtotal resection. bony metastasis were found at . months after diagnosis, and he died months after diagnosis. he initially received radiation, followed by nivolimuab. the second was a -year-old female with a tectal/pineal tumor and multiple spinal cord metastases, who had subtotal resection. she developed bony metastasis at . months after diagnosis and died months after diagnosis. her histologic diagnosis was pineoblastoma, revised to hgg after whole exome sequencing. she received craniospinal radiation followed by chemotherapy per acns (cisplatin, vincristine, and cyclophosphamide) for cycles. when she failed to respond satisfactorily to this therapy, wes of tumor was performed and the findings were consistent with hgg. treatment was transitioned to temodar and lomustine after hgg diagnosis was given. she had ongoing progressive disease despite this therapy as well as trials of nivolumab, everolimus, and vorinostat. neither patient had extraneural metastasis at presentation. in both tumors, whole exome sequencing identified the h f a k m mutation. both tumors also had additional known mutations associated with hgg but no other overlapping mutations. this case series represents the first description of the genetic alterations of pediatric hgg patients who developed extraneural metastases. while h f a k m is a common mutation in pediatric midline hgg, especially dipg, and is associated with more aggressive disease, there has not been an association with extraneural metastasis prior to this series. background: deferiprone-induced agranulocytosis is a well -known albeit rare side effect of the drug. incidence of agranulocytosis varies from . - . %, while milder neutropenia is reported in . % of patients treated with deferiprone. deferasirox is unknown to cause such a complication. clinical trials and post marketing side effect monitoring studied possible correlations between different risk factors and development of agranulocytosis. unfortunately, no studies directly addressed a special risk in a community with background of ethnic neutropenia, like oman. objectives: to report on the incidence of neutropenia among omani children with b thalassemia using different iron chelators design/method: a retrospective study conducted on patients < year-old with b thalassemia treated with different iron chelators. electronic patients records were reviewed to detect episodes of neutropenia either mild (anc . -< . /cmm), moderate (anc . -< ), severe < . , or agranulocytosis anc = ). data were collected including sex, age, personal or family history of ethnic neutropenia, iron chelating agent, infective complications, management and outcome. detailed clinical, laboratory ± radiological information were reported for patients who developed life-threatening agranulocytosis. among young patients with b thalassemia, treated between - in squh, neutropenia, was reported in patients ( . %).severe neutropenia was encountered on occasions in patients ( / : . %) ( on deferiprone including episodes of agranulocytosis, on defersirox, on combined chelation, and off chelation). moderate neutropenia was encountered in patients ( / : . %), on occasions: deferiprone ( ), deferasirox ( ), combined chelation ( ), and episodes off chelation. mild neutropenia was more prevalent, encountered in patients ( . %) on occasions ( on deferiprone, on defersirox, on combined chelation, and off chelation) of patients exposed to deferiprone, patients had neutropenia ( %), higher than previously reported. deferiproneinduced agranulocytosis was encountered in patients ( / = . %). three of them had life threatening complications. one patient developed pneumonia complicated by rupture of pulmonary artery aneurysm-massive hemoptysis, who recovered fully after catheter embolization. the second had facial cellulitis and treatment with gcsf was complicated by frequent ventricular extrasystoles. the third had sepsis, disseminated herpes simplex and required admission to icu for inotropic support. in a community with background ethnic neutropenia, neutropenia is more common to be encountered among thalassemic patients, both on and off chelation therapy. careful monitoring of anc and rational choice/modification of chelating agents is required for optimal management of iron overload and to avoid life threatening complications. objectives: this case control study aimed to evaluate the systolic and diastolic cardiac function in groups of children with ti: non transfused group and a group that received early regular blood transfusion comparing them to healthy controls. design/method: thirteen regularly transfused patients with ti with a mean age of . + . years were compared with eight patients who are non-transfused or minimally transfused (< rbcs transfusion/year); mean age . + . years and healthy controls with a mean age of . ± . years. clinical parameters and standard echocardiographic and tissue doppler imaging (tdi) were compared. results: young non-transfused ti patients had a statistically significant higher peak late diastolic velocity of the left ventricular inflow doppler, a mitral valve a wave duration over the pulmonary vein a wave duration ratio and the pulmonary s of s vein s/d velocities ratio compared to the transfused group with p values of . , . , . respectively. in addition, they have a lower e/a ratio of the mitral valve inflow and a larger left atrial to aortic diameter ratio compared to the control group with p values of . and . respectively. the diameters of the right and left outflow tract were significantly larger in the non transfused group with a trend to have a higher cardiac index compare to the transfused group. systolic function was similar in the studied groups and none of the patients had evidence of pulmonary hypertension. young patients with ti who are receiving early regular blood transfusion have normal systolic function. diastolic function assessment revealed indicators of an abnormal relaxation of the left ventricle in the non transfused group which indicate diastolic dysfunction. the abnormalities affected multiple diastolic function parameters which give an indication that the changes are clinically significant. a statistically significant increase in the diameters of the outflow tracts are likely attributed to high cardiac output status in nontransfused ti patients as they had a trend to have a higher cardiac index. these findings support the early commencing of regular blood transfusion therapy for ti patients to prevent serious cardiac complications in adult life. background: in the -week sustain study, crizanlizumab . mg/kg significantly reduced the frequency of scpcs versus placebo ( . vs . , p = . ) and increased the time to first on-treatment scpc ( . vs . months, p = . ) in patients with sickle cell disease (scd). to evaluate time to first scpc in sustain study subgroups and the likelihood of not experiencing scpc for the duration of the trial using post hoc analyses. design/method: sustain was a randomized, double-blind, placebo-controlled, phase study (nct ). inclusion criteria were: scd patients aged - years; - scpcs in previous months; concomitant hydroxyurea use permitted if ≥ months and stable dose for ≥ months. patients were randomized : : to receive intravenous crizanlizumab . mg/kg, . mg/kg, or placebo. study treatments were administered on days and , then every weeks to week , with the final assessment at week . median time to first scpc after first dose was summarized for crizanlizumab . mg/kg or placebo in these subgroups: - or - scpcs in previous months; scd genotype; and hydroxyurea use at baseline. hazard ratios (hrs) for crizanlizumab . mg/kg versus placebo were calculated based on cox regression analysis, with treatment as a covariate. descriptive statistics were used to summarize the frequency of patients who were scpc event-free for the duration of the study by prior scpc events, scd genotype, and hydroxyurea use at baseline. : patients received crizanlizumab . mg/kg and received placebo. there was a meaningful delay in time to first scpc with crizanlizumab . mg/kg versus placebo observed in the entire study population. the effect was present in both scpc subgroups, and the largest treatment difference was observed in hbss scd versus other genotypes ( . vs . months; hr: . ). in patients taking hydroxyurea who experienced - scpcs in the previous year, time to first onstudy scpc was longer with crizanlizumab . mg/kg versus placebo ( . vs . months; hr: . ). a greater proportion of patients treated with crizanlizumab . mg/kg were scpc event-free versus placebo in each of the analyzed subgroups. one third of patients who were taking hydroxyurea and treated with crizanlizumab . mg/kg were scpc event-free during the study versus . % with placebo, possibly suggesting an additive effect. with crizanlizumab . mg/kg, there was a clinically meaningful delay in time to first scpc and an increased likelihood of being scpc-free versus placebo in all subgroups investigated. cincinnati children's hospital medical center, cincinnati, ohio, united states background: shwachman-diamond syndrome (sds) is an inherited marrow failure syndrome associated with increased risk of myelodysplasia (mds) and acute myeloid leukemia (aml). objectives: this multi-institutional retrospective study investigated clinical features, treatment, and outcomes of sds patients who developed mds or aml by central pathology review. design/method: nine individuals presented with aml ( male, female), mds-eb / ( males, females, with mds ( male and female), and one male with isolated persistent somatic tp mutation. one mds-eb and mds patient progressed to aml. median age (years) at diagnosis of mds was (range . - ), mds-eb / was (range . - ) and aml was . (range . - ). complex cytogenetics were noted in / aml cases, with one having normal cytogenetics. complex clonal cytogenetic abnormalities were noted in of mds-eb /eb patients and clonal abnormalities in of mds patients. follow up was available for aml patients; are deceased. received chemotherapy with intent to proceed to hematopoietic stem cell transplant (hsct). four failed to achieve remission and died with disease without proceeding to transplant. one patient proceeded to hsct without prior chemotherapy. four of six transplanted subjects died with relapsed disease. treatment related mortality was largely infectious or gvhd. the sole surviving aml patient had normal cytogenetics, achieved remission with chemotherapy and underwent hscts with separate stem cell infusions due to two primary graft failures. he remains alive in remission more than years after diagnosis. of the mds-eb / patients, underwent ric hsct, three of whom are alive, one died of infection. the fifth patient has stable disease on continued decitabine monotherapy for . years. of mds patients with treatment data, had upfront hsct therapy, upfront chemotherapy and had no therapy. three patients required ≥ hscts all due to graft failure. follow up is available for , of whom are deceased, with relapsed disease. treatment related mortality was largely infectious or graft failure. one individual died of hepatic failure unrelated to mds. seven mds patients are alive in remission. in summary, prognosis is poor for patients with sds who develop aml due to resistant disease and treatment-related complications. better markers for risk stratification are needed to identify patients who would benefit from early transplant. novel therapeutic strategies are urgently needed to improve outcomes of sds patients with mds or aml. background: unlike primary myelofibrosis (pmf) in adults, which is associated with somatic mutations in jak , mpl, or calr, myelofibrosis in children is rare and the underlying genetic mechanisms remain elusive. here we describe families with autosomal recessive congenital macrothrombocytopenia with focal myelofibrosis (cmtfm) due to germline mutations in the megakaryocyte-specific immune receptor tyrosine-based inhibitory motif (itim) receptor g b-b. objectives: to characterize the clinical phenotype, histological features and identify the causative gene for cmtfm. we performed affymetrix snp . genotyping on the index family to identify shared regions of homozygosity by descent. whole exome sequencing (ws) was performed on all three pedigrees to identify potentially causative mutations. we studied affected children from families, with macrothrombocytopenia, anemia, mild leukocytosis and a distinctive pattern of bone marrow (bm) fibrosis centered around clusters of atypical megakaryocytes. affected children had mild to moderate bleeding symptoms and required platelet and red cell transfusions. none showed evidence of extramedullary hematopoiesis, and all were negative for mutations in jak , mpl, and calr. snp genotyping identified multiple statistically non-significant genomic loci, including the region of the major histocompatibility locus (mhc) on chromosome p (lod = . ). we focused on this region because affected individuals in two families shared a common homozygous human leukocyte antigen (hla) type and had congenital adrenal hyperplasia (cah) due to -hydroxylase (cyp a ) mutation; the cyp a and hla loci are located at p . and p . - p . . wes revealed homozygous frameshift mutations in the megakaryocyte and platelet inhibitory receptor g b-b, encoded within the candidate linkage region. we identified two distinct g b-b frameshift mutations (c. _ + dup; p. fs and c. inst; p. fs) in individuals within these three families. no other mutations that segregated with the phenotype were identified. to validate g b-b as a potential disease-causing gene, we evaluated g b-b expression in bm biopsy specimens from affected patient and control samples by immunohistochemical staining using a monoclonal antibody. g b-b was strongly s of s and selectively expressed in megakaryocytes of control samples, but completely absent in clinically affected individuals. a murine knockout that lacks g b-b has a strikingly similar phenotype with macrothrombocytopenia, myelofibrosis and aberrant platelet production and function, further affirming the causality of g b-b mutations. we showed that autosomal recessive loss-offunction mutations in g b-b cause cmtfm, uncovering the molecular basis of this rare disease. loss of g b-b-dependent inhibition of megakaryocyte activation likely underlies the distinctive focal myelofibrotic phenotype and might be important in other forms of marrow fibrosis. cardinal glennon, saint louis, missouri, united states background: intrauterine transfusion is the method of choice for management of fetal anemia due to red blood cell alloimmunization. despite the decrease in prevalence of anemia due to rhesus d alloimmunization with prophylactic administration of anti-rhd immunoglobulin in rh d negative patients, maternal red red blood cell alloimmunization with other type of red blood cell antigens remains an important cause of fetal anemia. newborn who received intrauterine transfusion for hemolytic disease may have prolonged postnatal transfusion requirement. objectives: -to evaluate clinical outcome of fetuses and newborns who received intrauterine transfusions. -to determine the need of packed red blood cell transfusions until months of age. we conducted a retrospective case series study of all intrauterine transfusions due to anemia secondary to red blood cell alloimmunization performed in our regional center ssm in st louis missouri, between april and january . we evaluated the indications, diagnosis, gestational age, and frequency of intrauterine transfusions, along with the infant's gestational age at birth, duration of admission, timing of blood transfusion and monitoring of hemoglobin. results: intrauterine transfusions were performed in patients. the most common causes of alloimmunization were due to d antibodies (n = , %) and kell antibodies (n = , . %). the median gestational age of the first intrauterine transfusion was . weeks, and the median pre-transfusion hemoglobin was . g/dl. the gestational age at the first intrauterine transfusions was found to be significantly correlated with the number of postnatal transfusions (r = . . p = . ). the median gestational age at birth was found to be weeks ( . - . weeks), with a hemoglobin of . ( . - . ). in our population, patients ( %) received postnatal transfusions, of which were during the first weeks of life, and close monitoring follow up with a hematologist was established in patients at their discharge from the nursery/nicu. one neonatal death occurred and severe morbidity due to severe anemia occurred in one infant. despite the continuing risk factor for persistent anemia, only patients had follow up hemoglobin monitored by their primary care provider. conclusion: infants with anemia due to red blood cell alloimmunization treated with intrauterine transfusion should be monitored closely via regular complete blood count for persistent anemia due to suppression of fetal erythropoiesis. sebastian hesse, piotr grabowski, juri rappsilber, christoph klein dr. von hauner childen's hospital, lmu university hospital, munich, munich, germany background: neutrophil granulocytes are the most abundant leukocytes in the peripheral blood. validated diagnostic options for these cells are limited, leaving many patients with functional neutrophil defects without a defined diagnosis. objectives: here we evaluate proteomics as a new diagnostic tool to investigate defects of neutrophil granulocytes. we analyzed neutrophil granulocytes from children with severe congenital neutropenia (scn) associated with elane mutations, children with chronic granulomatous disease (cgd) with cyba ( ) or cybb ( ) mutations and children with leukocyte adhesion deficiency (lad) due to itgb mutations. in addition we collected samples of children with genetically undetermined neutrophil defects. neutrophils from healthy individuals served as controls. cells were isolated from fresh venous blood using negative selection (purity > %). whole cell proteome analysis was done by data-independent acquisition. showed a correlation coefficient of ∼ . . principal component analysis demonstrated unequivocal separation of the proteome of healthy and diseased cells. differential expression analysis showed minimal proteome aberrations in lad with deficiency in cell surface receptors and upregulation of alpl (total downregulated proteins: / total upregulated proteins: ). analysis of neutrophils from cgd patients also showed limited proteome aberration. cyba and cybb were both diminished independent of genotype, whereas protein clusters around a stat / centered network were increased (total down: / up: ). neutrophils with elane mutations showed the gravest proteome disturbance (total down: / up: ) with an upregulated translational apparatus (srpdependent ribosomes and protein folding complexes) and increased mitochondrial proteins. proteins of each granule subset were dysregulated and metabolic pathways upregulated. a detailed analysis of the proteome from patients with genetically undefined diseases is currently ongoing. one patient with clinical phenotype of cgd was found to have no mutations of nadph oxidase members in whole exome sequencing but critically low levels of ncf on protein level. heterozygosity mapping showed autozytocity in the ncf region warranting current efforts to sequence promoters and intronic regions of the gene. mass spectrometry based proteomics promises exciting new insights into monogenic disease of neutrophil granulocytes and may offer new diagnostic options, in particular in synergy with genome sequencing. by virtue of our international care-for-rare alliance, open to new partners, we hope that our proteome focus may lead to better delineation of as yet unknown disease of neutrophil granulocytes. background: warm autoimmune hemolytic anemia (aiha) is an igg mediated disease. although it can be post-viral, it is often idiopathic and can also be a forme fruste for malignancy or an autoimmune disease. initial management includes steroids. it often relapses on steroid wean and can be refractory to the use of second line treatment such as rituximab. objectives: abatacept (ctla- -ig fusion protein, ctla- mimetic) has been used to ameliorate autoimmune manifestation associated with ctla- haploinsufficiency. we used abatacept as a novel therapeutic agent to manage patients with refractory aiha. design/method: a retrospective case series of two patients at phoenix children's hospital with severe refractory aiha. results: patient , a previously healthy year old female, presented with weeks of icterus, fatigue, and hemoglobinuria. spleen was enlarged cm below the costal margin. laboratory evaluation demonstrated: hemoglobin . g/dl, mild leukopenia /microliter, platelets , /microliter, reticulocytosis . %, positive direct coombs' test, mycoplasma igm and igg positive. bone marrow evaluation showed a hypercellular marrow. she continued to need packed red blood cell (prbc) transfusions despite receiving high dose steroids, ivig and rituximab from may-july . in august, she started sirolimus decreasing her transfusion requirement. after starting abatacept ( mg/kg/dose bi-monthly for three doses and then monthly) in october, she maintained hemoglobin of - g/dl without transfusion. patient , a previously healthy month old male, presented with one week of progressive fatigue, jaundice, and poor feeding. splenomegaly was absent. laboratory evaluation revealed hemoglobin . g/dl, leukocytosis , /microliter, platelets , /microliter, reticulocytosis . %, negative direct coombs' test, and non-specific reactivity on antibody screen. evaluation for inherited hemolytic anemia including a next generation sequencing panel was negative. further evaluation by blood bank showed + positive coombs' for c d due to a warm antibody. cold agglutinin disease was ruled out. bone marrow evaluation was normal. he received high dose ivig as a steroid sparing agent but continued to require prbc transfusions weekly. when prednisone did not seem to slow down hemolysis, treatment with abatacept was initiated and he has not required transfusions for two months. steroids are being weaned. we present successful treatment of two refractory aiha cases with abatacept. patient is steroid and transfusion free and continues on monthly abatacept and sirolimus. patient is also transfusion free and continues on a steroid taper. ctla- is crucial for suppressive function of treg cells. abatacept by binding to cd / seems to enhance treg activity ameliorating autoimmune hemolysis. children's minnesota, minneapolis, minnesota, united states s of s background: transfusional iron overload is common in patients receiving chronic red cell transfusions. as a result, iron chelation is required to minimize toxicity from iron overload. chelation with a single agent can be inadequate at controlling or reducing iron burden. when combination therapy is required deferoxamine may be added to oral chelation. deferoxamine is generally given subcutaneous over - hours for - days a week at - mg/kg/day. many patients struggle to remain compliant with this schedule which has prompted trials of intravenous high-dose (hd) deferoxamine. prior reports of short-term hd deferoxamine have shown minimal side effects however, prolonged use of hd deferoxamine has known toxicity. when compliance is a concern, our center has used hd deferoxamine infusions at mg/kg/hr x hours every to weeks. objectives: evaluate the safety and efficacy of hd deferoxamine at our institution to help guide future therapy. design/method: a retrospective review was completed of patients previously treated with hd deferoxamine between april and september at children's minnesota. final sample included patients ages to years with underlying diagnosis of thalassemia ( ) and diamond-blackfan anemia ( ). deferoxamine infusions were given for hours every - days with a mean length of treatment of days. results: all patients were on combination therapy with deferasirox, however deferasirox was held during deferoxamine infusion. mean pre-deferoxamine liver iron concentration (lic) was . mg/g and mean post lic was . mg/g (p = . ). ferritin mean pre-deferoxamine was ng/ml compared with mean post ng/ml (p = . ). two patients had possible allergy, leading to deferoxamine discontinuation. one patient developed hives, eye swelling and cough while the other had emesis and cough. another patient experienced facial nerve palsy of unclear etiology, which did not recur with resumption of deferoxamine. no respiratory complications were seen. results showed significant decrease in iron burden following combination therapy with high dose deferoxamine and deferasirox. no significant pulmonary, liver, renal, vision, or hearing toxicities were observed. three patients reported reactions to deferoxamine infusions. however, one of these was able to successfully continue deferoxamine without further incident. short-term, hd deferoxamine was effective at reducing lic in combination with oral chelation but requires further evaluation to assess for potential increased risk of toxicity. short-term hd deferoxamine may be considered in the setting of poor compliance of subcutaneous administration or inadequate chelation with single agent therapy. further studies are needed to clarify ideal dosing, timing and risk of toxicity. background: immune thrombocytopenia (itp) is the most common cause of symptomatic thrombocytopenia in childhood but remains a diagnosis of exclusion warranting further evaluation if atypical findings are present. two male children ( months and years old) with newly diagnosed immune thrombocytopenia (itp) were found on initial evaluation to have persistent elevations of lactate dehydrogenase (ldh), alanine aminotransferase (alt), and aspartate aminotransferase (ast). these serum enzyme abnormalities cannot be attributed to itp. in the setting of thrombocytopenia, elevated transaminases and ldh create diagnostic complexity for the hematology/oncology provider as their elevation raises concern for malignancy, hemolytic disease, and other systemic diseases. to raise awareness about an unexpected pattern of duchenne muscular dystrophy in patients undergoing evaluation for itp. to expand the differential of a hematologist/oncologist when abnormal labs support a nonhematologic diagnosis design/method: this case-series of two patients with their clinical and laboratory findings were discovered with retrospective chart review. results: after a thorough evaluation for hemolytic anemias, liver disease and infectious etiologies was negative, bone marrow and liver biopsies were considered. eventually, both children were found to have severely elevated serum creatine kinase (ck). skeletal muscle has the highest concentration of ck of any tissue. thus, significant ck elevation is almost exclusively attributable to muscle injury and is the most sensitive and specific enzyme for diagnosis of muscle disease. referral to a neuromuscular specialist and further genetic testing confirmed the diagnosis of duchenne muscular dystrophy in both children allowing initiation of appropriate interventions. to date, there is no clear genetic predisposition to itp in patients with muscular dystrophy although further investigation may be needed. hematology/oncology providers should consider obtaining a serum ck to rule out muscle disease in any male child with unexplained elevations of serum ldh and/or aminotransferases, as it provides an easy and inexpensive, non-invasive approach to screening. additionally, clinical history and physical examination can aid in the diagnosis of muscular dystrophy, with gross motor delay, abnormal muscle bulk, gower's sign, and proximal muscle weakness all possible findings. objectives: to identify the range of cbcs in patients with ds without infections, hematologic or immune disorders and to create more accurate reference ranges for total white blood count; hemoglobin; hematocrit; mcv; platelet count and absolute neutrophils (anc), lymphocytes, monocytes, eosinophils, and basophils. design/method: a retrospective investigation of healthy pediatric patients with ds who received a cbc between and as part of their medical care at a single, large, pediatric teaching hospital. the study group consisted of children with ds (male = , . %; mean age = . years, sd = . ) at time of blood draw. initially children were reviewed for possible participation in the study; however, patients were excluded due to not meeting the study's inclusion criteria. descriptive statistics were performed on demographic and clinical characteristics. kruskal-wallis h tests, anova, and t-tests were run to determine the significant associations between independent means. results: a significant difference in absolute neutrophils between racial groups, f( , . ) = . , p = . , was observed. there was an increase in anc from . +/- . with african americans to . +/- . in the other racial groups and to . +/- . with caucasians. differences were also found in anc in hispanics/latinos versus non-hispanic/latinos. the results were higher in non-hispanics and latinos, a significant difference of -. ( % ci, -. to -. ), t( ) = . , p = . . preliminary kruskal-wallis h tests run determined that there were significant differences between age groups for total white blood cell, hemoglobin, hematocrit, platelets, lymphocytes and anc. further studies are being run to evaluate in which age groups these differences lie and create reference ranges by age, race and sex. conclusion: among patients with ds, there are differences between racial groups and age groups. this data has been compared to previously established reference ranges for cbcs, but we are currently establishing healthy cbc controls which we will use to validate the reference ranges. these ranges will be published to help guide providers in workup and management of patients with ds. background: transfusion is a critical part of the care provided in the neonatal intensive care unit, but it is not without risks. low birth weight and premature infants can become anaemic from an immature haematopoietic system and frequent phlebotomy. these infants often receive multiple red blood cell transfusions. identifying infants more likely to require such intervention is important in ensuring the appropriate usage of this scarce resource. to determine whether birth weight, gestational age, gender, length of stay and mode of delivery can predict red cell concentrate (rcc) transfusion, units required, donor exposure and time to exposure. design/method: a retrospective chart review of all infants born below weeks gestation and/or birth weight less than , g who received a red blood cell transfusion between july and july in the cork university maternity hospital neonatal unit. results: infants met the inclusion criteria, ( . %) received a rcc transfusion. our study showed lower gestational age (p< . ) and lower birth weight (p< . ) infants are more likely to be transfused. donor exposure increases with a lower birth weight (p = . ). multivariate analysis showed infants with a lower gestational age (or - . per day; p< . ); lower birth weight (or - . per g; p< . ) and a longer length of stay (or . per day; p< . ) are more likely to receive a higher number of rcc transfusions. the time to first rcc transfusion is shorter in those with lower birth weight (or . per g; p< . ) and lower gestational age (or . per day; p< . ). gender and mode of delivery were not found to be predictors of red blood cell transfusion in this study. conclusion: low birth weight and premature infants are more likely to receive a rcc transfusion during admission to the neonatal unit. our study highlights predictors of rcc transfusion, donor exposure and time to transfusion. these can be used in identifying at risk infants, counselling parents and in anticipating transfusion requirements. emily southard, r. grant rowe, david williams, akiko shimamura, taizo nakano children's hospital colorado, aurora, colorado, united states background: the mecom locus encodes transcription factors that regulate hematopoietic stem cell self-renewal and maintenance. overexpression of mecom has been noted in - % of acute myeloid leukemia, several solid tumors, and denotes a poor prognosis. mutations that reduce mecom expression or that disrupt protein function, however, have been implicated in the development of bone marrow failure (bmf) through undefined pathways. an association between mecom mutations and radioulnar synostosis with amegakaryocytic thrombocytopenia (rusat) syndrome has been reported, however further characterization of this phenotype has yet to be explored. to characterize the phenotypic spectrum of a cohort of pediatric patients with novel mecom mutations. we performed a retrospective review of five patients with mecom mutations who were referred to hematology at children's hospital colorado or boston children's hospital. clinical, laboratory, and genetic data was collected on subjects and available family members. results: four of subjects were identified in infancy presenting with congenital cytopenias or physical dysmorphisms that prompted broad genetic screening. platforms for genetic detection included microarray, targeted genetic panels, and whole exome sequencing. three of subjects with cytopenias presented with congenital thrombocytopenia, of whom rapidly progressed to severe aplastic anemia. four of subjects presented with congenital anomalies, of whom demonstrated radioulnar synostosis. additional dysmorphic features identified include craniofacial (low set ears x ), cardiac (pda x , vsd x , aortic root dilation x ), pulmonary (pulmonary hypertension x , arteriovenous malformations x ), and developmental delay. one subject presented at age years with acute pancytopenia, hypocellular marrow, no dysmorphisms, and a mecom variant of unknown signif-icance. the identified mecom mutations include one . mb deletion involving several genes including mecom, one variant affecting a splice acceptor consensus sequence predicted to disrupt splicing, and three novel missense mutations, tyr cys, arg thr, and tyr cys, all of which were absent from public databases and were predicted in silico to be deleterious. we describe the phenotypic spectrum of patients with novel mecom variants. a subset of patients lacked radio-ulnar synostosis and had presence of additional systemic anomalies, demonstrating a varied clinical phenotype that is not isolated to rusat syndrome. a centralized publically accessible database to share clinically annotated mecom variants, together with analysis by experts in mecom function would advance our understanding of the clinical interpretation of mecom variants. mecom should be considered in the differential diagnosis of bone marrow failure and we advocate for the inclusion of mecom in targeted sequencing panels. cairo university, cairo, egypt background: beta thalassemia is regarded as a serious public health problem in the mediterranean region, southeast asia, and the middle east. however, very few studies have been conducted to assess the quality of life (qol) among thalassemia major patients. objectives: to assess the quality of life among b-thalassemia major patients using short form (sf)- questionnaire and to determine the factors associated with their quality of life. design/method: a cross-sectional study was conducted among thalassemia major patients who were attending the hematology outpatient clinic at cairo university hospital, during the study period. data were collected between october and march . the quality of life was assessed for patients aged ≥ years. the mean age of the studied group was . ± . years. the majority ( . %) had one monthly blood transfusion. the mean total score of sf- was . ± . . general health perception domain was the most affected domain with mean score, while vitality was the least affected one. there was no statistically significant difference between males and females regarding different quality domains except for vitality where the mean score was significantly higher in males than females (p = . ). age at onset of disease, and at first blood transfusion were the most documented factors positively correlated with the quality of life among the enrolled thalassemia patients. conclusion: the quality of life in thalassemia major patient was found to be compromised. all thalassemia patients should undergo assessment of the quality of life so that interventions focusing on the affected domains can be implemented. background: international adoption of children with special needs has become more prevalent in recent years leading to tremendous growth in the number of u.s. thalassemia patients adopted from foreign countries. currently % of the , thalassemia patients registered in the cooley's anemia foundation (caf) patient database have been adopted from foreign countries, primarily china. as this population continues to grow, further information is needed in order to provide these families with best supportive care. the primary goal of this study is to characterize the socio-demographics and health statuses of adopted children with thalassemia and their families. a secondary goal is to describe adoptive families' motivations, experiences, challenges, and support resources. design/method: a redcap survey was accessed by families of adopted children with thalassemia through the caf website and caf social media from january to august . following a four-question screen, eligible subjects were directed to complete an adoption questionnaire. families who had at least one adopted child with thalassemia receiving care at a participating thalassemia treatment center or hematology office in the u.s. were considered eligible. descriptive statistics were analyzed using sas . . respondents who were ineligible or who provided incomplete data were removed from the dataset prior to analysis. of survey respondents, qualified and completed the survey. these households had adopted a total of children with thalassemia ( . % male), most from china ( . %), where they had been living in orphanages ( . %). legal guardians identified primarily as christian ( . %). the majority had completed post-secondary education ( . %) with reported household incomes greater than $ , ( . %). most adoptive families were connected to an adoption group or community including online groups, local support groups, and adoption networks ( . %). commonly cited challenges were: ) volume of frequent medical appointments, ) insufficient support from their local care centers, and ) financial burdens. the reality of care for the population of adopted patients with thalassemia in the u.s does not seem to match the expectations set by their providers. we are hopeful this data will be used to assist adoptive families navigating the complexities of thalassemia care. the findings suggest that this population would benefit from additional outreach, education, guidance, and advocacy resources -especially in the early stages of adoption and during initiation of post-adoption medical care. background: in many higher-income countries, thalassemia major has become a chronic disorder; many outcomes are different in emerging countries with more limited resources. most analyzes of health-related quality of life (qofl) in thalassemia have been conducted in high-income settings. objectives: to assess the impact of health status on qofl in thalassemia patients in an emerging country. we assessed qofl in randomly-selected patients ( thalassemia major; with hemoglobin e thalassemia; five thalassemia intermedia) at the national thalassemia center in kurunegala, sri lanka where approximately patients are managed. treatment is free, but compared to north america/europe, access to tertiary staff and other resources are limited. overall, control of body iron as estimated by serum ferritin concentration (mean± sem, ± g/l) was not optimal in many patients. to understand the impact of health status on qofl, we used the sf v health survey, analyzing scores of physical function, pain, general health, social functioning, emotional and mental health, to generate overall physical and mental component scores. results: compared to reports from higher-income countries (american journal of hematology ; : - ), physical function scores (mean±sd, . ± . ) were similar in sri lankan patients; indeed, in three categories (physical role, social function, emotional role), sri lankan scores were slightly higher. by contrast, compared to scores from higherincome settings, those estimating bodily pain, general health, and mental health were significantly lower, resulting overall in a significantly lower physical component score in sri lankan patients. male sri lankan patients reported higher scores than females, and somewhat surprisingly, in four categories (physical function, physical role, social function and emotional role) reported higher scores than those obtained in higher-income settings. lower scores in physical functioning, leading to an overall lower physical component score, were recorded by females. patients with hemoglobin e thalassemia reported generally poorer qofl than those with thalassemia major. the lack of differences in qofl in patients with "high" and "low" hemoglobins was likely related to low pre-transfusion hbs (mean±sem, . ± . g/dl) in nearly all patients. these early data in a small cohort of thalassemia patients in an emerging setting suggest that in many patients bodily pain, reduced mental health, and poorer views of general health affect overall qofl. prospective studies in larger cohorts including evaluation of adequacy of transfusions and chelation therapy, complications, and overall accessibility of care may guide approaches to improve qofl in lower-income settings of thalassemia care. geetanjali bora, anand prakash dubey, tarun sekhri, mammen puliyel, aparna roy maulana azad medical college, new delhi, delhi, india background: in the last two decades, the presence of osteopenia has been described in optimally treated patients with transfusion dependent thalassemia, the pathogenesis of which seems to differ from osteopenia in non-transfused patients. the prevalence rate of low bone mineral density (bmd) in pediatric population is highly variable amongst studies done worldwide. furthermore, the role of metabolic and endocrine factors in determining bone mass in this population is not well understood. objectives: to assess bmd in subjects with transfusion dependent beta thalassemia by dual-energy-x rayabsorptiometry and find its co-relation with clinical, biochemical and hematological parameters. design/method: this is a comparative cross-sectional study and includes patients with transfusion dependent beta thalassemia between ages to years enrolled from a thalassemia day care center in the year - . at the time of enrollment age, sex, bmi z scores, pubertal staging, duration and type of chelation therapy were noted. enrolled subjects were scanned for bmd at lumbar spine l - and left femoral neck using dexa scan. the bmd was expressed in mean values and z scores. age, bmi, ethnicity and gender matched historic controls were used to generate z scores. ml of pre transfusion fasting venous blood samples were obtained to test for serum calcium, phosphate, alkaline phosphatase, pth, thyroid function panel, serum ferritin and serum igf- levels. mean values for pretransfusion hemoglobin and serum ferritin over last months were calculated. results: total no of subjects , median age . years, male ( %), female ( %), ethnicity % asian, bmi < rd centile ( %), pre pubertal %, all receiving transfusion and chelation therapy. prevalence of low (z score < - sd) and very low (< - . sd) bmd was %, % at l -l respectively and %, % at left femoral neck respectively. there was trend of lower bmd z scores with advancing age. statistically significant co-relation (p value < . ) was found between low bmd and low mean pretransfusion hemoglobin, serum phosphate, igf - and vitamin d levels conclusion: a sizable proportion of children and adolescents with transfusion dependent thalassemia have suboptimal bone mineral density and this decline may start as early as - years of age despite being on transfusion regimen highlighting the importance of yearly dexa screening and optimization of pre-transfusion hemoglobin, vitamin d and igf levels. vanderbilt university medical center, nashville, tennessee, united states background: it is well described that iron deficiency anemia (ida) can co-present with thrombocytosis or thrombocytopenia, though cases of thrombocytopenia are less frequent than thrombocytosis. prior reports of thrombocytopenia have included adult and pediatric patients with menorrhagia ( - ), menorrhagia due to uterine fibroids ( ), or other gynecologic abnormalities ( ). our cases highlight the pattern of ida, thrombocytopenia, and menorrhagia in the setting of significant menstrual clotting without observed gynecologic abnormalities in african-american adolescents. objectives: to describe the clinical course of three adolescent females with severe ida, menorrhagia, and thrombocytopenia. results: our cases included three female african-american patients ages - who presented with severe anemia and concurrent thrombocytopenia in the setting of menorrhagia. all three patients reported heavy and prolonged menstrual cycle bleeding with significant clots. two of the three were admitted for transfusions at presentation and noted to have significant menstrual bleeding with continued blood loss requiring additional transfusions until bleeding was controlled with estrogen therapy. these two patients were evaluated with pelvic ultrasounds revealing a prominent endometrium in both patients and hyperechoic material consistent with a clot in one patient. average hemoglobin on presentation was . gm/dl ( . - . ), average platelet count was , /mcl ( , - , ), and average mcv was ( - ). all had severe iron deficiency with an average ferritin of ng/ml ( - ) subsequently treated with oral iron. one patient had a prior history of ida that required transfusion and had subsequent normalization of her complete blood count. two patients had subsequent thrombocytosis before normalization of their platelet counts. two patients received platelet transfusions: one due to recent neurosurgical intervention with a higher goal platelet count and the other to help control menstrual bleeding after a nadir platelet count of , . a review of the clinical history and red cell indices pointed to ida and ongoing blood loss from menorrhagia as the reason for the bicytopenias. the thrombocytopenia in these cases may have been exacerbated by consumption of platelets in the significant clots all three patients reported. it is reasonable to treat with iron supplementation and supportive care which may include transfusions or management of menorrhagia with oral contraceptives or other hormonal methods. background: sickle cell disease is one of the most common inherited red blood cell disorders, yet many are not aware of their carrier status. the american college of obstetricians and gynecologists' guidelines recommend that pregnant women of african, mediterranean and southeast asian descent be screened for hemoglobinopathies with a cbc and hemoglobin electrophoresis . however, adherence to this practice and frequency of improper screening with sickledex is unknown. proper screening and counseling can impact families' knowledge, allowing for establishing relationships with pediatric hematology providers earlier. objectives: we sought to assess prenatal hemoglobinopathy screening practice patterns and methods of obstetrics & gynecology (obgyn) and family medicine providers in the nyc regional area. design/method: a cross-sectional electronic survey was administered to obgyn and family medicine practitioners from four nyc institutions. questions focused on prenatal hemoglobinopathy screening practices using case scenarios with variations on parental trait status and ethnicities. chisquare analyses were used to compare the two provider groups on categorical variables. there were total responses; surveys were complete, of which were obgyn and family medicine providers. respondents were mainly from academic medical centers, with the majority being faculty ( % of the obgyns and % of family medicine). no significant difference was found in frequencies of screening patients with a positive family history of a hemoglobinopathy. when asked about screening practices for patients without a personal/family history of a hemoglobinopathy, % of obgyns versus % of family medicine providers "always" screened for hemoglobinpathies (p = . ). when analyzed by ethnic background, there were significant differences by group in screening patients of white ( % vs %), black ( % vs %), mediterranean ( % vs %), and asian descent ( % vs %) (p≤ . for all). however, in cases where the hemoglobinopathy carrier status of both parents was known, there was no difference in screening with a hemoglobin electrophoresis. furthermore, > % of all respondents use sickledex for screening in the case scenarios. conclusion: this pilot survey highlights a difference in the methods and likelihood of prenatal hemoglobinopathy screening based on the type of prenatal care provider. screening differences can lead to variations in prenatal guidance, diagnostic procedures, informed decision-making and knowledge of families referred to pediatric hematology clinics. this is the first study analyzing prenatal screening for hemoglobinopathies in obgyn and family medicine. improving prenatal screening practices by collaborating with hematologists may decrease health care disparities and allow for earlier relationship building with pediatric hematology. . acog, opinion# , poster # hermansky-pudlak syndrome: spectrum in oman background: hermansky-pudlak syndrome (hps) is a rare autosomal recessive disorder, characterized by the triad of oculocutaneous albinism, a hemorrhagic diathesis resulting from storage pool-deficient platelets, and accumulation of ceroid/lipofuscin-like material in various tissues. before , nine different types of hermansky-pudlak syndrome were identified, which can be distinguished by their signs and symptoms and underlying genetic cause. in , a tenth type was defined based on mutations in the ap d gene. hps type is characterized in addition by severe neutropenia and recurrent sinopulmonary infection. the disease is more common in puerto rico, and this is the first report from oman. to describe the clinical, laboratory and genetic characteristics of hps sub-types in oman, including the first cases of hps type . design/method: this is a retrospective study, including cases with hps that had been suspected clinically and confirmed through genetic mutation analysis. clinical data included sex, age at presentation, initial clinical presentation (skin, eyes, development, neurological involvement, bleeding tendency, recurrent infections) and course of disease. laboratory data (complete blood counts, platelet and absolute neutrophil counts, coagulation screening, platelet function tests by platelet function analyzer, and platelet aggregation studies using different agonist had been recorded. pcr and next generation sequencing for genetic confirmation by testing mutations in hps , ap b , hps , hps , hps , hps , dtnbp ,, bloc s , bloc s genes had been done. results: seven omani cases with hps have been identified ( males and females). their age ranged between (at birth) to years. two patients had hps type , patient had type , while the other cases had hps type . no other sub-types were encountered in oman. all patients were products of consanguineous marriage. one patient had adrenal hge, while the others had mild hemorrhagic phenotype, characterized by recurrent bruising and mild epistaxis. laboratory testing confirmed variable platelet aggregation defects with different platelet agonists. all patients had characteristic hypopigmentation, iris transillumination, nystagmus, and foveal hypoplasia. both patients with hps type had the same homozygous mutation in the ap b gene (c. _ delta), and presented with severe neutropenia. early diagnosis and initiation of gcsf on one of them improved outcome and prevented the development of complications. late diagnosis in the other patient resulted in the development of bronchiectasis as a result of recurrent sinopulmonary infections. background: sickle cell disease (scd), a genetic disorder characterized by defective sickle hemoglobin (hbs), triggers red blood cell sickling, hemolysis, vaso-occlusion, and inflammation. ischemic injury from scd starts in infancy and accumulates over a lifetime, causing pain, fatigue, and progressive end-organ damage that culminates in early mortality. voxelotor (gbt ) is an oral, once-daily therapy that modulates hemoglobin's oxygen affinity, thereby inhibiting hemoglobin polymerization. objectives: to assess the safety, pharmacokinetics, and efficacy of voxelotor in pediatric patients with scd. design/method: this ongoing study is being conducted in parts: part a: a single dose of voxelotor mg in pediatric and adolescent patients; part b: multiple doses of voxelotor mg/d or mg/d for weeks in adolescents. part b's primary objective is to assess the effect of voxelotor on modifying anemia. secondary objectives include measuring other markers of disease modification, such as hemolysis; daily scd symptoms, using a patient-reported outcome (pro) measure; and safety. results: as of november , , patients ( females) had received voxelotor mg and patients ( females) had received voxelotor for ≥ weeks. the median age for the patients was years, % were receiving hydroxyurea (hu), and % had ≥ painful crises in the past year. data for hemolysis measures are available for patients who received voxelotor for weeks. six of the patients achieved a hemoglobin (hb) response of > g/dl increase. laboratory markers of hemolysis improved concordantly; the median reductions in reticulocytes and indirect bilirubin were % and %, respectively. ten of patients showed reduction in total symptom scores (tss) at week , with a % median reduction in tss from baseline. there were no treatmentrelated serious adverse events (aes) or drug discontinuations due to aes. voxelotor mg for weeks in adolescents with scd, the majority receiving hu, demonstrated consistent, sustained efficacy on hb levels and measures of hemolysis; > % of patients showed a > g/dl improvement in hb. improvement in tss in mildly symptomatic patients suggests that the pro is sensitive to treatment effect and supports use in the ongoing hope phase study. voxelotor's reassuring safety profile is consistent with results in adults. these interim results support ongoing clinical evaluation of voxelotor as a potential disease-modifying therapy for adults and children with scd. supported by global blood therapeutics. background: acute kidney injury (aki) is a common complication in sickle cell disease (scd), and a potential risk factor for sickle nephropathy. aki is associated with acute decline in hemoglobin (hb) during vaso-occlusive pain crisis and acute chest syndrome (acs). it is unclear which pathologic factor plays a stronger role in aki development during hb drop: increase in free heme during vaso-occlusive events secondary to hemolysis or hb decline itself. objectives: to investigate if hb decline alone is associated with aki, we tested if the renal function of patients with scd worsened during parvovirus b -induced transient aplastic crisis (tac), in the absence of accentuated hemolysis. design/method: with irb approval, a retrospective study of patients who had laboratory confirmed parvovirus-b was conducted. serum creatinine (scr), both during and within months from the tac event, was collected. comparisons of the clinical and laboratory characteristics were analyzed using the wilcoxon test for continuous variables. aki was defined as an increase in scr by ≥ . mg/dl or a % increase in scr from baseline. to evaluate differences in change in hb on aki risk, changes in scr during tac were compared to those during pain crisis or acs admissions by fitting a generalized linear mixed model for binary outcome. a comparative sample of acs events and vaso-occlusive pain crisis were used to estimate rates of aki according to hb levels. results: three ( %) of the patients with scd developed aki during tac. no association was identified between change in hb from baseline to tac event (p = . ). no cases of aki were identified until hb decreased < . g/dl or the change in hb was ≥ . g/dl from baseline. next, we developed a model to evaluate the impact of change in hb from baseline for patients admitted with tac, pain crisis or acs on aki. with a g/dl decrease in admission hb from baseline, patients with tac had a % probability of developing aki, while acute chest syndrome and pain crisis would have a % and % probability, respectively. our data suggest that aki is still prevalent during parvovirus b -induced tac. however, the risk of aki during a tac event is and times lower than that from severe anemia induced by acute chest syndrome and vasoocclusive pain events, respectively. hemolysis-induced anemia during scd crisis appears to have a more significant role in the development of aki as compared to agenerative anemia. background: the natural history of hemoglobin e beta thalassemia (hbethal), the commonest form of severe beta thalassemia worldwide, has been examined in very few longterm studies. previously, we reported findings in hbethal patients in sri lanka. objectives: to evaluate longterm requirements for transfusion and splenectomy, complications and death in hbethal patients. design/method: all available patients were reviewed - times annually over years. results: patients ( %) died, aged (mean ± sem) . ± . years; the (known) causes commonly included iron overload ( ) and infection ( ); patients surviving patients are aged . ± . years. of patients originally classified by severity (group the mildest, and group the most severe, phenotypes), ( %) were assessed as mild (groups and ), of whom transfusions had been discontinued in . ultimately, / ( %) resumed transfusions, often following shifts to increasingly severe phenotypes including increasing intolerance to anemia. age at resumption of transfusions (following a transfusion-free interval of . ± . years) was . ± . years; in the more severe groups and , regular transfusions were stopped in / patients and resumed in / ( %), at younger ages ( . ± . years) and after shorter transfusion-free periods ( . ± . years) than in "milder" patients. mid-parental height (mph) was ultimately achieved in %. patients ( %) were splenectomized; updated analysis of responses to splenectomy (originally "group " patients), showed that splenectomy (at . ± . years) was followed by an extended, but impermanent, transfusion-free interval ( . ± . years); % patients resumed transfusions, usually related to exercise intolerance or poor growth. in groups and , complications of anemia and ineffective erythropoiesis, including leg ulcers (in % and %) and gallstones ( % and %), were more frequent than in groups and ; fractures were observed ( - %) across all groups, except for regularly-transfused group patients ( %). pulmonary artery pressures > mm were recorded in % patients. evaluation of patients with hbethal requires observations over years, without which definition of patients as "mild" or "severe" may be misleading. while in many patients transfusions may be withheld or reduced in frequency, troublesome complications may surface with advancing age even in "milder" patients. although individual consideration of transfusion requirements is critical, the availability of effective chelation, where this can be provided without prohibitive cost, may alter the balance of risks and benefits of regular transfusions in hbethal. (premawardhena a. lancet ). background: social determinants of health (sdh) are environmental and socioeconomic factors, such as access to food and housing that affect health outcomes. pediatricians are increasingly screening for sdh as part of primary care visits, however less is known about screening for sdh in pediatric hematology. evidence suggests that sdh play a role in disease severity for children with scd, who face significant socio-economic and racial disparities. the goal of our quality improvement (qi) project was to increase the percentage of patients with scd who were connected to community resources for unmet social needs. design/method: we based our intervention on the successful implementation of wecare in our institution's pediatric primary care clinic. eligible patients were identified at the start of each clinic session. on arrival the parent was given a self-reported screening tool for six sdh (childcare, education, employment, food, utilities and housing). results were entered in the electronic health record by the physician or social worker who then printed a pre-existing resource list for patients with a positive screen. we used a series of plan-do-study-act (pdsa) cycles to study tests of change. we tracked process measures (percentage of patients screened, percentage of patients with an unmet social need who received a resource sheet), outcome measures (percentage of patients with an unmet social need who connected with a community resource) and balancing measures (staff, patient and provider satisfaction). run charts were reviewed weekly and then monthly to inform further tests of change. examples of pdsa cycles include who gave the paper survey to patients (social worker or physician versus medical assistant) and length of time between surveys ( to months). results: between august and december screening rates improved from % to %. of the patients screened, % report at least one unmet social need; of those % received a targeted list of community resources in the first month of the project, and % in the fifth month. finally, % of patients reached by phone had connected with a community resource within weeks of the clinic visit. we have successfully implemented universal screening for sdh for patients with scd in our urban pediatric hematology clinic without requiring extra staff. next steps include further pdsa cycles to connect more patients to appropriate resources, and tracking improvement in health care utilization outcomes from addressing sdh in this vulnerable patient population. background: the clinical manifestations of sickle cell disease (scd), chronic hemolytic anemia, and vaso-occlusion occur as a direct result of sickle hemoglobin (hbs) polymerization. voxelotor (gbt ) is a first-in-class, oral, oncedaily investigational agent designed to modulate hemoglobin's oxygen affinity in a targeted approach to inhibit hbs polymerization. objectives: to examine the pharmacokinetics (pk), safety, and dosing of voxelotor in children (aged - years) and adolescents (aged - years) with scd from part a of the gbt - study. design/method: gbt - is an ongoing, open-label, phase a study in patients aged - years with scd (sickle cell anemia or sickle beta zero thalassemia). part a of this study (the focus of this abstract) is examining pk of singledose ( mg) voxelotor. pk samples to measure whole blood and plasma voxelotor concentrations were collected up to days following single-dose administration. separate population pk (ppk) models were developed to describe the concentration versus time profiles of voxelotor in whole blood and plasma using nonlinear mixed effects modeling (non-mem, version . ). ppk modeling and physiologically based pk (pbpk) modeling were used to simulate voxelotor pk parameters and support dose selection for future evaluation in younger children. : part a included adolescents ( females; median age years [range - ]) and children ( females; median age . years [range - ]). mean weight was . kg (range - kg) and . kg (range - kg) in adolescents and children, respectively. voxelotor was well tolerated with no drugrelated grade ≥ adverse events (ae) or serious aes. a compartment model with first-order absorption best described the pk of voxelotor (and was the same model structure used for adults with scd). voxelotor pk exposures in adolescents were comparable to those observed in adults, but higher exposures were observed in children. ppk and pbpk modeling support the use of a weight-based dosing strategy in younger children (aged < years) in future trials. adult voxelotor doses can be used in adolescents. however, based on higher pk exposures, a lower weight-based dosing strategy is recommended in children. ppk and pbpk modeling provides an innovative approach to minimize experimental dosing in children and accelerate dose selection of voxelotor in ongoing and future clinical studies. this abstract is supported by global blood therapeutics. background: hydroxyurea (hu) reduces rates of acute complications, and improves long term outcomes in patients with sickle cell disease (scd) and is now fda approved for children. through previous work we have increased the number of eligible patients on hu in our clinic, however accessing a compounding pharmacy remained a significant barrier to hu adherence for infants and children who cannot swallow capsules. objectives: the objective of our quality improvement project was to improve adherence to hu among pediatric patients with scd at our urban safety net hospital by addressing barriers to obtaining liquid hu. design/method: to begin we met with the leadership of our outpatient pharmacy which offers mail order delivery. however, like most retail pharmacies, they do not have the necessary protective equipment to compound liquid hu. through a series of discussions, we began a unique partnership with our institution's inpatient chemotherapy pharmacy who compounds the liquid hu and delivers it to the outpatient s of s pharmacy, who then dispenses liquid hu to families. using a series of plan-do-study-act (pdsa) cycles we tracked adherence by calculating the medication possession ratio (mpr), defined as the percentage of days in a given period of time that each patient had their medication on hand. the mpr for liquid hu mpr among enrolled patients was tracked by pharmacy staff and reviewed monthly. additional pdsa cycles included adding automatic refills and reminder calls by pharmacy staff and improving communication about delivery. we also tracked patient satisfaction. results: between march and december , a total of thirty pediatric patients were enrolled in our program for on-site compounding and free mail order delivery of liquid hu. mpr for liquid hu is currently . % among enrolled patients, significantly higher than the mpr of % reported in the literature, and has risen steadily since the beginning of the project. families are highly satisfied with the program, specifically appreciating the convenience of mail order delivery, saving on delivery fees, and reminder calls when refills were due. by compounding and dispensing liquid hu directly from our institution's outpatient pharmacy we have significantly improved adherence to this hu therapy in our high-risk population. next steps include analysis of change in clinical outcomes for patients enrolled in this program. as adherence to hydroxyurea is associated with decreased acute care utilization and cost, programs such as ours could play a crucial role in reducing the excessive costs and ed utilization among this patient population. background: experience with the iron-chelator deferasirox is reported widely in higher-income settings. by contrast, real-life experiences in emerging countries are infrequently reported. objectives: to evaluate, in a non-trial setting, the real-life response to deferasirox in an emerging country. design/method: in sri lanka's national thalassemia center which manages patients without tertiary staff, quantitative evaluations of body iron or estimates of extra-hepatic iron, the records of patients who began deferasirox in / were retrospectively reviewed. results: baseline assessments (mean±sem) indicated substantial iron loading [serum ferritin (sf) , ± ug/l; serum alt ± . u/l (normal ≤ u/l)]. deferasirox was introduced at low doses ( . ± . mg/kg/day); many patients started at < mg/kg and, after months, doses remained ≤ mg/kg/day in % patients. after months, sf in % patients remained > , ug/l; only by months had (mean) sf declined to < , ug/l ( ± ; p< . ). similarly, mean alt normalized (to ± u/l) only by months. death and complications were not systematically recorded by staff who had been charged, without provision of additional resources, with the introduction of this new drug in hundreds of patients. these results contrast to those in sri lanka's tertiary thalassemia center where, in patients following the introduction of deferasirox ± . mg/kg/day, sf declined rapidly, even in relatively less ironloaded patients (from , ± to , ± g/l after months; p = . ). these findings underscore the importance, during the implementation of new drug regimens in lowerincome centers with marginal resources, for investments in methods to quantitate body iron burden, hands-on educational initiatives to guide day-to-day management by competent but non-expert staff, and data systems to record efficacy, effectiveness, toxicity and compliance. such investment is critical to optimising therapy and improving complications in thalassemia patients worldwide: even in sri lanka, where resources directed to thalassemia management are greater than in most of asia, results in the oldest living cohort (born - ) indicate under-treatment [elevated iron burdens (sf , ± ug/l) and high prevalences of diabetes ( %) and hypothyroidism ( %)]. even in a younger cohort (born - ) which has benefitted from improved treatments, the prevalence of many complications exceeds those reported from high-income settings. over the next decade, and two decades after the who declaration that the impact of thalassemia on global mortality and morbidity is underrecognized, increased investments by governmental and nongovernmental sources will be necessary to improve outcomes for asian patients with thalassemia. background: a major barrier to success in hydroxyurea (hu) treatment of patients with sickle cell disease (scd) is non-adherence. objectives: to optimize hu adherence in patients with scd. design/method: a care model was designed by the sickle cell (qi) team at children's hospital to improve hu adherence among scd patients. the original model included bimonthly family phone contact, monthly dispensing pharmacy phone contact and lab monitoring. adherence measures included obtaining hu from pharmacy monthly, completion of monthly labs, hb f percentage and mcv, and mtd achievement. from / - / , several pdsa cycles refined our care model. a one-year follow-up survey gathered feedback on the care model. the first-year data involved ∼ patients. the biggest improvements resulted from making pharmacy calls before patient/family calls, shipping liquid hu to outlying patients, and tracking call time/content. the qi goal was % hu adherence by / . the % baseline adherence rate increased to % by / , and has remained in that range. the completion rate of patient/parent phone calls increased from % the first month to % at six months. pharmacy prescription pick-up has increased from % to % per month. lack of liquid hu availability was overcome by shipping the medication to the patient's home. parental hesitance to share information by phone, especially with qi team members with whom they had no established relationship, was overcome by having the longtime sickle cell nurse do many of the early calls. however, survey feedback showed families became comfortable with several clinic personnel calling. the calls gave families the opportunity to ask questions about their child and/or get additional information about scd. the calls also provided an opportunity for seasonal flu shot or tcd testing reminders. the surveys gave information on the optimal time of day to reach each family, providing individualization and further increasing the percentage of completed calls. two families surveyed said they no longer needed two calls a month because they were now able to remember to pick up hu, administer it, and get labs on their own. this qi project has not only improved hu adherence, but also fostered health education/counseling, increased patient/parent satisfaction, and enhanced service utilization. medical team member and patient/family comments demonstrate that it has helped build relationships and trust between families and the medical care system. based on survey feedback, we will further individualize care to increase adherence rate and sustain improvements. cincinnati children's hospital medical center, cincinnati, ohio, united states background: the thalassemias are a heterogeneous group of genetic blood disorders caused by mutations that decrease or eliminate the synthesis of the -and/or -globin subunits of hemoglobin. the phenotype of thalassemia depends on the interaction of the -and -globin gene clusters, because both loci determine the -/ -chain balance. for example, a -thalassemia phenotype can be more severe than expected when coinherited with -globin gene triplication (copy number gain), which exacerbates the -/ -globin imbalance. objectives: describe four individuals with an incorrect diagnosis of -thalassemia trait who were later properly diagnosed by comprehensive genetic testing to have -thalassemia intermedia caused by heterozygous -thalassemia mutations coinherited with triplicated -globin loci. design/method: sequence analysis of the -globin (hba /hba ) and -globin (hbb) genes, and copy number variation analysis of the -and -globin gene clusters by multiplex ligand-dependent probe amplification. results: four unrelated individuals of northern european ancestry were evaluated for signs and symptoms not explained by a diagnosis of -thalassemia trait (previously made by a pediatric hematologist), including growth delay, splenomegaly, moderate anemia, marked elevation of hemoglobin f, thalassemic facies, reticulocytosis, and/or indirect hyperbilirubinemia. genetic testing revealed that all were heterozygous ( / ) for the same, single -globin mutation [hbb.c. c>t (p.q *)] and also heterozygous for an -globin triplication ( / anti- . ). their previous diagnoses of thalassemia trait had been made by complete blood counts, hemoglobin electrophoresis, and/or sequence analysis of the -globin genes only. these individuals' phenotypes ranged from moderate anemia only to multiple stigmata of thalassemia, demonstrating the phenotypic variation of a thalassemia genotype. correct diagnosis was made at an average age of . years. a trial of chronic transfusions was initiated for one patient for growth failure. all were educated about the potential for exacerbations of anemia, gallstones, osteoporosis, and iron overload (even without transfusions). parental genetic testing was recommended to assess reproductive risk, because inheritance of this complex genotype can be apparently autosomal dominant. conclusion: heterozygosity for a -thalassemia mutation does not necessarily indicate -thalassemia minor or "trait". when coinherited with -globin gene triplication, a symptomatic form of -thalassemia can occur. correct and timely diagnosis of thalassemia requires careful consideration of the degree of anemia and examination for organomegaly, bony changes, and jaundice. sequence analysis and copy number variation analysis of both the -and -globin gene clusters is key. hematologists need to be aware of this diagnostic possibility and how to test for it to prevent inaccurate or delayed diagnosis. background: the burden of healthcare costs for sickle cell disease (scd) is nationally estimated at over $ billion. the major components of these costs are inpatient and emergency center (ec) visits, many of which are potentially avoidable. in several chronic conditions, a subset of patients account for most of the avoidable encounters. identifying these patients is the first step in targeted care delivery. objectives: to measure and analyze scd patient utilization patterns in the ec and inpatient at texas children's hospital (tch). we identified all individuals under years old with any encounter at tch associated with an international classification of disease (icd)- or code for scd, including hgb ss, hgb sc, and hgb s/beta thalassemia. for each patient, we identified all inpatient and ec encounters in the days prior to their most recent encounter. finally, each encounter was classified as associated with pain, acute chest syndrome (acs), or "other" using an algorithm of discharge diagnosis codes and pharmaceutical delivery. the total number of scd-associated ec and inpatient encounters over the prior year was calculated for each patient. we stratified each patient according to their utilization patterns: low ( - encounters), intermediate ( - encounters), and high (≥ encounters). we identified unique patients with scd that had at least one encounter from july until june . there were , scd-related encounters in the days prior to their most recent encounter. most ( %, n = ) patients exhibited low-utilization patterns and % (n = ) were intermediate. finally, a small subset ( %, n = ) demonstrated high-utilization patterns and accounted for % of all encounters. high-utilization was associated with older age and public payment mechanisms. pain encounters were predominantly in pre-adolescents and teenagers with high-and intermediate-utilization patterns. acs was most frequent in pre-teens and younger teens in the intermediate-utilization group. finally, the youngest-aged high and intermediate users presented for other reasons such as febrile episodes and splenic sequestration. our findings reflect national trends in that a significant portion of encounters are attributed to a small subset of patients exhibiting a high-or "super-" utilization pattern. at our institution, scd super-utilization is associated with older age and pain. we also identified a group of infants and toddlers with frequent encounters for fever. to comprehensively address this burden, it will be important to design interventions targeted toward age and specific medical needs. background: background: the rarity of diamond blackfan anemia (dba) has hindered describing the spectrum of disease, identifying predictive correlations, and guiding datadriven recommendations. long-term toxicities from steroid or transfusion therapy that start in childhood remain the major clinical problems in patients with dba who do not receive stem cell transplant. objectives: objective: to define the dba patient population at st. jude children's research hospital including treatment responses and toxicities to help inform recommendations on treatment and monitoring. design/method: method: medical records were reviewed for all patients with dba treated at st. jude between and for diagnostic testing, treatment types and regimens, and outcomes. two-sample t-test or wilcoxon rank sum test was used to compare continuous variables in two groups depending on the normality of the data tested by shapiro-wilk test. results: a total of patients with dba were identified with a median age of . years (range months - years) at last follow up. a ribosomal protein gene mutation was identified in / patients ( %) with an rps mutation / ( %). thirteen different congenital malformations were described in / patients ( %). fourteen of twenty ( %) patients treated with corticosteroids had an initial response and of those achieved full remission. three patients became steroid-refractory and were unable to wean to an acceptable dose. five of twenty patients continue on lower-dose steroids. five patients currently require no therapy. univariate analysis revealed no statistically significant genetic predictors of response or remission, however, / rpl patients responded to steroids with / ( %) in long-term remission. ten patients are maintained on chronic transfusions and have undergone successful hematopoietic stem cell transplant. nineteen of treated patients ( %) had a treatment-related toxicity. patients on steroids were more likely to have short stature than patients on transfusions or in remission (p = . ). severe bone mineral density deficit occurred in / ( %) patients, in before age years. eight patients had hepatic iron overload, in one documented by age years. other severe toxicities included restrictive cardiomyopathy from iron overload, pathologic fracture, diabetes mellitus, and premature ovarian failure in one patient each. this genotypically and phenotypically heterogeneous dba cohort had a high rate of treatment-related toxicities, notably growth retardation, bone density loss, and hepatic iron overload even in very young children. these findings underscore the need for early standardized monitoring. background: patients with sickle cell disease (scd) face worsening morbidity and mortality between ages and , when they must transition from pediatric to adult healthcare.( ) an effective curriculum addressing disease knowledge, educational and vocational skills, self-efficacy, and social supports is critical to a successful transition. traditional didactic approaches have not led to durable knowledge retention. ( ) technology-based methods have been attempted, but the best educational approach remains unknown. objectives: . to understand how adolescent and young adult (aya) patients with scd view existing transition education. . to include patient preferences in improving our transition curriculum. we developed a qualitative survey to assess patient views of existing approaches for learning about scd and their opinions about preferred transition topics. thirty patients with scd aged to years old were recruited between january and december . responses were managed using redcap electronic data tools hosted at the university of rochester.( , ) qualitative and quantitative data analyses were performed, including independent t-testing to compare responses between age groups. results: approximately % of subjects were under years of age, while % were or older. seventy-one percent had a computer, and . % had a cell phone, with most reporting daily use. subjects reported greatest satisfaction with learning from their doctor during clinic visits ( . % agree or strongly agree) and websites on a cell phone ( . % agree or strongly agree); the least popular methods were online chat rooms and microsoft® powerpoint presentations. satisfaction was similar across age groups. recommended transition topics were viewed positively, with subjects ranking highest understanding their bloodwork ( . % agree or strongly agree) and understanding laws protecting students with chronic disease ( . % agree or strongly agree). older subjects ( - years old) agreed more strongly with learning about opioid addiction and understanding differences between adult and pediatric doctors than did younger subjects ( - years old) (p < . ). this pilot study was successful in helping us to understand the educational needs of aya patients with scd. preliminary data underscore the importance of education provided by the pediatric hematologist. our results also suggest that the optimal use of technology-based methods requires further investigation and that tailoring transition education by age group may be useful. background: similar to patients with transfusion-dependent beta-thalassemias (tdt-beta), survivors of hemoglobin barts hydrops fetalis (homozygous alpha- -thalassemia, tdtalpha) will require lifelong transfusions of erythrocytes. we have previously shown that a transfusion strategy that is based on the guidelines developed for tdt-beta (conventional transfusion) is suboptimal for these patients owing to the differences in the pathophysiology of anemia in the two conditions: in tdt-alpha, conventional transfusion strategy will lead to a gradual increase in non-functional hbh with subsequent tissue hypoxia and hemolysis. an aggressive transfusion strategy that was based on reduction of hbh and increase in "functional" hemoglobin level resulted in improvement of tissue oxygenation and reduction of hemolysis but was associated with significant increase in transfusional iron burden [amid et al, blood ] . objectives: to define the optimal chronic blood transfusion targets for hbh% and functional hemoglobin in patients with tdt-alpha. design/method: following research ethics board approval, longitudinal data of patients with tdt-alpha ( males, median age . ( . - . ) were retrospectively collected. variables of interest included total pre-transfusion hemoglobin, hbh%, and "functional" hemoglobin [measured as total hemoglobin x ( -hbh/ )]. outcome variables were lactate dehydrogenase (ldh, marker of hemolysis), and soluble transferrin receptor (str, marker of erythropoiesis). hemoglobin analysis was done using high-performance liquid chromatography and capillary zone electrophoresis. we examined the association of "functional" hemoglobin with str, and hbh% with ldh, using repeated-measures anova to adjust for the effect of multiple testing. we constructed receiver operating characteristic curve and calculated the area under the curve to define the best cut-off values for variables of interests. there was a strong association between functional hb and str, as well as hbh and ldh. the optimal cut-off for "functional" hemoglobin that was associated with str < . mg/l was g/l (auc = . , sensitivity and specificity of . % and % respectively). the optimal cut-off for hbh to supress ldh to < u/l was % (auc = . , sensitivity and specificity of . % and % respectively). the optimal pre-transfusion hbh% for reduction of hemolysis was % and the optimal "functional" hemoglobin to adequately supress erythropoiesis was g/l. to meet these hbh% and functional hb targets by simple blood transfusions, patients with tdt-alpha would require a hypertransfusion regimen with a minimum pre-transfusion total hb of g/l and consequently high transfusional iron burden. an alternative approach using exchange transfusion to reduce hbh% and improve functional hemoglobin would be associated with less volume of transfusion and potentially better long-term outcome. hospital sacre coeur, milot, haiti background: initial results of work developing a pediatric sickle cell disease (scd) clinic at the hôpital sacré coeur (hsc) in milot, northern haiti were presented at aspho . the purpose of this clinic is for a pediatrician with a special interest in scd to provide scd care, advising on trait and managing disease with penicillin prophylaxis (pcn) and hydroxyurea therapy (hu) for select patients. this clinic was started in collaboration with a us based hematologist and support from yale-new haven hospital. objectives: to describe the success and challenges of providing pcn and hu in the scd clinic at hsc through a review of patient records. design/method: since this clinic's inception, a database of patients, with basic clinical information has been kept and made accessible, through 'drop-box', to the us hematologist. the records of those that presented to the clinic were reviewed. the hemoglobin diagnosis was made either by clinical history and sickle cell prep or by hemoglobin electrophoresis through alpha laboratory, port-au-prince, haiti. results: ninety-nine individuals were seen in the first years of the program. fifty-six underwent a hemoglobin electrophoresis. of these , are ≤ years old. thirty-two were started on pcn vk, of which / ( %) were ≤ years old. eleven patients were started hu therapy. all patients on hu have shown progressive increases in hemoglobin. there have been no clinical complications of hu therapy. none of the patients taking hu have required hospitalization or transfusion in . three patients (not on hu) were hospitalized in for complications of scd (osteomyelitis, pain). in , with less than half the numbers in the program, there were admissions for severe anemia, pain, stroke and splenic sequestration. with ongoing external support and a local reputation for excellence in sickle cell care, the clinic at hsc has been able to expand services and improve the health of a growing number of patients with scd. early data suggests that pcn and hu therapies are helping to reduce complications and improve quality of life. challenges to date have included lack of funding for transportation to clinics, for hospitalizations and to cover the cost of electrophoreses. at the same time as continuing providing excellent care and gathering data, it is crucial to explore opportunities for collaboration and cooperation in ways that will assure that the clinic can become independently sustainable while continuing to improve the quality of life for the individuals it serves. background: ykl- is an inflammatory glycoprotein expressed by infiltrating macrophages in various inflammatory conditions. it has been found to be elevated in patients with different pathological conditions like acute and chronic inflammations, increased remodeling of the extracellular matrix (ecm), development of fibrosis and cancer. several studies have found elevated ykl- concentrations in sera of patients with liver diseases such as hepatic fibrosis by hepatitis c virus. it has been suggested that ykl- concentrations reflect the degree of liver fibrosis. to evaluate serum ykl- levels in patients with -thalassemia and its relation to viral hepatitis, liver stiffness as assessed by transient elastography (fibroscan, fs) and hepatic iron concentration. design/method: a prospective study included patients with -tm ( males and females) with mean age . ± . years (range: - years). serum ferritin level, liver enzymes (alt and ast), hbs ag, anti hcv ab and serum ykl- using elisa kit were evaluated. all patients were subjected to liver mri t * to detect liver iron content by the sequence and transient elastography (fibroscan, fs) to assess degree of liver stiffness. results: mean fibroscan value was ( . ± . ) kpa with a median . (range . to ) kpa. ( %) patients were categorized as f - and ( %) were stage f - , ( %) patients had severe fibrosis. their median serum ferritin was ng∖ml, with ( %) patients had values exceeding g/l. median cardiac t * was . with patients had values below ms, and the median lic was . mg/g dw with patients showed readings above mg/g dw. nyl- was evaluated as a marker of inflammation and liver fibrosis and showed mean value . (± . ) pg/ml, and range from to pg/ml. mean ykl- was significantly higher among males (p = . ), patients on chelation therapy (p = . ), patients on dfs (p≤ . ), in those with abnormal liver enzymes, splenectomised patients, patients with hbv sero-positivity, those with moderate elevation of t * and patients with high grades of liver fibrosis (p< . ). ykl- showed positive correlation with the rate of transfusion, lic, ferritin, alt and ast but negative correlation with weight, height and t *. roc curve analysis revealed that the cutoff value of ykl- at pg/ml could differentiate -tm patients with and without viral hepatitis with . % sensitivity and specificity of . %, area under the curve (auc) . , positive predictive value . and negative predictive value . (p< . ). roc curve analysis revealed that the cutoff value of ykl- at pg/ml could detect -tm patients with liver cirrhosis with . % sensitivity and specificity of . %, area under the curve (auc) . , positive predictive value . and negative predictive value . (p< . ). conclusion: serum ykl- levels are elevated in patients with -thalassemia and can detect patients with active viral hepatitis and liver stiffness. background: the most common splenic complication in pediatric patients with sickle cell disease (scd) is acute splenic sequestration (ass), which has often been managed with splenectomy. although splenectomy has been a treatment of choice for years, long-term vascular complications have not been thoroughly evaluated. pulmonary hypertension (phtn) is a severe complication of scd. in adults with scd, phtn has been associated with a -month mortality rate of approximately %. it has been reported that splenectomized patients with hemolytic disorders are at even greater risk of phtn. several medications exist to treat phtn, but with few studies of their efficacy or toxicities in patients with scd. additionally, these patients are often treated with either chronic prbc transfusions or hydroxyurea (hu) to raise hemoglobin, reduce hemolysis, and prevent vaso-occlusive events. objectives: to evaluate effect of chronic prbc or hu vs. no intervention, on tricuspid regurgitant jet velocities (trv) in pediatric patients with scd and history of splenectomy. design/method: retrospective chart review of splenectomized patients with hbss followed at marian anderson center at st. christopher's hospital for children, philadelphia, between and . we analyzed trvs ( hu, prbc, and from control group receiving neither treatment) from patients ( hu, prbc, neither). mean age at echo was . +/- . . data was analyzed with linear correlations and analysis of variance (anova), including the post hoc test of least significant difference (lsd) for all pairs of treatment groups. results: trv was not significantly correlated with age at time of assessment or with time between splenectomy and trv. univariate anova among groups yielded trv means of: . +/- . cm/s (hu), . +/- . (prbc), . +/- . (neither). we found a notable difference as the mean of the hu group was almost cm/s lower than the others, but no overall statistically significant association for any of the groups exists. however, when we performed post hoc tests to adjust for multiple comparisons and looked at all pairings within the anova, we found that the lsd between the hu and the prbc groups was statistically significant (p = . ), and that a trend exists between the hu group and the neither treatment group (p = . ). our data suggests that treatment with hu is correlated with a reduction in trv in pediatric patients with scd who underwent splenectomy. given these promising results, we believe our data warrants further study with larger treatment groups. nancy olivieri, gaurav sharma, susmita nath, rajib de, tuphan kanti dolai, prakas kumar mandal, abhijit phukan, amir sabouhanian, robert yamashita, angela allen, david weatherall, prantar chakrabarti background: hemoglobin e thalassemia (hbethal), which accounts for % of all severe beta thalassemia worldwide, has an estimated prevalence of . / , in west bengal, from which little information about clinical findings has been reported. objectives: to document clinical and laboratory findings in patients with hbethal, ultimately to improve resources for clinical management. design/method: we reviewed records from: a database recording patient names; clinic charts; "special" charts containing additional details; and, in transfused patients, transfusion day-care records. additionally, because in india's public hospitals original lab/imaging reports are commonly retained at home, % of families were interviewed to provide additional information. we excluded records of patients aged < years and patients aged < years who had not been reviewed since . results: while at least one visit had been recorded in , hbethal patients at nrs hospital, most patients are not regularly reviewed there. we examined charts [ ( %) aged ≥ years; ( %) aged - years; % male], representing approximately % of regularly-reviewed patients. most families ( . %) reported monthly incomes (< , indian rupees), below the monthly cost of living ( , rupees) in kolkata. mean (±sem) hemoglobin was . ± . g/dl. % patients were receiving eight or more transfusions per year; from , % had been treated with deferasirox, . ± . mg/kg/day. iron control estimated by serum ferritin concentration ( . ± g/l) was highly variable. a total of % patients were splenectomized. a substantial obstacle to documenting complications was the lack of recording, in any of the five sources, of many relevant parameters: for example, the status of sexual maturation (normal, delayed, or absent) was documented in less than %, and measurements of fasting blood glucose in less than %, of records. where recorded, complication rates were high: delayed/abnormal sexual maturation was recorded in % patients aged > years; in the patients aged > years and those aged - years, respectively, hypothyroidism was recorded in % and %, and elevated serum alt in % and %. in most evaluable patients > years, height was measured between the rd- th percentiles. cardiac findings, rarely documented, included pulmonary hypertension and reduced left ventricular ejection fractions in a few patients. despite dedicated attention to many aspects of thalassemia care, insufficient documentation limited a clear understanding of the current morbidity in hbethal patients. investment in personnel and technology will be critical to record relevant information, ultimately to improve clinical management, over the next decade. children's hospital of richmond at vcu health, richmond, virginia, united states background: sepsis is a common cause of death in children with sickle cell disease (scd). recommendations for care of fever in children with scd include immediate medical evaluation including blood culture and initiation of broad-spectrum antibiotic therapy. the increasing availability of pcr-based respiratory pathogen panels (rpp) provide the opportunity to rapidly identify viral causes of fever. the role for rpps in identifying the source of fever in children with scd and how it affects provider practice is not well studied. ( ) to determine the epidemiology of respiratory virus-associated fever in children with scd and ( ) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all children with scd seen in our emergency department (ed) with temperature ≥ . oc at home or in the ed from january , , through september , , as well as, all febrile children for whom rpps were sent since the introduction of rpps april . we reviewed the results of blood cultures, rpps, chest radiographs, and ed notes and discharge summaries to identify sources of infections. independent t test and chi-square analysis were used as appropriate to compare results using spss©. overall, the rate of bacteremia was %. there were no cases of bacteremia among children with positive rpps. % of children with negative rpps had true bacteremia. a positive rpp did not reduce the likelihood of bacteremia (p . ). patients with bacteremia had higher presenting temperatures than those without bacteremia ( . oc vs . oc, p . ). the most common rpp findings were rhinovirus/enterovirus ( %), human metapneumovirus ( %), and influenza a ( %). sending an rpp did not affect admission rate ( % and % respectively, p . ); however, likelihood of admission was lower in patients with positive rpps ( % vs %, or . [ . - . ], p . ). length of stay (los) was shorter in patients for whom an rpp was not sent ( . vs . days, p . ). as previously reported, bacteremia in febrile children with scd is very low, but remains a serious concern, particularly in the setting of high fever (> oc). a positive rpp did not reduce the odds of bacteremia, but did have a sta-tistically significant impact on both admission rate and los. more work is needed to understand how rpp results impact provider decision-making and care for children with scd. cincinnati children's hospital medical center, cincinnati, ohio, united states background: diffuse myocardial fibrosis is a common, if not defining, feature of the heart in sickle cell anemia (sca) that is strongly associated with diastolic dysfunction. we found diffuse myocardial fibrosis in every patient in a sca cohort (n = ) ranging in age from to years (niss ). the treatment and prevention of this complication of sca has not been studied before. objectives: because diffuse myocardial fibrosis must begin in early childhood, we hypothesized that early initiation and uninterrupted use of disease-modifying therapy for sca can prevent it. design/method: we use cardiac magnetic resonance imaging (cmr) to measure the myocardial extracellular volume fraction (ecv) to quantify diffuse myocardial fibrosis in individuals with sca who have been treated, uninterrupted, with hydroxyurea or chronic transfusion therapy since ≤ years of age. two comparison groups were used: individuals with sca who have not been treated with disease-modifying therapy since ≤ years of age (n = ) and controls without sca (n = ). results: we studied individuals ( m/ f) with a mean age of . years (range - ). mean age at the start of diseasemodifying therapy was . ± . years (range - ). only had evidence of mild diffuse myocardial fibrosis (ecv . ); the other had no detectable diffuse fibrosis (all had ecv < . , the upper limit of normal). mean ecv was . ± . , which was significantly lower than the ecv of individuals with sca who have not received early uninterrupted therapy ( . ± . ; p = . ) and not statistically different from normal controls ( . ± . ; p = . ). none had macroscopic fibrosis by late gadolinium enhancement or evidence of myocardial hemosiderosis by t * imaging. no patient had diastolic dysfunction by echocardiographic classification, right heart catheterization, or both. disease-modifying therapy for sca can prevent diffuse myocardial fibrosis, and possibly diastolic dysfunction, if started in early childhood. prospective trials of disease-modifying and anti-fibrotic therapy are planned to prevent diffuse myocardial fibrosis, which can be monitored noninvasively by cmr, and improve outcomes in sca. (niss, blood, ) . background: a statewide sickle cell surveillance system (sscss) was developed with the goal of determining the prevalence of sickle cell disease (scd) in indiana and the level of care that patients receive throughout the state. persons with scd are at high risk of infection, especially with encapsulated organisms, as well as at increased complications from influenza. utilizing sscss data, the relationship between vaccination status and mortality was explored. to determine if vaccination status is associated with mortality in persons with scd. the project was granted a waiver of consent by the st. vincent irb. death certificates were obtained to identify cause of death. deceased patients (cases) were matched by age, gender, and sickle genotype to living patients (controls). vaccination data were collected from the medical record and the children and hoosier immunization registry program (chirp) through the date of death for each case. cases and controls were assigned a point for completion of the pneumococcus, meningococcus and haemophilus influenza type b (hib) vaccine series and one point if the influenza vaccine was given within a year prior to death of the cases [max vaccine status score (vss): ]. total points were compared between the cases and controls. two tailed t-tests to compare means of continuous data and wilcoxon signed-rank test to compare ordinal data. one thousand forty-eight individuals were included in the sscss. six hundred and seven ( . %) were seen at one institution and included in this analysis (mean age = years). thirty-three of the ( . %) were deceased at the time of analysis. six point one ( . )% of controls and . % of cases received a vss of . the mean vss for cases was . ± . and . ± . for controls. thirty point three ( . ) % of controls had a vss of one or more, compared to % of cases (p = . ). patients who died of infection [streptococ-cus (n = ), pseudomonas (n = ) and unidentified organisms (n = )] were not up to date on vaccination against encapsulated organisms, but two had received the influenza vaccine in the year prior to death. in this sample, mortality occurred exclusively among adult patients, which is consistent with current patterns in developed countries. among these adults, vss and mortality rates were not related. limitations to the study include small sample size and potential incompleteness of vaccine records. vaccination rates and other standard of care indicators should be explored in a larger cohort of patients to determine associations with mortality. background: sickle cell disease (scd) is a genetic disorder resulting in acute and chronic complications, including delayed puberty. delayed puberty can have adverse physical and psychosocial effects on affected children and families. there are no published reports from ghana on pubertal timing in children with scd. the aim of this cross-sectional study was to describe pubertal changes in children with scd at korle bu teaching hospital (kbth), accra, and compare these findings to those in a control group without scd. design/method: children with scd and children with hb aa, ages - years, were consecutively recruited and matched for age, sex and socioeconomic status. investigator-administered questionnaires were used to obtain demographic data for all participants and information on menarche (girls only). pubertal status was assessed by physical examination using tanner staging. testicular volumes were determined in boys using a prader orchidometer. body mass index (bmi) and socioeconomic status (ses) of participants were analyzed to determine if there were any associations with tanner stage. of the with scd, ( . %) were hb ss and ( . %) hb sc. females comprised . % (cases and controls). mean age at onset of breast development was significantly delayed in girls with scd ( . ± . years) compared to controls ( . ± . years) but there was no significant age difference at onset of pubic hair development. mean age at menarche was significantly delayed in girls with hb ss ( . ± . years) and hb sc ( . ± . years), compared to those with hb aa ( . ± . years). in boys, the mean ages at onset of puberty were significantly delayed in those with scd ( . ± . years, for genital development and . ± . years, for pubic hair development), compared to those without scd ( . ± . years and . ± . years, respectively). mean testicular volumes were significantly lower in cases compared to controls, across all age ranges (p< . ). mean bmi in both cases and controls were similar at onset of breast development in girls. however, in boys with and without scd, mean bmi values were significantly different at pubertal onset. in univariate analysis, ses was not associated with tanner stage for both genital and breast development. mean ages at pubertal onset were significantly delayed in children with scd. longitudinal studies are needed to further characterize any associations with bmi and determine potentially modifiable risk factors affecting pubertal onset in scd. background: sickle-cell disease (scd) is a life-threatening genetic disorder associated with multiple chronic and acute complications. specific monitoring and treatment for children is a major part of the medical focus, but there remains a lack of real-world evidence of the disease burden and practice patterns among the pediatric scd population. objectives: to examine the clinical burden and management of scd among pediatric patients. design/method: a retrospective claims study was conducted using the medicaid analytic extracts database from jan - dec . pediatric patients (aged < years) with scd were identified using icd- -cm diagnosis codes ( . - . , . - . ). the first observed scd diagnosis during the identification period was designated as the index date. patients were required to have continuous medical and pharmacy benefits for at least months pre-and months post-index period. patient data were assessed until the earliest occurrence of the following events: disenrollment, death, or the end of the study period. patient demographic and baseline clinical characteristics, clinical outcomes (mortality, incidence of pain crisis, complications), scd management, and healthcare utilization were examined. all variables were analyzed descriptively. results: a total of , patients met the study inclusion criteria, with a mean age of . years. most patients were black ( . %) and had a charlson comorbidity index score of ( . %). mortality during follow-up was . in personyears, and the event rate of pain crisis in the inpatient setting was . in person-years. the three most common complications after pain crisis (highest rates in person-years) were fever ( . ), infectious and parasitic diseases ( . ), and asthma ( . ). rates of life-threatening complications were also examined in person-years, including acute chest syndrome ( . ), stroke ( . ), splenic sequestration ( . ), pulmonary hypertension ( . ), and pulmonary embolism ( . ). . % of patients were prescribed antibiotics during the one-year post-index period. other frequent medications utilized among children were folic acid ( . %), nonsteroidal anti-inflammatory drugs ( . %), opioids ( . %), and hydroxyurea ( . %). . % of patients had a blood transfusion within one year post-index date. patients had frequent health care utilizations in the inpatient ( visit), emergency room ( visits), office ( visits), and pharmacy ( visits) settings during the one-year follow-up period. pediatric scd patients are burdened with a high rate of complications including pain crisis. in addition, patients utilized a substantial amount of health care resources including outpatient office care and acute care visits. background: novel use of hydroxyurea in an african region with malaria (noharm, nct ) is a randomized controlled trial of hydroxyurea for very young children with sickle cell anemia living in uganda. during year , study participants received blinded study treatment of hydroxyurea or placebo; those receiving hydroxyurea had no increased risk of malaria, but had both laboratory and clinical benefits. during year , all study participants received openlabel hydroxyurea treatment. to assess the effects of open-label hydroxyurea treatment in a very young population of children with sickle s of s cell anemia living in uganda. study endpoints included the rates and severity of malaria infections, clinical sickle-related events, and laboratory effects. design/method: all children in the noharm trial were enrolled at mulago hospital sickle cell clinic in kampala uganda. during year , all children received open-label fixeddose hydroxyurea ( mg/kg/day) for months, after previously receiving either hydroxyurea or placebo for months. results: a total of children entered year of the noharm trial and received fixed-dose hydroxyurea, including males and females, at an average age of . ± . years. among children previously on placebo, there were malaria events in children, including with severity grade ≥ , and three deaths (two acute chest syndrome, one sepsis). clinical adverse event rates dropped from . to . per patient year, and hospitalizations were reduced from to . expected hematological benefits of increased hemoglobin, mcv, and fetal hemoglobin, along with decreased neutrophils and reticulocytes, were rapidly achieved. laboratory adverse events were infrequent at . events per patient-year, and only half of those were dose-limiting hematological toxicities. among children previously on hydroxyurea, there were malaria events in children, including with severity grade ≥ , and two deaths (one acute chest syndrome, one sepsis). clinical adverse event rates and hospitalizations were maintained at low rates, the hematological benefits of hydroxyurea continued throughout the extended treatment period, and dose-limiting toxicities remained infrequent. fixed-dose hydroxyurea treatment of young children with sickle cell anemia living in uganda is associated with no increased risk for malaria. clinical and laboratory benefits occur, including children previously on placebo who crossed-over to hydroxyurea treatment. future studies should focus on the optimal dosing and monitoring strategies, in an effort to determine the overall feasibility and safety of introducing hydroxyurea therapy across sub-saharan africa. background: acute chest syndrome (acs) is the second most common cause of hospitalization in patients with sickle cell disease and is a leading cause of morbidity and mortality. in mid- , an algorithm was implemented at cohen children's medical center to initiate transfusions within four hours of diagnosis of acs in order to improve patient outcomes. objectives: the aim of this project was to analyze the effect of early blood transfusion on the outcomes of patients with acs. we focused on the number of total transfusions, need for exchange transfusion, need for intensive care unit (icu) stay, and length of hospitalization. design/method: a retrospective chart review was completed on patients admitted to ccmc with a primary diagnosis of sickle cell disease and a secondary diagnosis of either acs or pneumonia during the years of - . data from the three years directly prior to implementation of the algorithm was compared to data from the three years directly after implementation of the algorithm. a total of patients were analyzed, of which belonged to the pre-algorithm group and to the postalgorithm group. patients from the post-algorithm group had a higher incidence of transfusions ( % with a mean transfusion number of . pre versus % with a mean of . post) as well as exchange transfusion ( % pre versus % post). the post-algorithm group had a shorter overall length of stay (mean of . days pre versus . days post). while the overall percentage of patients requiring an icu admission was similar in each group ( % pre versus % post), the post-protocol group had a lower likelihood of requiring an icu admission for reasons outside of line placement for exchange transfusion, most commonly for icu-level respiratory support ( % pre versus % post). despite a higher total number of transfusions, early recognition and transfusion for acs can lead to decreased lengths of hospitalization as well as decreased need for icu-level respiratory support. further studies comparing different center's clinical practice guidelines are necessary to improve the standard of care. background: novel use of hydroxyurea in an african region with malaria (noharm) was the first placebocontrolled randomized clinical trial of hydroxyurea in sub-saharan africa. in noharm, young children with sca received either hydroxyurea or placebo during year , followed by open-label hydroxyurea for all study participants during year . an ancillary noharm project was designed to determine if hydroxyurea treatment lowers transcranial doppler (tcd) velocities and possibly reduces stroke risk in this very young cohort. objectives: to perform tcd screening on the noharm cohort, measuring the time-averaged mean velocity (tamv) at the end of both year and year . we hypothesized that the maximum tamv would be lower for noharm study participants receiving hydroxyurea compared to those receiving placebo, and that key clinical and laboratory parameters would also influence tcd velocities. design/method: all children enrolled in noharm were eligible to undergo tcd examination at two study time points: month - when they were completing the blinded treatment phase, and again at month - at the end of the open-label treatment phase. tcd measurements included tamv readings from the main intracranial arteries: middle cerebral artery, distal internal carotid artery, and bifurcation on tcd. all tcd examinations were scored and classified as normal (less than cm/sec), conditional ( - cm/sec) or abnormal (greater than or equal to cm/sec), with higher scores correlating to greater risk of stroke. results: at the end of year , tcd exams were conducted of which were suitable for analysis ( hydroxyurea, placebo). based on the maximum tamv, the median velocity was cm/sec (iqr - ) for children on hydroxyurea and cm/sec (iqr - ) on placebo, p = . . maximum tamv values had negative correlations with hemoglobin concentration (- . ), fetal hemoglobin (- . ), and oxygen saturation (- . ); positive correlations were noted with age ( . ) and absolute neutrophil count ( . ). at the end of year , tcd exams were conducted and all were suitable for analysis; the median velocity was cm/sec on open-label hydroxyurea treatment, regardless of previous blinded treatment. all correlations with tamv were maintained except for age. conclusion: compared to placebo, hydroxyurea treatment for young children with sca living in uganda was associated with lower tcd velocities, which have been correlated in other studies with lower risk of primary stroke. tcd velocities were correlated with hematological and clinical parameters that can be improved by hydroxyurea therapy. children's hospital of richmond at virginia commonwealth university, richmond, virginia, united states background: acute chest syndrome (acs), defined by respiratory symptoms and a new pulmonary infiltrate, is a serious complication of sickle cell disease (scd). acs can occur during hospitalization for non-pulmonary conditions, such as a vaso-occlusive crisis or after surgery. nih clinical practice guidelines encourage incentive spirometry (is) which decreases the incidence of acs. it is additionally widely accepted that early, frequent ambulation in post-operative and pneumonia patients decreases the length of stay (los). to decrease acs events in children with scd at our children's hospital, we aimed for is use in % of ageappropriate pediatric sickle cell admissions. design/method: a multidisciplinary team examined inpatient acs prevention practices, including is, at children's hospital of richmond. key drivers were identified, including educational awareness of patients and healthcare staff, order placement, and documentation. we aimed for all scd patients ≥ months of age hospitalized with any admission diagnosis to participate in is with the use of a traditional incentive spirometer or similar age-and ability-appropriate devices (e.g. positive expiratory pressure devices, bubbles, and pinwheels). we secondarily aimed to increase activity events, specifically ambulation and out of bed time. educational and outreach tools included patient informational brochure and incentive program, and staff informational sessions and reference materials at workstations. a disease-specific order set was implemented including desired is and activity orders. data were collected prospectively may through november , during which pdsa cycles were conducted. admissions during the corresponding months of the previous year were reviewed for comparison. independent t-test analysis was performed using graftpad prism statistical analysis software. results: improvements reaching statistical significance included increase in is order placement from % to % of admissions (p < . ), and admissions with documented is use increased from % to % (p < . ). los decreased from a mean of . days to . days (p . ). post-admission development of acs also decreased from % to % of admissions, but did not reach statistical significance (p . ). there was an additional increase in appropriate activity order placement and documentation of activity events. conclusion: improving education and outreach to patients and staff, including implementation of a disease-specific order set, can improve is use and activity events. the decline seen in incidence of acs development during hospitalization, though not statistically significant, and the decreased los are encouraging, and efforts continue to improve on these trends. background: painful vaso-occlusive crises (voc) are a frequent and debilitating complication of sickle cell disease (scd) and are thought to occur due to progressive blockage of the microvasculature with rigid sickle shaped red blood cells. any trigger that decreases the microvascular blood flow (mbf) can promote entrapment of sickled cells in the microvasculature and progression to voc. exposure to cold wind and changes in weather are common triggers of voc and are associated with increased frequency of hospitalizations for pain in patients with scd. there is limited experimental data on the physiologic effects of these factors on peripheral perfusion in scd. to study the effect of graded thermal stimuli on the peripheral mbf in scd. design/method: scd and control (healthy or sickle trait) subjects aging to years were exposed to their individual threshold temperatures for heat and cold detection, heat and cold pain via tsa-ii thermode that was placed on the thenar eminence. mbf was measured on the contralateral thumb using photo-plethysmography (ppg). the vasoconstriction response within the complex ppg signal was detected using cross-correlation technique. mean mbf was derived from the ppg amplitude during each of these stimuli and compared to baseline mbf. cross correlation analysis showed that cold pain caused significant vasoconstriction response in % of the subjects, followed by heat pain ( %), cold detection ( %) and heat detection ( %).there was a significant drop in the mbf during cold pain (p < . ), heat pain (p < . ), heat detection (p = . ) and cold detection (p = . ) when compared to baseline mbf, with cold pain causing the greatest drop in mbf. thermal sensitivity and mbf responses were comparable between scd and controls. conclusion: exposure to graded thermal stimuli causes a progressive drop in mbf with exposure to cold pain eliciting the strongest vasoconstriction response. vasoconstriction occurred in the contralateral hand at an average of seconds after the stimuli, suggesting a neurally mediated mechanism. although there was no significant difference in vasoconstriction responses between scd and controls, the drop in mbf in patients with sickle cell disease can increase the likelihood of entrapment of the sickled red blood cells, leading to vaso-occlusion. these findings are consistent with extensive reports in literature that exposure to cold weather is associated with a higher frequency of voc. this suggests that neurally mediated vasoconstriction is likely an important factor in the pathophysiology behind cold exposure leading to voc in scd. background: vaso-occlusive crisis (voc) is a major cause of hospital admissions in children with sickle cell disease (scd). although the use of clinical biomarkers in voc has been studied, especially with regards to acute chest syndrome (acs), there is less data regarding overall voc severity prediction. in addition new biomarkers such as platelet to lymphocyte ratio (plr), neutrophil to lymphocyte ratio (nlr), and lymphocyte to monocyte ratio (lmr) have been little studied with regards to scd. objectives: to identify whether admission laboratory values, changes from well baseline laboratory values, and new biomarkers such as plr, nlr, and lmr could predict severity of vaso-occlusive crisis in children with sickle cell disease admitted with voc. design/method: this was a retrospective single center observational study of admissions of voc in children aged - years with hbss or hbs-b thal from september to november excluding those on hyper-transfusion protocol or having an admission diagnosis of acs. univariate analysis was done using student's t-test, mann-whitney non parametric test, or fischer's exact test as appropriate depending on the distribution between admission laboratory data of complete blood count (cbc), reticulocyte count, comprehensive metabolic panel, lactate dehydrogenase (ldh), change from well baseline cbc values within months previously, plr, nlr, lmr, and the development of complicated voc. complicated voc was defined as the development of secondary acute chest syndrome, prolonged admission duration > days ( hours), requirement of blood transfusion, and readmission within days. results: a total of admissions were studied. fifty-nine ( . %) were female. of the , ( . %) were complicated with no significant differences in sex (p . ) or age (p . ). univariate analysis revealed significant elevations in total bilirubin (p . ), ldh (p . ), and platelet count (p . ) in those with complicated voc. there is also significant difference in the percentage change of platelet count from baseline with greater decline in uncomplicated voc (p . ). there were no significant differences in plr (p . ), nlr (p . ), or lmr (p . ). conclusion: elevations in total bilirubin, ldh, and platelet count in admission laboratory values are associated with developing complicated voc. in addition, those with complicated voc present with significantly less decline in platelet count from baseline well cbc. plr, nlr, and lmr do not seem to be useful predictive biomarkers for severity of voc. background: sickle cell disease (scd) causes health problems of varying frequency and severity. the only validated biomarker for children with scd is transcranial doppler. if reliable predictors existed for scd severity, children with scd could be treated according to risk category. many patients with scd face psychosocial or economic hardships, but these factors have not been evaluated as risk markers for medical or functional severity of scd. objectives: the goal of this project was to develop and stratify a preliminary list of psychosocial risk factors for health outcomes that could be used as scd severity predictors. st. vincent institutional review board. a list of potential psychosocial risk factors for adverse health outcomes was compiled based on assessment materials utilized by the sickle safe program (indiana's hemoglobinopathy newborn screening follow-up program). this list of items was distributed to child abuse prevention ( ) and scd ( ) experts, who ranked each item on a likert scale of (least important) to (most important). mean scores were calculated using spss version ; assessments were retrospectively analyzed to determine psychosocial risk factor frequency. risk factors occurring in ≥ % of homes were considered high frequency events. overall, there was high agreement among experts on the risk factors that were considered the most important predictors of severe scd outcomes. the risk factor with the highest frequency ( %) was eligibility for public assistance programs. fifteen risk factors were rated ≥ by the experts. four ( . %) were high frequency events occurring in ≥ % of homes: a child with hbss or hbs thalassemia not taking hydroxyurea ( %); parent report that they had treated a fever (> ®f) at home in the past months ( %); tobacco use by someone in the household ( %); and the family reporting significant psychosocial stressors in the past year ( %). tobacco use in the home was significantly correlated with several other risk factors (smoking during pregnancy [r = . ], other health concerns in the child [r = . ], and child having health insurance [r = - . ]), suggesting that it is part of a constellation of health risk. in general, the risk factors that were rated as most important for health outcomes occurred less frequently in the sample. this study represents important progress toward identifying a group of psychosocial risk factors for scd severity, which is a necessary first step for future investigation of empirical relationships between candidate risk factors and scd outcomes. unitversity of cartagena, cartagena, bolivar, colombia s of s background: sickle cell disease is an autosomal recessive disorder characterized by a mutation in the -globin chain, which produces hbs. acute and chronic complications as aplastic crisis, acute chest syndrome, priapism, stroke, leg ulcers and primary/secondary prevention of stroke can be treated with simple transfusion or exchange transfusion. the latter offers advantages as lower iron overload, post-treatment hbs goal control, lower viscosity and improved microvascular circulation. but it is not a widely-used option because is associated with technical difficulties. objectives: standardization of a new partial exchange transfusion protocol in a group of patients with sickle cell disease, within the framework of a chronic transfusion program. design/method: this is a prospective descriptive study, which included patients under years with sickle cell disease ( hbss, hbs-tal), with indication of partial exchange transfusion in a chronic transfusion program, according to the institutional protocol; patients who fulfilled the inclusion criteria were enrolled in the study between february and december . a registry of the medical and technical complications was made in each of the procedures. a database was constructed in excel, and the graph-pad prism® version oc software was used for statistical analysis. the sequence is as follows: isovolemic phlebotomy and transfusion of packed red cells. depending of the recent hemoglobin level ( hrs), we do the phlebotomy there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg; isovolemic solution (ns , %) there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg and packed red cell transfusion there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg. the safety of this exchange transfusion protocol was analyzed in patients with sickle cell disease ( procedures). there were no differences in the sex distribution, and the median age was years. % of the population was homozygous. the indication of transfusion was . %( / ) primary stroke prevention, . %( / ) secondary stroke prevention and . %( / ) was other reason. a low percentage of complications was found ( . %); of which, those of medical origin (hypotension and nausea/vomiting) were only presented in . % of the total procedures. the standardization of this protocol was safe and its use could be extended to other low-income centers that treat patients with sickle cell disease that need chronic transfusion program including patient with hemoglobin level until gr/dl. we suggest do studies for measure the security and efficacy of this protocol in patients with acute complications. background: clinical trials that aim to achieve pain reduction have challenges achieving clinical endpoints as pain has no quantifiable biomarkers and may be unrelated to scd. furthermore, the threshold of seeking medical care differs between patients and vocs that occur at home are missed. we present a non-interventional, longitudinal study to identify vocs in patients with scd. objectives: to examine the longitudinal relationship between pros and biomarkers in subjects with scd before, during, and after a self-reported voc event, in order to build a model of in-home and clinical voc and to collect longitudinal pros and biomarker data from subjects that span voc events in the home, clinic and the hospital. design/method: longitudinal measures of pain, fatigue, function, activity, and biomarkers from scd patients in steady state and voc were studied over a six month period. patients self-reported pain, fatigue, function, and medication use using a novel epro tool. voc was reported in real-time, triggering a mobile phlebotomy team. blood was collected sequentially after self-reported voc (at home or hospital). blood samples were drawn two days after resolution of voc, as reported by the patient. during non-voc periods, blood was drawn every weeks to establish a baseline. biomarkers included leukocyte-platelet aggregates and circulating microparticles, cell and soluble adhesion molecules, cytokines, inflammatory mediators and coagulation factors. patients wore an actigraphy device to track sleep and activity and rest. results: twenty-seven of thirty-five patients experienced a total of days with voc > hr, of which only days resulted in healthcare utilization. voc days had significantly higher pain and fatigue scores. voc days were associated with significantly decreased functional scores, with significantly greater decreases during vocs requiring medical contact compared to at-home vocs. different activity profiles were identified for non-voc, at-home voc and medical contact voc days by actigraphy monitoring. at-home voc days exhibited increased daytime resting compared to non-voc days. medical contact vocs had decreased average and peak activity, and increased daytime resting compared to non-voc days. a sleep fragmentation index trended up for both at-home ( %) and medical contact voc days ( %). significant changes during voc days were observed in: c-reactive protein ( % increase), nucleated rbc ( % increase), monocyte-platelet aggregates ( % increase) and neutrophil-platelet aggregates ( % increase), interleukin- ( % increase), interleukin- ( % increase) and tnfalpha ( % increase). the identification and assessment of at-home vocs through use of epros, actigraphy and biomarkers is feasible as demonstrated by this innovative at-home study design. background: risk-stratifying sickle cell disease (scd) patients and demonstrating response to disease-modifying therapies is challenging due to the phenotypical heterogeneity of scd. a pathogenic role for procoagulant von willebrand factor (vwf) via excess vwf high molecular weight multimers (hmwm) has been proposed, with variable reports of increased vwf and hmwm in crisis vs. steady-state in adults, but less so for vwf in children with scd. moreover, vwf and multimers have not been studied in sickle trait. objectives: our pilot study evaluated the potential for vwf antigen (vwf:ag) and hmwm on densitometric tracings to serve as biomarkers for disease severity or treatment response in children and young adults with scd compared to sickle trait (hbas) siblings. design/method: we evaluated vwf:ag, vwf multimers and retrospective clinical data from hbss, hbsc and hbas subjects at steady state. one hbsc subject also had a crisis sample. median scd age was years ( . - . years). % were female. scd severity was judged by annual vasoocclusive and acute chest events, or stroke/elevated tcd. eight of ( hbss and hbsc) took hydroxyurea. four hbss subjects had severe scd, all of whom were chronically transfused. results: mean vwf:ag (normal - iu/dl) was higher for hbss ( +/- . ) and severe hbss ( +/- . ) compared to hbsc ( +/- . , p = . and . , respectively); however, lacked statistical significance when compared to hbas ( +/- . , p = . and . , respectively). vwf:ag was elevated in / ( %) steady-state, including / ( %) with "severe" disease on chronic transfusion and / ( %) taking hydroxyurea, in hbsc crisis but no hbsc / ( %) at baseline. vwf:ag was high in / ( %) hbas siblings. four ( %) had increased hmwm at baseline: hbss/severe disease/chronic transfusion, hbss/hydroxyurea and hbsc untreated. hmwm were increased only during vaso-occlusive crisis in hydroxyureatreated hbsc subject. no ultra-large hmwm were observed. in this preliminary study, in young scd subjects, vwf:ag trended higher in hbss vs. hbsc and in severe hbss participants at a single time-point, but serial evaluations at baseline, in crisis and with optimized diseasemodifying therapy are needed to determine the potential of vwf:ag and hmwm as biomarkers for severity or treatment response. surprisingly, vwf:ag was high in some sickle trait subjects. since hbas is associated with some health challenges such as increased thrombosis risk, further examination of vwf and endothelial dysfunction in sickle trait may provide novel insights into its role as a biomarker. background: the national heart lung & blood institute(nhlbi) guidelines for acute management of voe recommends rapid evaluation and treatment of pain, including administration of a parenteral opioid within -minutes of triage or -minutes from registration, pain reassessment & repeat opioid delivery within - -minutes. inf use has been increasing in peds due to its rapid onset and ease of administration. objectives: to evaluate ped utilization of inf & its effect on intravenous (iv) opioid administration and pain control for the treatment of voe. design/method: a retrospective review of emr was performed on children with scd± years presenting to a ped with voe (pain scores on a - scale) from jan-june . variables studied were median time (iqr, %ci) from ped arrival to first-parenteral-opioid-administration, time-to-first-iv-opioid, first & final pain score, disposition and readmission rate. time-to-first-iv-opioid was also compared to historical data (jan-dec ,n = ) prior to inf protocol initiation. . additionally, % patients received iv opioids within minutes of ed arrival in the inf+iv opioid vs. % in the iv opioids alone group (p< . ). no differences in -hour-returnrates were found in any of the groups, including inf alone group. conclusion: use of inf in the ped for voe is an excellent strategy to shorten time-to-first-parenteral-opioidadministration, improve pain scores & improve adherence to the nhlbi guidelines. however we had distinct unexpected findings: ( ) delays in iv opioid delivery after inf use & ( ) inf alone appeared to provide sufficient pain control without iv opioids for disposition home in % of voe patients. whether the latter reflects insufficient pain management or that there is a milder subgroup for whom inf alone is sufficient, requires further investigation. this study illustrates our experience with a ped-based inf protocol in terms of unanticipated delays in iv opioids and also discharges after inf alone. efforts are underway to further improve use of inf in voe management. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: folate supplementation is commonly included as standard management in patients with sickle cell disease. however, clear evidence supporting the clinical benefits of this practice is lacking. a single study demonstrated improvement on the occurrence of repeat dactylitis at a higher dose of folic acid. to compare clinical outcomes in pediatric patients with sickle cell disease treated with folate supplementation versus those who were not. design/method: this study was a retrospective chart review that included patients to years old with sickle cell disease type ss and s followed at st. christopher's hospital for children. data collected included information about folate supplementation, red cell indices and the presence or absence of clinical outcomes including vaso-occlusive crisis requiring hospitalization in the last six months, acute chest syndrome, infections, asthma, sleep apnea, nephropathy, cerebral vascular disease, stroke and avascular necrosis. analysis of variance (anova) was used to evaluate mean differences between age, number of infections, number of voc events, hemoglobin, reticulocyte count, and mean corpuscular volumes. additionally, chi square analysis was implemented to evaluate differences in folate and non-folate groups for left ventricular remodeling (lvr), sickle cell nephropathy, asthma, obstructive sleep apnea (osa), nocturnal hypoxia, and avascular necrosis (avn). mean differences between the folate and non-folate groups were compared for patients on and off hydroxyurea therapy. one hundred and seven patients met inclusion criteria following review of clinical data. of the patients included in the study, patients were found to be taking folate ( %), while patients were not ( %). statistical analysis showed that there were no significant differences in the incidence of clinical outcomes between patients on folate versus those who were not on folate. of the patients who were not on hydroxyurea, hemoglobin levels were significantly higher in patients on folate versus those who were not (p = . ), but not significantly different for the patients on hydroxyurea. this study suggests that folate supplementation makes no significant impact on the red blood cell indices of anemia nor on the incidence of adverse clinical outcomes in children with sickle cell disease. however, a larger prospective study is needed to guide future considerations for folate supplementation in sickle cell patients in the clinical setting. background: tanzania ranks rd globally for the number of infants born annually with sickle cell disease (scd) but lacks a national newborn screening program. the prevalence of sickle cell trait (sct) and scd is highest in the northwestern regions around lake victoria served by bugando medical centre (bmc) a teaching and consultancy hospital in mwanza. bmc also houses the hiv early infant diagnosis (eid) laboratory that tests dried blood spots (dbs) from hivexposed infants. dbs can be tested for hiv and then retested for sickle cell trait and disease. to determine the prevalence of sickle trait and disease by region and district in northwestern tanzania using existing public health infrastructure. secondary objectives explored associations between sct, scd, malaria and hiv. design/method: the tanzania sickle surveillance study (ts ) is a prospective year-long cross-sectional study of hivexposed infants born in northwestern tanzania, whose dbs collected by the eid program are tested at bmc and available for further testing of sct and scd. samples from children ≤ months of age were tested by isoelectric focusing (ief) and scored independently by two tanzanian staff as normal, sct, scd, variant, or uninterpretable. dbs samples scored as disease or variant were repeated. over the course of months, ief gels have been run. a total of , dbs samples have been scored, including , from children less than -months old. the overall prevalence of sct is . % and the prevalence of scd is . %, along with . % hemoglobin variants. quality of the laboratory results is extremely high, with only . % dbs samples yielding an uninterpretable result. geospatial mapping of the first , samples revealed a regional scd prevalence ranging from . % up to . % among the regions served by bmc. the prevalence of sct and scd is very high in northwestern tanzania. geospatial mapping will identify high prevalence areas where targeted newborn screening can be started using existing public health infrastructure with minimal start-up cost and training. further data will enhance the accuracy of the map to the district level. background: pediatric patients with sickle cell disease (scd) could develop obstructive, restrictive or mixed abnormalities of pulmonary function (pf). several publications report progressive worsening of pf over time, which could lead to severe morbidity in adult patients with sickle cell disease. in adults with sickle cell anemia up to - % of mortality is related to lung disease. early intervention aimed at improvement of lung function could significantly decrease morbidity and possibly improve life expectancy. among disease modifying approaches commonly used in scd are hydroxyurea (hu) and chronic prbc transfusions. both interventions lead to increase of hemoglobin, decrease of hbs fraction, leading to decreased hemolysis. reports of effect of hu on pulmonary function are conflicting with some suggesting no effect and others proposing a slower decline of pulmonary function. the goal of our study is to evaluate effect of disease modifying therapies, like hu and chronic prbc on change of pulmonary function in pediatric patients with sickle cell disease. design/method: this study utilized a retrospective chart review of children with scd who had multiple pfts. we analyzed pfts from patients done during clinic visits. scd patients were divided into three treatment groups: hydroxyurea, chronic transfusions or neither. data was analyzed with linear correlations and analysis of variance (anova). comparison were made between the three groups specifically observing the changes in absolute numbers on pfts over time using the first and last pft the patient had. results: there were a total of patients with multiple pfts (ranging from - ); control ( ), hydroxyurea ( ) and chronic transfusion ( ). the mean changes of the control, and hydroxyurea for the pft parameters fev (- . the chronic transfusion group demonstrated a small improvement in pfts over time for fev ( . ), fvc ( . ), fef - ( . ), however there was a decline in fev /fvc (- . ). however, there was no statistically significant (p-value < . ) in the difference in any pfts parameters between any of the groups. in children with scd there is a decline of pf parameters over time. although no significant differences were seen between the three groups it appears chronic transfusion may improve or limit the decline in pfts. larger studies need to be done to evaluate difference in pf decline in patients with scd patients. background: the use of mobile technology in health care has been a growing trend. patients with chronic diseases such as sickle cell disease (scd) require close monitoring to provide appropriate treatment recommendations and avoid complications. we conducted a feasibility study for patients with scd hospitalized for pain using our self-developed mobile application (tru-pain: technology resources to better understand pain) and a wearable activity tracker. subjective symptoms such as pain and objective data such as heart rate (hr) were measured. we aimed to ) correlate nursing recordings with mobile technology recordings; ) get feedback from patients about usability. design/method: we enrolled patients with scd > years old and < hours from admission for uncomplicated vasoocclusive crisis, excluding patients admitted to icu. patients were given an ipad and a wearable device. they were instructed to record in the application at least once per day and to keep the wearable on, removing only to charge. prior to discharge, patients completed a feasibility questionnaire. we enrolled patients, % females, median age . (range to ) who were admitted for a median days (range to ) for uncomplicated pain crisis. patients used the application throughout hospitalization and made one entry/day (range to ). pain scores recorded via tru-pain correlated well (r = . , p< . ) with pain scores recorded in emr. there was an average of , data points recorded per day, by the wearable, with a maximum of , data points/day. the median amount of hours of wearable data per day was . (maximum of . ). the hr recorded via the wearable correlated significantly with the hr recorded in emr (r = . , p-value < . ). as for usability, % of patients indicated never having a problem with the technology, % found tru-pain 'very easy' or 'somewhat easy' to use, and % were 'very satisfied' with their participation in the study, indicating that it helped them track their pain. our pilot study during hospitalization shows strong potential for using tru-pain for patients with scd. pain data from application and hr from wearable correlated well to the emr data. according to the feedback received, our application was easy to use and helped patients track their pain. despite limitations of battery life, the use of wearable technology is feasible, providing additional data such as activity. we are optimistic that we can continue to improve our tru-pain system to help improve care in patients with scd. background: hydroxyurea, chronic blood transfusion, and bone marrow transplantation can reduce complications, and improve survival in sickle cell disease (scd), but are associated with a significant decisional dilemma because of the inherent risk-benefit tradeoffs, and the lack of comparative studies. these treatments are underutilized leading to avoidable morbidity and premature mortality. there is a need for tools to provide patients high-quality information about their treatment options, the associated risks, and benefits, help them clarify their values, and allow them to share in the process of informed medical decision making. objectives: to develop a health literacy sensitive, web-based, decision aid (ptda) to help patients with scd make informed choices about treatments, and to estimate in a randomized clinical trial the acceptability and effectiveness of the ptda in improving patient knowledge, involvement in decisionmaking and decision-making quality. design/method: we conducted qualitative interviews of scd patients, caregivers, stakeholders, and healthcare providers for a decisional needs assessment to identify decisional conflict, knowledge, expectations, values, support, resources, decision types, timing, stages, and learning, and personal clinical characteristics, and to guide the development of a ptda. transcripts were coded using qsr nvivo . stakeholders completed alpha and beta testing of ptda. we conducted a randomized clinical trial of adults, and of caregivers of pediatric patients to evaluate the comparative efficacy of the ptda, vs. standard of care. results: ptda (www.sickleoptions.org) was developed per decisional needs described by stakeholders and finalized following alpha testing, and beta testing by and stakeholders respectively. in a randomized trial of subjects considering various treatment options, qualitative interviews revealed a high level of usability, acceptability, and utility in education, values clarification, and preparedness for decision making of the ptda. a median % rated the acceptability of ptda as good or excellent and provided narrative comments endorsing the acceptability, ease of use, and utility in preparation for decision making. the ptda met international standards for content, development process, and efficacy with the exception of having a full range of positive and negative experiences in patient stories. compared to baseline ptda group had statistically significant improvement in preparedness for decision making (p = . ) and informed subscale of decisional conflict (p = . ) but not for decisional self-efficacy, knowledge, choice predisposition, or stages of decision-making. a ptda for patients with scd developed following extensive engagement of key stakeholders was found to be acceptable, useful, easy to use, to improve preparedness for decision making, and decrease decisional conflict. background: painful vaso-occlusive crisis (voc) accounts for the majority of emergency department (ed) visits and hos-pitalizations in sickle cell disease (scd). we are interested in studying mental stress and associated autonomic nervous system (ans) imbalance that cause vaso-constriction as possible triggers of scd pain. to this end, we developed a mobile phone application (app) to record daily pain frequency and intensity as clinical endpoints that might be predicted by ans parameters measured in the laboratory. in particular, we think that the aura may represent ans instability that precedes or even triggers change in blood flow and voc. objectives: to assess the feasibility of using an app to evaluate frequency and severity of voc and its potential association with mental stress and presence of aura. design/method: an app was developed for both ios and android systems to allow patients to track pain, stress, and aura. the idea was to create an app that was easy to use with the intent to only capture pain episodes, rather than detailed description of the pain. all scd patients were eligible and a parent version was available for younger children. de-identified data was automatically transferred to a hipaa compliant database via a cloud-based server interfaced to the main research project database. a feedback questionnaire was implemented after at least a month of utilization to assess usability. of the scd patients enrolled, participants utilized the app and of the participants that provided feedback indicated the app was easy to navigate. the mean pain scale was out of (standard deviation . ) for those that entered they had pain that day. although the mean stress level was out of , there was a statistically significant correlation between increasing stress levels and increasing pain scores (p < . ). aura was reported by patients, with patients reporting more than episodes. moreover, on days aura was present there was greater incidence that pain was present as well (p < . ). however, there was no statistically significant association between pain intensity and presence of an aura (p = . ). conclusion: consistent with prior research, reported pain intensity is significantly associated with reported stress intensity. although there was an association between presence of aura and pain, it did not seem to correlate with pain intensity. this uniquely designed app can monitor scd pain clinically and help understand the role of sickle dysautonomia in the genesis of scd pain. university of florida college of medicine, gainesville, florida, united states background: evidenced-based guidelines recommend the emergent evaluation of fever in children with sickle cell disease (scd). as the prevalence of bacteremia has decreased, outpatient management has become more common. however, fever can sometimes herald other complications of scd, such as acute chest syndrome, vaso-occlusive pain crisis, splenic sequestration, or aplastic crisis. institutional practices regarding fever management in scd remain variable, and little is known about the clinical outcomes of children hospitalized for uncomplicated fever. objectives: the primary objective was to determine the rate of bacteremia or scd-related complications per febrile episode in children with scd admitted to a single institution between january and june for uncomplicated fever. this was a retrospective cohort study of febrile patients up to years of age with scd, any genotype, admitted to the university of florida during the defined study period. eligible patients were identified by a database search using admitting diagnosis codes for scd and fever based on the international classification of diseases th and th revisions. encounters were manually reviewed to confirm eligibility. patients were excluded if they had other indications for hospitalization apparent at the time of admission, such as an acute vaso-occlusive episode requiring parental narcotics, asthma exacerbation, or additional complications of scd. the database search identified encounters, of which were excluded based on confounding indications for hospitalization. sixty-three eligible patients accounted for hospitalizations. the median age was years (range weeks- years); . % were male. mean duration of hospitalization was . days (range - days). eight positive blood cultures were identified; six of these were classified as contaminants. bacteremia or the development of a scd-related complication was identified in ( . %) admissions. these included acute chest syndrome (n = ), bacteremia (n = ), splenic sequestration (n = ), and red cell transfusion (n = ). exploratory analyses of potential predictors of bacteremia or scd-related complications showed no association with the presenting white blood cell count or degree of fever (p = . ). of the patients classified as having a scd-related complication, % had hemoglobin ss disease and % had at least one prior documented complication. % of the patients transfused had at least one prior transfusion. conclusion: while improvements in preventative care have substantially lowered rates of bacteremia in children with scd, fever warrants careful evaluation for other acute scdrelated complications. providers should consider inpatient observation in select cases. additional studies are warranted to define subsets of patients suitable for outpatient fever management. background: children with sickle cell disease (scd) exhibit lower neurocognitive functioning than healthy peers, even in the absence of stroke. among the domains commonly affected, working memory (wm) seems particularly affected by disease processes and wm deficits have significant implications for academic achievement and disease selfmanagement. few interventions to improve working memory in pediatric scd have been evaluated. to determine the effects of cogmed, a homebased computerized wm training intervention, in children with scd using a randomized controlled trial design. design/method: participants (ages - ) with scd completed a baseline neuropsychological assessment and those with wm deficits were randomized to either begin cogmed immediately or enter an -week waitlist. cogmed is a homebased intervention completed on an ipad that consists of increasingly challenging exercises targeting visual-spatial and verbal wm, practiced over sessions. at the end of training, participants completed a post-intervention neuropsychological assessment, including tests of visual-spatial and verbal wm from the wechsler intelligence scale for children-fifth edition (wisc-v). results: ninety-one participants (m age = . , sd = . ; % female; % hbss) enrolled in the study; % (n = ) exhibited wm deficits and were randomized to either begin cogmed immediately or wait - weeks before starting cogmed. among those that have received the intervention and reached the end of their training period (n = ), participants ( %) completed at least cogmed sessions, ( %) finished at least sessions, and finished at least sessions ( %). the mean number of completed cogmed sessions was . (sd = . ). paired samples t-tests revealed significant improvements on the working memory index (t[ ] = - . , p = . ) and on the digit span (t[ ] = - . , p = . ), and spatial span-backward (t[ ] = - . , p = . ) subtests. improvements were especially pronounced for participants completing at least sessions. partial correlations controlling for respective baseline scores indicated that the number of cogmed sessions completed was positively correlated with post-test scores on digit span (r = . , p = . ) and spatial span-backward (r = . , p = . ) subtests. among participants who completed at least cogmed sessions, % scored in the average range or higher on the working memory index at the post-intervention assessment, compared to % at baseline. results support the efficacy of cogmed in producing significant improvements in wm. a dose-effect was observed such that participants who completed more cogmed sessions had greater improvements in wm. home-based cognitive training programs may ameliorate scd-related wm deficits but methods for motivating and supporting patients as they complete home-based interventions are needed to enhance adherence and effectiveness. background: sickle cell disease is associated with myriad complications that lead to significant morbidity and early mortality. hydroxyurea has been used successfully to reduce the incidence of these complications and has led to significant improvements in quality and duration of life. at children's minnesota we recommend hydroxyurea in all patients with hb ss/s thalassemia as early as months of age with a goal of starting all patients before months of age. objectives: the purpose of this study was to evaluate the use of hydroxyurea therapy in young patients with sickle cell disease, with particular attention to those children less than one year of age. design/method: a retrospective chart review was conducted on patients less than years of age with sickle cell disease who began hydroxyurea therapy between january , and december , . the study population was divided into three cohorts based upon age at hydroxyurea initiation: cohort ( - year), cohort ( - years), and cohort ( - years). outcomes included laboratory data, clinical events (hospitalization, dactylitis, pain crisis, transfusion, splenic sequestration, acute chest syndrome), and toxicity occurring in the first years of life. results: a total of patients were included in cohorts (n = , mean age . months), (n = , mean age . months), and (n = , mean age . months). patients in cohort had higher hemoglobin (p = . ) and mcv (p = . ) and lower absolute reticulocyte count (p = . ) when compared to cohort . the wbc (p = . , < . ) and anc (p = . , . ) were significantly lower compared to both older cohorts. however, no patient had therapy held because of neutropenia. the mean baseline hemoglobin f in cohort was . % compared to . % and . % in cohorts and respectively (p = . , p< . ). the mean duration of therapy in cohort was . months, compared to . months in cohort (p = . ) and . months in cohort (p = . ). during this time, hb f levels remained higher in cohort (mean . %) compared to cohorts and (mean . %, p = . and mean . %, p = . ). patients in cohort experienced fewer hospitalizations (p = . ), pain crises (p = . ), and transfusions (p = . ). there was no difference in toxicity between groups. hydroxyurea was used safely in infants to months of age and resulted in more robust hematologic responses and a decrease in sickle-related complications when compared with patients starting hydroxyurea later in life. children's national health system, washington, district of columbia, united states background: children with sickle cell disease (scd) have a significantly greater risk of silent or overt cerebral infarction than the general population. infarcts are associated with declines in cognitive functioning and academic achievement. while infarcts are reliably identified using mri, scans are expensive and occasionally necessitate sedation. moreover, mri's are not recommended for routine monitoring of cerebral infarcts. additional tools are needed for discriminating the presence of a cerebral infarct that are brief, noninvasive, inexpensive, and repeatable. objectives: to evaluate differences in performance on cogstate, a computerized neurocognitive assessment, in patients with scd with and without history of cerebral infarct. design/method: participants included children with scd ages - (m = . , sd = . ; % female; % s of s hbss) enrolled in a cognitive intervention trial. participants completed the cogstate pediatric battery, which measures processing speed, sustained attention, verbal learning, working memory, and executive functioning. history of silent or overt infarct was determined via health record review. participants also completed measures of intelligence (iq) and math fluency. results: participants' standard scores across most neurocognitive measures were lower than expected compared to the standardization sample (mean iq = . , sd = . ). thirty percent of participants (n = ) had a documented history of cerebral infarct. participants with a history of cerebral infarct scored lower on cogstate tasks measuring sustained attention (t[ ] = . , p = . ) and executive functioning (t[ ] = . , p = . ), as well as on a measure of math fluency (t[ ] = . , p = . ). receiver operating characteristic (roc) analyses demonstrated that the cogstate task measuring sustained attention was a fair discriminant of patients with and without a history of infarct (auc = . , ci = . - . , p = . ), whereas iq score was not (auc = . , ci = . - . , p = . ). cogstate processing speed and sustained attention tasks fairly discriminated between patients with at least average or below average intelligence (auc = . , ci = . - . , p = . and auc = . , ci = . - . , p = . , respectively). finally, the cogstate processing speed task was good at discriminating between at least average or below average math fluency (auc = . , ci = . - . , p< . ). multiple tasks in the cogstate pediatric battery appear to adequately identify patients with a history of cerebral infarcts. in addition, cogstate tasks appear to be fair predictors of impairments in iq and academic achievement outcomes. cogstate is inexpensive and can be easily administered in a medical setting with minimal training in approximately minutes. results support the potential for cogstate to be used as a screening tool for medical and neuropsychological abnormalities in children with scd. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: cardiovascular disease contributes to the morbidity and mortality of patients with sickle cell disease (scd). hydroxyurea therapy in scd has known clinical efficacy including improving anemia, decreasing episodes of vasoocclusive crisis and acute chest syndrome, and decreasing mortality. effect of hydroxyurea on cardiac function in children with scd is not well studied. an earlier study suggested the protective effect of hydroxyurea on left ventricular (lv) hypertrophy in scd. we hypothesized that hydroxyurea use would be associated with decreased lv remodeling and improved cardiac function. we aimed to evaluate the association between hydroxyurea use and lv remodeling and cardiac dysfunction in children with scd. design/method: we completed a retrospective study of patients with scd who were to years old, followed at st. christopher's hospital for children and had an echocardiogram completed in the past months. data collected included gender, bmi, scd genotype, hydroxyurea use, chronic transfusion use, and d and doppler echocardiographic parameters. cardiac structure, geometry, systolic function, and diastolic function echocardiogram parameters were included. analysis of variance (anova) tests were performed to assess for statistical significance of differences in cardiac parameters between patients with and without hydroxyurea use. analysis of covariance (ancova) tests were performed to control for age. results: demographic and echocardiogram data was collected on all patients who met inclusion criteria. of the patients included, ( %) were on hydroxyurea therapy. patients on hydroxyurea had significantly lower mean relative wall thickness (p = . ) and significantly higher mean peak early lv filling velocities (p = . ) and peak early lv filling/septal annuli early peak (e/ea) velocities (p = . ); however, only the e/ea velocities remained significant when controlling for age (p = . ). mean peak early lv filling velocities approached significance when controlling for age (p = . ). hydroxyurea therapy resulted in a significantly higher e/ea velocity, suggesting that these patients had worse diastolic function. it is possible that the patients initiated on hydroxyurea already had worse disease manifestations than those not on hydroxyurea, possibly accounting for the decreased diastolic function. when controlling for age, hydroxyurea use did not result in significant differences in cardiac structure parameters, systolic function parameters or cardiac geometry. prospective studies and larger sample size are needed to validate our findings, examine for additional statistically significant differences, and develop preventive strategies for cardiovascular disease in children with scd. background: acute chest syndrome (acs) is now the leading cause of death in children with sickle cell disease; mortality in the u.s. is reported to be - % and is mostly due to respiratory failure. early transfusion improves clinical outcomes. although patients with concurrent asthma are considered at increased risk for poor outcomes, risk factors for respiratory failure in pediatric acs have not been well-defined. to determine whether specific epidemiological and clinical features of children hospitalized with acs are predictive of the need for mechanical ventilation. design/method: data from the kids' inpatient database were reviewed to identify patients age < years with a discharge diagnosis of acs for the years , , , and . outcomes were defined by the international classification of diseases, ninth revision, clinical modification code. data were weighted to estimate total annual hospitalizations according to hospital characteristics in the united states. trends in healthcare costs, length of hospital stay, transfusion, and mechanical ventilation use were analyzed using multivariable linear regression. in addition, multivariable logistic regression was used to ascertain specific clinical or epidemiologic factors associated with mechanical ventilation use after adjusting for patient and hospital characteristics. the total hospitalizations for acs were , in ; , in ; , in ; and , in . reported use of mechanical ventilation ranged from . % to . % and was associated with non-black compared to black children (or, . ; %ci, . to . ) and the fall season (or, . ; %ci, . to . ), but not with age, preexisting asthma or hb-genotype. comorbidities of obesity (or, . ; %ci, . to . ), obstructive sleep apnea (or, . ; %ci, . to . ) and heart disease (or, . ; %ci, . to . ) were associated with mechanical ventilation use. the use of simple and exchange transfusion during all acs admissions ranged from . % to . % and . % to . %, respectively. among pediatric acs patients, those with obesity, obstructive sleep apnea or heart disease were at increased risk for respiratory failure and might benefit from early intervention (e.g., transfusion). surprisingly, asthma in children with acs does not appear to be a distinct risk factor for respiratory failure, and further studies are needed to clarify whether differences in treatment approach (e.g., addition of corticosteroids, bronchodilators) might impact on acs progression and/or severity even in high risk patients without asthma. objectives: to compare pulmonary functions between aa and k children with scd and to assess if a high hb f level contributes to better function. design/method: a cross sectional study was done on children with scd (hb ss disease) followed in comprehensive sickle cell programs. aa patients were followed at brookdale hospital, ny and k patients were followed in mubarak hospital, kuwait. children between the ages of and years who had pulmonary function tests (pft) done as a routine screening were enrolled. pft was done using spirometer and plethysmography. patients with congenital or anatomical lung abnormality, heart disease, pulmonary disease such as acute chest syndrome, acute asthma or pneumonia within weeks were excluded. results: there were children ( in each group) with scd,. restrictive pattern on pft was seen in / ( %) of aa vs. / ( %) of k (p> . ). obstructive pattern was seen in / ( %) of aa vs. / ( %) of the k group (p> . ). in both groups, children ( %) had normal pft. three/ ( %) in the aa group had a hb f> % as compared to / ( %) in the k group (p< . ). abnormal pft was noted in / children ( %) in each group. hbf was > % in / ( %) in the aa group vs. / ( %) in the s of s k group (p< . ). in patients with abnormal pft, mean hbf was . ± . in aa group, compared to . ± in k group (p< . ). conclusion: abnormal pft is highly prevalent among children with scd in both groups. aa children are more likely to have restrictive disease and k to have an obstructive pattern. level of hbf did not seem to protect k patients from abnormalities on pft. this finding should emphasize the importance of performing pft as part of the initial evaluation of all children with scd. background: sickle cell disease (scd) is a life-threatening disease with varied clinical spectrum and severity leading to premature death. there is a lack of validated prognostic marker in scd. recent evidence suggests that inflammation and platelet adhesion plays a critical role in the pathophysiology of vaso-occlusion in scd. elevated mean platelet volume(mpv) values are associated with a higher degree of inflammation in many disease states but it's effect on sickle cell disease or it's severity is unknown. objectives: to analyze the role of mpv in predicting disease severity/mortality in pediatric patients with scd. design/method: this is a single center retrospective study and included patients with sickle cell disease between months and years of age during a -year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . demographic information, lab data and clinical information including acute chest syndrome (acs), priapism, transfusions, sepsis, pain crisis, avascular necrosis were collected. all laboratory data were collected in steady state with no crisis in the recent past months. the disease severity score/probability of death was calculated using a validated model to predict risk of death in sickle cell disease (sebastiani et al. blood ) . pearson test was used to analyze correlation between mpv and probability of death. results: total no. of patients = ; male ( . %); female ( . %). median age is . years. all patients were of african-american origin. disease severity, hb ss - ( %); hb sc - ( . %) and sickle-beta thalassemia ( . %). patients on hydroxyurea has significantly lower mpv, p = . and this is independent of hb f levels. mpv has a significant positive correlation with the probability of death, p = . and correlation coefficient, r = . . on subgroup analysis, the correlation is even more significant in the age group between and years, p = . , r = . . using linear regression model, with probability of death as a dependent variable and hydroxyurea, mpv as independent variables, mpv maintains a significant association with probability of death (p = . ). conclusion: mpv is an independent biomarker predicting disease severity and probability of death in pediatric patients with sickle cell disease. hydroxyurea a known disease ameliorating agent is associated with lower mpv values. this effect is independent of the levels of fetal hemoglobin and may be due to anti-inflammatory effect of hydroxyurea or effect on the platelets. background: major success with initial qi projects by the sickle cell care team at children's hospital has precipitated ongoing inclusion of the qi approach to many other aspects of patient care. objectives: to optimize scd patient care utilizing qi processes. design/method: success of the scd qi team's initial project on transcranial doppler studies (tcds) and a second more complex project on hydroxyurea (hu) adherence, led to additional projects on completion of key immunizations, rbc phenotyping, and vitamin d level testing. using similar processes and principles from the hu adherence project, plan-do-study-act (pdsa) cycles were used to conduct smallscale tests of change. patient chart prep sheets, created for bi-monthly pre-appointment chart prep meetings, were significantly modified to include these focused care qi objectives. because of difficulty with emr database capability, data collected from the emr was tracked in excel spreadsheets or other unique tracking vehicles for the various parameters. for example, due to the clinic's diffuse, geographically scattered population, many separate non-shared primary care emrs, and lack of a mandatory state immunization registry; immunization records needed to be retrieved from pcps, outlying hospitals, public health departments, and fqhcs, and added to the emr and excel database. starting in / , all such data was collected and updated monthly. in one year's time ( - ) , the average immunization completion rate for seven key immunizations (pcv , pcv , hepatitis a, hepatitis b, meningococcal a, meningococcal b, and hpv) has increased by %. the biggest improvements were a % and % increase in completion for meningococcal a and meningococcal b, respectively. completion rate for rbc phenotyping rose from . % to . %. patients with at least one vitamin d lab test increased from . % to . %. since starting the tcd project in , the percent of patients who have completed their annual tcd has gone from a baseline of % to a sustained value of > %. conclusion: these qi projects have not only increased adherence to national recommendations for care of scd patients, they have helped establish a scd clinic methodology to create and implement sustainable processes. having the focused care initiatives prominently displayed on the patients' chart prep sheet serve as a reminder to medical team members to check the status of that item. this methodology is currently being used to formulate additional qi projects on annual renal function parameters and specialty visits, such as annual eye and dental exams. background: dominican republic has a high burden of sickle cell disease, and - % of children with homozygous hbss (sickle cell anemia, sca) will develop primary stroke. transcranial doppler (tcd) ultrasonography is an effective screening tool for primary stroke risk, but is not routinely available in dominican republic. hydroxyurea and blood transfusions are available, but no prospective screening and treatment program for stroke prevention has been implemented to date. ( ) to screen a large cohort of children with sca living in dominican republic, using tcd to identify elevated stroke risk; ( ) to determine the effects of treatments for stroke prevention (hydroxyurea for conditional velocities and transfusions for abnormal velocities). we hypothesized that both hydroxyurea and blood transfusions will decrease elevated tcd velocities and help prevent primary stroke. design/method: stroke avoidance for children with república dominicana (sacred, nct ) features a research partnership between cincinnati children's hospital and robert reid cabral children's hospital in dominican republic. the protocol, consent forms, and redcap database were prepared collaboratively and translated into spanish, and then irb approval was obtained at both institutions. in the initial prospective phase, children receive tcd screening over a -month period; those with conditional tcd velocities (maximum time-averaged velocity - cm/sec) receive fixed-dose hydroxyurea at mg/kg/day, followed by dose escalation to maximum tolerated dose, while those with abnormal tcd velocities (≥ cm/sec) receive monthly transfusions for stroke prevention. results: a total of children were enrolled in sacred, with an average age of . ± . years. initial tcd screening revealed ( . %) normal, ( . %) conditional, ( . %) abnormal, and ( . %) inadequate velocities. among children ( males, females, average age . ± . years) who initiated hydroxyurea at mg/kg/day for conditional tcd velocities, completed six months of treatment with expected hematological benefits including significant increases in hemoglobin concentration ( . to . g/dl) and fetal hemoglobin ( . to . %). no clinical strokes have occurred in the treatment group. repeat tcd examination after -months of hydroxyurea treatment revealed % ( / ) with previous conditional velocities had normal tcd velocities. the prevalence of conditional tcd velocities in the dominican republic is high, indicating an elevated stroke risk among children with sca. hydroxyurea treatment is associated with improved hematological parameters, lower tcd velocities, and probable decreased stroke risk. sacred is an important prospective and collaborative research trial providing epidemiological data regarding tcd screening, stroke risk, and hydroxyurea effects among children with sca. background: red blood cell aggregation is a rheologic property that explains the shear-thinning behavior of blood. at lower shear rate blood flow, red cells tend to aggregate, s of s whereas in higher shear rate blood flow, these aggregates are dispersed. this property is especially important in the venous system, where low shear rate blood flow predominates. there is inconsistent data in the literature concerning aggregation and aggregability in sickle cell disease (scd). objectives: because the lorrca and myrenne instruments have been shown to be similarly effective methodologies in red cell aggregation measurements, we aimed to determine whether the measurement of aggregation indices in scd, by myrenne and by lorrca, is consistent in our lab. design/method: we measured aggregation in blood samples corrected to % hematocrit. aggregability was measured using kda dextran in the myrenne but not the lor-rca. aggregation index using lorrca was measured in patients with scd and healthy subjects enrolled in a study of blood flow between and . aggregation and aggregability using the myrenne was measured in patients with scd and healthy subjects enrolled in a separate study of blood flow between and . results: using lorrca, we found that aggregation index in patients with scd was less than that of healthy subjects (p< . ). in the myrenne, aggregation at stasis was slightly higher in patients with scd compared to healthy subjects (p = . ) but aggregation at low shear rotation was not different. aggregability was higher in the patients with scd compared to healthy subjects at both stasis and low shear rotation (p< . ). red cell aggregation is an important determinant of low shear blood flow. deoxygenated venous blood is particularly important to low shear blood flow in patients with sickle cell disease. we found that two different aggregometers predict different aggregation results for scd. it is unclear why there is a systematic difference between the two methods, but there are some possibilities. first, the syllectogram in the lorrca is generated by the backscatter of light from the laser, while the myrenne measures transmitted light. second, the distance between the bob and cup in the lorrca is microns, while the gap between plates in the myrenne is microns, which might affect the disaggregation of red cells. further work is needed to understand the differences in red cell aggregation and aggregability when using these instruments, particularly when using aggregation as a predictor of blood flow and tissue perfusion. background: children with sickle cell disease (scd) are at risk of acute splenic sequestration crisis (assc). assc is a life-threatening complication characterized by splenomegaly, pain and severe anemia. assc most often occurs in young children with the most severe forms of scd and one-third of patients will have more than one episode. treatment is based primarily on expert opinion and includes blood transfusion and surgical splenectomy. objectives: we plan to assess the clinical practice patterns of physicians treating children with assc. design/method: a survey study was performed. the survey included six scenarios of severe scd with variation in age, hydroxyurea-use, and episode number of assc; questions focused on the acute and chronic management of assc. the survey was disseminated on three occasions over a six-month period, using an online survey tool, surveymonkey, to pediatric hematologist-oncologists participating in the american society of pediatric hematology-oncology hemoglobinopathy special interest group. the survey had a response rate of % ( / ). most respondents were recent graduates ( %; / ) practicing in academic urban centers with greater than sickle-cell patients. seventy-nine percent ( / ) recommended hydroxyurea initiation in - m/o with severe scd. prophylactic penicillin after surgical splenectomy was continued by % ( / ) after years. for the acute management of assc results did not vary despite patient age, hydroxyurea use, and the number of previous assc episodes. simple transfusion was preferred by % ( / ), with % ( / ) recommending slow transfusion and % ( / ) recommending routine simple transfusion. for the chronic management of assc, results varied based on patient age and the number of previous assc episodes. for a m/o after the first episode, % ( / ) recommended observation and % ( / ) hydroxyurea initiation. for a m/o with any prior episode of assc, % ( / ) recommended chronic transfusion therapy and % ( / ) surgical referral for splenectomy. for a y/o after the first episode, % ( / ) recommended surgical splenectomy and % ( / ) increasing hydroxyurea dose. for a y/o with any prior assc episode, % ( / ) recommended referral for surgical splenectomy. in this survey, we found most providers continue to recommend simple transfusions for assc and surgical splenectomy after two episodes. the majority of providers continue to delay referral for surgical splenectomy until age two, but earlier referral in children under two and use of chronic transfusion therapy were also reported. variability in chronic management highlights the need for further research of splenic sequestration. background: developing therapies for sickle cell disease (scd) is challenging in part because the accepted endpoint, vaso-occlusive crisis (voc), occurs infrequently, does not measure full disease burden, and is a measure of healthcare utilization. in phase / studies of patients with scd, voxelotor (gbt ) has demonstrated increased hemoglobin (hb) levels and reduced hemolysis and has been safe and welltolerated. voxelotor is being evaluated in the ongoing hope phase trial. objectives: to report the innovative phase / hope trial design with novel primary and secondary outcomes to accelerate drug development. design/method: hope (nct ) is a phase , randomized, placebo-controlled, multicenter study of oral voxelotor in patients with scd (aged - years) with baseline hb . - . g/dl and - episodes of voc in the prior year. to accelerate clinical trials to support drug development, the study combines a phase exploratory, dose-selection phase (group ) with a pivotal phase (groups / ). patients in group will be randomized : : to voxelotor or mg/day or placebo. analysis for dose selection will occur when the final patient has received weeks of treatment. group will continue enrollment with randomization : : until dose selection based on analysis of the group cohort. group will allow for a seamless transition into group , which will randomize patients : to the selected dose or placebo. the final data analysis set will include group patients who received placebo or the selected dose and all group patients. the primary endpoint is an objective laboratory measure and surrogate of clinical benefit, increase in hb > g/dl, from baseline to weeks based on voxelotor mechanism of action (inhibition of hb polymerization). this trial is the first to use a patient-reported outcome (pro), the -item sickle cell disease severity measure, as a secondary endpoint. this novel electronic pro, developed specifically for the hope study following fda guidance, will evaluate changes in scd symptom exacerbation and total symptom score from baseline to weeks. additional secondary endpoints include measures of hemolysis, rates of voc, transfusions, and opioid use. the study was designed to enable selection of pro-defined symptom exacerbations or traditionally defined voc as the key secondary endpoint after the group analysis. results: this study is ongoing. the hope trial, expected to complete enrollment by late , will evaluate the efficacy and safety of voxelotor compared with placebo in patients with scd. supported by global blood therapeutics. background: inflammation, coagulation activation, oxidative stress and blood cell adhesion are elements of sickle cell disease (scd) pathophysiology. patients with scd have low levels of the omega- fatty docosahexaenoic acid (dha) and eicosatetraenoic acid (epa) in plasma and blood cell membranes. dha is a bioactive fatty acid with anti-inflammatory, anti-blood cell adhesion and anti-oxidant properties. altemi-atm is a novel dha ethyl ester formulation with a proprietary delivery platform (advanced lipid technology® (alt®)) that enhances oral dha bioavailability. the scot trial investigated the effects of altemiatm in children with scd. objectives: to demonstrate the effects of altemiatm on blood cell membrane omega- index and selected biomarkers of inflammation, coagulation, adhesion and haemolysis associated with scd. s of s design/method: children with scd, aged - years (n = ), were enrolled. subjects were randomized to receive either placebo or one of three daily oral doses of altemiatm ( - , - or - mg/kg/day dha) for two months. the effects of altemiatm on red blood cell (rbc), white blood cell and platelet membrane omega- fatty acids index (total dha + epa levels) were assessed after four weeks of treatment. the effects of altemiatm on markers of inflammation, adhesion, coagulation, and hemolysis were assessed after eight weeks of treatment. cell membrane dha and epa concentration was determined by using lc-ms/ms method. the percent changes from baseline on blood cell membrane omega- index and select scd biomarkers were compared between the three dose groups and placebo using a mixed-model repeatedmeasures (mmrm) analysis with baseline blood cell membrane omega- index, hydroxyurea use, and treatment as fixed effects and patient as a random effect. after four weeks of treatment, blood cell membrane dha and epa levels were significantly increased in all altemiatm doses (p< . ). after eight weeks of treatment, significant reductions were observed in se-selectin (p = . ), and d-dimer (p = . ) in patients exposed to altemiatm dose level vs. placebo. hemoglobin was significantly increased at altemiatm dose level versus placebo. plasma high-sensitivity c-reactive protein, lactate dehydrogenase, soluble vascular cell adhesion molecule- and white blood cell count showed improvement after weeks of treatment in all three altemiatm doses levels but did not reach significance. conclusion: treatment with altemiatm enriches dha and epa in blood cell membranes of patients with scd and improves select sickle cell disease biomarkers of blood cell adhesion and thrombin generation. these findings provide insight into the mechanisms of action of altemiatm in sickle cell disease. brown university -hasbro children's hospital, providence, rhode island, united states background: despite clinical advances in the treatment of sickle cell disease (scd) in pediatric and young adult patients, pain remains a significant source of disease-related morbidity. physical therapy has been shown to be useful for the treatment of pain in children and young adults with various chronic illnesses of which pain is a significant component, however no data exists regarding potential benefits of physical therapy in pediatric and young adult patients with scd. objectives: to query healthcare providers and others involved in the care of pediatric and young adult scd patients regarding possible benefits of and barriers to physical therapy as a potential treatment modality. design/method: we conducted a web-based survey of healthcare providers within the new england pediatric sickle cell consortium (nepscc) in an attempt to identify potential benefits of and barriers to outpatient physical therapy in this patient population. results: nearly % of survey participants felt that physical therapy had the potential to be "somewhat beneficial" or "very beneficial" in pediatric and young adult patients with scd. a majority of physicians reported having referred patients with scd for physical therapy in the past. the most frequently identified perceived potential benefits included improved functional mobility, improvement of chronic pain symptoms, decreased use of opiates, improved mood symptoms, improved acute pain symptoms, and improved adherence with medications and clinic visits. significant perceived barriers identified included lack of transportation, time constraints, patient lack of understanding, and difficulty with insurance coverage. our study indicates that healthcare providers have an overwhelmingly positive view of the use of physical therapy in the management of pediatric and young adult patients with scd. significant barriers exist which need to be addressed. future research should focus on patient and parent perspectives regarding physical therapy, as well as a randomized controlled trial of a physical therapy intervention in this patient population. background: vitamin-d deficiency is fast becoming increasingly recognized in patients with sickle cell disease (scd). while it is estimated that these patients are five times more likely to develop vitamin-d deficiency, the exact clinical significance of this is largely unknown. given that this deficiency can be inexpensively and easily treated, our study sought to establish the prevalence of vitamin-d deficiency in our patient population and its relationship with disease severity. objectives: to estimate the prevalence of vitamin-d deficiency in patients with scd in our institution and to analyze their disease severity in relation to their vitamin-d level. design/method: through retrospective chart review we analyzed subjects that represent a cohort of patients followed at the adult and pediatric hematology services at university of miami with known diagnosis of scd that had a vitamin-d level drawn between january st, and august st, . we conducted a cross-sectional study and recorded the first vitamin-d level during this period. patient demographics, medical and social history information were collected along with laboratory data. the number of admissions for vaso-occlusive crisis (voc) and acute chest syndrome within one year preceding the collection the vitamin-d level was also recorded. results: a total of charts were reviewed, adult charts and pediatric charts. after exclusion, patients were enrolled. subclinical vitamin-d deficiency is only evident on laboratory blood testing of vitamin-d ( -hydroxy) and according to this laboratory result patients were classified as sufficient (≥ ng/ml), insufficient (< to ng/ml) and deficient (< ng/ml). out of the cases, . % ( / ) were deficient, . % ( / ) were insufficient and . % ( / ) were optimal. after statistical analysis two negative correlations were identified, increasing vitamin-d levels with decreasing white blood cell count (ci %- . (- . , - . )and decreasing incidence voc (ci %- . (- . , - . ). conclusion: this study confirms that there is a significant prevalence of vitamin-d deficiency in patients with scd. furthermore, the results of this investigation proved that vitamin-d deficiency is associated with acute pain and leukocytosis in patients with scd. given the multitude of confounding factors that affect vitamin-d absorption and intake, multivariate analyses are required to truly further investigate this relationship. texas children's hospital, houston, texas, united states background: hemophagocytic lymphohistiocytosis (hlh) is a rare but life-threatening condition of hyper-inflammation that is characterized by splenomegaly, cytopenias, hyperferritinemia, hypertriglyceridemia, hemophagocytosis and coagulopathy. although timely diagnosis is imperative, it is often challenging as these individual signs and symptoms may occur in a variety of clinical conditions. to report a case of undiagnosed sickle cell anemia presenting with severe ebv viremia and associated hemophagocytic lymphohistiocytosis results: a -month-old previously healthy male presented with respiratory distress, increased fatigue, and a focal seizure following a two-week history of cough and lowgrade fevers. physical exam was consistent with hypovolemic shock and revealed significant splenomegaly. laboratory testing revealed severe hypoglycemia, acidosis and electrolyte disturbances including hyperkalemia, hyperphosphatemia, and hyperuricemia. labs showed a leukocytosis (wbc , ), severely low hemoglobin ( . ), and platelets of , . coagulation testing revealed prolonged pt/inr and ptt, hypofibrinogenemia and a highly elevated d-dimer. additional workup was completed to determine etiology of acute presentation, given broad differential diagnosis. infectious studies were consistent with an acute ebv infection (plasma ebv pcr > , ). elevated levels of soluble il- and ferritin completed / criteria for the diagnosis of hlh. bone marrow evaluation showed trilineage hematopoiesis with no abnormal blast population or hemophagocytosis. results from hemoglobin electrophoresis sent from the initial cbc sample were notable for hbs . %, hbf . %, and hba of %, confirming the diagnosis of sickle cell disease. the patient was started on hydroxyurea and penicillin and splenomegaly resolved. with supportive care, he demonstrated gradual improvement in symptoms and laboratory abnormalities, including normalization of soluble il- , ferritin, cd , il- levels, immunoglobulins, and declining ebv titers. nk cell function has remained abnormally low, not eliminating the possibility of acquired hlh despite spontaneous improvement. conclusion: splenic sequestration associated with sickle cell disease in combination with acute infectious mononucleosis could have explained many of the presenting symptoms including anemia, thrombocytopenia, and splenomegaly. however, it does not explain the unusually high ebv titer and degree of inflammation meeting diagnostic criteria for hlh, which raises concern for an underlying immunologic abnormality such as x-linked lymphoproliferative disorder (xlp). although testing for xlp was negative, he will require s of s continued monitoring in the future for signs of relapse. this case illustrates the complexity of diagnosing lymphohistiocytic disorders and the significant overlap in presentation between these disorders and other medical conditions. background: vaso-occlusive crisis (voc) is one of the most distressing occurrences in patients with sickle cell disease (scd). patient controlled analgesia (pca) is recommended by nih and expert opinions favor its early use. we aim to review the use of pca in patients with voc and to evaluate if its early use is associated with faster pain control and reduced length of stay (los). design/method: this retrospective single center study included all pediatric patients admitted and treated with pca for a severe voc from to . "early" use was defined as start of pca within hours of arrival in the emergency department (ed) and "late" use after hours. time to reach adequate analgesia was defined as oucher, verbal scale or faces pain scale < / obtained twice consecutively in a -hours interval. time to reach adequate analgesia and los were compared between early-pca and late-pca groups. results: a total of patients presented episodes of voc treated with pca during the study. sixty-one episodes ( %) were treated with early-pca and ( %) with late-pca. both groups were comparable in terms of age ( . vs . years old), gender ( . % female vs . %), hemoglobin phenotype ( . % hbss vs . %), but median pain score at admission was higher in early-pca than in late-pca ( / vs / , median difference ( % ci , ). early-pca was associated with a median reduction in los of . days ( % ci . , . ) (median early-pca los . vs late-pca . days). time to reach analgesia could be evaluated only in a subset of patients ( in early-pca and in late-pca group). although time to reach adequate analgesia tended to be shorter in the early-pca group, it was not statistically different: median . hours vs . hours, difference of . ( % ci - . , . ). side effects were observed during ( . %) pca treatments ( / ( . %) episodes in early-pca, / ( . %) in late-pca group) among which ( . %) were significant adverse events. these were observed in patients who required interventions: desaturations requiring oxygen without intubation, neurologic abnormalities (hallucinations, visual abnormalities, no stroke), urinary retentions. conclusion: early use of pca for severe voc was associated with a reduced length of hospital stay despite that these patients had higher pain score on admission. prospective studies are needed to support these positive outcomes. background: acute chest syndrome is one of the leading causes of death in children with sickle cell disease - . while the cause of acute chest syndrome most commonly is not identified, fat embolism and infectious causes are believed to be most common. with an extremely high mortality rate, rapid identification and initiation of therapy is essential for survival. case presentation: we describe the case of an -year-old female with sickle cell sc disease who was admitted for vasoocclusive pain crisis and quickly progressed to multi-system organ failure due to fat embolism syndrome and parvovirus b infection objectives: the case highlights the presentation and diagnosis so other providers can optimize outcomes for those with this under-recognized syndrome design/method: her parvovirus studies returned after days which showed: parvovirus b dna pcr detected; parvo igg . (positive > . ); and igm . (positive > . ). the patient experienced an approximately . g/dl drop in hemoglobin( . to . g/dl/ hrs) with progressive thrombocytopenia (from , to , /ul) and a peripheral smear showed microcytic,normochromic red cells with nucleated rbcs and occasional nuclear budding, slight polychromasia, schistocytes, and polymorphic cells with toxic granules that suggested leukoerythroblastosis. she was emergently transferred to the regional quaternary care hospital for ongoing ecmo therapy where she experienced a change in her pupillary exam prompting a stat ct scan that showed severe, diffuse cerebral edema with transtentorial herniation. the decision was made to withdraw life-sustaining therapies and her family refused a post-mortem autopsy examination. fat embolism syndrome is a severe and uncommonly recognized complication of sickle cell disease, seen most commonly in those with a non-ss phenotype and previous mild disease course who present with severe, unrelenting vaso-occlusive pain episode and/or acute chest syndrome that progresses to respiratory distress with altered mental status and cutaneous changes. rapid identification and initiation of exchange transfusion therapy should be initiated with clinical suspicion because of the extremely high mortality rate. although previously considered rare, it needs to be considered in the differential diagnosis of more commonly encountered complications of sickle cell disease. background: patients with sickle cell disease (scd) experience vaso-occlusive crisis (voc), which results in extreme pain, often requiring opioids and admission. genetic and environmental factors affect the frequency and severity of these episodes. previous research has born conflicting evidence on whether environmental temperature is contributory. edmonton, alberta is the northern most city with a population over a million in north america. there is an increasing sickle cell population which is exposed to extreme winter conditions. this provides a suitable population and atmosphere to study the influence on cold external temperatures in scd. this study sought to identify if pediatric patients with scd, experience greater morbidity in cold external temperatures. board approved retrospective case control series. patients were identified through a clinical database, and emergency visit, phone call and admission data was collected over a fiveyear period. the average, minimum and change in temperature on day of presentation, and hours prior, was collected from the government of alberta, and was statistically analyzed using descriptive statistics, to determine the relation to vaso-occlusive events. results: one-hundred and eighteen patients were identified, and voc events reviewed. the mean patient age was . years of age with a range from . - years old. the female to male ratio was equivalent with female ( . %) and male ( . %) voc events. eight records ( %) had docu-mented cold exposures. the analysis between the temperature and the frequency of events did not yield significant correlation. average and minimum temperature on day of admission had the largest percentage of voc events occur at mild temperatures, from - . to • c and - . to respectively. change in temperature on day of admission, and hours had the largest percentage of voc events at a mild to moderate change in temperature of - degrees. data at & hours prior to admission showed similar results. secondary data analysis accounting for the lower proportion of extreme weather days in comparison to moderate temperate days showed no significant impact. there was no correlation of average, minimum or change in temperature on day of admission, or hours prior. multiple cofounding factors likely contribute to these results. as it was a retrospective study many confounding and precipitant factors may not be recorded or identified. a prospective study to better record specific cold exposure is warranted. children's national health system, washington, district of columbia, united states background: achieving optimal anticoagulation with unfractionated heparin (ufh) in pediatric patients receiving extracorporeal membrane oxygenation (ecmo) is often challenging due to antithrombin (at)-mediated heparin resistance (hr). intermittent at dosing during pediatric ecmo support does not maintain adequate at levels. continuous at infusion (cati) presents an alternative strategy to achieving consistent goal at levels and optimizing heparinization. however, cati during pediatric ecmo has not been adequately studied. objectives: to describe our center's experience with an ecmo cati protocol. design/method: in , we modified our ecmo anticoagulation protocols to include ufh titration according to anti-factor xa (anti-fxa) levels and cati in patients with at-mediated hr. the cati rate was calculated using baseline and goal at levels while accounting for the circuit volume. cati was administered with ufh into the circuit via a s of s y-infusion set. at and anti-fxa levels were monitored every hours. recombinant at (r-at) concentrate was used at our center until with subsequent transition to a plasmaderived at (pd-at) concentrate. due to the longer half-life of pd-at concentrate, the protocol was modified so cati is stopped once target at and anti-fxa levels are achieved. we conducted a retrospective study of all patients who received cati during ecmo support at our center. data are reported as median and interquartile range and compared using the mann-whitney u test. two-tailed p-value < . was considered statistically significant. since , patients [ males, age month ( . - )] on ecmo support received catis ( rat, pd-at) per our protocol ( patients received pd-at infusions during one ecmo run). the duration of cati was hours ( - ). cati administration led to significant increases in at and anti-fxa levels from baseline of % ( - ) and . units/ml ( . - . ) to the first level within goal of % ( - ) and . units/ml ( . - . ), respectively (p< . ). the respective times to achieve goal at and anti-fxa levels were hours ( - ) and hours ( - ). the respective peak at and anti-fxa levels were % ( - ) and . units/ml ( . - . ). during cati, no patient required circuit change, patient developed cannula thrombosis and patients experienced non-fatal major bleeding. conclusion: cati in pediatric patients receiving ecmo support with close monitoring of at and anti-fxa levels was associated with significant rapid increase in at, optimization of heparin effect, and reduction in thrombotic complications without increase in major bleeding compared to prior reports. a prospective study of this at dosing strategy is warranted. children's hospital of orange county, orange, california, united states background: inherited factor xiii (f ) deficiency is a rare bleeding disorder with wide heterogeneity in clinical manifestations ranging from mild bruising, and mucosal and umbilical stump bleeding to spontaneous, severe intracranial bleeding. the bleeding phenotype is influenced not just by zygosity of the fxiii mutation alone, but also by co-inheritance of variants in other clotting protein genes that also play a major role in clot formation and stability. we present a series of three siblings found with f a gene variant and platelet dysfunction linked to bleeding phenotype. design/method: retrospective chart review of the index case, coagulation studies and whole gene sequencing. the index patient presented at two years of age with a subdural hematoma after a fall, requiring emergent craniotomy. a week after initial evacuation, she re-bled, prompting an extensive work-up for potential bleeding disorders, including f activity, von willebrand profile, comprehensive fibrinolysis panel, pai- antigen level, platelet mapping thromboelastogram (plt-teg), and f genetic analysis. the patient's identical twin and older sibling, who had symptoms of bruising, underwent a similar evaluation. the index patient demonstrated consistently low f activity ( - %), and platelet function testing revealed decreased response to adp agonists. the twin and older sibling had normal f levels, and only slightly decreased response to adp in platelet studies. whole gene analysis of f and other genes on our next generation panel, revealed several intronic deletions in the index patient that were not shared by her siblings, which likely account for her decrease in circulating f levels. her symptoms have responded well to monthly treatment with factor concentrate. all three children shared the f variant, pro leu, previously described as a risk factor for intracranial hemorrhage. the f mutation, pro leu, has been associated with intracranial hemorrhage in young women, but the presence of the variant alone may not be enough to cause a severe bleeding phenotype. family studies identified novel deletions in the index patient which may account for her decreased f levels, which would have been overlooked with standard sequencing. future studies, including evaluation of 'platelet' f levels, should be performed when platelet dysfunction is detected. further laboratory and clinical evaluation is required to delineate the long term implications of the interaction of even mild f deficiency if present with additional clotting disorders such as the platelet function defect in these siblings. background: acquired hemolytic anemia can occur due to mechanical shearing of red blood cells and is classically seen in patients with prosthetic heart valves. there are reports of this same traumatic effect with other repairs, including annuloplasty. following valvular procedures flow disturbances can exist across the valve that lead to shear stress and hemolysis. although von willebrand disease (vwd) is typically seen due to an inherited disorder in the pediatric population, flow disturbances in the setting of valve abnormalities can lead to acquired von willebrand syndrome (avws). von willebrand factor multimers become unfolded and elongated in the setting of shear stress resulting in increased susceptibility to cleavage by adamsts- . specifically, loss of high molecular weight multimers (hmwms) can lead to a syndrome akin to type a vwd. objectives: to describe a case of mechanical hemolysis with acquired type a vwd design/method: a -month-old girl with history of hypoplastic left heart syndrome and severe tricuspid valve insufficiency underwent norwood procedure, blalok-taussig shunt placement and subsequently a bidirectional glenn and tricuspid valve annuloplasty. during the following month she requires weekly red blood cell (rbc) transfusions due to intermittent anemia. she also experienced bloody stools and dark urine. laboratory evaluation was notable for normocytic anemia, reticulocytosis, elevated lactate dehydrogenase, and low haptoglobin consistent with hemolytic process. immune-mediated hemolysis from transfusion reaction or presence of autoimmune or alloimmune antibodies testing was negative. to investigate gi bleeding, work up for vwd revealed normal vw activity and antigen but with loss of high molecular weight multimers consistent with acquired type a vwd. in consultation with cardiology, it was felt her tricuspid valve insufficiency jet could be leading to mechanical hemolysis and avws. a repeat echo showed persistent moderate tricuspid insufficiency but no other significant changes. due to the patient's continued need for weekly rbc transfusions she was subsequently trialed on pentoxifylline which is used in adult patients to decrease blood viscosity and increase erythrocyte flexibility in patients with mechanical hemolysis. her transfusion needs remained the same and the medication was discontinued after two weeks. she required one transfusion a week later but no transfusions since that time. although not commonly seen in pediatric patients, the diagnosis of mechanical hemolysis accompanied by avws should be pursued in a patient with congenital heart disease with significant anemia and/or bleeding. the work up in these patients is difficult as echocardiograms can be inconclusive thus an extensive hematologic evaluation is usually necessary. objectives: our aim was to assess incidence of and potential risk factors for central line-related dvt at our institution between - . additionally, our goal was to analyze if that incidence differed between the three central line types and identification of line-specific risks. design/method: a retrospective chart review of central line placements in pediatric patients at cleveland clinic between - was conducted. data included demographics, potential risk factors, line characteristics and any related thrombotic events. the study cohort consisted of lines in pediatric patients aged - years of age. there were . thrombi ( % ci . - . ) per , line days. statistically significant risk factors for thrombus include diagnosis group (liquid tumor highest rate of %, solid tumor lowest at %), type of line (picc %, broviac %, and mediport %), location of line, greater number of lines per patient, peg asparaginase ( % vs %), sepsis, and history of procoagulant state. line characteristics such as lumen size and number of lumens were not identified as a significant risk. there was a significantly higher rate of thrombus in than in the previous years when pooled ( % in vs . % from - , p = . ). the incidence of dvt in pediatric patients at our institution was highest with broviac lines, and significant risk factors in our patient population included liquid tumor, femoral vein location, peg asparaginase, sepsis, and history of a procoagulant state. the incidence of thrombi was highest in , and therefore highlights the urgent need for improvement in nationwide hospital practices to minimize risk of thrombi formation and early detection in the higher-risk s of s populations. there is still much to be learned regarding the characteristics specific to different central lines, which would influence thrombi formation. nyu winthrop hospital, mineola, new york, united states background: pediatric immune thrombocytopenic purpura (itp) is an autoimmune disorder with platelet counts < causing increased risk for significant hemorrhage. there is increased immunologic platelet destruction due to production of specific autoantibodies along with inhibition of platelet production. few randomized trials exist to guide management and ultimately each patient requires an individualized treatment plan. itp may be acute (diagnosis to m) or chronic (> months). one of the treatments of chronic itp is laparoscopic splenectomy (ls), which is very well tolerated. a rare complication of ls is splenosis, an autotransplantation or implantation of ectopic splenic tissue within the abdominal cavity or in any other unusual body compartment. splenosis is sometimes associated with relapsed itp due to preserved immune activity. the usual management of symptomatic splenosis is surgical resection. objectives: to describe medical management in a young patient with itp relapsed due to extensive unresectable splenosis following ls design/method: our patient was originally diagnosed at years with itp and was treated with ls at years of age for chronic severe thrombocytopenia and persistent bleeding not responding to first line therapies. she tolerated it well and had a complete response (cr) defined as a platelet count of > measured on occasions > days apart and absence of bleeding. she maintained a normal platelet count for twelve years after which she relapsed (loss of response after cr) with severe thrombocytopenia and hematuria necessitating high dose steroids. ct scans showed multiple wellcircumscribed soft tissue masses in the left lower quadrant adjacent to uterus and left ovary, involving left omentum and the anterior abdominal wall partly. findings were confirmed by damaged rbc nuclear scan to be splenosis. during laparoscopy the splenosis lesions were deemed too extensive and were not resected completely to avoid postoperative morbidity. she was started on sirolimus around the same time for treatment of her relapsed itp and steroids were weaned off. results: eight months since beginning sirolimus with therapeutic levels she remains in cr with no bleeding and has not required any steroids, immunoglobulins or anti d immunoglobulin. conclusion: sirolimus is a safe and effective steroid-sparing agent in treatment of chronic itp. this is the first instance of a patient with poorly resectable splenosis responding well to medications for itp. more data is needed regarding the longterm efficacy of such an intervention and whether it will eliminate the need for a second surgery in relapsed itp patients with extensive splenosis. background: storage pool disorders affecting platelets result in bleeding symptoms related to a deficiency or defect in alpha granules or delta granules. in delta-storage pool disorders (dspd,) there is a deficiency of the delta granules and their constituents, which results in the inability of platelets to properly activate as well as lack of proper constriction of blood vessels during bleeding episodes. amongst patients with dspd, females most commonly present with menorrhagia, while males tend to present with epistaxis and easy bruising. the international society on thrombosis and hemostasis (isth) developed a screening bleeding assessment tool (bat) for mild bleeding disorders, shown to be a validated tool in children. diagnosis of dspd is classically made with a platelet electron microscopy (pem) value < . delta granules per platelet (dg/pl), but recently lower diagnostic thresholds of dg/pl or even . dg/pl have been suggested. objectives: evaluate the correlation between pem and bleeding scores, and also examine various cut-off values used to diagnose and risk stratify patients with dspd. design/method: retrospective chart review of pediatric patients followed by hematology with a diagnosis of dspd was performed. clinicians obtained bleeding scores for each patient as standard of care in the hemostasis clinic. quartile ranges were established to appropriate three stages of severity based upon bleeding scores. statistical analysis was performed using software r and exploratory data analysis to evaluate for a correlation. results: amongst all patients, the average bat score was . and pem was . dg/pl. the average bleeding score for pem between . dg/pl and dg/pl was . , while the average bleeding score for pem below dg/pl was . . the correlation coefficient between pem and bleeding scores is . . using a threshold of dg/pl, % of patients would have met diagnostic criteria. quartile ranges for the bleeding scores are as follows: st quartile was - , nd quartile was - , and rd quartile was > . conclusion: patients with a more marked granule deficiency do not exhibit a more severe bleeding phenotype, suggesting proper platelet function is not solely determined by granule quantity in these patients. bleeding severity may be more appropriately assessed with bleeding scores rather than pem values, and using quartile ranges may aide in risk stratification and therapeutic interventions for dspd patients. further work remains to determine the optimal diagnostic threshold of pem dspd in pediatric populations. texas children's hospital, houston, texas, united states background: warfarin management has many challenging aspects including pharmacogenomics, food and drug interactions, lack of standardized dosing, patient compliance, tracking lab results from multiple lab locations, and the potential for significant bleeding or thrombotic complications. a literature review revealed limited data highlighting anticoagulation monitoring workflow and emr documentation and specifically, no data in the pediatric population. historically, the texas children's hospital cardiology and hematology centers were each documenting anticoagulation data within the epic tm system differently. epic's tm original design for anticoagulation documenting resulted in the necessity to duplicate documentation in order to see at-a-glance critical anticoagulation monitoring information. objectives: the objective of this project was to standardize inr documentation across departments to reduce the risk of patient safety events and improve workflow. design/method: a workgroup assembled consisting of nurses from the cardiology and hematology departments, along with staff members from the epic tm is support group. the workgroup identified current documentation practices, available epic tm tools, and brainstormed ideas to streamline and improve both documentation with the current epic tm tools. physician partners were identified in cardiology, hematology and coagulation laboratory to gain their input. a new anti-coag (ac) encounter was developed and first made available in an epic tm practice environment, then once approved, epic tm written education and training session were completed by both departments' staff. results: surveys were sent to health care providers in the cardiology and hematology centers prior to the new ac encounter, and also to health care providers six months after implementing the ac encounter. six responses were received for each survey. the pre-implementation survey showed the most problematic part of the documentation system for anticoagulation was no single place in the emr to find a complete anticoagulation picture. post ac encounter implementation survey results revealed more health care providers using the epic tm inr reminder pool, less time needed to compile a report of three months of anticoagulation information, less time needed to document individual encounters, less locations needed to document ac information and decreased amount of types of documentation used. standardized ac encounters improves workflow with less time needed to document and compile information, less types of documentation utilized and easier access to patients ac information. next steps include retrospective review of patients' inr time in therapeutic range to determine if there was an impact on patient compliance and continue to evaluate and modify the ac encounter to enhance user friendliness. caitlin tydings, jennifer meldau, christine guelcher, carole hennessey, eena kapoor, michael guerrera, yaser diab s of s children's national health system, washington, district of columbia, united states background: venous anatomic abnormalities (vaas) are considered a risk factor for developing deep vein thromboses (dvts) that occur as a result of significant alterations in venous blood flow. identification of predisposing vaas can be challenging. hence, diagnosis can be delayed or overlooked especially in pediatric patients. dvts in children or adolescents with predisposing vaas have been only described in sporadic case reports and small case series. objectives: to describe characteristics and outcomes of dvts in pediatric patients with underlying vaa treated at our center. design/method: we conducted a retrospective chart review of all pediatric patients with objectively confirmed extremity dvt treated at our institution over a -year period from to and identified all patients with underlying vaas. patients were managed according to standardized institutional protocols based on published guidelines. post-thrombotic syndrome (pts) was assessed at our center using the manco-johnson instrument. relevant data were collected and summarized using descriptive statistics. during the study period, of pediatric patients ( %) [ females, median age years (range - )] diagnosed with extremity dvt at our center were found to have an underlying vaa. vaas included may-thurner anomaly ( patients), venous thoracic outlet obstruction ( patients) and inferior vena cava (ivc) atresia ( patients). additional provoking factors were identified in patients at time of presentation. dvt locations included upper extremity veins ( patients), lower extremity veins ( patients) and lower extremity veins and ivc ( patients). the majority of dvts [ patients, ( %)] were completely occlusive. high risk thrombophilia (defined as inherited deficiency of antithrombin, protein c, or protein s, or antiphospholipid antibody syndrome) was present in patients ( %). all patients were treated with therapeutic anticoagulation with patients continuing indefinite anticoagulation. endovascular interventions were performed in patients and included percutaneous pharmacomechanical thrombectomy and/or catheter-directed thrombolysis ( patients), balloon angioplasty ( patients) and stent angioplasty ( patients). surgical interventions included thoracic decompressive surgery ( patients) and surgical thrombectomy ( patient vvas represent an important risk factor for developing extensive extremity dvt in adolescents. this special population is at risk for short-term and long-term com-plications. early identification and correction of vaas may improve outcomes. however, multicenter, prospective studies are needed for developing optimal evidence-based treatment approaches. alexander glaros, roland chu, sureyya savasan, meera chitlur, madhvi rajpurkar, yaddanapudi ravindranath children's hospital of michigan, detroit, michigan, united states background: acute budd-chiari syndrome (bcs) is a rare thrombotic emergency in children, and etiologies/treatment are less well-defined than in adults. in adults, a systematic approach including anticoagulation, relief of venous obstruction, and treatment of the underlying cause has proven successful. more recently treatment has tilted towards aggressive surgical interventions, which carry significant risk and are often not feasible. objectives: review our experience with three different patients with bcs and suggest a mechanistic based approach to treatment. the records of three patients with bcs were reviewed and their presentations, etiologies, treatment, and outcomes were reported. results: patient a was a -year-old female with paroxysmal nocturnal hemoglobinuria who presented with recurrent worsening abdominal pain over several months. narrowing of inferior vena cava (ivc) and hepatic veins was noted on imaging. liver transplant was not considered surgically feasible. she was treated with eculizumab, steroids, and anticoagulation with restoration of hepatic venous flow in weeks. patient b was a -year-old male with several weeks of right upper quadrant pain, fatigue, and pre-syncopal episodes, with a history of blunt abdominal trauma from football scrimmage weeks earlier. he was found to have near complete occlusion of the ivc and hepatic veins. liver transplant was not considered feasible. he was successfully treated with anticoagulation alone. patient c was a -yearold male with acute myeloid leukemia in induction cycle who developed severe pancytopenia; typhlitis was diagnosed and managed medically. days later he acutely decompensated, arrested, and was placed on extra corporeal membrane oxygenation, and imaging showed complete occlusion of the portal vein, hepatic veins, and ivc to the level of the atrium, with bilateral pulmonary emboli. emergency liver transplant or catheter based interventions was deemed not feasible. treatment with eculizumab was considered for presumed inflammation induced complement activation (c mg/dl [normal - ]; ch was u/ml [normal - ]) as a trigger for thrombosis, but the patient progressed quickly and died before it could be initiated. our experience with bcs shows that invasive interventional options and liver transplant may not be feasible in most patients for multiple reasons. rapid diagnosis and aggressive etiology-based medical management are paramount to successful treatment of this rare complication. eculizumab may be considered in treating bcs with complement activation not only due to innate disorders, but also secondary to acute inflammation when proper laboratory evidence is present. background: platelet aggregation studies are the gold standard for the diagnosis of platelet function defects during the evaluation of a patient with bleeding problems. the platelet aggregation test measures how well platelets clot in response to different concentrations of epinephrine, adenosine diphosphate (adp), collagen, arachidonic acid and ristocetin. because platelet function defects are often under-recognized and under-diagnosed in the pediatric patient, the true incidence is unknown. we report our experience in the diagnosis of platelet defects at our institution over a -year period in order to add some clarity to the limited pediatric data available. objectives: our primary objective is to document correlations/trends between less well-known platelet function abnormalities and clinically significant bleeding at our institution over a -year period. design/method: after appropriate irb approval obtained, we performed a retrospective chart review of all children who had platelet aggregation testing done from to . data collected included demographics (age, sex, race), personal and family history of bleeding, screening for coagulation defects and platelet aggregation test results. symptoms examined in our data were limited to epistaxis and heavy menstrual periods. for each of these symptoms, results were further analyzed to those with abnormal responses to adp and epinephrine. patients with existing bleeding diagnoses and those with incomplete medical records were excluded. we identified patients. of the patients with epistaxis, % had abnormal platelet aggregation testing while only % of those with heavy menstrual periods had abnormal results. within our population, abnormal platelet function assay (pfa- ) results or race did not appear to correlate with abnormal platelet aggregation testing. in the cases of epistaxis, sex was also noncontributory. our preliminary results suggest that platelet aggregation testing was more useful in predicting platelet defects in those with a clinical bleeding history of epistaxis as opposed to heavy menstrual periods. for other presenting symptoms, platelet aggregation testing did not offer diagnostic benefit. abnormal response to adp in the platelet aggregation test was the most common finding in our population; the clinical significance of which is not well understood. going forward, we plan to document whether abnormal results correlated significantly with the subsequent final diagnoses of our patients. background: decision making for severe hemophilia a in previously untreated patients (pups) has recently become a significant ethical debate. recombinant factor viii (rfviii) products previously were recommended to avoid transmission of blood borne pathogens associated with plasma-derived fviii (pdfviii) products. however, the increased incidence of fviii alloantibody inhibitors with rfviii products compared to pdfviii products has challenged this former standard of care. despite the support of the medical and scientific advisory council, recommendations considering pdfviii products for a pup remains controversial. design/method: we used a modified utilitarian approach involving clinical, public health, and research ethics. shared decision making permeates the framework to maximize understanding, minimize bias, respect informed consent or dissent, and provide care that aligns with patient and family values when medically and practically feasible. the framework has three tiers. first, it evaluates whether resources are scarce or abundant for equitable resource allocation. if fviii products are scarce, we s of s recommend developing a central supply for emergency use and then evaluating the needs of the severe hemophilia a patients. prioritization of who receives the factor products would be decided by a designated team based on the availability of the factor products and clinical scenarios, with no preference given to those on research trials. however, if resources are abundant, treatment for acute bleeding and standard of care prophylaxis measures, including primary prophylaxis, could continue. the second tier accounts for whether there is a new infectious epidemic or concern where a pathogen cannot be eliminated. if there is, healthcare and public health workers may limit the use of pdfviii products. if not, pdfviii and rfviii products are to be equally considered. the third tier evaluates whether the clinical scenario is emergent or not. if there is acute, emergent bleeding, the immediately available resource should be used, along with bypassing and/or adjuvant resources as needed until the bleeding has resolved or improved. to align with patient and family preferences, attempts to have both pdfviii and rfviii products available at similar costs in institutions would be ideal. this ethical framework endeavors to balance autonomy, beneficence, nonmaleficence and justice in helping guide discussions among providers, pups with severe hemophilia a, and their families. disclaimer: findings and conclusions are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention, emory university, or children's healthcare of atlanta. background: von willebrand disease (vwd) is a common bleeding disorder which affects up to % of the population without gender predilection. bleeding associated with this condition results from a deficiency or abnormality in von willebrand factor interfering with formation of primary hemostasis. ehlers-danlos syndrome (eds) is a group of rare inherited connective tissue disorders which may have an associated bleeding manifestation without abnormalities in coagulation testing. bleeding symptoms reported in eds result from capillary and tissue fragility. joint hypermobility syndrome (jhs) is an inherited condition which is nearly indistinguishable from eds iii. reports of coinheritance of vwd and eds or jhs are infrequent. the objective of this retrospective study was to review patients with coexisting vwd and eds or jhs at the indiana hemophilia and thrombosis center in order to describe the type and severity of bleeding symptoms, physical examination findings, and pertinent laboratory data. design/method: the electronic medical record database of the indiana hemophilia and thrombosis center was queried for patients with a diagnosis of vwd and one of the following descriptors: hypermobility syndrome, hypermobility, hypermobile joints, or ehlers-danlos syndrome. the records of identified patients were reviewed for demographics, type and severity of bleeding symptoms, beighton scores (bs), vwd antigen, ristocetin cofactor, factor viii levels, vwd multimer pattern, vwd subtype, genetic testing for eds, and family history of eds. results: a total of patients with dual diagnoses of vwd and eds and patients with vwd and hypermobility were identified with this query. two patients had completed genetic testing for eds, and one had a col a gene mutation identified. significant bleeding symptoms in the vwd and eds group included hematuria and postoperative hemorrhage. two of these patients had delayed wound healing postoperatively. seven of the patients identified to have type i vwd and jhs had moderately severe and somewhat unusual bleeding episodes reported including hematuria, hematemesis, and hemoptysis; of these patients had significant perioperative bleeding. females composed % of the vwd and eds group and % of the vwd and jhs group. conclusion: coinheritance of vwd and eds is an uncommon phenomenon. patients with vwd and eds or jhs may have atypical and moderately severe bleeding, especially with procedural intervention. incorporation of bs into the assessment of patients with bleeding disorders is useful to identify potential inherited collagen disorders, as diagnosis of these conditions may impact clinical management. in the year-long phase ii study (ro fd ), / khe patients responded. patients were followed for years after study completion, collecting data on growth and development, complications of therapy, unexpected toxicities, and need for continuing sirolimus. objectives: after study therapy treatment of one year, objectives include: . assess long term toxicity over the - year period after study therapy completion . assess unexpected toxicity . assess overall condition of the patient . assess need for restart or continuation of sirolimus therapy design/method: prospective follow-up of patients with a diagnosis of khe from institutions. inclusion criteria: follow-up for - years post-study. results: follow-up included data at year (n = ) and - . year (n = ) time points. average age at the start of treatment was months. of patients were available for follow up. four patients are no longer on sirolimus: one patient completed study therapy and remains off treatment (ot) ( years), required years of treatment and is now . years ot and required an additional treatment course prior to successful discontinuation now and months ot. of the patients still on sirolimus, all restarted medication for symptoms of pain, swelling and/or edema interfering with quality of life and have made an average of . attempts to discontinue sirolimus. no patient had reoccurrence of kmp. all patients had improvement in clinical and radiologic appearance of khe but all have residual lesions noted on imaging and/or clinical exam. no unexpected toxicity, growth delay, developmental issues or other long term toxicity of sirolimus was noted. conclusion: this is the first prospective data on long-term follow up of khe patients treated with sirolimus. although numbers are small, sirolimus is well tolerated; however, over half the patients were still on medication at - year follow up. this stresses the need for continued long term follow up in these young patients and investigation of the mechanism of sirolimus effect. nationwide children's hospital, columbus, ohio, united states background: recent studies have identified that adult persons with hemophilia (pwh) have a higher prevalence of hypertension and renal disease than the general population. while hematuria is a known complication of hemophilia a and b (ha, hb), its long-term impact on pwh is not currently known. by annually screening our patients with urinalysis, our pediatric center identified that just under half of our patients demonstrated hematuria over a four-year period. motivated by a desire to identify early markers of hypertension and renal disease, we sought to determine if this finding is reflected in the pediatric hemophilia population as a whole. objectives: establish the population-wide prevalence of hematuria in pediatric pwh. design/method: we used the pediatric health information system (phis) database, which contains clinical and resource utilization data for inpatients from hospitals nationwide, to analyze the prevalence of hematuria, hypertension, renal disease and related diagnosis codes in pediatric pwh who were admitted from january to september . results: during the five-year period, , unique pediatric pwh accounted for , admissions. while the majority of admissions were for bleeding or infectious concerns, ( . %) patients had an affiliated admission code for hematuria. for admissions as a whole, the median age was years with % of those admitted being infants, % toddlers, % children, % adolescents, % older than . we identified % of admissions were for ha with the remaining % were for hb. there were ( %) admits in which a bypassing agent was administered. the median length of stay for persons with hematuria was days compared to days for nonhematuria/other bleeding. there were ( . %) admissions with hypertension reported; though, only patients received an antihypertensive medication during that admission. additionally, only ( . %) admissions reported a diagnosis code of renal disease. our study demonstrated that pediatric pwh are experiencing hematuria. in general, only patients with persistent hematuria require hospital admission so we suspect this data underrepresents the numbers of pwh experiencing hematuria that is managed in the outpatient setting. we also suspect that hypertension is grossly underreported and undertreated in pediatric pwh. additionally, there are a low number of patients experiencing renal disease requiring hospital admission among this cohort. given that there is little research into the long-term impact of hematuria in hemophilia, we feel these findings support the need for further vigilance of our pediatric pwh. background: gla and gsd can aggressively destroy bone, with significant impact on morbidity and mortality. the mtor inhibitor, sirolimus has been shown to be effective in the treatment of these diseases. based on the addition of mtor inhibition to bisphosphonate therapy in metastatic cancer therapy, regimens have been used for refractory or high risk gla and gsd but there is heterogeneity of diagnosis, and variability of drug regimens and assessment of effectiveness. objectives: . assess the variability of clinical features of gla and gsd . assess the heterogeneity of diagnosis . assess drug regimens and response assessment across multiple institutions design/method: we conducted a retrospective review from institutions of cases of gla and gsd treated with sirolimus and a bisphosphonate for at least months with assessment of clinical features, treatment protocols, response regimens and side effects. results: patients included gla (n = ) and gsd (n = ). the average age at diagnosis was years. clinical features included effusions: gla (n = ), soft tissue lymphatic malformations: gla (n = ), gsd (n = ), multiple splenic lesions: gla (n = ), and soft tissue swelling at the site of bony lesion: gsd (n = ). the presenting symptom in patients was pain with patients (gla) presenting with shortness of breath. fracture was noted in patients: gla ( ), gsd ( ). diagnostic and/or response imaging included mri, ct, bone scan, skeletal survey and dexa scan. treatment consisted of: initial sirolimus use with the addition of bisphosphonate secondary to worsening disease (n = ), initial therapy with other agents (interferon, chemotherapeutic agents, radiation) and change to sirolimus and bisphosphonate secondary to toxicity (n = ), sirolimus and bisphosphonates (n = ) and sirolimus, bisphosphonates and interferon (n = ). seventeen patients had stable disease and patients had improvement of disease. sirolimus protocol was standard; however, bisphosphonate protocol varied in dosing and frequency. side effects were tolerable and expected with no grade iii or iv toxicity. sirolimus and bisphosphonates are a safe and effective therapy for gsd and gla. a consistent medication regimen, redefined response and an improved radiologic classification will be important for the development of a prospective clinical trial. background: hemophilia a is a bleeding disorder from the deficiency of clotting factor viii. the most significant sequelae of hemophilia a is the tendency to develop hemarthrosis that incites joint destruction. the prevalence of overweight and obesity has been increasing in the general and hemophilia population and leads to several morbidities including arthropathy. this is a particular concern for hemophilia a as arthropathy is a consequence of joint bleeding. objectives: the purpose of this study was to detect the relation between body mass index (bmi) and joint health endpoints in a pediatric hemophilia population. design/method: participants in this study included patients from the hemostasis and thrombosis center at children's hospital los angeles. participants were pre-screened and approached for this study during routine follow-up appointments. patients aged - years old who have been diagnosed with hemophilia a, including mild, moderate, and severe, qualified for the study. informed consent was obtained from the patients or parents before enrollment. joint health was objectively measured by physical therapists from children's hospital los angeles using the hemophilia joint health score (hjhs). an hjhs total score is calculated by assessing: swelling, duration of swelling, muscle atrophy, crepitus on motion, flexion loss, extension loss, joint pain, and muscle strength in major joints. subjective data was also obtained by patients recording their annual bleed rate within the past year. of the patients, ( %) were normal weight, ( %) overweight, and ( %) obese. we used chi-square analysis to compare joint scores across bmi classifications (chi square = . , df = , p-value = . ). although, this did not approach statistical significance, the average hjhs score in patients who had a hjhs > shows an increasing trend among bmi classifications: . in normal bmi patients, . in overweight bmi patients, and . in obese bmi patients. the average number of annual bleeds in those with positive values show: in normal bmi patients, in overweight bmi patients, and in obese bmi patients. although a positive effect of adiposity was found in the joints of hemophilia a pediatric patients, the effect shows there was not enough evidence to conclude a difference. future studies are needed to address whether obesity has an effect on hemophilia and to determine whether overweight/obesity can lead to further complications in hemophilic joints. background: stagnant blood flow in slow-flow vascular malformations (vm), particularly in their venous components, can lead to localized intravascular coagulation (lic) that is characterized by elevated d-dimer levels, low fibrinogen and decreased platelet count this coagulation derangement can lead to localized thrombosis or bleeding which can result in pain, functional limitations, and possible progression to disseminated intravascular coagulopathy (dic). the treatment of vm and their associated coagulopathy has proven difficult. patients with complex vm are frequently managed with sirolimus, an mtor inhibitor, and have clinical benefits, including reduction of pain and improvement in functional impairment. it is possible that some of these improvements from sirolimus could be secondary to improvement in the coexisting lic. objectives: this study assessed the use of sirolimus to manage the coagulopathy seen in slow-flow vm. design/method: we reviewed charts of patients with vm who are followed in the vascular anomalies center at arkansas children's hospital and were started on sirolimus. efficacy was objectively assessed through improvement of ddimer, fibrinogen and platelet count. three sets of lab values (pre-sirolimus, - months post-sirolimus, and most recent) were obtained for each patient when available. we identified a total of patients who had been prescribed sirolimus. eighteen were excluded based on underlying condition other than slow-flow vascular malformation and for inadequate medical records. a total of patients ( combined vascular, venous) were included in the study. all had elevated d-dimer levels (mean . mcg/ml feu, median . mcg/ml feu, range ( . - . )) prior to treatment. two patients had an associated low fibrinogen (below mg/dl), indicating severe lic. with treatment, ( . %) patients showed an overall decrease in d-dimer levels with an average decrease of . mcg/ml feu between pre-and post-sirolimus labs, and an average decrease of . mcg/ml feu between pre-sirolimus and most recent values. the two patients with low fibrinogen prior to treatment showed a decrease in d-dimer levels (mean decrease of . mcg/ml feu) and an increase and normalization in fibrinogen (mean increase . mg/dl) after beginning sirolimus. no patient had thrombocytopenia. we report that treatment with sirolimus was effective in improving coagulopathy associated with slowflow vm as evidenced by decreased d-dimer levels and increased fibrinogen and/or platelets. long-term use of this medication in this population may decrease the bleeding and thrombotic complications that these patients experience, especially following invasive vascular procedures. background: safety and efficacy of bay - , a sitespecifically pegylated b-domain-deleted recombinant factor viii, in previously treated adolescents and adults aged - years with severe hemophilia a was demonstrated in the phase / protect viii study and ongoing extension. objectives: this subanalysis examines the efficacy and safety of bay - in adolescents in protect viii and the ongoing extension study (data cutoff, january ). design/method: in protect viii, patients (including adolescents) received bay - on demand or as prophylaxis for weeks. prophylaxis regimens for weeks - were twice-weekly ( - iu/kg), every- -days ( - iu/kg), or once-weekly ( iu/kg) infusions based on bleeding during a -week run-in period of iu/kg twice-weekly prophylaxis. patients continued their prophylaxis regimens in the extension or changed regimens at any time. results: twelve patients aged - years were included in the protect viii intent-to-treat population; s of s additional patient discontinued after dose (included in safety population). for patients receiving prophylaxis before study enrollment, median (range) number of total and joint bleeds in the months before study entry was . ( - ) and . ( - ), respectively. ten patients ( . %) had target joints at baseline (median [range], [ - ] per patient). during weeks - of protect viii for the entire time patients remained on their designated prophylaxis dosing frequency, the median (quartile [q] ; q ) annualized bleeding rate (abr) for patients receiving twice-weekly (n = ), every- -days (n = ), and once-weekly prophylaxis (n = ) was ( ; . ), . ( ; . ), and . ( ; . ), respectively (overall prophylaxis [n = ], . [ . ; . ]). two patients switched from once-weekly to twice-weekly (n = ) or every- -days prophylaxis (n = ), and number of bleeds decreased from to in one patient and to in the other. all patients from the main study continued in the extension; mean abr in the extension was . and varied by dosing regimen (twice weekly [n = ], . ; every days [n = ], . ; once weekly [n = ], . ). two patients changed from every- -days to once-weekly prophylaxis during extension (mean abr, . ). one patient had a nonneutralizing antibody to bay - at baseline; end-of-study titers were negative. no patient developed anti-peg antibodies or factor viii inhibitors or experienced a serious adverse event related to bay - during the main study or extension. in previously treated adolescents with severe hemophilia a, bay - prophylaxis was effective in prevention of bleeds, with less bleeding overall versus prestudy, and was generally well tolerated. funded by bayer. cincinnati children's hospital medical center, cincinnati, ohio, united states background: vascular malformations (vms) consist of a heterogeneous group of congenital disorders characterized by the abnormal development of blood and/or lymphatic vessels, which cause a broad spectrum of clinical manifestations. although considered benign, vms are frequently associated with cutaneous complications that can cause significant morbidity such as nodular overgrowth, skin thickening, pruritus, oozing or bleeding of lymphatic blebs and secondary infection. oral sirolimus has shown to be effective in the treatment of complicated vascular malformations but has known side effects and need for frequent laboratory monitoring. currently, there are limited studies on the use of topical sirolimus for the treatment of cutaneous manifestations of vascular malformations. objectives: to evaluate the efficacy and safety of topical sirolimus in vms with cutaneous complications and propose indications for use. design/method: this is a retrospective review of medical records of patients with vascular malformations treated with topical sirolimus from january to december . response was determined by subjective and objective improvement. results: twenty-four patients, ( %) females and ( %) males, with vascular malformations and cutaneous manifestations were treated with topical sirolimus. age ranged from - years. indications for treatment were: blebs ( %, n = ) causing either leaking, bleeding, pain, pruritus, swelling or recurrent infection; nodular overgrowth % (n = ); pyogenic granuloma % (n = ); bleeding % (n = ) and cosmetic % (n = ). treatment course ranged from - months. no major side effects were reported. one patient reported burning and itching sensation. regarding clinical response: % (n = ) patients had improvement in cutaneous lesions; % (n = ) had a stable lesions; and % (n = ) stopped treatment due to side effects. for prior/concomitant treatment: % (n = ) had prior surgery, laser or sclerotherapy; % (n = ) had concomitant oral sirolimus. of the patients not receiving concomitant systemic sirolimus, only % (n = / ) had been on oral sirolimus. of these patients, % (n = / ) had a very good response to topical treatment. : topical sirolimus appears to be beneficial and well-tolerated with a minimal side effect profile for the treatment of cutaneous manifestations of vascular malformations as a single agent or as adjuvant therapy with systemic sirolimus when symptoms are not adequately controlled. further studies are needed to prospectively analyze efficacy and safety of topical sirolimus in this patient population. objectives: to evaluate the safety and efficacy of long-term romiplostim in children with itp. design/method: all patients received weekly sc romiplostim from - g/kg to target platelet counts of - × ( )/l. median (min-max) treatment for the patients was ( - ) weeks for a total of patient-years, or . years per patient. at baseline, median (min-max) age was ( - ) years; % were female; . % had prior splenectomy. median (min-max) average weekly dose was . ( . - . ) g/kg, including escalation to a stable dose; patients started on g/kg. reasons for discontinuing romiplostim (n = , %) included consent withdrawn (n = ), required other therapy (n = ), and ae (n = ) (asthenia, headache, dehydration, and vomiting in one patient and anxiety in the other; none treatment related). fifty four serious aes occurred in patients but were treatment related in one (concurrent grade thrombocytopenia, grade epistaxis, and grade anemia). anti-romiplostim neutralizing antibodies were detected in one patient who discontinued to receive other therapy; antibodies were absent on retesting. from week on, median platelet counts remained > × ( )/l; median platelet counts were > × ( )/l from weeks - . nearly all ( %, / ) patients had ≥ platelet response (platelet counts ≥ × ( )/l, excluding ≤ weeks after rescue medication). most ( %, / ) patients had a platelet response ≥ % of the time and % ( / ) did ≥ % of the time. sixty ( %) patients (or caregivers) self-administered romiplostim. fifteen ( %) patients had treatment-free periods of platelet counts ≥ × ( )/l for ≥ weeks (ie, remission); these patients ( girls, boys) had had itp for a median (min-max) of . ( . - ) years, none had prior splenectomy, and had received romiplostim for . ( . - ) years. all had platelet counts > × ( )/l for ≥ months and / for ≥ months; the median (min-max) duration of being ≥ × ( )/l was ( - ) weeks. of baseline characteristics such as sex, platelet counts, itp duration, and number of past itp treatments ( , , , > ), only age < years was predictive of developing treatment-free periods ≥ weeks (p = . ). in this seven-year open-label extension, > % of children with itp achieved a platelet response and romiplostim was well tolerated. importantly, % of patients were able to discontinue all itp medications for ≥ months. funded by amgen inc. background: sirolimus is an immunosuppressive drug that is widely used in solid organ and bone marrow transplantation, and more recently for the treatment of vascular and lymphatic anomalies. sirolimus has been associated with decreased immunity in the transplant setting in patients that have received other immunosuppressive drugs or were immunosuppressed from previous chemotherapy. the effects of sirolimus on the immune system in chemotherapy naïve children who have not received other immunosuppressive agents are not well understood, and there is variability in the approach to fever and pcp prophylaxis. to understand the effects of sirolimus on the immune system of patients with non-complicated vascular or lymphatic anomalies by evaluating anc, alc prior to and after sirolimus therapy. design/method: multi-institutional retrospective review was done to include patients with non-complicated vascular or lymphatic anomalies. those with effusions/ascites, multiorgan involvement, or history of vascular-anomaly-related infections prior to treatment were excluded. results: twenty patients with kaposiform hemangioendothelioma (n = ), generalized lymphatic anomaly (n = ), cloves syndrome ( ), and simple vascular malformation (n = ) were included. age at initiation of sirolimus treatment ranged from . - years. male to female ratio was : . sirolimus was initiated due to extensive disease, lack of response to steroids or bisphosphonates, pain, dment, lymphatic drainage, and prevention of ongoing overgrowth. prior to the start of sirolimus (sir- ) the mean anc was and alc was . the target level of sirolimus varied by indication and patient, and ranged from to . after the st steady state level, month after sirolimus (sir- ) the mean anc decreased to and alc was . at months after sirolimus (sir- ) the mean anc was and alc was . the first sirolimus levels (sir- ) mean was . ; and sir- level was . . nine patients were placed on pcp prophylaxis at the start of sirolimus. none of these patients had an infectious complication while on sirolimus at a median f/u of months. one patient had mild neutropenia (anc > ) which normalized after discontinuation of pjp prophylaxis. conclusion: in this small cohort of patients we found that the anc and alc level in patients with non-complicated vascular or lymphatic anomalies at sir- was not different from the sir- or sir- . prospective studies that specifically track anc, alc, igg, and lymphocyte function should be conducted to better understand the effects of sirolimus in the immune system. this data will allow for uniform recommendations regarding prophylaxis and management of febrile episodes. background: acute infections and the associated systemic inflammation can increase the risk of venous thromboembolism (vte) and in certain well-defined clinical scenarios may be the primary trigger of vte in pediatric patients. pediatric data on vte in the setting of acute infection are sparse. objectives: to describe characteristics and outcomes of vte in pediatric patients with acute infections. we conducted a retrospective chart review of all pediatric patients with objectively confirmed vte treated at our institution since and identified all patients in whom an acute infection was identified as a vte trigger. patients were managed according to standardized institutional protocols based on published guidelines. relevant demographic, clinical and laboratory data were collected and summarized using descriptive statistics. since , acute infection was identified as a trigger in of vtes ( %) diagnosed at our center. the median age at time of vte diagnosis in this group was . years (interquartile range . - ). males were more commonly affected than females, representing % of cases. neonatal vte events accounted for % of cases. sepsis was the most common acute infection to be identified as a vte trigger [ / cases ( %)]. most vte events ( %) associated with acute infections were considered hospital-associated vtes. at time of vte diagnosis, % of patients were critically ill. extensive vte (defined as completely occlusive thrombosis involving > venous segment) occurred in % of patients. acute infection was deemed to be the primary trigger for vte in / patients ( %). infection-associated vtes in this cohort included cerebral sinus venous thrombosis due to sinus or cns infection ( patients, %), septic throm-bophlebitis ( patients, %), lemierre's or lemierre's-like syndrome ( patients, %) and osteomyelitis-associated deep vein thrombosis ( patients, %). systemic anticoagulation was prescribed in / patients ( %). anticoagulationrelated major bleeding occurred in / patients ( %). vte complications included vte recurrence ( patients, %), vte progression ( patient), acute pulmonary embolism ( patients) and arterial ischemic stroke ( patients). our study indicates that acute infection is a common risk factor for pediatric vte, especially in critically ill children, and can be the primary trigger in a significant proportion of vte cases associated with acute infections. anticoagulation appeared to be overall safe in this population and was associated with low rates of serious vte-related acute complications. however, our study also suggests that this population may be at increased risk for vte recurrence and anticoagulation-related major bleeding. background: epithelioid hemangiomas (eh) are rare benign vascular tumors that occur in soft tissues and bone and present between the third and sixth decades of life. a subset ( %) of eh harbor fos rearrangement. eh has been described in children, but little is known about the long-term outcomes of pediatric eh. the main objective is to obtain data to be used for improved understanding of this rare disease in order to provide standardization of care and development of future research studies. board-approved retrospective review of clinical, pathologic, and radiographic characteristics, and treatment outcomes in patients diagnosed with eh between and . results: eight patients were male; mean age at diagnosis was . years (range: - ). lesions involved the lower extremities (n = ), cranium (n = ), pelvis (n = ), and spine (n = ). multifocal disease was identified in five patients. the most common presentations involved significant localized pain and neurologic symptoms: headache, cranial nerve injury, loss of consciousness. radiographic studies identified variable features, such as multifocal lytic bony lesions with sclerotic margins, enhancing soft tissue component, and surrounding inflammatory edema. histologically, all specimens were composed of vascular channels lined by epithelioid endothelial cells without significant cytologic atypia; solid cellular areas (n = ). endothelial cells were positive for cd and egr, and negative for camta . fos rearrangement was assessed in only one specimen and detected. mean follow-up time was days (range: - ). patients were treated with surgical resection, intravascular embolization, bisphosphonates, propranolol, interferon, and sirolimus. one patient treated with interferon and one with sirolimus exhibited partial response for mean follow-up of . days. although eh is a benign neoplasm, it is difficult to manage without standard protocols and portends considerable morbidity. our findings suggest medical management, particularly sirolimus, may benefit these patients; however, long-term follow-up is needed in treated children. novel fos inhibitors are in development and may benefit patients with fos rearrangement. penn state health children's hospital, hershey, pennsylvania, united states background: central venous catheters (cvc) are often required in critical care settings in order to provide a secure point of access for life sustaining care. clinical studies identify cvc presence as the single most important risk factor for deep vein thrombosis (dvt) in children. venous thromboembolic event (vte) incidence rates in critically ill children with a cvc range from . - % and . - . per catheter days depending on the population studied. per institutional protocol, the penn state health children's hospital picu (hershey, pa) utilizes a low dose continuous infusion of unfractionated heparin (ldufh) at units/kg/hr as prophylaxis against cvc-related vte and to maintain line patency. the efficacy of this approach has never been evaluated. to determine if ldufh for prophylaxis results in lower incidence of cvc-related vte, catheter dysfunction and central line associated blood stream infection (clabsi) without increasing morbidities. to determine if the incidence of catheter related vte is lower than historical published data, a retrospective chart review was conducted utilizing the institutional electronic medical record for all patients in , aged - . years, who had a cvc during a picu admission. secondary objectives such as the incidence of catheter dysfunction, clabsi, and any associated bleeding complications are also being analyzed. results: interim data analysis revealed cvcs ( nontunneled cvc, totally implantable devices, tunneled lines, peripherally inserted central catheters [picc] ) in total patients with a median age of . years. overall vte incidence was . % ( / ) with vtes associated with non-tunneled cvc and with piccs. sixty one percent of non-tunneled cvcs received ldufh and % ( / ) of the patients with vtes associated with non-tunneled cvcs did receive ldufh prophylaxis. vte incidence rate of nontunneled cvcs with ldufh was . % ( / ) and . per picu catheter days. the only other vte events identified within our study cohort were in the picc group where two patients experienced vte, one of which was receiving ldufh. clabsi incidence was . % ( non-tunneled cvc, tunnel cvc, picc). no major bleeding complications were associated with ldufh. preliminary data demonstrates ldufh is efficacious in preventing cvc-related vte in comparison to published reports. further analysis will compare another similar sized and acuity level picu which does not practice the same method. background: fibroadipose vascular anomaly (fava) is a rare, challenging disorder associated with pik ca mutations. fava often causes painful replacement of muscle and soft tissues with fibrotic and adipose tissue and is associated with ectatic draining veins. treatments for focal lesions are surgical excision, cryoablation or sclerotherapy and the role of medical therapy is unclear. some fava lesions are too extensive or directly involve neurovascular structure, resulting in refractory pain. objectives: to retrospectively evaluate the efficacy of sirolimus in patient with residual symptoms after procedural therapies for fava design/method: retrospective review of individual cases from institutions of fava refractory to other therapies treated with sirolimus for at least months. cases were s of s identified by polling member of the aspho vascular anomalies special interest group. results: all seven patients report improvement on sirolimus therapy. all patients had received prior procedures, including sclerotherapy ( patients), cryoablation ( patients) and/or resection ( patients). mean age at sirolimus initiation was y (range - y). mean length of therapy is . months (range - months). six patients were treated with bid dosing and one adult received daily dosing. goals of sirolimus were improvement in pain or musculoskeletal dysfunction. pain and function improved in all patients, including discontinuation of narcotic use and resumption of participation in sports. time to symptom improvement ranged from - weeks. in four patients for whom dose was lowered, pain recurred in all four and responded to restarting or increasing sirolimus dose. while all patients do not have pre-and postsirolimus imaging, decrease in fava lesion size is seen in cases with available imaging. sirolimus side effects are similar to prior reports, most commonly mouth sores, elevated lipids and acne. we report the first known data supporting a role of sirolimus in refractory fava cases. sirolimus is welltolerated and initial improvement is rapid, within weeks of initiation. whether sirolimus has a role in upfront therapy to reduce lesion size prior to procedures deserves further study. objectives: to assess platelet responses in children with itp receiving romiplostim. design/method: eligible children had itp for ≥ months, ≥ prior therapy, and screening platelet counts ≤ × ( )/l or uncontrolled bleeding. weekly dosing was from - g/kg to target platelet counts of - × ( )/l. bone marrow biopsies were evaluated in europe at baseline and after or years (cohorts and ). as of mar , patients received ≥ dose. at baseline, median (min-max) age was ( - ) years, itp duration was . ( . - . ) years, and platelet count was ( - ) × ( )/l; patients ( %) had had prior splenectomy. the median (q , q ) % time with a platelet response (platelet count ≥ × ( )/l, no rescue medications past weeks) in months - was % ( %, %) (primary endpoint). over the course of the study, % ( / ) of patients had a platelet response. four patients maintained platelet counts ≥ × ( )/l with no itp medications for ≥ weeks. median (min-max) treatment duration was ( - ) weeks for patient-years in total. median (min-max) average weekly romiplostim dose over the course of the study was . ( . - . ) g/kg; the median dose was g/kg at year (n = ) and g/kg at years (n = ). most ( %) patients initiated self-administration. sixty-four patients ( %) discontinued treatment, most frequently for lack of efficacy (n = ), patient request (n = ), and adverse event (ae) (n = ). fortyone ( %) patients had serious aes (saes) including epistaxis ( %) and decreased platelet count ( %). five patients had treatment-related saes: headaches, abdominal pain, and each of presyncope and neutralizing antibodies (ab). there were cases of neutralizing ab to romiplostim (of patients tested), but none to tpo; / had continued elevated platelet counts and in / cases ab were not found on retesting. for cohort , of patients with baseline bone marrow biopsies, had evaluable on-study biopsies scheduled for year; patient had an increase from grade to . there were no findings of collagen or abnormalities. in this interim datacut of a romiplostim openlabel study in children with itp, % of children had a platelet response. overall, the median dose was . g/kg; the median romiplostim dose over time reached g/kg. no new safety signals were observed over patient-years. funded by amgen inc. background: hepatic hemangiomas are benign vascular tumors without a medical home, managed by multiple specialties. the diagnosis has been assigned historically to various vascular lesions affecting the liver with completely different clinical presentations, resulting in difficult standardized management. objectives: the consensus steering committee identified an acute need of clear definitions and evaluation guidelines using the updated international society for the study of vascular anomalies (issva) classification. the goal was to formulate recommendations that will be adopted by all specialties involved in the care of children with hepatic hemangiomas. design/method: we used a rigorous, transparent consensus protocol, with input from multiple pediatric experts in vascular anomalies from hematology-oncology, surgery, pathology, radiology and gastroenterology. in the first section, we precisely define the subtypes of hepatic hemangiomas seen in children (congenital and infantile) using clinical course, histology and radiologic characteristics. inclusion and exclusion limits to the diagnosis are noted. the following two sections describe these subtypes in further detail, including complications to be considered during monitoring and respectively recommended screening evaluations. conclusion: while institutional variations may exist for specific clinical details, a clear understanding of the diagnosis of hepatic hemangiomas affecting the pediatric population and the possible complications that require screening during the monitoring period should be standard. as patients with hepatic hemangiomas are managed by different medical and surgical specialties, a multidisciplinary consensus based on current literature, on the data extracted from the liver hemangioma registry and on expert opinion was required and was accomplished by this manuscript. objectives: to investigate the association between routine prophylaxis with bay - and bleeding outcomes after adjusting for key patient and pharmacokinetic (pk) characteristics. design/method: the leopold kids study evaluated safety and efficacy of bay - prophylaxis in previously treated boys aged ≤ years with severe hemophilia a. patients received bay - - iu/kg x/wk (n = ) or > x/wk (n = ) and were followed up for - months. prophylaxis dose and frequency were assigned by investigators. pk parameters, including area under the curve (auc), half-life, and clearance, were derived from a population pk model and reflect predicted pk values with a -iu/kg dose. patient characteristics were compared between the x/wk and > x/wk groups using wilcoxon rank sum or chi-square tests. negative binomial regression was used to model the association between prophylaxis frequency and annualized bleeding rate (abr) for total bleeds, first without adjustment and then adjusting for age, pk parameters, and bleed history. results: mean ± sd age for patients in this analysis was . ± . years. patients receiving prophylaxis x/wk had more bleeding episodes in the months before study entry (mean ± sd, . ± . [median, . ] for x/wk vs . ± . [ . ] for > x/wk; p = . ) and were more likely to have been treated on demand ( % vs %; p = . ). pk parameters were similar between the x/wk and > x/wk groups. without adjustments, abr during the study was % higher in the x/wk group compared with the > x/wk group (rate ratio [rr], . ; % ci, . - . ; p = . ). abr was % lower in the x/wk group (rr, . ; % ci, . - . ; p = . ) after adjusting for age, auc, and number of bleeds in the prior months. conclusion: abr was numerically lower but not significantly different between the x/wk and > x/wk groups after adjusting for age and pk parameters. these findings suggest that even among patient groups that are homogeneous with respect to age, pk, and bleed history, further individualization of bay - prophylaxis based on other characteristics may help reduce bleeding episodes even at a lower treatment frequency. larger real-world studies are needed to verify these findings. funded by bayer. stanford, palo alto, california, united states s of s background: vascular malformations may be of lymphatic, arterial, venous or capillary endothelial origin. they may be simple or complex, with complex malformations being a combination soft tissue and skeletal overgrowth. although likely present at birth, these malformations often become symptomatic with puberty or infection, and range from little or no clinical impact to life threatening symptoms. in malformations primarily of venous origin, pain may be significant and hypothesized to be caused by phlebolith development (intra-malformation thrombi), inflammation, consumptive coagulopathy, vascular engorgement, and endothelial proliferation. anti-angiogenic and anti-platelet therapies have been reported to relieve pain. however, the use of anticoagulation for pain is not well described. objectives: to report clinical features and outcomes of patients with vascular malformations of venous origin treated with anticoagulation for pain. we performed a retrospective review of patients with vascular malformations followed by the hematology service between january and december who were treated for pain with anticoagulation. pain relief was determined both by wong-baker pain scales and patient report. clinical data were extracted from electronic medical records. we identified five patients with venous malformations (vm) who had received anticoagulation for pain. four patients were female and median age was years old (range to years old) at time of initiation of anticoagulation. all five patients had vm of the extremity, two with vm of the lower extremity, and three patients had vm of the upper extremity. two patients had concomitant coagulopathy and demonstrated decreased d-dimer after initiation of anticoagulation. four patients received enoxaparin, and one adult patient received rivaroxaban. all patients reported improvement in pain after administration of anticoagulation. one patient exhibited mild epistaxis and bruising at the injection site. there was no significant bleeding or other complications. pain is a significant complication in patients with venous malformations. our case series suggests that anticoagulation is a safe and effective therapy for pain relief in this population. further investigation is indicated to compare the effect of anticoagulation to other therapeutic interventions such sclerotherapy, surgery, and sirolimus in the treatment of pain associated with venous malformation. maria ahmad-nabi, christine knoll, sanjay shah, lucia mirea phoenix children's hospital, phoenix, arizona, united states background: estimates of the incidence of dvt in patients with osteomyelitis range widely from %- %, however risk factors and outcomes of dvt in this cohort have not been thoroughly established. objectives: this study aims to estimate the incidence of dvt in patients with osteomyelitis, and to assess risk factors and outcomes of dvt in this cohort. design/method: after irb approval, a retrospective chart review was conducted for patients aged - years seen at phoenix children's hospital between - with icd / codes for osteomyelitis. exclusion criteria included chronic recurrent multifocal osteomyelitis, and chronic dvt. demographics, clinical factors and outcomes were compared between osteomyelitis patients with and without dvt using the fisher-exact and wilcoxon-rank sum tests, as appropriate for the data distribution. results: a total of study subjects with osteomyelitis had a mean (standard deviation) age of . ( . ) years. dvt was present in ( % of ) patients, and ( %), ( %) and ( %) patients received anticoagulation for < , - and ≥ weeks, respectively. patients with vs without dvt were more likely to be male ( % vs %; p-value = . ), and had significantly higher rates of bacteremia ( % vs %; p-value = . ). rates of central lines were comparable between dvt and non-dvt patients ( % vs %; p-value = . ); however patients with dvt vs without dvt had significantly longer mean length of stay ( vs days; p-value < . ) and higher rates of icu admission ( % vs %; p-value < . ). the incidence of dvt among osteomyelitis pediatric patients was estimated at %, with risk increased by male sex and bacteremia. patients with dvt had significantly higher rates of icu admission and longer length of hospital stay. many of these patients had standard practice management of their dvt with - weeks of anticoagulation. our data highlights the need for recognition of high risk patients, and the need for future efforts targeting dvt prophylaxis. baylor college of medicine, houston, texas, united states background: lymphatic malformations (lm) frequently occur in the head and neck and can often be disfiguring and even life-threatening. management options include observation, surgery, sclerotherapy, and sirolimus. the optimal sequence of therapeutic interventions has not been determined due to the lack of comparative clinical trials or established guidelines. thus, prenatal planning with a multidisciplinary team is beneficial. we present a case series of ten children with head and neck lms evaluated in at our multidisciplinary vascular anomalies center. a chart review was performed to assess treatment modalities and recent trends. results: seven of patients ( %) with head and neck lms were diagnosed prenatally. six patients required an ex utero intrapartum treatment procedure. all patients were started on sirolimus at a median age of . months (range days - years). four patients most recently started on sirolimus were less than months of age at the time of initiation. six patients underwent partial excision of lm during the first year of life; none of whom received sirolimus prior to surgery. sirolimus was discontinued in one patient given chronic clostridium difficile infections, and non-compliance in another patient. five patients received sclerotherapy. tracheostomy was necessary in six patients; one patient was de-cannulated after months on sirolimus. all patients have had radiographic and clinical improvement of lm with varying treatment modalities. current clinical observations show improved response with sirolimus and demonstrate tolerability of sirolimus at a young age. conclusion: treatment of pediatric head and neck lms is challenging and a multidisciplinary approach is necessary. as the majority of patients are diagnosed prenatally, prenatal planning and discussion of potential use of sirolimus is beneficial. availability of vascular anomalies experts in the prenatal/neonatal period offers the best management results, and early initiation of sirolimus should be considered for complex lesions. long-term follow up is warranted to investigate the efficacy and timing of treatment options. yale school of medicine, new haven, connecticut, united states background: to mitigate transfusion of pathogencontaminated platelets, amotosalen, a synthetic psoralen compound, is added to sdp components. exposure to uv-a light activates amotosalen and crosslinks dna/rna base pairs, preventing replication of a broad spectrum of viral, bacterial, and other pathogens that may contaminate platelets. pr-sdps were fda approved for clinical use with no age restrictions in . we initiated use of pr-sdps in november of for all patients. we retrospectively analyzed usage of pr-sdp vs conventional (non-pr) platelets (cp) in neonatal and pediatric patients with thrombocytopenia to compare hemostatic efficacy and the incidence of transfusion reactions (tr) for these products, after one year of a dual platelet inventory. design/method: since pr-sdp were fda-licensed, no irb approval was required; pr-sdp and cp were both considered standard of care. we evaluated transfusions for all pediatric patients age - years who received any platelet transfusion between november and november . we determined the volume (mean ml ± sd) of each type of platelet component transfused, the number of platelet transfusion episodes, and reported trs based on cdc hemovigilance guidelines. a subgroup analysis was performed for thrombocytopenic neonates ( - months). results: patients - years who received only cps (n = ) received a total of , ml of platelets ( ± ml/patient) over transfusions ( . ± . episodes/patient). for comparison, in patients who received only pr-sdp, a total of , ml of platelets ( ± ml/patient, p = . ) were infused over transfusions ( . ± . episodes/patient, p = . ). for neonates ( - months, n = ) who received only cps, , ml of cps ( ± ml/ patient) were transfused over episodes ( . ± . episodes/patient). for comparison, those who received only pr-sdp (n = ), received , ml of pr-sdp ( ± ml/patient, p = . ), transfused over episodes ( . ± . episodes/patient, p = . ). for all recipients - years (n = ), including additional patients who received both cp and pr-sdp, there were three reported allergic trs over transfusion episodes, while no allergic reactions were reported with pr-sdp transfusions. one febrile tr was reported to cp transfusion, while three were reported for pr-sdp. in conclusion, pr-sdps, in our pediatric population age - years, were comparable to cp products in regards to volume and episodes of platelet transfusions, and incidence/type of transfusion reactions. pr-sdp were safe and effective for use in this pediatric patient population. background: vascular anomalies are classified as either vascular tumors or vascular malformations. fibro-adipose vascular anomaly (fava) is a newly described entity which presents with distinct clinical, radiographic and histopathologic findings. we present a case in which the diagnosis of fava was complicated by a persistent low platelet count secondary to immune thrombocytopenia (itp). to describe a challenging diagnosis of a novel vascular anomaly (fava) complicated by severe thrombocytopenia. a year old male presented to hospital with bruising and left thigh pain related to a remote sports injury. blood work revealed a platelet count of × /l, but with an otherwise normal complete blood count. the following were also normal: aptt and fibrinogen; d dimer levels were slightly increased. he was treated with one dose of ivig ( . mg/kg) for presumed itp and responded well with his platelet count increasing to × /l. he returned to hospital weeks later with recurrent thrombocytopenia and worsening leg pain. an ultrasound of the left thigh revealed a . cm x . cm x . cm lesion within the vastus medialis. the diagnosis of an intramuscular hematoma secondary to persistent thrombocytopenia was made. the patient presented with multiple episodes of thrombocytopenia over the next several months. his itp did not respond to oral prednisone ( mg/day for days). he continued to have short-lived responses to ivig requiring infusions every other week as his platelet count would fall below × /l. his leg pain progressed, restricting him to a wheelchair. further imaging by mri brought into question the diagnosis of a hematoma and a biopsy of the thigh lesion was performed. the results were consistent with a diagnosis of fava; this was subsequently excised. conclusion: this is a unique case where a vascular anomaly was misdiagnosed as a hematoma due to a patient's persistent thrombocytopenia and history of an injury. fava is a newer entity which, unlike other vascular anomalies, has not been linked to thrombocytopenia or a localized consumptive coagulopathy. after excision of the fava, the patient's chronic pain, and mobility resolved, though his itp persisted. objectives: this preliminary, exploratory analysis of realworld administrative data was conducted to determine units dispensed and factor replacement product-related direct expenditures associated with a currently marketed shl or ehl rfix product. design/method: de-identified claims data from the commercially available truven health marketscan® research u.s. claims database were used to identify direct expenditures and number of international units (ius) dispensed for all patients aged - years with a diagnosis code of icd- . /icd- d who used nonacog alfa or eftrenonacog alfa during the study period (june , to july , ). reference weight measurements from the centers for disease control and prevention national center for health statistics' (cdc nchs) anthropometric data were used to estimate product dispensation on an iu per kg basis. the nonacog alfa and eftrenonacog groups comprised and patients, respectively. the median [iqr] age in the two groups was . [ . ] and . [ . ] years, respectively. while of the patients in the eftrenonacog alfa group had > calendar quarter of available data, only of the patients in the nonacog alfa group had > available quarter. the median rfix product dispensation per quarter was , ius (iqr, , ius) in the nonacog alfa group and , ius (iqr, , ius) in the eftrenonacog alfa group. incorporating attributed weight values, the median rfix product iu dispensation per kg per week was . iu/kg/wk (iqr, . iu/kg/wk- . iu/kg/wk) in the nonacog alfa group, and . iu/kg/wk (iqr, . - . iu/kg/wk) in the eftrenonacog alfa group. applying wac prices (eftrenonacog alfa = $ . /iu; nonacog alfa = $ . /iu), the calculated estimates of $/kg/week were $ and $ in the nonacog alfa and eftranonacog alfa groups, respectively. conclusion: preliminary real-world data derived from a large u.s. claims database revealed differences in product dispensation and factor product-related expenditures among pediatric patients with any severity of hemophilia b to whom an shl or ehl rfix product was prescribed. refinements of these data, potentially to exclude instances of sporadic usage, may shed light on real-world dispensation of rfix products among pediatric hemophilia b patients. background: vascular malformations can be classified as simple (including capillary, venous, lymphatic, arteriovenous), combined, malformations of major named vessels or associated with other anomalies. multiple modalities including laser treatments, sclerotherapy, embolization, surgery and pharmacological intervention (with mtor inhibitors like sirolimus) have been used for treatment of vascular malformations. these interventions have been used alone or in combination with varied outcomes. we present our institution's experience with a multimodal approach to simple and combined vascular malformations. design/method: we performed a retrospective chart review of patients with vascular malformations who were referred to our center for an interventional radiology evaluation from june -july . we included patients (age at presentation: months - years), referred initially for interventional radiology procedures (irp) for vascular malformations. all patients had symptoms of pain and/or swelling/deformity. diagnosis of was based on vascular imaging (doppler ultrasound, mri/a/v). nine patients had venous malformations (vm), five had macrocystic lymphatic malformations (lm), six had lymphatic-venous malformations (lvm), and two arteriovenous malformations (avm). patients initially underwent interventional radiology procedures. all the vm patients responded to sclerotherapy alone. three patients with lm responded to sclerotherapy alone, remainder required surgical intervention. one avm patient responded well to embolization, the other needed surgical resection after embolization. four lvm patients underwent irp with minimal improvement in symptoms ( - procedures attempted), surgical resection was attempted in patients with poor response and patients were started on sirolimus ( . mg/m /dose twice a day). all lvm patients started on sirolimus have responded well (decreased pain and swelling); time to initial symptom response ranged from weeks - month from starting medication. in this case series, patients with simple vm responded well to sclerotherapy alone, avm and lm patients needed irp and/or surgery for complete response. complex lvm did not respond well to surgery or irp; . % had improvement in clinical symptoms with addition of sirolimus to the treatment regimen. response to various modalities of treatment varied based on the type of vascular malformation. a multidisciplinary approach to management of vascular malformations is essential to provide multimodal therapeutic options for rapid symptom relief and improve the quality of life of these fragile patients, especially those with complex malformations. background: von willebrand disease (vwd) is the most common bleeding disorder in humans, affecting ∼ % of the united states' population. desmopressin (ddavp) is a longacting vasopressin analog that induces vasoconstriction and release of vwf. ddavp is used in patients with vwd and as a surgical prophylaxis, but carries anti-diuretic properties. to avoid electrolyte imbalance and hyponatremia, fluid restrictions are recommended in the hours post-ddavp administration. objectives: this study sought to examine perioperative practices and outcomes following ddavp administration and a fluid restriction protocol in a population of pediatric patients with von willebrand disease. design/method: a retrospective chart review was conducted for patients with von willebrand disease who underwent surgical procedures at children's hospital of pittsburgh of upmc between january , and december , . patient age, sex, weight, diagnosis, surgical procedure, total fluids administered, and post-operative sodium level were recorded. the primary outcomes noted were the proportion of patients exceeding % of the recommended fluid consumption for the -and -hour periods post-ddavp s of s administration, as defined by local guidelines. secondary outcomes were the presence of any bleeding requiring an er visit or readmission or hyponatremic seizures within hours of ddavp administration. results: data was compiled for patients ( females, males). the mean age was . years (sd . years), median age was years (range to years). procedures included dental ( ), otolaryngology ( ), orthopedics ( ), gastrointestinal ( ), plastics ( ), neurosurgery ( ), ophthalmology ( ), dermatology ( ), general surgery ( ) and gynecology ( ). % of patients exceeded % of the fluid volume recommended for the first -hour period post-ddavp administration while still in the surgical setting. no patients exceeded % of the fluid volume recommended for the total -hour period post-ddavp administration. post-operative sodium levels were obtained in only of patients. no patients returned to the er or were admitted for bleeding in the hours post-ddavp administration. no patients returned to the er or were admitted for hyponatremia or seizures in the hours post-ddavp administration. maintenance of a fluid restriction protocol effectively deterred negative outcomes in this cohort. however, a significant fluid volume was administered in nearly a third of patients despite the restrictions. given the risk of hyponatremia, and limited compliance with fluid restrictions, postoperative sodium levels should be recorded in following ddavp administration to assess the possibility of a hyponatremia and to reinforce the importance of fluid restrictions and their communication. results: a male fetus required in utero insertion of a pleuroamniotic shunt for bilateral pleural effusions diagnosed antenatally by ultrasound. shortly after delivery at term, he developed respiratory distress and was found to have reaccumulation of the pleural effusions. blood work on day of life showed a platelet count of , / l, which then decreased precipitously. he demonstrated schistocytes on blood-smear, signs of consumptive coagulopathy with hypofibrinogenemia and high d-dimers, and compensatory reticulocytosis. he required multiple transfusions and admissions to the intensive care unit for respiratory support. investigations ruled out congenital ttp, neonatal alloimmune thrombocytopenia, and noonan syndrome. given high clinical suspicion for an underlying vascular lesion causing kmp, a full body mri without contrast was undertaken. this showed a focal area of suspicious signal intensity in the upper paraspinal musculature. an ultrasound and mri with contrast demonstrated an extensive infiltrative vascular lesion involving the paraspinal musculature, prevertebral space, posterior extrapleural space, mediastinum, and neck. the child was commenced on prednisone ( mg/kg/day) and rapamycin ( . mg/m twice/day). there was no clinical or laboratory improvement after one month. a biopsy was performed which confirmed khe. in the second month of rapamycin therapy, the platelet count gradually normalized and the patient was discharged from hospital at . -months of life. prednisone was weaned off at . months of life. a repeat mri at months showed significant reduction in the khe. he is now almost years into therapy and doing well. conclusion: this is a unique case of khe with kmp that initially presented with extensive and recurrent pleural and pericardial effusions. this case demonstrates the importance of suspecting an underlying vascular malformation in the presence of kmp. our patient had a delayed but overall good response to rapamycin. further studies investigating duration of rapamycin therapy is key for the optimal management of these patients. rosa diaz, donald mahoney, lakshmi srivaths, donald yee texas children's hospital, houston, texas, united states background: since von willebrand disease (vwd) is the most common inherited bleeding disorder, it must co-exist with other less common bleeding disorders in some dually affected patients. however, reports of combined deficiencies in factor viii (fviii) and von willebrand factor (vwf) are rare. objectives: to study the prevalence and bleeding phenotype of combined deficiencies of fviii and vwf in males with hemophilia a in a hemophilia treatment center. design/method: we retrospectively reviewed the electronic medical records of males with hemophilia a followed at our institution during the past years. the primary and secondary outcomes for the study were ( ) the prevalence of combined fviii and vwf deficiencies and ( ) the bleeding phenotype of these patients. we identified vwf deficiencies in % (n = ) of the patients with hemophilia a. most (n = , %) patients were tested for vwf deficiency as part of the initial hemostatic evaluation, but one-third were tested due to clinical concern for inadequate response to fviii concentrate. the median duration of follow up was . years (range . to . ). patients were referred to our clinic at a median age of months (range to years) for evaluation of easy bruising (n = , %), mucosal (n = , %) and surgical bleeding (n = , %). primary diagnoses included with severe, moderate and mild discrepant hemophilia a. secondary diagnoses included with low vwf activity, type vwd and with type unclassified. patients experienced episodes of musculoskeletal (n = , %), mucocutaneous (n = , %) and cns bleeding (n = , %). a total of patients received factor prophylaxis. half of the patients were initially treated with fviii concentrates but subsequently changed to combined fviii/vwf products due to the frequency of breakthrough bleeding despite good compliance. all patients are on combined fviii/vwf products at the time of this review. a total of ( %) of this cohort developed chronic joint disease manifest as decreased range of motion and/or abnormal mri findings. combined deficiencies of fviii and vwf were present in % of our center's hemophilia patients. these patients exhibited a severe bleeding phenotype as evidenced by the high frequency of hemarthrosis, need for prophylaxis and high prevalence of chronic joint disease. while the optimal treatment strategy remains to be elucidated, early recognition of a combined deficiency may have important clinical implications, particularly in patients who demonstrate a suboptimal response to fviii concentrate alone. background: childhood neutropenia is heterogeneous and may be congenital or acquired. cerebral cavernous malformation (ccm ) is a neurovascular malformation disorder where lesions consist of low flow, dilated capillary endothelial channels with increased permeability, predisposing to hemorrhage and thrombosis. programmed cell death protein (pdcd ) activity has been implicated in glia and neuron migration, and recently linked to the dysregulation of the actin and microtubule cytoskeleton, thereby affecting cellular morphology and migration. variants of pdcd encoding pdcd have been associated with ccm . ccm causes a greater and earlier disease burden than other ccms, with % presenting younger than years. some patients have associated extra-neuronal manifestations, suggesting that pdcd plays a role in other tissues. we describe a patient with significant blood cytopenias associated with ccm . design/method: retrospective chart review to obtain patient data. results: an -month old female presented with seizure and was found to have multiple intracranial cystic lesions and abscesses due to s. pneumonia serotype f. during her treatment, she developed anemia (hemoglobin . - . g/dl), thrombocytopenia (platelets , - , cells/l), and profound neutropenia (absolute neutrophil counts of zero). initial bone marrow evaluation revealed a normocellular marrow but with marked granulocytic hypoplasia and % hematogones on flow cytometry. florescent in situ hybridization excluded cytogenetic changes characteristic of myelodysplastic syndrome. further evaluation included testing for neutrophil antibodies, chromosome breakage, and telomere length and results were normal. whole exome sequencing excluded mutations affecting congenital neutropenia genes, but detected a de novo pdcd variant (c. + g>a), thereby diagnosing ccm . the neutropenia has responded well to granulocyte colony stimulation factor (gcsf), which is still needed at months of age. moreover, the thrombocytopenia has progressed, requiring periodic platelet transfusions. over time, the bone marrow hematogone population has decreased to % at months of age, though the granulocytic hypoplasia persists. conclusion: our case describes the first patient with neutropenia and thrombocytopenia associated with ccm . we hypothesize the pdcd variant is the etiology of bone marrow dysfunction due to its role in actin and microtubule cytoskeleton formation, akin to the pathophysiology of xlinked neutropenia. supportive features of an underlying genetic cause of marrow dysfunction include the persistence of cytopenias beyond infection resolution as well as presence of hematogones. hematogones were previously reported to occur in patients with other congenital neutropenia disorders, indicating they could be a feature of congenital neutropenia and may be reactive to surrounding cell apoptosis. further testing of pdcd role in hematopoiesis should be explored. background: - % of adult women will suffer from heavy menstrual bleeding (hmb) during their lifetime. % of women with inherited bleeding disorders suffer from hmb. there is a paucity of data about hmb among adolescents and young adults (aya), a population in which hmb may have large social and educational effects. objectives: to study the social and academic implications of hmb in an aya population. design/method: this is a questionnaire based survey conducted in a medium-sized city in california. we recruited females - years of age from one high school and from local university. the questionnaire was set up in research electronic data capture (redcap) at our institute which allowed us to obtain objective data about the respondents' menstrual cycles. a link was sent to the high school students via their online portal schoolloop and to the university students via social media and word of mouth. data was collected over weeks from may to august . we received replies, some were not complete. using regression analysis, data was analyzed from respondents in the age group of - (with a mean age of ) years. we developed a composite score for hmb based on factors including saturation levels, number of pads, duration of bleeding, soaking of a pad within two hours, passage of clots, size and number of clots, and gushing sensation. we conducted statistical analysis of the drivers and implications of hmb based on the composite score. results indicate that having a relative with hmb, having other bleeding problems, and having anemia are drivers of higher hmb score. the results also indicate that hmb adversely affects quality of life as measured by participation in sports, social activities, after-school activities, tiredness, absenteeism, and gpa. hmb is also associated with increased rates of anemia and use of anti-depressants. hmb-driven anemia further adversely affects gpa. under-represented minorities are more likely to have a higher hmb score, as well as an increased adverse effect of hmb on gpa. the results suggest that the social costs of hmb are pervasive in the aya population, and especially pronounced among minorities. a relative with hmb is a significant driver of heavy menstrual bleeding. a hemostatic screen should be included when assessing the aya population with hmb. johns hopkins all children 's hospital, st. petersburg, florida, united states background: propranolol is a non-cardioselective beta blocker medication frequently prescribed for hemangiomas and hyperthyroidism. propranolol inhibits types i and ii iodothyronine deiodinases, enzymes that convert bioinactive thyroxine (t ) into bioactive triiodothyronine (t ). hypothyroidism is a well-recognized complication of diffuse hepatic hemangiomas that produce type iii deiodinase, an enzyme that converts t into bioinactive reverse t and t into diiodothyronine. thyroxine is typically selected for replacement in this population, even though doses up to % above physiologic may be necessary. we hypothesized that low dose, nearly physiologic t would be safer and equally effective because it bypasses propranolol's impact on the pituitarythyroid axis. we report an infant with diffuse hepatic hemangiomatosis and acquired hypothyroidism successfully treated with propranolol, prednisone, and triiodothyronine. design/method: a mo healthy female presented with abdominal distension, poor oral intake, and hepatomegaly. mri confirmed diffuse hepatic hemangiomatosis, the largest lesion measuring . cm by . cm. thyrotropin (tsh) was elevated at . (reference range* . - mcgiu/ml), total t # (rr - ng/dl), and total t ^ . (rr - mcg/dl). treatment was started with prednisone ( mg/kg/day) for three weeks, propranolol ( mg/kg/day) and t ( . mcg/kg/day). the t dose was slowly titrated to a maximum of . mcg/kg/day. thyroid hormone levels rapidly improved on t replacement. after two weeks, the tsh was . , tt , and tt . . after eight months, the tsh was . , tt , and tt . . at twelve months, the tsh dropped to . , tt , and tt . , suggesting decreased tumor production of type iii iodothyronine deiodinase. liver mri confirmed fewer hemangiomas, largest being . cm by . cm. the patient's t dose was reduced. both propranolol and t were discontinued after twenty-four months of treatment. one year off all therapy, this child has normal growth and development, only two < . cm hepatic hemangiomas and no evidence of hypothyroidism (tsh . ; tt ; tt . ). conclusion: t at near physiologic doses corrects the consumptive hypothyroidism associated with diffuse hepatic hemangiomas. t replacement is preferable to thyroxine due to its lower risk of rebound hyperthyroidism as the hemangiomas involute and type iii deiodinase production declines. there are two prior case reports describing t use without t , one employing propranolol and the other utilizing steroids for hemangioma management. this is the first case report with long term follow-up of a child treated with multimodal therapy including propranolol, prednisone, and triiodothyronine. *rr = reference range; #tt = total t ;^tt = total t background: multifocal lymphangioendotheliomatosis with thrombocytopenia (mlt) is a rare congenital disorder first described in that is characterized by multiple vascular abnormalities commonly involving the skin and gastrointestinal tract as well as consumptive coagulopathy often resulting in gi bleeding in infancy( ). to describe an unusual presentation and successful management of mlt in a neonate. design/method: baby h was born at full term after a pregnancy complicated by maternal sinus venous thrombosis requiring anticoagulation beginning at weeks. at birth, she was diagnosed with multiple hemangiomas based on clinical exam. at two weeks of age, she developed melena and hematemesis. cbc revealed platelet count of and she was referred to the ed. abdominal ultrasound was concerning for abnormal hepatic waveform; cxr showed multiple pulmonary nodules. workup revealed no other lesions and no further hematologic abnormalities. biopsy of presumed hemangioma ultimately revealed a smooth muscle-lined vascular proliferation without glut- immunoreactivity, consistent with mlt. her early course was complicated by an acute hemodynamically significant gi bleed; esophagogastroduodenoscopy identified six bleeding vascular malformations within the stomach that were injected with epinephrine and sclerosed with successful hemostasis. she received multiple prbc and platelet transfusions. central access was obtained and she was started on oral sirolimus based on previous reports of successful use in management of vascular malformations given its antiangiogenic and immunosuppressive effects ( ). she has tolerated it well with no evidence of toxicity and has achieved a partial response with stable of hemoglobin > and platelet count > . cutaneous lesions have diminished in intensity and she has had no further signs of gi bleeding. she receives pentamidine for pcp prophylaxis. she continues to have appropriate growth and development. we describe here an unusual presentation of an already rare disease. while cutaneous and gi lesions are typical of mlt, pulmonary involvement is not well-described in the literature. early identification of tissue-based diagnosis enabled timely stabilization and treatment of the patient. five months later, she continues to tolerate sirolimus and has shown significant response with diminished coloration of cutaneous lesions, stable blood counts, and no further bleeding. mlt is a relatively newly-recognized disorder with significant phenotypic variability. given that bleeding secondary to a kasabach-merritt-type consumptive thrombocytopenia is the major cause of morbidity and mortality in the first year of life in children with mlt, it is essential to recognize the diagnosis and initiate appropriate treatment as early as possible. north, arch background: patients with generalized joint hypermobility (jhm) may experience easy bruising or bleeding given the association between these symptoms and abnormalities in collagen, a required component of primary hemostasis. heavy menstrual bleeding (hmb) is a common initial presentation for females with underlying hemostatic defects and may be the sole manifestation of a bleeding disorder. however, limited reports describe jhm as a cause of hmb, leading to under recognition. objectives: to describe the clinical characteristics and management of young women presenting with hmb in the setting of jhm. design/method: this study utilized our hmb research registry. we included subjects - years, seen in the nationwide children's young women's hematology clinic between february and november with both hmb and jhm. medical records were retrospectively reviewed for history of presentation, menorrhagia impact questionnaire (miq): a validated quality-of-life tool for females with hmb, medication profiles and relevant laboratory studies. results: twenty-five patients met inclusion criteria (median age years, range - ) with an average beighton score of . (range to ). participants presented an average of . years (range months to years) after menarche despite % of patients reporting heavy to very heavy menses since menarche. according to the miq responses, most participants expressed hmb-associated limitations in physical activities ( %), social activities ( %), and work or school activities ( %). of the participants, % reported bleeding symptoms in addition to hmb, most commonly easy bruising ( %), epistaxis ( %) and cutaneous bleeding ( %). forty percent of young women presented with anemia due to chronic blood loss. results of hemostatic testing were unremarkable, with the exception of one patient who was also found to have type von willebrand disease. additionally, % of females reported arthralgia, with knees and ankles the most commonly affected joints. at time of presentation, % of participants reported failure of initial therapies and most patients ( %) were managed long-term with oral hormone therapy. in a small population of young women found to have jhm who initially presented with hmb, patients were likely to have prior bleeding symptoms as well as substantial delays from menarche to timing of presentation at our young women's hematology clinic despite limitations in activities of daily life. greater awareness of the associations between bleeding symptoms and jhm, despite typically normal hemostatic laboratory results, is necessary so that patients can more easily be identified and receive appropriate therapy. the objective is to determine the impact of cl care practices involving the home environment on ambulatory clabsi rates. design/method: information for the pi was collected through a comprehensive survey that was completed annually by the ccbdn member hospitals. responses to the questions about cl care practices involving the home environment were selected from the pi for . ambulatory clabsi rates and ambulatory total bloodstream infection (bsi) rates were obtained from another ccbdn database. the proportion of hospitals that did or did not employ a particular cl care practice was tallied. the mean ambulatory clabsi rate and mean ambulatory total bsi rate of the hospitals that did or did not employ a particular cl care practice were compared using generalized linear model techniques assuming an underlying negative binomial distribution. results: twenty-five hospitals submitted responses to the questions about cl care practices involving the home environment. one hospital was excluded for lack of bsi data. sixty-three percent of the hospitals programmatically educated parents about all aspects of the cl care bundle. the mean ambulatory clabsi rate for the hospitals that educated parents was significantly lower than that of the hospitals that did not ( . infections/ cl days vs. . infections/ cl days; p = . ). the mean ambulatory total bsi rate was also significantly lower ( . infections/ cl days vs. . infections/ cl days; p = . ). the mean ambulatory clabsi rates and mean ambulatory total bsi rates were not significantly different for the other cl care practices. conclusion: an analysis of cl care practices involving the home environment reveals that parental education of all aspects of the cl care bundle is associated with a lower ambulatory clabsi rate and lower ambulatory total bsi rate. this finding highlights the importance of systematically teaching family members the proper method of handling cl. background: children undergoing chemotherapy are at a high risk for developing nausea. dr. amy baxter in collaboration with pediatric oncology patients and nurses, developed and validated a pictorial nausea rating scale for children aged - years, called the baxter retching faces (barf) nausea scale. staff nurses at a large, academic, pediatric hospital located within washington, d.c., have identified variability in nursing assessment and documentation of chemotherapy induced nausea and vomiting (cinv) in pediatric oncology patients. the purpose of this quality improvement project was to utilize the barf scale to standardize assessment and documentation of nausea in pediatric oncology patients receiving chemotherapy. the primary aims of this project were to: assess feasibility of the barf scale in clinical practice; increase nursing knowledge about cinv through education sessions; increase documentation of nausea assessments through the use of the scale. the secondary aim of this project was to: increase the recognition of nausea through the use of a standardized assessment tool. design/method: the pdsa model was used to guide the design and implementation plan. in the first phase of the project data was collected to identify the prevalence of nausea in patients admitted for chemotherapy in the prior three months. education sessions discussing cinv and the utilization of the barf scale were conducted. pre and post assessment of nurses' knowledge of cinv and documentation were assessed. in the second phase the barf scale was implemented into practice. nurses were asked to utilize the barf scale to assess and document nausea scores in patients, aged to years, receiving chemotherapy. at the end of the implementation period nurses were surveyed about the feasibility of the scale. post data was collected to identify the prevalence of nausea documented in the electronic health record. this project was undertaken as a quality improvement initiative at children's national and it does not constitute as human subjects research. as such it was not under the oversight of the institutional review board. results: all data has been collected; however complete data analysis will be conducted in the upcoming weeks. background: sickle cell disease (scd) is the most common inherited blood disorder in the united states (us); however, there are few quality measurements to evaluate scd practice. in , the nhlbi published guidelines that include two key interventions for children with sickle cell anemia (sca): the use of transcranial doppler (tcd) screening for stroke prevention and hydroxyurea (hu) to prevent scd pain crisis. we conducted a national survey of scd management sent to providers in over institutions in the us to better assess knowledge of the guidelines and barriers to hu counseling and tcd screening guideline implementation. it was hypothesized that the barriers to tcd screening are different than barriers to hu counseling and prescribing. a -question anonymous survey was sent to providers by mail (follow-up by email). survey themes included nhlbi guidelines knowledge and comfort with understanding and implementing both tcd screening and hu use. the response rate was % ( / ) however one survey was incomplete. thus, were analyzed in the final data set. all of the respondents are in active practice, % s of s in academics and all care for children with scd. the majority of providers ( %) felt "very" or "extremely" confident in their knowledge of tcd screening and interpretation. similarly, % of providers felt "very" or "extremely" familiar with hu dosing and management. for tcd screening, % of providers estimated their screening rates were > % and % providers felt their annual screening rates were - %. the two biggest barriers to tcd screening noted by providers (of moderate to extreme significance) included: lack of support staff ( %) and lack of time during a patient visit ( %). regarding hu prescribing practices, % of providers offered hu to at least % of children with sca over nine months of age. the biggest barrier to hu prescribing noted by % of providers was concerns about patient adherence or access to the medication. only % providers felt that lack of support staff was a moderately significant barrier to hu prescribing. the pediatric scd providers surveyed all have access to the nhlbi guidelines. despite widespread guideline knowledge, there are different barriers for tcd screening versus hu prescribing, which prevent optimal implementation. as a result, although both recommendations are from the same nhlbi guideline, they likely will require different implementation strategies (systems-based interventions for tcd screening; interventions to improve patient adherence for hu counseling) to improve outcomes. background: invasive fungal disease (ifd) is a major cause of mortality and morbidity among pediatric immunocompromised patients such as those who receive chemotherapy or hematopoietic stem cell transplantation. the current diagnostic 'gold standard' of ifd remains culture of infected tissue obtained by biopsy. noninvasive biomarker testing for galactomannan or , -beta-d-glucan (bg) can have low sensitivity and does not provide species-level identification. nextgeneration sequencing (ngs) of cell-free plasma is a promis-ing noninvasive approach to providing species-level identification of ifd via a blood test and can further guide specific treatment. objectives: describe the incidence of positivity for fungal specific pathogens on ngs analysis in a high-risk immunocompromised pediatric population and correlate results with other 'standard' infectious studies if performed. design/method: immunocompromised pediatric patients with suspected ifd were enrolled and plasma was collected at time of enrollment. ngs was performed on extracted dna in cell-free plasma (karius, redwood city, ca). after removing human reads, remaining sequences were aligned to a curated database including pathogens. organisms present at a significance-level above a predefined threshold were reported. results: twenty-seven samples from enrolled patients have been processed thus far. of these subjects, were enrolled for prolonged febrile neutropenia (≥ hours) despite broad-spectrum antibiotics, for recrudescent febrile neutropenia, for abnormal imaging, and with other findings. after evaluation of routine studies performed, patients met criteria for proven ifd, for probable ifd, and for possible ifd using eortc/msg guidelines. the ngs plasma test identified the same pathogen as cultured from infected tissue or blood in % ( / ) of the proven cases. in the probable cases, pneumocystis jirovecii was identified in a patient with a positive bg ( pg/ml) and pneumonia. among the possible cases, toxoplasma gondii was detected in a patient with prolonged febrile neutropenia and lung imaging suggestive of ifd. additionally, candida glabrata was isolated in a patient with prolonged febrile neutropenia but no other criteria for ifd. numerous pathogens were also identified that could explain the above clinical parameters, including hsv , cmv, vzv, hhv , ebv, bk polyoma virus, and ureaplasma parvum. the cell-free plasma ngs test can detect invasive fungal infections from blood. the test identified fungi from proven ifd, detected pathogens in both probable and possible ifd cases, and is a useful diagnostic tool in the evaluation of ifd. supplies and sample shipment and processing supported by karius, inc. baylor college of medicine, texas children's hospital, houston, texas, united states background: practicing medicine is a lifelong learning process. as noted in the institute of medicine's seminal report, 'to err is human,' adverse outcomes do not typically result from individual recklessness; rather, they result from faulty systems, processes, or conditions that provide an environment conducive to making a mistake, or failing to prevent one. learning to systematically review errors and translate lessons learned into quality improvement (qi) initiatives is a critical component of practice-based learning and improvement for practitioners at all career levels. objectives: to develop a methodical, self-reflective and nonthreatening approach to incident analysis and translation of lessons learned into qi initiatives. design/method: we used a validated, structured case audit approach, modified from szostek et al: ) review all documentation relating to the case and identify all health care providers involved; ) interview stakeholders, including those who directly provided and supported care; ) use a qi tool to conduct a root-cause analysis; ) identify a systems issue that contributed to the outcome; and ) propose systems-level interventions and prioritize initiatives based on effort-yield projections. results: pdsa cycle : plan: establish a committee to ) identify potential cases, ) triage cases for conference presentation, ) determine timing and frequency of conferences, ) develop a training manual, ) record identified qi initiatives. do: we established a quarterly section-wide meeting to which all members of the pediatric hematology/oncology service are invited, including administrative and nursing leadership. we developed a training manual and structured presentation template. prioritized cases were discussed in advance during multidisciplinary case review sessions, and presented by senior fellows who were instructed to focus discussion on potential opportunities for qi. study: we identified cases, meeting criteria for mmi presentation. qi initiatives identified from this conference resulted in a number of systemic practice changes; however, we encountered challenges to sustaining these changes over time. act: objectives for the next pdsa cycle are to ) establish a method for tracking the adherence to recommended changes in practice, ) maximize sustainability by integrating qi initiatives into institutional qi leadership and practice standardization committees. we have successfully implemented an mmi conference that meets out of institute of medicine quality domains: safety, effectiveness, patient-centeredness, timeliness, and efficiency. a standardized, consistent approach to mmi presentations that includes identification of contributing factors and specific qi implications has the potential for improving both provider education and patient care/safety. johns hopkins university, baltimore, maryland, united states background: receiving a cancer diagnosis is a life-changing event for patients and caregivers, although little is known about the experience. while some oncologists receive dedicated training in delivering this bad news, the initial conversation is often with a primary pediatrician, and these providers often feel they do not receive adequate training in the communication of a cancer diagnosis. objectives: our objectives were two-fold: first, to better define the experiences of caregivers/patients when told of a cancer diagnosis, and to query how caregivers/patients believe providers can improve the disclosure of this bad news. secondly, to assess what, if any, training primary pediatricians received in this skill, and to assess how comfortable providers in various settings and stages of training are with communicating cancer diagnoses. design/method: from november - , semistructured, in-depth interviews were conducted with pediatric oncology patients and caregivers of patients (n = ) diagnosed in the past year regarding their experiences receiving the diagnosis at our institution. in addition, pediatric residents (n = ), outpatient pediatric primary care physicians and pediatric emergency medicine physicians (n = ) were interviewed regarding their experiences delivering cancer diagnoses. interviews were analyzed following principles of thematic analysis. interviewers with patients and caregivers had two common themes: ) all emphasized their wish for direct and thorough information; ) both patients and caregivers emphasized the gratitude they felt for physicians who gave them hope by emphasizing the good prognosis of their child's cancer. lack of training in this area, as well as lack of comfort delivering this news was common will all providers. additionally, providers report variable approaches to giving bad news, including ) whether to tell caregivers separately or tell the child and parents together, and ) whether to give favorable prognostic information. additionally, attending physicians also differed significantly in their approaches to teaching residents. while some believed residents should give the news to gain experience, others felt that this is not appropriate if residents are inexperienced. only one resident reported ever receiving feedback on his communication skills in this type of discussion. conclusion: we plan to build on these interviews to develop a national survey of patients, caregivers, and providers to better understand the issues surrounding this discussion. we will use the findings to develop a communication curriculum for pediatric residents, focusing on the discussions that occur in the outpatient setting by primary pediatricians. background: human papilloma virus (hpv), common in both females and males, is responsible for pathologies ranging from benign genital warts to cervical and penile cancer. hpv strains and are responsible for , malignancies each year in the united states, and one third of them arise in men. pharmaceutical companies have now developed a vaccine that will help prevent the virus-associated malignancies. the cdc initially recommended that females ages - years receive the vaccine series, then starting in they expanded the eligibility to males ages - years. despite being widely available and highly publicized, only % of eligible females receive the full vaccine series. objectives: this study aims to assess the knowledge of hpv, the attitudes towards the hpv vaccine, and identify barriers preventing its full utilization. once identified, we aim to overcome the barrier(s) in order to improve vaccination rates in eligible adolescents. we distributed a standardized questionnaire to the parents of eligible female and male patients in our pediatric hematology-oncology clinic. it assessed the parents' knowledge of hpv and the vaccine, their views of the vaccine, and reasons why they may oppose it. results: approximately % of parents claim they have been educated about hpv, mostly by their primary care physician. however, % did not know what disorders hpv caused; % felt the vaccine should not be added to the typical vaccine schedule; % of parents do not intend to vaccinate their child. of those that opposed the vaccine, one-third were concerned about potential side effects and nearly % feel they do not have enough information. additionally, % of parents are not aware that the vaccine is available at their child's doctor and only % of parents have discussed the hpv vaccine with their child's doctor. the largest barrier to the utilization of the hpv vaccine that we have identified appears to be lack of educa-tion. as a result, we have begun distributing the cdc's hpv and vaccine patient guide to our patients' families as an intervention. we are currently in the process of re-administering our survey to these families after implementing the intervention to assess its success in increasing both knowledge and utilization of the hpv vaccine. cancer institute, chennai, chennai, tamilnadu, india background: rasburicase is a recombinant urate oxidase enzyme approved for use in tumor lysis syndrome (tls) and it acts by reducing serum uric acid levels. using rasburicase at the recommended dose of . mg/kg/day for days is expensive and it is not known whether this extended schedule is clinically beneficial compared to a single fixed dose of . mg. the aim of the present study was to evaluate the efficacy of single dose rasburicase . mg in prevention and management of tls. design/method: rasburicase is available as single use . mg vial. at our institution a single dose of rasburicase . mg irrespective of bodyweight has been used in adults and in children a dose of . mg/kg (maximum . mg) has been used since for prevention and management of tls and subsequent doses are given based on biochemical response and clinical condition. we retrospectively analysed the case records of patients who had received rasburicase from january to january . the study included patients with hematological malignancies who received rasburicase. children accounted for . % (n = ) patients and males comprised % (n = ). rasburicase was used prophylactically in ( . %) patients, for laboratory tls in patients ( . %) and for clinical tls in ( . %) patients. single fixed dose rasburicase prevented laboratory/clinical tls in % of the prophylactic group and prevented clinical tls in % of the laboratory tls group. none of the patients in prophylactic and laboratory tls group developed clinical tls. however, majority of the patients with clinical tls required more than one dose rasburicase. single dose of . mg ( vial) rasburicase is efficient in preventing and managing laboratory tls and is economically viable in resource constrained settings. nicole wood, lauren amos, nicholas clark, chris klockau, karen lewing, alan gamis children's mercy kansas city, kansas city, missouri, united states background: medication reconciliation for newly diagnosed oncology patients is complicated and cumbersome. these patients are often admitted on no medications, and leave on multiple. chemotherapy and supportive medications are crucial. despite numerous individuals overseeing this process, prescribing errors or omissions still occur. when reviewing the literature, improvement occurs when there is an interprofessional and standardized process to medication reconciliation. objectives: this project's aim was to improve the accuracy of the discharge medication reconciliation process from % to % from february -august . the process measure was the percentage of patients discharged with an accurate checklist. additional time for staff spent in completing the checklist and avoiding an increased error rate by changing the prescribing process were followed as balancing measures. we created a discharge medication checklist which included a list of required home medications prescribed by the resident, ideally hours prior to discharge. it required fellow or attending review and pharmacy to review the list and educate the family. checklists were collected monthly and reviewed against the electronic medical record (emr) for accuracy. results: six pdsa cycles were completed. there were errors during the data collection time frame. in pdsa cycle , a patient received acetaminophen for pain control which is avoided at home. in addition, this patient received diphenhydramine instead of ondansetron, which is preferred as an antiemetic. in pdsa cycle , a patient with a pending diagnosis was sent home with acetaminophen. of note, this patient did not have a checklist completed upon discharge. this project provides a novel and important method to standardize the discharge medication reconciliation process in a complex patient population. it clarifies which types of medications these patients need, provides pharmacy teaching to families which was not done previously, and prescribes discharge medications to families sooner. after the first medication reconciliation error, the checklist was revised. no further errors were made following revision, with the exception of one patient without a completed checklist at dis-charge. our accuracy rate increased from % at baseline to % following implementation. we are in the process of making the checklist electronic and accessible in the emr. in the interim between the end of data collection and implementation into the emr, a leukemia patient was sent home without an epinephrine pen, further demonstrating the importance of this standardized discharge process. for this reason, we have re-instituted the checklist until the electronic version is available. background: survivors of pediatric cancer are at risk of losing pre-existing protective antibodies to vaccine preventable diseases. in a prior study, % of children < years lost humoral immunity to measles as a result of chemotherapy induced alterations in immune system. measles in recipients of immunosuppressive chemotherapy has mortality rates up to %. because of volitional vaccine refusal, there has been a dramatic increase in measles infection from cases in to in , including several statewide outbreaks. small pediatric oncology practices frequently share floor/clinic space with the general pediatric patients putting them at risk for measles since virulence starts hours prior to symptoms. there is no standard protocol for revaccinating post-chemotherapy patients. to assess measles risk based on serial humoral immune status in a cohort of pediatric oncology patients receiving intensive chemotherapy design/method: patients < years age with known vaccination status receiving intensive chemotherapy between july -june at our institution's pediatric oncology practice were included in this prospective study. serial measles igg antibodies were measured at diagnosis, months and months after initiation of chemotherapy using elisa. measles immunity was defined per lab standards. a comparison of pre-chemotherapy and serial post-chemotherapy immunization titers was made for all patients by diagnosis. the study population consisted of children ( male); patients had all, non-hodgkin lymphoma, sarcoma and other solid tumors. two patients ( . %), both unvaccinated had non-protective measles antibody levels at s of s baseline. of the remaining patients, . % patients ( leukemia, lymphoma and sarcoma) lost protective antibody titers at months after initiation of chemotherapy and . % ( leukemia, lymphoma and sarcoma) at months after initiation of therapy. % of the remaining patients who retained measles antibody titers within protective range at months also demonstrated a steady decline in antibody titers at and months from therapy initiation. the loss of protective measles humoral immunity occurred significantly more often in patients with leukemia compared to other malignancies. oncology patients in our practice undergoing intensive chemotherapy demonstrated progressive waning of protective measles igg titers. our data suggests that it should be standard practice to check all patients for measles humoral immunity prior to starting chemotherapy and at completion. larger studies need to be performed to establish guidelines for revaccinating post-chemotherapy pediatric patients, an intervention that is easily applicable and of low cost. background: the accurate determination of glomerular filtration rate (gfr) is important to screen for acute kidney injury, to dose chemo-therapy, and to identify risk for chronic kidney disease.being correlated with inulin clearance, measured gfr by iohexol plasma disappearance (igfr) is a new gold standard for measurement of gfr in pediatric cohort studies. igfr is based on the clearance of an exogenous marker and is unaffected by endogenous compounds or a patient's muscle mass. we compared igfr with -hour urine creatinine clearance ( crcl) and gfr estimating equations using serum creatinine (scr) and serum cystatin c (cystc) in pediatric patients with cancer. we recruited participants who were ages to yrs, continent of urine, and diagnosed with a malignancy in the past years. eligible subjects had stable kidney function for at least two weeks prior to the assessment of igfr. consented subjects had baseline assessments including height, weight and vital signs. blood samples were obtained for serum chemistry, and time zero iohexol. igfr determined by ml iohexol solution infused over - minutes followed by ml of sterile saline. blood was drawn at , , and minutes.at the same time of igfr, the crcl was collected. igfr was calculated using a two-compartment model and area under the curve. we compared igfr to published gfr equations (schwartz et al, kidney int ). results: ten subjects ( female/ male) agreed to participate. the distribution of diagnoses for the subjects: all = , lymphoma = , brain tumors = and hepatocellular carcinoma = . six patients were off therapy. the lower gfrs are noted in patients who had malignancies other than leukemia, likely due to the use of cisplatin based therapy. the average igfr was ml/min/ . m^ whereas crcl was . ml/min/ . m^ ; demonstrating the crcl overestimates gfr compared to igfr. comparing igfr to univariate equations using scr, cystc, and the multivariate equation with both, the univariate cystc equation correlated well with igfr; the others overestimated igfr. we found that crcl overestimated igfr. the univariate cystc equation better correlated to igfr than equations with scr. the poor performance of scr based methods to assess gfr might be due to decreased muscle mass and inadequate nutritional status. creatinine-based determinations of gfr alone, may not be accurate in this population. further study is needed to determine if igfr should be a standard of care to assess gfr in children with cancer particularly who are receiving nephrotoxic medications and incontinent of urine. background: pediatric oncology patients undergoing chemotherapy through indwelling venous catheters are at increased risk for severe sepsis especially when neutropenic due to chemotherapy. rapid triage and early recognition are essential because delayed initiation of antibiotics and fluids in these patients or delayed transfer to higher level of care after initial stabilization is associated with poor clinical outcome. our pediatric oncology out-patient clinic is designated as an article unit whereby the providers can initiate and give treatment such as intravenous fluid, antibiotics, chemotherapy and blood products. objectives: global aim-optimize management of early sepsis and decreased morbidity, mortality and hospital length of stay in the high risk pediatric oncology patients. smart aim-improve timely management with initiation of fluids and antibiotics and transfer of septic patients to higher levels of care by % in months in above patients design/method: multidisciplinary team with physicians and nurses was created. retropective chart review of sepsis patients treated at the clinic from april to october was done using an audit sheet to identify the barriers in the delivery of care. three patients were identified and data analyzed prior to intervention; two were analyzed post interventions. a key driver diagram was created by the group to drive intervention. a process map was designed to identify the different steps in the care of these patients to pinpoint areas needing improvement. different timed data points were used starting from time of arrival to clinic, time to antibiotics and fluids and time to transfer to higher level of care. rapid pdsa cycles were done to improve the processes and delivery of care. run charts were created. there was an improvement close to the goal of % for all data points used. pdsa cycles for improvement included conducting frequent mock codes with appropriate feedback real time coaching and process planning with nursing staff. we partnered with pharmacy for close loop communication with clinic staff and we improved communication between physicans at different levels. conclusion: sepsis in neutropenic pediatric oncology patients is deadly and can be reversed with timely management at different levels. given the promising results of the above project, we want re-inforcement of the processes to be a part of the daily practice of first line clinical staff. eventually we will extend the principles learnt in management and triage of sepsis to other outpatient emergencies chemotherapy related anaphylaxis background: chemotherapy-induced nausea and vomiting (cinv) is a common side effect in children receiving antineoplastic chemotherapy. recommended prophylactic antiemetic medications are based on the classification of chemotherapy emetogenicity. however, despite appropriate use of these antiemetic agents, some patients will still experience nausea and/or vomiting. children's oncology group clinical practice guidelines recommend the addition of olanzapine to prophylactic regimens for management of breakthrough cinv. objectives: our pediatric hematology oncology center implemented a quality improvement (qi) project aimed to increase the use of olanzapine in pediatric cancer patients years of age and older receiving moderately or highly emetogenic chemotherapy and experiencing breakthrough cinv over a month period. design/method: this qi project was conducted utilizing plan-do-study-act (pdsa) cycles. for the first pdsa cycle, baseline data was collected through chart review to determine the rate of olanzapine use for breakthrough cinv over a month period from july to december . breakthrough cinv was defined as use of or more doses of antiemetic agents other than those given for cinv prophylaxis. guidelines for treatment of breakthrough cinv were reviewed with pediatric hematology/oncology attending physicians and fellows. flyers were created that listed chemotherapy regimens considered moderately and highly emetogenic. if a patient experienced breakthrough cinv, a flyer was to be placed in the patient's roadmap binder to signal olanzapine should be added to the next chemotherapy block. data was collected over a month period in september following this first intervention. the second pdsa cycle consisted of didactic education and training of pediatric oncology nurses as well as pediatric residents regarding the addition of olanzapine for breakthrough cinv. rates of olanzapine use were then collected from october through november . results: olanzapine use increased from . % at baseline to . % after the first pdsa cycle ( = . , p = . ). after the second pdsa cycle, olanzapine use increased another . % to . % ( = . , p = . ). the administration of olanzapine was successfully increased by modifying patients' roadmaps after patients experienced breakthrough cinv as well as with education and training of pediatric oncology staff, fellows, residents, and nurses. background: venous thromboembolism (vte) is increasingly affecting children. according to an administrative database study, there was a % increase in the incidence of vte among children admitted to free-standing children's hospitals in the united states from to . risk factors for hospital-acquired vte are well-known and well-studied in adults, with evidence-based preventative measures available. similar guidelines are lacking for children. objectives: there is an ongoing national-initiative to develop and institute methods for screening and preventing hospitalacquired vte in children. in / , nationwide children's hospital instituted an electronic screening form required for all patients admitted ≥ hours. patients were scored and riskstratified based on eight risk-categories. a summated score was used to determine the vte risk level, and used to make prophylaxis recommendations for patients ≥ years; as well as patients ≥ years who were admitted to an intensive care (icu), surgical, or trauma unit. the purpose of this irb exempt, quality improvement initiative was to retrospectively review our experience with this risk-stratification tool. results: hospital-acquired vte events occurred in unique subjects. median age at vte diagnosis was years. only ( %) vte occurred in children ≥ years of age. ( %) vte were deep vein thrombosis (dvt), and ( . %) involved pulmonary embolism. vte was most common in subspecialty units including the pediatric and cardiac icus ( . %); neonatal icu, ( . %); and hematologyoncology, ( . %). ( %) vte were associated with central venous catheters (cvc) and events ( %) were associated with altered mobility. congenital heart disease/heart failure was the most common chronic medical condition associated with vte ( ( . %) events); whereas infection and trauma/surgery were the most common acute medical conditions associated with vte ( ( . %) and ( %) events, respectively). during ( %) events, subjects scored a summated score ≥ . in summary, in this single institution, prospectively maintained database, cvc remains the most common risk factor for vte, followed by cardiac disease, infection and trauma/surgery. most subjects who developed vte scored high (score ≥ ) on our screening tool. only a small proportion of vte occurred in patients older than years and thus eligible for thromboprophylaxis. our results indicate that future vte prevention endeavors should include these age groups in addition to exploring more aggressive prophylactic modalities including pharmacological prophylaxis. background: pediatric fellows are required to have active engagement in quality improvement (qi) activities, and yet a national acgme review found most trainees had "limited knowledge of qi methods" and "limited participation in interprofessional qi teams". the twenty fellows in our pediatric hematology/oncology training program identified blood culture utilization as their qi priority. our institution recently introduced a hospital-wide decision algorithm to guide providers regarding when to obtain blood cultures. there is often a low threshold to obtain blood cultures in immunocompromised pediatric oncology patients, but these are often low-yield or result in falsepositives. our fellows spearheaded a project to implement the algorithm in the inpatient pediatric oncology population and improve the proportion of appropriately drawn blood cultures. we investigated how appropriately the algorithm was being utilized on the inpatient pediatric oncology floor prior to and after several educational steps aimed at disseminating the algorithm to members of the care team. our primary endpoint was to quantify the proportion of culture episodes drawn "inappropriately", with a goal of reducing inappropriate episodes to ≤ %. the algorithm was initially introduced to the nursing staff and residents covering the twenty-bed inpatient unit in september . qi project planning took place with upper level fellows in january . fellows and faculty received intensive training on the algorithm in july-august . we then conducted a retrospective chart review of blood culture episodes drawn between august and november . upper level fellows scored ∼ culture episodes as to whether the decision to culture and number of cultures drawn were "appropriate" or "inappropriate", and catalogued the indications for culture episodes and if applicable, why the episode was found to be inappropriate. additionally, fellows discussed inappropriate culture episodes with the team onservice, to provide direct feedback on where the algorithm failed. results: between august -december on average cultures/ patient-days were drawn. forty-nine percent of culture episodes were inappropriate. from january -october , following targeted education on the algorithm, the rate of blood cultures drawn decreased to cultures/ patient-days. the average proportion of inappropriate culture episodes fell to . %, representing a % decrease in inappropriate culture utilization. correct application of a decision algorithm for blood culture utilization can reduce total cultures drawn on an inpatient pediatric oncology unit. fellow-led education of the multi-disciplinary team decreases the rate of inappropriate culture episodes as well as provides active engagement in qi. background: inadequate understanding of sickle cell disease (scd) is common and can affect patients' compliance and therefore their morbidity and mortality, especially after transition to adult care. optimal clinical care for scd includes disease education, which can be difficult given the breadth of possible topics and limited time in clinic. it is unclear how best to provide personalized, efficient education for adolescents with scd. this quality improvement (qi) study aimed to implement a questionnaire-based system to improve patients' knowledge of their scd and documentation of education by the nurse or physician. the study objective was to improve provider documentation and patient knowledge about their scd by identifying patients' gaps in comprehension. by january , the study aimed to increase education documentation from % to %. by april , the study aimed to increase use of a smart phrase for education documentation from % to %. by june , the study aimed to increase patients' knowledge about their disease by %. design/method: twenty-one scd patients enrolled on an irb approved qi study, with twenty active patients. our comprehensive team generated a questionnaire with knowledgebased questions for two age groups: - and - years old. at each comprehensive visit, a questionnaire was distributed, with at least -month intervals. the provider scored questionnaires and reviewed two educational topics, with wrong answers taking priority. plan-do-study-act (pdsa) cycles included pdsa# : patients completed questionnaire. pdsa# : a smart phrase addressing questionnaire topics was created and shared with providers. pdsa# : patients received education handouts during clinic education. documentation in clinic notes was the process measure and questionnaire scores was the outcome measure. results: pdsa# is complete, pdsa# has four patients remaining, and pdsa# is ongoing. due to variable visit frequency, there are multiple concurrent cycles. after pdsa# , free text documentation was completed an average of % over the course of months. after pdsa # documentation increased to % within months and questionnaire scores increased from an average of % to %. of the questions that patients got wrong on their first visit, they were significantly more likely to improve on retesting if the topic was taught to them than if it was not addressed ( % vs. %, p = . ). we are currently completing pdsa# and collection of post pdsa# data. questionnaire-based scd education coupled with standardized smart phrases improves patients' scd knowledge and documentation by providers. further improvement in knowledge is expected with the addition of handouts. background: exposure to suffering can have a profound impact on the wellness of caregivers, often referred to as the "cost of caring". this cost is especially high in pediatric hematology/oncology. repeated exposure to suffering has the potential to negatively impact resilience and increases the risk of burnout, thus impacting quality of care and patient satisfaction. we have developed a peer support team utilizing the critical incident stress management (cism) model. this model has been successfully used in other professions that frequently face traumatic events such as fire fighters, police and emergency medical technicians. the h.o.p.e.s. team (helping our peers endure stress) consists of volunteer multidisciplinary staff members who have received training to provide both group and peer support following any 'critical incident' that may impact one or more staff members. we hypothesize that implementation of the h.o.p.e.s. team will improve staff resilience, decrease overall rates of burnout and improve compassion satisfaction. s of s design/method: we are using both empiric metrics and anecdotal reports to assess the impact of the h.o.p.e.s. team. prior to the activation of the team, all pediatric hematology/oncology clinical staff members were surveyed using validated tools to assess their levels of resilience, burnout, secondary trauma and compassion satisfaction (proqolv and brief resilience scale). they were also asked to rate the number of times they had experienced critical incidents, as well as their perceived level of distress after dealing with traumatic events. after the h.o.p.e.s. team has been functional for months, we will send the same survey to staff members to measure changes, paying special attention to resilience and rates of burnout and compassion satisfaction. results: enthusiasm for development of the team has been high. of people approached to volunteer their time to participate in the multidisciplinary team agreed, including attending physicians, fellows, nurses, nurse practitioners, child life specialists, social workers, clergy and psychologists. all volunteers participated in a -day training conducted by an instructor from the international critical incident stress foundation. engagement in the first staff survey has been high, with of responding to date. data collection is ongoing. clinical staff in pediatric hematology/oncology may be particularly vulnerable to burnout and decreased resilience by repeatedly witnessing suffering and trauma. peer support interventions following critical incidents may lead to increased resilience and compassion satisfaction while decreasing rates of burnout. enthusiasm for the development of a peer support team has been high. background: monthly blood transfusions are an indicated therapy for pediatric patients with sickle cell disease with certain complications. maximizing transfusion efficiency in a busy infusion clinic requires: ensuring that appropriate blood units are available in the hospital blood bank; laboratory specimens are obtained from patients in advance; and coordination of clinic appointment and nursing availability. we sought to improve clinic efficiency through identifying ways to better communicate with patients/families regarding upcoming laboratory and transfusion appointments, and to assess the efficacy of implementing a web-based personalized text reminder (pinger.com). we measured the baseline frequency with which transfusion appointments were missed by families, moved to later within the week, or delayed due to late labs. a convenience sample of patients receiving monthly transfusions received a questionnaire about patient/parent preferences for appointment reminders and barriers to keeping appointments. those patients/parents who did not opt-out of an additional text reminder received personalized texts from their care team reminding them of lab and transfusion appointments. rates of missed/moved/delayed appointments were compared between the group receiving the additional text messages and the group only receiving standard, hospitalgenerated appointment reminders (telephone call). results: forty-one families ( patients) responded to the survey, capturing information on % of patients receiving chronic transfusion therapy. thirteen families ( %) declined the additional text reminders. families reported a preference for text reminders ( %), more often than email ( %) or telephone ( %), and % of families wanted to receive reminders for both transfusion and laboratory appointments. the majority ( %) of families reported competing work/life priorities as the reason for missed/late appointments. other families noted transportation/travel ( %), fear/illness/pain ( %), and lack of reminders ( %) as the reason for missed appointments. at baseline (twelve weeks), . % of appointments were missed on a weekly basis (range - of available per week), . % were moved, and % of appointments were delayed. during our intervention period (twelve weeks), % were missed, . % were moved, and . % were delayed (combined, both groups). there was no difference in missed ( . % texted vs . % standard), moved ( . % texted vs . % standard) or delayed ( . % text vs . % standard) appointments. though families at our center reported a preference for a text-based reminder, personalized text reminders for appointments did not improve clinic efficiency as measured by missed, moved or delayed transfusion appointments. there was no improvement in appointment adherence in the group receiving personalized texts in addition to standard hospital reminders. university of utah, salt lake city, utah, united states background: childhood cancer outcomes have improved significantly, in large part due to multi-institution collaborative clinical trials run by the children's oncology group (cog). approximately half of eligible children with cancer will enroll on a therapeutic trial, but little is known about the factors affecting caregiver decision-making regarding enrollment or how well the required elements of informed consent are conveyed during the consent process. objectives: . assess coverage of ten of the required elements of informed consent for cog therapeutic trials. . describe factors affecting caregiver decision-making regarding therapeutic trial enrollment. we surveyed families of children who were offered enrollment onto a phase cog therapeutic study for an initial cancer diagnosis in the previous months. fisher's exact or wilcoxon rank-sum tests were utilized to compare demographic and other motivating factors related to enrollment decision-making. results: seventy participants were surveyed. regarding of the basic required elements of informed consent, % knew the trial involved research, % knew consent was required, % knew the enrollment length for the trial, % knew they could continue care independent of enrollment, % knew who to contact with questions, % knew there were options besides enrollment, % knew they could withdraw at any time, % knew the information was confidential, % knew there were risks associated with the trial, and % knew there were benefits. of all participants, % (n = / ) enrolled onto a therapeutic study. among enrollees, % (n = / ) of the primary caregivers had completed college compared to % (n = / ) of those not enrolled (p = . ). when asked about factors impacting their decision, % (n = / ) of those enrolled said they felt there were no risks or did not know if there were risks associated with the study compared to % (n = / ) of those choosing not to enroll (p = . ). of those enrolled, % (n = / ) reported the physician recommendation "somewhat" or "strongly" affected their decision to enroll compared to % (n = / ) of those not enrolling (p = . ). of those who enrolled, % (n = / ) reported feeling pressured to enroll while % (n = / ) of those not enrolled reported pressure (p = . ). of enrollees, % (n = / ) reported they did not have enough time to decide compared to % (n = / ) of those not enrolled (p = . ). failure to convey all required elements of informed consent highlights possible deficiencies in the consent process for cog therapeutic trials. caregivers' perception of being pressured and lack of time to make an informed decision may impact clinical trial enrollment. background: abnormal uterine bleeding (aub) is a frequent adolescent gynecologic complaint. however, limited research exists to guide management, and acute care varies. we sought to improve emergency care for adolescents with aub by developing a clinical effectiveness guideline (ceg) and assessing its impact on quality of care. design/method: a stakeholder engagement group consisting of members from the departments of hematology/oncology, adolescent medicine, general pediatrics, and emergency medicine designed a ceg algorithm for emergency aub management. pediatric residents received ceg training and their knowledge and attitudes were assessed using pre and post intervention surveys. icd- and codes identified electronic health record data for patients presenting to the pediatric emergency department (ed) for aub months before and after ceg implementation. pre-pubertal patients and those with vaginal bleeding from trauma were excluded. a weighted, -point scoring system consisting of prioritized aspects of history, laboratory studies and management was developed to quantify the quality of care provided. t-test, chi square test, wilcoxon rank sum test, and a run chart were used for analysis. of the patients identified, met inclusion criteria. there were % of patients currently using some form of contraception, while . % had bleeding related to a current or recent pregnancy. median aub quality care scores were pre-and post-intervention (p = . ). run chart data showed no shifts or trends (overall median score, -points). both pre and post-implementation, points were deducted most frequently for not assessing personal/family clotting disorder history and inappropriate use/dosing of oral contraceptives. we successfully designed and implemented a ceg and educational intervention for aub management in a pediatric ed. these data suggest our ceg may be an effective tool to improve emergency aub care for adolescents, though additional cycles are needed. background: high-dose methotrexate (hd-mtx) is a common chemotherapy administered inpatient at most centers. its administration is particularly susceptible to error due to the need for frequent drug levels with resulting changes in supportive care. errors can prolong patient stay and cause patient harm. objectives: global aim-to reduce the length of stay (los) of hd-mtx admissions. smart aims-to increase the percentage of patients whose pre-hydration fluids are started by am from % to % by / / , and to increase the percentage of patients who receive hd-mtx by pm from % to % by / / . we used rapid process improvement methods to target earlier methotrexate administration. a key driver of prolonged los was hypothesized to be drug levels returning overnight rather than in the day time due to delayed hd-mtx start. changes implemented have included scheduling hd-mtx patients as the first patients of the day for their exam in clinic and scheduling labs to pass for hd-mtx on the day prior to admission. there are ongoing pdsa cycles to change the location of pre-hydration start from the inpatient room to the clinic exam room in order to meet hd-mtx administration time goals. we are piloting two different education materials to improve patient experience. one explains hd-mtx levels in a red/yellow/green stoplight format and the other reminds patients how to prepare for the admission. other interventions regarding how we test urine ph and safety checks in the ordering process for history of delayed clearance are in the planning stage. the project is ongoing, but as of / / , we start methotrexate by pm % of the time which is improved from a baseline of %. when the project was started, pre-hydration was never started before am. now, fluids are started by am % of the time. pdsa cycles are ongoing and we have yet to sustain reductions in los, but some months have shown decreased los by as much as hours from baseline measurements. rapid cycle improvement can be utilized to decrease los hd-mtx admissions. this has important financial implications as well as the potential to reduce secondary harm from unnecessary time in the hospital. pediatric cancer centers should schedule hd-mtx admissions first thing in the morning so that data regarding kidney injury and drug clearance can be interpreted by the day team and children are not cleared for discharge in the middle of the night. background: education and training for interdisciplinary pediatric oncology providers requires training in principles of palliative and end-of-life (eol) care. the experiences of bereaved parents can inform and enhance palliative care educational curricula in uniquely powerful and valuable ways. the objective of this study is to present an innovative palliative care educational program for oncology providers facilitated by trained bereaved parents who serve as volunteer educators in local and national palliative care educational forums and to describe how incorporation of bereaved parents in these educational forums affects participant comfort with communication and management of children at the eol. design/method: survey tools were adapted to determine how bereaved parent educators affected participant experiences in different educational forums: institutional seminars on pediatric palliative and eol care, role-play based communication training sessions, and an international symposium on pediatric palliative oncology. pre-and post-session surveys with incorporation of retrospective pre-program assessment item to control for response shift were used in the evaluation of institutional seminars and communication training sessions. results from feedback surveys sent to all attendees were used to appraise the participants experience in the international oncology symposium. results: involvement of trained parent educators across diverse, interdisciplinary educational forums improved attendee comfort in communicating with, and caring for, patients and families with serious illness. importantly, parent educators also derive benefit from educational with interdisciplinary clinicians. integration of bereaved parents into palliative and eol care education is an innovative and effective model that benefits both interdisciplinary clinicians and bereaved parents. background: poorly controlled chemotherapy-induced nausea and vomiting (cinv) significantly impairs patients' quality of life and contributes to ongoing medical costs through increased length of stay in the hospital or readmissions and outpatient visits for control of nausea, vomiting or dehydration. lack of adherence to national evidenced-based guidelines that dictate antiemetic prescribing for variably emetogenic chemotherapy leaves patients vulnerable to increased cinv and its ensuing complications. objectives: to review our institution's antiemetic prescribing practices and their consistency with the antiemesis guidelines from the national comprehensive cancer network (nccn) and children's oncology group (cog)-endorsed supportive care guidelines and to further develop tools to increase adherence to these national-based guidelines to improve control of cinv. we performed a retrospective chart review of inpatient chemotherapy encounters. we evaluated emetogenicty of chemotherapy (high, medium, low), initial antiemetic regimen ordered, number of as needed medications required and adherence to national evidenced based guidelines tailored to each level of emetogenicity in the prescription of antiemetics. results: fifty-five total inpatient chemotherapy encounters were reviewed over months. eighteen of these encounters were considered to have been highly emetogenic chemotherapy (hec) with the remaining of these considered to be moderately emetogenic. only out of hec encounters completely included all guideline-recommended agents. there was a demonstrable lack of consistency across providers with dosing of aprepitant and most as needed medications. there was significant variation in order of first, second and third line anti-emetics ordered -with lorazepam and promethazine being used most frequently. with an aim of improving antiemetic prescribing practices for our patients, we are currently rebuilding chemotherapy treatment plans in our electronic medical record to incorporate antiemetic drug order sets that follow evidenced-based guidelines for variably emetogenic chemotherapy. this will be used in conjunction with an education initiative about best practices in supportive care for all prescribers of antiemetics. review of our department's recent inpatient chemotherapy encounters show we are falling short in following nationally recommended standards for appropriate antiemetic coverage during chemotherapy. identification of these deficiencies allows for implementation of quality initiatives to improve prescriber adherence to evidenced-based guidelines for better control of cinv. background: there are currently no consensus guidelines for the management of pediatric oncology patients presenting with fever without neutropenia. historically, these patients had been treated similarly to neutropenic patients with empiric antibiotics. while there has been a shift towards reducing unnecessary empiric treatment, there has been limited research into the outcomes associated with withholding empiric iv antibiotics in this patient population. we assessed the safety and efficacy of our institution's current protocol of observing well-appearing patients who present with fever without neutropenia and compared the outcomes of the patients who did and did not receive empiric iv antibiotics. design/method: this was a prospective, single-institution cohort study. patients were included if they were currently undergoing chemotherapy for an oncologic diagnosis and presented initially as an outpatient with fever and nonneutropenia (defined as anc ≥ cells/mm ). for each episode we recorded lab and blood culture results, signs and symptoms of initial presentation, and clinical outcomes, including antibiotic administration and hospital admission. results: a total of episodes of well-appearing patients with fever without neutropenia were identified. compliance with the institutional protocol was high; . % of patients were observed without receiving empiric iv antibiotics. the majority of patients were discharged home and there were no serious complications or infectious deaths. the incidence of positive blood cultures was low ( . % including several likely contaminants), despite the presence of central venous catheters in the majority ( . %) of patients. there were no significant differences in age, oncologic diagnosis, central s of s line access, anc value, or incidence of bacteremia between patients who did and did not receive empiric iv antibiotics. patients who were admitted to the hospital were significantly more likely to have received iv antibiotics (p < . ) despite documentation of a reassuring exam. however, admitted patients who initially received iv antibiotics were just as likely to discharge within hours compared to patients who were observed. we propose that empiric iv antibiotic administration in febrile, non-neutropenic, otherwise well-appearing patients is unnecessary. our study demonstrated no adverse consequences of observation and no significant differences in clinical outcomes between patients who did and did not receive iv antibiotics aside from rate of hospitalization. this supports the practice of observation without empiric antibiotics for such patients. background: children with hepatoblastoma (hb) undergo repetitive computed tomography (ct) scans to determine response to treatment and assess for relapse. this imaging exposes children to radiation, anesthesia, and imposes financial and emotional burden. objectives: review our institutional experience to determine if afp measurements are sufficient to assess response to treatment and detect relapse. we conducted a retrospective chart review of all patients diagnosed with hb at our institution between - . data collected included serum afp, total number and type of imaging studies during and post treatment, and how relapse or progressive disease was detected. results: thirty-one patients were diagnosed with afp positive hb. during therapy, ct scans were performed: to assess for response to therapy or surgical planning (average scans/patient) and due to concern for progression with rising afp. off therapy, surveillance ct scans were performed (average of . scans/patient) and ( %) included the chest in patients with no lung metastasis at diagnosis. relapsed patients averaged . surveillance scans, . of which were done before relapse was noted on imaging. there were no cases of radiographic evidence of relapse without a prior increase in afp. during treatment, response to therapy based on imaging correlated with a decline in afp in all patients, arguing that repetitive scans are not needed in this setting unless required for surgical planning. only of scans performed during off therapy surveillance displayed evidence of relapse, all of which were preceded by rise in afp. our study represents the largest cohort of hb patients. prior studies suggest similar results, but included fewer patients, lower stage of disease and less than years of surveillance monitoring. at our institution, the cost of a ct c/a/p is $ , with reimbursement varying from - %. in comparison, the cost of an afp measurement is $ . . many scans also require anesthesia and result in emotional toil for families concerned about this procedure as well as the results. thus, afp demonstrates greater sensitivity, with significant cost savings and decreased emotional burden, and should be used for monitoring both during and off therapy, replacing routine serial imaging. background: we observed that our practice of drawing daily blood cultures in hospitalized patients with fever and neutropenia was wasteful; it resulted in excessive negative cultures that did not add to patient care. the smart aim of this quality improvement project was to reduce the number of negative blood cultures drawn on hospitalized patients with fever and neutropenia by % in months. design/method: after reviewing published evidence suggesting drawing daily blood cultures in febrile neutropenic patients was unnecessary, a new blood culture guideline was implemented: cultures were drawn at presentation for fever with neutropenia and, if negative at hours, repeat cultures were not drawn except for clinical change, new fever after being afebrile > hours, or antimicrobials were being changed/broadened. to impact key drivers, we educated staff and changed blood culture order sets to require providers to select a reason for ordering the culture and to eliminate a nursing order to draw daily cultures with fever. we compared the number of blood cultures drawn per central linedays (/ -cld) and the proportion of positive versus negative cultures pre-guideline (july -may ) and postguideline (june -december ). we calculated the cost savings from reducing cultures. to assess patient safety, potential septic events without a corresponding positive blood culture were reviewed. data were analyzed by service (oncology and stem cell transplant). a chi-square test was used to compare rates. in stem cell transplant patients, pre vs. postguideline, there were vs. total cultures drawn/ -cld; vs. positive ( % decrease, p = . ) and vs. negative cultures/ -cld ( % decrease, p< . ). in oncology patients, pre vs. post-guideline, there were vs. total cultures drawn/ -cld; vs. positive ( % decrease, p = . ) and vs. negative cultures/ -cld ( % decrease, p< . ). the decreased positive culture rate among oncology patients may be due to decreased culture contaminants and/or the effect of a concurrent initiative to decrease clabsi in that group. there were safety concerns; however, chart review concluded that the guideline did not lead to missed infections in these patients. for the first months of the guideline, the total cost savings in blood cultures was $ , . . the implementation of our new blood culture guideline successfully led to a substantial reduction in the collection of negative cultures and a cost savings without compromising the detection of bacteremia in hospitalized pediatric patients with fever and neutropenia. background: there are various evidence-based guidelines for treatment of adult cancers, such as the nccn guidelines. previously, care was standardized for most new diagnosis pediatric cancer patients through enrollment on a clinical trial. with decreasing clinical trial availability and enrollment and few, if any, evidence-based guidelines for pediatric cancer, care standardization is challenging for pediatric cancers. objectives: to assess consistency of care, as determined by plan of treatment by diagnosis, for pediatric patients receiving chemotherapy for newly diagnosed cancer at a single center. design/method: patients with a new cancer diagnosis at a large, tertiary care pediatric oncology center in calendar year were identified through reports from the chemotherapy order entry (coe) system. reports included diagnosis (recorded through standardized options) and the plan of treatment. chart review was used to exclude patients who started treatment elsewhere and patients being treated for relapse, to clarify diagnosis if the standardized options in coe were unclear, and to clarify treatment plan if needed. data was entered and analyzed in a redcap database. specific diagnoses were clustered into higher level disease groups and the distribution of treatment plans for patients within each was determined. this project was deemed exempt from irb approval for human subject research as a qualifying quality improvement project. of the patients with a first chemotherapy order in , were excluded due to one or more reasons: stem cell transplant ( ), transfer of care ( ), relapse ( ), and other ( ). an additional patients were excluded because < patients/year/diagnosis. there was no cns tumor disease group with > patients. thus, patients with hematologic malignancies or non-cns solid tumors are the focus of this analysis. for patients with intermediate risk rhabdomyosarcoma, the plan of treatment was the standard arm of a cog protocol, arst for patients and arst for subsequent patient after protocol activation. for all other diseases including lymphoblastic leukemia/lymphoma (excluding infants), classical hodgkin lymphoma, aml (excluding trisomy and apml), stage iii/iv burkitt lymphoma/diffuse large b-cell lymphoma, posttransplant lymphoproliferative disease, wilms tumor, rhabdomyosarcoma, ewings sarcoma, osteosarcoma, neuroblastoma, and retinoblastoma, only one treatment plan per risk category was used. conclusion: this analysis demonstrates highly consistent chemotherapy treatment at a single center for patients with hematologic malignancies and non-cns solid tumors. next steps include exploring strategies to group diagnoses for cns tumors and assessing the quality of evidence supporting the treatments given. background: rapid initiation of empiric antibiotics in patients with fever and neutropenia has been shown to reduce morbidity and mortality. current practice guidelines call for the initiation of antibiotics in these patients within sixty minutes and time-to-antibiotic (tta) has been suggested as a quality-of-care measure. many institutions, including our own, face barriers to meeting this time limit. objectives: utilizing a quality improvement model, determine barriers and implement an intervention to reduce the time-to-antibiotics for pediatric febrile patients with suspected neutropenia who present to the emergency department (ed) at our institution. we have identified and implemented an intervention utilizing the plan-do-study-act model for quality improvement. a twelve-month retrospective review was conducted to evaluate the efficacy of the current practice algorithm at our large, academic tertiary-care hospital. subjects identified were pediatric oncology patients undergoing active chemotherapy who presented to the ed with febrile neutropenia. we identified two specific barriers, triage level assignments and delay in ordering antibiotics. to address these barriers, we have created a wallet sized "fever card" that patients were instructed to show upon arrive to the ed. in collaboration with the ed staff, efforts were also made to educate all pediatric staff on the use of the fever card. post-intervention data collection is currently underway and pre-and post-intervention antibiotic delivery times will be compared. the pre-intervention cohort consisted of thirty-three encounters with a mean time-to-antibiotic delivery of minutes, or seventy-five minutes greater than the accepted standard of care. only one patient received antibiotics within sixty minutes of arrival. post-intervention data collection is currently underway. since identifying two barriers to meeting the standard of care at our institution, we have implemented a quality improvement measure that empowers patient families to direct appropriate triage in the ed as well as simplifying the treatment protocol for ed providers. we expect to identify an improvement in time-to-antibiotics from the pre-intervention to the post-intervention period. background: sickle cell disease (scd) is a genetic disorder in which sickle hemoglobin (hbs) triggers multiple downstream effects, including red cell sickling, hemolysis, vaso-occlusion, and inflammation. scd, a lifelong disease initiated at birth with injury that accumulates over time, causes significant end-organ damage and clinical complications that are undertreated and associated with early death. homozygous mutation (hbss) causes the severe form of scd. individuals with scd are at increased risk of infection, stroke, and retinopathy. clinical guidelines for pediatric patients with scd recommend prophylactic penicillin use (ages - ), annual screening for stroke with transcranial doppler (tcd) imaging (ages - ), and annual ophthalmology exams to assess for retinopathy (ages ≥ ). there are limited real-world data on implementation of these nhlbi-based recommendations. objectives: to describe utilization of penicillin, tcd screening, and ophthalmology care in children with hbss disease. medicaid administrative claims databases were used to identify us patients aged - years at first indication of hbss recorded in each calendar year from to . patients were required to have medical and pharmacy benefits for the calendar year in which they were identified and for months prior to their first recorded hbss indication. prior year utilization of penicillin, tcds, and ophthalmologist visits was measured for each annual cohort. annual cohorts included - commercial (mean age . years, % female) and - medicaid (mean age . years, % female) patients with hbss disease. fewer than half of all patients had received a tcd scan in the previous year, with similar rates seen across all age groups for both payers. ophthalmologist visits increased as patients aged, and while patients aged - years had the highest proportion with an ophthalmologist visit in both payer populations, the overall implementation remained low. in contrast to the low use of tcd and ophthalmology visits, penicillin use was highest in the - year age group: > % use in any given year for both payers. conclusion: although our data demonstrated high penicillin use in the - year age group, consistent with guidelines there is an opportunity to improve implementation of other guidelines-based recommended screening. for example, tcd screening can identify children at risk of scd-related stroke in order to initiate preventive therapies. further research to understand potential barriers to proper screening and to evaluate strategies to improve awareness, adherence, and implementation of recommended screenings in children with scd is warranted. supported by global blood therapeutics. background: childhood cancer therapy has improved where there are many long-term survivors. while psychosocial difficulties in pediatric cancer survivors are recognized, the prevalence of these problems at initial survivorship presentation is unclear. objectives: to examine the prevalence of overall internalizing symptoms (e.g., depression/anxiety) in pediatric cancer survivors presenting to a survivorship clinic and to examine how this is mitigated by receiving psychological services and by evidence of parental depression/anxiety. design/method: pediatric cancer survivors attending their first visit at the reach for survivorship clinic at vanderbilt (ages - ) were included. survivors' parents ( % female) completed the child behavior checklist (cbcl), beck depression inventory-ii, and beck anxiety inventory. survivors > years completed a self-report. the wilcoxon rank-sum and pearson's test were used for univariate analyses. the effect size and % confidence intervals (ci) estimated from the multivariable linear regressions were reported. results: childhood cancer survivors a median of years old and . years off therapy were included. thirty one survivors ( %) showed at least borderline clinical internalizing problems (t score > ) on the cbcl, but only of these patients ( %) reported receiving psychological services. nine other survivors with normal t score ≤ also reported receiving psychological services. parental depressive and anxiety symptoms were correlated to the parental report of survivor overall internalizing symptoms (spearman = . , p = < . and = . , p = < . respectively), however they were not correlated to survivor selfreports. furthermore, parents with mild to severe depressive symptoms or mild to severe anxiety symptoms were more likely to rate their child as having higher overall internalizing symptoms (p = . ; p = . , respectively). multivariable linear regression showed that when adjusted for age, gender, cancer diagnosis and time off treatment, reported utilization of psychological services ( = . , % ci [ . , . ],p = . ), and parent depressive symptoms ( = . , [. , . ],p< . ) were significantly associated with child overall internalizing symptoms. in an otherwise identical alternate model substituting parental anxiety for parental depression, parental anxiety was also a significant risk factor ( = . , [. , . ], p< . ). alternatively, parent anxiety/depressive symptoms were not significantly associated with child self-report of internalizing symptoms. childhood cancer survivors have an elevated prevalence of experiencing internalizing symptoms but seldom report receiving psychological services. childhood cancer survivors' parents with anxious/depressed symptoms are more likely to rate their children as having more internalizing problems, compared to patient self-reports. ongoing longitudinal analyses will help clarify the best timing for potential interventions. background: life expectancy for adults with sickle cell disease (scd) has remained unchanged over the past years despite improvements in pediatric scd survival. at greatest risk are the adolescents and young adults (ayas) transitioning from pediatric to adult care. allen county ranks rd in scd incidence among the counties in indiana, and has board certified pediatric hematologist-oncologists. when children "age out" of the pediatric system, there are few providers knowledgeable about managing adults with scd in the region. a novel partnership between hematologists and the family medicine residency program in allen county was initiated to educate family medicine residents (fps) about scd, hydroxyurea (hu), and management of scd-related complications with the goal to increase the number of knowledgeable providers to care for adults with scd. to determine the effectiveness of online learning modules in educating fps about hu, best practices for aya scd care and transition. three online learning modules about scd (comprehensive care of ayas with scd, hu, best practices in aya transition) were developed and cme-accredited. electronic pre-and post-tests were distributed to fps with five questions for each module covering: contraception; screening tests; hu indications, dosing and monitoring; developmental milestones and scd knowledge assessments. the st vincent irb reviewed the protocol and granted a waiver of consent. results: twenty-six fps ( %) completed the pre-and posttests. over two-thirds correctly identified the clinical benefits of hu on both assessments. knowledge about the rationale for hu therapy increased after the completion of the hu module ( % correct on pre-test vs. % on post-test, p = . ). the proportion of correct responses increased for all comprehensive aya scd care post-test questions, but only the leading cause of death and the priapism-related questions reached statistical significance ( % vs. %, p = . ; % vs. %, p = . , respectively). the proportion of correct responses for of the transition-focused questions was unchanged ( % for both), while the proportion of correct post-test responses on the self-care assessment question significantly increased ( % vs. %, p = . ). after module completion, fps were able to correctly identify common scd complications and why hu is an effective treatment for individuals with scd. the best practices of transition clinic module may need modification to improve physician understanding of the intricacies in establishing and maintaining a scd transition clinic. overall, online training is effective at educating fps and could be used to increase the number of providers knowledgeable about scd care. background: survival rates for pediatric hodgkin lymphoma (hl) exceed % with contemporary therapy. studies of pediatric hl survivors treated in the s- s have shown increased risk for treatment-related chronic health conditions. risk-adapted therapy, including tailored radiotherapy, has been developed to reduce long-term morbidity while maintaining excellent survival. little is known about chronic conditions associated with contemporary therapy presenting during the first years from therapy completion (early outcomes). objectives: to analyze survival and early outcomes of pediatric hl patients treated with contemporary therapy. we conducted a retrospective review of hl patients diagnosed < years of age at our institution from - . three-year overall (os) and event-free (efs) survival were calculated with kaplan meier statistics using sas . . results of standardized screening for targeted toxicities that developed between - years from therapy completion were identified and graded per ctcae criteria. censoring occurred at date of death, years from therapy completion, or december , . data from the last collection point were used for prevalence calculations in cases with multiple evaluations. we identified patients ( % male; % non-hispanic white; mean age at diagnosis . ± . years) with a median time since therapy completion of . years (range . - . ). initial treatment included: ( %) chemotherapy only and ( %) multimodality treatment. all patients received anthracyclines (median dose mg/m ) and % received alkylating agents (median cyclophosphamide equivalent dose [ced] mg/m ). the -year os was % with an efs of % ( % chemotherapy only, % multimodality treatment; p = . ). patients with relapsed/refractory disease received salvage treatment including chemotherapy only (n = ), multimodality therapy (n = ), or multimodality treatment including stem cell transplant (autologous n = ; autologous+allogeneic n = ). no patients developed thyroid dysfunction, cardiac dysfunction, subsequent neoplasm, or male gonadal dysfunction during the study period. pulmonary dysfunction was limited to ctcae grade . anti-mullerian hormone (amh) below the normal range was found in / pubertal females who received ced ≥ mg/m compared to / females who received ced < mg/m . two of the females with low amh also had follicle stimulating hormone > iu/ml. this study is the first to evaluate early outcomes in pediatric hl survivors. the results indicate contemporary chemotherapy and a lower rate of radiotherapy utilization lead to excellent -year survival rates with minimal early toxicities. females exposed to ced ≥ mg/m are at increased risk for gonadal dysfunction and should be prioritized for fertility preservation approaches prior to initiation of cancer therapy. background: cancer is one of the leading disease-related causes of death among individuals aged < years in the united states. recent evaluations of national trends of pediatric cancer used data from before , or covered ≤ % of the us population. objectives: this study describes pediatric cancer incidence rates and trends by using the most recent and comprehensive cancer registry data available in the us. design/method: data from us cancer statistics were used to evaluate cancer incidence rates and trends among individuals aged < years during - . data were from states and covered % of the us population. we assessed trends by calculating average annual percent change (aapc) in rates using joinpoint regression. rates and trends were stratified by sex, age, race/ethnicity, us census region, county-based economic status, and county-based rural/urban classification, and cancer type, as grouped by the international classification of childhood cancer (iccc). we identified , cases of pediatric cancer during - . the overall cancer incidence rate was . per million; incidence rates were highest for leukemia ( . ), brain tumors ( . ), and lymphoma ( . ). rates were highest among males, aged - years, non-hispanic whites, the northeast us census region, the top % of counties by economic status, and metropolitan counties. the overall pediatric cancer incidence rate increased (aapc = . , % ci, . - . ) during - and contained no joinpoints. rates increased in each stratum of sex, age, race/ethnicity (except non-hispanic american indian/alaska native), region, economic status, and rural/urban classification. rates were stable for most individual cancer types, but increased for non-hodgkin lymphomas except burkitt lymphoma (iccc group ii(b), aapc = . , % ci, . - . ), central nervous system neoplasms (group iii, aapc = . , % ci, . - . ), renal tumors (group vi, aapc = . , % ci, . - . ), hepatic tumors (group vii, aapc = . , % ci, . - . ), and thyroid carcinomas (group xi(b), aapc = . , % ci, . - . ). rates of malignant melanoma decreased (group xi(d), aapc = - . , % ci, - . -- . ). this study documents increased rates of pediatric cancer during - , in each of the demographic variables examined. increased overall rates of hepatic cancer and decreased rates of melanoma are novel findings using data since . next steps in addressing changing rates could include investigation of diagnostic and reporting standards, host biologic factors, environmental exposures, or potential interventions for reducing cancer risk. increasing pediatric cancer incidence rates may necessitate changes related to treatment and survivorship care capacity. background: while childhood cancer treatment modalities have improved, the delayed effects of cancer treatment continue to compromise the quality of life in survivors. metabolic syndrome (ms) is diagnosed based on the presence of three of the following findings -obesity, dyslipidemia, hypertension and insulin resistance per the world health organization (who) criteria. the increased risk of ms among childhood cancer survivors was first reported in the 's and is known to increase the incidence of cardiovascular disease in these individuals. objectives: assess the frequency of ms in childhood cancer survivors at our institution. . we conducted a retrospective chart review on pediatric cancer survivors, - years of age, who had been treated at sri ramachandra medical institute and research foundation between august and august . patients who received at least one year of treatment with s of s chemotherapy and/or radiation and surgery were included. medical history, family history of diabetes, cardiovascular diseases, and hypercholesterolemia, tanner staging, weight for height (< y per who criteria), bmi (> y per indian academy of pediatrics iap), blood pressure (nhlbi criteria), fasting blood sugar levels and lipid profile were obtained from the charts. statistical analysis of the data was done using ibm spss statistical software (version ). results: patients were studied, . % were male. . % were under years of age, . % between - years and . % above years. leukemia survivors comprised . % of the sample and non-leukemic's were . %. . % were treated with chemotherapy alone, . % with radiotherapy and chemotherapy, and . % underwent surgery with radiotherapy and chemotherapy. hypertension was found in . % of the study group, dyslipidemia in %, impaired fasting blood glucose in . % and . % were found to be obese. % of the study group was diagnosed with ms based on who criteria. conclusion: % of our study population was found to have ms per who criteria. individual metabolic complications were detected in % of the population. acute lymphoblastic leukemia (all) survivors appeared to be at high risk in our population. ms has been known to increase cardiovascular complications in cancer survivors. a multidisciplinary team approach to management of these patients is important to closely monitor and manage the long-term complications related to ms such as type diabetes and atherosclerosis. such an approach is essential to decrease long term morbidity and mortality from ms in this vulnerable population. background: the -year survival rate for childhood cancer exceeds %. however, up to % of these children require admission to the pediatric intensive care unit (picu) within three years of diagnosis. these children account for approximately % of all picu deaths, with mortality being higher for those post-hematopoietic stem cell transplant (hsct). national guidelines recommend that providers share informa-tion regarding prognosis and treatment options within the first hours of icu admission. these prognostic goals of care conversations (pgocc) are critical to the care of children with malignancies, a subpopulation at risk for increased mortality. to determine the frequency of pgocc as well as describe differences in patient characteristics and critical care therapies by pgocc status. design/method: a retrospective cohort study was conducted using the university of michigan virtual picu system database. picu admissions lasting longer than hours for patients ages to years between july , and june , with an oncologic diagnosis and/or hsct were identified. data on pgocc, patient demographics, diagnoses, picu interventions, and outcomes were recorded and compared between children with pgocc and those without using chi square test for categorical variables and kruskal-wallis test for continuous data. of picu admissions, % were male; the mean age was . years. the leading diagnoses were acute lymphoblastic leukemia ( %), acute myeloid leukemia ( %), lymphoma ( %), neuroblastoma ( %), and brain tumors ( %), and % of patients were post-hsct. pgocc was documented in ( %) patients. in comparison with patients who did not have a pgocc, children with a pgocc were more likely to be readmitted to the picu ( % vs. %, p < . ) and more likely to have had relapse of disease ( % vs. %, p< . ). patients with a pgocc had higher severity of illness scores (p = . ), higher use of non-invasive ( . % vs. . %, p = . ) and invasive conventional ventilation ( . % vs. . %, p< . ), and high frequency ventilation ( . % vs. . %, p < . ). also, patients with pgocc were more likely to receive continuous renal replacement therapy ( . % vs. . %, p< . ), arterial catheterization ( . % vs. . %, p< . ), and cardiopulmonary resuscitation ( . % vs. . %, p< . ). in only in critically ill children with hematologic-oncologic disease is pgocc held. children with pgocc were sicker and received more critical care interventions. future research is needed to evaluate the content of pgocc. background: central nervous system (cns) tumors and autism spectrum disorder (asd) represent significant disease cohorts in the pediatric population. asd diagnoses in children have a prevalence of %, in every children in the united states. additionally, more than , cns tumors are reported in children age to years in the united states with brain tumors being the most common solid tumor and the leading cause of death among all childhood cancers. the genetic etiology of autism and cns tumors is complex. specific gene alterations present in certain cancers have similarly been described and suspected to play a role in asd subtypes. targeted therapy panels, like foundation one (fo), have been beneficial in guiding treatment for some cancers based on distinct gene alterations. given the genetic overlap, the potential for therapeutic benefit and crossover from such actionable gene target panels merit further exploration in asd and cns tumors. we aim to identify and describe genetic alterations with known actionable targets in cancer therapy from fo as potential diagnostic, therapeutic and research targets for neurodevelopmental diseases. we plan to discuss the common genetic alterations between our cancers and neurodevelopmental diseases described in the literature. fo data was extracted and compared to the literature. each reported gene alteration from fo plus the keywords "autism", "psych" were used on pubmed to search for a suspected association if any with a neurodevelopmental disorder. results: twenty-one patients representing a cohort of six unique (astrocytoma-five, ependymoma-six, gbm-four, glioma-three, nerve sheath tumor-one, etmr-two) cns tumors were investigated. fo produced eighty total with sixty unique gene alterations. thirty-one ( %) of these yielded at least one published, suspected association to a neurodevelopmental disorder. the most common gene alterations were tp -four, cdkn a/b-five and braf-four. the main functional categories were cellular: proliferation, structure, differentiation and degradation; chromatin modeling; histone transcriptional modification; dna methylation and repair; strna; and neural signaling. sixty unique gene alterations were found in our cns tumor set using foundation one. thirty-one ( %) of these discrete alterations paired with at least one description in the literature as having been similarly altered in an asd subtype. many of these alterations have actionable targeted therapies presented through foundation one for our cns tumors and may be a relevant guide in the future of targeted therapy and research in asd subtypes. monoclonal antibody therapy usage is associated with significantly improved survival in b-cell nhl aya patients. although the usage has increased in the aya population from to , the magnitude of the increase is low. factors that affect the use of mab include race and insurance s of s type. further research is warranted to identify why privately insured patients are less likely to receive these drugs. background: prevention of chemotherapy-induced nausea and vomiting (cinv) remains a challenge despite advances in pharmacotherapy and the development of cinv clinical practice guidelines by the pediatric oncology group of ontario (pogo) that have been endorsed by the children's oncology group. achieving control of cinv in pediatrics further is complicated by the difficulty young children have vocalizing their symptoms. use of a validated nausea-assessment tool in conjunction with improved adherence to evidence-based guidelines may result in better quantification of symptoms and reduction of both nausea severity and vomiting frequency for pediatric patients undergoing chemotherapy. the pediatric nausea assessment tool (penat) has been validated for children ages - , and its integration into clinical practice may help optimize cinv control. objectives: this single-institution study sought to improve control of cinv in patients admitted for chemotherapy by standardizing the antiemetic regimens prescribed by all providers according to an institutional cinv algorithm developed from the pogo guidelines. we hypothesized that treatment using a standardized guideline would improve cinv control in patients admitted for chemotherapy. a baseline cohort of admissions for chemotherapy completed penat assessments and cinv diaries prior to receiving chemotherapy, four times daily during each admission, and daily for days following completion of chemotherapy from may , to january , . providers then were provided an institutional cinv treatment algorithm based on the pogo guidelines and received education at departmental meetings on appropriate implementation of this algorithm. a second cohort of admissions completed penat assessments and cinv diaries in a similar fashion from july , to december , . results: complete control of vomiting markedly improved following cinv guideline implementation ( % vs %, p <. ) with treatment failure also significantly reduced ( % vs %, p <. ). after controlling for the degree of emetogenicity of chemotherapy received, a patient was . times more likely to vomit prior to guideline implementation (or . , ci . - . ). there was no difference in nausea control, even after adjusting for the emetogenicity of chemotherapy. conclusion: control of chemotherapy-induced vomiting (civ) improved following widespread implementation of an institutional cinv treatment algorithm at a single institution. the severity of nausea reported remained unchanged which may reflect the difficulty of assessing nausea or an inadequate sample size. future research may focus on cinv treatment management through the use of guidelines specifically for breakthrough cinv and delayed cinv. background: aspho's professional development committee (pdc) recognized pediatric hematologists-oncologists (phos) serving in the united states (us) military have unique professional development needs that may not be addressed by aspho or a similar professional society. these individuals may also encounter challenges when transitioning to a civilian career. however, barriers to professional development have not been systematically characterized. the objectives were to characterize the number of phos with current or prior military service (mphos) and to identify any unmet professional development needs. design/method: a working group consisting of pdc members and both senior and early career mphos was formed. initial comments were solicited by email from known mphos regarding potential gaps in professional development and interest in working with aspho to improve support of mphos. a survey was developed and piloted with four members of the advisory group, questions were revised based on their feedback, and a final version was distributed via the aspho website and online community forum. targeted emails were sent to mphos identified through aspho and military databases. eligibility to complete the survey included ) completion of a fellowship in pediatric hematologyoncology, and ) current or prior service as an active duty military provider. quantitative and qualitative information were collected, including demographic data and perceived barriers to professional development. responses were summarized using descriptive statistics. results: sixty-five mphos were identified and surveys were completed for a % response rate. respondents were engaged in a variety of professional activities; % were male, % were serving active duty commitments, and % felt there were professional development gaps. areas of concern were categorized into nine themes with the most concerning being ) limited civilian knowledge of mpho practices ( % of participants), ) inability to attend professional society meetings ( %), and possibility of deployment ( %). participants expressed a desire for educational products to meet their specific needs and for networking opportunities with civilian colleagues. qualitative analyses identified concerns about low patient numbers and practice size. a subset of mphos perceive significant gaps in professional development. additional research is needed to better define areas for intervention, but many of the concerns align with those of similarly sized civilian programs and may be addressed through professional society networking opportunities, such as an aspho special interest group. background: infertility is an established cause of distress and has a negative impact on quality of life among childhood cancer survivors. the american society of clinical oncology has established guidelines on fertility counseling for individuals of reproductive age diagnosed with cancer, with the goal of improving reproductive and psychosocial outcomes. studies have shown that instituting a fertility team that can provide counseling and discuss fertility preservation (fp) options results in improved patient satisfaction in patients with cancer. objectives: the goal of this study was to examine predictors of referrals to the multidisciplinary fertility team, and documented fp interventions among these patients. design/method: an irb-approved retrospective medical record review was performed at a large pediatric academic center. all patients with new cancer diagnoses receiving chemotherapy were included from january (when the fertility team was established) to present. a standardized abstraction form was used to collect information about: age at diagnosis, gender, cancer type, whether a fertility consult was placed, and documented fp interventions. data were summarized descriptively and comparisons were made using nonparametric statistical methods. results: patients met inclusion criteria, of which ( %) were male. cancer types were as follows: leukemia/lymphoma, cns tumors, sarcomas, embryonal tumors, and langerhan's cell histiocytosis (lch). the mean age was . years, (range < - years). overall, % of all patients had a consultation with the fertility team. patients were significantly less likely to have a fertility consult if they were younger (p< . ). further, there were differences in the consultation rate between diagnoses, with % of sarcoma patients completing a consult, compared to % of those with cns tumors, % of those with embryonal tumor, % of those with leukemia/lymphoma and none of the patients with lch. our findings show that many children, adolescents, and young adults newly diagnosed with cancer are still not receiving fertility counseling despite: ) an expanding body of literature supporting the need to provide this counseling, ) guidelines published by several organizations recommending discussions about infertility risk and fp options, and ) presence of a multidisciplinary fertility team. specific strategies need to be developed to improve access for younger children, and for disease groups in whom fertility consults are underutilized, such as youth with cns tumors, embryonal tumors, and leukemia/lymphoma. background: socioeconomic status (ses) has on impact on overall survival in the pediatric oncology population. unfortunately, data are insufficiently detailed to explain the mechanism behind this phenomenon. how parents handle the health management demands placed on them at the time of a child's cancer diagnosis may represent a point of differentiation in health outcomes. objectives: determine the association between socioeconomic factors, cancer literacy, and parents' understanding of home emergency management and their responses to instances of pain, nausea, and fever. in a prospective observational study of parents whose children were newly diagnosed with cancer, we obtained demographic information and, using a validated instrument, (dumenci, ) we evaluated cancer literacy. we tested understanding of the education parents received about home emergency management with a -item multiple-choice vignette-based questionnaire focused on actions needed in home scenarios. we then followed parents' actual behavior through periodic phone calls assessing instances of nausea, pain, and fever and their responses to these episodes. results: preliminary analysis of participants showed an average score of on the -item parental understanding questionnaire (range - ). variables associated with increased score were college-level education by . points ( % ci [. to . ]), private insurance by . points [. to . ] and adequate cancer literacy by . points [. to . ]. actual behavior reported by families indicated that married parents and those with income above $ , were less likely to treat instances of pain by % ( % ci [ to ]) and % [ . to ], respectively. white parents, those with college-level education, and those with adequate cancer literacy were less likely to treat instances of nausea by % [ to ], % [ to ] and % [ to ], respectively. no associations were found between socioeconomic markers and parental responses to instances of fever. our findings suggest an association between demographic and socioeconomic markers and improved parental understanding of home emergency management. paradoxically, the same markers show a decrease in treatment response to pain and nausea. larger prospective studies are needed to link this behavior pattern to health outcomes, and help inform the extent of ses impact on home emergency management. emory university/children's heathcare of atlanta, atlanta, georgia, united states background: cardiovascular disease is a leading cause of morbidity and mortality in childhood cancer survivors (ccs). previous research showed wide practice variation in referral patterns to cardiology from the survivor clinic and in recommendations from cardiologists about the need for further testing or exercise restrictions. to develop a cardio-oncology algorithm in order to standardize referrals to cardiology and provide guidelines for cardiologists evaluating pediatric ccs. design/method: survivorship and cardiology experts developed a weighted scoring system for pediatric ccs who received cardiotoxic therapy based on time since treatment and risk factors identified by the children's oncology group (cog) and american heart association (aha). the cardiooncology algorithm assigned a score of - . the score range was categorized to guide cardiology referral: screening echo only ( - ), consider cardiology referral ( - ), recommend cardiology referral ( - ), and regular cardiology follow-up (≥ ). the algorithm also provides recommendations to cardiologists for screening and exercise modifications based on the score. after establishment of the algorithm, a convenience sample of institutional survivor clinic patient charts were retrospectively reviewed from the first month of each quarter from april -march to validate the algorithm, evaluate referral patterns to cardiology, and assess cardiology recommendations. the retrospective chart review evaluated patients ( % male; % non-hispanic white; % leukemia survivors; median age at diagnosis . years [range - . ]; median time off-therapy . years [range . - . ]). patients ( %) received anthracyclines (median dose mg/m , range - ) and ( %) received cardiac radiation. assigned cardio-oncology scores resulted in: % echo only, % consider cardiology referral, % recommend cardiology referral, and % regular cardiology followup. when evaluating detection rates of late effects by cardiooncology score, survivors ( %) had an abnormal echo: / echo only, / consider referral, / recommend referral, and / regular cardiology follow-up. assessing referral patterns prior to initiation of the algorithm revealed forty-two survivors ( %) referred to cardiology: / echo only, / consider referral, / recommend referral, and / regular cardiology follow-up. of the patients seen by a cardiologist at our institution, had further diagnostic testing ordered (i.e., stress test) and received exercise restrictions. a cardio-oncology algorithm and guidelines will standardize cardiac care for survivors by assigning a score to guide referral and cardiology practice after referral. prospective clinical use has begun and review will occur in one year to determine changes in detection rates of cardiac late effects, referrals, and recommendations from cardiologists. oregon health and science university, portland, oregon, united states background: delirium affects - % of patients (pts) in pediatric intensive care units (picu) and is associated with increased length of stay, decreased attention in school, and post-traumatic stress disorder. the diagnostic and statistical manual of mental disorders (dsm v) defines delirium as a "disturbance of consciousness […] with reduced ability to focus, sustain or shift attention" due to an underlying medical condition. despite the medical complexity of the hospitalized pho population, there are no published prospective studies looking at delirium in these pts. hypothesizing that delirium is under recognized in the pho population, we designed a year-long prospective study using a validated screening tool to determine the frequency of delirium in hospitalized pho pts and to identify associated clinical factors. design/method: baseline frequency of pts with symptoms suggestive of delirium was determined through retrospective chart review using a data mining program of electronic medical records (emr). for the prospective study, pho and picu nurses were trained to use the cornell assessment for pediatric delirium and to record scores within the emr on all pho pts once every -hour shift. predetermined demographic and clinical variables were entered daily into a red-cap database on all hospitalized pho pts. results: baseline frequency of delirium, without active screening, was determined to be . % of hospitalized pho pts. in the first months of the prospective study, consecutive admissions occurred among unique pho pts: oncology, hematology, and stem cell transplant pts. pts had at least positive delirium screen, for a prevalence per admission of . %. statistically significant variables associated with delirium, at p < . by univariate logistical regression, included prolonged length of stay, pt location (picu vs pho unit), and fever. adjusting for length of stay, administration of benzodiazepines and opiates were also significantly associated with delirium, p = . and . , respectively. on average, nurses completed delirium screening in % of each pts' -hour shifts. study accrual ends in jan and final data analyses will be reported in the abstract presentation. conclusion: delirium does occur in the pho hospitalized population and screening by trained nursing staff is feasible. pts at highest risk appear to be pts with prolonged hospital stays, picu admissions, or frequent use of benzodiazepines/opioids. routine screening should improve our recognition of delirium and allow us to promptly intervene, or prevent delirium in an effort to avoid potential acute and long term consequences. background: with high survival rates for children and adolescents with hodgkin lymphoma (hl), treatment regimens are now designed to maximize cure while decreasing risk of long-term health outcomes associated with chemotherapy and radiation therapy. within contemporary treatment regimens, the comparison of toxicities experienced by patients receiving chemotherapy plus radiotherapy (crt) versus only chemotherapy (co) has not been studied extensively. objectives: this study examines select self-reported adverse health outcomes in survivors of contemporarily-treated pediatric hl to better understand the balance between efficacy and toxicity associated with chemotherapy and radiation therapy. (cog) ahod that evaluated a response-based treatment paradigm in pediatric hl. patient who received initial chemotherapy were randomized based on early response to continued chemotherapy, chemotherapy plus radiotherapy or augmented chemotherapy plus radiotherapy. patients completed self-report questionnaires on health problems at , , , and years following therapy. we examined selected patient-reported pulmonary, gastrointestinal (gi), cardiac and endocrine outcomes. kaplan-meier survival curves were used to determine probability of survival without the selected adverse health outcome. log-rank tests were used to compare the co versus the crt group. results: a total of , enrolled patients, patients in the co group and patients in the crt group, completed , questionnaires at a median of . years after s of s completion of therapy (q , q : . , . ) which were analyzed. the cumulative -year incidence of endocrine dysfunction was significantly greater in the crt group versus those in the co group ( % versus %; p< . ), driven by the incidence of hypothyroidism ( % versus %; p< . ). there were no significant differences in cardiac ( % versus %; p = . ), pulmonary ( % versus % p = . ), and gastrointestinal dysfunction ( % versus %; p = . ) between the co and crt patients. conclusion: this study demonstrates low cumulative incidence overall of organ dysfunction early post completion of contemporary therapy for hl. the addition of radiation therapy significantly increased risk for hypothyroidism, but with no higher risk noted for cardiac, pulmonary or gi dysfunction. limitations include self-report status, potential selection bias, and relatively short latency period following end of therapy. longer follow-up is needed to determine more delayed risks for organ dysfunction in order to best define the balance between therapeutic efficacy and long-term adverse health outcomes related to chemotherapy and/or radiation therapy. background: identification of an organism via bronchoalveolar lavage (bal) or respiratory tract biopsy (rtb) has historically been considered the gold standard for diagnosis of invasive fungal infection (ifi); however, data previously published by our group showed that these procedures infrequently lead to a change in management in children with an oncological diagnosis or undergoing hematopoietic stem cell transplant (hsct). there is also a paucity of data on the cost of ifi in this population. to compare the costs of work-up and management of pulmonary ifi diagnosed based on ct scan alone versus ct scan or chest x-ray prompting a bal or rtb. design/method: we collected cost data on patients at ann & robert h. lurie children's hospital of chicago undergoing chemotherapy or within months of hsct who were suspected of having an ifi between and . in order to include sufficient time to account for post-procedure compli-cations but avoid including costs unrelated to ifi, data were included for days from the day of their diagnostic scan or procedure. cost data was available for of the patients previously studied. thirty-six of these patients were diagnosed with suspected ifi based on ct only and patients underwent bal or rtb. when evaluating specific costs, inpatient beds costs were higher in the bal and rtb group (median $ , versus $ , , p = . ), yet there was only a trend towards higher costs for antifungal agents (median $ , versus $ , , p = . ) and respiratory support (median $ versus $ , p = . ). many of the initial ct scans were not captured in the -day evaluation period for the bal or rtb group based on the study design; however, even when accounting for ct scans up to a week prior these procedures, the total cost of ct scans was higher in the ct only group (median $ versus $ , p = . ), as they had more scans. despite this, total costs were significantly higher for patients who underwent bal or rtb versus ct scan only (median $ , versus $ , , p < . ). combined with our previous data that bal and rtb infrequently leads to a change in management in children with an oncological diagnosis or undergoing hsct suspected to have an ifi, the significantly higher costs associated with these procedures makes these invasive diagnostic techniques even less desirable. batra, pediatr blood cancer, . background: while infants > months of age with acute lymphoblastic leukemia (all) have a poor prognosis, infants with acute myeloid leukemia (aml) fare better despite more intensive therapy. there are limited data on this difference, particularly differences in supportive care requirements during induction therapy for infants. objectives: to compare induction mortality and resource utilization in infants relative to non-infants aged < years, separately for all and aml. design/method: we used previously established cohorts of children treated for new onset all or aml at children's hospitals in the us contributing to the pediatric health information system. patients with down syndrome were excluded. follow-up started on the first day of induction chemotherapy and continued until the earliest of: days after commencement of chemotherapy, start of the subsequent course, or death. high acuity of presentation, defined as icu requirements involving or more organ systems within the first hours following initial admission were compared using log binomial regression. -day inpatient mortality was compared using cox regression. resource utilization rates (days of use per inpatient days) were compared using poisson regression. results: a total of all ( infants, non-infants) and aml ( infants, non-infants) were included in the analyses. infants were more likely to present with high acuity compared to non-infants for both all ( % and %, rr = . , % ci: . , . ; p< . ) and aml ( % vs %; rr = . , % ci: . , . ; p = . ). infants with all had higher inpatient mortality compared to non-infants even after accounting for differences in acuity of presentation ( . % vs . %, adjusted hr = . % ci: . , . ; p = . ). in contrast, inpatient mortality was more similar for infants and noninfants with aml ( . % vs . %, adjusted hr = . % ci: . , . ; p = . ) and comparable to rates among infants with all. infants with all and aml had higher rates of utilization of fresh frozen plasma, cryoprecipitate, diuretics, supplemental oxygen, and ventilation relative to non-infants. infants with all also had higher rates of total parenteral nutrition, ecmo, and patient controlled analgesics compared to noninfants. infants with all experienced significantly higher induction mortality compared to noninfants, a difference not entirely explained by acuity at presentation. differences in ru among infants may reflect higher presentation acuity and greater treatment related toxicity. further work is needed to elucidate the contribution of treatment related toxicity to early mortality in infants with all. background: fever in a child with cancer is a medical emergency due to the significant risk of a serious bacterial infection. many attempts have been made to risk stratify these patients. the respiratory pathogen panel (rpp) is a panel of polymerase chain reaction tests that identify seventeen common respiratory viruses and three bacterial infections. samples are taken via nasopharyngeal swab. rpps are frequently sent, but we do not have data to determine whether a positive result can lead to stratification to a lower risk of bacterial infection. ( ) to determine the epidemiology of respiratory virus-associated fever in pediatric oncology patients ( ) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all pediatric oncology patients seen in our emergency department (ed) with fever from the introduction of the rpp in april to september , . we reviewed the results of blood cultures, rpp, chest radiographs, and discharge summaries to identify sources of infection. we also identified the patients' cancer diagnosis, age, absolute neutrophil count (anc), and absolute lymphocyte count (alc). results: positive rpps were found among pediatric oncology patients who presented to the ed with fever. the most common positive rpp findings were rhinovirus/enterovirus (rev) ( %), parainfluenza ( %), influenza ( %), coronavirus ( %), and polyviral ( %). among patients with a positive rpp, % had bacteremia compared to % bacteremia among all pediatric oncology patients with fever (or . [ . - . ], p . ). all cases of bacteremia were associated with rev. there was no bacteremia identified in patients with rpps positive for other viruses (or . [ . - . ], p . ). rev positivity did not confer a lower risk of bacteremia than rpp negative patients ], p . ). anc (p = . ) and alc (p = . ) less than , and number of patients with severe neutropenia (p = . ) were not statistically different between the rev and non-rev positive rpp groups. rpps positive for viruses other than rev reduced the likelihood of bacteremia in febrile pediatric oncology patients in the ed setting. patients with bacteremia may have concurrent infection with rev. a larger study is warranted to determine if positive rpp results can inform clinical management of a child with febrile neutropenia. emily mueller, anneli cochrane, seethal jacob, aaron carroll s of s background: the usage of mobile health (mhealth), which refers to the application of mobile or wireless communication technologies to health and healthcare, has grown exponentially in recent years. mhealth tools have been used by caregivers of other vulnerable populations, but little has been focused on caregivers of children with cancer. objectives: to conduct a survey to understand the mobile technology usage, barriers, and desired mhealth tools by caregivers of children with cancer. we conducted a mailed cross-sectional paper survey of caregivers of all children who were diagnosed with cancer at riley hospital for children between june, and june, . the survey contained questions, both fixed and open-ended, in both english and spanish. up to three rounds of surveys were sent to those who did not respond. of the respondents, they were primarily parents ( . %), median age was . years (range - ), and most were white ( . %) and non-hispanic/latino ( . %). the top three annual household income brackets included $ , to $ , ( . %), $ , to $ , ( . %) and under $ , ( . %). the majority had an education: . % college graduates, % graduate degree, and . % high school education or ged. nearly all respondents owned a smart phone ( . %) and . % owned a tablet. the majority used an ios operating system ( . %), while . % reported use of a device with an android operating system. all caregivers reported use of at least one mobile website/app regularly for their personal use. while . % of respondents reported no barriers to mobile technology use, the top barrier selected was "data limitations" ( . %). overall, . % wanted at least one medical managementrelated website/app: medical knowledge ( . %), healthcare symptom tracking/management ( . %), and medication reminders ( %). healthcare system-related desires were high, as . % wanted access to their child's medical record and . % wanted a website/app to facilitate better communication with medical providers. there were no significant associations between socioeconomic status (income or education) with barriers or types of websites/apps desired by caregivers. since the vast majority of caregivers use mobile technology with minimal barriers, future research should focus on designing an mhealth tool to address the medical management needs by caregivers of children with cancer. by supporting caregivers through this type of mhealth tool, it could positively impact patient clinical outcomes through greater adherence to medications and treatment protocols. background: in children with fever and neutropenia, early initiation of targeted antibiotic therapy improves outcomes, yet there are no standards for choice of empiric antibiotics. in our institution implemented an early empiric ceftriaxone (eec) protocol to reduce time to antibiotic administration in febrile hematology-oncology patients who are potentially neutropenic when the absolute neutrophil count is not yet know. ceftriaxone is given immediately after obtaining blood for culture and lab studies. in patients found to be neutropenic, ceftriaxone is discontinued and cefepime is initiated. the purpose of this retrospective study was to evaluate our eec protocol in neutropenic patients by assessing ceftriaxone sensitivity of positive blood cultures and comparing rates of adverse outcomes with a cohort of patients treated prior to implementation of the protocol. we are now conducting a prospective study to more thoroughly investigate antibiotic sensitivities of organisms isolated from blood cultures of neutropenic patients. design/method: hematology-oncology patients with at least one positive blood culture between january and december were identified. patient demographics, neutrophil count, antibiotic treatment, isolated organisms and sensitivities, and adverse outcomes (increased respiratory support, hypotension requiring intervention, and icu admission) were obtained by retrospective chart review. fisher exact test was used to compare dichotomous variables between patient groups. we are now prospectively identifying febrile neutropenic patients with positive blood cultures and performing antibiotic sensitivity testing to several antibiotics commonly used as empiric therapy for febrile neutropenia. results: retrospectively, we identified neutropenic patients with a total of bacterial isolates from blood cultures. of organisms isolated, were tested for sensitivity to ceftriaxone and ( %) were not sensitive, / ( %) of gram-positive cultures and / ( %) of gram-negative cultures. ten of ( %) eec patients had an adverse outcome versus / ( %) of non-eec patients (p = . ). notably, % of eec patients required icu admission versus % of non-eec patients (p = . ). thus far our data obtained prospectively is revealing similar rates of ceftriaxone resistance with / cultures not sensitive to ceftriaxone ( %, ci . %- . %). in our retrospective study, no statistically significant difference was seen in overall adverse outcome rate between the two cohorts, though icu admission rates were significantly higher in eec patients. ceftriaxone resistance rates were high in tested isolates, which is further supported by preliminary data from our ongoing prospective study. given these data, eec may not be effective at improving outcomes in febrile neutropenic pediatric hematology-oncology patients. background: approximately in children diagnosed with cancer will die of their disease, despite advances in treatment. results: two focus groups of six parents each met in june . the parents were predominantly female ( female, male) and had lost their children an average of . years prior (range - . years). two parents were in the same family. nearly all patients were offered palliative care ( / ), all were offered hospice and most died at home ( at home, in the icu). parent discussion uncovered six broad themes: beneficial provider qualities, optimal communication, helpful systematic supports, struggles to feel like a good parent, struggles with a loss of control and unmet needs. parents appreciated providers who were consistent, reliable and honest. parents desired communication that was sensitive to the needs of the patient and family with a balance of hope and realism. parents appreciated the tangible supports pro-vided by social work and the emotional support of child life both for the patient and their siblings. some parents struggled to define and advocate for their child's quality of life, especially when it led to disagreeing with the medical team. several parents expressed frustration with unfamiliar caregivers in the hospital, especially trainees. they expressed a strong desire for more anticipatory guidance about the end of life including how to discuss it with their children. they also wished for a cancer-specific support group for bereaved parents. conclusion: bereaved parents of pediatric oncology patients in our focus groups appreciated consistent, reliable providers who communicated with a balance of realism and hope. they appreciated the tangible and emotional support they received and wanted more anticipatory guidance at the end of their child's life. these results can help guide clinical care, especially in communities without strong palliative care support. further research is needed to develop interventions to improve end of life care. background: clinical trials involving human subjects depend on informed consent (ic) to ensure ethical protections for participants. parents of children with cancer often lack full understanding of the basic elements of ic for clinical trials. additionally, the stress of their child's cancer diagnosis may affect their decision-making capabilities. this is especially problematic as these children rely on parents to fully comprehend clinical trials and weigh their benefits and risks. physician communication is critical for effective family-centered care. the acgme mandates that training programs teach and assess trainees' communication skills. however, there are currently no published curricula aimed at training pediatric hematology/oncology fellows to deliver ic effectively for cancer clinical trials. to develop and pilot-test a simulation-based curriculum to enhance communication skills of pediatric s of s hematology/oncology fellows in the delivery of ic for cancer clinical trials. we developed, tested, and implemented the curriculum from to in two phases. in phase- , we reviewed literature on simulation-based curricula and completed a needs assessment to create a clinical scenario and full curriculum using standardized patients. using miller's pyramid model, fellows' assessments included: immediate de-brief, surveys to assess pre/post confidence and knowledge of the basic ic elements ("knows" and "knows how"), and -degree summative assessments compiled from fellow self-assessments, faculty, and standardized patients ("shows how"). after initial testing and refinements done with fellow, in phase- , we implemented the curriculum with our fellows. likert scale ( strongly disagree- strongly agree) and basic p values are reported. results: fellows gave high mean ratings for training relevance ( . ) and standardized patients' preparedness ( ). almost all ( . ) reported they have used the knowledge gained in their clinical practice. increase in self-reported confidence (pre/post) was noted in all domains: general -describing possible benefits of the clinical trial . / vs. . / (p = . ), risks and potential side effects . / vs. . / (p = . ), and explaining alternatives . / vs. . / (p = . ); research -discussing purpose of the clinical trial . / vs. . / (p = . ), and randomization . / vs. . / (p = . ); and family-centered -addressing emotions during ic . / vs. . / (p = . ), and delivering bad news . / vs. . / (p = . ). summative evaluation mean ratings for all fellows were . (range . - . ). our novel simulated-based ic curriculum, significantly increased fellows' self-reported confidence and skills during ic delivery. importantly, our ic curriculum addressed not just research-related content but also management of parental emotional needs during the ic discussion. next phase includes kirkpatrick model program evaluation and dissemination across other training programs in our institution. national kaohsiung normal university, kaohsiung, taiwan, province of china background: taiwan's childhood cancer foundation reported in that the -year survival rate of childhood cancer was %. as a result, many childhood cancer survivors were back in school after treatment. however, childhood cancer survivors' educational outcomes suffered because of their long-term absence from school and late effects of cancer and cancer treatment. a few school reentry protocols have been developed by the nursing professionals in taiwan to facilitate students' return to school but remained experimental in nature and hardly accessible. parents, students, and teachers were left to their own devices to make individual school reentry plans. objectives: this study aimed to examine and uncover the commonalities among three middle school students' successful school reentry experiences from their teachers' perspectives and to analyze the factors contributing to their success. design/method: this is a qualitative interview study. indepth semi-structured interviews were conducted with three middle school teachers in december about their perceptions, observations, and experiences working with adolescent childhood cancer survivors. the students were two boys with leukemia and one girl with bone cancer. they were diagnosed in the first year of middle school when they were - years old and returned to school for the third and the final year. these students met the following criteria for successful school reentry: regular school attendance, average/above average academic performance, friendship maintenance, and high school diploma. the theme -bring the class to the hospital was found to be the key to the adolescents' successful return to school. without a prescribed school reentry protocol and in the face of limited bedside education services, the homeroom teachers, as links between school, home, and hospital, brought the class to their hospitalized students. they doubled as bedside teachers conducting lessons at the hospital or students' homes, became friends with the parents, witnessed firsthand the students' pain and triumph during treatment, brought the students back to school for visits and celebrations, delivered the classmates' wishes and news to the students, encouraged and welcomed classmates' visits to the hospital, and, together with parents and other teachers, developed flexible school reentry schedules for the students. this on-going study demonstrated the critical roles and functions of homeroom teachers in successfully bringing the students back to school during and/or after cancer treatment. further analysis will be focused on how and why these three homeroom teachers were able to carry out this unexpected task on top of their already full workload. jennifer kesselheim, shicheng weng, victoria allen, collaborative group fellowship program directors dana-farber/boston children's cancer and blood disorders center, boston, massachusetts, united states background: a novel, -module, case-based curriculum entitled "humanism and professionalism for pediatric hematology-oncology" (hp-pho) aims to foster pho fellows' reflection on grief and loss, competing demands of fellowship, difficult relationships with patients and families, and physician well-being and burnout. in small group facilitated sessions, fellows work to identify coping strategies and explore how the challenges of fellowship influence both their own doctoring and the patient experience. objectives: to administer the hp-pho curriculum in a prospective, cluster-randomized trial, measuring whether exposure to this educational intervention, compared to standard conditions, fosters humanism and professionalism and improves satisfaction with training. design/method: pho fellowship programs (n = ) were cluster-randomized to deliver usual training in humanism and professionalism (control) or the novel curriculum (intervention) during the - academic year. the primary outcome measure was the pediatric hematology-oncology self-assessment in humanism (phosah). secondary measures included a -point satisfaction scale, the maslach burnout inventory (mbi), the patient-provider orientation scale, and the empowerment at work scale. participating fellows were pre-tested in summer and post-tested in spring . a change score was calculated for each study instrument. we compared each outcome between arms using mixed effect models adjusted for pre-test score as a fixed effect and site as a random effect. results: randomization yielded intervention and control fellows. the two arms did not significantly differ in distribution of fellow age, gender, or post-graduate year. the intervention sites successfully administered of ( %) modules. change scores on the phosah were not significantly different between the control and intervention arms (adjusted mean difference = . ; % confidence interval [ci] - . , . ; p = . ). compared to the control arm, fellows' exposed to the curriculum gave significantly higher ratings on several items within the satisfaction scale including satisfaction with their training on "physician burnout" (adjusted mean difference = . ; % ci . , . ; p< . ), "physician depression" (adjusted mean difference = . ; % ci . , . ; p< . ), "balancing professional duties and personal life" (adjusted mean difference = . ; % ci . , . ; p = . ), and "humanism overall" (adjusted mean difference = . ; % ci . , . ; p = . ). change scores on other secondary measures were not significantly different between study arms. conclusion: exposure to the hp-pho curriculum did not alter fellows' self-assessed humanism and professionalism. however, the curriculum proved feasible to administer and intervention fellows expressed higher levels of satisfaction in their humanism training, indicating the curriculum's positive impact both for fellows and their learning environment. background: recent work has documented significant levels of unmet needs among adolescents and young adults with cancer, particularly psychosocial challenges during the transition to adulthood, (e.g., abrupt disruption to school and social life, and social isolation). given that adolescents and young adults drive mobile app use, a mobile-phone may be an ideal way to deliver a psychosocial intervention to adolescents and young adults with cancer. to use a patient-centered approach to inform a mobile-based mindfulness and social support intervention for adolescent and young adult patients with cancer. design/method: participants were ten aya with sarcoma ( % female; % adolescents); parents of the five adolescents, and six healthcare providers (n = ). formative research involved three steps: ( ) in-depth interviews were conducted with ten aya with sarcoma; parents of the five adolescents, and six healthcare providers (n = ). ( ) adaptations were made to an existing mindfulness app which offers a program for youth. modifications included creating a -week "mindfulness for resilience in illness" program, with relaxation exercises, and the addition of videos featuring two sarcoma survivors as program hosts. content was informed by the mindfulness curriculum for adolescents, learning to breathe. ( ) a private facebook usability group was organized to (i) elicit beliefs about the mindfulness app and potential future enhancements, and (ii) promote social support. results of the in-depth interviews revealed themes around adolescents' functioning and coping, including body image concerns; recurrence-related anxiety; anger over loss; and being overwhelmed by medical information. themes from the interviews were incorporated into a demonstration version of the mobile app. a patient-centered approach is widely recommended in the development of mobile-based health behavior change interventions and may be a useful way to inform development of a mobile-based mindfulness and social support intervention for adolescents and young adults with cancer. background: medical trainees consistently report suboptimal instruction and poor self-confidence in communication skills. despite these deficits, few training programs provide comprehensive pediatric-specific communication education, particularly in the provision of "bad news." an in-depth survey to examine the historical experience and communication needs of pediatric fellows was conducted at a large academic pediatric center as the first step towards the development of a comprehensive communication curriculum. to determine the previous educational and clinical experiences of pediatric subspecialty fellows, assess their levels of comfort in the context of various communication topics, and query potential modalities and topics for future communication training. design/method: the needs assessment survey was developed using previously developed and validated questions and review of the literature. the survey was reviewed by internal and external pediatric oncology and palliative experts and pre-tested with a subset of trainees to enhance content validity. results: thirty-two out of a total of fellows completed the survey ( % completion rate), of which % were pediatric hematology-oncology or subspecialty fellows. most fellows had participated in previous teaching sessions ( %), including those involving role play or simulation ( %). however, few fellows had received feedback from senior clinicians on their communication skills ( % of fellows had received feedback ≤ times). on a scale of -x, with indicating "not well prepared," the mean score for of communication items was < . fellows felt least prepared to lead discussions around informed consent for experimental therapies, end of life care, and autopsy. fellows indicated that didactic educational sessions and additional coursework were less useful strategies for improving their communication skills, whereas small group role play sessions with faculty and/or bereaved parent educators were most useful. fellows' overall communication preparedness score was not correlated with post-graduate year but was positively associated with the number of times they previously had delivered bad news to patients and families. fellows requested additional training on many topics, with greatest interest in learning skills to optimize communication with an angry patient or family. additional topic requests included placing limitations on resuscitation, withdrawing/withholding further therapy, and ageappropriate inclusion of patients in difficult discussions. despite self-report of prior communication skills training, pediatric subspecialty fellows felt underprepared to participate in difficult discussions with patients and families. learners identified role-playing and coaching with real-time feedback from other physicians and bereaved parents as more useful training strategies as compared to didactic sessions. background: when children die of cancer, parents must adjust to their child's absence amidst the lingering turmoil of what preceded their death: witnessing their child undergo painful treatments, making difficult decisions, and anticipating a devastating loss, all the while hoping for a recovery. adjustment to a child's death, as depicted by current bereavement literature, necessitates making meaning of one's loss. professional care staff can help parents make sense of their child's illness, and in turn, of their own parental experience during treatment. however, the extent to which relationships with professional care team members influence parents' ability to make sense of, and successfully cope with, their loss has not been examined. objectives: to examine how bereaved parents' interactions with their deceased child's pediatric oncology professional care team have impacted their grief symptoms design/method: to better understand how interactions with professional care staff relate to parents' grief outcomes, we conducted a mixed-methods study examining staff impact on parental grief. thirty participants whose children died of cancer one to three years ago completed an in-depth interview and psychometrically validated surveys measuring meaningmaking, depression, and grief symptoms. results: correlational analyses of the measures found that an increase in meaning making was associated with lower depressive and grief symptoms. a content analysis of the interviews found that many participants regarded staff "like family," had on-going relationships with staff after their child died, and described various ways staff interactions during treatment and after the child's death helped them make sense of their loss. in particular, participants described how interactions with staff have helped them find benefits in their loss and learn to create a new relationship with their child despite their physical absence. quantifying the interview data and statistically analyzing it along with the measures found that participants' increased frequency of describing staff's positive impact on their grief correlated with higher meaning-making scores and lower grief symptom scores. our study found that bereaved parents who lost their children to cancer were articulate in sharing their experiences of staff engagement and communication during treatment, offering numerous examples of how staff aided them in making meaning of their loss that were reliably associated with their subsequent grief. we hope the results of this mixed methods research encourage further study of the importance of staff interaction with families during the critical period of their children's care, and the lasting impact this can have regardless of the treatment outcome. memorial sloan kettering cancer center, new york, new york, united states background: although resiliency has been recognized as necessary for healthcare professionals, trainees feel unprepared for the emotional challenges inherent in caring for sick and dying patients. compounded by long hours, challenging work environments, and lack of formal training on handling emotionally difficult situations, many institutions are recognizing the need for interventions to reduce trainee distress. the goals of this fellow-led quality improvement initiative were: ) to determine whether there is a need for emotional support amongst pediatric hematology and oncology fellows, ) to provide formal resiliency and debriefing sessions, and ) to measure feasibility, acceptability and effectiveness of implemented curriculum. design/method: an anonymous survey to determine need for resiliency and debriefing sessions following a traumatic event was distributed to active pediatric hematology & oncology fellows at memorial sloan kettering cancer center in january . once need was established, an intervention consisting of a formal curriculum was developed and initiated in june , involving: ) scheduled and ad hoc debriefing sessions in response to traumatic events (including patient death, codes, interpersonal conflicts, end-of-life care); led by a psychiatrist and social worker with fellows and a pediatric oncologist mentor in attendance, and ) a resiliency didactic curriculum, led by a palliative medicine specialist, focused on skills such as contesting cognitive distortions and mindfulness. the effectiveness of these sessions will be measured using follow-up anonymous surveys at months (currently underway) and months post-initiation of intervention. the initial survey demonstrated most trainees ( / ) were present at or more deaths during their training, while less than half of respondents had attended a post-event debriefing session. % of respondents felt there was not sufficient emotional support from the institution for physicians caring for dying patients. a separate pre-intervention survey found all respondents ( / ) expressed a need for regular debriefings, and nearly all anticipated that they would benefit from such debriefings. concerns identified by trainees that would preclude participation in the curriculum included preference to deal with emotional situations privately and time constraints. trainees identified a need for formal debriefings and resiliency skill development. the program was easily implemented, and is both feasible and acceptable with good attendance. feedback received at the -month mark will determine deficits and possible improvements to the curriculum. the -month survey will measure effectiveness of the program and whether it should be continued. background: acute kidney injury (aki) is a common but under-recognized complication among patients with leukemia. it is associated with prolonged hospital stays, increased mortality, progression to chronic kidney disease, and delays or changes in cancer therapy which may affect a patient's prognosis. however, data on aki in pediatric patients with cancer is still lacking overall. we investigated the incidence of aki in patients who were newly diagnosed with all at our center from january to september . we performed a retrospective chart review of all patients who were newly diagnosed with all from neonate to years in our facility. we determined the incidence of aki in our population using the kidney disease: improving global outcomes (kdigo) diagnostic criteria. we also assessed for nephrotoxic exposures, nci all risk stratification and risk of aki, and tumor lysis syndrome (tls). we identified patients diagnosed during the study period who met inclusion criteria. median follow-up time was . months (range . - . ). the cohort was predominantly male ( . %) and hispanic ( . %). our analysis showed . % had aki by kdigo criteria ( % grade , . % grade , and % grade ), . % had aki on presentation, and % had multiple aki episodes during the study period. older age and longer length of hospitalization were associated with aki (p = . and p = . , respectively). there was no association between aki and nci all risk classification, contrast exposure, hyponatremia, elevated white blood cell count, uric acid levels, antimicrobial therapy, or diuretic use in this study. conclusion: aki was a common finding in our study population. the majority had grade aki by kdigo criteria. however, aki was associated with older age and a longer length of stay. further study is needed to determine the short-and long-term impact of aki on pediatric patients with all. st. jude children's research hospital, memphis, tennessee, united states background: in some regions, the availability of trained pediatric oncologists is a limiting barrier for the care of children with cancer. in , the unidad nacional de oncología pediátrica (unop) and the universidad francisco marroquín school of medicine in guatemala established a pediatric hematology/oncology fellowship program sponsored by st jude children's research hospital to provide central america and the caribbean with well-trained specialists. a systematic analysis of the impact of fellowship programs in pediatric oncology has never been done, especially in the context of a regional education program. objectives: this study sought to analyze the impact of the unop fellowship program based on the regional number of providers, pediatric cancer centers and patient volume. in addition, it sought to characterize the jobs and scientific output of the graduates. the impact will be evaluated in the context of a cost analysis. to define the volume of providers, pediatric cancer centers and patients, the directors of pediatric cancer centers in central america were sent an online survey to obtain these data. all the centers contacted maintain an updated hospital-based patient registry. in addition, the graduates of the fellowship program were also sent an online survey, asking about their job at graduation, current role and scientific productivity. the cost analysis will include assessment of direct costs including salaries and stipends for away rotations, as well as the indirect costs of faculty time spent teaching. since the establishment of the unop fellowship program, the region has more providers for pediatric cancer (p< . ) and centers treat a larger volume of patients (p< . ). two new centers have opened with graduates of the program. all but one graduate practice pediatric oncology ( / ) and the majority do it in their country of origin ( / ). no graduate practices outside of this region. almost half of the graduates ( %) hold a leadership role at their institution. the majority of their time is spent in the public sector (> %). the majority of graduates participate in clinical research ( %) and have participated in the creation or implementation of therapeutic protocols ( %). on average, the graduates have published peer-reviewed articles since completion of training. the unop fellowship program has had a favorable impact on pediatric cancer care in the region, contributing to the capacity to treat a larger volume of patients. graduates practice pediatric oncology in the region in the public sector, frequently hold leadership roles and are scientifically productive. background: abandonment of treatment is a major cause of treatment failure and poor survival in children with cancer in low-and middle-income countries. the incidence of abandonment in peru has not been reported. objectives: the aim of this study was to examine the prevalence and associated factors of treatment abandonment in pediatric patients with cancer of peru. we retrospectively reviewed the sociodemographic and clinical data of children referred between january and december to the two main tertiary centers for childhood cancer, located in lima, peru. definition of treatment abandonment was used from the siop (international society of paediatric oncology) podc (paediatric oncology in developing countries) abandonment of treatment working group recommendation. results: data of children diagnosed with malignant solid tumors and lymphomas were analyzed, of which ( . %) abandoned treatment. univariate logistic regression analysis showed significant higher abandonment rates in children living outside the capital city, lima (p< . ); prolonged travel time to a tertiary center (> hours; or . , p = . ); living in a rural setting (or . ; p< . ) and lack of parental formal job (or . ; p = . ). according to cancer diagnosis, children with retinoblastoma were more likely to abandon compared with other solid tumors. in multivariate regression analyses, rural origin and lack of formal parental employment were independently predictive of abandonment. conclusion: treatment abandonment prevalence in our country is high and closely related to socio-demographical factors. treatment outcomes could be substantially improved by strategies that help prevent abandonment of therapy based on these results. st. jude children's research hospital, memphis, tennessee, united states background: to improve the quality of a pediatric hematology/oncology fellowship program, a systematic assessment must be performed that can evaluate its current state and identify areas of opportunity, as well as modifications over time. unfortunately, widely agreed-upon metrics of quality for pediatric hematology/oncology fellowship programs currently do not exist. this is particularly important in this field due to the global shortage of specialists. for this reason, an assessment instrument that is applicable throughout the world must be created. objectives: the st. jude global education program assessment tool (epat) is a novel instrument that seeks to evaluate pediatric hematology/oncology fellowship programs around the world in systematic and objective way. epat will help determine key performance indexes that are relevant for quality education in pediatric hematology/oncology fellowship programs and establish the framework for improvement. design/method: firstly, key domains to be evaluated for program assessment were identified a priori based on the continuum of pediatric hematology/oncology fellowship programs in the context of geography and educational structure. subsequently, questions were formulated to evaluate these key domains, seeking to assess elements involved in ensuring competence in clinical practice, academic productivity and regional impact. due to the novelty of this tool and the lack of defined metrics of quality, epat relies on expert opinion in a two-step process: internally in the department of global pediatric medicine at st. jude children's research hospital and, subsequently, from a panel of experts in global pediatric oncology and medical education from around the world. ten key domains were identified to evaluate all aspects relevant to training programs around the world, regardless of educational and geographic context. questions have been created to assess these domains and, to make epat quantitative, these have assigned weights with a value reflective of their relative importance. this grading system allows for a score in each key domain, permitting monitoring of changes over time. epat is currently at the stage of external expert review, and subsequently will be piloted in five fellowship programs around the world to provide different geographical and patient care contexts for its validation. once epat is finalized, it will be distributed to pediatric hematology/oncology fellowship programs around the world to be applied. epat proposes a novel strategy to assess training programs in a systematic way that includes all aspects relevant for a training program in a global context. this tool will help guide improvements in pediatric hematology/oncology fellowship programs and assure a well-trained workforce. background: with the improvement in pediatric oncology patient survival and outcomes in the past several decades, monitoring for recurrence and long-term effects of therapy has become even more important. the utilization of personalized treatment summaries and survivorship care plans (scps) is one way to communicate this information with patients and families. the american college of surgeons commission on cancer (coc) created a standard regarding provision of scps to % of eligible patients by december , as a metric for accreditation of all cancer centers. the standard applies to all patients with stage i, ii, and iii cancer diagnoses and requires creation of the scp within one year of diagnosis or six months of completing treatment. during implementation at our pediatric cancer center, we identified barriers to use of the guidelines in the childhood cancer setting. objectives: define eligibility for an scp for pediatric oncology patients to include all patients with curative intent and to deliver scps within six months of finishing therapy. design/method: using chart review and a cancer center registry query, we identified childhood cancer patients potentially eligible for an scp by collecting stage, goal of therapy, and dates of treatment. all patients with curative intent were deemed eligible for an scp regardless of stage i-iv. patients being followed in the oncology clinic for posttreatment surveillance and care were included even if they had received an scp in the survivorship program or were greater than six months off therapy at time of implementation. as expected in the pediatric oncology population, acute lymphoblastic leukemia (all) was the most common diagnosis comprising . % of patients. all is stratified into risk groups instead of surgical staging categories, and treatment duration is greater than one year, unlike many adult-onset malignancies. these differences required interpretation of the guidelines to apply to our pediatric population for all and other pediatric diagnoses with non-surgically based staging. our pediatric oncology clinic has to date provided scps to of eligible patients by adapting the guidelines to focus on patients with curative intent to receive an scp by six months off therapy. cancer staging guidelines and goals for curative intent as well as lengths of treatment vary between the pediatric and adult populations. the coc guidelines require adaptation for optimal applicability to the pediatric oncology population. background: education in communication for fellows in fields that require difficult discussions with families are few in nature. adult learning pedagogies such as role play are under-utilized in medical education, and have been shown to be as effective as traditional teaching methods such as lecture. an -module course for fellows in hematology/oncology, hospice and palliative medicine, radiation oncology, and pediatric hematology/oncology was implemented in january/february . fellows participated in the program. topics covered including fundamentals of communication, coping and spirituality, delivery of bad news, communicating with families, sexual dysfunction during treatment, palliative care/death and dying, and burnout. objectives: overall goal of this course is to foster holistic physicians who views their patients as people with cancer, not cancer patients, and physicians that can communicate effectively with their patients throughout the disease continuum. by the end of the course, learners should be able to practice the fundamental principles of good communication. design/method: fellows initially participated in a pre-course osce to establish baseline skills. osce was facilitated by the center for learning and innovation at northwell, and included actors portraying a pediatric patient and family member to whom the fellow had to break bad news. two months later, the course was carried out over the span of eight weeks and included didactic sessions followed by minutes of role play scenarios. five of the eight modules included role play, with faculty members serving as simulated patients. after the course, a second breaking bad news osce was held. both osces were filmed, and feedback was given by the on-site actors. additionally, faculty members were given access to the videos in an on-line format and were given an evaluation tool to assess the fellows' performance pre-and post-intervention. fellows were given subjective surveys pre-and post-course as well. results: subjective data from participants showed a noticeable increase in comfort level in all areas on the pre-and post-course survey. data obtained from osce videos showed improvement in communication skills as assessed by sps and faculty members using a new evaluation tool developed by faculty. initial first-run data shows that this course is successful in improving communication skills as well as increasing fellows' comfort level across several domains of communication. future directions for our course include improving and validating our assessment tool, expanding our topic base to include more aya and pediatric scenarios, faculty development for improved role play, and investigating impact on practice after course completion. background: acute lymphoblastic leukemia (all) is the most common form of childhood cancer with approximately children diagnosed each year. survival rates have improved significantly over the past several years. children with all are at risk for developing musculoskeletal complications during and after completion of treatment, which can contribute to impaired activity, elevated body mass index (bmi), and risk for complications. interventions involving physical activity could improve musculoskeletal strength as well as overall health in these children. the aims of this study are to examine the feasibility of a directed physical activity program for children with newly diagnosed all during the initial intensive phase of therapy and to evaluate the overall health and quality of life of children participating in the directed physical activity program. design/method: all subjects will receive education materials about the importance and safety of physical activity and a nutrition handout. all subjects will also participate in the directed physical activity program under the supervision of a trained physical therapist for at least minutes every week for weeks. the program will entail four stations including a cardiovascular, balance/proprioception, strength and flexibility, and coordination and cardio. feasibility will be assessed by tracking the participation rate throughout the study period. other assessments will be made at study entry, at the end of weeks of physical activity initiative and months after completion of the intervention. assessments include overall strength and flexibility, weight, height, bmi, blood pressure and performance scores. descriptive statistics will be used for this study. results: a total of patients, male and female, enrolled in the study over a . month period. patient ages ranged from - years. half of the patients enrolled have completed the week program and all patients had stability or improvement of their physical functioning scores. further data collection and analysis is ongoing. patients in the early intensive phase of all therapy are at risk for complications that can affect their physical functioning. a directed physical activity protocol may improve their overall physical functioning. patients may not need specific physical therapy; however a directed physical activity program appears to be beneficial for these patients. the main roadblocks to successful completion of the program were difficulty with scheduling, strain on the parents and patient from treatment, unplanned admissions for fever, as well as nausea and fatigue at time of visit. albany medical center, albany, new york, united states background: communication skills are a core competency highlighted by the acgme. increasing resident confidence in delivering difficult news has been shown to lead to more s of s effective communication. currently, the majority of residency programs lack formal training in communication skills. our objective was to demonstrate feasibility and efficacy of integrating a standardized-patient based training program for communication skills into the curriculum of pediatric residents design/method: to date, pediatric and medicine/pediatric residents have participated in the program during the intern year. the program consists of three, two-hour long sessions, in which each resident is given several opportunities to act out case scenarios with a standardized patient. scenarios included informing a parent of their child's new cancer diagnosis and disclosure of a positive hiv test to a teenager. residents received post hoc peer to peer, and preceptor to learner feedback. pre and post-program surveys were completed by residents. results: following course completion residents reported an increase in confidence in multiple areas of communication including giving a difficult diagnosis (p< . ), discussing a poor prognosis (p< . ), responding to different patient/family member emotional responses i.e. crying or anger (p< . ), and organizing vital information to be relayed (p< . ). in conclusion, communication skills training of pediatric residents is feasible and provides a platform for developing valuable skills not taught elsewhere within the curriculum. background: for children with cancer, transitioning back to school during or after treatment can be challenging. literature supports the need for school re-entry programs to ease this transition. however, these programs vary widely among pediatric cancer institutions with little data addressing their program components. data from this study provides information on current school re-entry programs across these institutions. objectives: one objective of this study was to assess for correlation between the presence of a school re-entry program and other factors, such as geographic location and institution size. a second objective was to establish a list of differences between institutions' school re-entry program components. finally, we aimed to describe current school reentry practices, as well as program benefits and perceived areas for improvement. states with membership in the children's oncology group were offered enrollment in this study. a member of each institution was invited to participate in a survey established by the research team. this person was closely associated with the institution's school re-entry practices. each interview queried institution demographics, as well as program components (e.g., participants, target audience, resources). comment was also collected on program benefits and potential for improvements. analysis of transcripts was performed using pearson's correlation to assess for relationships between institution size, geographic location, and program presence. grounded theory was used for analysis of benefits and improvements. results: thirty-nine of forty-one pediatric institutions who were offered enrollment participated in this study. twentynine institutions ( %) indicated the presence of a school reentry program, and ten ( %) stated they had none. no correlation was found between institution size and the presence of a school re-entry program (p = . , ns). there was also no correlation found between institution location and the presence of a school re-entry program (p = . , ns). a major theme surrounding the benefits of having a program included education for the returning student's peers. for those with programs, perceived improvements included increasing staffing and the ability to offer more services. the results do not support the hypothesis that the presence of a school re-entry program is influenced by the size and geographic location of the treating institution. however, data seem to suggest that available staffing may influence the presence of a program. future studies are needed to address other potential influences, as well as to take an evidence-based approach to determine the effectiveness of the interventions present in these programs. cohen children's medical center/ zucker school of medicine at hofstra-northwell, new hyde park, new york, united states background: genetics/genomics is evolving at an extremely rapid pace. current advances lead to individual algorithms toward disease treatment for each disease with multiple branch points. fellows learn only a fraction of the knowledge and there is no formal approach to teaching critical analysis of information and application algorithms toward disease. additionally, as knowledge evolves extremely rapidly, any approach must teach self-acquisition and application of evolving discoveries. objectives: to create, implement and evaluate a novel curriculum for genetics/genomics targeted toward pediatric hematology/oncology fellows design/method: the curriculum includes four components: ) genetic and genomic medical knowledge, with one initial team-based learning session and weekly online multiple choice questions; ) essential pathways, which will teach molecular pathways common in oncogenesis and relevant to targeted therapy in microteaching sessions with using auditory, visual and tactile learning; ) knowledge acquisition and clinical judgment, to allow learners to gain experience into researching data available, then developing and prioritizing potential treatment plans using problem-based learning sessions in which they will stage a patient, research treatment options, prioritize and present findings; and ) synthesis to demonstrate independent ability to research and recommend therapy through an independent project in which the learner, given a case, will present the case and research findings, genetics/genomics, molecular pathways and make recommendations for therapy in molecular tumor board for faculty and fellows. to evaluate, we plan to recruit to institutions, match for size of programs and implement in half and evaluate nd and rd year fellows in both groups by mcq exam and satisfaction surveys. the creation of a multi-module, adult-learning based curriculum for genetics and genomics in pediatric oncology is feasible. implementation and evaluation are necessary to demonstrate efficacy. background: neuroblastoma is the most common extracranial solid tumor in children. chimeric anti-gd antibody ch . (dinutuximab) therapy has improved the survival of children with newly diagnosed high-risk, neuroblastoma patients as well at the time of first relapse/progression. acute neuropathic pain is a well-documented side effect of dinutuximab administration. however, additional adverse effects including sensorimotor neuropathy, ocular symptoms, and behavioral changes have been described. the incidence and severity of these effects are currently not well-documented in pediatric patients. with improved long term survival of patients receiving this modality, it is important to look for the potential late effects of dinutuximab. objectives: to determine the incidence and severity of neurologic, ophthalmologic, or behavioral changes after dinutuximab administration at our institution. we performed a retrospective chart review using our electronic medical record. we included all patients with high-risk neuroblastoma between the ages of and years at our institution diagnosed between and who received dinutuximab. patients with history of opsoclonus-myoclonus syndrome or gross sensorimotor neuropathy prior to receiving dinutuximab were excluded. we examined clinical documentation for subjective reports and objective exam findings of neurologic, ophthalmologic, or behavioral changes. we also looked for referrals made to neurology, ophthalmology, physical medicine & rehabilitation (pm&r), and psychology. : twenty-two patients met inclusion criteria. at the time of chart review, patients were alive and were deceased. eighteen patients received dinutuximab per anbl ; patients received dinutuximab per anbl . of these patients, patients reported symptoms of interest and reported multiple symptoms. six patients reported symptoms that began at least months after completing dinutuximab. nine patients had objective findings on exam, including decreased deep tendon reflexes, abnormal pupils, and nearsightedness. for patients, referrals were made to ophthalmology, pm&r for neuropsychologic testing, or neurology. two patients who reported symptoms of interest were not referred to a specialist. conclusion: neurologic, ophthalmologic, and behavioral symptoms were commonly reported and demonstrated on exam among pediatric patients with high-risk neuroblastoma who received dinutuximab. it is important to identify these effects so that appropriate specialist referrals can be placed for adequate management of these changes. we recognize that these symptoms may not be solely due to dinutuximab as these patients receive other agents including opioids, so a prospective trial is needed to further evaluate the long-term effects of dinutuximab and to determine how best to screen for these effects. akron children's hospital, akron, ohio, united states background: pediatric cancer is the leading cause of diseaserelated death in children in the united states (u.s.). in , over fifteen thousand children were diagnosed with cancer in the u.s. this population is at high risk for malnutrition due to the multimodal therapies they receive: surgery, chemotherapy, radiation therapy, antibody therapy, and/or bone marrow transplant. adverse effects of these therapies include taste changes, loss of appetite, diarrhea, vomiting, and/or mucositis, making it difficult for the children to be able to consume adequate amounts of nutrition during therapy. there is no "gold standard" measurement tool for identifying patients at risk for malnutrition. nutritional status is not frequently evaluated as a component of clinical trials. assessment of anthropometric measurements (weight, height, z-scores) at diagnosis, as well as over the duration of treatment, can assist in the early identification of malnutrition. the incidence and prevalence of malnutrition in this population is unknown at akron children's hospital. the purpose of this study is to describe the nutritional status and provision of nutritional support therapies in pediatric patients during their first year post new oncologic diagnosis. objectives: identify the incidence and prevalence of malnutrition across oncologic diagnostic categories over the first twelve months post diagnosis. we performed a retrospective records review of all patients newly diagnosed with cancer in at akron children's hospital. demographic and anthropometric data was collected at time of diagnosis and nutritional status categorized by z score. anthropometric and nutrition support data was then collected every two months for the first year after diagnosis along with incidence of unplanned inpatient admissions. results: a total of patients were included in the analysis, with . % malnourished at time of diagnosis; . % developed malnutrition the first year. patients with solid tumors represented % of patients with pre-existing or acquired malnutrition. overall, % of patients received at least one nutritional support modality. patients with pre-existing or acquired malnutrition had a non-significant increase in unplanned admissions (p = . ). our study demonstrated that patients with solid tumors were found to be at increased risk of pre-existing and acquired malnutrition, followed by leukemias, and experienced higher incidence of unplanned admissions in the time period observed. prospective, multi-center replication of this study, including detailed collection of nutrition therapies is recommended to guide development of diagnosis specific nutrition support guidelines. background: pediatric and young adult oncology patients treated with intense chemotherapy have a high incidence of transfusional iron overload. iron deposition can lead to heart failure/arrhythmias, liver abnormalities, endocrine dysfunction, ineffective erythropoiesis, and increased cancer and mortality risk. however, there is a paucity of data regarding recommendations for management of transfusional iron overload in these cancer survivors. consequently, long-term complications of transfusional iron overload specific to these patients have not been assessed. objectives: to assess screening and phlebotomy-based treatment algorithms for this population. design/method: a retrospective chart review of pediatric and young adults who completed oncology management, had iron overload, and initiated phlebotomy treatment was conducted. tiered screening occurred in patients that received at least packed red blood cell (prbc) transfusions. patients were recommended for evaluation and possible phlebotomy if: ( ) liver iron concentration (lic) > mg of iron/gram dry weight liver tissue by ferriscan and/or ( ) cardiac mri t * < ms. during phlebotomy, iron status was assessed quarterly and phlebotomy discontinued with lic < or normalization of ferritin/imaging lic verification. descriptive statistics were employed to report the characteristics of the study population. spearman correlations were utilized to describe associations between transfusions, lic, ferritin, iron saturation and number of phlebotomy sessions. results: twenty five survivors underwent phlebotomy. the mean age was . years (sd . ) and ( %) were female. oncologic diagnoses: all ( %), aml ( %), nhl ( %), ewing sarcoma ( %), osteosarcoma ( %), neuroblastoma ( %) and cns ( %). patients received a median of . (iqr - ) transfusions. median number of phlebotomy sessions was (iqr - ) over . years (iqr . - . ). prior to phlebotomy, median lic was . mg/g (iqr . - . ) and ferritin was . ng/ml (iqr - ) . no patients demonstrated abnormal cardiac t * mri (n = ). ( %) patients completed phlebotomy. one discontinued due to poor vascular access. no patients developed iron deficiency. lic was reduced by a median of . mg/g (iqr . - . ) and ferritin by ng/ml . correlation between number of transfusions and phlebotomy sessions was poor (r = . ). conclusion: management guidelines are lacking for transfusional iron overload in pediatric and young adult survivors of cancer. we demonstrate a phlebotomy algorithm that is effective and tolerated. correlation between number of transfusions received and phlebotomy treatments was poor, necessitating serial assessments. using this management algorithm, prospective studies can evaluate the effect of iron removal on iron overload complications in this patient population. penn state children's hospital, hershey, pennsylvania, united states background: cancer therapy leads to an impaired immune system that takes time to recover. it is important to ensure that these survivors have adequate immunity to prevent common yet potentially severe childhood illnesses. no validated guidelines currently exist for surveillance testing or re-immunization in this population. retrospective analysis involving a small cohort of pediatric cancer patients treated at penn state children's hospital showed % of patients screened for varicella immunity after therapy completion did not have adequate disease titers. to determine the proportion of pediatric cancer survivors who have lost humoral immunity to previously received vaccines; to determine the rate of response to single dose boosters or full vaccine series in seronegative subjects after one booster. design/method: pediatric cancer survivors treated at the children's hospital who are at least months from completion of cancer therapy are prospectively tested for antibody levels to hepatitis b, tetanus, varicella, measles, and strains of pneumococcus ( , b, v, c, f, and f). samples are analyzed by the cdc for measles and varicella avidity. seronegative subjects by commercial studies, are eligible to receive booster vaccines. titers are rechecked at least weeks after boosters to re-evaluate immunity; if still seronegative, subjects will receive the entire vaccine series. titers are finally tested at least weeks after the final dose of the vaccine series. immunity analyzed after therapy, after boosters, and after vaccine series. results: of pediatric cancers survivors who completed therapy, % were non-immune to hepatitis b, % nonimmune to > % of pneumococcal strains tested, % nonimmune to measles, % non-immune to varicella, and % non-immune to tetanus. of subjects who received mmr vaccine after therapy and prior to study enrollment did not have protective antibodies to measles. of the subjects who received varicella vaccine after end of therapy and prior to study enrollment, did not maintain protective antibody levels. cdc results for measles and varicella are pending, as well as repeat studies after vaccine boosters and series. conclusion: a significant percentage of pediatric cancer survivors do not retain immunity to hepatitis b, pneumococcus, measles, and varicella. after one booster, a high percentage of subjects did not develop protective immunity to varicella. only subject did not have immunity to tetanus, which is consistent with the high immunogenicity of tetanus toxoid. formal guidelines are needed to protect this population from vaccine-preventable illness post-therapy. children's hospital of richmond at virginia commonwealth university health system, richmond, virginia, united states background: childhood cancer survivors are at risk for being overweight. diet and physical exercise are important in maintaining a healthy lifestyle and weight; however, it has been reported that cancer survivors are less active than their peers. one reason for this may be that there are no clearly established risk-based exercise recommendations for cancer survivors. another reason may be that providers tend to focus s of s recommendations for exercise more towards patients who are overweight. objectives: to describe changes in physical fitness of childhood cancer survivors who exercise. design/method: 'moving forward' is a wellness and physical fitness program that the center for care beyond the cure at chor offers in partnership with the ask childhood cancer foundation and the ymca. the program is available for any childhood cancer survivor between y and y age, being seen at our center. survivors define their fitness or wellness goals and then work with a trainer once a week (at least) for min sessions throughout the year to achieve these goals. baseline and ongoing measurements for core strength, endurance, overall strength and balance were collected. the average of each of the parameters of all participants were compared from the beginning to the end of the program. over the year, there was a % increase in endurance as measured by the average of the miles walked in minutes, % increase in core strength as measured by the average number of sit-ups in secs, an % and % increase in overall strength as measured by the average weight lifted by leg press and the average weight lifted by chest press, and a % increase in balance as measured by the average number of seconds balancing on a single leg. in addition, each child had actually gained weight in the process with an approximately % increase in the average of the weights of all children. there are benefits to regular exercise beyond weight control, and improvements in physical fitness can be seen even without weight loss. regular physical exercise results in improved physical fitness and should be universally advocated to all patients. determining insulin resistance, measuring changes in fatigue and wellness perception following exercise are future directions that we intend to explore. dana-farber cancer institute, boston, massachusetts, united states background: improvements in adolescent and young adult cancer patient (aya) survival rates and quality of life outcomes have lagged behind those of children and older adults, highlighting a need for research targeting this unique population. current literature supports the value of strong ayaclinician communication, notably in facilitating therapeutic alliance, however little is known about aya communication priorities during cancer care and barriers to optimal ayaclinician communication. objectives: to explore aya and oncology clinician communication priorities and to identify barriers and facilitators to aya-oncology clinician communication. design/method: semi-structured interviews were held with aya cancer patients and survivors (ages - years) from a single large academic institution and oncology clinicians (physicians and nurse practitioners) from academic institutions in the northeastern united states. interviews were conducted in english by phone or in person. all interviews were audio-recorded and transcribed verbatim. analyses were aided by nvivo software. ayas identified a wide range of topics as important to discuss with clinicians. the most frequently identified topics were ) side effects of treatment (with an emphasis on physical appearance and function, n = ), ) social issues (including friendship, family, and school, n = ), ) looking ahead to the future (n = ), and ) sexual & reproductive health (including future fertility, contraception, and romantic relationships, n = ). clinicians prioritized ) cancer treatment and side effects (n = ), ) emotional and psychological health (n = ), and ) sexual and reproductive health with a focus on fertility risk and fertility preservation (n = ). aya reported facilitators to good communication including an open and long-established relationship with the clinician (n = ) and clinician engagement in age-appropriate and patient-directed conversations (n = ). barriers included parental presence during visits (n = ). clinicians reported barriers including ) clinician discomfort (not feeling wellequipped to discuss psychosocial topics such as sexual health, spirituality, and relationships with peers, n = ), ) presence of parents/family (n = ), and ) perceived patient discomfort discussing specific topics (such as sexual health, n = ). clinicians acknowledged the need for collaborative efforts with additional team members (i.e. nurses, psychosocial providers) to assist in meeting aya communication needs. conclusion: aya and clinician-reported communication priorities are largely aligned. however, ayas emphasize some topics, such as social function, appearance, and sexual health that are not highly prioritized by clinicians, which may result in gaps in care for ayas in treatment and in survivorship. these data identify opportunities for intervention, including clinician education, patient and family education, clinic-based intervention, and systems-based changes that can be developed and tested. background: primary care physicians (pcps) cite lack of knowledge and inadequate communication with the oncology team as major barriers to providing recommended surveillance for late effects of treatment to childhood cancer survivors. a standardized telephone handoff to pcps posttherapy is a potential strategy to increase survivorship care by pcps through interactive communication. to determine the feasibility of a structured telephone communication using the situation, background, assessment, and recommendation (sbar) communication tool delivered by a trained oncology nurse to increase pcp knowledge and willingness to provide survivorship care. design/method: from / / to / / , a registered nurse expert in childhood cancer survivorship attempted to contact by telephone the pcps of the most recent patients attending yale's childhood cancer survivorship clinic that were < years old, english-speaking, and ≥ years posttreatment. all pcps had been previously sent an individualized survivorship care plan (scp) that listed the patient's previous treatment history and recommended surveillance tests. upon successful contact and after confirming receipt of the scp, the nurse explained the definition of late effects, description of patient's diagnosis and treatment history, and associated potential late complications and schedule of recommended surveillance tests. the pcp was also asked about his/her ability and willingness to provide needed surveillance for late effects in the future. overall, of pcps were successfully contacted with a median of phone call (range: - ) that lasted a median of minutes (range: - ) after a median of business day (range: - ). no pcps ended the call mid-conversation. all pcps were receptive and expressed appreciation for the call. twenty-five of ( %) pcps expressed an understand-ing of the material discussed and endorsed belief in their ability and willingness to provide late effects surveillance for their patients. no pcps questioned discussing their patient's care with a nurse versus a physician. interactive, structured communications between nurses and pcps by telephone are feasible and are associated with high-levels of pcp confidence in providing survivorship care. background: childhood cancer (cc) admissions account for % of non-newborn pediatric hospitalizations. these hospitalizations are longer and more expensive than other hospitalizations. admission payer (medicaid or commercial) reflects both health policy and sociodemographic status. the objective of this study was to determine if length of stay (los) or cost of cc admissions differed by payer. we used the kids inpatient database, a sampling of all pediatric hospital discharges in the united states. analysis for this study was limited to admissions containing a cancer diagnosis in any discharge icd- codes. admissions were further subcategorized by discharge codes according to diagnosis (leukemia, lymphoma, solid tumor and brain tumor) and reason for admission (chemotherapy, procedure, infection, non-infectious toxicity or "other"). charges were converted to costs using cost-to-charge ratios. multivariable linear regression models were performed to control for age, gender, race, reason for admission, and diagnosis. results: there were , weighted admissions for children with a cancer diagnosis in . of these admissions, . % had medicaid, . % had commercial insurance, and less than % had other payers. the mean los for medicaid admissions was . days ( % ci . - . ), compared with . days ( % ci . - . ) for commercial insurance. surgical admissions accounted for the largest difference in length of stay with medicaid admissions being . days longer than those covered by commercial insurance ( . days vs . days), however, the difference was significantly different for all reasons for admission. in multivariable analysis admissions associated with commercial insurance were % shorter s of s (p< . ), accounting for approximately one hospital day, than admissions associated with medicaid after controlling for other variables including race. the mean overall cost for medicaid admissions was $ , ( % ci - ), compared with $ , ( % ci - ) for commercial insurance. in the multivariable model, cost was collinear with race. conclusion: los and cost of admissions associated with medicaid differed from those associate with commercial payers. medicaid admissions were % longer on average than commercial insurance, accounting for a difference in length of stay of approximately one day although the difference varied with the reason for hospitalization (chemotherapy, surgical procedure, infection, other toxicity, other). costs of admissions were not independent of race. further investigation into potential explanations for this difference including differential access to home care needs, outpatient reimbursement differences, social indications for prolonged hospitalization, and provider biases, is warranted. background: pediatric cancer is a major cause of morbidity and mortality among children surpassed only by accidents. despite improved outcomes in high income countries (hic) survival rates remain poor in the developing word. there are various diagnostic and therapeutic limitations contributing significantly for the survival gap. the main objective of the study is to to evaluate the outcomes of pediatric cancer in armenia and identify diagnostic and therapeutic limitations in the country. we conducted a retrospective study among (≤ years old) children with cancer (solid tumors and hematological malignancies), who were diagnosed and treated at the clinic of chemotherapy of muratsan hospital complex of yerevan state medical university between and . those patients, who didn't receive chemotherapy for any reason were not included in the study cohort. epidemiological, social, medical information was collected through the patient charts review. this included patient age at diagnosis, sex, place of residence (city vs village), the educational level and employment status of parents, type of cancer, stage, presentation of symptoms, first medical specialty consulted and the time consulted, initial work-up, the type of treatment received, information on the diagnosis/treatment received abroad. results: at our clinic during the mentioned period of time the majority of patients presented with hematologic malignancies- %. ( . %) patients had information on diagnosis delay. average delay in diagnosis was about days. in % of cases the first contact with "healthcare system" was through pediatrician, and in % with surgeon. out of relapsed patients received salvage treatment in armenia and abroad. from those who stayed for treatment in armenia patients survived. majority of relapsed patients had acute lymphoblastic leukemia. from leukemia patients immunophenotyping and cytogenetics were available for ( . %) patients; the majority of missing cases were between and , when these diagnostic modalities were not available or affordable in the country. ( %) patients received part of diagnosis and/or treatment abroad. the most frequent reason for going abroad was bone marrow transplantation, otherwise none available in armenia. out of patients were lost to follow-up, patients had a fatal outcome. patients were in remission at a median follow up of . years. conclusion: unavailability of cancer registry and several essential diagnostic/treatment modalities, luck of multidisciplinary care and palliative support, high rate of out-of-pocket expenses were among the main challenges of pediatric cancer care in armenia. background: adverse drug reactions (adrs) are increasingly recognized as important and sometimes irreversible complications of cancer treatment. anthracyclines and cisplatin are effective chemotherapeutic agents, but their use can be limited by cardiotoxicity (anthracyclines) and ototoxicity (cisplatin) in up to % of patients. genetic variants that can be used to predict who is most at risk of developing these adrs have been discovered and replicated. objectives: to create pharmacogenetic risk prediction models for anthracycline and cisplatin toxicities and discuss results with oncologists to facilitate incorporation into treatment decision-making when appropriate. design/method: risk prediction models were developed from the linear regression of strongly-predictive genomic variants (odds ratios ≥ ) discovered and replicated in at least three patient populations. these models were used to assess an individual patient's genomic risk of developing cardiotoxicity from anthracyclines or hearing loss from cisplatin. risk results were returned to oncologists showing where the specific patient's genetic risk of toxicity lies on a continuum between the lowest and highest risk groups across all studied patients using a multi-gene model. interviews were conducted with patients, families, and oncologists to determine how results were valued and utilized. results: patients have been genotyped and had their genetic risk results returned to their oncologists. the first patients have been characterized to determine the impact these test results have had on their clinical care. results were described as being useful in decision-making by patients and/or oncologists in % of cases. additionally, for patients in the most extreme risk groups (highest and lowest risk), a change in treatment plan was ordered % of the time for cisplatin patients and % of the time for anthracycline patients. this included increased cardiac and audiological monitoring, the addition of a protective agent, or choosing an alternative treatment protocol if the risk outweighed the benefits of remaining on the current treatment plan. in interviews, patients indicated that they felt more involved in decision making, and felt reassured by understanding their genetic risk of toxicities. genetic risk prediction models for anthracycline cardiotoxicity and cisplatin ototoxicity were highly utilized by patients and oncologists in decision-making. results were found to be an important tool for informing patients of the risk of adrs during cancer treatment, and resulted in patients and their families feeling more involved in decision-making. background: childhood cancer survivors are at increased risk of developing executive dysfunction, and low socioe-conomic status (ses) has been identified as one of the mediators of executive functioning. previous studies have used traditional measures of ses, such as parents' education level, family annual income and occupation. but more recently, area based socioeconomic measures like block group poverty status are deemed to be more useful in monitoring of social inequalities in health in the united states. block groups are statistical divisions of census tracts and generally contain between and , people. the current study aims to understand the association of block group poverty status (percentage of households in family's block group of residence living below the federal poverty level) with executive functioning among cancer survivor children. design/method: we used a retrospective cohort of childhood cancer survivors. relevant information was collected from the medical record, administrative data sets and parent-filled surveys. address information was geocoded using arcgis . to obtain data on the block group poverty status. a priori cut-points were set to represent block groups with families living below poverty level at %, . % to . %, and ≥ . %. executive functioning were assessed through a parent-rated instrument, the behavior rating inventory of executive functions (brief). multiple linear regressions were used to determine the relationship between block group poverty status and the brief scores. results: data was examined from families of childhood cancer survivors, ranging in age from to years. in this sample, . % families reported an annual income <$ , , . % reported income between $ , and $ , while . % reported annual income ≥$ , . primary care giver of . % of cancer survivors had more than more high school education, and . %, . % and . %, of families were living in a block groups with %, . - . % and ≥ % poor households respectively. block group poverty level was not significantly associated with annual income levels (spearman's rho = . , p = . ), or parental education level (spearman's rho = - . , p = . ). in a step-wise multiple linear regression, there was no statistically significant association seen between block group poverty status and executive functioning after adjusting for co-variables in the final model. future prospective study with a bigger sample size, longer follow up period and more robust measures of the executive functioning like a clinician administered test are needed to understand the effect of block group poverty status on executive functioning. to d completion was . days (range - ). all parents strongly agreed/agreed that d was helpful and would recommend d participation to another family. ten parents ( %) reported time spent on d was "just right." no parent felt more worried due to the intervention, though parent found d participation stressful. this interim analysis suggests that parents have a favorable d experience and recommend the intervention. to date, < % of enrolled parents fail to participate. d shows promise as an acceptable interdisciplinary communication intervention targeted to the early treatment period for childhood cancer. children 's hospital and research center oakland, oakland, california, united states background: screening echocardiograms are recommended by children's oncology group (cog) guidelines to assess for anthracycline-induced left ventricular (lv) systolic dysfunction. the yield of screening echocardiograms during chemotherapy and in the immediate post-therapy period is uncertain. objectives: to assess the incidence of lv dysfunction detected by screening echocardiograms during chemotherapy and in the immediate post-therapy period, defined as - months off-therapy. design/method: children diagnosed with cancer between january -march who received anthracycline chemotherapy were identified. echocardiograms were performed as per protocol, institutional and cog guidelines, and were reviewed retrospectively. lv dysfunction was defined as fractional shortening (fs) < % or ejection fraction (ef) < % ( ) results: in this cohort (n = , median age years), the most common diagnosis was all ( . %), followed by aml ( . %). of echocardiograms, ( . %) were performed during treatment and in the immediate posttreatment period. thirty-eight ( . %) patients had a > % decrease in fs compared to their pre-treatment echocardiograms. none of these patients required any treatment modification or cardiac medications. only patient ( . %) had echocardiogram-proven lv dysfunction discovered on a screening echocardiogram during her treatment course. she eventually died due to multi-organ failure following septic shock. this patient was receiving treatment for aml and had received mg/m of doxorubicin-equivalent anthracyclines at the time of the abnormal echocardiogram. one patient with metastatic ewing sarcoma had borderline lv dysfunction with a fs of % detected a month before completion of therapy. she had received mg/m of doxorubicin equivalent anthracyclines at the time of the abnormal echocardiogram. she did not require any therapy modification or additional cardiac medications. serial echocardiograms done on this patient have shown stable ventricular function. no off-therapy screening echocardiograms identified lv dysfunction. in our experience, the yield of echocardiograms to detect anthracycline-related cardiac dysfunction during treatment and in the immediate post-therapy period is very low. one patient developed lv dysfunction during treatment and one had borderline fs, while no lv dysfunction was identified within months of completing chemotherapy. though fs decreased in % of patients, none required intervention. further study is needed to optimize the use of echocardiography screening in children treated with anthracyclines. references: . landier w et al. jco . background: platinum-based chemotherapy increases the risk of sensorineural hearing loss in children with cancer. little is known about the impact of hearing loss on cognitive and emotional functioning in survivors. to determine the association of severe/profound hearing loss after platinum-based chemotherapy with ) cognitive impairment and ) emotional distress (i.e. anxiety and/or depression). cross-sectional study of all patients attending yale's childhood cancer survivorship clinic ≥ years off therapy for cancer diagnosed at < years and treated with cisplatin and/or carboplatin, but with no history of cns tumor, cranial radiation, congenital hearing loss, or developmental delay. hearing loss severity and hearing aid data were abstracted from audiograms and detailed clinical history. cognitive impairment was defined as behavior rating inventory of executive function t score ≥ , assessment by neuropsychologist, and/or history of special education. emotional distress was determined by brief symptom inventory t score ≥ (global or two subscales) or behavioral and emotional screening system t score ≥ , psychologist interview, and/or history of psychotropic medication/psychotherapy. the most recent available patient data were used. logistic regression with sas software, version . was performed. results: overall, patients ( % female, % white) met eligibility criteria with a median age of . years (iqr = . ) at diagnosis and . years at evaluation (iqr = . ) after a diagnosis of sarcoma ( %), neuroblastoma ( %), or other ( %) for which % received cisplatin and % received carboplatin. fifteen patients ( %) had severe/profound hearing loss in at least one ear. patients with severe/profound hearing loss had a significantly increased risk of cognitive impairment (or = . ; % ci = . - . ), but not emotional distress, compared to patients without severe/profound hearing loss. there was no significant association between age at diagnosis, current age, time since diagnosis, sex, race, ethnicity, or diagnosis with either cognitive impairment or emotional distress. similarly, there was no significant interaction between ) age at diagnosis and hearing loss or ) sex and hearing loss with either cognitive impairment or emotional distress. ten of the ( %) patients with severe/profound hearing loss in at least one ear were recommended hearing aids, of which ( %) reported compliance most of the time. we conclude that severe/profound hearing loss is significantly associated with cognitive impairment, but not emotional distress, in childhood cancer survivors. our data supports the need for interventions to improve hearing in these patients, including compliance with hearing aids. background: who grade anaplastic astrocytoma is a high grade glioma dependent on vascular endothelial s of s growth factor (vegf) mediated angiogenesis for its growth and infiltration. bevacizumab is a recombinant humanized monoclonal antibody which binds vegf-a and inhibits angiogenesis. common adverse effects of bevacizumab are hypertension, proteinuria, thrombosis and bleeding. while animal model based studies have shown that bevacizumab may impair ovarian function the effects of bevacizumab therapy on human fertility are not clear. since the physiology of pregnancy involves neovascularization/angiogenesis it is recommended that conception be avoided for at least months following exposure to bevacizumab. to describe the course of a young adult who became pregnant after receiving bevacizumab and radiation therapy for treatment of an anaplastic astrocytoma. a year old woman diagnosed with a localized hemispheric who anaplastic astrocytoma was treated with chemotherapy and radiation (temozolomide/ . gy) followed by cycles of bi-weekly bevacizumab/temozolomide. patient opted not to pursue fertility preservation prior to initiation treatment. she experienced bevacizumab-associated proteinuria and hypertension during treatment but received all protocol mandated doses (cumulative doses: bevacizumab = mg/kg; temozolomide = . gm/m ). she had a spontaneous unassisted pregnancy months after completing treatment. her pregnancy was uneventful and she was normotensive throughout. fetal ultrasonography at , , , weeks revealed no abnormality of the brain, heart, great vessels, kidney, extremities, placenta and umbilical cord. at weeks she delivered a female infant via cesarean section (birth weight: grams, apgars: and ) excessive post-partum hemorrhage was not reported. placenta was bi-lobed and weighed g. histological analysis revealed normal placental villous development and maturation and two small infarcts. conclusion: exposure to bevacizumab in our patient had no detrimental effect on fertility and on placental/fetal vascular development. we hope this report will add to the existing data on the effects of bevacizumab therapy on fertility. children's healthcare of atlanta, emory university school of medicine, atlanta, georgia, united states background: reports of malnutrition incidence and prevalence in young cancer patients are variable and not well established. previous research suggests children, especially less than years old, treated with intensive cancer-directed therapy are at higher risk for malnutrition. however, no standardized assessment has been used to evaluate risk in this population. objectives: we aim to assess the trends of weight-for-age for patients following cancer diagnosis. this study will be the first to use a standardized measure of treatment intensity (intensity treatment rating scale, itr- ) and will assist in targeting interventions for identification and treatment of malnutrition. design/method: this observational, retrospective study obtained data through the center's pediatric cancer registry and electronic medical record. patients were classified by tumor type (brain or non-brain tumor) and treatment intensity (itr- ). itr- incorporates diagnosis, chemotherapy, radiation, and surgery, beginning with lowest intensity ( ) to highest intensity ( ). inclusion criteria included new cancer diagnosis - at less than years old, with weight obtained and available within days of therapy start date. incomplete data, alternate growth charts, or treatment intensity of , were excluded. weight was obtained at start of therapy and through years after treatment initiation (approximately days) and converted to z-scores adjusted for age and sex. weight trajectories were modeled using generalized linear mixed models with subject-specific random intercepts and spline functions. separate functions were constructed for subgroups of interest (tumor type and itr). results: there were patients included: patients with brain tumors ( . %) and with non-brain tumors ( . %). of included patients, had treatment intensity of ( . %), of ( . %) and of ( . %). over the observation period, , valid weights were recorded. at initiation of treatment, no difference existed between z-score by tumor type (p = . ) or by intensity ( vs. , p = . ; vs. , p = . ; vs. , p = . ). tumor type did not affect z-score through the follow up period. z-scores were higher for intensity rating vs. and vs. (p = < . and p = . respectively) at days after the start of treatment and persisted through days (p = . and p< . respectively). higher treatment intensity is associated with decline in z-score and failure to return to baseline. future directions include further analysis on specific risk factors and timing of weight loss, longer-term follow-up of weight trends, and targeted interventions for identification, prevention, and treatment of malnutrition. objectives: asses the pt requirements for bleeding episodes in a prospective cohort of pcp using a < × e threshold compared to a < × e /l threshold in a historical cohort. we collected pt data in all pcps treated at our center between january/ through december/ . diagnosis, prescription for pt (prophylaxis vs bleeding disorder), plt count and transfused units were assessed for each pt. pcps treated from january/ through june received prophylactic pt with a < × e threshold (cohort a), and pts treated from july/ through december/ received prophylactic pt with a < × e threshold. pts done for procedures and pts with concomitant hemorrhagic pathology were excluded. we compared the number of pts prescribed as prophylaxis vs bleeding episode between cohorts. data analyzed: graphpad prims . ®. statistical analysis: percentages with confidence interval (ci); t-student test (parametric variables) and mann-whitney test (nonparametric variables). statistical significance: p< . . we reviewed pts ( in cohort a, cohort b) in patients. % had acute leukemia, % received and auto or allo hsct. diagnoses and the proportion of patients undergoing hsct was comparable in both cohorts. the average number of pts per patient was , in cohort a and , in cohort b (p = ns), but a significant difference was found when hsct patients were excluded from this comparison ( , pt per patient in cohort a vs , in cohort b, p = , ), which resulted in an estimated , % reduction in pts prescription. furthermore ( , %) pts were prescribed for bleeding episodes in cohort a versus ( , %) in cohort b (p = ns). patients receiving hsct in the entire group ver-sus those not receiving hsct had similar pt requirements for bleeding episodes ( % vs , % p = ns) conclusion: a < × e plt count threshold for prophylactic pts is safe in pcp in chemotherapy and hsct. it can result in a significant reduction in pt usage. key words: platelets, transfusions, prophylaxis, cancer, childhood. ucsf benioff children's hospital oakland, oakland, california, united states background: transition of care for adolescent and young adult (aya) survivors of childhood cancer from pediatric to adult-oriented long-term follow-up (ltfu) is complex. loss to follow-up is common, and little is known about the success rates among different models. the survivors of childhood cancer program (sccp) at ucsf benioff children's hospital oakland employs a community-based model for transitional care. our multidisciplinary team provides aya survivors a comprehensive treatment summary and recommendations, then facilitates transition to primary care or adult oncology ltfu programs. evaluate the success rate for transition of care among aya survivors of childhood cancer in our ltfu program, and identify barriers to successful transition. design/method: aya patients seen from november to august in the sccp with intent to transition were asked by email or telephone if they had followed up with their designated provider. the primary outcome was successful transition, defined as establishing care within months of their visit. patients were also asked about barriers to transition and to rate the new provider's familiarity with their cancer history and ltfu needs. results: transition was intended for patients. eightyseven were contacted and responded. of these, ( %) successfully transitioned, while ( %) were lost to followup. ages ranged from to years, at to years since completion of therapy. ten ( %) transitioned to a primary care provider, ( %) to an adult oncology ltfu program, and ( %) to a pediatrician. patients rated their new provider's knowledge above average ( . ) on a -point scale from poor ( ) to excellent ( ). survivors lost to follow up indicated the following barriers to transition: loss/change of insurance ( ), inability to find a provider ( ), too busy/forgot ( ), problems with transportation ( ), concerns about cost/copay ( ), and s of s other ( ). twelve patients requested further assistance with transition. conclusion: two-thirds of responding patients successfully transitioned. more work is needed to overcome various barriers to transition for one third of aya survivors. albany medical center, albany, new york, united states background: the transition from active treatment, to offtherapy follow-up, is a stressful event for parents of children with cancer. the psychosocial needs of parents after therapy have received limited attention in the united states with only published quantitative studies, the largest with parents. we have secured funding for and recruited a transition care coordinator (tcc) to investigate this further. objectives: our objective is to assess and screen parents at the end of their child's treatment, and to develop interventions to support parents during this time and thereafter. design/method: after informed consent, a standardized questionnaire, the psychosocial assessment tool (pat . ), was administered to parents at end of therapy (t ), months later (t ) and year later (t ). the tcc provided "universal" intervention to all families with an end of therapy binder containing a treatment summary, follow-up roadmaps, information on late effects, and survivor scholarships. based on their pat . scores, some parents were provided intervention specific to symptoms (targeted intervention for scores - . ) or referred to a behavioral health specialist through the clinic social worker for counseling (for scores > ). results: analysis of pat data showed that % of parents (n = ) scored in the targeted or clinical ranges; % of parents scored in those ranges at pat . significant gender differences were revealed with the mean score for men of . and for women of . . this was confirmed by showing statistical significance (p = . ) when analysis was conducted for only a subgroup of data composed of couples (n = ). analysis of pat data by couples (n = ) showed the mean score for men was . and for women was . (p = . ). gender differences were most apparent in caregiver stress reaction questions that focused on ptsd symptoms. when the subgroup of couples' scores (n = ) for caregiver stress reaction at pat was analyzed, there was a significant difference (p = . ) in caregiver stress reaction with a mean of . for men versus . for women. [note: subcategory scores range from to ]. this study was initiated in october using a tcc and the pat . screening tool. the results suggest greater stress on mothers after therapy, with a substantial proportion of parents having symptoms of ptsd after therapy. background: hodgkin lymphoma (hl) is a common childhood cancer characterized by an inflammatory microenvironment. chemotherapy and radiation may exacerbate this inflammation and contribute to the development of late effects (pneumonitis or pulmonary fibrosis). in a heterogeneous cohort of childhood cancer survivors exposed to pulmonarytoxic therapy, no association between pro-inflammatory cytokines and late pulmonary dysfunction was observed. our objective was to test this association in a relatively uniform cohort of survivors of hl, given the well-recognized proinflammatory background of this disease. objectives: to characterize off-therapy pulmonary function in survivors of hl treated with contemporary therapy, and to investigate its association with persistent systemic inflammation. design/method: blood samples, clinical data, and pulmonary function tests were obtained from survivors of hl ≥ months off therapy. lung function score (lfs), a validated method for assessing degree of pulmonary dysfunction on a scale of i to iv, was determined from diffusion capacity and forced expiratory volume in one second (fev ). for a control group, blood samples from patients with benign, noninflammatory hematologic conditions were used. plasma concentrations of inflammatory cytokines were measured on a luminex platform (emd millipore). associations between clinical features or cytokine levels and lfs i (normal) vs. ii-iv were evaluated using logistic regression or wilcoxon rank sum tests, respectively. results: of survivors (mean age at diagnosis: years, range: - ; mean time off therapy: . years, range: . - ), % were categorized as lfs ii (mild dysfunction), % as lfs iii (moderate dysfunction), and no survivors as lfs iv (severe dysfunction). higher lfs was associated with female sex (p = . ) but not other demographic, disease, or treatment factors. forty-eight survivors had blood samples collected at a mean age of . years (range: - ) with a mean time since treatment completion of . years (range: . - . ). of controls, the mean age at time of blood collection was years (range: - ). survivors did not have significantly elevated cytokine levels compared to controls. female survivors of hl ≥ months off therapy are at increased risk of pulmonary dysfunction. neither evidence for pulmonary dysfunction, as measured by lfs, nor duration of time off therapy were related to systemic inflammation in this study. pulmonary function deterioration and clinical pulmonary symptoms are rarely observed immediately following therapy but increase over time. future studies may consider exploring the contribution of systemic inflammation to pulmonary late effects in survivors farther off therapy, when risk for this late effect is greater. background: thyroid carcinoma is a very rare tumor in pediatrics, accounting for . - % of childhood carcinomas in the united states and europe. we aim to detect the risk of second malignancies among pediatric thyroid cancer survivors. the cohort analysis consisted of pediatric cancer patients aged less than years diagnosed with a primary thyroid cancer and identified by site code icd- - : c , reported to a seer database between and . they were followed up by death or the end of the study period (december , ) . out of patients diagnosed primarily with thyroid carcinoma, there were patients who had incidences of subsequent malignancies. the mean age of patients at initial diagnosis of thyroid cancer was years. females ( . %) had significantly higher incidence of second malignancies (sm) than males ( . %). the overall standardized incidence ratio (sir) of sm in thyroid pediatric patients was higher than expected (sir = . ). some specific sites showed significantly higher incidences: salivary gland (sir = . ), gum and other mouth (sir = . ) and kidney (sir = . ). the overall risk of sm in patients received radioactive iodine was higher than expected (sir = . ). the cumulative inci-dence of sms from the initial diagnosis of thyroid cancer was calculated with the survival methodology of competing risk, death treated as a competing event. cumulative incidence of sm was . % [ % ci ( . , . %)] at years and substantially expanded after years, reaching . % [ % ci ( . , . %)] at years. the cumulative incidence of each tumor type at years was . % [ % ci ( . , . %)] for breast cancer, . % [ % ci ( . , . %)] for salivary gland, . % [ % ci ( . , . %)] for each one of kidney and cervix uteri and . % [ % ci ( , . %)] for each one of ovary and melanoma of the skin. cumulative incidence of sm was stratified based on race, gender and radiotherapy exposure, but there was no statistical difference in each of them. conclusion: race, gender, histological subtypes, and radioactive iodine may play an important role as prognostic factors for developing sm among pediatric thyroid cancer survivors. identification of underlying mechanisms that raise the risk of sm is important for both treatment and follow-up strategy. background: the ethical practice of informed consent requires it be both voluntary and understood by the research participant. in pediatric oncology, parents must undergo informed consent to enroll their child with cancer into clinical trials, but often it can be difficult to understand especially for parents with low english proficiency. previous research has shown that parents of children with cancer have difficulty understanding voluntariness, and that parental satisfaction with informed consent does not always correlate with adequate comprehension. objectives: to examine socio-demographic and contextual correlates of comprehension of informed consent, voluntariness, and satisfaction in parents who consented to participation of their child in a cancer clinical trial. we focused on characterizing differences between non-hispanics and hispanics, the fastest growing ethnic group in the u.s. design/method: parents/guardians (n = ) of children aged - years with newly diagnosed cancer, who had consented to participation of their child in a clinical trial for cancer treatment at rady children's hospital-san diego were s of s prospectively recruited. parents completed questionnaires assessing comprehension, voluntariness, satisfaction, health literacy, socio-demographics, and acculturation level, if hispanic. comprehension was surveyed at baseline and longitudinally at months. comprehension, voluntariness and satisfaction outcomes were analyzed by socio-demographics, health literacy, and acculturation level using logistic regression. results: of the participants surveyed, ( . %) were hispanic and ( . %) were non-hispanic. we found that higher health literacy was associated with greater objective comprehension (p< . ), voluntariness (p< . ), socioeconomic status (p< . ), and acculturation (p< . ). hispanics reported lower objective comprehension (p = . ), voluntariness (p = . ), health literacy (p< . ) and ses (p = . ) compared to non-hispanics. spanish-speakers reported lower voluntariness (p = . ), health literacy (p< . ), and acculturation (p< . ) compared to englishspeakers. at the -month follow-up, comprehension in hispanics significantly improved (p = . ) compared to their baseline comprehension. satisfaction was moderately high across all subgroups and was not significantly impacted by socio-demographics, health literacy, or acculturation. in this study, with equivalent numbers of hispanic and non-hispanic participants, we found that hispanic and spanish-speaking parents of children with newly diagnosed cancer had inadequate informed consent comprehension, voluntariness and health literacy despite high satisfaction. our study suggests that hispanics and individuals with limited english proficiency are not making truly informed decisions for their child with cancer. to ensure the ethical practice of research in pediatric oncology, the informed consent and decision-making process must be improved with culturally and linguistically interventions for these underserved populations. memorial sloan kettering cancer center, new york, new york, united states background: pediatric oncology patients undergo repeated bone marrow aspirations and biopsies (bma/bx). these potentially painful procedures can exacerbate anxiety and distress. standard practice at memorial sloan kettering (msk) department of pediatrics is to use propofol, which has amnestic but no analgesic properties. we sought to evaluate whether the addition of local anesthetic would improve patient experience with bma/bx. the purpose of reppair: reducing procedural pain and improving recovery of quality of life (qol) (nct ) is to evaluate the efficacy of local anesthesia with ropivacaine in reducing procedural pain and improving post-procedure qol in pediatric neuroblastoma patients undergoing bma/bx with general anesthesia. reppair is a prospective, randomized, crossover clinical trial that opened for enrollment october . eligible patients were - years old with neuroblastoma. participants were observed on trial for two sequential bm procedures; one procedure with intervention a: propofol alone (pa), and the other with intervention b: propofol plus ropivacaine (p+r). participants were randomized to intervention sequence ab or ba and were blinded to the order of interventions. participants and recovery room (rr) nurses, who were also blinded, followed a standardized postprocedure pain management algorithm. the primary endpoint was percentage of participants requiring opioid analgesia in the hours post-procedure. secondary endpoints included total opioid in hours, non-opioid analgesia use, pain scores, time to first opioid, and short-term qol. qol was assessed by a parent-proxy metric that evaluated pain interference with sleep, physical, emotional, and social recovery. as of january , patients were assessed for eligibility and patients were randomized ( have completed both procedures). for the primary endpoint, a slightly higher proportion of participants required opioid for pa than p+r ( % versus %, p = . ). pain scores in the rr were significantly higher for pa than p+r (median [ th, th percentile]: [ , ] versus [ , ], p = . ). there were no statistically significant differences in total opioid or non-opioid analgesia, -and -hour pain scores, median time to first opioid, or pain interference scores. there were no adverse events. conclusion: preliminary findings of the reppair trial suggest that local anesthesia does not reduce the need for opioid analgesia or improve short-term qol in pediatric patients undergoing bma/bx with general anesthesia. local anesthesia did improve pain scores in the immediate recovery period. final results of this study will help establish evidence-based guidelines and optimize the experience of pediatric patients with bone marrow procedures at our center. background: children with advanced cancer experience a range of symptoms throughout treatment or at end of life, some of which are poorly controlled. minimizing suffering, including effective symptom management, in children with advanced cancer is a central value for pediatric oncology clinicians. patient-reported outcomes have been used in symptomrelated research in pediatric oncology patients; however the majority of literature specific to symptoms during palliative care and end of life for children and adolescents with advanced cancer is based primarily upon medical record reviews and to a lesser extent, patient self-report. the purpose of this study was to prospectively describe symptom frequency, severity, and level of distress in children/adolescents with advanced cancer using patient selfreport and parent proxy. design/method: a prospective cohort design was used for this study. five pediatric oncology institutions from across the united states participated. children and adolescents were eligible to participate if they were - years of age, englishspeaking, and had a diagnosis of advanced cancer, defined as a -week history of progressive, recurrent, or non-responsive disease or a decision not to pursue curative-focused therapy. a modified version of the memorial symptom assessment scale (msas) was used to measure symptom frequency, severity, and level of distress and was administered to child/parent dyads electronically via smartphones every two weeks. information regarding disease status and cancer treatment was collected concurrently. data was analyzed using descriptive statistics and univariate logistic regression analysis. results: a total of children and adolescents and parents participated in the study. the median age of child participants was years, with half being male. the median age of parents was years. the child participants had a variety of primary diagnosis, including: leukemia/lymphoma (n = , %), solid tumor (n = , %), and brain tumor (n = , %). the most frequently reported symptoms by children with advanced cancer and parents were pain (n = / , . %), lack of energy (n = / , . %), and nausea (n = / , . %). presence of disease (p = < . ), recent disease progression (p = . ), and receiving cancer therapy (p = . ) were significant factors on the presence of pain. high intensity cancer therapy was a significant factor on pain frequency (p = . ) and level of distress (p = . ). it is feasible to collect data prospectively in children with advanced cancer regarding symptom frequency, severity, and level distress. clinicians' increased understanding of the symptom experience may promote communication with children and adolescents and timely intervention. more research is needed to understand symptom clusters in children with advanced cancer. vanderbilt children's hospital, nashville, tennessee, united states background: febrile neutropenia (fn) is a frequent occurrence in children undergoing chemotherapy. though guidelines recommend adding a second antibiotic to broad-spectrum antipseudomonal coverage in specific scenarios, augmenting empiric therapy with a second antibiotic is common practice. additional empiric antibiotic (aea) use increases the risk of antibiotic toxicity and future antimicrobial resistance. data clarifying the indications for aea are limited in pediatric patients. objectives: to identify risk factors for gram-positive (gp) and gram-negative (gn) bacteremia in patients presenting with fn to determine situations in which aea use is warranted. design/method: a retrospective chart review was conducted of pediatric severe fn with absolute neutrophil count < / l occurring at a single institution between and . potential a priori risk factors based on clinical reasons for antibiotic expansion were chills, hypotension, mucositis, skin or soft tissue infections (sstis), recent administration of highdose cytarabine (hdac), and a diagnosis of acute myeloid leukemia (aml). potential factors for gn bacteremia were chills, hypotension, mucositis, and abdominal pain. the association between each potential risk factor and gp or gn s of s bacteremia was identified. logistic regression was used for multi-variable analysis. the review yielded episodes. gp bacteremia was isolated in cases ( . %) and gn bacteremia in episodes ( . %). in multivariable analysis, hypotension (or . ( % ci . , . ), p = . ) and sstis (or . ( . , . ) , p = . ) were independently associated with increased risk of gp bacteremia, while mucositis (p = . ), recent administration of hdac (p = . ) and chills (p = . ) were not. ten patients with aml didn't receive hdac, thus the association between aml and gp bacteremia could not be reliably estimated. hypotension (or . ( . , . ), p< . ) and chills (or . ( . , . ), p< . ) were independently associated with a higher risk of gn bacteremia, while mucositis (p = . ) and abdominal pain (p = . ) were not. of the gn infections, ( %) were resistant to cefepime, the empiric agent of choice at our institution. patients with fn with sstis, hypotension, or recent hdac had increased risk of gp bacteremia indicating potential benefit of empiric vancomycin in these settings, while mucositis and chills were not associated with gp bacteremia. hypotension and chills were associated with gn bacteremia, potentially warranting empiric antibiotic expansion, while mucositis and abdominal pain were not. identifying specific indications for aea use in pediatric severe fn use may improve antimicrobial utilization, decrease unnecessary antibiotic use, and improve patient outcomes. background: for children/young adults with incurable high grade gliomas (hggs), like diffuse intrinsic pontine glioma (dipg) or glioblastoma multiforme (gbm), oncologists endeavor to align therapy with patient/family goals of care, but may be influenced by providers' preferences or limited resources. ethical challenges can arise around the perceived purpose, risks and benefits of therapy options, provider conflicts of interest, access to care, deciding decisional priority between patients and families, and conflicts around end-oflife care. objectives: evaluate factors that play into longitudinal decision making for children and young adults with hggs, their families and oncologists using a qualitative approach with ethnographic elements. design/method: eligible patients were aged - with dipg, gbm, or secondary hgg. patient exclusions included: non-english speaking, in state custody, death prior to diagnosis, seen by oncology once, or an oncologist declined participation. key decision making visits (e.g. mri reviews) were serially audio-recorded, along with subsequent : semistructured interviews with patients and/or parents about the decision making process. field notes from clinician meetings, chart notes, and oncologist questionnaires were obtained. discussions and interviews were transcribed and independently coded by three investigators. inter-rater reliability was assessed during code book development. discrepancies were discussed until consensus met. constant comparison analysis with maxqda software continued until thematic saturation. results: twenty-two of eligible patients were approached; agreed to participate. one withdrew upon transferring care. mean age was . years (sd . ); % male, % caucasian, % african american, % hispanic, and % asian. four encounters, ( . hours), were recorded on average per patient. parent/patient interview themes included: ) hope (for a cure, prolonged life, and quality of life), ) importance of physician recommendations, ) importance of support systems (family, community, social media), ) food (as cancer etiology, intervention) ) finances (personal, research funding), ) communication (with medical providers, family, community), ) death, and ) god (beliefs, prayer, existential questions). oncologists desired prolonged quality of life, while patients/families transitioned to that hope from hope for a cure. decisions made in the setting of hggs are multi-factorial, ultimately reflecting the competing values of decision makers. optimism about treatment efficacy is held in tension with poor prognosis, allowing for functional hope. acknowledging patients' and families' shifting hopes allows for changes in goals of care and shared decision making. future work is needed to ) develop preference tools for pediatric patients and families to inform medical providers and ) provide training in communication and shared decision making with oncologists. emory university, atlanta, georgia, united states background: bone marrow transplantation (bmt) is a potentially curative but underutilized treatment for scd. our previous work has shown that there is variation in physician philosophy and practice in considering bmt as a treatment option for patients with scd, and physicians may not discuss this with patients and families as a potential treatment option. in a randomized clinical trial to test the effectiveness of a decision aid for disease modifying therapies for sickle cell disease, adult patients with scd as well as caregivers of adult/pediatric patients were interviewed about how they seek or have sought information related to scd, made decisions about treatments for scd, and identified a treatment option they were interested in learning more about using the decision aid tool. we performed a secondary analysis of these baseline data to understand patient information needs and attitudes regarding bmt as a treatment option for scd. the goals of this analyses was to understand patient and caregivers' attitudes and perceived information needs regarding bmt as a treatment option for scd. we performed an analysis of baseline interviews from caregivers of patients with scd or adult patients from a randomized control trial for a decision aid tool for scd. of the interviews belonged to caregivers of patients with scd. in addition to reviewing interviews for discussion of bmt, we interrogated for mention of terms such as 'bone marrow transplant' or 'cure' or 'stem cell transplant'. interviews were coded using nvivo and analyzed for emerging themes. results: of the baseline interviews, interviews met selection criteria. thirteen of the interviews were with caregivers of pediatric patients, and the remainder were with adult patients, including young adult patients with scd. the majority of participants want to learn about bmt or curative options. in many participants, this was expressed despite knowledge that they were not a likely candidate for transplant. desired information about bmt included eligibility, benefits, risks, long-term effects, quality of life and financial aspects related to bmt. of the patients who discussed how they learnt about bmt, approximately half mentioned that their healthcare provider had not previously mentioned this to them. we then examined knowledge of bmt and attitudes with demographic and clinical variables. patients and caregivers of pediatric patients with scd want to learn about bmt as a treatment option. healthcare providers should consider discussing bmt with their patients with scd. natasha frederick, anna revette, alexis michaud, jennifer mack, sharon bober dana-farber cancer institute, boston, massachusetts, united states background: adolescents and young adults (ayas) consistently identify the need for improved patient-clinician communication on sexual and reproductive health (srh) issues. however, oncology clinicians do not routinely integrate srh conversations with ayas through disease treatment and survivorship. little is known about why these conversations do not take place. objectives: explore aya perceptions of and receptiveness to srh communication with oncology clinicians and to identify barriers and facilitators to these conversations. design/method: semi-structured interviews were held with aya cancer patients and survivors (ages - years, men, women). twelve participants were on active treatment and were within years of treatment completion. interviews were conducted in english by phone or in person. the interview transcript underwent pre-testing with ayas. all interviews were audio-recorded and transcribed verbatim. transcripts were analyzed and summarized by two trained qualitative researchers according to standard comprehensive thematic qualitative analysis methods. analyses were aided by nvivo software. results: ayas perceived existing srh communication between ayas and oncology providers as inadequate. all ayas reported a need for improved srh communication with oncology providers, and three key areas of need emerged: ) general education; ) addressing specific srh issues experienced during treatment and survivorship; and ) understanding the long-term impact of cancer and treatment on srh. ayas felt that current srh discussions are limited and too narrow in scope and scale. ayas reported that most srh conversations focus exclusively on fertility (n = ), usually taking place at the start of treatment. other additional yet limited communication reported was about sexual activity (n = ), contraception (n = ), sexual function (n = ). no ayas reported conversations about potential treatment complications related to sexuality other than infertility. key barriers to srh conversations include patient discomfort initiating conversation (n = ) and presence of family members (n = ), with additional reported barriers including perceived provider discomfort (n = ), lack of rapport with provider (n = ), and age/gender differences (n = ). ayas felt that s of s communication tools such as handouts, brochures, and websites would be helpful facilitators to direct communication from the oncology clinician, and wanted conversations to start before treatment initiation and to continue through treatment and survivorship conclusion: ayas identify a key role for pediatric oncology providers in srh care from diagnosis through survivorship, however multiple barriers interfere with discussions about srh on a regular basis. identified barriers suggest that future efforts should focus on provider education and training in srh and srh-related communication in order to optimize care provided to this unique patient population. background: peripherally inserted central venous catheters (picc) provide secure vascular access in pediatric patients for the delivery of necessary therapies. the ease of placement in the inpatient and outpatient settings has expanded their utilization. however, recent data analyses show a significant increase in venous thromboembolism (vte) risk with the use of picc lines. with its rising use, modifiable risk factors need to be understood for preventative measures. objectives: in this study we aim to understand patient and catheter specific characteristics in relation to the development of vte. design/method: with irb approval, a retrospective interrogation of the electronic medical record and a picc database, at rainbow babies and children's hospital, was completed. the study cohort contained patients < years of age who had a picc line placed between january of and december of . data collected included indication for line placement, line dwell time, location of insertion including blood vessel and extremity, number of attempts at line placement, lumen size and indwelling line length. in addition, we collected number of days to vte formation, associated symptoms and location of vte. chi-squared analyses and fischer's exact test were used where appropriate for statistical analysis. we analyzed ( neonatal) newly placed picc lines. fifty line-associated vte events were found, for an incidence of . %. all vte occurred with the placement of the first picc line. intravenous therapies were the most common reason for line placement. no statistical significance was found between various indications for placement. the most common symptom of vte manifestation was extremity swelling, follow by extremity pain. right extremity picc was found to have a higher incidence of vte. larger catheter lumen sizes (> french) had a higher incidence of vte. we found a mean time of . days to vte detection. we were unable to find any clinical, patient or line specific factors leading to increased vte formation after statistical analysis. special consideration should be given to the duration of picc line use as this may reduce the incidence and comorbities associated with vte. there is still much to be understood about catheter associated vte formation as our analyses indicates the need for prospective data collection on a larger scale in hopes to create guidelines related to catheter use in pediatrics. background: the decision to transfuse a patient is a complex one and is never based solely on a number; however, certain hemoglobin or platelet count thresholds have been proposed in aiding physicians make transfusion decisions. in our hospital, the thresholds for packed red blood cell (prbc) and platelet transfusion in pediatric oncology patients are hemoglobin levels below . g/dl and platelet counts below , /mm (< , for brain tumors), respectively. recently, these thresholds have been questioned and we were asked whether we could safely lower the thresholds to < . g/dl of hemoglobin and < , /mm platelet count objectives: to investigate platelet and hemoglobin transfusion thresholds for oncology patients at children hospital of michigan design/method: retrospective chart review over a -month period, examining platelet and hemoglobin pretransfusion levels for each prbc and platelet transfusion given to oncology patients results: over the course of months, eligible oncology patients (median age years) received transfusions ( prbc transfusions and platelet transfusions). the mean pretransfusion hemoglobin level was . ± . g/dl (range . - . ) (n = ) for total prbc transfusions and this was not different among disease categories (p = . ). patients who had anemia symptoms and signs (n = ) had a slightly lower hemoglobin level compared to those who did not (n = ): . ± . vs . ± . g/dl (p = . ). the mean pretransfusion platelet count was , ± , /mm (range , - , ) for total platelet transfusions (n = ); , ± , /mm in patients with brain tumors (n = ); , ± , in patients with leukemia (n = ); and , ± , in patients with solid tumors (n = ). the mean pretransfusion platelet count was significantly higher in transfusions for brain tumors compared to that in the other disease groups (p< . for both). the mean pretransfusion platelet count was not different among those patients who had bleeding/bruising symptoms ( , ± , , n = ) versus those who did not ( , ± , , n = ) (p = . ). the bleeding/bruising rate was slightly but insignificantly higher in those who had platelet counts < , vs those who had ≥ , ( . % vs . %, p = . ). since most patients develop symptoms of anemia at hemoglobin above g/dl and about / of patients develop bleeding/bruising symptoms at platelet counts above , /mm , our current policy so far reflects a safe threshold for transfusion, and further lowering of the thresholds should be investigated in prospective studies. background: renal impairment is an important complication of childhood cancer and its treatment. serum creatinine level is frequently used as a screening test to monitor renal function; however, patients can have significantly decreased glomerular filtration rate (gfr) with normal serum creatinine. to determine the prevalence of chronic kidney disease (ckd) among children with cancer diagnosis, based on calculated gfr. to compare the difference between using serum creatinine value alone versus gfr in detecting ckd. design/method: retrospective review of medical records of patients, age - years, diagnosed between / - / with solid tumors were analyzed. serum creatinine and calculated gfr using schwartz formula were recorded. ckd as classified by the foundation of kidney disease and outcome quality initiative was used: ckd stage : gfr ( to ml/min per . m ) ckd stage : gfr ( to ml/min per . m ) statistical analysis using spss software v. . chi-squared test for proportions within group, and pearson chi-squared and fisher exact tests for statistical differences between groups. p-value < . was considered to indicate significance results: out of the records reviewed, ( %) were males and ( %) females, with mean age of . ± . years. ( . %) patients received one or more of nephrotoxic chemotherapy drugs; cisplatinum, carboplatinum, or ifosphamide mainly in the non-wilms solid tumors group ( . %) compared to ( . %) in the wilms tumor (wt) group. based on calculated gfr (by schwartz formula) ckd stage /or was diagnosed in ( %) patients with overwhelming majority ( %) were in the mild stage ckd, only ( . %) of those patients had abnormally high serum creatinine levels (p = . ). . % of patients who received nephrotoxic chemotherapy developed ckd, compared to . % in those who did not receive it, (p = . ). despite that only / ( %) of wt group patients received nephrotoxic chemotherapy, yet this group had higher percentage of ckd ( . %) compared to non-wt group ( . %) p = . . significantly lower mean gfr . ± was noticed in the wt group compared to . ± in non-wt group (p = . ) conclusion: high prevalence of mild ckd was found among solid tumor patients. using serum creatinine alone as measure of renal function significantly under estimates renal impairment in those patients. early identification of ckd is easily achieved by using calculated gfr, which can helps providers and care givers to avoid potential nephrotoxic antibiotics, contrast media, nsaids and dehydration that may further deteriorate renal function the university of texas southwestern medical center, dallas, texas, united states background: children with down syndrome (ds) have increased risk of developing leukemia. pediatric patients with ds-associated acute lymphoblastic leukemia (ds-all) are known to have significant toxicities with reinduction chemotherapy and historically poor outcomes with stem cell transplant (sct). anti-cd chimeric antigen receptor (car) t-cell therapy, tisagenlecleucel, demonstrated high rates of durable complete remission (cr) and a manageable safety profile in children with r/r b-cell acute lymphoblastic leukemia (b-all). objectives: characterize the efficacy and safety of tisagenlecleucel in pediatric/young adults with ds-all. design/method: pooled data from single-arm, multicenter, phase trials of tisagenlecleucel in pediatric/young-adult patients with r/r b-all (eliana, nct ; ensign, nct ) were analyzed. eight patients with ds-all were enrolled (data cutoff: eliana, november ; ensign, february ). seven were infused with tisagenlecleucel; patient died from all progression and intracranial hemorrhage before infusion. no manufacturing issues occurred during production. / infused patients were male, / had prior sct (age range, - years). / patients achieved cr or cr with incomplete blood count recovery (cri) by day (d) (cr+cri, %); died before d and was not evaluable. analysis of minimal residual disease was negative in bone marrow in responding patients. two patients had cd negative relapses at and months. ongoing remissions in patients without relapse ranged from to months. the safety profile (n = ) appears similar to that in patients without ds in the same trials (n = ). grade (g) / cytokine release syndrome occurred in % ( / ) of patients with ds and in % without ds. rates of other g / adverse events of special interest did not appear to favor a consistent trend between patients with/without ds (febrile neutropenia: % vs %; neurological events: % vs %; tumor lysis syndrome: % vs %). g / infections were not observed in patients with ds ( % vs %). one patient died after infusion due to intracranial parenchymal hemorrhage on d associated with ongoing coagulopathy. time and extent of tisagenlecleucel expansion and long-term persistence were similar between groups. conclusion: this is the first analysis of car t-cell therapy in pediatric patients with r/r b-all and ds. these data suggest that toxicities appear similar to those in patients with b-all without ds, remission rates in ds-all are high, and longterm outcomes with sustained persistence appear promising. further exploration of tisagenlecleucel as an alternative to sct in children with r/r ds-all is warranted. sponsored by novartis. background: hispanic adolescence and young adults are twice as likely to develop acute lymphoblastic leukemia (all) with high risk features as non-hispanic whites. they also have poor prognosis and % higher death rate. b-all with crlf overexpression caused by genetic alteration of the cytokine receptor, crlf is five times more common in this subgroup. approximately % of crlf b-all cases also have ikzf genetic alterations. ikaros is involved in transcriptional regulation of several important genes involved in leukemogenesis. overexpressed casein kinase ii (ck ) impairs functions of ikaros. objectives: understand the molecular mechanisms that regulate crlf expression in crlf b-all. here we present evidence that ikaros-mediated repression of crlf transcription in b-all in hispanic children is regulated by ck . design/method: primary b-all patient samples from hispanic children were used. ikaros retroviral transduction, ikaros shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: ikaros binding to promoter of crlf was confirmed using quantitative chip. functional experiments such as overexpression of ikaros in b-all primary cells results in transcriptional repression of crlf whereas ikaros silencing using shrna resulted in increased transcription. these results suggest that ikaros negatively regulates crlf expression. molecular inhibition of ck with shrna targeting the ck catalytic subunit, as well as pharmacological targeting of ck with cx resulted in transcriptional repression of crlf . ck inhibition was associated with increased ikaros dnabinding to the promoter of crlf . however, the ability of cx to repress crlf is lost or severely reduced, in cells with shrna silencing of ikaros, as compared to cells with intact ikaros. moreover, similar results were noted following treatment with cx in leukemia cells obtained from high risk b-all patients with deletion of one ikzf allele. ikaros binds poorly to promoters of crlf gene in these cells. treatment with cx restores ikaros dnabinding to the promoters of crlf , which is associated with its strong repression. serial qchip analysis of the epigenetic signature at the crlf promoter showed that increased ikaros binding to the crlf promoter, following ck inhibition, is associated with enrichment for the h k me histone modification, which is a marker of repressive chromatin. results demonstrate that crlf expression is epigenetically regulated by the ck -ikaros axis .cx show antileukemic effect via restoration of ikaros tumor suppressor function, resulting in crlf repression suggesting advantage of using ck inhibitors as potential therapeutic approach in crlf altered b-all. results: hypodiploid all (modal chromosome number < and/or di < . ) was identified in patients ( . % of all patients; . % of nci standard risk (sr) and . % of nci high risk (hr)), who were removed from frontline protocol therapy post-induction. overall -year efs and os were . %± . % and . %± . %. transplant status was retrospectively available for / ( %), of whom underwent hsct in cr . five-year efs with hsct was . %± . % vs. . %± . % without (p = . ). -year os with and without hsct was . %± . % vs. . %± . % (p = . ). when corrected for the median time to hsct ( days), there were no significant differences in -year efs or os rates with and without hsct: . %± . % and . %± . % vs. . %± . % and . %± . %. no nci risk group or mrd subset benefitted significantly from cr hsct. sr patients (n = ) had -year efs and os of . ± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. hr patients (n = ) had -year efs and os of . %± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. for those with end-induction mrd < . % (n = ), -year efs and os were . %± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. end-induction mrd-positive patients (n = ) fared poorly with both year efs and os of . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. multivariate regression analysis including nci risk group, mrd, and cr hsct, showed only mrd negativity was significantly associated with efs (hr . , p< . ) and os (hr . , p< . ). patients with hypodiploid all fare poorly, particularly those with end-induction mrd ≥ . %. while cr hsct is a standard treatment approach, it does not confer significant benefit. we were unable to assess bridging therapy prior to hsct, and comparator groups are small. taken together, however, new strategies are urgently needed for these patients. background: ras-pathway mutations are known to play a pivotal role in a significant proportion of myeloid malignancies, including upwards of % of pediatric aml cases. ras-pathway mutations in myeloid malignancy commonly co-occur with mutations of epigenetic regulators, suggesting cooperative leukemogenesis. among the epigenetic modifiers most frequently mutated in myeloid malignancy are regulators of dna methylation. this indicates that the alteration of dna methylation contributes to leukemogenesis. the ten-eleven translocation (tet ) is an epigenetic regulator that plays an important role in regulation of dna methylation through its action of hydroxylation of -methylcytosine, which ultimately leads to passive de-methylation of dna cytosines. in myeloid malignancy, loss of function tet mutation is one of the most frequently co-occurring lesions in ras mutated malignancy. how specifically the altered methylation patterns in ras-pathway driven diseases promotes leukemogenesis is unclear. objectives: we hypothesize in mice with a ras-pathway mutation, that when an epigenetic modifier co-occurs, such as loss of function of tet , this primes stem cells and/or early differentiating progenitors for transformation by preventing the repression of stem cell self-renewal genes, inhibiting differentiation, enhancing ras signaling and leading to leukemogenesis. we have generated a novel murine model with constitutive deletion of tet (tet -/-) combined with an inducible activating krasg d mutation (krasg d/wt). mice have been tracked for evidence of hematologic malignancies and compared to mice with corresponding single genetic lesions. cooperative leukemogenesis will be demonstrated by decreased latency to disease onset, impact on malignancy lineage, in addition to investigating mechanistically through which pathways leukemogenesis may be promoted. results: krasg d/wt/ tet -/-mice demonstrate statistically significant differences in peripheral white blood cell count, hemoglobin, and platelet levels as early as -weeks post ras-pathway activation. peripheral cell lineage analysis demonstrates early skewing toward myeloid differentiation and marked splenomegaly in mice harboring both genetic lesions compared to wild type or mice with single genetic lesions. phospho-flow cytometric analysis reveals increased perk and ps activation in krasg d/wt/ tet -/-sca- enriched bone marrow cells compared to either genetic lesion alone. our study utilizing a murine model to examine how in ras-pathway mutations the addition of a co-occurring epigenetic lesion demonstrates that these lesions appear to cooperate to promote early myeloid differentiation with attendant changes in signaling pathways. this exploration to elucidate the mechanics of ras-pathway mediated disease lay the foundation for identification of patients who may benefit from existing therapies, such as dmtis, or identify new signaling targets for therapeutic exploration. background: the humoral immunogenicity of car , a chimeric antigen receptor (car) with a murine scfv domain developed for treatment with tisagenlecleucel in relapsed/refractory (r/r) pediatric/young-adult acute lymphoblastic leukemia (all), was evaluated in studies. little is known about the presence/impact of preexisting/treatmentinduced anti-murine car (mcar ) antibodies in patients treated with car therapy. objectives: patients from eliana (nct ; n = ) and ensign (nct ; n = ) were evaluated before and after tisagenlecleucel infusion to determine the impact of anti-mcar antibodies on cellular kinetics, efficacy, and safety. design/method: anti-mcar antibodies were determined by flow cytometry and reported as median fluorescence intensity. assay validation included evaluation of the interferences of intravenous immunoglobulin (ivig) treatment with the anti-mcar antibody assay. impact of preexisting and treatment-induced immunogenicity on cellular kinetics, efficacy, and safety was determined. treatment-induced immunogenicity was defined by a positive increase in anti-mcar antibody levels over baseline and was assessed by calculating the fold-change between preexisting (ie, baseline) and postinfusion levels. results: % of patients displayed preexisting anti-mcar antibodies; a similar incidence was detected in healthy volunteer samples during method validation. % of patients developed treatment-induced anti-mcar antibodies. no relationship was identified between tisagenlecleucel expansion (auc - d) and preexisting/treatment-induced anti-mcar antibodies (r < . and r = . , respectively); similar results were seen for cmax. presence of treatment-induced anti-mcar antibodies did not appear to impact transgene persistence or response. kaplan-meier estimates showed that preexisting/treatment-induced anti-mcar antibodies did not appear to impact duration of response or event-free survival. strip plots showed consistent levels of preexisting/treatment-induced anti-mcar antibodies across patients with safety events, including cytokine release syndrome, neutropenia, thrombocytopenia, and neurological events. there was no apparent relationship between treatment-induced anti-mcar antibodies and b-cell recovery categories (≤ months, > and ≤ months, > months, and ongoing sustained aplasia). no association existed between time of b-cell recovery and presence of treatment-induced anti-mcar antibodies. b-cell aplasia requiring ivig occurred following tisagenlecleucel in the majority of patients. the tisagenlecleucel concentration-time profiles in patients with treatment-induced anti-mcar antibodies were categorized by time following ivig administration. time of ivig administration had no impact on in vivo transgene expansion and persistence. we report the first comprehensive assessment of the impact of anti-mcar antibodies on clinical endpoints with car therapy. pediatric/young-adult patients with r/r all had a high frequency of baseline anti-mcar antibodies, and preexisting/treatment-induced anti-mcar antibodies did not impact the cellular kinetics, safety, and efficacy of tisagenlecleucel. cell-mediated immunity studies are ongoing. sponsored by novartis. background: adoptive immunotherapy, using cd engager (cd -eng) t-cells, has shown success in preclinical studies, recognizing and killing acute myeloid leukemia (aml) blasts in vitro and in vivo. cd -eng t-cells secrete bispecific molecules that recognize cd (t-cells) and cd (aml blasts), and are able to direct transduced t-cells and recruit bystander t-cells to kill cd -positive blasts. however, cd -engs do not provide costimulation and have not shown the capability for sequential killing of targets in vitro. we are seeking to improve the expansion, persistence and sequential killing capabilities of cd -engs by genetically modifying these cells with an inducible costimulatory molecule, which can be activated by a chemical inducer of dimerization (cid). we generated a retroviral vector encoding cd -eng and the inducible costimulatory molecule myd .cd linked by a a sequence (cd -eng. a.imc). cd -eng and cd -eng.imc t-cells were generated by retroviral transduction, and their effector function was compared with and without cid. we used flow cytometric analysis to assess transduction efficiency, chromium release assays to evaluate cytolytic activity, and elisa to determine cytokine production. we successfully generated cd -eng.imc tcells and achieved a mean initial transduction efficiency of % that was maintained above % throughout our study period. cd -eng.imc t-cells +/-cid and cd -eng t-cells readily killed cd -positive aml blasts (molm and kg a) in cytotoxicity assays when compared to the cd -negative control (k ). in co-culture assays, cd -eng.imc t-cells secreted increased il- and ifn-gamma in the presence of cid and cd -positive targets (kg a and molm ) when compared to co-culture with cd -positive targets in the absence of cid. in addition, cd -eng.imc t-cells displayed enhanced sequential killing capabilities and ifn-gamma secretion when stimulated weekly with cid and tumor cells at a : ratio when compared to cd -eng t-cells. conclusion: cd -eng.imc t-cells are able to recognize and kill cd -positive aml blasts in an antigen dependent manner. cd -eng.imc t-cells have improved effector function in the presence of cid as judged by cytokine production and their ability to sequentially kill cd -positive target cells. thus, inducible myd and cd costimulation is a promising strategy to improve the effector function of cd -eng t-cells, and warrants further active exploration in preclinical studies. background: eliana (nct ; n = ) is a pivotal multicenter study testing the efficacy of tisagenlecleucel, anti-cd car-t, in children/young adults with r/r b-all. tocilizumab (toci) has been used for management of moderate/severe (grade / ) crs in ≈ % of patients treated with tisagenlecleucel at equivalent doses used in approved nononcological pediatric indications (< kg received mg/kg; ≥ kg received mg/kg [ mg max dose]).( ) crs onset, as graded by the penn grading scale, generally occurred at a median of days (range, - ) after infusion, requiring administration of - toci doses in some patients via a protocol-specific treatment algorithm. toci is a humanized monoclonal antibody that inhibits il- receptor (il- r) signaling. the pharmacokinetics (pk) and pharmacodynamics (pd) of toci in pediatric patients with b-all with carassociated crs have not previously been described. objectives: characterize toci pk/pd for crs management following tisagenlecleucel infusion and describe its impact on cellular kinetics. design/method: toci pk and levels of soluble il- r (sil- r) were determined from serum and quantified using validated assays. maximum toci concentration (cmax) was derived using noncompartmental methods. sil- r, proinflammatory cytokines, and crs resolution time were characterized to describe toci pd. summary statistics and graphical analyses of tisagenlecleucel exposure by number of doses were performed to describe the impact of toci on tisagenlecleucel kinetics in patients responding to tisagenlecleucel infusion. : / patients with crs received the first toci dose at a median of days (range, - ) after crs onset. seventeen patients received dose (range, . - mg/kg); received doses ( - mg/kg); received doses ( - mg/kg), per the crs treatment algorithm. first-dose mean cmax (sd) was ≈ ( . ) g/ml; second dose, ≈ ( ) g/ml. individual patient pd concentration-time profiles showed increased sil- r levels after the first toci dose which remained elevated following the second dose. following toci administration, median time to crs resolution (including fever resolution) was days (range, - ). crs onset coincided with tisagenlecleucel expansion, followed by a peak in serum cytokines, including il- . the geometric mean auc - day and cmax of tisagenlecleucel transgene (by pcr) were % and % higher in tisagenlecleucel-responding toci-treated patients. conclusion: crs symptoms resolved within a median of days after toci administration. toci levels achieved in patients with b-all were similar to reported pediatric nononcological indications (tocilizumab label) and resulted in concentration/time-dependent sil- r increases. transgene continued to expand and persist following toci administration. these data support treatment with toci for crs management. ( ) buechner, eha, . sponsored by novartis. background: in acute myeloid leukemia (aml), mesenchymal stem and stromal cells (mscs) in the bone marrow microenvironment contribute to extrinsically mediated chemo-resistance and are therefore important potential therapeutic targets. the study of patient-derived mscs is at a competitive disadvantage, however, because traditional means of isolating mscs from a bone marrow aspirate interferes with isolating the more highly prioritized leukemic cells. many opportunities to study mscs are therefore missed. objectives: to develop a novel method of isolating mscs using the otherwise discarded portion of a bone marrow aspirate, thereby de-coupling the isolation of primary mscs from the isolation of leukemia cells. design/method: aml patient bone marrow aspirates were obtained prospectively from the children's oncology group. healthy patient marrow was purchased. experimental mscs were isolated from the bottom-most layer (rbc-layer) produced by density-gradient separation of a bone marrow aspirate, which is typically discarded. control mscs were isolated from the buffy coat (mnc layer). non-adherent cells were removed after hours, and adherent cells were cultured at % co with mem-alpha containing % fbs. growth curves were obtained by seeding -well plates with , cells per well. cells were stained using oil red o to observe adipocyte differentiation. results: rbc-layer mscs grow successfully following overnight shipment of the aspirate. identical to mnc-layer mscs, rbc-layer mscs exhibit a fibroblastic morphology and are adherent to plastic. rbc-layer mscs persist in culture up to passages before senescence. they exhibit a slower growth curve relative to mnc-layer mscs, but their overall doubling time is similar at approximately hours. surprisingly, mscs from the rbc-layer exhibit adipocyte differentiation on stimulation, revealing their stem-cell like qualities. we present a method of isolating mscs from the discarded portion of a bone marrow aspirate that does not interfere with the isolation of leukemia cells from the same patient. this portion of the aspirate can be shipped, or can sit for at least hours, without sacrificing its mscs. rbclayer mscs are nearly identical to mscs obtained conventionally. perhaps most importantly, rbc-layer mscs retain a stem-cell like capacity, showing them to be a highly valuable cell population in aml research. future plans include investigating potential selective enrichment of stem-cell mscs in the rbc-layer, which could explain the unexpected difference in growth kinetics. aml researchers now have the opportunity to study this exciting component of the bone marrow microenvironment without sacrificing valuable leukemic cells in the process. background: neutropenia is one of the most frequent side effect of chemotherapy associated with an increase in the risk of infection, especially in the cases when the depth and duration of neutropenia are extended. some genes, as variations of darc, gsdma and cxcl are known to influence white blood cell and neutrophil counts. our previous study conducted in children with acute lymphoblastic leukemia (all), showed that polymorphisms in these genes might play a role in the onset of chemotherapy complications during consolidation and maintenance treatment. objectives: in order to support our previous finding, we have expanded the study to the induction period in a cohort of all children treated at the sainte-justine university health center between july and july . design/method: previous associated single nucleotide polymorphisms (snps) in darc, gsdma and cxcl genes were analyzed for an association with the complications occurring during induction including the duration of low neutrophil count (pnn) and low absolute phagocyte count (apc), proven infections and delay between induction and consolidation phases. results: significant effect was found for all studied polymorphims. minor alleles of darc rs , cxcl rs and gsdma rs were all associated with higher risk of complications during induction treatment, whereas that of darc rs (particularly gg genotype) had a protective effect. the gg genotype of rs was associated with a lower risk of post-induction delay (p = . or = . , %ci . - . ), less frequent febrile episodes (p = . ) and lower number of days with apc/pnn count reduction (p = . for apc< . and p = . for pnn< . ). in contrast, the minor t allele of another darc polymorphism (rs ), was associated with longer apc/pnn count reduction (p = . for apc< . and p = . for pnn < . ), as it was the tt genotype of gsdma rs (p = . for apc< . and p = . for pnn< . ). the patients with the gsdma rs had also a higher risk of documented febrile episodes (p = . or = . %ci - . ). the aa genotype of rs cxcl was associated with a higher risk of post-induction delay due to infection (p = . , or = . , % ci . - . ). conclusion: this complementary study confirmed our previous results, showing overall that variations in darc, gsdma and cxcl genes influence the onset of chemotherapy complications in pediatric all, regardless of treatment phases. these polymorphisms might be useful pharmacogenetics markers possibly guiding an adjustment of chemotherapy intensity. background: pediatric acute myeloid leukemia (aml) has a poor survival rate of about % and there is an urgent need for newer targeted therapies. car t-cell based therapies are effective against all but similar therapies against aml are still under development. recent clinical trials have highlighted the concerns about toxicity and therapy related deaths from car t-cells. antigen selection is the key factor determining the specificity, efficacy and toxicity of car t-cells. while contemporary adoptive t-cell therapies use monoclonal antibodies against tumor associated antigens we employed the naturally occurring flt ligand (fl) to target aml cells expressing flt receptors. flt receptor is expressed on multipotent and myelomonocytic progenitors as well as myeloid leukemia cells. to generate fl containing chimeric tlymphocytes designated flcar t-cells and to evaluate their efficacy against aml cells. design/method: flcar was constructed by fusing the coding sequences of the human fl, cd costimulatory domain, and cd -zeta chain (intracellular region) in series. it was then cloned into the phiv-egfp lentiviral vector for expression in cell lines and primary t cells obtained from healthy donors. the empty phiv-egfp vector was used as a negative control. flcar was expressed on both cd + and cd + t-lymphocytes, confirmed by western blot. cell cytoxicity was evaluated by co-culturing flcar t-cells and aml cells followed by flow cytometric analyses. cytokine production was assessed by analyzing expression of interleukin- using quantitative rt-pcr. results: flcar t-cells were generated from cd + jurkat and cd + tk- cell lines with up to % lentiviral transduction efficiency. the efficiency for primary t cells was lower ( - %). flcar was expressed as a ∼ kda protein in cells and was partially phosphorylated on tyrosine. the expression of flcar on lymphocytes lead to increased basal il- expression in the cells. this was further augmented (by > folds) upon co-incubating flcar t-cells with flt expressing target cells. jurkat cells, tk- cells and primary human t cells expressing flcar suppressed the growth of flt -expressing aml cell lines and primary aml cells in vitro. notably, flcar t-cells generated from healthy donors caused strong inhibition of aml cells even at a lower transduction efficiency. in vivo experiments using nsg-sgm mice xenografted with human aml cells are underway. our data demonstrate that flcar can be effectively expressed on t-lymphocytes and mediate potent cytotoxicity against flt -expressing aml cells in vitro. being a completely human derived chimeric protein, it represents a promising candidate for further therapeutic development. holly pacenta, kelly sullivan, ahwan pandey, kelly maloney, joaquin espinosa children's hospital colorado, denver, colorado, united states background: individuals with down syndrome (ds) have a -fold higher risk of developing acute lymphoblastic leukemia (all) than the typical population. there are several important differences between all in individuals with ds (ds-all) and all in individuals without ds (nds-all): first, patients with ds-all have a lower percentage of favorable cytogenetic features compared to nds-all. second, patients with ds-all are more likely to have activating mutations in jak , crlf overexpression, and ikzf deletions. despite these clear genotypic differences, this knowledge has not yet been exploited for therapeutic purposes in ds-all. when outcomes for ds-all are compared to nds-all with similar cytogenetic features, the survival rates are similar. however, individuals with ds-all have an increased risk of treatment-related mortality (trm). current therapy for ds-all is similar to that for nds-all, with the exception of small changes to decrease toxicities that are more prevalent in ds-all. it was recently identified that interferon signaling is constitutively activated in healthy individuals with t . we hypothesize that aberrant interferon signaling could play a role in the unique leukemias observed in ds patients. objectives: to identify differences in gene expression and intracellular signaling cascades that are unique to individuals with ds-all, relative to both nds-all and healthy individuals with ds that can be exploited for therapeutic use. design/method: bone marrow samples were obtained from ds-all patients and matched nds-all patients based on clinical characteristics and genetic features. rna sequencing of these samples was performed and a total of samples were used for the transcriptome analysis ( ds-all vs. nds-all). the differential expression data was generated by deseq and analyzed using ingenuity pathway analysis. the analysis revealed that the chromosome genes that have been implicated in leukemogenesis are not differentially expressed in the ds-all samples, relative to nds-all. an inflammatory signature was identified, which included interferon gamma as an upstream regulator with predicted activation in ds-all. this finding is consistent with prior observations from healthy individuals with ds. other examples of results with potentially actionable targets include the upregulation of several genes in the ras pathway and genes involved in histone methylation. the increased interferon signaling seen in healthy individuals with ds was also identified in ds-all. this may contribute to the development of mutations in inflammatory pathways such as jak and crlf in ds-all. targeting these common pathways with small molecule inhibitors may have a therapeutic benefit in ds-all. cincinnati children's hospital medical center, cincinnati, ohio, united states background: next-generation sequencing (ngs) guides precision medicine approaches in oncology using therapies targeting molecular alterations found within an individual cancer. increased availability of ngs coupled with a proliferation of targeted drugs in development heightens the need for reliable pre-clinical animal models. here we report a patientderived xenograft (pdx) system with integrated molecular profiling for pre-clinical testing of conventional cytotoxic and novel targeted agents. objectives: to utilize ngs from patients with pediatric leukemia to guide rational pre-clinical trials in pdx leukemia avatars, and to determine pdx mice tolerance of and response to cytotoxic and targeted therapies. pediatric acute lymphoblastic leukemia (all) samples were obtained in adherence to an irb-approved protocol and xenografted into nod/rag/interleukin- (il- )rg (nrg) mice. ngs was performed clinically using the foundationone® heme panel. a de novo all sample bearing mutations involving jak , crlf , ntrk , cdkn a/b, ptpn and wt was used for pre-clinical testing. thirty-seven nrg mice were transplanted with million patient cells/mouse via iv injection. standard -drug induction chemotherapy was administered consisting of vincristine, dexamethasone, pegaspargase, and daunorubicin [vxpd, n = mice], in comparison to vehicle control [n = ]. parallel pdx cohorts were treated with single agent targeted therapies based on ngs findings, including ruxolitinib [n = ], crizotinib [n = ] and loxo- [n = ]. the four-week treatment period began on day + from transplant after confirmation of engraftment. following completion of therapy, residual disease burden was analyzed by flow cytometry (hcd +, mcd -cells) in the bone marrow [bm] . to date, pdx models have been established using over thirty ngs-profiled pediatric all samples, including six samples bearing philadelphia (ph) chromosome or phlike mutations. pre-clinical testing was performed in a repre- conclusion: ngs reveals concomitant mutations in ph-like all that may represent additional targets for therapy, or predict tyrosine kinase inhibitor (tki) resistance. we show that all xenograft nrg mice can tolerate a -week multi-agent cytotoxic chemotherapy induction regimen, as well as rational targeted agents, and serve as a robust pre-clinical model for precision medicine trials. background: osteonecrosis is a well-characterized all therapeutic toxicity attributed to glucocorticoids, asparaginase, and methotrexate that disproportionately affects adolescents. in ccg- , alternate-week dexamethasone during double delayed intensification (di) reduced osteonecrosis vs continuous dexamethasone with single di in rapid early responders (rer) ≥ y. to compare efs and os between hr-all patients with vs without osteonecrosis. design/method: hr-all patients - y on aall ( - ) received cog augmented therapy with a × randomization to: ( ) induction dexamethasone ( mg/m d - ) vs prednisone ( mg/m d - ), and ( ) interim maintenance (im) high-dose methotrexate (hdm) vs escalating-dose methotrexate/pegaspargase (ema). rer received single, and slow early responders (ser) double, im/di. initially, all received monthly dexamethasone maintenance pulses, patients ≥ y received di alternate-week dexamethasone, and patients ≤ y received di continuous s of s dexamethasone. there were osteonecrosis-related amendments: after / all patients ≥ y received di alternateweek dexamethasone; after / all patients ≥ y were assigned to induction prednisone, and all patients received di alternate-week dexamethasone and maintenance prednisone pulses. results: osteonecrosis was confirmed in / patients. the y cumulative incidence (ci) was . % overall and increased with age: - y . %, - y . % (alternateweek dexamethasone . % vs continuous dexamethasone . %; p< . ), ≥ y . % (p< . ). among randomized rer patients ≥ y, ci differed by glucocorticoid (dexamethasone . % vs prednisone . %; p = . ) but not methotrexate assignment (hdm . % vs ema . %; p = . ). among randomized ser patients ≥ y, ci was . % with no difference by regimen. results were similar for patients ≥ y. in the entire study population, patients with osteonecrosis had superior y efs ( . % vs . %; p< . ) and os ( . % vs . %; p< . ) than those without osteonecrosis. y efs was significantly higher among randomized patients ≥ y with vs without osteonecrosis ( . % vs . %; p< . ); this finding was present in different age ranges (≥ y, ≥ y, ≥ y) and rer/ser subsets within each, especially in the ≥ y rer ( . % vs . %; p = . ) and ser ( . % vs . %; p< . ) cohorts. across groups, asparaginase allergy was significantly associated with reduced osteonecrosis risk (≥ y: hr . ; p = . ). patients who develop osteonecrosis have significantly increased efs and os, suggesting host differences that increase sensitivity to develop osteonecrosis and render all cells more chemo-responsive. pennsylvania state university, hershey, pennsylvania, united states background: cdc (cell division cycle protein ) belongs to rho family of small gtpases in ras-oncogene superfamily. pro-oncogenic role of overexpressed cdc in ras driven solid tumors are well known. however, role of cdc in leukemia is yet to be established. ikzf encodes ikaros protein which has important role in regulation of lymphoid development and tumor suppression in leukemia. casein kinase ii (ck ) oncogene is overexpressed in leukemia. ck impairs ikaros function which can be restored by using ck inhibitors. objectives: to investigate role of cdc in leukemia and regulation of cdc by ikaros and ck in b-cell acute lymphoblastic leukemia (b-all). shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: cdc is identified as one of the ikaros target genes by analysis of genome-wide dna binding of ikaros using chip-seq and qchip in b-all primary cells. expression of cdc was also noted to be higher in all patient samples compared to normal bone marrow. functional experiments showed that ikaros overexpression via retroviral transduction results in transcriptional repression of cdc . ikaros silencing using shrna resulted in increased expression of cdc . these data suggest that ikaros negatively regulates transcription and expression of cdc . ck directly phosphorylates ikaros and impairs its function as transcription factor. we noted that molecular inhibition of ck via sirna as well as treatment with specific ck inhibitor, cx also decreases expression of cdc . treatment with cx of primary b-all with ikaros haploinsufficiency restores ikaros binding to cdc promoter and represses cdc expression. however, this effect is evident only in presence of ikaros. treatment with cx in ikaros silenced (ikaros shrna) cells showed no change in expression of cdc . these results emphasizes the importance of ikaros in regulating cdc expression. furthermore, we analyzed the changes in epigenetic signature at the cdc promoter following treatment with cx . results show that loss of histone marker of open chromatin (h k ac) and increased histone marker for repressive chromatin (h k me ), at the cdc promoter. these data suggest that ikaros transcriptionally represses cdc via chromatin remodeling. a specific cdc inhibitor, ml showed cytotoxic effects on primary b-all cells. conclusion: cdc may have important role in hematologic malignancies. expression of cdc in b cell all is regulated by ikaros and ck . these results suggest that targeting cdc could be a potential therapeutic strategy in leukemia. caitlyn duffy, laura hall, justin godown, koyama tatsuki, scott borinstein monroe carell jr. children's hospital at vanderbilt, nashville, tennessee, united states background: systemic corticosteroids are widely used as treatment of acute lymphoblastic leukemia (all) and lymphoblastic lymphoma. there are anecdotal reports of bradycardia in pediatric patients receiving corticosteroids, but a more extensive analysis of this effect is needed. objectives: the aim of this study was to describe the incidence, severity, and timing of steroid-induced bradycardia and document any adverse events associated with bradycardia. design/method: we performed a retrospective review of all newly diagnosed patients at our center ( - ) with all/lymphoblastic lymphoma who received corticosteroids (dexamethasone - mg/m /dose or prednisone mg/m /dose) during induction chemotherapy. patients were excluded if they had a pre-existing cardiac abnormality or if they received prior corticosteroids. the average hour heart rate (hr) was assessed for the period prior to initiating steroid therapy and for the hour period surrounding the nadir following steroid administration. the degree and time of steroid induced bradycardia was assessed. adverse patient events and concomitant medication use was documented to identify other contributing factors to bradycardia. a total of children ( females, males, months- years) were included in the analysis with demonstrating a decrease in mean hr following steroid administration. median hr decrease was . beats per minute (quartiles . - ) from prior to initiating steroids to surrounding nadir. sixty one percent developed bradycardia less than or equal to the st percentile for their age range. nadir occurred doses (range - ) into treatment, which corresponded to hours ( - ) after initiation of therapy. of patients who experienced bradycardia, % were associated with dexamethasone rather than prednisone. hr nadir was not associated with other vital sign abnormalities. after completion of induction chemotherapy, % of patients had documented resolution of bradycardia with hr greater than the th percentile for age. it was observed that the children who continued to have relatively low hr were often younger ( months- years old). examination of nadir hr during subsequent hospitalizations in which steroids were not being administered (excluding hr during procedural sedation) did not demonstrate a significant incidence of bradycardia. concomitant opioid, beta-blocker, or other medication exposure did not contribute to the incidence of bradycardia. corticosteroid-induced bradycardia is extremely common in children, teenagers, and young adults with all receiving induction chemotherapy. bradycardia was not associated with clinical adverse events and resolved after completion of corticosteroid treatment. therefore, further cardiac assessment may not be warranted in the presence of bradycardia suspected to be secondary to steroid administration. baylor college of medicine, houston, texas, united states background: survival in newly diagnosed pediatric acute myeloid leukemia (aml) is approximately %; however survival falls dramatically if a patient relapses. currently, approximately one-third of patients with pediatric aml relapse on standard chemotherapy regimens. aml cells are exposed to proteotoxic stress at baseline due to their rapid and inefficient metabolism; proteotoxic stress increases after chemotherapy due to accumulation of reactive oxygen species resulting in misfolded proteins. this leads to activation of cell stress pathways, such as the unfolded protein response (upr) in the endoplasmic reticulum. because an activated upr can make cells more sensitive to proteotoxic stress, we hypothesize that upr activation correlates with response to chemotherapy. objectives: determine the status of upr in pediatric aml and its correlation with chemosensitivity; design/method: peripheral blood samples from pediatric patients with aml were collected at the start of induction chemotherapy, - hours (h) and h post initiation of systemic chemotherapy. tumor cells were sorted from peripheral blood mononuclear cells. expression of upr proteins was determined by chemiluminescence using an automated capillary electrophoresis system. clinical correlations were performed using an annotated database. we measured five upr proteins: grp (glucose regulated protein kda), phospho-eif , inositol-requiring enzyme (ire ) and activating transcription factor (atf ). patients with aml had - times higher expression of upr proteins (except atf ) at baseline than normal controls. grp -the key upr driver-had the highest level of protein expression in myeloid blasts. there was a wide variability in the level of baseline upr expression. eight out of samples expressed > fold increase in grp above those with the lowest grp levels. similarly, and patients respectively, had a > fold increase in peif and ire , compared to patients with low basal expression of these upr proteins. in our limited sample set, there was a trend towards lower overall survival (os) and event-free survival in patients with low baseline grp and ire . conclusion: upr has a variable expression at baseline in pediatric aml, with a trend towards lower os in patients with a low basal grp and low ire expression, suggesting less chemosensitivity in this subgroup. conversely, it is possible that blasts with an upregulated upr prior to chemotherapy manage proteotoxic stress less effectively, having faster apoptosis and hence a better response to chemotherapy in patients with a high basal upr. we are currently expanding our findings in a larger cohort of patients enrolled in the children's oncology group aaml protocol. background: children with newly diagnosed acute lymphoblastic leukemia (all) undergo chest x-ray (cxr) evaluation during initial diagnostic workup to ensure safe airway management. however, to our knowledge, no systematic assessment of cxr findings has been reported. objectives: to evaluate cxr findings at diagnosis of all and their associations with clinical characteristics. we reviewed the cxr findings at diagnosis of all in patients treated on the total xv and xvi protocols at st. jude children's research hospital. findings were evaluated for associations with clinical characteristics at presentation, and the clinical management of mediastinal masses was reviewed. mediastinal masses were seen in ( . %) of patients evaluated and were more common in older patients (mean age, . years) than in younger patients (mean age, . years) (p = . ), in males than in females (p = . ), and in patients with t-all than in those with b-all (p< . ). also associated with mediastinal masses were a higher white blood cell count (wbc) at diagnosis (mean, . × /l) (vs. a lower wbc; mean, . × /l) (p< . ), cns involvement (vs. no involvement) (p = . ), and standard/high-risk disease (vs. low-risk disease) (p< . ). other cxr findings included pulmonary opacity ( patients [ . %]), bronchial/perihilar thickening ( patients [ . %]), cardiomegaly ( patients [ . %]), and osteopenia/fracture/periosteal lesions ( patients [ . %]). pulmonary opacity was more common in younger patients (mean age, . years) than in older patients (mean age, . years) (p = . ) and in those with t-all (vs. b-all) (p = . ). bronchial/perihilar thickening, cardiomegaly, and osteopenia/fracture/periosteal lesions were also more common in younger patients than in older ones (p< . , p = . , and p< . , respectively) and in those with low-risk disease (versus standard/high-risk disease) (p< . , p = . , and p = . , respectively). of the patients with a mediastinal mass on cxr, underwent a confirmatory chest ct scan, and ( . %) were confirmed to have a mediastinal mass. notably, patients ( . %) had airway compression, and compression of venous structures was identified in of patients ( . %) who received iv contrast. the clinical course was evaluated for patients with mediastinal masses detected by cxr. fifty patients ( . %) required icu admission (mean stay, . days). general anesthesia was used for only patients ( . %), and patients ( . %) had a less invasive peripherally inserted central catheter. no deaths occurred in the acute phase. conclusion: cxr at the time of all diagnosis can detect various intrathoracic lesions and is helpful in planning initial diagnostic workup and management. background: mertk is a receptor tyrosine kinase that is aberrantly expressed in % of pediatric primary aml samples. mertk inhibition with the small molecule tyrosine kinase inhibitor (tki) mrx- decreases tumor burden and prolongs survival in aml xenografts. while treatment with mrx- reduces leukemia in the peripheral blood, it is less effective in the bone marrow, suggesting a role for the marrow microenvironment in therapeutic resistance. the jak/stat pathway has been implicated as a mediator of bone marrow derived resistance to tkis and inhibitors of this pathway are in clinical development for the treatment of aml. to determine the role of the bone marrow stromal niche in mediating resistance to mertk inhibition and to evaluate the efficacy of combined mertk and jak/stat inhibition. design/method: aml cell lines were cultured with or without the hs stromal cell line or hs conditioned medium, then treated with mrx- +/-the jak/stat inhibitor ruxolitinib, or control. induction of apoptosis and cell cycle arrest in aml cells was measured by flow cytometry. expression of h ax and total and phosphorylated stat were determined by immunoblot. results: co-culture with stromal cells significantly reduced aml cell death and g /m phase arrest in response to treatment with nm mrx- compared to no co-culture (cell death: . % versus . %, p< . ; g /m arrest: . % versus . %, p< . ). g /m arrest was accompanied by an increase in h ax expression which was similarly abrogated in co-culture. conditioned medium did not provide protection from mrx- induced apoptosis, g /m arrest, or h ax induction. mrx- inhibited stat phosphorylation but direct co-culture and conditioned medium potently increased basal stat phosphorylation which was not inhibited by mrx- . to determine whether the observed induction of stat phosphorylation was functionally relevant, cocultures were treated with both mrx- and ruxolitinib. while ruxolitinib potently inhibited the phosphorylation of stat in the presence of co-culture, combination treatment did not overcome stromal mediated protection from mrx- induced apoptosis. similarly, the addition of exogenous gm-csf induced stat phosphorylation but did not yield protection from mrx- functional effects in the absence of co-culture. together these data support a model whereby direct cell-cell contact with stromal cells in the bone marrow niche protects leukemia cells from mrx- induced apoptosis, cell cycle alterations, and dna damage. while co-culture potently induces phosphorylation of stat in leukemia cells, this is neither necessary nor sufficient for stromal-cell mediated protection from mertk inhibition and combined treatment with jak/stat inhibitors is unlikely to be therapeutically efficacious. background: mercaptopurine ( -mp) is an immunosuppressive thiopurine drug that is a key component of acute lymphoblastic leukemia (all) treatment. -mp is metabolized into -thioguanine ( -tgn), which is responsible for anti-leukemic effects, as well as -methylmercaptopurine nucleotides ( -mmpn/ -mmp), which are associated with hepatotoxicity. some patients preferentially metabolize -mp to -mmpn/ -mmp, increasing their risk for hepatotoxicity and potentially reducing anti-leukemic effects. hepatotoxicity can cause interruptions or delays in therapy that may jeopardize cure rates. allopurinol has been increasingly used in patients with inflammatory bowel disease (ibd) to shunt -mp metabolism toward -tgn and away from -mmpn to minimize hepatotoxicity and preserve therapeutic effects. objectives: this retrospective chart review expands upon our previously published case series of three patients with all in whom allopurinol was successfully used to redirect -mp metabolism. twelve additional patients have subsequently received allopurinol and -mp combination therapy at texas children's hospital. data from this larger patient sample, with longer follow up, is being analyzed to increase knowledge of the effectiveness and longitudinal effects of adding allopurinol to -mp to reduce risk of hepatotoxicity. design/method: data were abstracted from the electronic medical records of patients with all treated at texas children's hospital from to present, who had been found to have evidence of altered -mp metabolism and in whom allopurinol was added to -mp therapy due to concern for risk or recurrence of hepatotoxicity. metabolite levels, -mp dose, and alanine transaminase (alt) prior to initiation of allopurinol and approximately weeks later were compared. wilcoxon signed-rank test was applied for statistical analysis. : after the addition of allopurinol, patients experienced a significant decrease in mean levels of -mmpn (p = . ), correlating with a significant decrease in mean alt (p = . ). with the initiation of allopurinol, the mean -mp dose was decreased from to mg/m /day over an -week period. mean -tgn levels increased (p = . ). in follow up beyond weeks, no patients had further holds in -mp due to hepatotoxicity. addition of allopurinol appears to shift metabolism from -mmpn toward -tgn, with increases in mean -tgn levels despite a decrease in mean -mp dose. this may limit negative side effects, thus resulting in fewer gaps in therapy and possible improved outcomes. further analysis of -mp dose titration and effects on anc over time as well as effects on overall survival is ongoing. prospective background: alterations in epigenetic patterning are a fundamental feature in acute myeloid leukemia (aml). treatment with dna methyltransferase inhibitors (dnmti) yields responses in aml, but the molecular mechanisms underlying this effect are poorly understood. in prior work, we demonstrated induction of genes involved in the pirna rna (piwi) silencing pathway as a common gene feature of aml cell lines treated with decitabine. the piwi pathway is an rna silencing system, distinct from classical small rna transcriptional silencing, responsible for transposon-silencing in gametogenesis; emerging data suggest a role for this system in somatic cells. based on these data, we postulate that piwi induction plays a crucial role in aml recovery following demethylation and that disruption of this pathway would modulate response and/or recovery from decitabine treatment. to assess the effect contribution of the pirna pathway response following dnmti treatment in aml. design/method: to choose target genes in the pirna pathway for disruption, molm cells were first treated with escalating doses of decitabine. using quantitative rt-pcr, the dose-dependent expression of several pirna-associated genes were analyzed. two genes, mael and piwil , were selected for disruption experiments based on preliminary data suggesting decitabine dose-dependent responses. molm cells were transduced with shrna targeting these genes using a lentivirus delivery system with selection in puromycin. knockdown efficiency was assessed by rt-qpcr. to determine how gene disruption affected cell growth, knockdown cells were treated with decitabine nm. proliferation was assessed by celltiter glo assay following decitabine treatment. clonogenic potential was assessed by colony forming assays of transduced cells after treatment with decitabine at nm and nm. results: following decitabine exposure in molm , there was a markedly increased expression of mael and piwil compared to untreated cells ( : and : , respectively) . thus, these were the candidate genes chosen for disruption. of mael shrna constructs, two resulted in a % relative expression of mael compared to controls. of the piwil shrna constructs, the best knockdown showed % relative expression. there were no significant differences in proliferation or clonogenicity of stably selected mael or piwil knock-down molm cells following decitabine treatment. using gene knockdown procedures, mael and piwl do not appear to have a marked effect on growth and response to decitabine treatment in molm . however, these results may be limited by inefficient knockdown using shrna targeting methods. further work using a cas /crispr based inactivation of these genes is ongoing. children cancer hospital cairo, egypt background: hypodiploidy < chromosomes is very uncommon and have particularly poor outcomes in childhood acute lymphoblastic leukemia (all). it is subdivided into: near-haploid ( - chromosomes), lowhypodiploid ( - chromosomes) and high-hypodiploid ( - chromosomes). to determine if minimal residual disease (mrd) can identify a group of patients with better prognosis in the hypodploid population who can be treated with intensive chemotherapy alone. design/method: a retrospective study that included all patients under age of diagnosed as hypodiploid b-precursor all during the period between january -december and treated at children's cancer hospital egypt on sjcrh total study-xv for ir/hr all. sixteen patients had < chromosomes ( nearhaploid and low-hypodiploid), constituting % of all pediatric patients with b-precursor all during the study period. patients with near-haploid all had a median age of years (range - ), initial leukocyte count (wbc) median of . × /l (range . - . ), ( . %) were males and / ( . %) had hr-nci criteria. four patients ( . %) are alive in complete remission(cr) (range - months, median ), one died in induction and ( . %) had hematological relapse (range . - months, median ). patients with low-hypodiploid all had significantly older age (median years, range - ), median wbc . × /l (range . - . ), / ( . %) were males. one patient ( . %) is alive in cr, one died in induction, one failed to achieve cr post-induction and patients( %) had hematological relapse (range . - . months, median . ). mrd< . % by flow-cytometry on day- and end of induction was achieved in / ( . %) and / patients( %) with near-haploid, compared with / ( . %) and / patients( %) with low-hypodiploid; respectively (p = . , p = . ;respectively). allogeneic transplantation was performed during initial remission only in mrd negative patients (one relapsed and one is in cr) and in the patient with induction failure (relapsed post-transplant). five of the total six patients who had negative mrd on day- and end of induction are alive in cr ( / with chemotherapy alone). all patients with negative mrd at end of induction but with mrd levels≥ . % on day- (range . - . %) relapsed as well as all patients with detectable mrd at the end of induction. the difference in relapse was statistically significant in relation to negative-mrd on day- (p = . ), but not at end of induction(p = . ). conclusion: children with hypodiploid all and negative mrd on day- of induction are highly curable with intensive chemotherapy alone, while patients with negative mrd at the end of induction and detectable mrd on day- had dismal outcome. background: overall survival in pediatric acute myeloid leukemia (aml) has plateaued between - %, with death during induction chemotherapy seen in - % of patients. respiratory complications contribute to morbidity and mortality in pediatric aml induction, however the incidence, patterns, and predictors of respiratory adverse events (aes) during this period are unknown. to estimate the incidence of respiratory aes during induction therapy for de novo pediatric aml, to characterize and grade these respiratory aes, and to identify predictors of respiratory ae development. we conducted a retrospective longitudinal study from presentation to day in institutional de novo pediatric aml patients (≤ years) between march and december . outcomes included any nci ctcae grade - respiratory ae or death from another cause. demographic, disease, and treatment-related data were abstracted. the most specific, best-fitting ctcae category and grade for each ae was determined. descriptive statistics, survival analysis, multivariable logistic regression analysis, and time-toevent distributions were performed (sas v . , cary, nc) . among eligible patients, . % (n = ) experienced discrete respiratory aes. incidence of grade - aes was . % (n = ). a bimodal time-to-event distribution demonstrated peaks at treatment days and . induction death occurred in . % (n = ) including deaths from respiratory failure associated with disseminated fungal disease. in univariate analysis, those experiencing aes differed significantly in regards to older age at diagnosis (p< . ), higher initial wbc (p = . ), higher initial peripheral blast percentage (p = . ), coagulopathy at diagnosis (pt (p = . ), d-dimer (p = . )), fluid overload status (p< . ), occurrence of infection (p = . ), and occurrence of tumor lysis syndrome (tls) (p = . ). patients with hyperleukocytosis (p = . ), fluid overload (p< . ), and fab m morphology (p = . ) each had a significantly decreased probability of completing the follow up period without experiencing a respiratory ae. on multivariable analysis, fluid overload (aor . [ % ci: . - . ) and older age (aor . [ % ci: . - . ) were significantly associated with ae occurrence when gender, hyperleukocytosis, tls, and infection status were held constant. we describe a high incidence of respiratory aes during pediatric aml induction. fluid overload and older age at diagnosis are independently associated with ae development when controlling for other proposed risk factors. interventions focused on conservative fluid management and offset of fluid overload should be explored in newly diagnosed pediatric aml in an effort to reduce respiratory complications during induction. overall, all survival rates are outstanding and have continued to improve with risk-adapted therapy. the most striking improvement occurred in t-all where -year os rates now exceed % and parallel b-all. survival improvements, however, have not been observed uniformly across all subgroups. while the gap in outcome differences narrowed among blacks, outcomes for hispanics have remained static. further, no improvements in survival were observed in infants or ayas and new treatment approaches have been implemented for these populations. background: acute myeloid leukemia (aml) accounts for approximately % of new childhood leukemia cases. chest x-ray (cxr) is performed in all newly diagnosed aml cases to evaluate the safety of airway management for anesthesia during diagnostic procedures; however, cxr results in pediatric patients with aml have not been described. objectives: the primary objective was to evaluate cxr findings at diagnosis in patients with aml. the secondary objectives included assessing associations between cxr findings and clinical characteristics, with the overall goal of aiding in the evaluation of the use of cxrs as an initial diagnostic study in pediatric patients with aml. design/method: cxr findings and clinical characteristics were evaluated in patients with newly diagnosed aml who were enrolled in one of three protocols at st. jude children's research hospital (aml , aml , and aml ). the findings were categorized based on radiologic reports. further, the associations of these findings and clinical characteristics were evaluated. we evaluated cxr findings in a total of patients: from aml ; from aml ; and from aml . common cxr findings were pulmonary opacity (n = , . %), bronchial/perihilar thickening (n = , . %), splenomegaly (n = , . %), mediastinal mass and lymph nodes (n = , . %), pleural effusion/thickening (n = , . %), demineralization/fracture/periosteal lesions (n = , . %), scoliosis (n = , . %), and granulomatous disease (n = , . %). three cxr findings were associated with younger age at diagnosis: pulmonary opacity (median age, . years in patients with positive findings vs. . years in those with negative findings, p< . ), bronchial/perihilar thickening (median age, . years vs. . years, p< . ), and demineralization/fracture/periosteal lesions (median age; . years vs. . years, p = . ). two cxr findings were associated with older age at diagnosis: scoliosis (median age, . years vs. . years, p< . ) and granulomatous disease (median age, . years vs. . years, p = . ). higher white blood cell counts (wbcs) at diagnosis were associated with cxrs showing pulmonary opacity (median wbc; . × ^ /l vs. . × ^ /l, p = . ) or splenomegaly (median wbc; . × ^ /l vs. . × ^ /l, p = . ). french-american-british (fab) m /m subtypes were more frequently associated with pulmonary opacity compared with others (p< . ). we did not find significant differences between female and male patients. conclusion: cxr in patients with newly diagnosed aml showed a variety of thoracic, abdominal, and bony lesions that are important for the initial evaluation and management. pulmonary opacity was the most common finding and was frequently seen in patients who were younger or had higher wbcs at diagnosis or fab m /m . background: children diagnosed with acute lymphoblastic leukemia (all) require a central venous catheter (cvc) to administer chemotherapy safely. both external and internal cvcs carry risks of complications including thrombosis, infection, and possible replacement. internal catheters, such as a port, are generally used for the majority of patients for the duration of treatment since therapy lasts for several years. many institutions place a port at the time of diagnosis. other institutions prefer to start induction therapy via placement of a peripherally inserted central catheter (picc) and defer port placement until the completion of induction therapy due to concerns of increased risk of infectious complications with port placement. objectives: to compare rates of common cvc associated complications by type of cvc placed at start of induction therapy in children treated for newly diagnosed all at the jimmy everest center (jec) at the university of oklahoma health sciences center. design/method: a retrospective chart review analyzed data from newly diagnosed all patients treated at the jec between - . data was collected on complications including thrombosis, bacteremia, insertion site infection, cvc malfunction and need for removal. data collection began at the start of induction and was completed at the end of induction therapy. statistical analysis used a univariate and multivariate logistic regression model to compare complication rates between those who had a port versus those who had a picc placed at start of induction. results: data was collected on patients. fifty-six patients had a port placed at start of therapy while had a picc placed. fourteen percent of patients had a cvc associated complication. univariate analysis showed no statistically significant difference in rates of cvc associated complications between the groups (port %, picc . % p = . ). the rates of hospitalization for cvc associated complications were similar between both groups (port %, picc % p = . ). rates of cvc removal were also similar between both groups (port %, picc % p = . ). multivariate model that included baseline patient characteristics including type of all, patient body surface area, gender, ethnicity and age continued to demonstrate no significant difference in cvc associated complications between both groups. conclusion: this single institution study showed that there was no significant difference in cvc associated complications between port and picc line placement at the start of childhood all induction therapy. port placement can be considered as a safe option at the start of induction therapy. complete remission [cr] or cr with incomplete blood count recovery [cri]) within treatment cycles - . interim data are reported (nct ). results: seventeen patients were enrolled and received ≥ dose of lenalidomide; median age was years (range - ); patients were female. patients received median prior regimens (range - ). nine patients had previously undergone bone marrow transplantation (bmt). four patients had relapsed aml and were refractory to immediate prior treatment. median duration of study treatment was weeks (range - ); patients completed a median of treatment cycle (range - ). all patients were evaluable for primary outcome; achieved morphologic cri after cycles (no patients achieved cr). the responder was a -year-old male with history of r/r aml after first-and second-line treatment, bmt, and salvage chemotherapy. at baseline, he had a complex cytogenetic karyotype (monoallelic − q . , − q, − q . , − p ) with no identifiable molecular mutation; he was also positive for del( q) (− q , − q ). his post-treatment karyotype showed no abnormalities. sixteen patients experienced treatment failure; due to resistant disease, of indeterminate cause, and had treatment failure before a post-baseline assessment was performed. all patients experienced ≥ grade - treatment-emergent adverse event (teae). the most commonly reported were thrombocytopenia (n = ), anemia (n = ), febrile neutropenia (n = ), and hypokalemia (n = ). fifteen patients experienced ≥ teae related to lenalidomide. all patients discontinued treatment; remain in follow-up. the study is now closed to enrollment. ten patients died on study: during treatment, during follow-up. all deaths were attributed to aml or complications due to aml. conclusion: third-line lenalidomide monotherapy was associated with clinical response in of pediatric patients with r/r aml; however, treatment exposure was limited. safety data are consistent with the known profile of lenalidomide. lenalidomide was not an efficacious treatment for r/r pediatric aml. funding: celgene corporation, summit, nj, usa. cook children's medical center, fort worth, texas, united states background: it is well documented that pediatric patients with acute lymphoblastic leukemia (all) often experience significant weight gain during induction therapy and later struggle with obesity. however, some patients experience unintended weight loss during induction therapy; since this issue is not well reported, it often goes undertreated. although malnutrition is reported to be associated with decreased survival, increased risk of infection, and loss of lean body mass, there remains a scarcity of in-depth analysis of prevalence and risk factors that contribute to this problem. our study attempts to address this critical yet unmet need. objectives: our aim was to identify the clinical risk factors and outcomes associated with weight loss during induction therapy for pediatric all. design/method: this was a retrospective chart review of patients between and years of age diagnosed with all at cook children's medical center from / / to / / . for each patient, we collected height, weight, age, body mass index (bmi) z-scores at diagnosis and end of induction therapy, risk stratification, and whether consolidation was delayed. patients with a bmi > th percentile at diagnosis were categorized as being overweight or obese. using logistic regression analyses, we examined which variables predicted whether the patient had an increase or decrease in bmi z-score throughout induction. a critical alpha level of . indicated statistical significance. results: ninety-six patients met our inclusion criteria. of these, % experienced a decrease in bmi during induction therapy. compared to patients whose bmi increased during induction, patients with a decrease in bmi were more likely to be overweight or obese at diagnosis ( % vs. %; p< . ), to be ≥ years of age ( % vs. %; p< . ), to have a high-or very-high-risk stratification ( % vs. %; p< . ), and to experience a delay in the start of consolidation therapy ( % vs. %; p< . ). conclusion: this research highlights a risk not previously identified in the literature that may impact outcomes. patients treated on high-or very-high-risk protocols, who are overweight or obese at diagnosis, and who are ≥ years of age at diagnosis should be monitored closely for weight loss during induction therapy. patients who experience weight loss should receive prompt intervention. it is our hope that this information can be used for future prospective studies and to help develop evidence-based guidelines. background: p abnormalities have been observed in some patients with hematologic malignancies. loss of p function as a tumor suppressor gene in the chromosome plays an important role for development of leukemia. these patients usually have poor outcome due to the chemotherapy and are associated with poor prognosis. objectives: this study aimed to identify frequency of p abnormalities between iranian children and adult patients with aml (acute myeloid leukemia) malignancy. design/method: the p abnormalities were analyzed via bone marrow karyotyping and fish method in acute myeloid leukemia patients. in this study, p abnormalities were observed in ( %) patients out of diagnosed cases. a significant strong correlation between p abnormalities and other high risk factors (poor risk cytogenetic) were observed. from patients with aml malignancy ( p abnormalities), ( %) patients have complex karyotype, ( %) patients monosomal karyotype and ( %) patients have monosomal karyotype accompanied with a complex karyotype. overall, p abnormalities are independent risk factor in acute myeloid leukemia and evaluation of these abnormalities by fish or other complementary techniques prior to treatment, might help for better risk stratification of high risk aml patients. background: hepatotoxicity in treatment of acute lymphoblastic leukemia (all) is well studied and transiently affects most patients receiving antimetabolite therapy. rarely, patients develop liver injury severe or prolonged enough to undergo a liver biopsy. little is known about how these patients differ from patients that develop transient hepatotoxicity. we sought to describe disease and treatment characteristics for all patients that developed hepatotoxicity severe enough to undergo liver biopsy. we also looked for pre-dictive factors for liver biopsy, including signs of early hepatic injury from the initial treatment protocol. design/method: pathology reports of all patients from the liver biopsy database at children's healthcare of atlanta were collected. controls were matched : for age, all subtype, and treatment protocol. demographics, treatment protocols, and overall outcomes were collected through the electronic health record. hepatic lab results for transaminases, coagulation, and albumin were collected for induction, consolidation, interim maintenance, delayed intensification, and maintenance. results: sixteen patients diagnosed between - (median age at diagnosis years, range - ; % male; % pre-b all) were included in the case series. the median time from diagnosis to liver biopsy was . years (range - ). eight patients ( %) were in maintenance at the time of biopsy; none had active disease. eight ( %) were postbone marrow transplant. biopsy results included: steatosis ( ), acute inflammatory/infectious ( ), liver infiltration ( ), fibrosis ( ) and graft-vs-host disease (gvhd) ( ). six patients were deceased; -year all-cause mortality from diagnosis was %. thiopurine methyltransferase (tpmt) status was known in % cases and % controls. all cases had intermediate or wildtype status, which did not differ from controls (p > . ). patients requiring liver biopsy did not have evidence of acute hepatotoxicity (ast/alt > × normal values) during their initial treatment protocol. hepatotoxicity requiring liver biopsy is a rare outcome of all treatment. these patients had elevated rates of relapse, bmt, and -year all-cause mortality, suggestive of a more severe disease process. however, it is difficult to sort out the temporality of relapse, bmt, and hepatoxicity requiring biopsy in this limited sample. additionally, patients with bmt preceding liver biopsy have other confounding factors that makes them difficult to include in the analysis. finally, our limited descriptive data show no notable correlation between early hepatotoxicity and later indication for liver biopsy. future cohort or case-control studies with larger sample sizes are required to further explore early predictive factors for severe hepatotoxicity requiring liver biopsy. nathan gossai, joanna perkins, michael richards, yoav messinger, bruce bostrom background: the majority of chemotherapeutic agents used to treat hodgkin lymphoma are teratogenic. pregnancy screening prior to the start of chemotherapy is supported by clinical guidelines and baseline testing is a standard component in therapeutic trials. there is limited data available on the incidence of pregnancy screening prior to the start of hodgkin therapy but previous studies suggest that pregnancy screening, especially at pediatric institutions, is not consistently completed. objectives: the objective of this study is to evaluate the incidence of pregnancy screening and contraceptive counseling prior to the start of therapy in females diagnosed with hodgkin lymphoma. design/method: a retrospective chart review was performed for all female patients newly diagnosed with hodgkin lymphoma from to at the hospital for sick children in toronto, ontario. all patients who were intended to receive multi-agent chemotherapy were included, regardless of age. data collected included demographic and disease information, chemotherapy regimen and enrollment on clinical trial. all pregnancy testing within two weeks prior to the start of therapy was captured, as well as type of pregnancy test performed, documentation of menstrual status, contraceptive counseling and contraceptive provision. univariate and multivariate analyses were used to describe factors influencing the incidence of pregnancy testing. results: a total of female patients with newly diagnosed hodgkin lymphoma between the ages of and years were identified. sixty patients ( %) had pregnancy testing done prior to the start of therapy. testing modalities included serum and urine screens as well as quantitative beta-hcg measures. older age (p = . ), documentation of menstrual status at diagnosis (p = . ) and diagnosis between and (p = . ) were associated with higher incidence of screening. enrollment on a therapeutic trial was not associated with a higher incidence of screening (p = . ). contraceptive counseling was documented for patients ( %) and patients ( %) were prescribed contraceptive medications during therapy. pre-chemotherapy pregnancy testing was completed on % of females with newly diagnosed hodgkin lymphoma. improvement is required and interventions, including clarification of institutional standards, modification of chemotherapy order sets and staff education, are planned. (rao et al., cancer, ) . university of louisville, louisville, kentucky, united states background: granulocytic sarcomas (also known as chloromas or leukemia cutis) were first described by a. burns in . they are solid tumors comprised of immature granulocytic cells and represent extramedullary manifestations of underlying leukemia. chloromas are most commonly associated with acute myeloid leukemia. they may arise in other myeloproliferative disorders but are rarely seen in b or t cell acute lymphoblastic leukemia (all). objectives: although patients with all rarely have chloromas, it should remain on the differential for patients with unusual swelling or masses. design/method: we present a case series of two patients from our institution diagnosed with b cell all who had a chloroma as the presenting symptom. the first patient is a yo who presented to his primary provider with nasal congestion and a one-week history of bilateral eye swelling and was referred to an allergist when the symptoms did not resolve with anti-histamines. his review of systems was otherwise negative. he was referred urgently to ent two months later for a × cm mass palpated along the medial border of the left eye. an mri showed a left facial mass surrounding the zygoma and extending into the anterior inferior left orbit. biopsy revealed b cell acute lymphoblastic lymphoma, and bone marrow aspirate and biopsy confirmed the diagnosis as b cell all. the second patient is a yo who presented to his primary doctor for rapid growth of a scalp nodule that had been present for about months. he was referred to dermatology and treated for a supposed kerion from tinea capitis. the lesion continued to grow and became more irritated with this treatment. punch biopsy revealed a complicated phenotype of lymphoblastic lymphoma. however, after a lymph node biopsy and bone marrow aspirate and biopsy, the diagnosis was confirmed as b all. his only other positive point on review of systems was a questionably pathologic -pound weight loss and an area of matted cervical lymph nodes. for both of our patients, the chloromas completely disappeared during induction therapy. it is worth noting that both of these patients presented with the chloroma as the only symptom of the underlying leukemia. this led to initial misdiagnosis and delay in identifying their leukemia. therefore, while it is very rare for a patient with b all to present with a chloroma, our experience shows that all should be on the differential for patients presenting with unusual swelling or masses. background: hodgkin lymphoma (hl) is a lymphoproliferative neoplasm that commonly presents with history of adenopathy and a predictable pattern of disease involvement with or without systemic symptoms of fever and/or weight loss. in the hands of an experienced oncologist the diagnosis of hl is usually not a challenge. occasionally a diagnostic challenge is presented by a patient who has an atypical presentation which is suggestive of an alternative diagnosis. we describe a case series of patients diagnosed with hl whose initial clinical presentations lead to a diagnosis different form hl. honduras, nicaragua and the united states. results: six pediatric oncology centers from the american continent conducted a retrospective review of patients diagnosed with hl since . patients that had an initial presentation not suggestive of hl or who were initially diagnosed with a disease other that hl were included for a total of patients. argentina n = , guatemala n = , honduras n = , nicaragua n = , united states n = . five patients were female and male. patient's ages ranged from to years. most patients (n = ) were older than years. three patients ( %) presented with non-immune cytopenias without overt lymphadenopathy, of those one had active hemophagocytic syndrome. five patients ( %) were suspected to have localized solid tumors: ewing sarcoma n = , rhabdomyosarcoma n = , hepatocellular carcinoma n = , and soft tissue tumor of the cheek n = . two ( %) metastatic solid malignancy as they presented with disseminated pulmonary nodules. five ( %) with autoimmune disorders: hashimoto thyroiditis n = , autoimmune hemolytic anemia n = , nephrotic syndrome n = . ten ( %) with chronic infectious processes: brucella n = , tonsillar abscess n = , splenic abscess n = , and tuberculosis (tb) n = . patients with suspected tuberculosis were diagnosed outside of the united states. six of patients were ultimately diagnosed as having both tb and hl. seventeen patients had ann-arbor stage iii or iv, seven patient had stage ii with either b symptoms or bulky disease. patients were treated with various chemotherapy regimens according to the treating center: abvd, abve-pc oepa-copdac, avpc, beacopp. two patients had recurrent disease, one died of disease progression and one died from causes not related to hl conclusion: a small proportion of hl patients have atypical or unusual presentations. hl should be included in the differential diagnosis of solid tumors, autoimmune disorders, infections or cytopenias. the most common atypical presentation is an infectious process. background: acute lymphoblastic leukemia (all) represents the largest group of pediatric malignancies. the high cure rate of childhood all represents one of the most remarkable success stories in the war on cancer. in a lower middle income country (lmic) like the philippines, we reviewed the five year survival in a tertiary referral center. objectives: this retrospective cohort study aims to determine the survival of children - years old with all treated at a tertiary referral center for childhood cancer in the philippines from january to december . design/method: this is a retrospective cohort study that reviewed medical charts of newly diagnosed all ages to years old from january to december . a total of subjects were included in the study. the year overall survival (os) and event free survival (efs) were . % and . %, respectively. the year os for standard risk all was . % and for high risk patients was %. the year os for the patients on remission was . % and for those who relapsed was . %. univariate and multivariate by cox proportional hazards regression revealed wbc count at diagnosis, risk classification, immunophenotyping, and development of relapse showed significant prognostic impact for mortality. age and gender were reported with no prognostic significance. the -year os and efs were lower compared to developed countries but are comparable with other lmics. the prognostic factors for relapse and mortality were compatible with the literature. overall, the adopted treatment protocols for childhood all in this institution showed acceptable results. relapse has a significant prognostic impact for mortality. development of accessibility to care, increase awareness, early detection and resources at hand should be achieved. improvement in the follow up protocol to prevent delays in the treatment, patient education to prevent non-compliance and psychosocial support, to developed better supportive care, and expand facilities should be given emphasis to further improve survival and prevent relapse. objectives: here, we seek to further characterize this entity by describing the pathologic and clinical features of pediatric cases of burkitt-like lymphoma with q aberration. we collected pathologic and clinical data from the medical record on all pediatric high grade b-cell lymphoma (hgbcl) cases diagnosed at our institution over a -year period ( - ) . for those cases classified as neither burkitt lymphoma nor diffuse large b-cell lymphoma (dlbcl), fish for myc, bcl- and bcl- , as well as array comparative genomic hybridization (acgh), were performed. we identified cases of hgbcl, including cases of burkitt lymphoma presenting as purely leukemic phase. of the hgbcl cases, had burkitt lymphoma as defined by myc rearrangements, and had dlbcl. collectively, the majority of these patients had primary disease outside of the head/neck, and most patients presented with advanced stage (iii-iv) disease. of the remaining cases, q aberration was identified in cases using acgh. all cases histologically and immunophenotypically resembled burkitt lymphoma but lacked myc rearrangement, instead showing proximal gains in q -q and telomeric losses in q . qter. all cases involved primary disease in the cervical lymph node and/or tonsil. three of these cases were localized (stage ii), and the fourth case involved a few metabolically active but non-enlarged lymph nodes in the chest and abdomen (stage iii). all patients achieved complete remission with standard therapy for mature b-cell lymphoma, and were alive with no clinical evidence of disease at a median follow-up of months. although the number is small, our results suggest that the majority of non-burkitt, non-dlbcl cases of pediatric hgbcl carry q aberrations. in addition, patients with q aberrations appear to be more likely to present with lower stage disease, thus requiring less intensive therapy, and also tend to have primary disease in the head/neck. these findings further support the classification of burkitt-like lymphoma with q aberration as a distinct pathologic and clinical entity, and we propose that all pediatric non-burkitt, non-dlbcl cases of hgbcl regularly undergo further workup for possible q aberrations. marie claire milady auguste, joseph bernard st damien hospital, port-au-prince, port-au-prince, haiti background: hodgkin lymphoma (hl) and non-hodgkin lymphoma (nhl) account for % of cancers in the united states pediatric population ( , ). in central america and the caribbean, they are in second position among all types of pediatric cancers ( ). a previous study on pediatric cancers in haiti showed that the lymphomas were in fifth place after the leukemias, wilms tumor, retinoblastoma and the sarcomas ( ). the main objective of this study is to present the epidemiological profile of lymphomas managed at a haitian pediatric hospital. design/method: this is a retrospective study conducted on the cases of lymphoma diagnosed and managed at st damien hospital from january to december . key variables such as age, gender, stage at diagnosis, histopathological types and outcome were collected to present the characteristics of this retrospective cohort. of the cases of cancer diagnosed during the study period, ( . %) had the diagnosis of lymphoma. the sex ratio was . ( males for females) and the average age was . years [ - years]. there were cases of hl ( . %) and cases of nhl ( . %). . % of the patients were diagnosed at stages iii and iv. among the hl cases, ( . %) were nodular sclerosis lymphoma, ( . %) with mixed cellularity and ( . %) with lymphocytic predominance. for the nhl cases, ( . %) were burkitt's lymphoma and ( . %) lymphoblastic t-cell lymphoma. among the patients for who immunohistochemistry was found, the cases of hl were cd -positive and out of cases of nhl were cd -negative. only patient was hiv-positive, and patients had a confirmed exposure to epstein-barr virus. patients ( . %) were lost to follow-up, ( . %) were in remission, ( %) relapsed, ( . %) were still in treatment and ( %) were deceased. university of chicago, chicago, illinois, united states background: due to the adoption of risk-adapted therapy, pediatric and adolescent acute lymphoblastic leukemia (all) is associated with high cure rates. despite excellent outcomes in most children, patients with certain blast cytogenetic features do not fare as well. furthermore, african american, native american, and hispanic patients have worse outcomes than caucasian patients. while the outcome discrepancies are certainly multifactorial, and blast cytogenetics are related to age, it remains unclear whether ethnicity and blast cytogenetics correlate. the diverse patient population at the university of chicago provides an opportunity to evaluate for such a correlation. objectives: to describe cytogenetic findings in a racially and ethnically diverse population of patients of all age groups diagnosed with all at university of chicago from to and determine if there is a correlation between race/ethnicity and blast cytogenetics. results: a total of newly diagnosed patients with all between the ages of - from - were included in this study. of those, patients ( . %) had b-all, had t-all ( . %), one had early t-cell precursor all and one had mixed phenotype all (b/t). caucasians accounted for % of patients, african americans (aa) %, hispanics . %, asians . %, and % were of other races. age distribution had a bimodal pattern, with a peak in incidence at and another at years of age, consistent with published data. cytogenetic categories included: t( ; )(p ;q ), q rearrangements (kmt a), iamp , t( ; )(q ;p . ), t( ; ) (q ;q ), hypodiploidy, hyperdiploidy and double trisomy of chromosomes and . aa and hispanic patients with b-all presented more frequently between the ages of - years compared to caucasians (p = . and . , respectively). in aa patients, t( ; ) (q ;p . ) was overrepresented (p = . when compared to caucasians), and was mainly observed in patients between - years. caucasian patients were more likely than non-caucasians to have hyperdiploidy (p = . ), especially in patients aged - years. the rate of t( ; )(q ;p . ) was significantly higher in aa patients in our cohort, in particular in patients between the ages of - years. hyperdiploidy was more likely in caucasians aged - years. these findings may suggest that varying blast cytogenetics could contribute to outcome differences between races. ahmed elgammal, yasser elborai, mohamed fawzy, asmaa salama, eman d el-desouky, lobna shalaby national cancer institute, cairo, cairo, egypt background: hodgkin lymphoma (hl) in children is one of the malignancies that have a high chance of cure. stage iv hl remains a challenge for getting good clinical outcome as in other stages. many treatment protocols used to give combination chemotherapy while combined modality treatment is the mainstay in other treatment protocols. objectives: we aimed in to assess the outcome using consolidation radiotherapy to chemotherapy (combined modality treatment) versus combination chemotherapy alone in treatment of stage iv hl. design/method: we included patients with stage iv hl and whose data were retrieved from the medical records of the pediatric oncology department, national cancer institute, cairo university, egypt from till june and were followed till august . treatment was either to give cycles of abvd (adriamycin, bleomycin, vinblastine, dacarbazine) only or to give cycles of abvd followed by consolidation radiotherapy. the study included cases; were males and were females. mean age was . years ranging from to years. the histopathology subtype was nodular sclerosis in the majority of cases ( cases) followed by mixed cellularity ( cases) then only one case of lymphocyte rich. nine cases were initially bulky while cases were not. constitutional manifestations were present in cases while it was absent in cases. bone marrow was involved in only cases. radiotherapy was given after completion of chemotherapy to cases while cases received chemotherapy only. the -year overall survival for patients who received radiotherapy was superior to those who received chemotherapy alone; % versus . % respectively with statistical significance (p = . ). the -year progression free survival was also higher with radiotherapy than others; % versus . % (p = . ). patients with stage iv hl who received consolidation radiotherapy apparently had a better outcome than those who received chemotherapy only. this suggests that radiotherapy contributes significantly with chemotherapy to the cure rate for those patients. the feinstein institute for medical research, manhasset, new york, united states background: microrna (mirnas) are short non-coding rnas that play a decisive role in cancer biology, including leukemia. exosomes are microvesicles ( - nm) produced by most cells in biological fluids. exosomes represent the fingerprint of the parental tumor and are loaded with bioactive markers such as mirnas, which may regulate tumor growth. exosomal cargo can be transferred into target cells changing their biological properties. our study investigates a functional role for exosomal mir- a in pediatric acute lymphoid leukemia (p-all). objectives: / to demonstrate that p-all exosomes induce cell proliferation / to confirm that exosome-induced cell proliferation is disease-stage specific / to analyze exosomal mir- a expression profiles in p-all / to authenticate that inhibition of exosomal mir- a reduces leukemia proliferation design/method: exosomes were isolated by ultracentrifugation from healthy donors (hd) & p-all serum and conditioned medium (cm) of sup-b , jm , and cl- (control) human cell lines. cell lines were exposed to different sources of leukemia-derived exosomes in a paracrine or autocrine fashion for hrs in triplicates. proliferation was assessed by microscopic cell counting and confirmed by gene expression for proliferation, pro-survival and pro-apoptotic genes. mirna profiling was performed with the human cancer pathway finder microarray (qiagen). silencing of exosomal mir a was carried out by a mir- a inhibitor (qiagen), utilizing exo-fecttm exosome transfection reagent (sbi, system biosciences). further, exosomal mir- a silencing was confirmed by q-pcr. cellular uptake of texred-sirna (sbi, system biosciences) was confirmed by flow cytometry. transfer of exosomal mir a to the target cells was evaluated by q-pcr. we elucidated that cm-derived exosomes from sup-b and jm cell lines induce cell proliferation in sup-b , jm (autocrine and paracrine) and cl- cells (paracrine) (p< . ). serum p-all exosomes promote paracrine cell proliferation in all cell lines compared to hdderived exosomes (p< . ). heatmap analysis of mirna profiles of leukemia exosomes (all cell lines and p-all) identified mir- a significantly upregulated in leukemia exosomes compared to controls. mir- a was also upregulated in all cell lines after exposure to leukemia exosomes that induced proliferation. moreover, exosomal mir- a inhibition reduces leukemic proliferation in pediatric all. our data suggest that all exosomes induce cell proliferation of leukemic cell lines in both paracrine and autocrine fashion. exosomes regulate these phenomena in a highly orchestrated way, by transfer of functional exosomal mirnas such as mir- a. the results of this study suggest s of s that exosomal mir- a inhibition can act as a novel way for growth-suppression of pediatric leukemia. results: a total of disease sites were detected at pet/ct, while sites were detected at contrast-enhanced ct and bone marrow biopsy (bmb). pet/ct showed improved detection of nodal lesions (p < . ) (kappa value = . ), extranodal lesions (p < . ) (kappa value = . ) and bone marrow (p < . ) (kappa value = . ) compared to contrast enhanced ct and bmb. pet/ct had upstaged cases ( %) and down-staged cases ( . %) (p < . ) (kappa value = . ). among the upstaged cases, patients ( . %) were upstaged from stage ii to iii, based on residual in pet/ct not seen in contrast enhanced ct after abdominal mass excision. four patients ( . %) were upstaged from stage iii to iv based on bone marrow uptake in fdg-pet without positivity in bma or bmb.regarding response assessment, sensitivity was % for pet and % for contrastenhanced ct (p = . ). specificity was % for pet and % for ct (p< . ). positive predictive value for pet was %, while was % for ct scan (p< . ). negative predictive value for both pet and ct was % (p = . ). five patients had nd biopsy to confirm viability of the residual lesions, lesions were negative in pathological examination (all of them were metabolic negative in pet/ct; deauville score below ). one lesion was positive in pathological examination (was positive in pet/ct; deauville score of ). conclusion: pet/ct detected additional sites compared with contrast-enhanced ct and resulted in changing stage of disease. pet scan is significantly more specific than ct in the management of children with burkitt lymphoma. background: deep sequencing of the immunoglobulin heavy chain (igh) locus indicates that each b all is composed of innumerable subclones. in many cases, subclones exhibit differing phenotypic qualities. however, it remains unclear whether subclones demonstrate distinct tissue distribution within a patient. objectives: . to quantify the extent of clonal heterogeneity in diagnostic b all specimens; . to identify variability in clonal composition between bone marrow (bm) and peripheral blood (pb) disease sites. design/method: igh sequencing was performed on purified dna from pairs of matched bm and pb patient specimens. multiplex pcr was used to globally amplify the igh locus; next generation sequencing (ngs) was performed using illu-mina® miseq. index clones (defined as ≥ % of all sequence reads in a specimen) and their subclone progeny (defined by shared nucleotide bases immediately upstream of a common jh, or n_jx) were identified using igblast-determined vh and jh alignments (http://www.ncbi.nlm.nih.gov/igblast/) and an established in-house computational pipeline. results: up to index clones per specimen were discovered in of the samples. in the remaining ( bm/pb pairs), pair did not reveal a clonal igh and was eliminated from analysis; in the other, clone frequency did not reach the % index threshold, but predominant clonal precursors were inferred by the prevalence of their subclone progeny. subclone counts ranged from to , per index clone. a combined , subclones derived from pb index clones were observed; in contrast, bm index clones gave rise to only subclones. subclone heterogeneity was observed between all paired specimens. in bm/pb pairs, index clones existed in equivalent proportions between disease sites. in contrast, bm/pb pair demonstrated high-frequency index clones in the bm ( . % & . %) with limited representation of these clones in the pb ( . % & . %, respectively); in this case, the most prevalent clone in the pb ( . %) matched the least frequent index clone in the bm ( . %). similarly, another pair showed a predominant index clone in the pb ( . %) which was below index threshold ( . %) in the bm. in paired patient specimens, index clone predominance was discovered to be overtly distinct between bm and pb. among all pairs, the extent of subclone progeny derived from each index clone showed marked variability, with far higher subclone frequency in the pb than in the bm. our data indicate that b all clonal composition differs between disease sites. valley children's healthcare, madera, california, united states background: tuberculosis (tb) presenting with hodgkin lymphoma (hl) is rare. their coexistence could lead to delay in diagnosis of both tb and hodgkin lymphoma due to the similarities in signs and symptoms of presentation. most cases have been reported in the adult literature. we describe a case series of children that were suspected to have tb and were found to have coexisting tb and hl. results: a retrospective review of hl patients in guatemala and argentina over six years, uncovered patients with simultaneous diagnosis of tb and hl. eight patients were from guatemala (incidence of . %) and from argentina (incidence of . %). there were females and males. age ranged from - years (mean . years, media years). nine patients were suspected to have tb at presentation by the referring physician. two patients were found to have tb at the time of relapse through routine tissue culture. initial systemic symptoms included fever (n = ), weight loss (n = ), and night sweats (n = ). six patients had a second systemic symptom in addition to fever. time for referral to oncology center ranged from weeks to months. nine patients were diagnosed with tb and hl through a tissue cultures and with serum quantiferon. one patient was found to have hl without tb. two patients had no systemic symptoms and the diagnosis of tb came to light through routine tissue culture. five patients had stage iiib and ivb, two stage iia and one iib at diagnosis. hl treatment was given according to the insti-tutional standards depending on stage and risk with abvd, oepa/copdac +/-radiation therapy, and ice for relapse. five patients started anti tb treatment (isoniazid, rifampin, pyrazinamide +/-ethambutol for months followed by isoniazid and rifampin for - weeks) simultaneously with chemotherapy, and three others after completing cycles. the two relapsed patients started tb treatment after cycles of chemotherapy. seven patients are alive and have been followed for months - years. one patient died during therapy, another died for causes not related to tb or hl and one is currently receiving treatment. conclusion: tuberculosis can coexist with hl. in areas were the prevalence of tb is high, microbiology investigations of biopsy specimen should be strongly considered. therapy for tb can be given simultaneously with chemotherapy. coexistence of tb and hl does not appear to affect outcomes. the children's hospital affiliated to the capital institute of pediatrics, united states background: the pi k/akt signaling pathway plays a central role in cell growth, proliferation and survival in physiological conditions. this signal pathway is considered to be an innovative targeted therapy of cancer, and its abnormal activation has been proved to be related to t-cell acute lymphoblastic leukemia (t-all). despite improved treatment strategies, such as multi-drug combination, high-dose chemotherapy and all kinds of application and popularization of hematopoietic stem cell transplantation, children with drug resistance or relapse t-all are still rather worse and its overall outcome and prognosis are much poorer than the more common b-lineage all. objectives: to explore the relationship between the pi k/akt pathway and the pediatric t-all, so as to probe the exact molecular mechanisms of t-all and provide more directions for its treatment. design/method: cases of new or recurrent acute t lymphocyte leukemia children with clinical information were collected in the children's hospital affiliated to the capital institute of pediatrics from dec. to oct. , with age and gender matched healty children as control (all was informed consent). the expressions of key genes in pi k pathway were s of s analyzed by western blot rt-pcr analysis, the pi k enzyme activities were detected by elisa,and the ccrf -cem's proliferation and its apoptosis were tested by mtt and flow cytometry technology on t-all cell lines ccrf-cem in different treatment group. the results of t-all children in clinical showed that pi k protein and gene expression level were higher apparent than the control group (p< . ), and pi k enzyme activity increased as well (p< . ); pi k inhibitor ly made a significant inhibition of cell proliferation and promoted cell apoptosis. ly also enhanced the effectiveness of clinical commonly used chemotherapeutic drug dnr. in combination ly and dnr treatment group cell viability dramatically declined, apoptosis and the apoptosis relation protein casepase expression in t-all patients was obviously higher than the control and the single drug group; pi k/akt signaling pathway related proteins and gene expression level, pi k, akt, gsk transcription in ccrf-cem were significantly higher than the control (p< . ), while pten transcription was significantly lower than the control (p< . ). the abnormal activation of pi k/akt signaling pathway might play an important role in pediatric t-all patients, especially in the cell proliferation or apoptosis. the results might provide new train of thought and direction in targeted suppress this signal pathway or in combination with other chemotherapy drugs therapy in looking for the more effective and less cytotoxic treatment of pediatric t-all. cleveland clinic children's hospital, cleveland, ohio, united states background: non-hodgkin lymphomas (nhls) are a heterogeneous group of lymphoproliferative diseases which comprise % of all childhood malignancies. nhls can be divided in to b cell lymphomas and t cell/natural killer (nk) cell lymphomas depending on immunophenotype, molecular biology, and clinical response to treatment. although nk/t cell lymphomas occurring in childhood and adolescence comprise a small portion of all lymphomas, they present many diagnostic and therapeutic challenges. the role of angiogenesis in lymphoma pathogenesis is becoming more evident. high molecular weight kininogen (hk) is a central compo-nent of the kallikrein-kinin system. it has been previously reported that cleaved hk (hka) induces apoptosis of proliferating endothelial cells and inhibits angiogenesis in matrigel plug and corneal angiogenesis models. however, the role of endogenous kininogen in regulation of angiogenesis is in tumor microenvironment is unknown. objectives: to elaborate the role of hk in lymphoma angiogenesis, we used a murine t-cell lymphoma model and compared angiogenesis and tumor growth between wild-type and kininogen deficient (mkng -/-) mice. we also evaluated the effect of hka on lymphoma cell proliferation. design/method: el- murine t-cell lymphoma cells ( × ^ ) were implanted into wild-type and mkng -/-mice. tumor size was measured using calipers and tumor volume was calculated using the formula volume = length × width^ × . . seventeen days after cell implantation, tumors were harvested and processed by immunoblotting and immunofluorescent staining. cell proliferation assays (mts) were performed to investigate any possible inhibitory effect of hka on el- cell growth, with human umbilical vein endothelial cells (huvec) were used as a positive control. results: el- lymphomas grew more rapidly and to larger sizes in mkng -/-mice compared to wild-type mice, with significant differences apparent by day after tumor implantation (p< . ). by day , the volume of tumors in mkng -/-mice was approximately . -fold larger than in wild-type mice (mean volume ± standard deviation; ± vs. ± mm , respectively, p< . ). mts assays showed that hka does not directly inhibit the proliferation of el- cells in vitro, though it does significantly impair the viability of ecs studied simultaneously. conclusion: these findings suggest that hk is an important endogenous regulator of angiogenesis and tumor growth in this t-cell lymphoma model, and suggests that hka specifically modulates endothelial proliferation in tumor microenvironment. further work is needed to understand the mechanisms underlying these findings and provide future anti-angiogenic approaches to increase the therapeutic options for patients with nhl. bruce bostrom, jack knudson, nathan gossai, joanna perkins, michael richards, jawhar rawwas, susan sencer, julie chu, nancy mcallister, yoav messinger children's minnesota, minneapolis, minnesota, united states background: osteonecrosis causes significant pain and morbidity in older patients treated for acute lymphoblastic leukemia. besides altering the schedule of dexamethasone in delayed intensification there is no other intervention known to reduce the incidence of symptomatic osteonecrosis. pamidronate has been shown to reduce bone pain from osteonecrosis but not to prevent joint collapse when advanced. objectives: to compare the incidence of symptomatic osteonecrosis in patients who received prophylactic pamidronate compared with concurrent controls. to describe any increase in side effects from the use of pamidronate. design/method: patients age to years at time of all diagnosis were given intravenous pamidronate monthly for one year at the discretion of the primary oncologist starting in the first year of therapy. concurrent controls were patients age to who did not receive pamidronate. all patients were treated according to the concurrent cog protocols and received intermittent dexamethasone during delayed intensification. patients with bcr-abl all were excluded as the use of imatinib may increase the risk of osteonecrosis. imaging was only done if osteonecrosis was suspect based on clinical symptoms. patients were censored at the time of relapse. data were analyzed as of / / . this retrospective study was approved by the children's minnesota irb. of the patients evaluated % were male and % female, % had b-cell and % t-cell. the median followup is . years with a range of . to years. pamidronate was given to patients with developing symptomatic osteonecrosis. there were concurrent controls with developing osteonecrosis. there was no significant difference in the leukemia lineage, gender distribution or body mass index (bmi) at diagnosis between groups. for all patients the median bmi was with a range of to . the age at diagnosis was significantly higher in the pamidronate group with a median of . years vs. . in the controls (p = . ). by kaplan-meier analyses the incidence of symptomatic osteonecrosis was significantly lower in the pamidronate group at % vs. % in controls. the log-rank p-value was . and the breslow p-value, which is more sensitive to early events, was . . there were no untoward side-effects from pamidronate. pamidronate infusions significantly reduced the incidence of symptomatic osteonecrosis in patients over the age of compared to concurrent controls who did not receive pamidronate. arahana awasthi, dina edani, janet ayello, christian klein, mitchell cairo new york medical college, valhalla, new york, united states background: mature b-nhl, including bl and pmbl express cd +/cd b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ % yr. efs, cairo et al. blood. ; gerrard/cairo et al., blood, , goldman/cairo et al. leukemia, . pv has been demonstrated to possess significant preclinical activity against indolent cd b+nhl (polson et al. can. res. ). we previously observed that obinutuzumab (anti-cd mab) significantly enhanced cell death and increased overall survival against bl (awasthi/cairo et al., bjh ) in xenografted nsg mice. however, additive/synergistic effects of pv with obinutuzumab against mature pmbl/bl are unknown. to determine the efficacy of the pv or obinutuzumab/rtx alone or in combination against pmbl and rituximab (rtx) sensitive/resistant bl cell lines. design/method: raji rh (provided by m. barth, md, roswell park cancer institute) and raji/ karpas p (atcc, usa) were cultured in rpmi. tumor cells were incubated with pv, and/or anti-cd b, mmae (generously supplied by genentech inc.) with obinutuzumab /rituximab ( ug/ml) for hr with nk cells at : e: t ratio and cytotoxicity was determined by delfia cytotoxicity assay. six to week old female nsg (nod.cg-prkdcscid il rgtm wjl/szj), were divided into groups: pbs, isotype control, pv, anticd b mab and mmae ( mg/kg). mice were xenografted with intravenous injections of luc+ bl and pmbl cells and tumor burden was monitored by ivis spectrum system. results: os of mice receiving pv alone was significantly increased compared to anticd b ab or isotype control in raji ( . vs. vs. . our preliminary data indicates that pv significantly increased survival in bl and pmbl nsg xenografts compared to anti-cd b ab alone. furthermore, pv in combination with obinutuzumab significantly enhances in-vitro cytotoxicity in bl and pmbl compared to obinutuzumab or pv alone. results: maximal grades (g) / , , and crs occurred in , , and patients, respectively. median lowest fibrinogen levels were . , . , and . g/l in patients with maximal g - , , and crs, respectively. %, %, and % of patients with maximal g - , , and crs had lowest reported fibrinogen levels of ≥ to < . g/l. eight patients (all with g crs) had very low fibrinogen levels (< g/l), which occurred before (n = ) or during (n = ) maximal crs grade or at time of improvement (n = ). no patients with maximal g - crs had < g/l fibrinogen levels. at the onset of < g/l fibrinogen levels, patient had concurrent g , and had g - increased international normalized ratio and activated partial thromboplastin. cryoprecipitate was the primary treatment in the us, and fibrinogen concentrate (fc) guidelines for tisagenlecleucel-associated coagulopathy were developed for other countries because administration of fresh frozen plasma can be problematic. fc was available at / sites for infused patients: / (g crs) and / (g - crs). cryoprecipitate was available at / sites for infused patients: / (g crs), / (g crs), and / (g - crs). risk of bleeding increases in pediatric patients with comorbid thrombocytopenia and anticoagulant treatments. / patients had g / decreased platelets within day of < g/l fibrinogen levels. fatal case of intraparenchymal cranial hemorrhage occurred during resolving crs with g hypofibrinogenemia, ongoing thrombocytopenia, and continuous veno-venous hemofiltration with citrate. hypofibrinogenemia was observed more frequently in patients with higher crs grades during/when crs was improving or resolving. fc and cryoprecipitate treatment guidelines were developed. frequent monitoring and fibrinogen replacement are needed in patients with g / crs. sponsored by novartis. its prolonged cns half-life, may allow a reduction in the number of intrathecal injections. objectives: to safely reduce the burden of therapy by reducing the number of it injections and reducing the total dose of doxorubicin with the addition of liposomal cytarabine and rituximab. design/method: patients ( - years) with cd + b-nhl with fab group b good risk (=stage i/ii and stage iii with ldh < xuln), fab group b intermediate risk (=stage iii ldh ≥ xuln and stage iv {bm blasts < %}) and fab group c high risk were eligible. patients received fab backbone therapy with the addition of six rituximab ( mg/m ) doses; two doses prior to each of two induction courses and one dose prior to each of two consolidation courses. cumulative doxorubicin was reduced from to mg/m in gr patients. after systemic methotrexate clearance, patients received age based dosing of it liposomal cytarabine. it injections were reduced from nine to five. the primary outcome is safety and toxic deaths among evaluable patients with an estimated -year survival above %, monitored by an independent dsmb. results: to date, evaluable patients, fab group b and group c ( cns positive), median age years (range - ), males, burkitt/ dlbcl with gr, ir and hr have enrolled. there has been one grade anaphylactic reaction to rituximab and one grade facial nerve palsy. no other serious adverse events were attributable to protocol therapy. there has been death from progressive disease and relapse at a median follow up of months. efs and os are % and %, respectively. our initial results show excellent efs and os, consistent with published standard of care outcomes, with the addition of rituximab and intrathecal liposomal cytarabine despite the reductions in therapy. further enrollment is ongoing and continued long term outcomes are needed to confirm early results. future randomized studies are needed to examine both short term (mucositis, infections, hospitalization days) and long term (late cardiac toxicity) endpoints. . goldman etal, leukemia, . cairo etal, jco st. jude children's research hospital, memphis, tennessee, united states background: bereaved parents identify significant spiritual needs around time of death and throughout their bereavement journeys. spirituality has been identified as a primary means by which bereaved parents can find meaning in their losses, and this ability to find meaning is associated with lower maladaptive grief symptoms. the use of spiritual coping strategies has been associated with improved coping and mental health outcomes among bereaved parents. objectives: to better understand how bereaved parents' experiences with spirituality throughout bereavement effects objective measures of grief, depression, and meaning-making. design/method: thirty participants whose children died of progressive cancer or related complications one to three years prior to participation completed an in-depth semi-structured telephone interview about their experiences with grief. participants were prompted to describe the impact of their spirituality on their bereavement processes. additionally, participants completed surveys related to grief (prolonged grief disorder questionnaire, pg- ), depression (beck depression inventory, bdi), and meaning-making (integration of stressful life experiences scale, isles). results were analyzed using a mixed methods approach including semantic content analysis of qualitative content and kruskal-wallis h test and post-hoc analyses of quantitative data. results: correlation analyses demonstrated significant differences between participants with positive and negative spiritual experiences of bereavement. participants with negative experiences of bereavement had a statistically significant increase in scores on the pg- compared to those with positive spiritual experiences signifying greater symptoms of prolonged grief. participants with negative spiritual experiences with grief had significantly lower scores on the isles, suggesting a lesser degree of adaptive integration of their losses. there were no significant differences in depression scores between groups. conclusion: bereaved parents that have a negative spiritual experience of bereavement are at increased risk for prolonged grief symptoms and are less likely to find meaning in their children's deaths than bereaved parents that describe a positive spiritual experience of bereavement. providers should consider exploration of spiritual beliefs and provision of spiritual care for parents of children facing life-limiting illnesses during treatment and bereavement. background: langerhans cell histiocytosis (lch) is an inflammatory myeloid neoplasia characterized by frequent relapse, with treatment failure associated with higher risk of death and neurodegenerative disease (lch-nd). activating somatic mutations in mapk pathway genes have been identified in almost all cases, with braf-v e in approximately % of lesions. targeted therapies have been successful in treating other refractory cancers with braf v e mutations (such as melanoma). given the central role of mapk pathway activation in lch, mapk pathway inhibition may be an effective therapeutic strategy for children with lch. objectives: the purpose of this study was to report the efficacy and toxicity profile of a retrospective cohort of patients with lch treated with mapk pathway inhibitors. design/method: medical records from pediatric patients with lch (systemic and/or lch-associated neurodegeneration) who were treated with a mapk pathway inhibitor were retrospectively reviewed from five institutions. all patients had failed at least one prior systemic therapy and had a proven mapk pathway mutation. results: all patients in this series were less than years old (median = . years; range: - years) with a median of three prior treatments (range: - ). at the time of initial mapk inhibitor use, nine of the patients had lch-nd diagnosed clinically and/or by radiographic imaging; the remaining three patients had systemic disease. patients were treated for a median of months (range: - months) with various reasons for discontinuation. three patients received combination mapk inhibitor therapies and three patients received other concurrent lch-directed therapies. four of the twelve patients had a grade or toxicity reported and three of these patients required dose reduction in order to be able to successfully resume therapy. overall survival was % with median month follow-up (range: - months) with only one patient achieving transient complete response. the remaining ten patients had partial response or stable disease and four of these patients developed progressive disease while on therapy. conclusion: mapk pathway inhibitors may be a relatively safe salvage therapy for refractory systemic lch and lch-nd but the efficacy and durability of this strategy remains to be defined. combination with cytotoxic chemotherapies may be required in order to eradicate the disease-causing cell. future prospective trials of mapk pathway inhibitors for patients with refractory lch are needed in order to directly compare their efficacy and toxicity relative to other current salvage strategies. cincinnati children's hospital medical center, cincinnati, ohio, united states background: medication adherence during maintenance therapy has been shown to have a direct relationship with disease relapse in pediatric leukemia. previous research determined that patients who are ≤ % adherent to mercaptopurine ( mp) have a greater risk for relapse. the primary aim of the present study is to examine the relationship between metabolite profiles of mp with behavioral adherence rates obtained via electronic monitoring at , , and days. it is hypothesized that patients demonstrating low levels of thioguanine (tgn) and methylated mercaptopurine (mmp) will have lower behavioral adherence rates prior to the blood draw. design/method: in a multisite, prospective study of patients ages - years diagnosed with acute lymphoblastic leukemia (all) or lymphoblastic lymphoma (lbl), mp adherence was measured across months of maintenance therapy using behavioral adherence (electronic monitoring) and pharmacological (metabolites) measures of mp. mp is metabolized into mmp and tgn. cluster analysis was used to generate three mutually-exclusive profiles of mp adherence. behavioral adherence rates were calculated for , , and days prior to the blood draw. results: this study identified three metabolite profiles of mp across months. previous research indicated that low levels of both metabolites suggest nonadherence to medication. low levels of one metabolite with high levels of another metabolite indicate adherence to mp. in this study, . % of the low tgn-low mmp group had -day behavioral adherence rates ≥ % (mean = %); . % had adherence rates < % (mean = . %). in the high tgn-low mmp group, . % had a mean -day adherence of %; . % had adherence rates < % (mean = . %). the low tgn-high mmp group had % of patients with a mean -day adherence level of %; % had adherence rates < % (m = . %). at and -days, to % of patients in the low tgn-low mmp group had adherence rates < %. conclusion: these findings suggest that electronic monitoring and metabolite concentrations can be used to monitor mp medication adherence during maintenance therapy. it is notable that there is a sub-sample of pediatric patients who are identified as being nonadherent to mp based on electronic monitoring, however, metabolite levels indicate adherence to mp. similarly, a sub-sample of patients were identified as being adherent based on electronic monitoring, but metabolite profiles indicated sub-therapeutic levels of mp. our findings underscore the clinical significance of using both objective measures of medication adherence to inform clinical decision making. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hemophagocytic lymphohistiocytosis (hlh) is a life-threatening hyperinflammatory syndrome characterized by non-remitting fevers, rash, hepatosplenomegaly, cytopenias, liver dysfunction and coagulopathy, and can include central nervous system involvement. several genetic diseases cause hlh by impairing normal lymphocyte or macrophage function. the hlh panel at the cincinnati children's genetics laboratories includes genes associated with hlh and other lymphoproliferative diseases, including the genes that cause primary hlh (prf , unc d, stxbp , stx , rab a), x-linked lymphoproliferative diseases (sh d a, xiap), itk deficiency (itk), hermansky-pudlak syndrome types and (ap b and bloc s ), chediak-higashi syndrome (lyst), cd deficiency (cd ), xmen syndrome (magt ) and lysinuric protein intolerance (slc a ). deletion/duplication analysis is available as a reflex test for all genes, as copy number variations (cnvs) are not directly assessed by sequencing. objectives: the prevalence of cnvs among large groups of patients with hlh in north america is unknown. we assessed the frequency of cnvs in the genes on the hlh panel through a retrospective review of orders for deletion/duplication analysis performed after next-generation or sanger sequencing: orders for all genes on the panel, and orders of - genes from the panel. deletion/duplication analysis was performed on a custom × k microarray annotated against ncbi build (ucsc hg , march ). deletion/duplication analysis resulted in a confirmatory diagnosis in of cases ( . %). pathogenic or likely pathogenic cnvs were most common in the three x-linked genes: sh d a ( deletions), xiap ( deletions, duplication), and magt ( deletions). hemizygous deletions in xlinked genes in male patients were typically suspected after amplification failure during previous sequencing. of the autosomal recessive genes, pathogenic cnvs were observed once in each of three genes: rab a (heterozygous), lyst (heterozygous), and stxbp (homozygous). in the two heterozygous cases, a second change was not identified by sequencing, so deletion/duplication analysis did not offer a confirmatory diagnosis. in patients, deletion/duplication analysis was performed after a pathogenic or likely pathogenic variant was identified in an autosomal recessive gene during sequencing; however, in no case was a second mutation uncovered by cnv analysis. we recommend that deletion/duplication analysis be routinely performed in all male patients with hlh who lack a genetic diagnosis after sequencing of hlh-associated genes, especially if any regions failed to amplify. deletion/duplication analysis may be performed in female patients after sequencing if a genetic form of hlh is highly suspected, but the yield is expected to be low. cleveland clinic children's hospital, cleveland, ohio, united states background: the development of post-transplant neoplasia, typically from lymphoproliferative disease (ptld), is a severe complication in transplant recipients and affects approximately % of pediatric solid organ recipients. rates of lymphoma in adult heart transplantation patients are comparatively low, at less two percent at ten years. there are few published reports of the long-term outcomes of neoplasia after pediatric heart transplantation. we aimed to identify the subsequent malignancies that occurred in pediatric heart transplantation patients in a large single institution, and describe their treatment and subsequent clinical course. we performed a retrospective chart review of all pediatric heart transplant recipients followed at the cleveland clinic children's hospital from january to october . we excluded patients who died within days of heart transplantation. we reviewed in depth the history and clinical course of subjects who developed neoplasms. results: between and , patients underwent heart transplantation and survived at least days post transplantation. nine patients ( . %) developed a subsequent malignancy. in this case series, the median age at heart transplant was years old and the median time to develop neoplasia was . months. primary neoplasia included monomorphic ptld ( ), polymorphic ptld ( ), burkitt lymphoma ( ), hodgkin's lymphoma ( ), plasmacytoma-like lymphoma ( ) and epstein-barr virus-associated smooth muscle tumor (ebv-smt) ( ). one patient with hodgkin lymphoma subsequently developed monomorphic ptld, one patient with polymorphic ptld subsequently developed ebv-smt and later, an undifferentiated gastric cancer. one patient with monomorphic ptld developed an ebv-smt. evidence of epstein-barr virus was present in six of nine patients at diagnosis of first malignancy. four of nine patients received reduction in immunosuppression as a primary intervention for the initial malignancy, with two complete responses (cr), one partial response, and one with progressive disease. five patients were treated with chemotherapy, with four cr and one with progressive disease. three patients died of malignancy (recurrent ebv-smt, undifferentiated gastric cancer, and monomorphic ptld post-hodgkin disease) and two patients died of other transplant related complications. conclusion: secondary malignancies represent a significant disease burden to survivors of cardiac transplantation. as expected, much of the malignancy burden is driven by ebv. despite aggressive histology, many malignancies can be successfully cured in this setting with a multidisciplinary approach. stanford university school of medicine, palo alto, california, united states background: current treatment of langerhans cell histiocytosis (lch) is based on extent of organ system involvement and if high risk systems are affected. gastrointestinal (gi) involvement is diagnosed in about % of lch patients, and classically presents in children under years of age with malabsorption, failure to thrive, bloody diarrhea and anemia. although the gi system is considered standard risk, a mortality rate over % occurring within years of diagnosis has been reported. this study was performed due to this discrepancy and the limited number of published cases. objectives: to review the clinical course and outcomes of patients diagnosed with gi lch. design/method: a retrospective chart review of patients with histologically confirmed gi lch diagnosed in the last years identified from the bass center histiocytosis clinical database was performed. two other pediatric hematology/oncology centers (ucsf benioff children's hospital oakland and san francisco) were queried for additional cases. results: four patients with biopsy proven gi lch [ subjects ( . %) from database records and l from center queries] were identified. failure to thrive, hypoalbuminemia, bloody diarrhea and rash were the most common presenting symptoms. lch of the skin was found in all patients. risk organ systems were involved in patients. of note, subjects were of african racial background. the median age at diagnosis was . months ( . months to years), mean albumin . g/dl ( . - . g/dl), mean esr of mm/hr ( - mm/hr). all patients initially received combination therapy per lchiii protocol (vinblastine, prednisone, and mercaptopurine). two patients had recurrent disease and received second line therapy (cytarabine, cda, and local radiation therapy). all patients are alive without active disease at last follow-up ( to months after completion of therapy). a systematic approach to evaluate gi involvement should be performed in children diagnosed with lch. from our experience, combination chemotherapy for patients with lch involving the gi tract is an effective intervention for active disease. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bhatia indicated that rates of mp adherence ≥ % have better clinical outcomes. those with adherence rates ≤ % have an increased risk for disease relapse. the present study investigated patterns of mp medication adherence using group-based trajectory modeling in a large sample of pediatric patients. to describe patterns of behavioral adherence during the maintenance phase of therapy for a cohort of pediatric patients ages - years who were diagnosed with acute lymphoblastic leukemia or lymphoblastic lymphoma (n = ). previous research has documented the relationship between optimal levels of medication adherence with positive health outcomes. it was hypothesized that three groups would be identified: optimal adherence, deteriorating adherence, and chronic nonadherence. it was hypothesized that patients in the optimal adherence group would have adherence rates ≥ %. those with poor adherence would have adherence rates ≤ %. design/method: the present study was a longitudinal, multisite study investigating adherence to -mercaptopurine in a pediatric cohort of patients using electronic monitoring devices. daily adherence rates (electronic monitoring of mp) were examined across -months. health outcomes were measured at quarterly intervals through medical chart reviews. results: unconditional growth curve modeling indicated that the mean percentage of behavioral adherence was . % at baseline and declined to . % at -months. three trajectories of mp behavioral adherence were identified: ) optimal adherence ( % of patients): averaging % behavioral adherence across months; ) moderate adherence ( %): relatively stable nonadherence with rates of % across months; and, ) chronically nonadherent ( %): adherence decreased from % to %. with respect to patterns of medication adherence and relationship to clinically-relevant health outcomes, there were no significant differences in health outcomes between patients in the adherent versus nonadherent trajectories, including mean absolute neutrophil counts (anc), risk for infection as measured by anc, healthcare utilization, or risk for disease relapse. although longitudinal patterns of mp behavioral adherence were not related to health outcomes, it is notable that only % of the current sample had adherence rates ≥ %. in fact, % of the current sample demonstrated adherence rates ≤ %. our findings are important for development of future adherence promotion studies in pediatric cancer. our findings underscore the relative significance of tailoring adherence promotion interventions to subgroups of patients, including those with problematic patterns of adherence. patients who demonstrate adequate levels of adherence could still benefit from less intensive, preventative interventions to sustain and improve adherence. sophie gatineau-sailliant, pascale grimard, marie-claude miron, guy grimard, anne-sophie carret, jean-marie leclerc chu sainte-justine, montreal, quebec, canada background: vertebral involvement in langerhans cell histiocytosis (lch) is still a subject of interest, due to its low frequency and the absence of management's guidelines. objectives: to provide additional information on presentation, treatment and morbidity of pediatric lch vertebral lesions, we report cases of children with vertebral lesion of biopsy-proven lch, between january st and december st , at sainte-justine university health center (montreal, quebec, canada). we conducted a retrospective study by reviewing charts and imaging of vertebral lch in a population of children (median age of . years at lch diagnosis), followed for a median duration of months. symptoms at presentation, treatment modalities and morbidities were collected. results: vertebral lesions were present at lch diagnosis in of cases. they were usually diagnosed secondary to back pain in of cases and were asymptomatic in only one case. despite an epidural extension in of cases, no child developed neurological symptoms. lesions frequently involved vertebral body ( of cases) and were rarely unstable ( of cases). out of vertebral lesions, most of them had a dorsal localization ( of lesions) and of patients had lch in multiple vertebrae. at diagnosis, median vertebral height loss was . % compared to % at last imaging control. most used imaging modalities were pet-scan and plain x-rays. treatments were diverse and consisted in chemotherapy in all children but three and bisphosphonates in only cases. radiation therapy was not used in any patient. six out patients did benefit of an orthosis. a lch recurrence was observed in patients and involved vertebrae in cases. one patient with treatment-resistant lch disease had relapses, and required multiple lines of treatment. all children were alive and disease-free at their last follow-up, patients having radiological vertebral sequelae and only had clinical sequelae. our study is consistent with the epidemiological data described in larger cohorts of children with vertebral lesions of lch and the favorable prognosis associated with such lesions. nevertheless, aggressive treatment and long term follow-up seemed to be essential as recurrences are s of s not rare and spontaneous bone regeneration often incomplete. plain x-rays appears to be a good follow-up tool for vertebral lesions as it allows reliable measures, less exposure to radiation at lower cost. national cancer institue, giza, giza, egypt background: acute lymphoblastic leukemia (all) is the most common type of childhood cancer and also the most complicated in the treatment, so it requires many interventions for both treatment and to alleviate suffer form side effects. pancreatitis is one of the toxicities, which is more common in all as it appears in about % of the patients. it occurs in many drug combinations which induce pre-pancreatitis and even direct destruction of pancreatic tissues. pancreatitis can be induced by many drugs used in the treatment such as chemotherapeutic agents or supportive treatment. lasparaginase is the backbone drug of the treatment of all in which to doses are required to achieve complete remission status in the induction phase of treatment and to doses in the maintenance phase.it is an enzyme that destructs the l-asparagine amino acid into aspartic acid and ammonia thus deplete the asparagine from the extracellular matrix . many drugs are investigated for their effect on treatment of induced pancreatitis such as interleukin- , nsaid as antiinflammatory, glycerin tri nitrates as improvement of microcirculation, tnf-alpha antibody, paf inhibitor as specific anti-inflammatory and low molecular weight heparin .none of the drugs was investigated for their ability to prevent the occurrence of pancreatitis. objectives: this study was designed to evaluate the protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis design/method: acute pancreatitis was induced in rats by intra-muscular injection of l-asparaginase ( i.u/kg) given daily for five days. enoxaparin was given subcutaneous ( i.u/kg) and diclofenac was given intra-peritoneal ( mg/kg) daily for five days. then, markers of pancreatic injury, lipids, immune cell infiltration and oxidative stress were analyzed with histo-pathological examination of the pancreatic tissue results: during acute pancreatitis, oxidative stress markers were significantly changed as indicated by reduced tis-sue glutathione and increased malondialdehyde levels. this was accompanied with significant increase in immune cells infiltration as indicated by high levels of myeloperoxidase and pro-inflammatory cytokine tnf-alpha. triglyceride only showed increase level. treatment with enoxaparin and/or diclofenac restored levels of biochemical markers including serum alpha-amylase, reduced glutathione, malondialdehyde, pro-inflammatory cytokine tnf-alpha, myeloperoxidase and triglyceride. histological injuries of pancreatic tissues as vacuolation and necrosis of epithelial lining pancreatic acini, inflammatory cells infiltration and focal pancreatic hemorrhage were also reduced by treatment with enoxaparin and/or diclofenac. the present study emphasizes the potential protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis background: rosai dorfman disease (rdd), or sinus histiocytosis with massive lymphadenopathy (shml), is a rare condition of immune dysregulation of unknown etiology arising from the massive accumulation of non-langerhans type histiocytic cells inside lymph nodes. the disease classically presents as bulky, painless lymphadenopathy often associated with infection showing distension of lymph node sinuses by abundant histiocytic cells (cd a(-), s- (+)/cd (+)). in some cases, the disease can be self-limiting, but in cases with a prolonged chronic course of exacerbations and remissions, those with extranodal involvement, or disease that threatens vitals structures, treatment may be necessary. there is no treatment consensus. to describe a case of life-threatening, unresectable, recurrent rdd successfully treated with langerhans cell histiocytosis (lch) -inspired therapy. design/method: we compared this case to the current literature on chemotherapeutic treatments for rdd. we searched pubmed, ovid, and google scholar for similar cases. we believe this to be the first reported case of using lch therapy to successfully treat rdd. an -year-old male presented to an outside hospital with two years of massive neck swelling causing torticollis. biopsy confirmed rdd. he was intermittently treated with courses of antibiotics with partial response. surgical removal of the affected lymph nodes was unsuccessful due to proximity to the spinal cord. two years later, the patient presented to our institution. he was initially treated with prednisone with a fast tapering dose, but after a second relapse the decision was made to try chemotherapy following the lch- protocol of weekly vinblastine ( mg/m ), -mp ( mg/m ), and high dose steroid bursts. he experienced two additional relapses off therapy at ages and years old, including cmv(+) associated septic shock and cytokine storm requiring rapid response, picu admission, and ionotropic support. this last episode was treated with a more prolonged induction and maintenance therapy. an extended and slowly tapered maintenance therapy regimen of . years of daily -mp, monthly vinblastine and steroids with a slowly tapered dose during his fourth remission has resulted in -months of continuous complete remission-the longest stretch of his life. no similar cases were found. literature search demonstrated no consensus regarding the most effective treatment of rdd, with no previous cases being successfully treated following lch chemotherapy protocols. we hypothesize that the multi-agent relatively mild lch- therapy mitigates the immune dysregulation of rdd. this case suggests that lch- therapy can be used to treat cases of rdd that is not amendable to surgery or observation. nicklaus children's hospital, miami, florida, united states background: central venous catheters (cvc) are necessary in the management of patients with malignancies, especially children. patients with acute leukemia (al) have higher rates of central line associated complications such as bloodstream infections compared with other malignancies. objectives: to examine the choice of placement of cvc and the differences in outcome between peripherally inserted central catheters (picc) and ports in patients with leukemia during induction. design/method: retrospective chart review of patients with newly diagnosed leukemia at nicklaus children's hospital between and . results: ninety four patients with a new diagnosis of leukemia undergoing induction chemotherapy were identified. the average age was . years. overall, ( . %) patients had a port placed and ( . %) had a picc placed. the decision for picc or port was subjective and physician based. the main outcome measures were local inflammation/infection, bacteremia, thrombophlebitis, blocked catheter and premature removal. the most common complication was bacteremia ( . %). in a multiple logistic regression analysis for predicting whether patients had at least one complication, results showed that having at least one complication is . times the odds in patients with aml compared to patients with all (p = . ). when comparing picc vs. ports, patients with picc had more frequent episodes of blocked catheters ( . %) and premature removal ( . %) compared to the patients with ports ( . % and . %) (p = . and p = . respectively) during induction. local inflammation, bacteremia and thrombophlebitis were not statistically different (p = . , p = . and p = . respectively). the most common place for port placement was the right subclavian vein ( %). there was no significant association between port location and having at least one complication (p = . ). acute lymphocytic leukemia subgroup analysis: fourteen patients ( %) in the picc group had at least one complication and ( %) in the port group but that was not statistically significant (p = . ). our series showed a higher incidence of blocked catheters and premature removals with picc compared to ports in patients with leukemia during induction. the choice of placement of picc vs port was subjective and physician based. patients with all, despite receiving steroids and asparaginase during induction, did not show a statistically significant increase risk in thrombosis or infection but larger numbers may be needed in future studies. university of california, san francisco, san francisco, california, united states background: hemophagocytic lymphohistiocytosis (hlh) is classically a disorder of young children meeting systemic hyperinflammation criteria. presentation in late adolescence is uncommon. furthermore, though cns signs occur in - % of cases, initial isolated neurologic presentation is rare, frequently resembling encephalitis or demyelinating disorders. these cns signs can be isolated or precede systemic disease, delaying hlh diagnosis. hlh declaring in adolescence with predominant psychiatric features has not been well documented. objectives: to describe a case of cns hlh presenting with neuropsychiatric features in absence of classic hlh criteria. design/method: retrospective review of clinical, radiologic, histologic, immunophenotypic, and molecular features of a patient with cns hlh. a -year-old female presented with acute-onset headaches following nine months of progressive anxiety, short-term memory loss, emotional lability, perceptual disturbances, and hypomania. brain mri demonstrated numerous enhancing t hyperintense supratentorial and infratentorial white matter lesions in the left thalamus and caudate head. brain biopsy showed histiocyte-rich inflammation and associated demyelination. extensive evaluation including universal microbial pcr failed to reveal underlying infection or malignancy. past medical history was notable for presumptive pulmonary sarcoidosis diagnosed months prior with progressive respiratory failure with associated granulomatous pulmonary nodules which responded to systemic immunosuppression. at presentation of her neuropsychiatric symptoms, she had normal sil- r, ferritin, fibrinogen, and triglycerides. there was no pancytopenia, coagulopathy, bone marrow hemophagocytosis, fevers, or splenomegaly. given the possibility of partial immune suppression of systemic symptoms and the prominent neurologic symptoms, hlh screening labs were sent and notable for decreased natural killer and cytotoxic t lymphocyte function, normal granzyme expression and cd a mobilization, and absent perforin expression. genetic testing confirmed compound heterozygous mutations in prf (c. g>a, c. a>c) and familial hlh type . she was treated with low-dose dexamethasone and intrathecal chemotherapy per hlh- . due to lack of evidence of systemic inflammation, vp- and high-dose steroids were held. within one week of initiating therapy, she had decreased anxiety and improved cognition, with sustained, incremental neuropsychiatric improvement with additional intrathecal treatments. she tolerated dexamethasone tapering without symptom flare. mri also demonstrated parenchymal lesion improvement. for definitive treatment, she underwent unrelated allogeneic hematopoietic cell transplantation and remains at neurologic baseline as of eight months post-transplant with ongoing imaging improvement. conclusion: this case of familial hlh with compound heterozygous perforin mutations in an adolescent with isolated neuropsychiatric symptoms illustrates that cns hlh may be an underrecognized phenomenon in absence of systemic signs. standard hlh therapy may effectively reverse these symptoms with associated radiologic responses. rush university children's hospital, chicago, illinois, united states background: posterior reversible encephalopathy syndrome (pres), a recognized complication of pediatric leukemia treatment has been reported in up to % patients in various series. hypertension, chemotherapy and cortical spreading depression have been implicated in the pathophysiology. due to the combinations used, it is difficult to identify the offending drug, several have been implicated. since delay of chemotherapeutic treatment in children with high risk leukemia is unfavorable, it is important to recognize the characteristic radiologic findings, manage appropriately and reintroduce the treatment as soon as possible. pharmacoethnicity is now recognized as an important factor for variation in neurotoxicity in children with all. ethnic differences in reported pres events in pediatric patients with all has not been well described in literature. to describe the factors associated with pres in a cohort of high risk pediatric all patients at a single institution. design/method: a total of children with an average age of years ( - years) diagnosed with all between - were retrospectively reviewed for the occurrence of pres. various demographic factors, therapy received, clinical features, radiology related findings and management were reviewed. a search for all published articles on pres in leukemia was conducted using pubmed databases. results: five ( %) children (average age . years) developed pres during days - of induction. % of the patients that developed and % of those that did not develop pres were hispanic. all the patients that developed pres and % of those that did not were diagnosed with high risk all. all patients received vincristine, % received daunomycin and intrathecal methotrexate and % received asparaginase in the week prior to the event. mri findings confirmed pres in all patients with no evidence of methotrexate related leukoencephalopathy or leukemia. at the time of pres all patients were in remission based on mrd and spinal fluid cytology. two-thirds of the patients had seizures and hypertension at the time of the event with no prior history of either. all patients had complete recovery of normal mental status after resolution of pres. a higher incidence of pres than previously reported was noted in our series. hispanic ethnicity, high-risk all and exposure to vincristine, daunomycin and intrathecal methotrexate in induction were associated with pres in our cohort. a new association that emerged was that of hispanic ethnicity with pres .larger studies to understand the importance of pharmacoethnicity in pres may help in individualization of chemotherapy based on ethnic differences. children's hospital of illinois, peoria, illinois, united states background: hyper ige syndrome is a primary immunodeficiency characterized by susceptibility to skin and lung infections as well as increased propensity for malignancy. hemophagocytic lymphohistiocytosis (hlh) is a syndrome characterized by overwhelming activation of t lymphocytes and macrophages occurring as either primary hlh caused by genetic abnormalities or secondary hlh associated with infectious, malignant, metabolic, or immunodeficiency causes. we describe the first case to our knowledge of hlh in a patient with hyper ige syndrome. to describe a case of hlh in a pediatric patient with hyper ige syndrome. results: a -year old caucasian male with known autosomal dominant hyper ige syndrome (stat mutation) was transferred to the pediatric intensive care unit secondary to concern for septic shock. the patient had persistent slow bleeding from oral lesions and central catheter sites despite the addition of aminocaproic acid and recombinant factor viia. he also required numerous blood product transfusions sec-ondary to anemia and thrombocytopenia. clinical suspicion was high for hlh and the patient met criteria for diagnosis of hlh with the following: ferritin > , ng/ml, triglycerides mg/dl, decreased nk cell function with the sample only containing % nk cells, elevated soluble il- receptor at u/ml, splenomegaly, and fever. infectious workup was remarkable for a positive ebv qpcr with , copies/ml suggestive of ebv driven secondary hlh. familial hlh testing was unable to be completed. therapy was initiated based upon the hlh- study. the addition of ruxolitinib and anakinra were considered but the patient declined rapidly prior to treatment. ct of the head was concerning for a stroke with signs of edema and increased intracranial pressure likely leading to the development of symptoms consistent with brain stem herniation. the decision was then made to withdraw care. conclusion: to our knowledge, this is the first report of hlh in a patient with hyper ige syndrome. diagnosing hlh requires a high index of suspicion in critically ill patients, and prompt initiation of therapy is essential. this challenging case of hlh in a patient with hyper ige syndrome highlights the diagnostic challenge, variable presentation, and need for effective therapy in this vulnerable patient population. background: adolescents and young adults (ayas) with cancer are at risk for psycho-social as well as physical symptom burden during cancer therapy. the purpose of this study is to explore psychological and physical symptoms endorsed by aya while receiving therapy for cancer design/method: surveys were given in both inpatient and outpatient settings during cancer therapy. symptom screening in pediatrics tool (sspedi) and memorial symptom assessment scale (msas). symptoms severity was rated by teens on a point likert scale. spss , used for statistical analysis. results: : a total of aya on cancer therapy (age range - . years) % female, % male, . % acute leukemia, . % solid tumors, and . % diagnosis was not reported. % of aya on cancer therapy reported at least or more symptoms, % reported > symptoms cluster. of the physical symptoms that were reported as most distressing to the teens, mouth sores and headaches were the top causes. of the physical symptoms that were most frequently endorsed; fatigue was on the top ( %), followed by change in appetite %, vomiting %, and pain %., the least was bowel habit changes. aya rated sadness as the most frequent psychological symptom %, followed by feeling angry %, and scared %. statistically significant difference was noticed based on gender difference with more females reported symptoms (p = . ), while type of cancer (acute leukemia versus solid tumors) was not statistically different. conclusion: aya with cancer reported multiple physical and psychological symptoms with significant distress. females seem to report more symptoms compared to males. screening aya for cancer therapy related symptoms is feasible during routine visits and adds important information about the aya well-being. background: sinus histiocytosis with massive lymphadenopathy (shml), also known as rosai-dorfman disease, is a rare histiocytic proliferative disorder of unknown etiology. many treatment modalities have been employed; however, no uniform guidelines exist. objectives: literature review of treatment options for shml. design/method: chart review was performed on pediatric patients diagnosed with shml at the children's hospital at montefiore between and after irb approval. inclusion criteria included children between the ages of and years with shml. exclusion criteria included children with cutaneous shml. four cases of shml seen at montefiore are described. a comprehensive review of the literature identified additional cases published between and . manuscripts that did not include the treatment modality or outcome were excluded. results: many of the patients with shml responded to observation alone. of patients, patients were observed, with ( %) having resolution of disease, five having stable disease, and five being lost to follow-up. one patient received subsequent systemic therapy. surgical management was con-ducted upfront in patients. of those, ( %) had resolution of disease, one had stable disease, and one had recurrence with no further therapy noted. of the remaining nine patients, % were successfully treated with systemic therapy, consisting of either steroids ( ) or steroids and chemotherapy ( ). systemic therapy was used as first-line therapy in patients. steroids alone or in conjunction with chemotherapy resulted in resolution of disease in / and / patients ( / , %), respectively, with four patients having stable and three with progressive disease. chemotherapy without steroids resulted in resolution of or stable disease in / patients. radiation was ineffective. conclusion: shml is a rare disease with no published guidelines for treatment. from the results of the cases and a detailed review of the literature, it can be suggested that observation may be considered as first line management in patients providing there are no significant symptoms. for patients who are symptomatic or have significant progression, surgery may be considered. in patients with recurrence or refractory disease, steroids and/or chemotherapy may be used. the presence of nodal or extra-nodal disease did not seem to have a significant impact on the course of treatment. given the rarity of the disease, it is difficult to conduct a randomized control trial. further work, involving collaboration between centers and cooperation with the international rare histiocytic disorders registry would be helpful. boston children's hospital, boston, massechusettes, united states background: increasing census and intensified work compression on the inpatient oncology service at our institution was identified as leading to resident dissatisfaction, impaired resident learning and decreased perceived quality of patient care. objectives: to evaluate the impact of a redesign of a pediatric inpatient hematologic malignancy (ihm) service on resident perceptions of the educational value of the rotation and safety of patient care. design/method: during the - academic year, we initiated a bundled intervention on the ihm service. modifications included ) decreased patient volume: the ihm service was divided into two teams, utilizing an extra attending -a teaching service consisting of residents and fellows and a team comprised of nurse practitioners. ) intentional patient team assignment: patients were deliberately assigned to a care team based on educational opportunities and provider skill sets. ) intentional attending faculty selection: attending faculty with deeper clinical and teaching experience were selected to supervise on the teaching team. ) increased weekend staffing. after completing the service, junior residents completed an electronic survey to evaluate their perceptions of the educational value of the rotation, as well as their ability to deliver safe care while on the rotation. fisher's exact tests were used to compare responses from residents in who experienced the redesign to residents in , whose experience results: survey completion rates were % ( / ) in and % ( / ) in . intervention residents were significantly more likely than comparison group residents to choose the answers "very good" or "excellent" to describe both the overall quality of the rotation ( % intervention vs. % comparison, p< . ) and the educational experience on rounds ( % intervention vs. % comparison, p< . ). intervention residents also reported caring for fewer average primary patients daily on weekdays as compared to comparison residents ( . vs . patients, p< . , % ci - . to - . ). furthermore, intervention residents were more likely than comparison residents to "agree" or "strongly agree" that they could provide safe patient care on weekend days ( % intervention vs. % comparison, p< . ) and on nights ( % intervention vs. % comparison, p< . ) while on the oncology service. a redesign initiative of an oncology service with the development of a new teaching service led to improved resident perceptions of the educational value of the rotation and ability to provide safe care to patients. this approach could be useful to other services and institutions to promote similar outcomes in resident education and patient care. background: alk-positive histiocytosis is a rare histiocytic proliferative disorder that has been reported in three infants presenting primarily with hepatosplenomegaly, anemia, and thrombocytopenia. given the rarity of this disease, there are no standard treatment algorithms for this diagnosis and the disease course and outcomes remain largely unknown. the published series describes treatment ranging from monitoring alone to multi-drug chemotherapy regimens. there was ulti-mately resolution of presenting symptoms in all three cases despite varying treatment strategies. objectives: to report a newly diagnosed case of alkpositive histiocytosis that was treated with a novel approach using cytarabine monotherapy. results: a full term male infant presented at birth with difficulty feeding and hyperbilirubinemia. over the first few weeks of his life, he subsequently developed thrombocytopenia, transaminitis, and profound hypoalbuminemia. by six weeks of life, he was experiencing significant abdominal ascites requiring repeat paracenteses, massive hepatosplenomegaly, respiratory distress secondary to abdominal distension, anemia, and coagulopathy. he underwent numerous diagnostic tests, including a liver biopsy followed by a bone marrow biopsy that showed alk-positive histiocytic infiltrates in both sites. treatment was initiated with cytarabine mg/kg/day x days, repeating every weeks. throughout his course of five cycles of treatment, he experienced intermittent fevers and mild nausea with no other adverse events. by the end of five cycles, his hepatosplenomegaly resolved, his blood counts normalized, he demonstrated weight gain on oral feeds, and his liver enzymes normalized. he is currently months post completion of therapy and remains well with a normal physical exam and laboratory values. conclusion: treatment of alk-positive histiocytosis with lose dose cytarabine resulted in complete resolution of our patient's symptoms with minimal treatment related adverse effects, and few long-term treatment related risks. given the rarity of the diagnosis, the reporting of effective novel treatment options is important for future patient care. background: adult patients with melanoma or lung cancer harboring braf v e have benefitted from the development and subsequent approval of specific braf inhibitors. as such, delineating the subset of similarly targetable pediatric oncology patients may spur development and rational use of these inhibitors in children. importantly, other point mutations and fusions of braf may also be targetable in s of s children analogous to recent emerging data in adult cancer patients. objectives: to define the genomic landscape of known and novel braf alterations and raf fusions in pediatric malignancies and report index cases with clinical response to braf or mek inhibitors. design/method: dna was extracted from microns of ffpe sections of , tumors from pediatric (< years of age) oncology patients, and cgp was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of x for up to cancer-related genes plus introns from genes frequently rearranged in cancer. genomic alterations (ga) included base substitutions, indels, copy number alterations and fusions/rearrangements. a total of ( . %) braf-altered pediatric malignancies were identified. ( . %) harbored a single kinaseactivating braf short variant, indel, or fusion. an alteration resulting in reduced braf kinase activity was identified in ( . %) tumors while ( . %) tumors harbored multiple braf alterations, of which contained at least a single activating short variant. the remaining tumors ( . %) contained functionally uncharacterized variants. kinaseactivating braf alterations were identified in diverse tumor spectra comprised of brain tumors ( . %; subtypes), carcinomas ( . %; subtypes, with melanoma constituting % of cases), hematological malignancies ( . %; subtypes), sarcomas ( . %; subtypes), and extracranial embryonal tumors ( . %; subtypes). seventy-two ( . % of braf-altered cases) braf fusions were identified, ( . %) of which were kiaa -braf; involved the novel fusion partners: stard nl and khdrbs . seven ( . %) raf fusionpositive cases, predominantly brain tumors ( ), were identified; involved the novel fusion partners: tmf and sox . index cases of response to therapy of intracranial tumors will be presented. we describe a population of pediatric patients with targetable braf alterations predominantly enriched in primary intracranial tumors, but spanning diverse solid tumor types and hematologic malignancies. we additionally report a cohort of raf fusion-positive patients. an index case and multiple previous reports suggest raf or mek inhibitors may benefit pediatric patients with either intracranial or extracranial disease, and development of such drugs in pediatric indications is strongly warranted. background: diffuse midline gliomas (dmg) with h k m mutation, including diffuse intrinsic pontine glioma (dipg), are the leading cause of brain tumor-related deaths in children. there are no effective therapeutic strategies and the median survival remains dismal. genomic studies have identified a recurrent mutation in the majority of dmgs involving a lysine to methionine substitution (k m) in histones . and . , resulting in changes in the epigenetic landscape that dysregulate gene expression and promote gliomagenesis. panobinostat, a multiple histone deacetylase (hdac) inhibitor, was found to be one of the most effective agents against dipg patient-derived cell cultures and xenograft models in previous studies and is presently in clinical trial for dipg. hdac inhibition with panobinostat may also exhibit activity against h k m+ diffuse midline gliomas of the thalamus and spinal cord. to evaluate the effect of panobinostat as a single agent against patient-derived thalamic and spinal cord h k m+ diffuse midline glioma cell cultures and in an orthotopic xenograft murine model of h k m+ spinal cord glioma. design/method: patient-derived thalamic and spinal cord h k m+ diffuse midline glioma cell cultures were treated with single agent panobinostat at a range of concentrations. cell viability was evaluated using the celltiter-glo assay. panobinostat was systemically administered to orthotopic xenograft murine models of luciferase-expressing spinal cord h k m+ diffuse midline glioma. response to panobinostat was evaluated with ivis in vivo imaging. results: hdac inhibition with panobinostat significantly decreases cell proliferation with an ic of nm and nm in the spinal cord and thalamic glioma patient-derived cell cultures respectively. panobinostat slowed tumor growth in murine models of spinal cord glioma by . -fold in the brain (p = . , n = ) and -fold in the spinal cord (p = . , n = ) when compared to vehicle controls after week of administration. panobinostat is in clinical trials for dipg. this study suggests that hdac inhibition with panobinostat may also be beneficial for patients with thalamic and spinal cord diffuse midline glioma h k m mutants. background: brain tumors are the most common solid tumor of childhood and the leading cause of childhood cancer deaths. while medulloblastoma is the most common malignant brain tumor of childhood with a -year survival - %, children with high-grade gliomas (hggs) such as glioblastoma multiforme (gbm) fare much worse with a -year survival of - %. implicated in this poor outcome is the presence of treatment resistant brain tumor stem-like cells. gbm stem-like cells (gscs) have been implicated in tumor growth, treatment resistance and patient relapse, making them a key therapeutic priority. antipsychotic drugs (apds) have been used for decades in various psychiatric clinical settings and are associated with a lower incidence of cancer, including malignant brain tumors. currently, atypical apds are being evaluated for their potential to alleviate cancer and treatment induced side effects. furthermore these drugs may have direct anti-tumor effects, potentially via inhibition of dopamine d receptors (drd ). objectives: determine the anti-cancer effects of atypical apds on gbm stem-like cells design/method: the anti-cancer effects of apds (quetiapine and risperidone) were evaluated on gbm stem-like cell lines developed in our laboratory (glio and ) and the group medulloblastoma cell line hdmbo . cell proliferation/viability was determined using trypan blue exclusion and mts assays. the effect of apds on cancer stem cell self-renewal was determined by neurosphere assay. receptor expression and apds effect on cell cycle proteins were examined by western blot analysis. results: western blot analysis of gscs and hdmbo demonstrated robust drd expression indicating a viable therapeutic target. both apds induced dose dependent cell death of all cell lines tested. treatment with only um of either apd for days significantly reduced cell proliferation by % (hdmbo ) and - % (gscs). consistent with these findings, we observed an increase in cell cycle inhibitors p and p . furthermore at day both apds induced a robust increase in gsc death, approximately % compared to only % in non-treated controls. lastly, um apds significantly reduced gsc neurosphere formation compared to untreated controls by up to % suggesting inhibition of gbm stem cell self-renewal. our data indicates that clinically relevant concentrations (low micromolar) of these apds induce anticancer effects in both gscs, which are enriched with tumor initiation/propagation properties, and in the group (myc amplified) medulloblastoma cell line. these apds represent strong candidates as potential adjuvant therapies for the treatment of these brain tumors. background: while the poor prognosis for high risk neuroblastoma (hrnb) underscores the need for new treatment strategies, the elucidation of specific biologic subsets of neuroblastoma suggests a way to improve disease management. the identification of agents that target specific molecular pathways associated with the development or progression of diseases holds promise. dfmo, an inhibitor of odc, has been shown to decrease lin and mycn and target cancer stem cells in preclinical studies. currently % of patients undergoing immunotherapy relapse. dfmo is in studies to prevent relapse after immunotherapy and may be helpful during immunotherapy as well. the hypotheses for this study were that: ) the incorporation of a targeted therapy, selected based upon upfront tumor genomic interrogation, into standard induction chemotherapy for hrnb is safe, feasible and may increase the pr/cr/vgpr response rate at the end of induction therapy; and ) the addition of dfmo as maintenance during immunotherapy is safe and feasible and may decrease the relapse rate for hrnb. a multicenter feasibility pilot trial in subjects with newly diagnosed hrnb within the beat childhood cancer consortium. at diagnosis, patients' tumors underwent dna exome and rna sequencing which were analyzed within a molecular tumor board to identify the single best drug of targeted agents to be added to cycles - of induction chemotherapy. after consolidation with asct and radiation, the patients received dfmo along with standard dinutuximab and retinoic acid and dfmo for years after immunotherapy. patients were evaluated for additional toxicities with the addition of targeted agents and dfmo in addition to induction response. results: the pilot study of eligible patients has shown this process to be feasible. all patients have completed induction portions of the study. the combination of targeted agent with chemotherapy was shown to be safe without any unexpected toxicities. delays between induction cycles were < weeks and related to surgery, infection, or thrombocytopenia. the induction response demonstrated % cr/vgpr/pr rate, which suggests improvement over historical %. in addition, patients were eligible for the combination of dfmo with dinutuximab and retinoic acid was well tolerated and safe without additional toxicities due to dfmo. the pilot study of patients has shown the process of genomic sequencing and addition of a targeted agent to upfront chemotherapy and addition of dfmo to dinutuximab and retinoic acid maintenance therapy in newly diagnosed hrnb patients and is feasible and safe without any unexpected toxicities. background: identifying sub-populations of medulloblastoma tumors with stem cell-like properties holds promise for reducing disease recurrence, but there is no known unifying marker of medulloblastoma cancer stem cells. the granulocyte stimulating factor receptor (gcsf-r or cd ) is well understood in the context of hematopoiesis, but its role in solid tumor pathogenesis is less clear. neuroblastoma and melanoma subpopulations expressing gcsf-r have cancer stem cell properties of chemoresistance and increased tumorigenicity, and are enriched in tumors after chemotherapy. gcsf-r activation leads to signaling through the jak-stat pathway, suggesting a potential therapeutic target. we hypothesized that a subpopulation of medulloblastoma cells would express the gcsf-r and that this subpopulation would demonstrate chemoresistance and response to inhibitors of the jak/stat pathway. objectives: our objective was to identify a subpopulation of medulloblastoma cells expressing the gcsf-r and determine their relative growth rates, tumorigenicity, and responses to chemotherapy and jak/stat inhibition. design/method: medulloblastoma cell lines were sorted via flow cytometry for gcsf-r surface expression. subpopulations of gcsf-r-positive and -negative medulloblastoma cells were then monitored for growth by continuous live cell imaging. responses to chemotherapy were measured in subpopulations of gcsf-r-positive and -negative medulloblastoma cells using continuous live cell imaging to measure percent cell confluence and cell viability assays. ic values were calculated for each cell line and each agent. parental medulloblastoma cell lines and isolated gcsf-r-positive and -negative subpopulations were also treated with the jak / inhibitor ruxolitinib and growth rates, viability, and ic values were calculated. results: gcsf-r surface expression was identified on . - . % of medulloblastoma cell lines. isolated gcsf-r positive cells demonstrate a slower growth rate compared to gcsf-rnegative or parental unsorted medulloblastoma cells. gcsf-r positive cells are more resistant in vitro to vincristine, etoposide, and carboplatin, when compared to the gcsf-r negative population and an unsorted population of the same cell line. ruxolitinib is cytotoxic to medulloblastoma cells in vitro, with higher ic values noted in gcsf-r positive cells compared to unsorted and gcsf-r negative cells. we show that a subpopulation of gcsf-r positive cells are present in multiple medulloblastoma cell lines via flow cytometry, and that isolated gcsf-r-positive cells have a slower growth rate than gcsf-r-negative or unsorted populations. we also show that ruxolitinib has in vitro activity against medulloblastoma cell lines. we propose that jak inhibition may represent an adjunct therapy targeting overall tumor burden and specifically targeting the gcsf-r-positive subpopulation of medulloblastoma cells that may drive tumor recurrence. we investigated the efficacy of intensified adjuvant chemotherapy in osteosarcoma patients. design/method: we retrospectively analyzed the medical records of children with osteosarcoma treated at asan medical center between and . all patients received a -drug induction consisting of cycles of cisplatin and doxorubicin along with cycles of methotrexate (map), and proceeded to surgical resection. adjuvant ct was map or map with the additional ifosfamide and etoposide (mapie), and mapie was mainly considered for poor responders (tumor necrosis below %) or patients with metastases. results: among patients, patients had metastases at diagnosis. surgery was conducted in patients who responded to induction ct, and showed over % tumor necrosis. among patients who proceeded to adjuvant ct, and patients received to map and mapie protocols. with a median follow-up of months, the -year overall survival (os) and event-free survival (efs) rates of all patients were % and . %. of those patients, patients recurred, and of them died of disease progression. relapsed patients received salvage ct and/or surgery, and were rescued after autologous stem cell transplantation (sct). three patients developed treatment-related acute myeloid leukemia, and they are alive after allogeneic sct. according to the response to neoadjuvant ct, the os rates of good responders (n = ) and poor responders (n = ) were % and . % (p = . ), and efs rates were . % and . % (p = . ). of the poor responders, patients received map as adjuvant ct, and the other received mapie. the os rates of map and mapie group were . % and . % (p = . ), and efs rates were . % and . % (p = . ), respectively. when patients were classified into three groups: . localized disease & necrosis ≥ % (n = ), . localized disease & necrosis < % (n = ), . metastatic disease (n = ), survival rates were in the order of group > > (os = %: . %: . %, efs = . %: . %: %). in each group, intensified adjuvant ct by mapie did not improve survival outcomes. conclusion: initial metastatic disease and poor histological response to neoadjuvant ct were major risk factors for poor survival in osteosarcoma patients. we found that adding ifosfamide and etoposide to map did not improve survival outcomes of patients with adverse risk factors. more effective adjuvant therapy for these patients is needed. background: circulating cell-free dna (cfdna) that shed from tumors into circulation have been used for noninvasive molecular profiling in adult cancers but little is known about its utility in pediatric cancers. pediatric patients with metastatic and refractory solid tumors are known to have poor survival rates, and a key challenge in their management is obtaining biopsy samples especially at times when disease is widely spread or the patient is physically unfit for sampling. the development of a noninvasive profiling strategy is critical for optimizing molecularly guided therapy and assessing response to treatment. in this study, we want to determine the utility of cfdna to noninvasively analyze the molecular profiles of pediatric solid tumors such as neuroblastoma (nb), osteosarcoma (os), and wilms tumor (wt). design/method: tumor, plasma, and matched controls were collected from patients with nb, wt, and os, at diagnosis or time of disease progression. cfdna was extracted from the plasma and analyzed through multiple methodologies including a targeted next generation sequencing panels and shallow whole genome sequencing (swgs). results: fifteen nb patients, os patients, and wt patients had tumor molecular profiles known from different targeted next-generation sequencing platforms. in the cfdna of / nb patients, somatic mutations and copy number alterations previously reported in the tumors were detected, including recurrent nb drivers such as mycn amplification, alk, and atrx mutations. mutations not detected in the original tumor were also found in / nb patients including nras, mll , arid b, some of which are potentially actionable. in os, mutations known from the tumor were found in the cfdna of of patients, including atrx and notch mutations, as well as copy number alterations such as cdk amplification, which has targetable therapeutics available. of the two wt patients analyzed, cfdna revealed the same mutations as tumor in one patient, however in a cohort of patients where tumor was not available, cfdna revealed recurrent driver mutations such as amer , dicer . it is feasible to noninvasively identify somatic mutations and copy number alterations in cfdna of patients with pediatric solid tumors. establishing a platform using cfdna to identify molecular profiles of these tumors can serve as a powerful tool for guiding treatment and monitoring response to treatment. background: despite multi-modality therapy, the prognosis for patients with metastatic osteosarcoma remains poor necessitating development of novel targeted therapies. immunotherapy can be exploited to target osteosarcoma with exquisite specificity but remains limited by insufficient tumor specific targets. objectives: to overcome the dearth in tumor specific antigens, we have explored the use of tumor derived mrna (representing a tumor specific transcriptome) for development of personalized nanoparticle vaccines. design/method: rna-nanoparticles (rna-nps) can be amplified from limited amounts of biopsied tissue for induction of tumor specific t cells against osteosarcoma. since local vaccination strategies are mired by poor overall immunogenicity, we assessed the feasibility, immunogenicity and antitumor activity of intravenously administered rna-nps (tumor mrna complexed to dotap nanoliposomes) in pre-clinical murine and canine tumor models. we identified a clinically translatable np formulation for the delivery of rna to antigen presenting cells (apcs) that induces in vivo gene expression and preserves rna stability over time. tumor derived rna-nps induced antigen specific t cell immunity and mediated anti-tumor efficacy in several pre-clinical solid tumor models (i.e. b f , kr b). when administered intravenously, rna-nps increased expression of co-stimulatory molecules (i.e. cd , cd , cd , ccr ) and pd-l on cd c+ cells throughout reticuloendothelial organs (i.e. spleen, liver, bone marrow) and within the tumor microenvironment; this phenotype was strictly dependent on type i interferon. targeted inhibition of type i interferon signaling (via infar mabs) abrogated anti-tumor efficacy mediated by rna-nps. we enhanced the immunogenicity of this platform by simply combining mrnas encoding for immunomodulatory molecules (i.e. hcv-pamps, gm-csf) or by combining rna-nps with immune checkpoint inhibitors. addition of checkpoint inhibitors (pd-l mabs) to rna-nps increased tumor infiltrating lymphocytes, and intratumoral mhc class i/ii expression, and mediated synergistic anti-tumor activity in settings where pd- or pd-l inhibition alone did not confer therapeutic benefit. we then explored the feasibility of rna-nps in a large animal osteosarcoma model. in ongoing studies for canines with osteosarcomas, we have shown that sufficient amounts of rna can be extracted, amplified, and manufactured into personalized rna-np vaccines. conclusion: rna-nps reprogram systemic immunity and mediate anti-tumor activity providing near immediate immune induction without the complexity of cellular immunotherapy. the immune correlate of preclinical response to rna-nps is hallmarked by interferon dependent pd-l expression on activated apcs (cd c+ mhcii+ cd + cells). based on these findings, we are exploring the preclinical safety, efficacy and immunologic effects of rna-nps targeting canine osteosarcoma before first in-human evaluation. background: ewing sarcoma is an aggressive bone tumor affecting mainly adolescent and young adults. treatments are based on compressed schedule chemotherapy combined with local control (surgery and/or radiation). prognosis is poorer for patients with metastatic disease, older age and central primaries. survival when disease recurs within two years of diagnosis is < %. the ews-fli fusion gene t( ; ) (q ; q ) has been well characterized as a dominant ews driver-gene. the most common variation is ews exon with fli exon ( % of fusion positive patients). we designed a novel pbi-shrna tm ews/fli type lpx which has demonstrated, safety and efficacy in animal model (rao et all). the pbi-shrna strategy silences target gene expression by concurrently inducing translational repression and p-body sequestration as well as post-transcriptional mrna cleavage. to determine the safety and maximum tolerated dose of intravenous administration of pbi-shrna tm ews/fli type lipoplex in patients advanced ews. design/method: phase i study × escalation cohort. testing pbi-shrna tm ews/fli type lpx (starting iv dose of . mg/kg) on patients (≥ age ) with advanced ewing's sarcoma, all with a type translocation. intravenous infusion was given twice a week for weeks with the following escalation schema: % → % → % → % → %. required kps > % and adequate organ function. cytokines induction pre and post-infusion was analyzed (il- , il- , tnf-alpha, il ra). first cohort of patients has been enrolled (ages between - years). three relapsed patients had > lines of therapy and patient had refractory disease, patients received a complete cycle of pbi-shrna tm ews/fli type lpx with twice a week infusions. a total of doses were given. the most prominent related toxicity has been hematological, patient developed transient g neutropenia, another patient developed g anemia that required prbc transfusion, and of note this patient had significant bone and bone marrow involvement. one patient only received two lpx infusions; she developed a fatal rsv pneumonia. other reported grade toxicity includes fatigue and headache. evaluable patients (n ) had stable disease between and months before progression. one patient had sustained response for month before progression, two patients are still alive. our preliminary experience supports the safety and potential efficacy of pbi-shrna tm ews/fli type lpx as novel treatment for advanced ews with limited toxicity. il- increase correlates with higher bi-shrnai ews/fli lpx infusion rate and clinical symptoms. further clinical testing is indicated. background: as more children with cns malignancies (bt) are surviving, the late effects of the therapies they receive are better described. studies show that radiation therapy is particularly harmful to neurocognitive functioning, specifically processing speed, working memory, and attention span. these deficits have negative effects on quality of life, especially in academic and professional settings. a large proportion of s of s adult survivors of bt are unable to reach adult milestones such as living on their own, holding a steady job, and getting married. proton beam radiation therapy (pbrt), is touted for the potential to have fewer and less severe side effects than traditional photon radiation therapy (xrt). because of the properties of protons, the amount of damaging energy released in non-target healthy tissue is reduced when compared to xrt. although a study comparing iq testing between pbrt and xrt found no difference between the two therapies, no studies have compared the specific neurocognitive domains. it would be valuable to evaluate full neurocognitive testing scores (nct) since the specific domains, particularly processing speed (psi), appear to be most vulnerable to radiation therapy. objectives: our primary aim was to assess differences in psi for patients with bt who underwent pbrt versus xrt. a secondary aim was to assess differences in iq (fsiq) and working memory (wmi). we retrospectively evaluated all patients treated for bt at the jimmy everest cancer center within the past years who received rt and had nct post radiation. we examined the full nct results for both subsets of participants to evaluate differences in the specific domains of processing speed, working memory, and iq by measuring percentiles scored in these domains. objectives: we report our experience on imaging children with mm treated uniformly on an institutional melanoma trial. we retrospectively reviewed the clinical and imaging findings of patients with ajcc stage iic-iv cutaneous mm treated on our institutional mel protocol. brain mri/ct, pet/ct, ct chest, abdomen, and pelvis (ctcap) were performed at diagnosis in all patients. on treatment, stratum a patients (peg-interferon; ajcc iic, iiia, iiib) (n = ) had the same imaging repeated every months; stratum b (peg-interferon and temozolomide; unresectable measurable disease metastatic, or recurrent) (n = ) had pet scans every months and brain imaging every months; those in stratum b (peg-interferon and temozolomide; unresectable non-measurable, metastatic, or recurrent) (n = ) had the same imaging performed every months. off therapy all patients continued same imaging every months for years. results: there were patients ( female; median age years). eleven had spitzoid and conventional melanoma. primary sites included head/neck (n = ), trunk (n = ), and extremities (n = ). patients with spitzoid melanoma had imaging studies ( pet, ctcap, ct chest, ct brain, and mri brain) with a median of , , , and studies/patient respectively. median cost per patient was $ , . thirteen studies ( . %) showed suspicious lesions with additional scans and diagnostic biopsies of which one only was positive stratum a with tert promoter mutation and died from disease). for conventional mm, studies ( pet, ctcap, ct chest, ct brain, and mri brain) were performed with a median of , . , , , studies/patient respectively. median cost per patient was $ , . twenty ( %) showed suspicious lesions with additional scans and diagnostic biopsies; four were positive (two at diagnosis); both died of disease; the other two recurred locoregionally and were detected clinically; both are alive and disease free; one patient had diffuse metastases and died shortly after enrollment. after a median follow up of . years (range . - . ) patients are alive and disease free. children with spitzoid melanoma should have minimal imaging at diagnosis and follow-up given the low risk of recurrence and low yield and high cost of aggressive imaging protocols. patients with conventional mm should be imaged according to the adult guidelines. nationwide children's hospital, columbus, ohio, united states background: the role of infections in the long term outcome of patients with bone tumors is controversial. two retrospective studies have shown increased survival in osteosarcoma patients who had a post-operative wound infection, while another showed no changes in overall survival. to determine the relationship between wound infections and/or bloodstream infection (bsi) on survival in pediatric and young adult patients with osteosarcoma and ewing sarcoma treated at a tertiary children's hospital. design/method: a retrospective chart review was performed for patients with diagnosis of osteosarcoma or ewing sarcoma from - . patients received standard chemotherapy regimens for their disease type and stage. local control included surgical resection and/or radiation therapy. presence of infection was determined by bsi or wound cultures while receiving treatment for primary tumor. the median age of patients was (range - years) at diagnosis. % had a diagnosis of osteosarcoma and % had ewing sarcoma. of these, % of patients developed an infection during treatment; % had bsi, % had wound infections, and % had both. patients with bsi had a year os of . %, compared to % in those without bsi (p = . ). those with both bsi and wound infections had the poorest overall survival of %, compared to . % for patients without any infection. patients with wound infections alone had a year os of . %, compared to % of patients without a wound infection. our analysis revealed decreased os in patients with bsi; however, this could be due to other confounding factors in the presence of bsi. those with bsi or bsi and wound infections had the poorest survival. wound infections without bsi were associated with a slight increase in survival; however, this study was limited by the number of patients that had local wound infections. with the use of newer surgical techniques, availability of antimicrobials and routine use of prophylactic antibiotics, the incidence of infections while undergoing treatment is low. however, the importance of this clinical observation indicates a likely enhanced immune system associated with infection, supporting the role of immunotherapy for treatment of these aggressive tumors. background: hypoalbuminemia is a well-recognized effect of cancer and other chronic illnesses and is often regarded as a marker of malnutrition. in adults, hypoalbuminemia has been associated with adverse outcomes in patients with cancers of the lung, pelvis, head and neck, gastrointestinal tract, and bone marrow, as well as in some pediatric patients with ewing sarcoma and hodgkin lymphoma. hypoalbuminemia has not been well studied in children with cancer. to determine the incidence of hypoalbuminemia (using age-specific references) in children with cancer receiving chemotherapy at baseline (prior to starting chemotherapy) and to determine whether hypoalbuminemia is associated with inferior -year overall survival. design/method: we performed a single institution, irbapproved, retrospective review of pediatric oncology patients diagnosed between and . five-year survival was estimated using the kaplan-meier method; groups were compared using cox regression. we identified pediatric patients with a first diagnosis of cancer, brain tumor, or other condition possibly requiring chemotherapy. of these patients, were excluded for reasons including not receiving chemotherapy and missing data, leaving patients who had a serum albumin level within days prior to starting chemotherapy. the mean age was . years (sd . years); % were male; % were non-hispanic. the most common diagnosis was acute lymphoblastic leukemia ( of ; %). one hundred thirty nine of ( %) had hypoalbuminemia prior to starting chemotherapy. there was no statistically significant difference in -year overall survival between those with and without hypoalbuminemia ( % vs. %, respectively; hazard ratio . , % c.i. . - . ). conclusion: hypoalbuminemia at baseline in pediatric oncology patients requiring chemotherapy is common (one in five), and was not associated with inferior -year overall survival in this cohort. leptomeningeal metastases at diagnosis. standard treatment for completely resected, non-anaplastic supratentorial ependymomas is close observation. treatment for anaplastic or incompletely resected non-anaplastic ependymomas is maximal safe surgical resection followed by focal radiation. however, up to % of localized ependymomas recur. the role of chemotherapy in treating ependymomas is under investigation. extraneural metastases of anaplastic ependymomas have rarely been reported and the outcome is dismal. objectives: to report extraneural cervical node metastases of a non-anaplastic ependymoma and successful treatment with surgical resection, radiation, and systemic chemotherapy. design/method: retrospective review of patient medical records, including radiographic imaging and tumor tissue pathology, and comprehensive literature review. results: a previously healthy -year-old girl underwent gross total resection (gtr) of an isolated right parietal lobe ependymoma (who grade ii). at age years, magnetic resonance imaging (mri) revealed an isolated localized recurrence. she underwent gtr followed by observation. at age years, she again experienced isolated localized recurrence and underwent gtr followed by . gy focal conformal photon radiation. at each recurrence, pathology revealed a non-anaplastic ependymoma, and cerebral spinal fluid (csf) cytopathology and spine mri were negative. at age years, she developed an enlarged right posterior cervical chain lymph node. subsequent mri revealed a large rim-enhancing, t hyperintense lymph node and multiple abnormally enhancing regional nodes consistent with metastases. biopsy revealed a non-anaplastic ependymoma. mri of the brain and spine, computed tomography of the chest, abdomen, and pelvis, and csf and marrow evaluations were unremarkable. chemotherapy according to acns was initiated. mri after course demonstrated significant node size reduction. she underwent right neck node dissection. only one right level ii lymph node showed metastases. she was treated with . gy irradiation to the neck and additional courses of chemotherapy. she remains in remission months and months after diagnosis of metastatic disease and end of therapy, respectively. literature review reveals rare reports of extraneural metastatic disease of anaplastic ependymomas to bone, lung, or liver, and only involving lymph nodes, all associated with a poor outcome despite multimodal therapy. to our knowledge, this is the first report of extraneural metastases of a non-anaplastic ependymoma. extraneural metastases should be considered in children previously treated for non-anaplastic ependymomas who experience systemic symptoms, even in absence of cns relapse. multimodal treatment offers potential long-term disease control with acceptable toxicity. arun gurunathan, joel sorger, andrew trout, joseph pressey, rajaram nagarajan, brian turpin cincinnati children's hospital medical center, cincinnati, ohio, united states background: pigmented villonodular synovitis (pvns) is a benign neoplasm of the synovium. standard treatment is surgery, but post-operative recurrence rate is as high as %. radiation therapy can be used for local control, but is associated with late effects. while pvns is rarely fatal, aggressive disease and/or extensive surgery can result in substantial functional impairment. colony stimulating factor- (csf ) overexpression, often due to chromosomal translocation involving csf , drives pvns through recruitment of synovial-like mononuclear cells expressing the csf -receptor. tyrosine kinase inhibitors such as imatinib are active against the csf -receptor, and have shown benefit in the post-surgical relapse setting. however, questions remain regarding the broader application of imatinib and regarding optimal response assessment. to present three patients with pvns, each with different clinical scenarios, who demonstrate clinical response to imatinib monitored by changes in metabolic activity (maximum suv) on pet/ct. results: three patients with pvns demonstrate pet/ct response to imatinib, guiding management of their challenging clinical scenarios. patient is a year-old female with left hip pvns and high grade articular cartilage loss, with decrease in metabolic activity (suvmax . to . in months) on neoadjuvant imatinib, enabling total hip replacement surgery planning. patient is a year-old female with left knee pvns with recurrences after synovectomies, spared subsequent surgical control attempts after clinical improvement correlating with pet/ct response to imatinib (suvmax . to . in months). patient is a year-old male with right knee pvns that recurred after total knee replacement, now with clinical improvement correlating with pet/ct response to imatinib (suvmax . to . in months). all patients would have been characterized as stable disease by response evaluation criteria in solid tumors (recist). in each of these patients, imatinib has been tolerated well, with no therapy interruptions and absent or easily managed side effects (one patient takes dronabinol for decreased appetite, one patient takes prn immodium for diarrhea). all patients are currently still taking imatinib, with therapy length ranging from five to eleven months. in our series of three patients with pvns, imatinib shows promise for disease management in neoadjuvant and adjuvant settings with a tolerable side effect profile. imatinib should be considered in the treatment of pvns to spare surgical and radiotherapy related morbidity, and treatment effect can be monitored by pet/ct. background: metastatic rhabdomyosarcoma (rms) carries a poor prognosis with three-year event free survival rates ranging between %- % (depending on oberlin risk factors) due to the lack of significantly effective breakthroughs in the recent past. there is an urgent and unmet need for new treatment strategies against this disease. metastatic rms cell lines exhibit increased expression of the erm family membrane-cytoskeleton linker protein ezrin. knockdown of ezrin expression using sirnas decreases the metastatic potential of these cells, whereas forced expression of ezrin results in increased degree of metastasis. the activity of ezrin is controlled by its phosphorylation at the threonine (thr ) residue at the c-terminus of the protein, suggesting that alteration of ezrin phosphorylation may control rms growth and metastasis. our goal was to determine if pharmacological inhibition of thr phosphorylation in ezrin affects the growth, survival and metastasis in rms in vitro as well as in vivo. design/method: rms cell lines representative of the alveolar and embryonal histological subtypes were used. rms cells were treated with a small molecule inhibitor of ezrin, nsc , which specifically dephosphorylates ezrin at the thr residue. baseline expression of ezrin and perm levels as well as the effect of nsc on perm levels in the rms cell lines was determined by western blotting of cell lysates. viability of cells was assessed by trypan blue exclusion, and morphology visualized by bright field microscopy. the extent of apoptosis was detected by imaging caspase / activation using fluorescent microscopy. motility of rms cells was examined by performing a wound-healing assay. subcutaneous and orthotopic xenografts were established in nsg mice using rd cells (embryonal rms). mice harbor-ing xenografts were treated with intraperitoneal injections of nsc or dmso. results: ezrin is constitutively phosphorylated at the thr residue in a majority of the rms cell lines examined. nsc dephosphorylates ezrin at the thr residue in these cell lines. treatment with nsc inhibits growth, induces apoptosis and inhibits the migration of rms cell lines in vitro. further, treatment of nsg mice bearing subcutaneous or orthotopic embryonal rhabdomyosarcoma xenografts with nsc significantly impedes tumor progression without any obvious adverse effects. our findings suggest that dephosphorylation of ezrin at the threonine residue may have the potential to be a novel therapeutic strategy for rms patients. all india institute of medical sciences, new delhi, new delhi, delhi, india background: the role of laparoscopy in the management of pediatric intra-abdominal solid tumors is yet to be established. the safety of laparoscopic management of pediatric intra-abdominal tumors is still questionable. we study the results of the initial case series of pediatric intraabdominal tumors managed laparoscopically at our institute from july onwards. design/method: total children ( -males, females) who presented to us with pediatric intra-abdominal tumors were included. the tumors included wilms tumor (n = ), neuroblastoma(n = ), adrenal cortical tumor(n = ), ovarian teratoma(n = ).children were between months - years and received neo-adjuvant chemotherapy. a -port laparoscopic nephrectomy and lymph node sampling for wilms tumor and adrenalectomy for adrenal tumors was performed. the tumors were removed in-toto with no rupture (except in one). specimens were retrieved through a lumbar incision (n = ) or an inguinal incision(n = ). all the children are under regular follow up. two children with wilms tumor had recurrence. the neuroblastoma child underwent open surgery for recurrence later. conclusion: laparoscopy/laparoscopic assisted removal of pediatric intra abdominal tumor is a feasible and safe option. it has the advantage of less postoperative pain, shorter hospital stay and a better cosmetic result. proper patient selection, port placement and laparoscopic experience are contributory. background: targeting of proteins and cell surface antigens specific to cancer cells with monoclonal antibodies has proven to be an effective form of treatment in many forms of cancer. gd is a cell surface disialoganglioside that is expressed on the cell surface of some normal tissues including nerve cells, melanocytes, and mesenchymal stromal cells and is overexpressed in some pediatric cancers like neuroblastoma and osteosarcoma. dinutuiximab is a chimeric monoclonal antibody that is fda approved for the treatment of patients with high risk neuroblastoma and under investigation for the treatment of relapsed osteosarcoma. little is known about the patterns of gd expression in other pediatric malignancies. objectives: we sought to describe the patterns of gd expression in the following pediatric sarcomas: synovial sarcoma, rhabdomyosarcoma and ewing sarcoma. design/method: synovial sarcoma (n = ), rhabdomyosarcoma (n = ) and ewing's sarcomas (n = ) formalin fixed, paraffin embedded cores were obtained from the seattle children's research institute tissue microarray (tma) biorepository. tma blocks consisting of melanoma cores stained with and without gd antibody were used as positive and negative controls, respectively. slides were incubated with anti-ganglioside gd antibody clone q (ab from abcam) diluted : in % normal goat serum and % bsa in tbs overnight at ˚c. the negative control of human melanoma section was incubated in % normal goat serum and % bsa in tbs without primary antibody. the expression of gd was indicated by characteristic brown diaminobenzidine staining. the intensity and location of tissue staining were assessed and compared to positive and negative controls. staining was considered positive (+++) if the intensity of the staining was consistent with that of the positive control with - % of cells staining positive. classification of intermediate gd expression (++) was assigned to slides in which - % of cells stained positive. slides were classified as sporadic staining (+) if - % of cells stained positive. tissue was considered (-) if there was complete absence of staining, similar to the negative control. objectives: to evaluate the clinical presentation, management and treatment outcomes of children with malignant germ cell tumor at our institute design/method: a prospective study was conducted from june to dec in the department of pediatric surgery in a tertiary care institute in a developing country. all patients were evaluated for local disease and metastatic disease by imaging and tumor markers. risk stratified chemotherapy was used with low risk tumor receiving no chemotherapy, intermediate risk: courses of peb chemotherapy and high risk: courses of peb + courses of pe. upfront resection of the primary or the residual disease after neoadjuvant chemotherapy if feasible was performed. follow up was done with monthly tumor markers for months and imaging studies every - months for initial years. five year overall survival and disease free survival was calculated. results: during the study we treated children who formed the study group. of these ( %) were gonadal ( ; % testicular and ; % ovarian) and the remaining ( %) were extragonadal with sacrococcygeal (sct) being the most common site ( %). one hundred and thirteen children ( %) presented to us primarily while the remaining had received treatment elsewhere. stage or stage disease at presentation was present in ( %) children. recurrence was noted in ( %) patients. respectively. patients with testicular mgct and children with age - years and males had significantly poor rfs rates. conclusion: patients with mgct should be staged correctly and adjuvant chemotherapy is advisable to all patients except stage i endermal sinus tumor of testis. awareness regarding the same is still lacking in our country. meticulous follow up is needed as more than % of will recur. cure rates are dismal in children with recurrent mgct especially those who are not chemotherapy naïve. nemours children's specialty care, jacksonville, florida, united states background: radiotherapy for pediatric head and neck tumors often results in mucositis, limiting oral intake and compromising patients' nutritional status. this may be reduced through the improved conformality offered by proton therapy. despite widespread use of enteral tube feeding through a percutaneous gastrostomy (peg) or nasogastric tube (ngt), there is little data available regarding overall incidence of ngt/peg placement and perspectives of pediatric patients and caregivers. objectives: to (a) estimate the need for ngt/peg support and (b) characterize patient and caregiver perceptions surrounding enteral feeding in children with head and neck tumors undergoing proton therapy. design/method: dependent on development stage, patient (n = ) or parents (n = ) filled out a series of customized surveys according to a prospective irb approved study. seventythree percent of patients also received concurrent chemotherapy. questions addressed their current feeding route and perception, for example, "what aspect(s) of tube feedings are beneficial to you?" and "what aspect(s) of tube feeding worry or scare you?" fifty-five surveys were distributed before and after radiation, and with any change in feeding route. results: at the start of proton therapy, patient had a ngt and patients had peg. of these, patients ( %) had a ngt/peg in place exclusively for the administration of medication; only patient ( %) needed a ngt/peg for nutrition. in those patients without ngt/peg, % would "consider" enteral feeds. in patients without ngt/peg, the most commonly cited benefit was "maximizing my nutrition" ( %) and the most common negative aspect was "fear" of tube placement ( % of patients). all sub-populations ( % of patients) cited change in appearance as a negative aspect. in patients without ngt/peg at the start of proton therapy, % of patients/caregivers felt enteral feeding to be "unnecessary," and % of these patients would not "consider" ngt/peg even if their "physician advised it." over the course of proton therapy, the patients/caregivers who deemed enteral feeding "unnecessary" decreased from % to %. at completion of treatment, patients ( %) were using a ngt/peg tube for nutritional support but only one ( %) patient relied exclusively on their enteral feeds. two patients (without ngt/peg) ( %) required parenteral support. our data does not support prophylactic placement of ngt/peg in of children with head and neck tumors undergoing proton therapy. ongoing research is needed to identify which patients will need ngt or peg to supplement their diet. in this cohort, anticipatory counseling should focus on pain, cosmesis, and utility. children's national medical center, washington, district of columbia, united states background: ovarian sex cord-stromal tumors (osct) are rare neoplasms that typically present with signs/symptoms of an adnexal mass and signs of hormonal production approximately % of ovarian sex cord-stromal tumors in children are sertoli-leydig cell tumors (slct) with median age of presentation years overall. to our knowledge the youngest reported case in the literature describes a -month old female in china with a slct that was treated with oophorectomy alone. some studies have found an association in families between pleuopulmonary blastoma and osct with a germline mutation leading to dicer syndrome, which has been associated with a younger age at diagnosis. , objectives: to describe an unusual case presentation of slct in an infant results: -month old, twin female, ex- week premature infant presented to the emergency department on multiple occasions for abdominal distention and feeding intolerance initially thought to be related to previous omphalocele repair and umbilical hernia. an ultrasound demonstrated an × cm mass arising from the right ovary with large volume ascites. she required admission to the intensive care unit due to s of s respiratory distress from her significant ascites. serum tumor marker including hcg, afp and ldh were negative. patient underwent right oophorectomy with tumor capsule noted to be open at time of surgery. further imaging post operatively demonstrated no other sites of disease. the patient was classified as figo stage ic due to the presence of her significant abdominal ascites that was presumed to be malignant pre-operative tumor rupture. the pathological diagnosis was challenging and eventually resulted as a mixed germ cell sex cord stromal tumor with pattern of sertoli cell tumor with neuroendocrine differentiation. based on the staging of figo ic with pre-operative rupture, the decision was made to treat with a standard platinum based regimen as there is a higher incidence of relapse in stage ic patients when compared to ia treated with observation alone. our patient tolerated four cycles of chemotherapy well and end of therapy scans showed no evidence of disease. interestingly, her dicer mutation genetics performed by ion torrent tm next generation sequencing was negative in germline and tumor studies. to our knowledge, our patient is the youngest described with slct. she will continue to be followed with serial imaging alone as she had no evidence of elevated tumor markers at diagnosis. , due to young age and unusual diagnosis, she was referred to cancer genetics team. background: approximately % of patients with wilms tumor (wt) have metastatic disease at diagnosis and often have a grave prognosis. limited cell lines are available for the study of metastatic wt and long-term passaged cell lines do not always recapitulate the human condition. focal adhesion kinase (fak) is a non-receptor tyrosine kinase that controls cellular pathways involved in the tumorigenesis of pediatric renal tumors. using a novel patient-derived xenograft (pdx) model from a patient's primary wt (coa ) and matched isogenic metastatic wt (coa ), we previously demonstrated that fak is expressed and its inhibition led to decreased tumorigenicity of both the primary and metastatic pdxs. kinomic profiling is an innovative, high-throughput method used to investigate kinase signaling to identify potential therapeutic targets. to date, the kinomic profile of primary and metastatic wt has not been examined. objectives: investigate baseline kinomic differences between primary and metastatic wt and evaluate kinases upstream and downstream of fak as potential targetable therapies. design/method: cells from coa and coa were treated with pf- , (pf), a small molecule fak inhibitor. protein from cell lysates of treated and untreated coa and coa were combined with kinase buffer, atp, and fluorescently labeled antibodies and loaded into a phosphotyrosine kinase or serine-threonine kinase pam-chip® per the uab kinome core protocol. phosphopeptide substrate analysis with the pamstation® kinomics workstation (pamgene® international), pamchip® protocol using evolve software, and bionavigator v. . were used to analyze kinases upstream and downstream of fak. the primary wt had increased epha , ror sgk and decreased pdgfrb relative to the paired metastatic wt at baseline. treatment with pf increased ron, pdgfrb, p s kb, mak, camk g, vacamkl, camk d, ck a and pskh in the primary wt. treatment with pf decreased tnk , lmr , cck , epha , pdk , sgk , lkb and increased pskh in the paired metastatic wt. primary wt displayed a different kinomic profile compared to metastatic wt in a matched isogenic pdx model. these data reveal that alternative therapies to specifically target metastases are needed. furthermore, fak inhibition resulted in diverse kinomic alterations between primary and metastatic wt. inhibitors targeting many of these pathways, such as pdgfrb inhibitors, are currently available and potentially could be combined with fak inhibitors in the treatment of wt. the results of the current study indicate that kinases upstream and downstream of fak in primary and metastatic wt warrant further investigation. background: use of high-dose methotrexate (hd-mtx, g/m^ ) is a mainstay of standard therapy for pediatric osteosarcoma (os) in north america. in pediatric os, there is a narrow therapeutic window for hd-mtx, with decreased tumor response rate with mtx concentrations < m and decreased survival due to severe toxicity with concentrations > m. risk factors for hd-mtx toxicity have been defined in adults, including body mass index (bmi) and male gender, but such studies have not been conducted in children. we sought to examine the relationship between mtx levels and toxicities during hd-mtx infusion for pedi-atric os, thereby identifying risk factors for increased toxicity and providing a framework for therapeutic drug monitoring. design/method: this retrospective chart review included patients treated at texas children's hospital with hd-mtx as first-line therapy for os from - . data abstracted from electronic records included patient characteristics, bmi and body surface area (bsa), baseline and post-treatment laboratory values, mtx levels and hours after dose given ( h, h), hour mtx cleared (mtx < . um), grade / mucositis, myleosuppression, persistent lft elevation (ctace v . ), and % tumor necrosis. correlation between h mtx level and other covariates was summarized using descriptive statistics. we reviewed hd-mtx infusions corresponding to patients. bmi was found to significantly impact h mtx level (p< . ). female gender was also significantly associated with higher h mtx level (p< . ). percent necrosis (available in patients) was associated with h mtx levels at near-statistical significance (p = . ). h mtx level was not found to contribute to toxicities or associate significantly with mtx clearance. analysis in a larger cohort is ongoing. we have identified at least one patient factor (bmi) that significantly impacts h mtx levels and is of potential use for future modeling, as current models incorporate bsa only. our findings concord with studies in adult os in that bmi significantly impacts h mtx level but diverge in that female gender is associated with higher h levels. importantly, these data support targeting h mtx levels to ensure that minimum concentration for adequate tumor necrosis is reached. these results do not suggest that monitoring h levels would prevent toxicities, thus necessitating further characterization of any intrinsic patient factors that associate with toxicity. overall, our definition of the clinical factors that associate with h mtx levels contributes to a framework for therapeutic drug monitoring in pediatric os. children 's mercy hospital kansas city, kansas city, missouri, united states background: post consolidation immunotherapy with dinutuximab, aldesleukin (il- ), granulocyte macrophage colony stimulating factor (gmcsf) and isotretinoin is standard of care for children with high risk neuroblastoma. dinutuximab is combined in alternating cycles with s of s gmcsf or il , followed by a th cycle with isotretinoin alone. il- is administered as a hour continuous infusion on days - at miu/m /day followed by a higher infusion dose, . miu/m /day, in combination with dinutuximab on days - of cycles and . the miu/m /day dose may be administered inpatient or in the ambulatory setting. objectives: to retrospectively compare the incidence of inpatient and outpatient side effects and complications associated with low dose ( miu) il to provide the tolerability data necessary to evaluate these venues for future administration options. design/method: this study was a descriptive, singlecentered definitive study utilizing a retrospective convenience sample population of children with high risk neuroblastoma who received low dose il either as an inpatient or an outpatient without exclusion from may to june . subjects were identified by a tumor registry query post irb approval. electronic and paper medical records were reviewed for the dates and location of the infusions, the home health company used if applicable and all documentation regarding clinical status, side effects and toxicity. demographics was limited to age and gender. results: infusion venue was chosen by provider preference. twenty-six infusions, inpatient and outpatient via separate home health companies were all administered in entirety and without interruption. there were males and females ranging from - years of age. two children received a single outpatient infusion due to intolerance of il when combined with dinutuximab and received therapy in both settings. fever, inpatient and outpatient was the only common side effect. no source of infection was ever identified. there was one incidence of diarrhea and one patient with pruritus in both the outpatient and inpatient settings respectively. no planned outpatient infusions required subsequent admission however the outpatient fever did necessitate an er evaluation. conclusion: low dose il can successfully be administered outpatient. the medication has minimal side effects with fever occurring in %, none of which were associated with infection. no outpatient infusion required a subsequent admission. no patients who received cycle infusions outpatient opted to receive the next cycle inpatient. baylor college of medicine, houston, texas, united states background: metastatic ewing sarcoma (es) has an extremely poor overall survival, necessitating investigations into molecular mechanisms to identify novel targets and develop new therapies. we previously performed an in vivo study, using our mouse model, designed to provide insights into transcriptomic and proteomic signatures for metastatic es to identify potential therapeutic targets. comparing profiles of primary tumors to corresponding metastatic lesions, we identified aberrant expression of integrin ß (itgb ) and downstream activation of integrin-linked kinase (ilk) in metastatic lesions compared to primary tumors, implicating this pathway as a key regulator in the ability of es to establish and enhance metastasis. our hypothesis is that upregulation of itgb and its downstream signaling events play a key role in es metastasis and are viable therapeutic targets. objectives: to investigate the role of itgb and its downstream signaling pathways in driving the establishment and enhancement of metastasis in es and to investigate this pathway as a potential therapeutic target. to investigate the role of itgb and ilk in es metastasis, we used sirna to knock down itgb and ilk expression in established es cell lines and then performed functional assays in vitro, including cell proliferation and invasion/migration assays. we also tested inhibition of this itgb signaling pathway using available small molecule inhibitors targeting itgb , ilk and the downstream target ap- , using cilengitide, compound and sr , respectively. we are currently using these small molecule inhibitors as treatment in vivo and assessing rates of metastatic tumor formation. we generated stable itgb and ilk overexpression and knockdown cell lines, which we are using for similar in vitro and in vivo investigations. knockdown of itgb and ilk in our sirna cell lines resulted in decreased cell proliferation and decreased invasion and migration compared to controls. we also found significantly decreased cell proliferation using each of the small molecule inhibitors in vitro. our preliminary studies using compound in vivo established a safety profile and dose escalation is underway to assess the effectiveness of inhibiting es metastasis. these results support our hypothesis that itgb and its downstream signaling events play a key role in the ability of es to establish metastatic foci and may serve as a potential therapeutic target. we continue to investigate this pathway in vitro. we are also using our small molecule inhibitors and itgb and ilk overexpression and knockdown approaches to study these effects on metastatic tumor development in vivo using our mouse model. background: neuroblastoma (nbl) is characterized by phenotypic heterogeneity. outcome is excellent for patients with low-(lr) and intermediate-risk (ir) disease, whereas only % of high-risk (hr) patients will survive. -hydroxymethylcytosine ( hmc) is an epigenetic marker of active gene transcription, and hmc profiles are prognostic in many types of adult cancers. we hypothesized that hmc profiles will serve as robust biomarkers in children with nbl tumors, refining current risk stratification. objectives: analyze genome-wide hmc in nbl tumors and correlate hmc deposition with chromosomal copy number and gene expression. design/method: hmc was quantified by nano-hmc-seal-seq from the dna extracted from hr, ir and lr nbl tumors. read counts and clinical data were analyzed with deseq to identify genes with differential hmc patterns between risk groups. chromosomal copy number was assessed by chromosomal microarray analysis (cma) in a subset of samples ( lr and hr). expression of genes located on chromosome p was evaluated using publically available microarrays (e-mtab- ) of hr nbl tumors with known p loh status. results: globally, lr tumors had more hmc peaks ( , ) than ir ( , , p = . ) tumors, or hr tumors ( , , p = . ). , genes had different patterns of hmc deposition in hr versus lr tumors. ( %) of these genes mapped to chromosome p and had decreased hmc in hr versus lr tumors (padj < . ). in the cma analysis p deletion was detected in of the tumors tested. in the tumors with p loss, genes that map to p showed decreased hmc deposition compared to the hr tumors without p loss (p< . ). further, compared to the tumors without p loss, the expression of of the p genes was decreased (p< × - ), including chd , camta , and arid a, known and proposed tumor suppressor genes in nbl. conclusion: different patterns of hmc accumulation are associated with neuroblastoma risk classification. nano-hmc-seal-seq is sensitive to copy number variations and has the potential to identify these changes in patient tumors. our results suggest that hmc deposition contributes to the silencing of tumor suppressor genes in p and may also regulate the transcription of other genes that drive tumor phenotype. background: metastatic osteosarcoma has a -year survival rate of - %. pulmonary metastases remain a major treatment challenge in osteosarcoma. current treatment with conventional chemotherapy shows inadequate activity towards metastases and has toxic systemic side effects. chloroquine is a widely used anti-malarial drug and has been shown to have promising anti-cancer and anti-metastatic activity. polymeric drugs have been shown to have multiple advantages over their small molecular parent drugs, including enhancing the therapeutic efficacy, an improved pharmacokinetics profile and decreased systemic toxicity. we hypothesized that by developing chloroquine into a polymeric drug and combining it with conventional chemotherapy it will improve the treatment of metastatic osteosarcoma. objectives: to identify the optimal combination of polymeric chloroquine (pcq) with conventional chemotherapy active in osteosarcoma as a new means of treating metastatic disease in a murine osteosarcoma model. we synthesized and developed pcq and evaluated its anti-invasive activity using an osteosarcoma cell migration and invasion assay. we evaluated the efficacy of cell killing using combination drug therapies with pcq and a panel of conventional chemotherapy agents (doxorubicin, docetaxel, cisplatin and paclitaxel) using celltiter blue cell viability assay. to develop the murine osteosarcoma model, we intravenously injected luciferase-expressing human osteosarcoma cells b into nsg mice. we administered the drug combination that showed the strongest in vitro synergy to the mice and evaluated their anti-cancer and anti-metastatic effects in vivo. tumor growth and suppression were evaluated using whole body bioluminescence imaging. results: we successfully synthesized pcq that contains . % chloroquine with a molecular weight of . kd. pcq was also found to decrease the toxicity of the parent chloroquine. pcq showed strong inhibition of osteosarcoma cell migration with % inhibition compared to % by chloroquine. we screened the combination drug therapies and found the combination of pcq and doxorubicin to show the strongest synergism. the pcq/doxorubicin combination is currently being evaluated in the murine model. combination drug therapy using pcq and doxorubicin showed synergistic cell killing and inhibition of cell migration in vitro. the combination represents a promising treatment strategy for pulmonary metastatic osteosarcoma. emory university/children's healthcare of atlanta, atlanta, georgia, united states background: survival for relapsed high-risk neuroblastoma (rnb) is < %, underscoring the critical need for novel therapies. rnbs have increased ras/raf/mapk mutations and increased yes-associated protein (yap) transcriptional activity. yap is a transcriptional co-activator that binds with tea-domain (tead) transcription factors to regulate cellular proliferation, self-renewal, and survival. we found that shrna inhibition of yap decreases nb cell proliferation and sensitizes ras-mutated nbs to mek inhibitors, supporting yap as a tractable therapeutic target. verteporfin (vp), a photodynamic drug used for macular degeneration, is the only drug found to inhibit yap expression or yap:tead binding to kill tumor-derived cells. peptide is a mer yap peptidomimetic that also disrupts yap:tead interactions. we sought to determine whether these compounds are potent in nb via yap direct effects. design/method: yap expressing (nlf, sk-n-as) or yap null (ngp, lan , sk-n-as-shyap) human-derived nbs were incubated with vp, with and without direct light exposure, or with peptide . celltiter-glo and immunoblots were used to assess for cell death and yap-downstream protein expression, respectively. results: without direct light exposure, vp inhibits yap expression at nm dosing, yet no nb cell death was observed at equal or higher concentrations. egfr and erk / were inhibited along with yap, confirming yap/ras pathway coregulation. when vp was exposed to direct incandescent light for minutes, > % nb cell death occurred in all nbs tested, even those lacking yap. peptide caused no cell death or yap inhibition up to um. neuroblastomas are resistant to vp at doses sufficient to inhibit yap expression. in macular degeneration, light-activated vp produces reactive oxygen species, which we hypothesize is the off target mechanism killing nbs independent of yap. given the off target effects and the need for light activation, vp is not an ideal preclinical or clinical yap inhibitor. accordingly, peptide has poor cell permeability and low tead affinity, leading to its lack of efficacy. given the relevance of yap in rnb and other cancers, we are chemically optimizing a yap peptidomimetic with enhanced permeability, nuclear localization, and tead affinity to create a bonafide yap inhibitor for preclinical and clinical application. kayeleigh higgerson, aaron sugalski, rajiv rajani, josefine heim-hall, jaclyn hung, anne-marie langevin ut health san antonio, san antonio, texas, united states background: osteosarcoma is the most common bone malignancy in children, adolescents, and young adults. most study cohorts have to % hispanic patients that encompass many different hispanic backgrounds. the university of texas health science center at san antonio (uthscsa) sarcoma team serves a latino population that is predominantly mexican american, thus providing a unique opportunity for evaluation this population. this study expands on previous data collected from january to december from the same institution, providing increased insight into outcomes of mexican american children, adolescents, and young adults with osteosarcoma. objectives: to further understanding of osteosarcoma in latino children, adolescents and young adults. design/method: a retrospective analysis of demographics, tumor characteristics, response to treatment, and survival outcome of all localized osteosarcoma of the extremity patients below years of age diagnosed and treated by the uthscsa sarcoma team between january and june was performed. results: in our original cohort from january to december , we observed a significantly decreased -year eventfree survival (efs) in patients diagnosed before age (preadolescent) relative to patients diagnosed between ages and ( % vs. %, p< . ). patients had a -year overall survival (os) and event-free survival of % and % respectively. in our expanded cohort from january to june we evaluated sixty-six patients with a median age of (range, to y) with localized high-grade osteosarcoma of the extremity. the expanded cohort was % mexican american, with a median follow-up of months (range, to ). the analysis of our expanded cohort is ongoing and we postulate that the findings will hold true, as we increase the cohort size and length of follow-up. conclusion: analysis of our previous cohort, predominantly of mexican american ethnicity, showed that preadolescent patients had an increased rate of relapse when compared with previous large studies. we also showed a trend towards decreased efs for the entire cohort. we hypothesize that we will further validate these findings with this expanded cohort and this will support further investigation into potential causes of poor outcome in this vulnerable latino population. background: neuroblastoma in infants has the potential to regress or mature spontaneously. growing literature showed that some cases subjected to initial observation didn't show inferior outcome compared to actively treated similar categories. objectives: we investigated whether early active treatment can be safely avoided/deferred in selected favorable cases at the children's cancer hospital-egypt (cche). design/method: patients enrolled on the watch and see strategy (w&s) at cche had small primary tumor; inss stage - , uncomplicated stage s or stage infants (< days). tissue biopsy was not mandatory for infants below months of age with localized adrenal mass (stage - ). on progression, immediate intervention took place according to stage and risk of disease after biological characterization. results: thirty four nbl patients were enrolled on w&s strategy; m/f: . / . eighteen patients had stage s disease, patients had stage - and were stage . primary adrenal site was reported in patients ( . %), patients ( . %) had small mass measuring ≤ cm in its largest diameter. the -year os & efs were . ± . % and . ± %, respectively, with months median follow-up (range: - months). spontaneous total/near total resolution of mass occurred in / patients ( %). median time to eliciting regression was . months (range: . - . months), and . months (range: - months) till complete resolution. only / patients ( . %) witnessed progression ( local, distant and combined local and distant progression); median time to progression was months (range: - months) with / deaths after starting chemotherapy. watch and see strategy is a safe approach in localized and uncomplicated stage s neuroblastoma. progressive cases could be rescued. baylor college of medicine, houston, texas, united states background: ga- dotatate binds to somatostatin receptor expressed in neuroendocrine tumors (nets). it was approved by fda in for use with pet/ct scan for localization of somatostatin receptor positive nets in adult and pediatric patients. pediatric approval was based mainly on extrapolation of data from adults. objectives: to describe the use of ga- dotatate pet/ct scan in children with neuroendocrine tumors and compare with other imaging modalities. design/method: patients with nets enrolled in texas children's rare tumor registry between february and october were reviewed and those patients who underwent ga- scan were included. results: four patients with nets underwent ga- scans without any adverse reactions. first patient was a -yearold female with small bowel net with multiple liver metastases. mri abdomen and fdg pet at diagnosis showed s of s multiple liver metastases but could not identify the primary lesion. ga- scan was able to accurately identify the enlarged lymph nodes in the small bowel and was better than fdg pet in delineating the liver metastases. second patient was a -year-old female with recurrent small bowel net with liver, lung and paraspinal metastases. the lesions were initially detected by ct scan. octreotide scan failed to show any uptake in the identified lesions while ga- was taken up by the liver lesions, lung lesions > cm in size and the paraspinal lesion. third patient is an year-old male with pancreatic net with peripancreatic lymphadenopathy, multiple liver metastases and cardiophrenic lymph node involvement. the primary lesion in the pancreas could not be identified by ct scan, ct angiogram, mibg scan, or octreotide scan. in addition, there was uncertainty about involvement of the enlarged cardiophrenic lymph node. in addition to clearly identifying the primary lesion, ga- scan was able to detect multiple peripancreatic lymph nodes not detected by other scans and revealed uptake in the cardiophrenic lymph node confirming its involvement by the tumor. fourth patient is a -year-old female with malignant abdominal paraganglioma with solitary lung metastasis. both mibg scan and ga- scan were able to identify the primary lesion. ga- scan was performed after the lung metastasis was removed and thus its ability to detect it could not be confirmed. background: neuroblastoma is the most common extracranial solid tumor of childhood, with overall survival for high-risk patients (hrnbl) near %. the outcomes of hrnbl have improved with high dose chemotherapy followed by autologous stem cell rescue (abmt). data about factors influencing the rate of hematopoietic recovery following abmt in hrnbl is lacking in the literature. our objective was to identify factors influencing the rate of hematopoietic recovery following abmt in hrnbl. design/method: this was a retrospective chart review of patients with hrnbl treated at texas children's hospital from to . neutrophil engraftment was considered the first of three consecutive days with post-transplant neutrophil count greater than cells/ul. red blood cell and platelet engraftment were considered at a hemoglobin greater than g/dl and platelets greater than , /ul three days after the last transfusion. race and conditioning regimen were analyzed using one-way anova; amount of infused cells was analyzed using pearson correlation coefficients; chemotherapy delay and bone marrow (bm) involvement after cycle of induction chemotherapy were analyzed using independent sample t-tests. the study included males and females with a median age at diagnosis of . years. thirtyeight patients were caucasian, african-american, hispanic, asian, and did not have race documented. the mean dose of infused cd + cells was . × ^ cells/kg. forty-five patients received conditioning therapy with carboplatin/etoposide/melphalan (cem), received busulfan/melphalan (bu/mel), and received thiotepa/cyclophosphamide (thiotepa/cpm). the conditioning regimen administered was significant (p = . ) for time to engraftment of neutrophils, with bu/mel at . days, cem at . days, and thiotepa/cpm at days. a delay of chemotherapy during induction (n = ) was significant (p = . ) for time to platelet engraftment of greater than , /ul and trended towards significance (p = . ) for time to neutrophil engraftment. bm involvement at diagnosis and after cycle of induction was not significant for time to engraftment. dose of stem cells infused was the only variable significant for hemoglobin engraftment. background: osteosarcoma (os) is the most prevalent aggressive primary malignancy of the bone affecting children and young adults. approximately % to % of patients have metastatic disease at initial presentation, and % of those patients have isolated pulmonary metastases. although overall survival in patients with os has improved with advances in therapy, there have been no significant improvements in survival outcome in patients with metastatic disease. recent studies suggest that tumor-associated vascular cell adhesion molecule (tvcam- or cd ) plays a critical role in the metastatic progression of various tumors. indirect evidence from these studies suggest that vcam- / integrin signaling promotes tumor survival and metastatic progression by changing the tumor niche and associated immune response. to determine if interfering vcam- / signaling between pulmonary metastatic osteosarcoma (pos) and macrophages (macs) by down-regulating vcam- , depleting macs or blocking vcam- / signaling will reduce pos and improve overall disease-free survival. design/method: we used a pair of spontaneous, high-grade murine os cell lines from balb/c mouse (h- d), k and k m (derived from in vivo k metastasis). we used lentiviral shrnas to knockdown vcam- mrna and protein expression in k m (vcam- kd). we introduced luciferase into k , k m and various k m shrna cell lines to follow lung metastasis by bioluminescence (bli). we depleted macs by intranasal administration of liposomal clodronate formulation. we tested the ability of k and k m supernatants to polarize m macs into m or m phenotype in vitro. we also administered anti- monoclonal antibody (anti- mab) intranasally to assess the outcome of functional blockade of vcam- / signaling. results: k m over-expressed vcam- compared to k . mac depletion in k m -bearing animals exhibited reduced pos. weekly administration of anti- mab resulted in % tumor-free rescue among mice with established k m pos. interestingly, supernatant from k m but not k preferentially induced m -like macs, suggesting a novel integrin-mediated mechanism of m differentiation. validation data with additional os cell lines will be presented. despite aggressive multimodal therapy, overall outcome for patients with pos remains dismal at - %. for this reason, novel and directed therapy approaches are desperately needed. molecular targeted approaches for therapy are challenging, due to the complex genetic heterogeneity of os. immune-modifying therapy is a promising new alternative approach for pos. university of chicago, chicago, illinois, united states background: only half of all patients diagnosed with high-risk neuroblastoma achieve long-term survival. imetaiodobenzylguanidine (mibg) scans are routinely used to evaluate disease at diagnosis and following treatment, and the extent of disease is quantified using the curie scoring system. a previous study by yanik et al., has shown that for high-risk patients with mycn non-ampliified tumors, scores less than versus greater than following cycles chemotherapy are associated of superior survival, whereas scores less than versus greater than were prognostic in patients with mycn-amplified tumors. however, the prognostic significance of specific sites of metastatic disease at diagnosis is not known. to determine if site of metastatic disease determined by i-metaiodobenzylguanidine (mibg) imaging in high-risk patients at the time of diagnosis was associated with outcome design/method: we performed a retrospective chart review of high-risk neuroblastoma patients treated at comer children's hospital and lurie children's hospital in chicago between and with positive mibg scans at the time of diagnosis. we collected imaging data as well as other clinical data including bone marrow status. sites of disease were defined as curie regions with any positive value. kaplan-meier analysis was performed to evaluate the association with disease sites and survival. pearson correlation coefficients were calculated to compare bone marrow disease to sites of positivity on mibg scan. the cohort consisted of high-risk patients. had skull disease, and had pelvic disease. the presence of mibg positive disease in the skull and in the pelvis trended toward worse efs. efs at years for patients with disease in the skull at diagnosis was ± % and for patients without skull disease was ± % (p = . ). efs at years for patients with and without pelvic disease was ± % and ± % (p = . ). consistent with prior data, we found that the presence of bone marrow disease was associated with worse survival with year efs of ± % and ± % with and without marrow disease at diagnosis (p = . ). there is the highest correlation between pelvic disease on mibg scan and bone marrow disease with pearson coefficient . . pelvic disease noted on mibg scan likely reflects underlying bone marrow disease. in patients with high-risk neuroblastoma, skull disease and pelvic disease on mibg scan at diagnosis may predict worse event free survival. background: osteosarcoma is one of the deadliest cancers in the pediatric population with little progress in morbidity and recurrence rates since the 's. oncolytic herpes simplex- virus (ohsv) is an attenuated virus that has shown encouraging results against certain solid tumors. programmed cell death protein (pd)- -mediated t cell suppression via engagement of its ligand, pd-l , is also of particular interest due to recent successes in selected cancers, especially those with high genetic mutational loads. most pediatric cancers do not have a wide variety of mutations; however, osteosarcoma has a chaotic genome, prone to genetic mutations. it has been shown through numerous other studies that pd- inhibition alone is not sufficient to result in statistically significant tumor growth delays in osteosarcoma models and patients. we hypothesize the addition of ohsv therapy as an immunologic stimulus to pd- inhibition is efficacious for osteosarcoma. ( ) to determine whether ohsv therapy enhances response to pd- inhibition in immunocompetent murine models of osteosarcoma and ( ) to quantify and characterize the anti-tumor t-cells infiltration after treatment with ohsv and pd- inhibition individually and in combination. we utilized an immunocompetent transplantable murine model using a cell line derived from a spontaneous metastatic osteosarcoma (k m , balb/c background). we transplanted established tumor wedges subcutaneously and monitored tumor volume by caliper measurement. once tumors reached - mm , we administered intratumoral injections of hsv ( × plaque-forming units) every other day for a total of injections. we then gave intraperitoneal injections of ug anti-pd- or control antibody twice weekly, up to weeks, starting from the last dose of virus treatment. we monitored tumor growth via calipers twice weekly until tumors reached mm or cm diameter. we quantified and characterized innate and adaptive immune cell infiltrates in tumors using flow analysis. we found significantly prolonged survival with our combination therapy group compared to all other groups. we found that anti-pd- by itself had little impact on t cell recruitment while the combination group had higher influx of cd + cells with a reduced amount of t-regulatory cells (cd +foxp +cd +). we also found an increase in cd + effector memory cells. osteosarcoma is a deadly cancer with therapeutics remaining unchanged for the last years. here, we describe prolonged murine survival after treatment with combination of pd- inhibition and ohsv injection. the combination treatment changed the microenvironment to be more inflammatory. our data support further preclinical and clinical studies. background: neuroblastoma is the second most common cause of cancer related death in children. treatment for high-risk neuroblastoma has improved significantly over the past twenty years, however cure rates remain below %. immunotherapy has emerged as an effective therapy for neuroblastoma, however new modalities and targets are needed to improve outcomes. objectives: our lab has developed a chimeric antigen receptor (car) that targets b -h (cd ), an immune checkpoint molecule overexpressed on many cancers, including neuroblastoma. we hypothesized that b -h would be a good target for car based immunotherapy for neuroblastoma. design/method: neuroblastoma tissue microarrays of primary patient samples were screened for b -h expression by immunohistochemistry and cell lines were screened using flow cytometry. b -h car t cells were tested in vitro by measuring tumor cell killing and cytokine production after coculture with tumor cell lines and in vivo in an orthotopic model of neuroblastoma. results: b -h expression was detected by ihc on % of the screened neuroblastoma patient samples. b -h was expressed at high levels ( + or +) in more than half of these samples ( %). almost all cell lines screened were homogeneously positive for b -h by flow cytometry. retrovirally transduced b -h . - bb. car t cells were cocultured with three b -h positive neuroblastoma cell lines (sk-n-be , kcnr, and chla ) and robust tumor cell killing was demonstrated using an incucyte assay. supernatant from the co-cultures was harvested after hours and both interferon gamma and il- production were detected by elisa.in an orthotopic subrenal capsule xenograft model of neuroblastoma, mice treated with b -h car t cells show significant reductions in tumor growth and prolonged survival compared to those treated with untransduced control t cells. however, the treatment is not always curative.b -h car t cells express high levels of exhaustion markers (pd , tim , and lag ) when compared to cd car controls. in order to overcome inhibition from exhaustion, b -h car t cells were co-cultured with neuroblastoma cell lines and pd- blocking antibody. nivolumab significantly increased the production of il- and interferon-gamma by b -h car t cells. further studies are underway to determine if b -h car t cell activity is enhanced in vivo by treating animals with pd- blockade along with car t cells. conclusion: b -h is expressed on a majority of neuroblastoma samples and appears to be a promising candidate for car t cell therapy. b -h car t cells demonstrate activity against neuroblastoma xenografts that may be enhanced by the addition of pd inhibitors. helen devos children's hospital, michigan state university, grand rapids, michigan, united states background: osteosarcoma is the most common bone tumor in children. it is often metastatic at diagnosis and in this scenario less than % of children survive. polyamines, small molecules found in all cells, are involved in many cell processes including cell cycle regulation, immune modulation, cell signaling and apoptosis. they are also involved in tumor development, invasion and metastasis. in neuroblastoma, inhibition of the polyamine biosynthesis pathway with odc inhibitor alpha-difluoromethylornithine (dfmo) results in decreased cell proliferation and differentiation. these finding have led to multiple phase i and phase ii multicenter clinical trials in pediatric neuroblastoma patients. dfmo is an attractive drug as it is oral, well-tolerated, can be given for prolonged periods and is already used in pediatric patients. the polyamine pathway has not been evaluated in osteosarcoma. objectives: evaluate effect of inhibition of polyamine biosynthesis with dfmo on osteosarcoma proliferation and cell differentiation. design/method: up to three osteosarcoma cell lines were used: mg- , u- os and saos- . cells were exposed to mm dfmo for days with replacement of media and dfmo on day . intracellular polyamine levels were measured by high performance liquid chromatography (hplc). cell numbers were obtained with a hemocytometer using trypan blue. flow cytometry cell cycle distribution (facs) and propidium iodide were used to evaluate for cell cycle arrest. the protein expression of several osteosarcoma differentiation markers was measured by sds-page and western blot using differentiation specific antibodies. a bioluminescent cell viability assay was used to measure cell recovery over several days after dfmo was removed and replaced with standard media. results: dfmo exposure resulted in significantly decreased cell proliferation in all cell lines. after treatment, intracellular spermidine levels were nearly eliminated in all cells. cell cycle arrest at g was observed in u- os. cell differentiation was most pronounced in mg- and u- os cells as determined by increased osteopontin levels. remarkably, cell proliferation continued to be suppressed for several days after removal of dfmo. conclusion: based on our findings dfmo is a promising new adjunct to the current osteosarcoma therapy for high risk patients. it is a well-tolerated oral drug that is currently in phase ii clinical trials in pediatric neuroblastoma patients as a maintenance therapy. the same type of regimen may also improve outcomes in metastatic or recurrent osteosarcoma patients for whom there have been essentially no medical advances in the last years. background: recent studies demonstrate that lower levels of the ews-fli fusion oncoprotein are associated with enhanced metastatic capability in ewing sarcoma. the nf-kb transcription factor is a critical mediator of cxcr and cxcr -driven metastasis in multiple cancers, and increased cxcr and cxcr expression have each been associated with increased metastasis and poor prognosis in ewing sarcoma. we thus sought to investigate the impact of ews-fli on cxcr /cxcr -dependent nf-kb signaling in ewing sarcoma. objectives: the goals of this study are ) to determine the impact of cxcr /cxcr signaling on metastasis-associated nf-kb target gene expression in ewing sarcoma and then ) to investigate how the ews-fli fusion oncoprotein modulates this response . design/method: we utilized multiple ewing sarcoma cells lines including a , chla , chla , tc and tc . cxcr /cxcr cell surface expression was determined by flow cytometry. ews-fli level was modulated using sirna and expression levels were confirmed by western blot and rt-pcr. p dna binding was measured via elisa. nf-kb target gene expression was assessed via rt-pcr. results: consistent with ihc analysis of primary and metastatic patient tumor samples, the paired primary and metastatic ewing sarcoma cell lines chla and chla showed dramatic differences in cxcr and cxcr expression, with the metastatic chla line demonstrating much higher expression of both receptors. other cell lines (nonpaired) showed variable cxcr /cxcr expression. genetic knock-out of cxcr lead to significant decrease in expression of both cxcl /sdf- and il- , two nf-kb transcriptional targets known to play a key role in tumor metastasis. knock-out of cxcr did not alter endogenous ews-fli mrna levels. conversely, lowering the level of ews-fli using sirna lead to enhanced nf-kb signaling, indicated by an increase in p dna binding. consistent with this observation, treating ewing cell lines with ews-fli sirna also resulted in significantly increased nf-kb target gene expression compared to control cells and target gene expression was then further enhanced upon cxcr /cxcr receptor stimulation with the receptor ligand cxcl /sdf- . our findings indicate that the ews-fli oncoprotein negatively modulates cxcr /cxcr -dependent nf-kb signaling. this suggests that ews-fli low, cxcr /cxcr high cells, which are associated with enhanced metastasis and poor prognosis, would be anticipated to exhibit enhanced expression of key nf-kb target genes. importantly, the nf-kb pathway is a druggable target that could potentially serve as an "achilles heel" in this subset of high risk tumors. current work is evaluating nf-kb inhibition as an approach to treating metastatic and refractory ewing sarcoma. background: acute graft versus host disease (agvhd) is a major cause of morbidity and mortality following allogeneic bone marrow transplant (bmt) in pediatric patients. gastrointestinal (gi) agvhd is the most serious manifestation. recently, decreased paneth cell (pc) in a predominantly adult cohort was shown to correlate with agvhd clinical grading and response to treatment. we aim to demonstrate the relationship between pc counts and gi agvhd stage and response to therapy. design/method: charts of patients who underwent endoscopy following bmt between - were reviewed. for repeated biopsies during the course of agvhd, only the first was included for analysis. one pathologist retrospectively reviewed the biopsies and counted pcs in high powered fields; the average pc count was analyzed. twenty-six percent of biopsies were reviewed by a second blinded pathologist. statistical associations between pc counts and day (d ) response, agvhd stage, and other study covariates of interest were gauged using general linear regression. agreement in pathologist pc counts was quantified by intraclass correlation (icc). the research was approved by the children's healthcare of atlanta irb. results: seventy-eight biopsies were included in the analysis. mean age at transplant was . years ± . (range: months - years). most patients underwent transplant for hematologic malignancies ( , %). the majority of transplants used a matched unrelated donor graft -including cords ( , %) and myeloablative conditioning regimens ( , %) - % received total body irradiation. of these, % were diagnosed clinically with gi agvhd (stage , %; stage , %; stage , %; stage , %). icc showed good agreement ( . ) between the pathologists. mean pc was . for patients with no gut agvhd, . for stage , . for stage , . for stage and . for stage (p = . ). on multivariate analysis pc was strongly associated with gi agvhd stage (p< . ) after controlling for age, preparative regimen intensity, and diagnosis (malignant vs. non-malignant). mean pc counts were significantly lower in patients with no response to steroid therapy at d (complete response (mean . ) vs. persistent disease ( . ) vs. partial response ( . ) (p = . )). patients diagnosed with gi agvhd with pc counts less than had a higher risk of mortality (hr . , % ci: . , . ; p = . ). lower pc count correlated with stage gi agvhd, refractory disease at d , and mortality. incorporating pc count in pathology review during gi agvhd work-up may help in agvhd risk stratification. background: there have been increasing discussions pressuring health care teams and institutions for potentially bearing the cost of clostridium difficile infections (cdi) as a health care-associated infection in the recent years. the pediatric oncology patient population, though small, accounts for significant portion of all cdi with - -fold increased risk. hematopoietic stem cell transplant (hsct) recipients constitute a unique subset with distinct risk factors, such as severe immune deficiency state and graft versus host disease (gvhd). although there is ample data on cdi in adult hsct recipients, reports on pediatric experience are limited. objectives: to evaluate the incidence and patterns of cdi among pediatric hematology, oncology and hsct inpatients at our institution. a retrospective review of all clostridium difficile (cd) stool tests performed using toxin enzyme immunoassay and later, polymerase chain reaction targeting toxin genes between and in a large, urban academic children's hospital was performed. the data were analyzed for hematology, oncology, hsct inpatient population and all the other cases separately and statistical comparisons were performed. results: a total of samples were submitted to the microbiology laboratory for cd testing during the study period. while hematology patients constituted . %, oncology . %, hsct . % and others . % of the cases on whom cd testing was done; per patient average test number was . , . , . , and . , respectively. of all the cd tests per-formed, . % were positive. test positivity was higher in hsct ( . %) and oncology ( . %) cases tested compared with hematology ( . %) and other cases ( . %) with statistical significance (p< . ). overall recurrence rate was . %; hsct patients had the highest recurrence with a rate of % followed by oncology ( . %), hematology ( . %) and other ( . %) cases, again reaching statistical significance (p< . ). again, hsct patients had the highest average number of recurrences at . ( - ) followed by oncology . ( - ), general . ( - ) and hematology . ( - ) groups. there was no seasonal variability in the incidence of cdi among populations analyzed. prolonged hospital stay/antibiotic use and persistent diarrhea due to gvhd are the likely reasons for higher rate of cd testing in hsct as a result of increased monitoring and thus might have even caused underrepresentation of positive cd test frequency. higher incidence and frequencies of recurrence underscores the inevitable nature of cdi in hsct population as a consequence of the current therapies and may lead to future radical treatment approaches like fecal implantation. background: viral infections remain a challenge to treat post hct in children, and significantly contribute to morbidity and mortality. virus specific t cells (vsts) have shown tremendous clinical efficacy in treating viral infections post-hct, with minimal toxicity and long term efficacy. we have used donor-derived vsts in individual patients, however not all donors are agreeable to the process, and numerous patients may benefit from vsts who do not have an identified donor/have other disease indications objectives: we sought to actively build a third-party vst bank, for "off the shelf" use in eligible patients. design/method: vsts targeting cmv, adenovirus and ebv were manufactured using one of techniques. initially ebv transformed b cells were genetically modified with an ad f pp vector and used as antigen presenting cells (apc) to stimulate and expand ebv, ad and cmvpp specific t cells. more recently, vsts were expanded using s of s apc pulsed with commercially available peptide pools (pep-mixes) to expand ebv/cmv/ad specific t cells. products were entered into the "bank" via two mechanisms: a) left over products from our "donor-derived" protocol when patients no longer required vsts or were not at risk of developing viral infections, or b) by targeting regular blood donors based on their hla typing to ensure an appropriate mix of high frequency hla types for optimal patient matching and antigen presentation based on current knowledge of antigen presentation. results: a total of products are currently in the thirdparty vst bank ready for use. twenty seven of these are from our donor derived protocol, and three from targeted donors. all vst products met safety and in vitro efficacy testing. thirteen vst infusions have been given to patients. eleven infusions have been given for cmv and two for adenovirus. five out of seven patients responded to thirdparty vst infusions, with a median of vst infusions per patient (range - ). the median hla matching was out of per patient (range to ) no patients experienced adverse reactions, gvhd or other toxicity related to the vst infusion. a third-party vst bank is feasible and produces clinically appropriate vsts for use in patients with viral infections. hla typing and matching of vst products is essential to reduce toxicity and promote appropriate antigen presentation and expansion of vsts in vivo. further work is underway to further characterize the vsts using epitope mapping to better define the hla restriction and immunogenicity of each vst product. akron children's hospital, akron, ohio, united states background: acute graft-versus-host disease (agvhd) is a well-known complication of hematopoietic stem cell transplant (hsct) and a major cause of post-transplant related morbidity and mortality. first line therapy of agvhd involves corticosteroids and calcineurin inhibition. in patients with severe refractory gvhd, mortality can reach up to %. currently, there is no standard of care for the treatment of steroid refractory agvhd. many centers have looked at the use of antibody mediated control of agvhd to competitively inhibit the inflammatory cascade. basiliximab, a chimeric monoclonal antibody against the t-cell il- receptor, has been used in adults with steroid refractory agvhd. patients receiving this medication have demonstrated complete and partial responses to therapy with minimal toxicities. objectives: report the successful use of basiliximab in the treatment of agvhd in a -year-old following matched unrelated (mud) hsct. design/method: a -year-old male underwent mud transplant for high risk aml with monosomy . conditioning regimen included busulfan, fludarabine and equine atg. his clinical course was complicated by fever, mucositis and agvhd (stage skin; stage gi-biopsy proven). gvhd prophylaxis included tacrolimus and methotrexate, however with progressive skin rash, diarrhea, and early satiety, gvhd treatment with corticosteroids was initiated. as the patient continued to have worsening symptoms, basiliximab therapy was started. the patient received doses ( mg) iv basiliximab on two consecutive days and then received weekly therapy for a total of doses leading to initial improvement. the patient further developed acute on chronic gvhd on day + , and subsequently received a second course of basiliximab. after initial administration of basiliximab, the patient had near complete resolution of symptoms. however, with a small wean in his tacrolimus dose, the patient experienced another skin gvhd flare prompting the second basiliximab course. the patient was subsequently weaned off all immunosuppression by day + . the only acute complication the patient experienced while receiving basiliximab was right toe paronychia and asymptomatic low ebv titer. the patient is currently off all immunosuppression at the time of report without evidence of cgvhd. conclusion: this single case report, in a young pediatric patient, demonstrates the use of basiliximab may be a safe and efficacious treatment for pediatric patients with agvhd. university of california, san diego, la jolla, california, united states background: clinical outcomes after allogeneic hematopoietic stem cell transplantation (hsct) depend on restoration of t lymphocyte populations. association between recovery of cd +foxp + regulatory t cells (tregs) and protection from chronic graft versus host disease (cgvhd) has been described in adult hsct. in adults, t cell recovery is driven by expansion of donor t cells and treg reconstitution is hypothesized to result from peripheral conversion. restoration of t cells in pediatric patients has a larger contribution from thymopoiesis, however, the relationship between thymopoiesis and treg recovery is undefined. objectives: we hypothesized that effective thymopoiesis is important for restoration of treg populations and protection from cgvhd in pediatric hsct patients. design/method: we performed longitudinal flow cytometry of peripheral blood t cells from pediatric hsct patients and age-matched healthy donors. laboratory data were correlated with clinical outcomes to evaluate impact. recovery of tregs occurred in / ( . %) patients by post-transplant day . day treg frequency in patients that developed cgvhd ( . ± . % of cd + t cells) was reduced compared to cgvhd-free patients ( . ± . %). failure to restore tregs to > . % of cd + cells by day was associated with increased risk of cgvhd in the first year post-hsct (rr = . , p = . ). a majority ( . ± . %) of tregs from patients recovering the peripheral treg compartment expressed helios, a marker of thymic-derived tregs; only . ± . % of tregs expressed helios in patients failing to restore adequate tregs. this prompted examining the relationship between defects in thymopoiesis and inability to restore tregs. we evaluated thymic function by flow cytometry quantification of cd ra+cd +ptk + recent thymic emigrant (rte) cd + cells (confirmed by qpcr for trec content). most ( / , . %) hsct patients had detectable rtes by day post-hsct. thymic production of rtes was persistently absent in patients that developed cgvhd (< / ^ cd + cells in / patients), compared to cgvhd-free patients ( / patients > rte/ ^ cd + cells by day , average . ± . / ^ cd + cells). post-hsct thymic activity as measured by rte enumeration correlated with treg restoration; / ( %) rte+ patients restored tregs, compared to / ( %) of rte-patients. conclusion: failure to restore tregs after allogeneic hsct results in increased risk for cgvhd. in pediatric patients thymic generation of new t cells is an important contributor to restoration of the treg compartment. this data supports further investigation into mechanisms impairing post-hsct thymopoiesis and suggests peripheral blood tregs may be a prognostic biomarker for cgvhd. background: haploidentical stem cell transplantation (haplo sct) is riddled with unique challenges. objectives: we present our experience in the use of haplo sct with post-transplant cyclophosphamide (ptcy) and the adaptations required for each disorder for optimal outcome. design/method: we performed a retrospective study at the pediatric blood and marrow transplant unit, apollo cancer institutes, chennai, india. children up to years of age, diagnosed to have benign disorders and underwent haplo sct with ptcy from to july were included. results: ptcy was used in i.e. % haplo transplants for children with benign disorders. the underlying conditions included fanconi anemia , severe aplastic anemia , mds , jmml , hemoglobinopathy , prca , xld and primary immunedeficiency disorders (pid) . source of stem cells was peripheral blood in %, bone marrow in %. conditioning included fludarabine with treosulphan or cyclophosphamide for pids and aplastic anemia respectively. neutrophil engraftment by day+ - with a durable graft was noted in % transplants with graft versus host disease in %, cmv reactivation in %. mortality rate was % with infants less than months of age developing severe fatal cytokine release syndrome. the median follow up is year with years being the longest. no significant late effects have been noted with chronic skin gvhd in children. survival rate was superior among children with pids with survival of % in this group. haplo sct with ptcy is a feasible and costeffective option for cure in children with life-threatening benign disorders with no compatible family or matched unrelated donor. careful patient selection, reducing cyclophosphamide related free radical toxicity with the use of n acetylcysteine, limiting t cell numbers by capping cd at × /kg, post-transplant viral monitoring protocols are required to reduce morbidity and mortality. we have been working on universal access to care for children from s of s all socioeconomic background and incorporating innovations to reduce the cost of hsct without compromising outcomes. haploidentical hsct using tcr / depletion costs usd as compared to ptcy priced at usd. children with severe aplastic anemia and pids can be transplanted using reduced intensity conditioning and ptcy. in hemoglobinopathies, pretransplant immunosuppression is required to prevent graft rejection. graft versus host disease remains the main cause of mortality in children with fanconi anemia. mortality in infants less than months after ptcy has been high, tcr / depletion would be superior in this cohort. cincinnati children's hospital medical center, cincinnati, ohio, united states background: fanconi anemia (fa) is a congenital bone marrow failure syndrome with hsct the only curative option for associated bone marrow failure. patients with fa undergoing hsct may experience increased toxicity related to either their underlying disease, or the effects of medications, resulting in the inability to tolerate prophylactic medications or sideeffects from anti-microbial therapy. objectives: we postulated that increased cd cell dose would be associated with a rapid immune reconstitution and therefore early withdrawal of anti-infective prophylactic medications. design/method: patients with fa transplanted at cchmc from an unrelated donor had peripheral blood stem cell grafts collected and cd selection performed. where possible, patients had serial measurements of their immune system performed at varying intervals post hsct. we defined immune reconstitution as normalization of lymphocyte subsets-cd , cd , cd and cd cells, as well as a normal response to mitogen stimulation including phytohemagglutinin, concanavalin a and pokeweed. the first measurement of either normal cell number or mitogen response was recorded for each patient. results: a total of patients underwent hsct for fa at cchmc between and . patient demographics included a median age of years at hsct, the vast majority of patients having a fully matched or one anti-gen mismatched donor, and the majority of patients transplanted for bone marrow failure. there was a statistically significantly decreased time post-transplant to immune cell recovery in patients receiving > × /kg cd cells (median . ) compared to those receiving < × /kg cd cells (median . ). the median time to normalization of cd count was days (cd count > /kg) versus days (cd count < /kg), cd count days (cd count > /kg) versus days (cd count < /kg), cd count days (cd count > /kg) versus days (cd count < /kg) and cd count days (cd count > /kg) versus days (cd count < /kg). time to normalization of mitogen response was decreased posttransplant in those patients receiving increased cd cell dose at time of transplant, though this was not significant, reflecting low number of patients with evaluable responses. no patients in either group experienced gvhd or graft failure. patients with fa who are transplanted with higher cd cell doses have quicker immune reconstitution than those who receive lower cell doses. along with benefit to patients including less risk of infection and early termination of immune-prophylaxis medications, this supports the use of high dose cd selected grafts in this vulnerable population. background: parvovirus b (pvb ) infection after transplantation was first reported in . since then, numerous cases of pvb infections after hematopoietic stem cell transplantation (hsct) and solid organ transplantation (sot) have been reported. most report anemia as the predominant clinical manifestation. however, pvb has been associated with pancytopenia, hepatitis, myocarditis, and allograft rejection. we present a patient with acute lymphoblastic leukemia who developed bone pain and pancytopenia following hsct in the setting of pvb infection. to describe an unusual presentation of pvb in a patient with acute lymphoblastic leukemia following hsct. design/method: a search of the english-language medical literature was performed using pubmed and medline databases. a review of the patient's medical history was performed. a year old male with relapsed b-cell all and history of "fifth disease" in infancy presented four months after hsct with focal left arm pain and difficulties fully extending the arm. bone mri showed enhancement of the medullary space centered within incomplete transverse cortical fracture interpreted as pathologic fracture due to neoplastic involvement of the ulna with no history of inciting injury. subsequently, peripheral blood counts decreased from low normal values to wbc . k/microl, anc /microl, plt k/microl, and hemoglobin . g/dl. the patient's chimerism remained % donor. a bone marrow biopsy and aspirate were performed to assess for recurrent leukemia given persistence of bone pain and developing pancytopenia. marrow findings included morphologic cytopathic effects with erythroid precursors and strong parvovirus staining with no signs of red cell aplasia or recurrent b-cell disease by morphology or flow cytometry. pvb was detected in blood by pcr and immunoglobulins with resolution of cytopenia and bone pain. this case highlights an unusual constellation of symptoms following hsct in a child with all. unexplained bone pain and medullary infiltrates with pancytopenia suggestive of recurrent leukemia were likely triggered by pvb infection. the question remains if he had reactivation of pvb , a primary infection by a new strain, or the virus was aquired through stem cells. bone biopsy could not be justified in light of clinical improvement. so far, bone lesions have only been described with congenital pvb infection. pvb appears to be uncommon after hsct, with a review of literature yielding pediatric cases. however, it may be underestimated due to lack of routine screening. our patient's presentation supports that evaluating for pvb may be warranted in hsct patients presenting with symptoms suggestive of relapsed leukemia. background: cardiac injury may occur during hematopoietic stem cell transplant (hsct) in pediatric patients and can be asymptomatic for many years. recommendations for screening are available for patients who received anthracyclines or chest irradiation, but no guidelines exist for unexposed longterm survivors. we sought to define the prevalence of echocardiographic abnormalities in long-term survivors of pediatric hsct and determine the need for screening in asymptomatic patients. design/method: we analyzed echocardiograms performed on long-term survivors (≥ five years) who underwent hsct at cincinnati children's hospital between and . we analyzed echocardiograms for left ventricular ejection fraction (ef), end-diastolic dimension (lvedd), septal thickness, posterior wall thickness, and global longitudinal strain (gls). we normalized linear measurements for age and patient body surface area. we included for further analysis patients who had echocardiogram obtained for routine surveillance. results: a total of patients underwent hsct and were alive more than years after transplant in , with having an echocardiogram obtained ≥ five years postinfusion. those with an echocardiogram were transplanted more recently (median vs. ). however, no difference between screened and unscreened individuals was noted for age at transplant, sex, transplant indication, anthracycline exposure, chest irradiation, or cyclophosphamide based preparative regimen. indications for echocardiograms included: cardiac symptoms ( . %), congenital cardiac anomalies ( . %), hypertension ( . %), known cardiac or pulmonary disease ( . %), routine post-hsct surveillance ( . %), and unknown ( . %). the mean time post-hsct was . years. among routine surveillance echocardiograms, the mean ef z-score was - . . mean lvedd zscore was - . , mean septal thickness z-score - . , mean posterior wall thickness z-score - . , and mean gls - . %. for patients that had echocardiogram performed for routine surveillance, / patients ( . %) had ef measured, and / ( . %) had ef z-scores ≤ - . (abnormally low). patients exposed to anthracyclines had a mean z-score ef of - . vs. unexposed patients - . (p = . ). among individuals who received neither anthracyclines nor tbi only / ( . %) was found to have an abnormal ef, . % (z-score - . ) or gls (- . %). only one patient who had a normal ejection fraction (z-score - . , ef . %) had an abnormal gls, - . % (normal ≤ - . ). long-term survivors of pediatric hsct who are asymptomatic and did not receive radiation or anthracyclines likely do not require surveillance echocardiograms, unless indicated by clinical symptoms. patients exposed to anthracyclines or tbi require close echocardiographic s of s screening and clinical monitoring for the development of cardiac complications. duke children's hospital, durham, north carolina, united states background: children undergoing pediatric blood and marrow transplants (pbmt) experience significant symptom distress. mobile health (mhealth) technologies can be leveraged to collect and monitor patient generated health data, and subsequently enhance our understanding of pbmt symptom clusters, patterns, and trajectories. better understanding of symptom complexity can foster development of precision health strategies to improve patient outcomes. however, limited research exists in integrating mhealth technology into pbmt management. we aimed to explore the feasibility, acceptability, and usability of using a pbmt specific mobile application to collect and monitor symptoms and wearable technology (apple watch) to measure objective data such as heart rate (hr) and activity. design/method: an exploratory mixed method design began in october to monitor pbmt symptoms for patients using real-time data from: ) a self-developed mhealth application (app) to collect subjective symptom data; and ) apple watch to collect physiologic measures such as heart rate and number of daily steps. data is collected pre-transplant through days. acceptability will be assessed through satisfaction surveys at study completion. we have enrolled patients to date who are all currently using the app and watch. patients' average frequency of daily charting in the app %. the wearable average daily recorded measurements are for hr and for step count. most common symptoms recorded within the app include fatigue and pain. we have noted trends in data including a decrease in activity following transplant and gvhd and an increase following engraftment. patients have stated "the app is helpful to keep track of how my pain is doing day to day" and "i try to take more steps each day than the day before". patients often remove the watch for charging, then forget to put it back on, but consistently put it on upon reminder. finally, parents often were required to make app entries with patients too sick to record. we continue to enroll patients with enthusiasm from both patients and parents to use mhealth during pbmt. preliminary findings suggest feasibility of using the mhealth devices is strongly correlated to the patient's post-transplant stage and is facilitated by caregiver participation with device management (charging devices, reminders to wear watch and record in app). patients reported satisfaction and ease of use with devices, but found it difficult to keep up with charging and charting. these findings indicate using mobile devices may be useful methods to collect patient generated health data. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bacterial bloodstream infections (bsi) are a common complication following hematopoietic stem cell transplantation (hsct) in both pediatric and adult populations, and are associated with poor outcomes. there is limited data describing the outcomes and characteristics of patients who develop three or more bsi after hsct. objectives: to describe the characteristics and outcomes of pediatric patients who develop three or more blood stream infections in the first-year post hsct. design/method: we performed a retrospective chart review of consecutive patients who underwent hsct at our institution from through to compile this case series. data were collected through the first year post-hsct including: patient demographics, underlying disease and therapy characteristics; and transplant complications such as thrombotic microangiopathy (tma), graft versus host disease (gvhd) and overall survival. bsis were classified according to current center of disease control guidelines. results: of patients, ( %) developed or more bsi in the first-year post transplant (total bsi cases = including all patients). of the cases, the majority underwent allogeneic hsct (n = / ; %). most cases were from unrelated donor (n = / , %). more than half of patients had grade - gvhd (n = / , %). sixteen ( %) had tma. of these cases, tma preceded the first bsi in n = / ( %). the majority of bsis were classified as central line-associated bloodstream infections (clabsis, n = / , %), followed by mucosal barrier injury laboratory-confirmed bloodstream infections (n = / , %) and secondary bsi (n = / , %). the majority of isolated organisms ( %) were associated with mucosal barrier injury pathogens. one-year overall survival in the cohort was % (n = / ). pediatric patients undergoing hsct who develop or more bsis in the first-year post transplant demonstrated an increased rate of tma compared to the overall institutional incidence of roughly %. tma diagnosis preceded the first bsi in over half of patients, suggesting that tma may predispose to recurrent bsi. improved strategies for early detection and treatment of tma as well as prevention of clabsis may help reduce the number of bsis ultimately leading to decreased morbidity and mortality in this patient population. background: in neutropenic pediatric patients, infection remains a significant cause of morbidity and mortality. while granulocyte transfusions have been utilized for decades to treat infections, including in the pediatric population, the efficacy of this intervention remains poorly described. previous guidelines have primarily utilized information from adult populations. furthermore, recruitment of donors typically involves friends or relatives of the patient with periodic involvement of community donors. the use of a readily available local donor population to improve availability has yet to be well described. as the immunocompromised population is particularly susceptible to worsening infection and clinical deterioration, the ability to rapidly harvest and deliver granulocytes warrants further investigation. to investigate the efficacy, safety, and outcomes of severely immunocompromised patients receiving granulocyte transfusions from a local altruistic granulocyte program in a pediatric tertiary care center. design/method: a retrospective review was performed to evaluate the context for receiving a transfusion as well as primary outcomes including infection clearance, survival to discharge, and overall mortality. the indiana blood bank assisted with timing the interval from initial order placement to onset of first granulocyte infusion. results: among the patient population reviewed, patients received separate granulocyte regimens. ages ranged from - years with a mean neutrophil count of at time of first transfusion. indications for transfusions included bacteremia (n = ), fungal pneumonia (n = ), and fungemia (n = ). primary outcomes included clearing infection ( %) and surviving to discharge ( %). the median time from initial order placement to infusion was hours, although there was no significant difference between responders who cleared the infection and non-responders who did not. however, additional investigation found that ward patients had a % chance of surviving to discharge while patients in the icu at time of initial transfusion had a % chance of survival to discharge. the readily available granulocyte transfusion program allows patients to quickly receive therapy in neutropenic settings. this is beneficial for patients as transfusion prior to clinical decompensation correlates with increased likelihood of infection clearance, and subsequently improved mortality. further investigation is needed, likely as a prospective study, to better explore circumstances that are beneficial for granulocyte transfusions. background: donor lymphocyte infusions (dli) are composed of immune cells to treat relapse after hematopoietic cell transplantation (hct). to date, data regarding its efficacy is limited in pediatric populations. furthermore, while outcomes related to cd content have been characterized, to our knowledge, the relationship between outcomes and other cellular content in dli has never been reported. objectives: determine whether the primary hematological malignancy, presence/absence of graft-versus-host disease s of s (gvhd), and unique phenotypic content of each dli impact overall survival (os) in pediatric patients with hematological malignancies. design/method: irb-approved, retrospective study investigating all consecutive dlis given to patients at the children's hospital of wisconsin. analyses were conducted using mann-whitney, fisher's exact, and chi-square. from from - patients ≤ years old with hematologic malignancies [myeloid (aml/ mds/cml/jmml),n = ; lymphoid (all),n = ] underwent dlis ( %% ≥ dlis). the median time between hct and dli was . (range, . - . ) years. there were significant differences between the lymphoid and myeloid groups, respectively, in regard to median age at hct ( . vs . yrs, p = . ) and at first dli ( vs years, p = . ). ultimately, there were no statistically significant differences in gvhd or os in products with either higher or lower cd , cd , cd , cd , or cd cellular content. however, the median cd /kg content was more than double in the patients who developed gvhd as compared to patients who exhibited no gvhd after dli ( . × vs . × , p = . ). patients receiving one dli had a -year os of ± % vs those receiving + dli of ± % (p = . ). with a median follow-up of . (range, . - . ) years, the year estimated os of patients in the lymphoid group was higher at ± % vs ± % in the myeloid group, although not significant (p = . ). our results indicate a survival benefit when using dli in a subset of patients who relapse after hct. unlike adult studies demonstrating little effect of dli in lymphoid diseases, many children with all achieved durable remission. while our analysis did not demonstrate that dli cellular content had a statistically significant effect on gvhd or os, it is possible that differences could be found if a larger population and more targeted cell doses were studied. more data will be needed to further define these relationships and identify patients who stand to benefit most. cincinnati children's hospital medical center, cincinnati, ohio, united states background: many arabic speaking muslim parents of children requiring bone marrow transplantation (bmt) receive medical care in the united states. providers may not understand the impact of islamic parents' religious beliefs and practices on their health care experience. objectives: to explore how islamic parents used religion in decision making and to understand the impact of their religious beliefs and practices on their overall health care experience. design/method: we used grounded theory, an inductive method gathering data from interviews and analyzing text, to identify core themes. ten caregivers of bmt children from middle eastern countries were interviewed by an arabicspeaking provider; interviews were coded by an interdisciplinary team. we identified key themes: . patience is a core belief in islam. patience results from the acceptance of allah's will. behaviors showing patience include praying rather than questioning and crying. . al qur'an provides comfort, healing, and protection. families listen to recitations of al qur'an in the patient's room because they feel that this practice not only comforts them but promotes healing as well. for some, certain portions of the qur'an were especially meaningful such as surat al-baqara, which explains that while we may think something is bad for us, allah will know it is good for us. . religious care in the medical center helped families feel respected. religious care in the medical center included interactions with chaplains, who were understood to be "religion experts," and provision of space for prayer and religious resources. . seeking religious consultation. religious consultation from imams or religious scholars (muftis or sheikhs) provides interpretations of the qur'an applied to the family's specific situation helps families make difficult decisions and follow allah's plan. . muslim beliefs guided decision making; muslim practices brought comfort, strength, and peace. drawn from the parents' understanding of islam. parents who addressed this topic said they would only do what islam allowed. they did indicate that most aspects of healthcare were understood to be allowed within islam. additionally, muslim practices of prayer, reading/listening to qur'an, and giving alms all provided comfort, strength and peace. we identified several recurring themes through our interviews that allowed us to understand how families use their muslim faith to deal with their children's illnesses and how it influences their decision making. we believe this better understanding will allow for more informed conversations about patients' health care and decision making, and shows respect for religious beliefs and practices. nemours/dupont hospital for children, wilmington, delaware, united states background: virtually all children will be infected with human herpesvirus (hhv- ) by the age of two. hhv- reactivation after stem cell transplantation causes multiorgan toxicities, including encephalitis, with inflammation and destruction of the temporal lobes and hippocampi, memory loss, and seizures. catatonia is characterized by posturing, immobility, mutism, and autonomic instability, and it's associated with various psychiatric and medical conditions. we describe a patient with hhv- encephalitis and unusual neurologic sequelae, including cognitive and neurobehavioral dysfunction and catatonia, which may impact our understanding of the pathophysiology of hhv- reactivation encephalitis. objectives: describe a case of hhv encephalitis with practice implications for stem cell transplantation. results: our patient was diagnosed with acute myeloid leukemia at age . within years, he relapsed and received two stem cell transplants. on the th day after his second transplant, he developed hyponatremia and refractory seizures. brain mri showed edema in the medial right temporal lobe with linear ischemic change. eeg showed diffuse encephalopathy. cerebrospinal fluid (csf) demonstrated white blood cells, red blood cells, and hhv- by pcr. his prophylactic antiviral was switched to foscarnet and ganciclovir. repeat mri showed abnormal signals in bilateral medial temporal lobes and the right insula. three months later he developed episodes of diaphoresis, hypothermia, agitation, mutism, and unusual posturing, recurring almost daily, recognized as catatonia. mri showed improvement of the abnormalities in the bilateral medial temporal lobes and hippocampi. eegs showed diffuse slowing. after months of antiviral therapy, csf was negative for hhv- . over the ensuing years, he had numerous episodes of diaphoresis, hypertension, hypothermia, pruritis, confusion, agitation, cogwheel rigidity, and bizarre posturing. dopamine blocking agents did not help. clonazepam helped reduce their frequency, and hot showers helped break acute episodes. further mris showed generalized cortical volume loss. he suffered from depression and severely impaired sleep and cognitive function. we describe a novel, debilitating outcome of hhv encephalitis which may provide diagnostic considerations as we continue to improve our understanding of the breadth of possible neurologic sequelae in transplant patients. hhv- is understood to infect and destroy the temporal lobes and hippocampi, but our patient's autonomic dysfunction indicate involvement of the hypothalamus and basal ganglia. antidopaminergic agents may worsen catatonia, and they were not effective for our patient. treatment of catatonia includes benzodiazepines; electroconvulsive therapy was not attempted in this case but may also be useful. background: epstein-barr virus (ebv)-related posttransplant lymphoproliferative disorder (ptld) is a lifethreatening complication in patients following hematopoietic stem cell transplantation, with a frequency estimated at . % and a cumulative incidence of mortality estimated as high as %. studies of ebv have hypothesized that the tonsils are critical for propagating this infection, as tonsillar epithelial cells have been shown to be the site of primary viral infection and continued viral shedding; however, to date no studies have been performed assessing the role of tonsillectomy in patients with ebv ptld. objectives: identify patients with localized ebv ptld treated with tonsillectomy to identify prognostic factors that may be able to help guide future treatment decisions. design/method: patients treated at memorial sloan kettering cancer center who had received hematopoietic stem cell transplantation and had billing codes for both ebv and tonsillectomy were eligible for inclusion in this study. a retrospective chart review was performed, assessing patient demographics, transplant characteristics, laboratory values, tonsillar pathology, and clinical course. any patient who did not have unilateral or bilateral tonsillectomy performed or who had non-localized disease (defined as disease involvement outside of the oropharynx and neck) was subsequently immunodeficiency; % (n = / ) fanconi anemia (fa); % (n = / ) hemoglobinopathy; % (n = / ) non-fa marrow failure and % (n = / ) a metabolic disorder. seventy one percent (n = / ) had normal amh for age pre-transplant, % (n = / ) had low amh for age pre-transplant; of these, % (n = / ) had an oncologic diagnosis; % (n = / ) had fa; % (n = / ) had previously treated hlh; % (n = / ) had non-fa marrow failure; one had a metabolic disorder and one a hemoglobinopathy. of the patients with post-transplant amh measurement % (n = / ) had low levels. of the patients with previously normal pre-transplant amh % (n = / ) underwent myeloablative conditioning (mac) regimen with a % (n = / ) having low amh levels post-transplant compared to %(n = / ) who underwent reduced intensity conditioning (ric) regimen with % (n = / ) having low amh levels post-transplant (p . ). fifteen percent (n = / ) had low levels pre-transplant and underwent mac regimen with % (n = / ) remaining low; % of these patients (n = / ) had fa. nine percent (n = / ) had low levels and underwent a ric regimen with % (n = / ) of amh levels remaining low; % (n = / ) of these patients had hlh treated prior to transplant. conclusion: amh levels can be used for detection of premature ovarian failure and fertility counseling. there is a higher risk of premature ovarian failure with mac regimens and prior chemotherapy vs ric regimens. follow up of this cohort will provide more information to understand the effects of hsct in ovarian function and the usefulness of amh as a predictor of fertility potential. background: there are no proven strategies to prevent blood stream infections (bsi) secondary to oral mucosal barrier injury after hematopoietic stem cell transplant (hsct). additionally, we recently reported progressive gingivitis and dental plaque accumulation in hsct recipients despite our current oral standard of care (three times daily oral rinse). xylitol is a non-fermentable sugar alcohol that reduces dental caries, plaque accumulation, and oral disease progression by inhibiting bacterial growth. we hypothesized that the addition of xylitol to standard oral care will decrease dental plaque accumulation, gingivitis and bacteremia from oral flora. objectives: identify a clinically effective strategy to improve oral health and prevent bsi secondary to bacterial translocation through the oral mucosa in patients undergoing hsct. we are conducting a prospective randomized control study to test our hypothesis. those in the intervention arm receive our current standard of care (three times daily oral rinse) in addition to daily xylitol wipes; controls receive oral standard of care alone. oral exams are performed at baseline and weekly for the first days post hsct. metagenomic shotgun sequencing (mss) of gingival samples is performed at all time points to evaluate microbiome diversity and pathogenic bacterial load. finally, we performed whole genome sequencing of pathogenic bacterial isolates causing bacteremia to assess for genetic relatedness to corresponding strains present within the patient's oral microbiome preceding the infection. : preliminary interim analysis of patients demonstrates improved oral health in patients receiving xylitol (n = ) over those receiving standard of care (n = ), measured by the oral hygiene index (p = . ) and gingivitis index (p = . ). in the nine patients having complete oral mss analysis, xylitol appeared to be associated with decreased streptococcus mitis/oralis domination in the oral microbiome. finally, patients receiving xylitol had no incidence of streptococcus mitis/oralis bacteremia through the first days compared to three patients ( %) in standard of care arm. interestingly, streptococcus mitis/oralis comprised % of the oral microbiome in one child who subsequently developed a streptococcus mitis/oralis bsi. we expect to complete this study in the next months (n = ). the addition of xylitol to oral standard care appears to decrease dental plaque and gingivitis in patients undergoing hsct. xylitol may also impede streptococcus mitis/oralis dominance in the oral microbiome with potential reduction in blood stream infections. (range: - days). twenty-one mdli ( %) were administered because of lymphopenia, fourteen of them ( %) in patients with concomitant viral/opportunistic infections. mixed chimerism/graft failure was the motive of % of the mdli (n = ) and six ( %) were administered to accelerate immune reconstitution. all infusions were well tolerated without appearance or worsening of gvhd. an increase in t-cell counts was observed following six mdli ( . %), although it was a transitory response ( - weeks) in five cases. viral/opportunistic infections were controlled in five cases ( . %), requiring a median of mdli to achieve this response. none of the mdli administered in cases of mixed chimerism/graft failure were effective in reverting this situation. our preliminary data suggests that mdli, is a safe adoptive immunotherapy strategy even with high dose of t-cells without infusion side effects or gvhd complications. some efficacy has been observed in patients with lymphopenia and opportunistic infections, with no positive results in patients with mixed chimerism/graft failure, up to date. however, to determine the real efficacy of this strategy, prospective studies are required. jun zhao, kristen beebe, lucia mirea, alexandra walsh, shane lipskind, alexander, ngwube phoenix children's hospital, phoenix, arizona, united states background: male adolescents undergoing myeloablative hematopoietic stem cell transplantation (hsct) develop infertility with impaired spermatogenesis with reported rates ranging from % to %. in nonmalignant diseases, myeloablative regimens have been replaced with reduced intensity conditioning (ric) with the hopes of better survival rate, less organ toxicity and improved quality of life. despite the increased use of ric regimens for hsct, the effects of ric on fertility remain unknown. objectives: to assess fertility following ric hsct in young adult males. we assessed gonadal function and semen characteristics in adolescent males (> years) who received a single ric hsct at phoenix children's hospital for nonmalignant diseases during - . male patients who were a minimum of year from ric hsct and had postpubertal development at tanner stage iii or above were eligible for this study. gonadal status was assessed by measuring fsh, lh, testosterone, and inhibin b levels, and semen anal-yses assessed fertility indicators (semen volume, sperm concentration, motility, viability, forward progression, morphology, and total count). results: hormone levels and semen analysis have been obtained for patients thus far. the median time between transplant and semen analysis was years. post hsct, ( %) patients showed abnormally elevated lh levels, but fsh, testosterone (total and free), and inhibin b levels were within normal range for all patients. sperm morphology and viability testing were not able to be performed due to low concentrations and volumes. as a result, the total motile sperm count, the most useful estimate for fertile potential, is essentially for all patients. conclusion: recruitment is ongoing, but so far our limited results suggest that ric hsct may have detrimental longterm effects on male fertility. a multi-institutional trial may be appropriate due to small patient numbers at each institution. we are currently exploring options to expand to other centers. further consideration is warranted regarding decisions made by providers, ways to improve anticipatory counseling provided to patients and their families prior to transplant, and how to augment the preventive care of these patients in longterm follow-up. currently all male patients being considered for ric transplant should be counseled to sperm bank prior to transplant. background: a previous systematic literature review identified all published studies of defibrotide treatment for patients of all ages with vod/sos. to assess day+ survival for defibrotidetreated pediatric patients (≤ or ≤ years, per study) all patients exhibited infectious complications with at least viral infection. four patients also had bacterial infections. of note, no patient developed evidence of fungal infections. conclusion: early institution of ecp in patients with high risk acute gvhd (grade - ) was very effective at treating agvhd, allowed for an aggressive steroid taper and contributed to excellent overall survival rates ( %). infectious complications were primarily viral and bacterial, with no fungal infections in this very high risk population. background: vod/sos is a life-threatening complication of hsct conditioning. vod/sos with multi-organ dysfunction (mod) may be associated with > % mortality. defibrotide is approved to treat hepatic vod/sos with renal/pulmonary dysfunction post-hsct in the us and severe hepatic vod/sos post-hsct patients aged > month in the eu. there are few published data on survival of neuroblastoma patients with vod/sos post-hsct. objectives: to report day+ survival and safety post hoc for patients with neuroblastoma and vod/sos post-hsct in the defibrotide t-ind trial. design/method: vod/sos was diagnosed by baltimore or modified seattle criteria or biopsy, with/without mod, after hsct or chemotherapy. defibrotide treatment ( mg/kg/day) was recommended for ≥ days. this post hoc analysis is based on adult and pediatric patients receiving ≥ dose of defibrotide, including with mod. results: among patients with neuroblastoma, developed vod/sos after hsct. for these post-hsct patients, . % were male and . % were female, median age was years (range - years): . % aged - months, . % - years, . % - years, and patient > years. day+ survival data were available for / of these neuroblastoma patients ( with mod and without mod); had autologous and had allogeneic transplants. kaplan-meier estimated day+ survival for the neuroblastoma group was . % ( % confidence interval [ci] , . %- . %). for the mod and no mod subgroups, kaplan-meier estimated day+ survival was . % ( % ci, . %- . %) and . % ( % ci, . %- . %), respectively. in the overall t-ind hsct population aged ≤ years (n = ) and pediatric autologous hsct subgroup (n = ), kaplan-meier estimated day+ survival was . % and . %, respectively. treatment emergent adverse events (teaes) occurred in . % (n = / ), with serious teaes in . % ( / ; most common: multi-organ failure, . % [ / ]). teaes lead to treatment discontinuation in . % (n = ; most common: pulmonary hemorrhage, n = ); death occurred in . % (n = ; > %: multi-organ failure, . %; vod/sos, . %). treatment-related adverse events, as assessed by investigators, occurred in . % (n = ; most common: pulmonary hemorrhage, . %). this post hoc analysis found kaplan-meier estimated day+ survival of . % in patients with neuroblastoma and vod/sos post-hsct, which was consistent with outcomes in pediatric patients after autologous hsct. the safety profile of defibrotide in neuroblastoma patients was consistent with the overall hsct population in this study and other defibrotide studies in pediatric patients. cincinnati children's hospital medical center, cincinnati, ohio, united states background: blood stream infections occur in nearly % of patients undergoing hematopoietic stem cell transplant (hsct) and fever is often the first symptom. timely administration of antibiotics is associated with improved outcomes, thus, early recognition of fever is paramount. current standard of care (soc) includes episodic monitoring of temperature in hospitalized patients, which may delay fever detection. therefore, continuous real-time body temperature measurement may detect fever prior to the current soc. temptraq is a food and drug administration cleared class ii medical device and consists of a soft, comfortable, disposable patch that results: of patients, were started on a pca in the days post hct. % were male with median age of y. % had all, and % aml. matched related donors were used in % and % received tbi. pca was initiated median d+ . oral mucositis alone was the most common indication ( %). a majority of patients were started on hydromorphone ( %); % started on morphine and % started on fentanyl. % started on continuous infusion. pca was used for a median of days (range - days). median pain score was highest d+ of pca use, however, there was inconsistency in charting of numerical pain scores. on d+ , patients had insufficient data to determine efficacy of pain control; of the remaining patients, % had good pain control while % had moderate and % had poor pain control using our devised scale. the most common toxicity observed was respiratory depression (∼ %), however, etiology was often multifactorial and not due to opiates alone. analysis is ongoing to assess variables predicting pca use as well as efficacy of pain control and correlation between current reporting scales and patient perception. conclusion: pca use is common in pediatric hct yet pain control remains inadequate. there's a need for better evaluation of pca management, especially uniform assessment of pain, thereby improving quality of life post hct. children's national health system, washington, district of columbia, united states background: actinomycosis is a rare invasive anaerobic gram-positive bacterial disease caused by actinomyces spp. that may colonize the oropathynx, gastrointestinal tract and urogenitial tract and can lead to abscesses. respiratory tract actinomycosis is characterized by pulmonary cavities, nodules, consolidations and pleural effusions. although actinomyces are nearly always sensitive to penicillin they are frequently resistant to cephalosporins and variable sensitives to fluoroquinolones. although rare in children, immunosuppressed patients are at increased risk for actinomycosis. to describe a case of next-generation sequencing identification of actinomycosis. a -year-old male with a history of very high risk b-cell acute lymphoblastic leukemia who was months status post a / matched unrelated donor bone marrow transplant complicated by prolonged fevers, persistent weight loss, and splenic lesions, treated with posaconazole and levofloxacin developed fever and cough in the setting of neutropenia. blood cultures demonstrated staphylococcus epidermidis. ct showed micronodules and effusion not consistent with s. epi, prompting bronchoscopy. all bacterial cultures were negative. patient was prescribed a three-week course of vancomycin with rapid improvement. design/method: s next generation sequencing (ngs) from bronchoalveolar levage sample was performed at the university of washington laboratory results: ngs assay from bronchoalveolar lavage showed major abundance of actinomyces most closely related to meyeri or oodontolyticus. demonstrated actinomyces. the patient was started on a six month course of amoxicillin with continued clinical improvement. in retrospect, the splenic nodules that were presumed fungal disease were likely actinomycosis, partially treated with levofloxacin. this case highlights the potential utility of ngs in the diagnosis of rare diseases in immunocompromised patients. actinomycosis was only demonstrated through ngs and led to a change in treatment regimen and durable clinical improvement. because actinomyces often mimics malignancy, tuberculosis or nocardiosis, the use of this novel test both targeted appropriate therapy and reduced the exposure to unnecessary medications to treat the differential diagnosis. finally, we highlight that actinomyces should be considered in patients who present with unexplained fevers, weight loss, and night sweats. haneen shalabi, cynthia delbrook, maryalice stetler-stevenson, constance yuan, bonnie yates, terry j. fry, nirali n. shah center for cancer research, national cancer institute, national institute of health, bethesda, maryland, united states background: car-t therapy, while effective, may not be durable for all, and antigen negative escape is a growing problem. hct, in relapsed/refractory all, can be curative, particularly for those in an mrd negative remission. we demonstrated that cd directed car-t therapy effectively rendered patients into mrd negative remissions (by flow cytometry) and the leukemia free survival post-hct was high . in pastorek, jesssica bruce, michael a. pulsipher, chloe anthias, peter bader, andre willasch, jennifer sees, jennifer hoag, wendy pelletier, brent logan, pintip chitphakdithai, lori wiener university of pittsburgh, pittsburgh, pennsylvania, united states background: more than , pediatric hscts are performed in north american and europe each year. the ethics of exposing a healthy child to donation procedures which have some risks and no direct medical benefits continue to be a topic of debate. pediatric donors may experience psychological distress and poorer quality-of-life during and after donation compared to healthy controls. although there are fact/jacie requirements related to the management of pediatric donors, it is unclear what standardized practices exist for psychosocial assessment/management of this group. objectives: to describe transplant center practices for psychosocial evaluation/ management of pediatric donors (< years) and to examine differences in practices by location (cibmtr/ebmt) and number of harvests (volume). design/method: data were collected via a single crosssectional survey distributed electronically to cibmtr and ebmt centers between / / and / / . : / ( %) of cibmtr and / ( %) of ebmt centers completed the survey. most centers had written eligibility guidelines for pediatric donors ( %). most also had a process for ensuring that donors were freely assenting to donate ( %), managed by a transplant physician ( %). a single physician often jointly managed donor/recipient care ( %). half of centers had a pediatric donor advocate ( %), who was most often a physician ( %) or social worker ( %). cost was the largest barrier to having a donor advocate ( %). most centers performed psychosocial screening of donors ( %) but rarely declined donors based on psychosocial concerns ( %). less than half of centers provided post-donation psychosocial follow-up ( %). comparisons by center location indicated that ebmt centers were more likely to have a physician doing joint donor/recipient care ( % vs. %; p = . ), less likely to have a psychosocial assessment policy ( % vs. %; p = . ), less likely to have a donor advocate ( % vs. %; p = . ), but marginally more likely to do post-donation psychosocial follow-up ( % vs. %; p = . ). large volume centers were more likely to have a psychosocial assessment policy than their medium/smaller counterparts ( % vs. %, %; p = . ) â€"there were no other differences on key psychosocial management variables by volume. although most centers have written guidelines for pediatric donor eligibility and mechanisms for ensuring assent, substantial numbers of donors do not undergo psychosocial assessment, are jointly managed with the recipient by a single physician without an assigned donor advocate, and do not receive psychosocial follow-up. the field would benefit from guideline development for the psychosocial management of pediatric donors. background: germline mutations in samd and samd l genes cause mirage (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy) and ataxia-pancytopenia syndromes, respectively, and are associated with chromosome deletions, mds and bone marrow failure (bmf). there are limited data on outcomes of hct in these patients. to describe outcomes of allogeneic hct in patients with hematologic disorders associated with samd /samd l mutations. results: seven patients underwent allogeneic hct for primary mds (n = ), congenital amegakaryocytic thrombocytopenia (camt)(n = ), and dyskeratosis congenita (n = ). retrospective exome sequencing revealed gain-of-function mutations in samd (n = ) or samd l (n = ) genes. constitutional mosaic monosomy was present in cases. two samd patients had features of mirage syndrome. unusual findings of panhypopituitarism, laryngeal cleft, and glomerulosclerosis were noted in one case. in another case with a samd mutation hypospadias & bifid scrotum were the only findings. the remaining patients had no phenotypic abnormalities. median age at hct was y (range: . - . ). patients received transplants from bone marrow (matched unrelated (n = ) & hla identical sibling (n = )), or unrelated cord blood (ucb) (n = ). five mds patients received myeloablative s of s conditioning (busulfan-based (n = ) or tbi-based (n = )); patients (mds (n = ); camt (n = )) received reducedintensity conditioning (ric) (fludarabine, cyclophosphamide, with ratg or alemtuzumab). syndrome-related comorbidities (diarrhea, infections, malnutrition, electrolyte imbalance, lung disease and hypoxia) were present in both patients with mirage syndrome. one patient with a familial samd l mutation, mds and morbid obesity failed to engraft following ric double ucbt. she died one year later from refractory aml. all other patients achieved neutrophil and platelet engraftment, at a median (range) of ( - ) and ( - ) days, respectively. posttransplant complications included severe hypertension (n = ), pericardial effusions (n = ), veno-occlusive disease of liver (n = ), and recurrent aspiration pneumonias (n = ). one patient developed grade iii agvhd which resolved with treatment. one patient developed mild skin cgvhd and suffers from chronic lung disease. all surviving patients had resolution of hematological disorder and sustained peripheral blood donor chimerism ( - %). overall survival was % with a median follow-up of years (range: . - . y). patients with hematological disorders associated with germline samd /samd l mutations tolerated transplant conditioning without unusual, or unexpectedly severe toxicities. allogeneic hct led to successful resolution of mds or bmf, with excellent overall survival. more data is needed to refine transplant approaches in samd /samd l patients with significant comorbidities, and develop guidelines for their long-term follow-up. shyamli singla, tiffany simms-waldrip, andrew y. koh, victor m. aquino background: steroid-refractory acute graft versus host disease (agvhd) is a potentially fatal complication of allogeneic hematopoietic stem cell transplantation (hsct). basiliximab (anti-il -r monoclonal antibody) as a single agent or in combination infliximab (anti-tnf-monoclonal antibody) has demonstrated efficacy in adult cohorts with steroid-refractory agvhd, but has not been well studied in the pediatric population. we adopted the use of basiliximab and infliximab as our institutional standard of care for steroid-refractory agvhd in pediatric hsct patients. to determine the response and survival of hsct children who received basiliximab and infliximab for the treatment of steroid-refractory agvhd. design/method: we retrospectively reviewed children who received basiliximab and infliximab for steroid-refractory agvhd refractory between september and december . complete response (cr) was defined as resolution of all clinical signs of agvhd. partial response (pr) was defined as at least one grade reduction in one target organ (e.g. skin, gut or liver) without increased grade in another target organ. no response was defined as either no improvement or progressive worsening of agvhd in at least one organ. baseline demographics, transplant details, laboratory findings, and treatment outcomes were also evaluated. results: of the evaluable hsct patients, children (median age yrs, range mo- yrs) with steroid-refractory agvhd received combination monoclonal antibody (mab) therapy. the median time from the start of steroid therapy to initiation of mab was days. the overall glucksberg grade of agvhd at the time of initiating mab therapy was grade i (n = , . %) ii (n = ; %), iii (n = ; %) or iv (n = ; %). the overall response rate was %, with ( %) patients achieving cr, ( . %) patients achieving pr, and ( . %) patients with no response at days following the start of mab therapy. the median overall survival was , , and days for patients who exhibited cr, pr, and no response, respectively. the overall survival at year following start of mab therapy was %. background: the role of high dose chemotherapy (hdc) and autologous stem cell rescue (ascr) in patients with high risk (advanced metastatic or relapsed) soft tissue sarcomas is controversial. despite multimodal chemotherapy, radiotherapy, and local control measure advancements, prognosis of patients with advanced metastatic or unresectable and relapsed sarcomas remains poor, with less than % years disease free survival. objectives: to determine if consolidation with myeloablative hdc and ascr improves relapse free (rfs) and overall survival (os) outcomes in a high risk patient subgroup. we performed retrospective review of all high risk soft tissue sarcoma patients who underwent hdc and ascr at the children's hospital at montefiore, bronx, ny between october and january . the protocol was approved by albert einstein college of medicine institutional review board. results: patients ( primary metastatic high risk disease, relapsed or recurrent disease) received hdc with ascr. primary diagnoses were rhabdomyosarcoma (rms) (n = , alveolar histology), primary site nasopharynx (n = ) and lower extremity (n = ). ewing's sarcoma (ews) (n = ), axial site (pelvic) in patients ( %). median age years (range - years), ( %) were male. all patients were in complete metabolic remission before transplant. median pre transplant comorbidity index was (range - ). patients ( rms and ews) received conditioning with carboplatin, etoposide and melphalan. remaining patients with ews received conditioning with busulfan, melphalan and topotecan. all patients received peripheral blood mobilized hematopoietic stem cell transplantation. stem cell mobilization achieved with high dose filgrastim in all patients except one who required addition of plerixafor. median cd +/kg s of s recipient body weight cell dose infused was . × ^ (range . - . × ^ ). median times to neutrophil and platelet (> , / l) engraftment were (range - ) and ( - ) days respectively. patients ( %) developed bk viuria (one with grade iii hemorrhagic cystitis); ( %) developed cmv viremia; and one patient ( %) had asymptomatic ebv viremia. there was no graft failure, sinusoidal obstruction syndrome or transplant related mortality. median follow up post-transplant was days (range - days). year probability of os and rfs were % and % respectively. hdc with ascr is a promising therapeutic strategy to consolidate remission and improve survival in select high risk soft tissue sarcoma patient subgroups. prospective clinical trials will inform the impact of disease status prior to hdc and ascr on outcome, optimal conditioning and long term relapse free and overall survival. background: absence of minimal residual disease is paramount for cure of pediatric acute lymphoblastic leukemia (all). the testis may harbor occult leukemia and this disease may result in treatment failure. objectives: the purpose of this study was to assess the longterm outcomes of boys with or without testicular leukemia pre-hematopoietic stem cell transplantation (hsct). design/method: retrospective analysis of boys with high-risk de novo ( with hypodiploidy all) or recurrent/refractory all was conducted. flow cytometry of bone marrow mononuclear cells was used to determine remission status. testicular evaluations were performed by physical examination and wedge biopsy pre-hsct. the median age at time of transplant was . years. all patients were in remission by flow cytometry of bone marrow mononuclear cells at the time of transplant and none had evidence of clinically apparent testicular disease. testicular leukemia was detected in patient and he underwent bilateral orchiectomy. he developed acute graft versus host disease (gvhd) of the duodenum and sigmoid colon which resolved, and the leukemia remains in second complete remission and he is free of hsct-related morbidity . months post-hsct. of the patients without testicular leukemia died a median of . months (range, . to . ) post-hsct ( with adenovirus infection and each with thrombotic microangiopathy and aspergillus pneumonia); experienced infection (staphylococcus species, corynebacterium, enterococcus, klebsiella, citrobacter, e. coli, epstein barr virus, adenovirus, bk virus, human herpesvirus- , candida albicans, fusarium, aspergillus, yeast, and other fungus); experienced gvhd ( of the gi tract, of the skin, of the liver, of the eyes, of the mouth, and of the lungs); and developed a second neoplasia (right lower leg leiomyosarcoma). one patient developed bone marrow minimal residual disease ( . % phenotypically abnormal cells detected months after / matched sibling hsct). reinduction therapy comprised weekly doses of rituxan, courses of blinatumomab and donor lymphocyte infusions with il- . two subsequent bone marrow evaluations were minimal residual disease negative. thirteen months post-hsct residual disease recurred ( . %) and he will receive inotumumab. overall median survival post-transplant of the boys is . months (range, . to . ) and of the surviving boys is . months (range, to . ). conclusion: testicular biopsy can detect occult leukemia pre-hsct. testicular leukemia pre-hsct does not appear to increase the risk of subsequent relapse or other hsct-related adverse events compared to those without it. yaya chu, nang kham su, sarah alter, emily k. jeng, peter r. rhode, mathew barth, dean a. lee, hing c. wong, mitchell s. cairo new york medical college, valhalla, new york, united states background: rituximab has been widely used in frontline treatment of b-nhl including burkitt lymphoma (bl), however, some patients retreated with rituximab relapse, which limit patient treatment options. novel therapies are desperately needed for relapsed/refractory b-nhl patients. several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering immune cells with chimeric antigen receptors (car), as well as second-generation anti-cd antibodies. nature killer (nk) cells play important roles in the rejection of tumors. however, nk therapy is limited by small numbers of active nk cells in unmodified peripheral blood, lack of tumor targeting specificity, and multiple mechanisms of tumor escape of nk cell immunosurveillance. our group has successfully expanded functional and active peripheral blood nk cells (expbnk). b t m was generated by fusing alt- , an il- superagonist, to four single-chains of rituximab. b t m displayed tri-specific binding activity through its recognition of the cd molecule on tumor cells, activated nk cells to enhance adcc, and induced apoptosis of b-lymphoma cells. objectives: to examine if b t m significantly enhances the cytotoxicity of expbnk against rituximab-sensitive and -resistant bl cells. design/method: expbnks were expanded with lethally irradiated k -mbil - bbl and isolated using miltenyi nk cell isolation kit. alt- and b t m were generously provided by altor bioscience. nk receptors expression and cytotoxicity were examined as we previous described. ifng and granzyme b levels were examined by elisa assays. equal doses of rituximab, alt- , rituximab+alt- , obinutuzumab (obinu) were used for comparison. igg was used as controls. anti-cd car expbnk cells were generated as we previously described by mrna electroporation. rituximab-sensitive raji andresistant bl cells raji- r and raji- rh, were used as target cells. results: b t m significantly enhanced expbnk cytotoxicity against rituximab-sensitive raji cells, rituximab-resistant raji- r cells and resistant raji- rh cells compared to the controls igg, rituximab, alt- , rituximab+alt- , obinu (p< . , e:t = : ). furthermore, we confirmed the enhanced cytotoxicity by measuring ifn-g and granzyme b production. b t m significantly enhanced ifn-g and granzyme b production from expbnk against raji, raji- r and raji- rh compared to igg (p< . ), rituximab (p< . ), alt- (p< . ), rituximab+alt- (p< . ), and obinutuzumab (p< . ). when compared to anti-cd car expbnk cells, b t m + expbnk had the similar cytotoxicity against raji, raji- r and raji- rh as anti-cd car expbnk cells did (p> . ). conclusion: b t m significantly enhanced expbnk activating receptor expression and in vitro cytotoxicity against rituximab-sensitive and -resistant bl cells. the in vivo functions of b t m with expbnk against rituximab-sensitive and -resistant bl cells using humanized nsg models are under investigation. background: cardiac dysfunction, including left ventricular systolic dysfunction (lvsd), is a known complication in stem cell transplant (sct) survivors. while detection of lvsd by echocardiography is important in this population, there has been minimal research to determine if subclinical cardiac dysfunction exists in sct patients. cardiopulmonary exercise testing (cpet) is a valuable tool to assess cardiac function, and to determine how the heart responds to the stress of exercise. no studies have been performed to determine if sct patients with normal lvsd on standard echocardiography may have abnormal cpet. to determine the feasibility of cpet, as well as additional echocardiographic parameters, to detect dysfunction in sct patients with a normal ejection fraction on echocardiogram. design/method: we performed a cross-sectional analysis of sct survivors who were at least years post sct, years of age or older and with an ejection fraction > % (low end of normal range) on echocardiogram. we assessed the exercise capacity of all patients with cpet, and sub-clinical cardiac dysfunction through tissue doppler and strain analysis from the echocardiogram. results: seven patients ( male) have qualified and completed this study so far with an average age of . ± . years. the median time from transplant is . ± . years. all seven patients had a normal ejection fraction, however four patients had abnormalities on their cpet. these abnormalities included abnormal predicted peak oxygen consumption (vo ) ( %± . , normal > %) (the best predictor of functional capacity), predicted oxygen pulse ( %± . , normal > %) (measure of cardiac stroke volume) and ventilatory efficiency (ve/vco slope) ( ± . , normal < ). submaximal exercise data, used when patients are unable to complete a maximal effort test, demonstrated low-normal predicted vo at anaerobic threshold ( . %± . %, normal > % of was . days while patients who received autologous infusions had a mean number of days to engraftment of . . engraftment after hsct needs to be prompt to minimize duration of neutropenia and maximize survival rates . our data demonstrates that the infusion of hematopoietic stem cell products with a syringe or iv pump is an effective method of delivery for stem cell products and does not delay the time to engraftment. the median days to neutrophil engraftment was . days. this is comparable to data from the nmdp, which reports engraftment occurs within - days. the main limitation to this study was its small sample size due to the number of transplants done at our center. however, it does provide evidence to support that infusion of stem cell products via pump mechanism is a safe alternative to the infusion by gravity method in the process of the hematopoietic stem cell administration. johns hopkins all children 's hospital, st. petersburg, florida, united states background: leukemic relapse remains the most common cause of treatment failure after allogeneic hematopoietic cell transplant (allohct) for myeloid malignancies. most children who relapse post-allohct will die of their disease, making interventions to minimize this risk a high priority. objectives: to evaluate the safety and efficacy of posttransplant azacitidine for relapse prevention in children undergoing allohct for myeloid malignancy. design/method: we retrospectively reviewed the charts of children undergoing allohct for myeloid malignancies between february and november at johns hopkins all children's hospital. results: during the study period, children (ages to years, median ) underwent allohct for myeloid malignancies: de novo acute myeloid leukemia (aml), ; mixed phenotype acute leukemia, ; treatment-related aml, ; juvenile myelomonocytic leukemia with aml transformation, ; and myelodysplasia/aml, . thirteen were in first complete remission, were in cr or greater. most patients ( / ) received fludarabine/melphalan/thiotepa conditioning; received hla-identical related or unrelated donors, and received haploidentical bone marrow grafts with post-transplant cyclophosphamide. three patients never received planned azacitidine ( early relapse; early trm), leaving evaluable patients. azacitidine ( mg/m /dose for days, in -day cycles for up to cycles) was started at a median of days post-transplant (range - ). two-thirds ( / ) of patients received eight or more cycles. of five patients who stopped therapy early, only one was due to toxicity; other reasons included severe gvhd ( ), parental preference ( ), and relapse ( ). cycle delays occurred in patients, with a median cycles delayed per patient, mostly for mild myelosuppression with early cycles. no patient required blood product transfusion during therapy, but g-csf was used in three patients to maintain anc> / l. dose-modifications were made in patients (renal tubular acidosis, acute kidney injury, and myelosuppression). there were relapses ( %), two of which occurred in patients in cr , for a relapse incidence of % in patients in cr , with a median follow-up of months (range . to ). no patients who received azacitidine died of transplant-related mortality. conclusion: administration of azacitidine in children undergoing allohct for myeloid malignancies is safe and feasible, with most patients successfully receiving all planned cycles. toxicity was acceptable and there was no trm or secondary graft failure. despite the limitations of a small cohort, relapse incidence-particularly in patients transplanted in cr suggests a potential benefit in disease control that warrants investigation in follow-up studies. background: despite significant improvements in the success rate of hematopoietic cell transplantation (hct), graft failure remains an important complication in patients transplanted for severe aplastic anemia (saa). second allogeneic hct can salvage patients, but -year overall survival (os) rates have been reported as low as % . objectives: identify patients who developed dropping donor chimerism, graft rejection, and/or graft failure after first hct for saa, necessitating additional hcts or cellular boosts (defined as stem cell products infused without preceding chemotherapy), and evaluate treatment-related complications and os. with vod/sos with and without multi-organ dysfunction (mod) pubmed and embase databases were searched for "defibrotide and retrospective chart reviews; excluded publication types were: case reports (< cases); meta-analyses; reviews; animal, modeling, pharmacokinetic, chromatography, and adult-only studies; guidelines; articles; and letters. resulting reports were screened for exclusion criteria. full-text articles were then reviewed for eligibility. study characteristics of selected publications were summarized, and publications were categorized by patients' mod status. when necessary, additional data tables were requested. a random effects model was used for pooling data for efficacy. interstudy heterogeneity was assessed with cochran's q-test. percentage of total variation across studies due to heterogeneity (i ) was evaluated we quantified ∼ proteins in each sample. reproducibility for one donor at different time points children 's minnesota, minneapolis, minnesota, united states background: pediatric and young adult hodgkin lymphoma (hl) has five-year survival rates > %. chemotherapy required to achieve this rate is associated with a lifetime risk of cardiac deaths, second malignancies, pulmonary disease and infertility. as effective salvage therapy exists, outcomes may be improved by de-intensifying initial therapy to lessen toxicity.objectives: we piloted a regimen in low and intermediate risk hl patients using agents without known association to significant late effects. this retrospective chart review was approved by children's minnesota irb.design/method: the bvg(p) regimen incorporated bortezomib ( . mg/m day , , , ); vinorelbine ( mg/m day , ); gemcitabine ( mg/m day , ) every days and prednisone ( mg/m /dose bid x days). we treated newly diagnosed patients, ages - years, with non-bulk stage iia (n = ) or iib (n = ) hl. two patients received bvg and received bvgp with the addition of prednisone.results: newly diagnosed patients were all pet negative after the first or second cycle and remained pet negative at end of therapy, cycles. nausea was well controlled with -ht antagonists and scopolamine. pegfilgrastim was not necessary due to the high absolute neutrophil count nadir [median . and minimum . × /l]. there were no episodes of febrile neutropenia, infection or transfusion need. no patients experienced alopecia. one patient developed sensory neuropathy after the eighth dose of bortezomib that was controlled with gabapentin and a switch to subcutaneous bortezomib administration. of the five newly diagnosed patients, four remain in remission at , , , days; relapsed at previous disease sites at days and subsequently achieved remission with bvgp with the addition of brentuximab. this series provides early evidence to stimulate expansion of this pilot experience and subsequent multiinstitutional study leading to a randomized trial of bvgp and current chemotherapy for low and intermediate hl. st jude affiliate clinic at st francis hospital, tulsa, oklahoma, united states background: symptoms suggestive of morning hypoglycemia has been noticed in children receiving all chemotherapy. only few small studies looked at this therapy related complication. factors increase risk of hypoglycemia in all patients include accelerated starvation, steroid induced adrenal suppression, mercaptopurine therapy and prolonged fasting for procedures.objectives: to study the prevalence and risk factors for hypoglycemia during all therapy design/method: medical records of of children (up to years old) treated for all between - ( patients) were studied for evidence of morning hypoglycemia defined as blood sugar (bs) < mg/dl. statistical mean differences between the subgroups were analyzed with spss using a nonparametric mann-whitney u test.results: fifty two percent ( %) of patients developed hypoglycemia during all treatment, with an average of . episodes/patient. % were males and % females. almost / ( %) of patients with hypoglycemia were in maintenance phase of therapy. % of hypoglycemic episodes occurred in % of patients. majority of hypoglycemic episodes ( . %) occurred on the day of procedure when patients were fasting overnight. . % of hypoglycemic episodes occurred in children ≤ years, with . % in ≤ years. patients who developed hypoglycemia were significantly younger (mean age at time of diagnosis of all was . ± . at the hypoglycemia group versus the non-hypoglycemia ( . ± . ) p< . . no statistically significant difference was found regarding sex, or tpmt genotype. % of hypoglycemic children-all < years of age-presented with life threatening hypoglycemia symptoms including seizure and loss of consciousness. this study showed high prevalence of hypoglycemia during childhood all therapy. younger age, especially ≤ years, is associated with higher risk of hypoglycemia as well as life-threatening episodes. to decrease fasting hypoglycemia during therapy for childhood all, we recommend that children under the age of years receive bed time snack high in proteins and complex carbohydrates, and to get them up early the day of procedure to take clear sugary drink. hospital for sick children, toronto, ontario, canada ann & robert h. lurie children's hospital of chicago, chicago, illinois, united states background: childhood brain tumors are the most common solid malignancy and the leading cause of cancer-related mortality in children. the most aggressive type of pediatric central nervous system (cns) tumors is diffuse intrinsic pontine glioma (dipg). despite decades of clinical trials, there has been no substantial improvement with respect to therapeutic outcomes with most children eventually succumbing to the disease. research on adult high-grade gliomas has shown a targetable pathway through the inflammationinduced expression of indoleamine , dioxygenase (ido ) and its recognized ability to suppress the anti-tumor immune response. a limited understanding into the role of ido in pediatric central nervous system tumors serves as the foundation of this research project. furthermore, the integration of nanotechnology is a fundamental step for the investigation and targeting of ido . spherical nucleic acids (snas) composed of nanoparticles have been shown to transverse cellular membranes, exhibit stability in physiological environments, escape from degradation, and create precise targeting in brain tumors.objectives: the purpose of our project is to delineate the role of ido in pediatric dipg, and develop small inhibitory (si)rna oligonucleotides and snas aimed at therapeutically inhibiting the gene expression of immunosuppressive ido . our specific aims are to: ( ) confirm the gene expression ido in different human dipg cell lines; ( ) generate and characterize sirna oligonucleotides targeting human ido in vitro; and ( ) generate and characterize gold nanoparticles for targeted inhibition of ido .design/method: unique patient-derived dipg cell lines were grown in culture, stimulated with increasing concentrations of the proinflammatory cytokine, ifn , and analyzed for mrna levels. sirna specific to ido was transfected into cells. sna generation is in progress.results: ido is expressed in multiple human pediatric dipg cell lines. sirna targeting ido among exons and results in a significant decrease in overall ido expression by dipg cells. sna generation for targeting ido with improved penetration & stability is ongoing, with preliminary results demonstrating a robust ability to inhibit ido expression. the grim prognosis of children with dipg, the lack of effective therapies, and the expression of ido by human dipg cells emphasize the importance of developing the treatment capability to inhibit ido gene expression, as a excluded from this study. the remaining patients were analyzed using descriptive statistics.results: a total of patients meeting inclusion criteria were identified. of these, patients ( . %) received tonsillectomy alone, ( . %) underwent tonsillectomy and decreased immunosuppression, ( . %) received tonsillectomy and rituximab, and another ( . %) received tonsillectomy with additional therapy (including ebv-specific cytotoxic tlymphocytes, donor leukocyte infusion, and chemotherapy). of the patients who received tonsillectomy with or without a decrease in immunosuppression, all were diagnosed with high-grade lymphoma and achieved clinical remission following tonsillectomy with no evidence of relapse to date. on further analysis looking at ptld risk factors, all patients were under years of age, all received t-cell depleted grafts, and none had significant graft-versus-host disease (gvhd) at the time of ptld diagnosis. we have identified a population of patients with localized ebv ptld that achieved clinical remission with no evidence of recurrence following tonsillectomy, suggesting that tonsillectomy alone may be an adequate treatment for localized ebv ptld in a specific subgroup of patients. further analysis is needed to identify characteristics of this subgroup to determine which patients would be most likely to respond to this treatment. university of rochester, rochester, new york, united states background: malignant central nervous system (cns) tumors in young children have a poor prognosis and pose a significant therapeutic challenge. consolidation therapy with carboplatin and thiotepa was piloted in ccg- , cog acns , and cog acns with the goals of intensifying therapy and omitting or delaying radiation.objectives: to document outcomes for patients undergoing carboplatin/thiotepa consolidation with autologous stem cell rescue (ascr) and to demonstrate the feasibility and toxicity of this regimen.design/method: patients up to years old (median age: months) with malignant cns tumors treated at the university of rochester from - with at least one cycle of carboplatin ( mg/kg/day x days) and thiotepa ( mg/kg x days) followed by peripheral blood ascr were included in retrospective analysis. data were recorded on time to engraftment (defined by absolute neutrophil count (anc) recovery to > . × ^ /l), length of hospitalization, toxicity with each consolidation cycle, progression free survival (pfs) and overall survival (os). stem cell harvest data were also collected.results: eleven patients with malignant cns tumors ( atypical teratoid/rhabdoid tumor, primitive neuroectodermal tumor, glioblastoma multiforme, and pineoblastoma) received a total of cycles of carboplatin/thiotepa. of these, underwent stem cell harvest at our institution, with complications limited to procedure-related hypotension for patient with known autonomic instability, and catheter-associated deep vein thrombosis (dvt) for patient. four patients were in complete remission (cr) /status-post gross total resection, was in cr , and had residual tumor at the time of consolidation. nine patients received planned consolidation cycles, patient (of ) planned cycles, and patient of an anticipated cycles thus far. average time to engraftment for these cycles was . (+/- . ) days, with a mean hospital length of stay of (+/- . ) days. fever occurred in of cycles ( %); infectious toxicity included documented bacterial infection in cases (enterococcus faecalis bacteremia in , klebsiella pneumoniae in ). there were no regimenrelated deaths. with a mean follow-up of months, survivors have not yet completed all therapies, and patients have relapsed ( have died of disease). of the survivors, have been disease-free for > months. background: autologous hematopoietic stem cell transplantation (auto-hsct) has resulted in improved survival for patients with high-risk neuroblastoma. treatment intensification is however associated with greater complications. data on early infectious complications in low-and-middle income countries are limited.objectives: to review the early infectious complications following auto-hsct in patients with high-risk neuroblastoma.design/method: a retrospective chart review of pediatric patients with high-risk neuroblastoma who underwent auto-hsct at the american university of beirut medical center between and was conducted. infectious complications during the first days post-transplant were reviewed.results: forty-three patients ( males and females) with a median age at diagnosis of . years [range: . - . ] years underwent auto-hsct during the above-mentioned period. conditioning regimen consisted of melphalan, etoposide and carboplatin. all patients received antiviral and antifungal prophylaxis. median time for neutrophil engraftment was days [range: - ]. bacteremia and clostridium difficile infections occurred in ( %) and ( %) patients respectively. seven ( %) patients developed enterocolitis diagnosed by imaging, were adenovirus induced. cmv viremia was diagnosed in ( %) patients, of whom required treatment. varicella zoster reactivation, parvovirus viremia, toxoplasmosis encephalitis, bk virus cystitis ( patients) and central nervous system ebv related post-transplant lymphoproliferative disorder were diagnosed in different patients. there was no invasive fungal infection. sixteen ( %) patients have died, of whom died in the early post-transplant period, due to disease progression and ( . %) due to infectious complications. among the patients who died due to infection, developed toxoplasmosis encephalitis, developed severe enterocolitis, of which were adenovirus related. the mean igg level within one week post-transplant was lower in patients with clinically significant viral infection compared to others ( vs . mg/dl, p: . ). the mean igg level at the time of clinically significant bacterial infection was lower in infected patients compared to others ( . vs . mg/dl, p: . ). neither absolute lymphocyte count nor absolute neutrophil count at day post-transplant affected the incidence of clinically significant infections. our results show that the rate of infections during the early post auto-hsct period is higher than what has been described in developed countries and has a significant impact on mortality. prevention, early detection and improvement in the treatment is required to improve outcome. university of miami, miami, florida, united states background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative treatment for many malignant and non-malignant (bone marrow failure, immunodeficiency, or metabolic diseases) in pediatrics. despite advances in medicine, graft-versus-host-disease (gvhd) remains a significant cause of non-relapsed morbidity and mortality, specifically in those with malignant diseases.objectives: to highlight the complexity to acute gvhd management and seldom-described treatment approach. a year male with a history of high risk acute myeloid leukemia (aml) due to failed induction therapy. he received a matched ( / ) unrelated donor hsctmarrow product-conditioned with busulfan, fludarabine, and anti-thymoglobulin (atg). his post-transplant course was complicated by hhv- viremia, pres (prompting a change from prograf to cyclosporine), mucositis, and grade iii acute gvhd (skin s , gut s , liver s ) around post transplant day , which later morphed to ocular involvement by d+ . he was started on mg/kg steroids with good response but flared up with each attempt to taper steroid dose. a course of rituximab and later atg were tried without success in weaning off steroids. switching cyclosporine to sirolimus did not provide any additional benefit either. extracorporeal photopheresis (ecp) was started times a week. he initially responded well, yet was not able to wean off steroids. in addition, he developed a flare when ecp session was reduced to days per week. ecp was therefore increased to days per week, which appeared to stabilize skin lesions. a trial of weekly methotrexate was attempted to wean off steroids and photopheresis, which provided no response. finally, a trial of bortezomib on days , , , and of a day cycle as published in a case series of multiple myeloma patients who developed post hsct gvhd. skin lesions improved remarkably however dose had to be reduced due to related pancytopenia. given the response to therapy, he was continued on a weekly dose of bortezomib, receiving a total doses, which has permitted the slow taper of prednisone that has since been discontinued without a major flare. he however is currently maintained on ecp times per week, which is now been slowly withdrawn.conclusion: management of acute gvhd in pediatric patients after hsct can be challenging with no definite options for those who fail steroids or become steroid dependent after initial response. in these situations, bortezomib could be a valid therapeutic option. background: neuroblastoma (nbl) is the second most common solid tumor in children and despite recent treatment advances, overall survival for high risk nbl remains < %. the addition of immunotherapy has improved survival and includes anti-gd antibody therapy. the success of antibody therapy in neuroblastoma is primarily due to natural killer (nk) cell mediated antibody dependent cellular cytotoxicity. we previously demonstrated that nk cells from patients with high risk nbl can be successfully isolated and expanded to large numbers and exhibit potent anti-tumor effects against nbl ( ). thus, infusions of autologous expanded nk cells in high risk nbl in combination with anti-gd antibody are being studied in clinical trials. toll-like receptors (tlr) present on the surface of leukocytes are responsible for pathogen recognition, and activation of these receptors stimulate the production of cytokines that critically link innate and adaptive immune responses. the tlr agonist, poly(ic) is a synthetic analog of dsrna that has previously been shown to directly stimulate cytokine production and improve cytotoxicity in primary nk cells through activation of genes regulated by interferon-response elements (ire) ( ). we hypothesized that ex vivo activation of tlr pathways in nk cells during our normal -day expansion using k feeder cells expressing membrane bound il- would enhance their function.design/method: nk cells were isolated from peripheral blood mononuclear cells and expanded with our previously described expansion protocol in media containing il- and ug/ml poly(ic) ( ). at the end of the -day expansion, nk cells expanded with poly(ic) were compared to controls using a calcein cytotoxicity assay to measure cytotoxicity against high risk neuroblastoma and cytometric bead array to measure cytokine production. : surprisingly, the addition of poly(ic) during nk cell expansion did not improve proliferation, cytokine production or cytotoxicity compared to our standard expansion method. rnaseq demonstrated that our standard expansion method results in a modest decrease in tlr expression at the transcriptional level, but significant upregulation of several ireregulated genes. we conclude that either our standard approach interferes with tlr signaling or saturates the innate immune response pathway such that co-stimulation with poly ic does not produce an additive effect. we are performing expression analysis on nk cells receiving poly(ic) during expansion to further explore this hypothesis. background: gonadal dysfunction leading to infertility is a complication after hematopoietic stem cell transplant (hsct). anti-müllerian hormone (amh) is a marker of ovarian reserve; it is not controlled by gonadotropins and has minimal inter-cycle variations, therefore, it can be used as a marker of ovarian reserve and aid in fertility counseling.objectives: assess ovarian reserve in hsct patients utilizing amh levels. background: tgf beta is an immune suppressive cytokine frequently elevated in the tumor microenvironment causing tumor immune evasion. acute tgf beta treatment potently inhibits nk cell cytotoxicity, cytokine secretion, and proliferation. however, tumor infiltrating nk cells receive chronic inhibitory tgf beta signals in conjunction with activating signals from tumor cells. objectives: to this end, we hypothesized that long-term tgf beta-cultured nk cells would induce functional and phenotypical changes on nk cells that differ from short-term tgf beta treatment.design/method: to explore this, primary human nk cells were cultured with the leukemia cell line, k , alone or with exogenous tgf beta for weeks. : surprisingly, nk cells cultured in tgf beta proliferated faster, and upon challenge with a variety of cell line targets they secreted much greater quantities of ifnÎ ( -to -fold increase against / cell lines) and tnf ( -to -fold increase against / cell lines). further, the high cytokine secretion induced in these nk cells was no longer inhibited by adding additional tgf beta. degranulation was also increased ( / cell lines), however cytotoxicity was not enhanced in a -hour cytotoxicity assay. after resting in il- , the cytokine hypersecretion of tgf betacultured nk cells was maintained for several weeks suggesting this functional change might involve cellular reprogramming. we investigated the mechanism behind these functional changes and profiled genes involved in tgf beta signaling. we found significant reduction of smad transcription which corresponded to a striking decrease in smad chromatin accessibility. we also found significantly increased smad and decreased tgfbr expression. phenotypic analysis revealed that tgf beta also induced remodeling of the nk receptor repertoire with decreased nkp , cd , and klrg and upregulation of trail. the functional consequences of these tgf beta-induced changes on in vitro and in vivo nk cell function are currently under investigation. background: the use of t-cell depleted grafts in haploidentical stem cell transplantation (hsct) has been associated with a delay in early t-cell recovery which increases the risk of viral infections, relapse or graft rejection. conventional donor lymphocyte infusion (dli) after hsct transplantation is effective but conditioned because of a high prevalence of gvhd. the infusion of selected lymphocyte subpopulations with low aloreactivity is emerging as an effective strategy to rectify this issue. the depletion of cd ra+ naive lymphocytes, preserving cd ro+ memory t-cells, could provide a safe source of functional lymphocytes with anti-infection, antileukemic and anti-rejection properties, and lower rates of adverse effects. our objective is to present data of patients that have received cd ro+ memory t-cells dli (mdli) and assess its safety and outcome. we present data of mdli performed after hsct in cases of mixed chimerism, persistent lymphopenia, viral/opportunistic infections or as a strategy to accelerate immune reconstitution.results: fifteen patients with diagnosis of all (n = ), aml (n = ), mds (n = ), saa (n = ), sideroblastic anemia (n = ) and cgd (n = ), received mdli after hsct. a total of forty-three mdli were infused. the median dose of cd ro+ memory t-cells infused was . × /kg (range: . × - . × /kg), with a median dose of cd ra+ naive t-cells of . × /kg (range: - . × /kg). the mdli were infused at a median of seventy-seven days after hsct (range: - days), with a median interval between mdli of thirty-four days results: eight published studies reported survival outcomes for pediatric vod/sos patients (n = ), across all defibrotide doses. estimated day+ survival ( % confidence interval) was % ( %- %). for vod/sos with mod, studies were identified (n = ) with pooled estimated day+ survival of % ( %- %). only one openlabel expanded-access study, the treatment-ind, reported outcomes separately for pediatric vod/sos patients without mod (n = patients aged ≤ years). the day+ kaplan-meier estimated survival for those patients was % ( %- %). safety results were not pooled due to differences in reporting methodology; however, study results were consistent with the safety profile of the phase historicallycontrolled trial in vod/sos patients with mod ( % pediatric), in which / defibrotide-treated patients and all controls experienced ≥ ae. hypotension was the most frequent ae ( %, defibrotide; %, controls); common hemorrhagic aes (ie, pulmonary alveolar and gastrointestinal hemorrhage) occurred in % of defibrotide-treated patients and % of controls. in this pooled analysis of studies with defibrotide-treated pediatric patients with vod/sos, estimated day + survival was % (without mod, %; with mod, %). safety results in individual studies were generally consistent with the known safety profile of defibrotide. taken together, these results show a largely consistent defibrotide treatment effect in pediatric patients treated with defibrotide for vod/sos, with or without mod. results: six patients met inclusion/exclusion criteria. all patients were started on ecp while concurrently receiving . to mg/kg steroid therapy for agvhd plus a calcineurin inhibitor. patients had initiation of ecp within a maximum of weeks from initial diagnosis of agvhd (range - days). patients had grade - agvhd ( / patients with grade ) with skin, liver, and gi gvhd represented. patients received ei-ecp - times per week for the first weeks and then had ei-ecp frequency tapered based on initial response.after weeks of therapy patient had a decrease in overall gvhd grade by grade. all patients were able to have steroids tapered, with doses decreased by an average of % ( % - % decrease).at weeks of therapy, one patient with grade agvhd died of mof associated with infections. three patients had complete resolution of agvhd and patients decreased by grade. steroid doses were decreased by an average of % ( % - % decrease). continuously measures axillary temperature and wirelessly transmits real time-time data. the primary aim of the study was to evaluate the feasibility, safety and tolerability of continuous temperature monitoring in hsct patients using temptraq. we are performing a prospective observational study of pediatric patients ( - years of age) undergoing hsct at cincinnati children's hospital in cincinnati, ohio. enrolled patients wore a temptraq patch for days. a - rating scale survey was completed by the parent/guardian at the end of the study to determine tolerability, ease of use, satisfaction and desire for future use in the inpatient and outpatient setting. temperature data from the temptraq patch was compared to the standard episodic temperature monitoring to determine detection of febrile episodes. seven of ten patients have completed screening. we anticipate completion of the study in early february. the temptraq patch was well tolerated by study subjects (mean tolerability rating of . / ). one patient developed skin breakdown at the site of the temptraq patch attributed to recent thiotepa. the patch was easy to apply with an easy of application rating of . / . parents were overall satisfied (rating . / ) and would like to use the temptraq patches in future hospitalizations (rating . / ) and at home (rating . / ). temptraq patch identified fever (≥ . • f) in patients. the fever was never detected by episodic monitoring (soc) in patients and significantly delayed in the other patients (> hours). temptraq was well tolerated in pediatric hsct patients. timely fever detection was improved in temptraq over the current soc. background: serotherapy is commonly used in patients undergoing hematopoietic stem cell transplant (hsct) to reduce the incidences of engraftment failure and graft versus host disease. however, one well-known side effect is fever. as children undergoing hsct have compromised immune defenses, fever may also be an early indicator of bloodstream infection, which would warrant prompt use of broad-spectrum antibiotics. in a subset of patients with serotherapy-associated fever, antibiotics, which may induce antibiotic resistance and increase costs, may be unnecessary. we aimed to determine the incidence and characteristics of serotherapy-related fever, as well as the likelihood of concomitant bacteremia, in our institutional experience. a -year retrospective chart review was conducted of pediatric patients who received serotherapy as part of hsct conditioning at the university of minnesota. one-hundred sixty eight consecutive hsct patients who received serotherapy -either atg (n = ) or alentuzumab (n = ) -were identified. the median age at hsct was -years (range, . - years). a total of patients ( %) developed fever while on serotherapy (atg = , alentuzumab = ). one-hundred sixteen patients presented fever following the first infusion, and the median onset of fever was hours after commencing infusion (range, . - hours). fever resolved at a median hours (range, - hours). one hundred and fourteen patients ( %) underwent blood cultures. only seven patient were not started on ( %) empiric antibiotics, while % (n = ) were on antibiotic treatment prior to serotherapy for previously known or suspected infections. nine patients ( % of febrile patients, % of all patients) had positive blood cultures (atg = ; alentuzumab = ). no infection-associated deaths were observed.conclusion: while fever is common during serotherapy conditioning in children undergoing sct, episodes of concomitant bloodstream infection are rare. ongoing analysis identified potential risk factors for bacteremia as recent history of infection, first episode of fever following second or subsequent infusions, and previous central line placement. further analysis is being conducted to identify subgroups of patients for whom close monitoring alone may be safe. background: hsct is potentially curative for caya with high-risk leukemias; however, most lack an hla-matched aspho abstracts related donor. the risk of gvhd is increased with unrelated (urd) or partially matched related (pmrd) donors. selective t-cell depletion based on the elimination of t cells carrying and chains of the t-cell receptor may greatly reduce the gvhd risks, while allowing the maintenance of mature donor-derived alloreactive nk cells and / (+) t cells, which may augment the anti-leukemia effect.objectives: this is a prospective study of caya with acute leukemia who underwent hsct with mmrd or urds and tcr / /cd depletion. outcomes included engraftment, toxicities, viral reactivation, and relapse.design/method: this study included caya with acute leukemia transplanted between october and may . all received a myeloablative preparative regimen with targeted busulfan (n = ) or tbi ( cgy/ fractions) (n = ), with thiotepa ( mg/kg) and cyclophosphamide ( mg/kg). atg ( mg/kg x ) was given to those receiving haploidentical grafts and to the first who received urd grafts. immune suppression was not given post-hsct. the stem cell source was mobilized peripheral blood stem cells (pscs), which then underwent tcr / /cd depletion utilizing the clinimacs device under gmp conditions in the chop cellular immunotherapy lab.results: median age was (range . - . ). diagnoses included all ( -b-cell, -t-cell) and aml ( ; -secondary aml). urd were used for ; were / allele matched and were / matched. haploidentical donors were used for . median cd (+) dose - . × , / (+) cd (+) cells - . × , and b cells - . × . all patients achieved an anc at a median of d+ ( - ), and % had platelet engraftment at median d+ ( - ). nine patients ( %) developed acute gvhd (all skin, grades i-iv). five developed chronic gvhd (skin, gut, lung): limited in , extensive in . viral reactivations included: adenovirus ( , %), bk virus ( , %), cmv ( , %), and hhv ( , %). nine ( %) patients relapsed at a median of days (range - ) post-hsct, including aml patients ( . %) and all patients ( . %). transplant-related mortality was %; causes included sepsis ( ) and ards ( ). os was %; efs was % (gvhd-free efs %, lfs %). hsct with tcr / /cd depletion demonstrates excellent engraftment kinetics with limited gvhd without immune suppression. elimination of post-hsct immunosuppression may offer an excellent platform to augment anti-leukemic immune therapy or to enhance immune reconstitution. background: hematopoietic cell transplantation (hct) is the only curative treatment available for patients with sickle cell disease (scd). low bone mineral density (bmd) has been described in scd, but little is known about the impact of curative hct on this outcome. to determine the prevalence of low bmd and variables associated with low bmd in scd patients after hct. we conducted a retrospective chart review of scd patients who underwent hct at children's healthcare of atlanta (choa) between / and / and survived ≥ year post-hct. transplant characteristics, post-hct dual-energy x-ray absorptiometry (dexa) scan results, vitamin d levels, graft-versus-host-disease (gvhd) status, and fsh levels were reviewed. for patients - years of age, height corrected z-scores were calculated using a nihvalidated calculator, with t-scores used for older patients. bmd was categorized as low if between - and - sd below the mean and clinically significantly low if >- sd, in accordance with the children's oncology group long-term follow-up guidelines. vitamin d levels < ng/mol were considered deficient, and fsh levels > miu/ml suggestive of premature ovarian failure. fisher's exact test was used to compare variables in those with normal versus abnormal dexa scan results, with p< . considered significant.results: hct was performed on patients with scd, with surviving ≥ year post-hct. dexa scans were obtained in patients ( % female), with mean time from hct to dexa scan being years ( . - . years) and mean age at time of dexa . years ( . - . years). patients with and without dexa scans did not differ by sex, donor source, age at transplant, or vitamin d status. low bmd was noted in patients ( . %), with these patients more likely to be > years (pubertal; . versus . %, p = . ). acute gvhd was more common in patients with low bmd ( . versus . %), but not statistically significant (p = . ). clinically significant low bmd was noted in patients ( . % of those with dexa scans). these patients were older ( . years at testing), were more likely to be male ( . %), and all had acute and chronic gvhd, while none had evidence of gonadal failure.conclusion: clinically significant low bmd is uncommon after hsct for scd. patients at risk for low bmd include older patients and likely those with gvhd. this preliminary data suggests routine dexas may not be indicated for all patients who undergo hct for scd, but further data is needed. background: causes of renal dysfunction after hematopoietic cell transplantation (hct) include damage from radiation, nephrotoxic medications, graft vs. host disease (gvhd), hepatorenal syndrome, viral infections, or transplant associated microangiopathy. we sought to investigate the incidence of, and risk factors for, acute kidney injury in pediatric hct patients and associated risk with mortality.design/method: data from patients who underwent hct between and at a single institution were sequentially retrospectively captured on irb approved protocol. acute kidney injury (aki) was defined at multiple time points post-hct using the standardized criteria: kidney disease: improving global outcomes (kdigo). interval differences between values were analyzed using wilcoxon rank sum testing and categorical variables were analyzed using chi-square analysis.results: ninety-eight patients were included in the study: allogeneic (n = ) and autologous (n = ), mean age . years, of whom % were african american, % asian, % caucasian, % latino, and % mixed race. forty-seven percent of patients developed aki within the first years of hct. increased risk for aki was associated with a lower pre-transplant creatinine level (p = . ), abnormal pretransplant bun (p = . ) and an unrelated donor (p = . ) while preparative regimen intensity, race, or primary disease were not. twenty-six percent of patients developed aki within days of hct. of those with aki, % were exposed to either cidofovir, aminoglycosides, and/or ambisome for at least days versus % without aki and % were exposed to vancomycin compared to % without aki. evaluating outcomes at year after hct, of those with stage aki: % had reduced gfr and % died, while % had reduced gfr and % had died for patients with aki stage or . the absence of aki by day was associated with % reduced gfr and % death at -year after hct. overall, those with aki at any time in the first year post-hct had a . fold increased risk of death compared to those without. for patients who required renal replacement therapy (rrt, n = ), the risk of death was . fold greater compared to those who did not. in the % of patients who survived rrt, both recovered renal function within years.conclusion: acute kidney injury is common after pediatric hct, and may be associated with low creatinine, abnormal bun, unrelated donor pre-hct, and renal toxic medications. early-onset aki post hct is associated with an increased risk of mortality. these data should be validated in a larger prospective study but may offer opportunities to intervene and enhance outcomes. background: myeloablative hematopoietic stem cell transplant (hct) for pediatric malignant disease is associated with significant morbidity with % patients experiencing mucositis. patient controlled analgesia (pca) utilizing opioids is an effective strategy for pain management. we sought to describe and analyze pca use in d+ days post myeloablative hct for malignancies at lurie children's hospital of chicago from - .design/method: utilizing retrospective chart review, pca details were collected: indication, initiation day, pca duration, team managing pca (anesthesia or palliative), medication and dose in morphine equivalents, and pca toxicities. efficacy of pca was evaluated on pca day + , + , + , + using demands %, maximum pain score (rflacc, faces, vas) and subjective patient, parent and/or pain team perception of pain control. we devised a scale based on the above to designate pain control as good, moderate or poor. variables being analyzed include recipient age, sex, donor type, source, diagnosis, tbi use, gvhd/trm. this analysis, we analyze the depth of remission, car-t persistence, and post-transplant gvhd on our phase i anti-cd car-t protocol (nct ) to better understand the role of car-t in the peri-hct setting.design/method: children and young adults with relapsed/refractory cd + all treated on our phase i anti-cd car-t protocol were analyzed. mrd was assessed by flow cytometry (fc) in all, with pcr-based mrd analysis using igh or tcr testing assessed in select patients. hcts were performed at each patient's local institution based on standard of care and included varying conditioning regimens, donor types, stem cell source, and gvhd prophylaxis.results: on our cd car trial, patients were treated, the majority of patients (n = ) having relapsed following a prior hct. / patients ( %) attained a cr, of whom were mrd negative by fc. concurrent pcr based mrd analysis available in patients demonstrated that all patients achieved pcr based negativity. in , this was simultaneous with the month mrd negative fc, and in , pcr negativity was achieved over time (fc remained negative). patients proceeded to hct at a median time of days (range: - days) post-car-t, which was a first hct in . these two patients remain in an mrd negative cr, year post-car-t. no patients developed acute or chronic gvhd. car persistence was seen in patients who had detectable car-t cells on the pre-hct marrow suggesting the possibility of ongoing anti-leukemia surveillance prior to initiation of the conditioning regimen.conclusion: by inducing pcr negativity, car-t therapy may have a synergistic role with hct to improve leukemia free survival, prior to emergence of antigen negative leukemia, without an increased risk of gvhd. while the sample size is small, car-t therapy may offer an effective bridge to hct, particularly for those who are pcr negative, and those who have not had a previous transplant. given the underlying risk of hct related trm, pre-hct car may potentially allow for hct conditioning de-intensification as it may not be needed to eradicate residual disease. lee dw, ash abstract , background: post-transplant lymphoproliferative disease (ptld) is a complication after solid organ transplantation (sot) that is frequently due to epstein -barr virus (ebv) as a decrease in ebv-specific t cell immunity due to immune suppression allows for uncontrolled proliferation of ebv-infected b cells. outcomes for ptld are suboptimal with relapse rates approaching %. however, ebv-infected b cells in ptld express the ebv antigens lmp and lmp that can be targeted with immune therapy.objectives: we hypothesize that third party "off the shelf" lmp-specific t cell products may improve outcomes and decrease associated co-morbidities for patients with ptld by not only target the lymphoproliferating ebv-infected b cells but also restoring ebv-specific immunity.design/method: lmp-specific t cells (lmp-tcs) are manufactured from eligible donors with a broad range of hla types in our gmp facility to be used in a children's oncology group (cog) trial (anhl ) for patients with ptld after sot. lmp-tc products are manufactured from healthy donors using autologous monocytes and lymphoblastoid cell lines (lcl) transduced with an adenoviral vector expressing Δlmp and lmp as antigen presenting cells. lmp-tc products undergo comprehensive characterization by ifn-elispot assay to determine lmpspecific epitopes, class i and/or ii response, and hla restriction to guide selection of lmp-tc product for each patient.results: thus far, lmp-tc products have been manufactured. lmp-tcs were active against lmp (mean: sfu/ × ^ cells; range: - ), lmp ( ; - ), and lcl ( ; - ) as determined by ifn-elispot assay. at the time of cryopreservation, the lmp-tc products comprised a mean of % cd + t-cells, % cd + t-cells, and % nk cells. no b cells or monocytes were detected in the final products. thus far, we have identified novel lmp epitopes (lmp specific: n = ; lmp specific: n = ). approximately % of the lmp-tc products have lmp-specific activity through multiple hla alleles, and % have a mixed class i and class ii response. conclusion: thus, lmp-specific t cell products can be expanded from healthy donors to creat a third party bank, and identifying epitopes and hla alleles with lmp activity will facilitate selecting the most appropriate product for patients. while lmp-specific t cells have previously demonstrated safety and efficacy in phase i studies, anhl is the first trial using cellular therapy within a cooperative group setting. children's cancer hospital at the university of texas md anderson cancer center, houston, texas, united states background: in , the united states food and drug administration (fda) approved the first chimeric antigen receptor t cell (car-t) therapy; tisagenlecleucel. this cd -directed genetically modified autologous t cell immunotherapy has shown response rates of almost % among children and young adults with b-cell precursor acute lymphoblastic leukemia (all) that are refractory or in second or later relapse. cytokine release syndrome (crs) and car-t cell related encephalopathy syndrome (cres) are well described toxicities associated with car-t therapy. crs is a systemic inflammatory response and is typically characterized by fever, hypoxia, tachycardia, hypotension and multi-organ toxicity. cres may occur concurrently or following crs, or without any associated crs symptoms and is characterized by encephalopathy, delirium, seizures and rarely cerebral edema. almost half of patients who receive tisagenlecleucel may require pediatric intensive care unit (picu) support. crs and cres are generally reversible but may be associated with fatal outcomes. pediatric specific management guidelines, comprehensive training of multidisciplinary staff, effective communication and phased infrastructure ensure that adequate resources are available to facilitate early diagnosis and appropriate management of pediatric patients with crs and cres and allow for optimal patient outcomes and accreditation by the foundation for accreditation of cellular therapy (fact).objectives: develop a comprehensive program to ensure safe administration of immune effector cell (iec) therapy to pediatric patients.design/method: an inter-disciplinary pediatric cartox (car t cell therapy associated toxicity) committee consisting of cell therapy and picu physicians, neurologists, fellows, nursing leadership, advanced practice practitioners, pharmacists, registered nurses and social workers was created to monitor patient toxicity and establish specific clinical guidelines and diagnostic and treatments algorithms for pediatric patients receiving iec therapy. educational modules were developed as (i) live in-services and (ii) an online module with a competency based assessment. electronic medical record (emr) order sets and documentation and warning systems were also developed by the committee. the pediatric cartox committee developed a diagnostic and treatment algorithm for patients receiving iec therapy. emr orders and flowsheets were developed to support adherence to the algorithm. inter-disciplinary staff training and competency assessments were closely tracked. almost % of identified staff have completed training and achieved competency including, pediatric cell therapy staff, emergency center, picu, outpatient clinic/triage, neurology and sub-specialty staff and nocturnalists.conclusion: an inter-disciplinary approach can assist in institutional readiness for an iec program, promote quality assurance and perhaps fact iec accreditation. future directions include a program for ongoing staff competency assessments. predicted peak vo ) and abnormal oxygen uptake efficiency slope at the anaerobic threshold ( . ± . . , normal ± ). additionally, on echocardiogram three patients had evidence of diastolic dysfunction as evidenced by an elevated e/a ratio ( . ± . ) on tissue doppler. three patients demonstrated depressed longitudinal peak systolic strain (- . ± . ), indicating dysfunction not captured by ejection fraction. in this feasibility study, sct patients without evidence of lvsd on standard measures by resting echocardiogram can demonstrate abnormal exercise capacity. additionally, they can demonstrate systolic and diastolic dysfunction by measures not always included in standard echocardiography. these data suggest the need for a more thorough screening of survivors, and will be further validated as additional patients are recruited for this study. background: in hematopoietic transplantation, the t lymphocytes of the inoculum play a determining role in promoting hematopoiesis, transferring immunity to pathogens and acting as mediators of the graft-versus-leukemia effect (gvl). however, they are also responsible for graft-versus-host disease (gvhd), the main cause of post-transplant morbidity and mortality. the depletion of cd ra lymphocytes, by eliminating naive t lymphocytes from the inoculum, aims to conserve the gvl without producing gvhd.design/method: since april , patients ( boys and girls), with a median age of years, have undergone an allogeneic hematopoietic transplant from an hla donor identical with cd ra/cd depletion. the indication for transplant was: acute lymphoblastic leukemia ( ), acute myeloblastic leukemia ( ), myelodysplasia ( ) and medullary aplasia ( ). the donor was familiar in cases and unrelated in . the conditioning regimen was with fludarabine, busulfan and thiotepa. the median of cd + cells infused was . × / kg. on the day , + and + a programmed infusion of × / kg lymphocytes cd ra-was performed.results: all the patients grafted with a median leukocyte (> . × / l) and platelet (> × / l) engraftment time of and days, respectively. only one patient has developed acute gvhd grade i and no patient has developed chronic gvhd. immune reconstitution was early and rapid in all t cell subsets no patient has relapsed so far and only patient with myelodysplasia has developed an aml. she has received a nd transplant and has died of relapse. there was no case of toxic mortality. the event-free survival (sle) was ± % with a median follow-up of months. at present, patients are alive, out of immunosuppressive treatment and doing well. allogeneic transplantation with cd lymphocytes ra depletion resulted on very encouraging results, with a very low incidence of acute and chronic gvhd, but preserving the gvl effect by infusing cd ra-donor lymphocytes. miami children's health system, miami, florida, united states background: hematopoietic stem cell transplantation (hsct) using autologous or allogeneic progenitor cells is a potentially curative treatment for patients with high-risk malignancies and nonmalignant conditions. the american society for blood and marrow transplantation developed a task force to establish consensus guidelines for defining patient care in hsct and advocated for further studies to delineate safe procedural steps as an increasing amount of hsct are being offered to patients. there is limited evidence to support engraftment in recipients who receive their infusions via iv or syringe pump. we present novel data from patients who achieved neutrophil engraftment following hsct by a pump mechanism.objectives: to provide evidence supporting the use of pump (intravenous or syringe) infusion method in hematopoietic stem cell transplantations.design/method: a retrospective review was completed for patients who underwent hsct between and . inclusion criteria included patients who had received hematopoietic stem cell transplants between and and who were ages months to years old. the main outcome measure was days to neutrophil engraftment (defined as the first of three consecutive days with an anc > × /l).results: among patients who received infusion of hematopoietic stem cell products via pump mechanism, patients ( . %) received autologous products and ( . %) received stem cells from allogeneic donors. neutrophil engraftment (anc > × /l) occurred in a median of . days after stem cell infusion. the mean number of days to engraftment for patients who received allogeneic infusions s of s design/method: a retrospective chart review was performed at the children's hospital of wisconsin. statistical analyses included kaplan-meier estimate for os, mann-whitney test for comparing outcomes between subjects, and descriptive analyses.results: from - , patients with a median age of . ( . - . ) years at st hct were identified. patients were conditioned with cy/atg (n = ), cy/flu/atg (n = ), or cy/flu/atg/tbi (n = ) and received marrow (n = ) or cord blood (n = ) with median cd /kg dose of . ( . - . ) × . two patients developed grade i acute graftversus-host disease (gvhd); none developed chronic gvhd. due to dropping chimerism, graft rejection, or graft failure, nd hct (n = ) or boost (n = ) was offered. the median cd chimerism prior to hct/boost was ( - )%. median time between st hct and nd hct or boost was days ( days- . years). in patients receiving nd hct, used the same donor, of which used the same stem cell source (marrow) and switched to peripheral blood stem cells (pbsc). in patients who switched donors, used pbsc and used cord blood. most patients receiving nd hct underwent a uniform conditioning regimen of cy /flu /equine atg/ gy tli (n = ) or cy /flu /rabbit atg/ gy tbi (n = ); one received cy/atg. acute and chronic gvhd (limited seen in %) developed in % and % of patients, respectively. four patients required additional boosts and additional hct. after final intervention, cd and whole-blood chimerism at last follow-up was between - % (n = ) and - % (n = ), respectively. with a median follow-up of . ( . - . ) years, of patients are alive with an estimated -year os of . ± . %, having performance status ≥ % (n = ) or % (n = ). one patient developed chronic extensive gvhd and died of fungal infection . years after nd hct. our single-center experience demonstrates excellent ability to salvage patients who develop graft failure after initial hct. transplant-related complications such as gvhd and infections remain significant concerns.