Background: Scientists and regulatory agencies strive to identify chemicals that may cause harmful effects to humans and the environment; however, prioritization is challenging because of the large number of chemicals requiring evaluation and limited data and resources.Objectives: We aimed to prioritize chemicals for exposure and exposure potential and obtain a quantitative perspective on research needs to better address uncertainty in screening assessments.Methods: We used a multimedia mass balance model to prioritize > 12,000 organic chemicals using four far-field human exposure metrics. The propagation of variance (uncertainty) in key chemical information used as model input for calculating exposure metrics was quantified.Results: Modeled human concentrations and intake rates span approximately 17 and 15 orders of magnitude, respectively. Estimates of exposure potential using human concentrations and a unit emission rate span approximately 13 orders of magnitude, and intake fractions span 7 orders of magnitude. The actual chemical emission rate contributes the greatest variance (uncertainty) in exposure estimates. The human biotransformation half-life is the second greatest source of uncertainty in estimated concentrations. In general, biotransformation and biodegradation half-lives are greater sources of uncertainty in modeled exposure and exposure potential than chemical partition coefficients.Conclusions: Mechanistic exposure modeling is suitable for screening and prioritizing large numbers of chemicals. By including uncertainty analysis and uncertainty in chemical information in the exposure estimates, these methods can help identify and address the important sources of uncertainty in human exposure and risk assessment in a systematic manner. The primary objective of chemical assessment programs is to identify and regulate chemicals that may cause harmful effects to humans and ecosystems. Tens of thousands of chemicals require evaluation; however, data and resources are limited . In partMethods to screen and prioritize chemicals for more comprehensive evaluations include “separate” persistence, bioaccumulation, and toxicity (PBT) classification categories and “holistic” multimedia, multipathway mass balance exposure and risk assessment models that simulate key processes in the source–receptor relationship. The PBT method employs multiple bright-line pass/fail criteria and mass balance and QSA(P)R models for data generation. The pass/fail criteria are variable  and the multiple binary scoring results reduce consistency and transparency in decision making . AlthougIn this article, we describe the parameterization and application of a mass balance model to screen and prioritize > 12,000 organic chemicals using four far-field human exposure and human exposure potential assessment metrics. The propagation of uncertainty in model input parameters is included through the model calculations, thus providing uncertainty estimates for the four assessment metrics. A primary objective was to use these results as a case study to obtain a better quantitative perspective of the relative uncertainties in chemical information required for screening-level exposure and risk assessment. In addition, we consider external and internal human exposure metrics and discuss recommendations for future research needs to improve assessments.Multimedia, multipathway far-field human exposure models and metrics. EA; kilograms per hour or nanograms per day) provide actual exposure estimates such as chemical concentrations in humans  and human intake rates . For each exposure pathway , the intake rate is the product of the environmental medium intake, or contact rate , and the chemical concentration in the corresponding medium . Aggregate exposures are the sum of all of the exposure pathways considered in the assessment. Intake rates do not account for chemical absorption, distribution, metabolism, and excretion (ADME) processes in the receptor of interest; however, internal concentrations do account for ADME processes. Actual exposure estimates are applicable in risk-based chemical assessments by comparing concentrations or intake rates with concentrations or rates of intake associated with effect or no-effect levels; however, it is often difficult to obtain reliable actual emission rate information. A consistent, arbitrary unit emission rate  for all chemicals can be used to provide estimates of relative exposure potential for screening and prioritizing chemicals in a hazard-based context. The chemical intake fraction  and the unit emission rate–based concentration  are examples of relative exposure potential metrics that are independent of the actual chemical emission rate. We selected the iF, iRA, CU, and CA metrics to consider a range of possible far-field human exposure assessment objectives.Various studies have proposed metrics for assessing human exposure and human exposure potential e.g., . Exposur2) environment. Using these concentrations in the physical environment, RAIDAR calculates bioaccumulation in aquatic, terrestrial, and agricultural food webs to estimate exposures of, and potential risks to, humans and representative ecological species . The model assumes chemical emissions are continuous from diffuse, nonpoint sources, and losses from the regional environment can include reaction  and advection . Primary producers and invertebrates bioconcentrate chemicals from their ambient environment of air, water, soil, or sediment, whereas vertebrates bioaccumulate chemicals from their ambient environment and from their diet. The bioaccumulation models consist of a mass balance equation formulated over a single compartment for each vertebrate species (including humans) accounting for major processes of chemical uptake  and elimination . RAIDAR calculates concentrations in outdoor air, water, soil, sediment, and biota, including humans, using either EU or EA for a particular simulation. For this case study, RAIDAR version 2.0 was coded in Visual Basic for Applications hosted in Excel  to quantify the propagation of uncertainty in chemical properties (model input parameters) in the calculated iF, iRA, CU, and CA exposure metrics .Various multimedia, multipathway, mass balance exposure models can be used to calculate some of these far-field human exposure metrics e.g., . We usedCase study chemicals. We compiled a database of 12,619 organic chemicals including organic chemical substances with reported production in Europe, the United States, Canada, Japan, and other countries that participate in the Organisation for Economic Co-operation and Development. This database comprises a broad range of chemical properties and production volumes and is considered to represent much of the diversity of current-use organics .Model parameterization and high throughput screening applications. The RAIDAR model input parameters required for assessing exposure potential are molar mass , octanol–water partition coefficient , Henry’s law constant , degradation half-lives  in air, water, soil, and sediment and primary biotransformation HLs in vertebrates. The dimensionless air–water partition coefficient (KAW) is calculated by dividing H by the gas law constant  and absolute system temperature . The model uses the octanol–air partition coefficient  for processes such as aerosol–air partitioning and bioaccumulation in air-breathing organisms; however, KOA is not a required input parameter because it is calculated internally by the model as KOW/KAW. Chemical mode-of-entry information is required  for Level III  fate simulations. For assessments of actual exposure, an estimate of the regional-scale actual emission rate  is also required.Kas) and publicly available high throughput models for parameterizing diverse types of ionogenic chemicals. Further details on the selection of partitioning properties are in the Supplemental Material, http://dx.doi.org/10.1289/ehp.1205355). Further details on the selection of degradation half-lives are in the Supplemental Material, EU) of 1 kg/hr for all chemicals to estimate human exposure potential . Estimated “actual” regional-scale emission rates (EA) are needed for estimates of actual exposures and Level III fate calculations require mode-of-entry information. We used production volume estimates and the European Union Technical Guidance Document (EU TGD) emission factor scenarios ]. The EU TGD emission factors R models and expert judgment (S) of an input parameter (Pi), such as KOW, on model output, such as iF, can be approximated asjudgment . The senS = (ΔiF/iF)/(ΔP/Pi), [1]iF is the change in the iF value and ΔP is a fixed change to a selected input parameter value . The contribution to variance  of the chemical input parameters on an exposure calculation for that particular chemical (CVi) can be evaluated as a function of the variance (uncertainty) σ2i and sensitivity (Si) of the individual input parameters (Pis) for all model input parameters (n) for that particular chemical aswhere ΔCf), also referred to as distribution factors  as described by Thus, the variance in the log-normal distribution increases with an increase in udgment” . Cƒs canCƒ = elog[1.96 σXln(10)]. [4]Cƒs using professional judgment. We used SDs from the EPI Suite™ (U.S. EPA 2011) QSA(P)R training and testing sets to guide the application of professional judgment for calculating and assigning screening-level Cfs using Equation 4. In general, the relative uncertainty in chemical information progresses as follows: measured data < predicted data “within the defined domain” < predicted data “outside the defined domain.” Cfs for model input parameter categories .We assumed model input parameters to be log-normally distributed, and because of data gaps, we assigned iRA and CA) and iF and CU). The upper tails of the rankings suggest that all methods have the capacity to differentiate chemicals with relatively higher exposure and exposure potential from all chemicals in the database; however, the actual exposure metrics show larger ranges than the comparative exposure potential metrics because of the greater range of possible EA values. Estimates of actual exposures span approximately 15 and 17 orders of magnitude for iRA and CA, respectively. Estimates of exposure potential span approximately 7 and 13 orders of magnitude for iF and CU, respectively.CV for modeled exposure metrics (output) as a function of model input parameters for all chemicals. Clearly the greatest contribution to uncertainty in estimates of actual internal  in mammals is the input parameter with the second greatest contribution to variance in CA calculations. This is because the primary biotransformation HL in the human is a key determinant of the overall residence time in the body for many parent chemicals. Chemicals with log KOW > 2 and log KOA > 6 have high bioaccumulation potential in humans when biotransformation is assumed to be negligible . For iF simulations, a number of input parameters contribute to variance including  degradation HL in water, biotransformation HL in fish, degradation HL in air, KOW, and KAW. For certain chemicals the CV on iF can be 1 for most input parameters (as noted by the maxima points), the only exceptions being for the degradation HLs in soil and sediment. These two input parameters are generally shown to have low CV in human exposures. In general, KOW is shown to have a greater CV in exposure metrics than KAW despite a relatively greater degree of uncertainty in KAW than in KOW  maximize the value of biomonitoring data by quantitatively linking emission rates and exposure pathways to internal doses (b) complete model evaluations through comparisons with biomonitoring data . Most notably, the biotransformation HL parameter generally contributes a substantial amount of uncertainty in the calculated human concentrations.An internal exposure metric may be the most biologically and toxicologically relevant because the site of toxic action is typically located inside the body. Calculated human concentrations can be integrated with toxicity data such as tissue concentrations, providing direct linkages to high throughput toxicity test data for screening-level risk assessment . Biomonial doses , and b) ing data , that isEA, a high level of error is expected and these errors in some cases may be beyond what we have attempted to quantify. In particular, the EU TGD emission scenarios are considered “realistic worst case” for screening-level objectives; therefore, the “actual” exposure estimates are expected to be conservative, particularly for high production volume chemicals that are used as intermediates or not released to the environment following the assumed emission scenario estimates.We emphasize that the screening results for “actual” exposures should be interpreted with some skepticism. Because of the complex issues in estimating CU and iF provide guidance for exposure hazard potential and the relative information can be useful for benchmarking chemicals before considering industrial production  necessarily required professional judgment because of substantial data gaps in measured and predicted chemical information. Because of the data gaps and the current screening-level approach, some codependence in uncertainty in model input parameters may occur in some instances; however, given the overall limitations in obtaining robust uncertainty estimates for all input parameters and the generally large uncertainties in model output, the issue of codependence is best addressed at higher tier assessments or when better data are available to characterize uncertainty. We expect uncertainty estimates for information used in chemical evaluations to evolve with the availability of more measured data and improved measurement techniques, better QSA(P)R models, and experience.Approximately 33% of the chemicals have predicted partitioning properties outside of the range of current measurements. This suggests that a large number of chemicals that are being evaluated may have highly uncertain predicted properties; however, there is no regulatory guidance for assessing such chemicals and for addressing the uncertainty of these predictions. It is stressed that the HLs and biotransformation HLs. If uncertainties in other chemical properties are reduced through refined property measurement and QSA(P)R development, chemical assessments will still be highly uncertain because of the prevailing contribution to uncertainty of EA. Although continued academic research may result in some modest improvements in estimating EA and the associated uncertainty in this parameter, any substantial improvements to reduce this key source of uncertainty in exposure and risk assessment will ostensibly require coordinated efforts with the chemical regulatory, manufacturing, and use communities. It must be recognized that the same general level of uncertainty in screening and prioritization results shown in this case study can be expected using other methods . Improved information on chemical production and usage and addressing key sources of uncertainty such as biotransformation and environmental degradation HLs and continued model refinement are required to improve chemical screening and prioritization efforts.This study has provided a quantitative perspective on the uncertainty in exposure data to better address uncertainty in screening-level exposure and risk assessment. The results indicate that more measured data and models are needed for environmental degradation (147 KB) PDFClick here for additional data file.


Integrating knowledge from across the natural and social sciences is necessary to effectively address societal tradeoffs between human use of biological diversity and its preservation. Collaborative processes can change the ways decision makers think about scientific evidence, enhance levels of mutual trust and credibility, and advance the conservation policy discourse. Canada has responsibility for a large fraction of some major ecosystems, such as boreal forests, Arctic tundra, wetlands, and temperate and Arctic oceans. Stressors to biological diversity within these ecosystems arise from activities of the country's resource-based economy, as well as external drivers of environmental change. Effective management is complicated by incongruence between ecological and political boundaries and conflicting perspectives on social and economic goals. Many knowledge gaps about stressors and their management might be reduced through targeted, timely research. We identify 40 questions that, if addressed or answered, would advance research that has a high probability of supporting development of effective policies and management strategies for species, ecosystems, and ecological processes in Canada. A total of 396 candidate questions drawn from natural and social science disciplines were contributed by individuals with diverse organizational affiliations. These were collaboratively winnowed to 40 by our team of collaborators. The questions emphasize understanding ecosystems, the effects and mitigation of climate change, coordinating governance and management efforts across multiple jurisdictions, and examining relations between conservation policy and the social and economic well-being of Aboriginal peoples. The questions we identified provide potential links between evidence from the conservation sciences and formulation of policies for conservation and resource management. Our collaborative process of communication and engagement between scientists and decision makers for generating and prioritizing research questions at a national level could be a model for similar efforts beyond Canada.Generación de Preguntas de Investigación Prioritarias para Informar a las Políticas y Gestión de la Conservación a Nivel Nacional Although we recognize the benefits of ongoing curiosity-driven research, we believe there is clear value in aligning research in the natural and social sciences more closely with the information needs of decision makers. Scientific research can inform decisions regarding domestic environmental protection and enhance a country's ability to contribute to international solutions to global environmental challenges . In CanaTargeted research can have instrumental effects , which dRecent projects have been undertaken at the global  and natibiodiversity, defined here as “the variety of genes, species and ecosystems and the ecological processes that allow them to evolve and adapt to a changing world” , environmental nongovernmental organizations (n= 3), industry associations (n= 2), policy analysis or advisory organizations (n= 6), and academia (n= 7). The core team of eight organizers and four student assistants brought the total number of participants to 40. Twenty-eight of the participants attended a workshop in Ottawa, the national capitol, in April 2010. Although provincial and municipal governments also have substantive, often paramount, responsibilities for ecosystem management, involving participants from a full range of regional government organizations was not feasible logistically. Academic participants were drawn from across the country and many team members had extensive regional policy experience.A core team of eight organizers (the eight lead authors) designed and implemented the project. A larger group—who participated in solicitation and synthesis of questions—included policy advisors and decision makers in governments and nongovernmental organizations  and academics with expertise in conservation science. Of 57 invitees to that group, 43 responded and 28 accepted. The final group of participants was drawn from federal government agencies with national responsibilities . Questions were sought from policymakers and their advisors in government, policy specialists in the nongovernmental and private sectors, Aboriginal leaders and science advisors, researchers in the natural and social sciences, and the philanthropic community. We used a bilingual (English and French) website established for this exercise to collect questions over 5 weeks.yes, no, or it depends response, (5) contain a subject of the intervention, an intervention, and a measurable effect if related to interventions and effects, and (6) increase the effectiveness of policy about, and management of, resource use and biodiversity in the face of environmental stressors if answered.Instructions for individuals who wished to submit questions were similar to those for other priority-setting exercises , although policy applications were stressed. Individuals were asked, “What research question, if answered, would substantially advance the development of effective policies and management strategies for species, ecosystems, and ecological processes in Canada?” Aspirational criteria for questions were that they (1) be answerable through a realistic research design, (2) be answerable on the basis of facts rather than value judgments, (3) be of a spatial and temporal scale that could be addressed realistically by a research team or program, (4) not be answerable simply with a A total of 396 questions were received in 271 submissions. Questions were submitted by individuals with diverse organizational affiliations . Over 62% (159 of 255) of individuals who voluntarily provided background professional information had more than 10 years experience in natural resource management. Core team members culled candidate questions that failed to meet the aspirational criteria and combined and refined questions to avoid substantial redundancies. This process reduced the list to 242 questions for participants to consider in the workshop. All questions are included in the Supporting Information.At the workshop, participants selected the top 40 questions from those submitted through a process developed for similar priority-setting exercises in the United Kingdom and United States . The workshop opened with a plenary session that included participant introductions, an overview of how participants were selected, highlights from recent exercises, an explanation of the policy emphasis of this exercise, and a review of workshop processes and ground rules. The balance of the day was spent in three breakout sessions (75–90 minutes each) composed of three concurrent groups per session. Each breakout group discussed one loosely themed set of 25–30 candidate questions, narrowing the set to four candidates for the top 40 and two alternates. Participants in breakout groups sometimes combined candidate questions, developed alternate questions that captured key ideas from candidate questions, and referred to the original list of 396 questions. The 54 questions that resulted from the first day  were discussed during a plenary session on the second day, during which the top 40 were selected by consensus.After the workshop, the core team and participants, including those individuals unable to attend the workshop in person, refined the questions by consensus-based email dialogue. During the refinement phase, it became apparent that some questions were quite similar. Combining three pairs of questions allowed us to include other candidates that were not on the initial list of 40. Participation of scientists helped ensure that questions were sufficiently narrow to form the basis of a research program. Participation of policymakers helped ensure that the final questions were relevant.We grouped the top 40 questions into themes: ecosystem structure and function, land cover and habitats, populations and species, resource-based industries, parks and protected areas, environmental change, environmental values, economic benefits and costs, individual and community well-being, adaptive management, and policy and governance. The organization of these themes was drawn from the adaptive-management logic framework outlined in Ecosystem attributes that provide benefits to humans  are increasingly a focus of biodiversity policy . The alt1. To what extent can ecological function and the supply of ecosystem services be predicted on the basis of ecosystem composition and structure?2. How does managing ecosystems for particular ecological functions or services affect other elements of biodiversity?3. How can transition points or thresholds among ecosystem states best be identified and predicted?Current approaches to ecosystem management seek to link land-cover types, species’ habitats, and ecosystem stability and services in part to understand how natural and human-induced changes in land cover might affect ecological, social, and economic well-being e.g., .4. How are populations of animals and plants affected by different configurations of land cover?5. At what spatial and temporal scales are management or policy interventions most likely to achieve various objectives for conservation of land cover?6. What ecological principles and information standards employed in the identification of critical habitat under the Species at Risk Act are most likely to contribute to species recovery?7. To what extent can habitat restoration or rehabilitation compensate for loss of quantity or quality of existing species’ habitat?Many ecosystem functions and services depend on the maintenance of particular populations or species e.g., . Populat8. What investments and management interventions will be required to maintain or increase the abundance of harvested populations when harvesting is one of multiple stressors acting on those populations?9. What are the cumulative demographic and genetic effects of harvest on target and nontarget populations and species?10. How do transboundary migrations of terrestrial and aquatic animals affect efforts to manage populations of those species?Natural-resource industries account for a substantial portion of Canada's economic activity . Where past policies have failed to effectively manage biodiversity there have sometimes been enormous economic and social impacts. For example, collapse of the Newfoundland cod fishery led, in 1992, to the largest single layoff of workers in Canadian history . Policy 11. What are the potential effects of large-scale energy exploration, development, and extraction projects on ecosystems and species?12. How does management of terrestrial and aquatic food-production systems affect species, ecological functions and services, and economic benefits?13. What are the magnitude and extent of effects of terrestrial resource-development activities on aquatic ecosystems?14. How do extractive and nonextractive industrial activities affect ecological functions and biodiversity in aquatic ecosystems?Canada has an extensive system of terrestrial parks and other protected areas  and a growing system of freshwater and marine parks for which federal, provincial, territorial, and regional governments all have responsibilities. Parks are managed to meet different objectives and mandates to protect species and ecosystems, and provide recreational opportunities that sometimes conflict. There are gaps in knowledge of how networks of parks can and should be designed, connected, and managed to meet ecological, social, cultural, and economic objectives.15. To what extent can species be maintained over the long term within existing parks and protected areas in the absence of proactive, external management?16. What are the ecological consequences of existing human activities in parks and their implications for future management?17. What are the impacts of alternative configurations of, and management strategies for, aquatic reserves on ecological function, species, and social well-being?18. What are the effects of different configurations of, and management strategies for, protected areas on biological diversity across landscapes and beyond the boundaries of terrestrial protected areas?Canada is already affected by climate change, especially in northern regions . Underst19. How is climate change likely to affect the abundance, rates of growth, and distribution of and interactions among populations?20. How can undesirable ecological, social, and economic effects of future changes in freshwater availability be reduced or mitigated?21. How will northern coastal ecosystems respond to changes in climate and industrial activity if reduction in ice cover increases human access to those ecosystems?22. What policies and management strategies most effectively mitigate the undesirable effects of non-native invasive species, potential disease vectors, and emerging pathogens?23. What are the effects of different policies and management strategies on the capacities of humans and other species to adapt to environmental change?Environmental values are influenced by demographic, cultural, experiential, and educational variables . They ca24. How does the integration of conservation science into public education influence environmental values, ecological literacy, and behaviors that affect the environment?25. How does urbanization and immigration affect environmental values, behavior, and conservation outcomes?26. How do individual and societal values about collective responsibility for common- pool resources affect private conservation initiatives and stewardship efforts?International agencies, governments, and researchers have highlighted the economic value of biodiversity. Accounting for the full costs and benefits of ecological change is central for credible economic analyses . The way27. What are the true direct and indirect contributions of ecosystems to economic well-being and how are these benefits likely to change in the future?28. What are the comparative ecological, economic, and social impacts of economic instruments versus input or output controls for ecosystem management?29. What are the benefits and costs of horizontally and vertically integrating policies and regulations within and across different policy domains such as environment, health, and trade?Policymakers are concerned with aspects of individual, community, and societal well-being beyond economics. Environmental change affects human health e.g., , communi30. How do terrestrial and aquatic conservation policies directly or indirectly affect human health?31. What are the effects of conservation initiatives on the health, livelihoods, traditional cultures, and identities of Aboriginal peoples?32. What are the effects of different strategies for building community capacity on levels of citizen engagement in environmental stewardship, restoration, and conservation?Canadian governments are committed to working together with citizens using “an ecosystem and adaptive management approach to achieve shared outcomes” . Ecosyst33. How can local and traditional knowledge be most effectively communicated and synthesized with scientific knowledge to inform conservation science and management?34. What forms of scientific evidence, risk assessment, and knowledge transfer most effectively and increase the probability of achieving ecosystem-management objectives?35. What monitoring methods can effectively and efficiently assess long-term, incremental, and cumulative environmental changes?36. What are appropriate indicators to evaluate the effects of public, private, and nongovernmental-sector policies, programs, and initiatives on biodiversity?Canada is a parliamentary democracy with a complex federal structure and diverse citizens who expect to participate in collective decisions. This situation complicates the formation and implementation of national evidence-based and results-oriented policy. It is difficult to develop consistent policies across jurisdictions and to determine the most-appropriate level of governance to address a given environmental problem. It is also difficult to balance the legitimacy of governance (a function of structure and inclusive processes) with explicit action to conserve biodiversity as required by laws, international commitments, and moral values.37. What factors influence the likelihood of compliance with environmental legislation and regulations?38. How do policy, legal, or institutional arrangements shape the effectiveness of integrated management for terrestrial watersheds and adjacent coastal environments?39. How do the actions of Aboriginal governments and organizations influence conservation strategies and the assignment of responsibilities for environmental governance in Canada?40. How can environmental laws, policies, and regulations be integrated and harmonized across multiple jurisdictions and sectors to achieve conservation objectives?Answers to questions about conservation policy can be informed by scientific evidence about the ecological, social, and economic effects of governance interventions. The Some of the themes identified in this Canadian exercise coincide closely with those identified in the United States . Relative to the United States, however, there was less emphasis on environmental stressors (27% vs. 55% of the 40 questions) and more emphasis on governance and management issues (67% vs. 32% of the 40 questions). In Canada there was strong emphasis on the effect of aboriginal governance on federal and provincial conservation policy and, conversely, on the effect of governmental conservation policy on individual and community well-being for Canada's Aboriginal population.We suspect that the contrasting priorities within North America arise from cultural and political differences . In other fields of study, political context has been identified as an important factor influencing the effect of research , so it wThe questions we identified provide potential links between evidence from the conservation sciences and the formulation of environmental policies for conservation and resource management. Integrating knowledge from across the natural and social sciences is necessary to effectively address societal tradeoffs between use and conservation of biodiversity. The collaborative process of communication and engagement between scientists and decision makers can change the ways that decision makers think about evidence, enhance levels of mutual trust and credibility, and potentially help advance the conservation policy discourse in Canada and internationally.


Rabies, an acute progressive, fatal encephalomyelitis, transmitted most commonly through the bite of a rabid animal, is responsible for an estimated 61,000 human deaths worldwide. The true disease burden and public health impact due to rabies remain underestimated due to lack of sensitive laboratory diagnostic methods. Rapid diagnosis of rabies can help initiate prompt infection control and public health measures, obviate the need for unnecessary treatment/medical tests, and assist in timely administration of pre- or postexposure prophylactic vaccination to family members and medical staff. Antemortem diagnosis of human rabies provides an impetus for clinicians to attempt experimental therapeutic approaches in some patients, especially after the reported survival of a few cases of human rabies. Traditional methods for antemortem and postmortem rabies diagnosis have several limitations. Recent advances in technology have led to the improvement or development of several diagnostic assays which include methods for rabies viral antigen and antibody detection and assays for viral nucleic acid detection and identification of specific biomarkers. These assays which complement traditional methods have the potential to revolutionize rabies diagnosis in future. Rabies virus (RABV) and other Lyssavirus species of the family Rhabdoviridae. Rabies, one of the oldest and most feared zoonotic diseases known to mankind, is an acute, progressive, and almost fatal encephalomyelitis caused by the Despite the lack of accurate data on the global burden of neglected tropical diseases, the estimates of direct mortality due to rabies, transmitted most commonly through the bite of a rabid animal, are among the highest. The annual number of human rabies deaths globally, in 2010, is estimated to be 61,000 , with the vast majority of deaths (84%) occurring in rural areas. The estimated annual cost of rabies is US$ 6 billion , with almost US$ 2 billion due to lost productivity after premature deaths and a further US$ 1.6 billion spent directly on postexposure prophylaxis [Most of the human deaths due to rabies occur in Asia and Africa. Estimates of human mortality due to endemic canine rabies in Asia and Africa annually exceed 30,000 and 23,000, respectively [Canine rabies has been eliminated from western Europe, Canada, the United States of America (USA), Japan, Malaysia, and a few Latin American countries. Australia is free from carnivore rabies, and many Pacific Island nations have always been free from rabies and related viruses. In these areas, human deaths from rabies are restricted to people exposed while living or travelling in areas endemic for canine rabies , 4. HoweLaboratory diagnosis and surveillance for animal and human rabies are severely constrained in much of the developing world where rabies is endemic. The true disease burden and public health impact due to rabies remain underestimated due to lack of simple, sensitive, and cost-effective laboratory methods for rabies diagnosis. This may be one of the important reasons why rabies remains a neglected zoonotic disease in many developing countries in Asia and Africa , 7. Two distinct forms of rabies—furious and paralytic—are recognized in humans. Diagnosis of the classical furious  form, which constitutes about 80% of human rabies cases, is based on its distinctive clinical signs and symptoms and rarely poses diagnostic difficulties. However laboratory assistance may be required in some cases wherein characteristic clinical features like aerophobia or hydrophobia are lacking. In clinical practice, the paralytic or atypical forms, which constitute about 20% of human rabies cases, pose a diagnostic dilemma. These cases are often clinically indistinguishable from Guillain-Barre syndrome (GBS) and also need to be differentiated from neuroparalytic complications due to Semple-type antirabies vaccine which is still being used in few countries like Mongolia, Myanmar, and Pakistan . The sitRapid diagnosis of rabies is vital for initiating prompt and appropriate infection control and public health measures. Early diagnosis can obviate the need for unnecessary treatment and medical tests and also help in prognostication, institution of barrier nursing, timely administration of pre- or postexposure prophylactic vaccination to family members of the patient and the treating medical and nursing staff, and case closure and grief counselling with family members. Laboratory tests negative for rabies can indicate the presence of another infectious agent or a noninfectious aetiology, and assist in appropriate medical management. Laboratory diagnosis of rabies can also help specific characterization of the causative agent and suggest the potential source of infection, especially when a history of exposure to an animal is lacking, and identification of other individuals who may have been exposed to the same source of infection , 9, 12.Reports of rabies being acquired through organ transplants –15 highlAlthough human rabies is known to be almost 100% fatal, the reported survival of a teenager who developed rabies following a bat bite in USA using the “Milwaukee Protocol” in 2005  has reviContinual surveillance and laboratory confirmation in clinically suspected cases of rabies are imperative in countries which have recorded a decline in human rabies cases in recent years  and are working towards rabies elimination in the near future. Geographical boundaries cannot restrain the rabies virus; as long as foci of wildlife or canine rabies exist anywhere, and international travel and global trade of livestock, pets, and wildlife continue, the threat of reintroduction of rabies exists even in countries which have been rabies-free for many years. For instance, Bali, an Indonesian island, was considered rabies-free until late November 2008. However, an island-wide rabies outbreak has since occurred and rabies has been confirmed in both dogs and humans, causing 141 human deaths by the end of 2012 [Laboratory techniques in rabies were started as early as 1800 BC when for the first time Zinke demonstrated that the infection could be transmitted to a normal animal after inoculating with saliva from a rabid animal. The landmark discovery of Negri bodies by Adelchi Negri in 1903 and demonstration of their diagnostic significance by his wife Lina Negri-Luzzani in 1913 paved the way to laboratory confirmation of rabies. Laboratory Techniques in Rabies [and the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals [A definitive diagnosis of rabies can be made only with the appropriate laboratory methods. The basic techniques are described in the WHO publication n Rabies  and the  Animals .μm to as large as 30 μm and are generally circular or oval and deeply eosinophilic with characteristic basophilic granules, often arranged in the form of a rosette, within the eosinophilic matrix. Infected neuronal cells reveal aggregates of viral particles “Negri bodies” which are intracytoplasmic inclusion bodies specific to rabies encephalitis, demonstrated by histological tests (Seller's Technique) on smears taken from various areas of the brain. Negri bodies vary in size from as small as 3 Though it is a simple, rapid test, Seller's method on unfixed tissue smears has a very low sensitivity and is only suitable for fresh specimens. Techniques that stain sections of paraffin embedded brain tissues are time consuming, less sensitive, and more expensive. Histological techniques are much less sensitive than immunological methods, especially in the case of autolysed specimens, and are no longer recommended for primary diagnosis, both in humans and animals , 18, 19.The most widely used test for postmortem rabies diagnosis is the fluorescent antibody test (FAT), which is recommended by both World Health Organization (WHO) and World Organization for Animal Health (OIE). Developed by Goldwasser and Kissling in 1957, this test is still the gold standard for rabies diagnosis , 21. It Reliable results are obtained only when fresh brain tissue is used; however, FAT can also be applied to specimens preserved in 50% glycerol saline after rigorous washing of the specimens with normal saline. If the specimen has been preserved in a formalin solution, FAT may be used only after the specimen has been treated with a proteolytic enzyme . HoweverObtaining a postmortem brain biopsy/autopsy continues to be a challenge due to religious, cultural, and other factors, with declining rates of autopsy being a common problem in both developed as well as developing countries. Availability of methods of antemortem diagnosis of rabies in samples other than brain tissue is very critical and would circumvent the need for invasive brain biopsies/autopsy and the associated logistics and safety procedures. FAT can also be performed on corneal smears and nuchal skin biopsy in suspected cases; however it has been found to have limited reliability and low sensitivity for antemortem diagnosis of rabies , 25. BesFurthermore, the need for an expensive fluorescence microscope, which requires maintenance as well as skilled personnel to interpret the test, limits its use in many developing countries.The rabies N antigen can also be detected by applying immunohistochemical techniques as well as enzyme immunoassays. An ELISA-based technique was developed by Perrin et al. in 1986 which is known as rapid rabies enzyme immunodiagnosis (RREID) . This teVirus isolation is required for confirmatory diagnosis, especially when FAT gives an uncertain result and more importantly for molecular characterization of viruses in a geographical location and for tracing the origin of the virus if rabies occurs in a rabies-free area. Two techniques can be employed for this purpose: the mice inoculation technique (MIT) and rapid tissue culture infection test (RTCT) , 30. Three-to-ten mice, 3-4 weeks old (12–14 g), or a litter of 2-day-old newborn mice, are inoculated intracerebrally with the clarified supernatant of a 10–20% (w/v) homogenate of brain material in an isotonic buffered solution containing antibiotics. The inoculated mice are observed daily for 28 days; they develop typical signs and symptoms of rabies any time after 5–7 days depending on the incubation period. These consist of initial ruffling of hair, hunch back, and dragging hindlimbs followed by paralysis of hind- and forelimbs. Further confirmation of the diagnosis can be made by extracting the brain of the diseased mouse and subjecting this to FAT. The disadvantage of MIT is the long interval before a diagnosis can be made since the inoculated mice need to be kept under observation for 28 days as some wild viruses may have a very long incubation period. If cell culture facilities exist in the laboratory, consideration should be given to replacing the mouse inoculation test with cell culture whenever possible as it avoids the use of live animals, is less expensive, and gives more rapid results. However, advantages of MIT are that when the test is positive, a large amount of virus can be isolated from a single mouse brain for strain identification purposes and that it can be easily and practicably applied in situations where skills and facilities for other tests  are not available . As compared to MIT, virus isolation in cell culture is fast and results can be given in 24–48 hours. The cell lines most suitable for virus isolation are of neural origin and the most commonly used cell line is the murine neuroblastoma cell line Neuro-2a. Other cell lines which are used but may not be as sensitive as Neuro-2a include chicken embryo-related (CER) and baby hamster kidney (BHK 21) cells. The suspect clinical specimen or the brain homogenate is inoculated onto the cells grown in a shell vial or 96-well plates, incubated for 24 h and stained by direct FAT after acetone fixation. Recently, a new cell line, human embryonic kidney cell line (HEK 293), was evaluated for isolation of both fixed and street viruses and was found to be as sensitive and specific as Neuro-2a cell line  Figure . RTCT isThe demonstration of antibody in the serum in the absence of a history of vaccination for rabies or in CSF offers indirect evidence of rabies infection. Interpretation of test results may be difficult since the host immune response may vary among individuals. The negative predictive value of serological tests for rabies diagnosis is considered poor . SerologAs neutralizing antibodies are considered a key component of the adaptive immune response against rabies virus, virus neutralization (VN) assays in cell cultures are prescribed tests for checking vaccination responses. Results are expressed in international units relative to an international standard antiserum. The Mouse Neutralization Test (MNT), Rapid Fluorescent Focus Inhibition Test (RFFIT), and the Fluorescence Antibody Virus Neutralization Test (FAVN) have been described for this purpose. The widely used virus neutralization test in mice (MNT) developed in 1935 by Webster and Dawson , 34 is nThe adaptation of the “challenge virus standard (CVS) strain” of rabies virus to grow in cell culture has led to the development of alternative assays in which the replication of nonneutralized virus in culture is detected by the observation of plaques, infected cells stained with fluorescent antibody, or the colour developed with enzyme-labeled anti-rabies antibody. The RFFIT is one of the most widely used substitutes to the Mouse Neutralization Test (MNT) and is a rapid test that requires only 20 hours for completion and is slightly more sensitive than the MNT for detecting virus neutralizing antibodies (VNAs) in postvaccinal sera , 36. TheIn a recent laboratory study on diagnosis of human rabies, rabies viral neutralizing antibodies were detected using RFFIT in 4/11 (36.3%) CSF samples received for antemortem diagnosis and 7/13 (53.8%) CSF samples obtained postmortem, which represents the terminal stage of illness. An inverse correlation was found between detection of neutralizing antibodies and presence of viral RNA in CSF samples . AntibodThe RFFIT is considered the gold standard assay and has been used to estimate the titre of rabies virus neutralizing antibodies for several years. However the test requires trained manpower, cell culture and fluorescent microscopy facilities, and adequate biosafety measures to handle live virus. The RFFIT is sensitive to cytotoxicity in poor-quality sera  and nons2O2 and amino ethyl carbazole). The rabies N antigens, if present, are found as brownish red clusters within the neuron, along the axons and scattered all over the brain smears  developed at Centers for Disease Control (CDC), Atlanta, USA . The tesn smears . The whoThough refrigerated reagent storage is required for dRIT, one of the main advantages of this newly developed test is the relative ease of interpretation using an ordinary light microscope. This simple test will enable developing countries to enhance rabies epidemiologic surveillance at greatly reduced cost and without the need for prohibitively expensive fluorescent microscopic equipment along with the expertise and financial input needed to maintain them. The cost-effectiveness of the dRIT will allow knowledge and technology transfer to remote areas of the developing world where rabies incidence data are difficult to obtain. It could be also valuable in guiding decisions regarding rational use of postexposure prophylaxis for rabies , 44.The WHO recommends further development of direct rapid immunohistochemistry tests as an alternative to the direct fluorescent antibody test for improved decentralized laboratory-based surveillance . HoweverRecently, an indirect rapid immunohistochemistry test (IRIT), for the detection and differentiation of rabies virus (RABV) variants, was evaluated by traditional light microscopy. Fresh frozen brain touch impressions or cell culture monolayers fixed in buffered formalin are stained with a panel of murine anti-nucleoprotein monoclonal antibodies (mAb-N) and commercially available biotin-labeled goat anti-mouse antibody. Ninety-six (96) RABV isolates, representing 20 RABV variants previously determined by antigenic typing using a panel of mAb-N and the indirect fluorescent antibody test (IFA), and genetic sequence analysis were characterized by IRIT. IRIT results revealed distinct reactivity patterns associated with current and historical RABV reservoir hosts similar to IFA test and genetic sequence analysis.IRIT does not require specialized equipment and can be performed in a field setting. Further, commercially available labeled secondary antibodies permit the use of a standard panel of unlabeled primary mAbs, without the need for fluorescence microscopy. IRIT can therefore be used as a cost-effective diagnostic test, which can also help to study the prevalence, distribution, and transmission of rabies virus among reservoir hosts in rabies enzootic areas .Another recently described method for the detection of rabies virus antigen from postmortem samples is the rapid immunodiagnostic test (RIDT), a useful method for rabies diagnosis without the need for laboratory equipments. This immunochromatographic lateral flow strip test is a one-step test that facilitates low-cost, rapid identification of viral antigen. Briefly, processed suspect material is added to the test device and conjugated detector antibodies attached to two different zones on a membrane indicate the presence of viral antigen . AnotherLyssaviruses belonging to all seven genotypes circulating in Europe, Africa, Asia, and Oceania [A simple, reliable and rapid sandwich ELISA (WELYSSA) can be used for detection of  Oceania , 52. Oth Oceania  and an e Oceania . The colorimetric enzymes used in rabies viral antigen detection are usually coupled to an antibody by chemical means using cross-linking reagents, which can lead to heterogeneous conjugates, sometimes with reduced activity and specificity. Chimeric bifunctional molecules in which the variable domains of an antibody are linked to unrelated protein tracers by genetic engineering can help circumvent this problem. A recent study described the successful production of a bifunctional chimeric protein based on alkaline phosphatase-fused anti-rabies virus glycoprotein scFv antibody fragment. It was found to be a novel in vitro tool for detecting rabies viral antigen in brain smears .Nucleic acid amplification and detection techniques have revolutionized the diagnosis of rabies in recent years and have an important role in the antemortem diagnosis of human rabies. Currently, several polymerase chain reaction (PCR) based assays have been evaluated as an adjunct to conventional tests for antemortem and postmortem rabies diagnosis. Most assays target the highly conserved rabies viral nucleoprotein gene for amplification. Several conventional gel-based reverse transcriptase PCR (RT-PCR) assays with nested/heminested protocols for detection of rabies viral RNA on clinical samples have been described , 56–60. Lyssaviruses [Real-time PCR based assays allow for the detection and quantification of genome copies and a considerable reduction in cross-contamination is achieved due to the “closed-tube” nature of these assays. Real-time PCR using the SYBR Green chemistry has been evaluated on human saliva samples for antemortem rabies diagnosis  and as aaviruses . Though aviruses . Real-tiaviruses , 63–66, aviruses . In a reaviruses . Despite several advantages, viral genetic heterogeneity may prove to be an impediment to the development of TaqMan probe based PCR since mismatches between the target and the probe can lead to false negative results or decreased sensitivity , 65. HowOther Molecular Assays. The nucleic acid sequence-based amplification (NASBA) technique uses three enzymes to generate multiple copies of the RNA under isothermal conditions. The automated NASBA enables easy and rapid testing of samples and has been reported to have a higher sensitivity than conventional PCR assays for detection of rabies viral RNA in antemortem saliva and CSF samples [ samples . Loop-Me samples , 69. Sin samples .Nucleic acid detection tests can be performed on a range of biological samples like CSF, saliva, tears, urine, skin biopsy, extracted hair follicles, and brain tissue for antemortem and postmortem diagnosis of human rabies. These assays have been found beneficial for diagnosis of rabies in decomposed and archival samples , 70–72 aHowever, a major limitation of these assays is the need for stringent quality control measures to avoid false positive results and the lack of international standards and universal protocols to be used for diagnosis. Currently, molecular assays are not recommended for routine postmortem diagnosis of rabies; if brain tissue is available FAT should be performed. However, they can be used for antemortem diagnosis of human rabies and for epidemiological surveys in laboratories with strict quality control procedures and with experience and expertise in using such techniques .Lyssaviruses using lentiviral pseudotypes. Pseudotypes, which are viruses that carry the genome and core of one virus and the envelope of another, are a safer alternative to live virus neutralization tests and can be used for rabies surveillance studies [Though the RFFIT is considered the gold standard to assess the rabies viral neutralizing antibodies, it has several drawbacks as mentioned earlier. Wright et al. investigated antibody neutralization of  studies . Enzyme-linked immunosorbent assay (ELISA) based methods are being used as an alternative to RFFIT. Though they are only antigen-binding assays and not functional assays which can detect neutralizing antibodies, unlike RFFIT, they are a simple, easy, safer, and rapid alternative to RFFIT. Assays that do not require live virus and high-containment facilities and produce rapid results have been validated and found to correlate well with RFFIT , 76. An Recent advances in genetic engineering have led to further improvement in ELISA based assays. A novel double sandwich ELISA utilizing recombinant antigen preparation, for detecting antibodies in dogs and other species, was developed recently . Nimmaga1HNMR) spectroscopy identified several metabolites that differentiated rabies survivors from those who subsequently died [A recent study reported quantitative proteomic analysis in human brain tissues obtained at autopsy from confirmed cases of encephalitic and paralytic rabies to identify signature proteins that are differentially regulated using high resolution mass spectrometry. Several proteins were differentially expressed, which included karyopherin alpha 4 (KPNA4) overexpressed only in paralytic rabies, calcium calmodulin dependent kinase 2 alpha (CAMK2A) which was upregulated in paralytic rabies, and glutamate ammonia ligase (GLUL) which was overexpressed both in paralytic and encephalitic rabies. These molecules need to be further investigated in body fluids like cerebrospinal fluid in a larger cohort of rabies cases to determine their potential use as antemortem diagnostic biomarkers in rabies . Anothertly died . FurtherRabies still remains one of the most neglected zoonotic diseases worldwide. The low level of commitment to rabies control is partly attributable to lack of accurate and extensive surveillance data to indicate the disease burden, frequent misdiagnosis of rabies, and an absence of intersectoral coordination. Traditional methods for antemortem and postmortem rabies diagnosis are fraught with several limitations. Though rabies is almost always fatal, a few human survivors have been reported in recent years. Hence an early, antemortem laboratory confirmation of rabies provides an impetus for clinicians to attempt experimental therapeutic approaches, especially in patients with paralytic rabies who may have a longer survival. Recent advances in diagnostic methods, including molecular methods, complement the conventional diagnostic approaches and have a potential to revolutionize rabies diagnosis. Though financial and logistical barriers may prevent the routine use of molecular diagnostic assays, the cost/benefit ratio should still be measured. The rapid reduction in turnaround time and cost with these tests indicates that in the near future they will become a viable technology in diagnostic and reference laboratories globally , 88.


The 2013 “Vaccine and Drug Ontology Studies” (VDOS 2013) international workshop series focuses on vaccine- and drug-related ontology modeling and applications. Drugs and vaccines have contributed to dramatic improvements in public health worldwide. Over the last decade, tremendous efforts have been made in the biomedical ontology community to ontologically represent various areas associated with vaccines and drugs – extending existing clinical terminology systems such as SNOMED, RxNorm, NDF-RT, and MedDRA, as well as developing new models such as Vaccine Ontology. The VDOS workshop series provides a platform for discussing innovative solutions as well as the challenges in the development and applications of biomedical ontologies for representing and analyzing drugs and vaccines, their administration, host immune responses, adverse events, and other related topics. The six full-length papers included in this thematic issue focuses on three main areas: (i) ontology development and representation, (ii) ontology mapping, maintaining and auditing, and (iii) ontology applications. Drugs and vaccines have been critical to prevent and treat human and animal diseases. Work in both (drugs and vaccines) areas is closely related - from preclinical research and development to manufacturing, clinical trials, government approval and regulation, and post-licensure usage surveillance and monitoring. Many drug and vaccine related ontologies have already been or are being developed for different use cases and applications. The 2013 “Vaccine and Drug Ontology Studies” workshop (VDOS 2013) workshop series aims to become an international forum for researchers to identify, propose, and discuss solutions for important research problems in ontology representation and analysis of vaccine and drug formation and preparation, administration, function mechanisms, and induced host immune responses. The immune responses can be positive responses for prevention and/or treatment of a disease, or can be negative responses, i.e., adverse events. This workshop aimed to support the deeper understanding of vaccine and drug mechanisms and effects.VDOS 2013 was held on July 7, 2013, at Montreal, Qc, Canada. This workshop was part of the fourth International Conference on Biomedical Ontology (ICBO 2013). The workshop attracted interest from many international attendees, including paper presenters, senior academic and government scientists, postdoctoral fellows, and graduate students. After a rigorous peer review process , six full-length papers and three short-length papers were accepted for proceeding paper publications and oral presentations in the workshop. After one additional round of independent peer reviewing by the workshop co-organizers and the journal editors, the selected six full-length papers were extended and accepted for publication in the current issue of the Journal of Biomedical Semantics (JBMS).The VDOS-2013 workshop is the 2nd in this series. The first workshop of the series was organized as the “Vaccine and Drug Ontology in the Study of Mechanism and Effect” workshop (VDOSME 2012)[The six papers selected for this thematic issue are extended versions of the original full-length papers presented at the VDOS 2013. These pIn the area of ontology development and representation, Lin and He introduced their Ontology of Genetic Susceptibility Factors (OGSF) for representing the susceptibility factors for post vaccination events using a formal ontological mechanism. OGSF isIn the area of ontology mapping, Winnenburg, et al. tried to map the terms in  and Medical Subject Headings (MeSH) through the Unified Medical Language System (UMLS). Both leIn the area of ontology applications, Doulaverakis et al. present a semantic framework to discover drug-drug and drug-disease interactions. They usIn the workshop, the six full-length papers described above were orally presented. In addition, three short papers were accepted for short oral presentations. Zhu et al. introduces their work on building a drug and drug class network derived from multiple drug terminological resources, such as ATC, National Drug File Reference Terminology (NDF-RT), RxNorm, and Structured Product Label (SPL). He et aDuring the discussion session, we discussed two main areas – (a) mapping between different drug models (6 papers), and (b) adverse events detection and analysis (3 papers). The major focuses on drug ontologies themselves (including models that contribute greatly in this area), their goals, and the challenges in aligning them, show that some of the preparatory work still needs to be done to continue the research into adverse events. A number of different approaches to drug mapping were discussed – lexical, ingredient based, using chemical structures and by exploiting networks (e.g. via the UMLS). While each provided their unique benefits, there was concurrence on the need for using more than one method to improve the quality of mapping. Furthermore, these papers also demonstrated the need for enhancing the definitions  of the terms and the relations both for improved human understanding as well as for better integration between them. The two papers focusing on adverse event detection and analysis also highlighted some of the gaps and shed light on areas for further ontology development efforts. These studies also demonstrated promising usages and advantages of ontology in standardizing, integrating, and analyzing adverse event data.Overall, the VDOS 2013 workshop provided an ideal platform for ontology researchers and users to present and discuss the progresses and issues in the development and applications of ontologies related to vaccines and drugs. Positive feedbacks were obtained.The authors declare that they have no competing interests.


To the Editor: Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic virus in the family Bunyaviridae, genus Phlebovirus, which affects mainly domestic ruminants and humans on continental Africa, Madagascar, and the Arabian Peninsula   in 1979, outside an epizootic period  rats  rats . In addioramanga .Serum samples were tested for IgG against RVFV by ELISA, as described  and introduced bushpigs  could be candidates, and their role in RVFV maintenance should be investigated (Serologic and virologic results from rodent and tenrec samples collected during and after the epizootic 2008–2009 periods were negative for RVFV; 72.8% had been collected in municipalities where RVFV cases were reported. This finding does not indicate a role of native Rodentia and Afrosoricida mammals in the epidemiology of RVFV in Madagascar, nor does it indicate evidence of infection of Taxonomy of small terrestrial animals sampled in Anjozorobe, Madagascar, October 2008 and 2009, March 2009 and 2010


Many nephrology observational studies use renal registries, which have well known limitations. The Canadian Kidney Disease Cohort Study (CKDCS) is a large prospective observational study of patients commencing hemodialysis in five Canadian centers. This study focuses on delineating potentially reversible determinants of adverse outcomes that occur in patients receiving dialysis for end-stage renal disease (ESRD).The CKDCS collects information on risk factors and outcomes, and stores specimens  at baseline and in long-term follow-up. Such specimens will permit measurements of biochemical markers, proteomic and genetic parameters (proteins and DNA) not measured in routine care. To avoid selection bias, all consenting incident hemodialysis patients at participating centers are enrolled, the large sample size (target of 1500 patients), large number of exposures, and high event rates will permit the exploration of multiple potential research questions.Data on the baseline characteristics from the first 1074 subjects showed that the average age of patients was 62  years. The leading cause of ESRD was diabetic nephropathy (41.9%), and the majority of the patients were white (80.0%). Only 18.7% of the subjects received dialysis in a satellite unit, and over 80% lived within a 50 km radius of the nearest nephrologist's practice.The prospective design, detailed clinical information, and stored biological specimens provide a wealth of information with potential to greatly enhance our understanding of risk factors for adverse outcomes in dialysis patients. The scientific value of the stored patient tissue will grow as new genetic and biochemical markers are discovered in the future. More than 1 million patients with end-stage renal disease (ESRD) receive renal replacement therapy (RRT) worldwide in the form of dialysis or transplantation, and this number is projected to reach 2.2 million by the year 2030 . DevelopThe widespread use of RRT to prolong life for people with ESRD has been one of the major advances in medicine . NonetheObservational studies are important sources of information to close knowledge gaps in every clinical discipline. However, most observational studies in ESRD have collected either good quality information on a small number of individuals, or poorer quality data on a much larger number of individuals. Many existing observational data are derived from renal registries such as the United States Renal Data Systems (USRDS) , EuropeaThe Canadian Kidney Disease Cohort Study (CKDCS) is a large prospective observational study of patients commencing dialysis in five Canadian centers serving ethnically mixed populations . This novel study focuses on identifying the potentially reversible determinants of the major complications associated with dialysis in an incident cohort of adult patients.The key research objectives for the CKDCS are:1. To assemble a prospective cohort of incident Canadian hemodialysis patients and associated database which contains research-quality information on patient characteristics, comorbid diseases and routine and novel laboratory markers and genetic data.2. To utilize data from the cohort to test specific hypotheses about the independent relation between biochemical and genetic markers and cardiovascular disease (CVD) risk and outcomes such as mortality, hospitalization and quality of life measures3. To rapidly capture information on the characteristics and health history of incident hemodialysis patients, using a structured patient interview and review of medical records.4. To operate a specimen management and storage system so that diverse specimens  can be collected at dialysis initiation and eventually be assayed by new biochemical and genetic assays.5. To make the data available to other academic investigators and collaborators if required.In order to have the most representative registry, the CKDCS enrolls all incident adult (≥18 years of age) hemodialysis patients from the five participating renal programs. The sole exclusion criterion is failure to provide informed consent.All subjects commencing chronic hemodialysis (HD) are approached by study staff within 8 weeks of commencing dialysis therapy. A variety of options for participating in the study are offered, depending on the interests of the patient (Appendix 1).Consenting participants undergo a structured interview to collect detailed data on demographic characteristics, medical history, social history and satisfaction with care. Information from the clinical record is used to supplement the history. At the time of the first annual follow-up visit, study subjects in Edmonton, Calgary and Vancouver independently complete a Kidney Disease Quality of Life Questionnaire (KDQOL), Health Utility Index questionnaire (HUI version 3) and an Attitudes Toward Transplantation Questionnaire. To avoid unnecessary expenditures on laboratory tests, routine test results  are captured in the study database using either a direct data link or entered manually by study personnel Table .Study visits are conducted at baseline, month 6, year 1, year 2, year 3, year 4, year 5 and then every 5 years thereafter. The baseline study visit is conducted within 8 weeks of initiating chronic dialysis. Follow-up visits are completed ± 2 month of the scheduled visit date  are available in English, French, Chinese and Spanish and may be completed by hand or electronically.Blood, dialysate effluent, fingernail and hair samples are obtained at baseline, 6 months, 12 months and at each study visit thereafter, according to standard protocols. Blood samples are immediately divided into multiple aliquots, from which plasma, DNA, RNA and/or cells are extracted and separately stored. Dialysate specimens are aliquoted to allow future measurement of genetic and cellular material. Specimens are frozen at -80 degree Celcius. Specimens are sent to the Canadian Biosample Repository in Edmonton and are labeled using a unique study identifier to maintain confidentiality.Source water used for dialysate is collected in triplicate four times annually from each dialysate unit . Patients are also asked to bring a sample of their drinking water from home on two separate days during one week at baseline and month 6. Source water specimens are shipped to the study laboratory and frozen for future analysis.A subset of 500 subjects will undergo imaging to determine the presence and extent of Coronary Artery Calcification (CAC) and Cardiac geometry including Left Ventricular Hypertrophy (LVH). CAC will be assessed by multi-slice computed tomography at two points during the study: baseline  and 12 months after dialysis initiation. All CT scans will be reported locally by an experienced reader for the presence, distribution and amount of CAC, using dedicated software (manufacturer dependent). Calcium mass, plaque volume and Agatston score will be calculated .To ensure uniformity and high quality image acquisition, all sites must: (1) use ≥64-slice multidetector Computed Tomography Scan (MDCT) technology or better (2) led by level 3 accredited cardiologist/radiologist. Before the start of the study, each site will send ten anonymized CAC reports and data sets to the core lab for assessment of image quality/data transfer/reproducibility of results. For sites demonstrating excessive variation, the core lab will review the site protocol/analysis tools and work with the site to reach the standard needed.Cardiac geometry (including LVH) will be assessed by cardiac magnetic resonance imaging (CMRI). Scanning is performed at baseline and at 12 months after dialysis initiation in these 500 participants. Left ventricular (LV) mass, and change in LV mass are analyzed as both quantitative traits and dichotomous end-points. All CMRI scans will be analyzed using dedicated software (manufacturer dependent) and reported locally by an experienced reader. Absolute and normalized to body surface area (BSA) left ventricular muscle mass, end diastolic blood pool volume, end systolic blood pool volume, ejection fraction, cardiac output and stroke volume will be calculated. Results forwarded to the core lab together with image data. To ensure uniformity and high quality image acquisition; all sites must: (1) use 1.5T CMR sequences (2) led by a level 3 CMRI certified radiologist/cardiologist. Before the start of the study, each site will send ten anonymized LV function data sets and results to the core lab for assessment of image quality/transfer capability and reproducibility of results. For sites demonstrating excessive variation, the core lab will review the site protocol/analysis tools and work with the site to reach the standard needed. An additional two studies will be requested to ensure the site fulfils all requirements.All participating renal programs perform ongoing surveillance of bacteremia in HD patients as an infection control initiative. Renal programs routinely record the date and microbiological species associated with positive blood cultures, as well as patient-specific information such as health card number and demographic data. Data on the management of bacteremia are also collected including: whether antibiotics were prescribed, antibiotic type/duration, whether catheter removal/exchange occurred and whether the event was resolved. Recurrent bacteremia is defined as a repeat positive blood culture with the same species within 6 months of the index event, according to current guidelines . PeriodsStudy subjects who die are identified by monthly follow-up by study coordinators; date of death is obtained from the renal programs. A prospectively defined method is used to ascertain the cause of death as previously published ,21. A stWhenever possible, other data sources are utilized by linking patient identifiers for analysis of outcomes and their determinants Figure . All datContain detailed information on pre-dialysis, dialysis and kidney transplant patients. Data linkage will provide information on medication use, laboratory results, bacteremia incidence and management, and access blood flows.The use of administrative data sources permit determination of disease incidence and prevalence using validated algorithms for common medical conditions such as hypertension , diabeteCensus data includes the population counts of each geographic area, as well as information, for example, on whether it is in a rural location (Statistics Canada definition), averageComputerized provincial databases are used to obtain laboratory data recorded outside of the renal programs.Study data are stored in a Microsoft SQL database created specifically for this project. Wherever possible, free text fields, blank values and open-ended questions have been eliminated from the database. "Not available" and "not applicable" values are permitted. Data from enrolled subjects are either entered directly into the database using a Microsoft Access interface through dedicated, password controlled laptop computers or are first collected on paper forms. All paper forms identify subjects exclusively by initials and study numbers. Quality assurance reports are generated on a monthly basis for the purposes of ensuring standard and compliance with the study protocol. Working copies of the database used to perform analyses are stripped of uniquely identifying information such as provincial health care number.The institutional review boards at the participating centers approved the study, and it is conducted according to the Helsinki declaration for medical research in humans.The sample size and large number of exposures together with the high event rate will permit the exploration of multiple potential research questions. In addition, the availability of stored blood samples will allow the testing of hypotheses raised by data which becomes available in the future. The target sample size is 1500 subjects.This study will provide a combination of cross-sectional and prospective clinical data and biological specimens of huge potential for conduct of high quality observational studies and clinical trials Figure .Potential Applications of the Cross-sectional data:1. Investigating the role of anemia, bone metabolism, inflammatory markers, endothelial dysfunction, genetic factors, and socio-demographic factors  on CVD at dialysis inception2. Assessment of pre-dialysis care and its impact on adverse outcomes3. Access and utility of kidney transplantationPotential Applications of the Prospective data:1. Determinants of all-cause mortality2. Determinants of dialysis access failure and consequences3. Determinants of poor quality of life and functional status4. Assessment of care delivery quality, impact and satisfaction5. Bacteremia screening: data on episodes and treatment6. Potential for biomarker discovery and validation using array technologyData on the baseline characteristics of the first 1074 subjects enrolled are presented in Table The CKDCS uses a unique study design to assemble a cohort of subjects recruited at hemodialysis inception in Canada to prospectively collect detail clinical and laboratory data as well as novel biochemical and genetic markers. While clinical trials are considered best evidence to guide clinical care and policy, this study will inform important knowledge gaps for several reasons . First, An ideal cohort study in ESRD should consist of a well-characterized population of incident patients to avoid prevalence-incidence bias ,30,32. TThe additional strength and novelty of this study is the collection and storage of materials  so that new risk factors such as the impact of trace elements on outcomes can be measured and studied in the future as they arise. The storage of genomic DNA and plasma for future analyses is a strength given the emerging importance of genetic and proteomic analysis -35. ThisThese unique characteristics make this study novel, topical and an extension of the existing observational databases on ESRD population. The largest such database is the USRDS, which although extremely useful, derives much of its information from billings data, which has significant limitations . For exaIn conclusion, the CKDCS will capitalize on the unique demographic and multiethnic characteristics of the Canadian dialysis population, a supportive Canadian regulatory climate, and the established and productive collaborations between Canadian nephrology investigators to address the limitations of existing databases. It will also provide novel information on determinants of adverse outcomes in hemodialysis patients, because of its unique combination of detailed prospectively collected information and the availability of stored biological specimens. This will provide new platforms for clinical and translational observational studies and clinical trials among dialysis patients in the future.ANZDATA: Australia and New Zealand Dialysis and Transplant; BSA: Body Surface Area; CAC: Coronary Artery Calcification; CHOICE: Choices for Healthy Outcomes in Caring for ESRD; CKDCS: Canadian Kidney Disease Cohort Study; CMRI: Cardiac Magnetic Resonance Imaging; CBC: Complete blood count; CT: Computed tomography; CVD: cardiovascular disease; DOPPS: Dialysis Outcomes and Practice Pattern Study; ERA-EDTA: European Dialysis and Transplant Association; ESRD: End-stage renal disease; EUTox: European Uraemic Toxins; GIS: Geographic Information System; HD: hemodialysis; HUI: Health Utility Index questionnaire; KDQOL: Kidney Disease Quality of Life Questionnaire; LVH: Left Ventricular Hypertrophy; MDCT: multidetector Computed Tomography Scan; PTH: Parathyroid hormone; RRT: Renal Replacement Therapy; USRDS: United States Renal Data Systems.The authors declare that they have no competing interests.All authors have made substantial contributions to the development of the manuscript, and have all been involved in revising it for important intellectual content and approved the final version.All subjects wishing to participate in the study are required to sign the Main CKDCS consent form which permits collection of health history and/or clinical laboratory data at baseline and during follow-up and will allow future analysis of stored blood specimens for non-genetic laboratory assays to examine "factors that influence the cause or treatment of heart or kidney disease".Genetic Sample Consent - The Genetic Sample Consent form will permit proteomic and genetic testing (plasma and DNA) from stored peripheral blood and other available biological samples such as tissue, to examine the same research questions.1. Both consent forms will explicitly state that:• data and specimens may be obtained and used after the death of the subject• provincial and national databases can be accessed to provide obtain other information about their healthControl Genetic Sample consent - Whenever possible, control blood samples are collected from a family member or acquaintances of the study subject. These samples are used as controls for the genetic analyses. Eligible family members or acquaintances will include those who do not have kidney disease and are willing to provide samples with the understanding that they are used in an anonymous fashion as the control in genetic analyses. The same safeguards are taken to protect the privacy of these control subjects, as is taken for participants treated with dialysis.2. CT consent - The Coronary CT consent permits a multi-slice coronary CT scan at baseline.3. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/12/10/prepub


Rift Valley fever (RVF) is a viral zoonosis that primarily affects animals resulting in considerable economic losses due to death and abortions among infected livestock. RVF also affects humans with clinical symptoms ranging from an influenza-like illness to a hemorrhagic fever. Over the past years, RVF virus (RVFV) has caused severe outbreaks in livestock and humans throughout Africa and regions of the world previously regarded as free of the virus. This situation prompts the need to evaluate the diagnostic capacity and performance of laboratories worldwide. Diagnostic methods for RVFV detection include virus isolation, antigen and antibody detection methods, and nucleic acid amplification techniques. Molecular methods such as reverse-transcriptase polymerase chain reaction and other newly developed techniques allow for a rapid and accurate detection of RVFV. This study aims to assess the efficiency and accurateness of RVFV molecular diagnostic methods used by expert laboratories worldwide. Thirty expert laboratories from 16 countries received a panel of 14 samples which included RVFV preparations representing several genetic lineages, a specificity control and negative controls. In this study we present the results of the first international external quality assessment (EQA) for the molecular diagnosis of RVF. Optimal results were reported by 64% of the analyses, 21% of the analyses achieved acceptable results and 15% of the results revealed that there is need for improvement. Evenly good performances were achieved by specific protocols which can therefore be recommended as an accurate molecular protocol for the diagnosis of RVF. Other protocols showed uneven performances revealing the need for improved optimization and standardization of these protocols. Rift Valley fever (RVF) is a zoonotic viral disease posing an increasing threat to animals and humans worldwide. Recent severe outbreaks of the disease in animal and human populations in endemic regions and outside the disease's traditional geographic boundaries necessitate the need for evaluating the diagnostic performance of RVF expert laboratories. Molecular methods are increasingly used for a rapid and accurate detection of viral nucleic acid. In this study we present the results of the first international external quality assessment (EQA) for the molecular diagnosis of RVF. Such EQA studies allow participating laboratories to monitor the quality and identify possible weaknesses of current diagnostic methods. Participants to this RVF EQA were 30 expert laboratories from 16 different countries worldwide. The study demonstrated that optimal results could be achieved by the majority of laboratories. Specific protocols showed evenly good performances and can therefore be recommended to all expert laboratories. However, other methods showed uneven performances suggesting the need for improved optimization and standardization of these protocols. Rift Valley fever (RVF) is a mosquito-borne viral zoonosis that primarily affects animals but also has the capacity to infect humans. An epizootic of RVF is usually first indicated by a wave of unexplained abortions as infected pregnant livestock abort virtually 100% of fetuses. The disease is less fatal to humans as most human infections are asymptomatic and when clinical symptoms appear they are in majority influenza-like. Nevertheless, some cases may develop a severe RVF disease with variable clinical signs. More severe cases occur in 2% of the RVF cases and fall into three categories: liver necrosis with hemorrhaging, retinitis with visual impairment and meningoencephalitis Phlebovirus of the Bunyaviridae family. The number of identified viral lineages of RVFV has increased from 3 in an early analysis th century, but it was first identified in 1930 during an outbreak of abortions and deaths among sheep in the Rift Valley region of Kenya. In 1977–78, several millions of people were infected and more than 600 died during a severe epidemic in Egypt The causative agent of RVF, the RVF virus (RVFV), is a negative-stranded RNA virus, a member of the genus Aedes mosquitoes with the capacity for transovarial transmission Anopheles and Culex species The emergence or re-emergence of RVFV activity is periodic and associated with exceptionally heavy rainfalls which allow massive breeding of flood-water Unavailability of effective antiviral drugs and commercial vaccines for human or animal use outside endemic countries, including the US and Europe, and the recent spread of RVFV beyond its usual boundaries has resulted in increased international demand for qualified diagnostic tools for a rapid and accurate diagnosis of RVF.Diagnostic methods for RVFV detection include virus isolation http://www.enivd.org) in 2012. Using the results of this study, the ENIVD can also provide support and advice to all laboratories performing RVFV molecular diagnosis.The performance of the different techniques applied for molecular diagnosis of RVFV may vary between laboratories. External quality assessment (EQA) studies to assess the quality of RVFV molecular diagnostics have not been performed until now. The EQA study allows the participating laboratories to monitor the quality of current diagnosis, identify possible weaknesses of particular diagnostic methods and evaluate their capacity for surveillance activities. Therefore the first EQA study for the molecular diagnosis of RVFV was organized by the European Network for Diagnostics of ‘Imported’ Viral Diseases (ENIVD)  to assure their non-infectivity. A serum sample spiked with Toscana virus, another phlebovirus, was included as a specificity control as well as two negative controls. The RVFV positive samples selected for this EQA panel are detailed in All virus material used for the preparation of the EQA panel was obtained from cell culture and not from clinical samples of infected patients. Therefore, there is no requirement for any ethical statement in this study.All samples were diluted with fresh thawed human plasma previously confirmed as negative for RVFV. Aliquots of 100 µl were number-coded, freeze dried for 24 h  and stored at 4°C until dispatched.Before dispatching the panels, 3 different sets of EQA samples were tested and validated by 2 expert laboratories. For validation, the samples were resuspended in 100 µl of water and the RNA extracted using the QIAamp viral RNA minikit . The number of RVFV genome copies present in these samples was determined by qRT-PCR.Panel samples were shipped by regular post at ambient temperature. We requested participant laboratories to resuspend the samples in 100 µl of water and to analyze the material as serum samples for nucleic acid detection of RVFV following their routine protocols. The EQA panels were distributed to participants with documentation including full instructions and an evaluation form to fill in their results. Participants were also asked to report information on the adopted protocol, the type of RVFV strain and the number of genome copies in each sample when possible as well as any problems encountered concerning the shipment or the packaging of the samples.To guarantee anonymous participation, an individual numerical identification code was assigned to the results reported by each laboratory. This number was followed by a letter  when distinguishable data sets of results based on different methods were sent.The results were scored in reflection of analytical sensitivity and specificity as in previous EQA studies performed by the ENIVD Results were classified as:Optimal when all results were correctAcceptable when all correct results are reported except one false-negative resultNeed for improvement when one or more false-positives and/or several false-negative results were reported.We obtained from the invitees a response rate of 91% representing a total of 30 participating laboratories from 16 different countries :CODA-CERVA, Department of Virology, Epizootic Diseases Section, Uccle, Belgium; ANSES, Virology Unit, Laboratory of Lyon, France; CIRAD, Department BIOS «Control of exotic and emerging diseases», Montpellier, France; IRBA-IMTSSA, Virology Unit, Le Pharo, Marseille, France; BNI, National Reference Centre for Tropical Infectious Diseases, Hamburg, Germany; Bundeswehr Institute of Microbiology, Munich, Germany; Institute for Novel and Emerging Infectious Diseases Friedrich-Loeffler-Institut, Germany; Robert Koch Institute, Berlin, Germany; Institute of Virology, Georg-August University, Gottingen, Germany; Central Virology Laboratory, Ministry of Health, Public Health Laboratories Sheba Medical Center, Israel; Army Medical and Veterinary Research Center, Rome, Italy; Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy; Padiglione Baglivi National Institute for Infectious Diseases “L. Spallanzani”, Rome, Italy; Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Italy; Center for Vectors and Infectious Diseases Research, National Institute of Health, Aguas de Moura, Portugal; King Fahd Medical Research Center, King Abdulaziz University, Saudi Arabia; Arboviruses and viral hemorrhagic fever Unit, Institut Pasteur de Dakar, Senegal; Defense Medical & Environmental Research Institute, DSO National Laboratories, Singapore; Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Slovenia; Onderstepoort Veterinary Institute, South Africa; Deltamune (Pty) Ltd, Centurion, Gauteng, South Africa; Special Viral Pathogens Laboratory, National Institute for Communicable Diseases, South Africa; Laboratory of Arboviruses and Imported Viral Diseases, National Center for Microbiology, Instituto de Salud Carlos III, Spain; National Institute for Agricultural Research and Experimentation (INIA), Madrid, Spain; Viral Diseases Unit, CReSA, Barcelona, Spain; Swedish Institute for Infectious Disease Control, Sweden; Virology group, Spiez Laboratory, Switzerland; Laboratory of Virology, University Hospitals of Geneva, Switzerland; WHO Collaborative Centre for Virus Reference and Research (Arboviruses & VHFs), Health Protection Agency, United Kingdom; Viral Special Pathogens Branch, Infectious Diseases, CDC, Atlanta, United States of America.A total of 39 datasets were received including 5 double sets from laboratories using 2 methods  and 2 triple sets from lab #5 and #14. Methods used by the same laboratory could differ from the type of technique, the protocol used for a specific technique or the type of instrument used for a specific protocol.Performances varied among laboratories and scores ranged from 7 to the maximum value of 14. Optimal results were reported by 64% (n = 25) of the analyses; 21% (n = 8) of the analyses achieved acceptable results due to the inability to detect one positive sample, and 15% (n = 6) revealed several false negative and/or one or more false positive results indicating that there is still need for improvement  and 3.RVF reference laboratories responded keenly to this EQA study (91% response rate), including laboratories situated in RVFV endemic countries such as South Africa and Saudi Arabia. Nonetheless, there is still a need to encourage more laboratories situated in RVF-endemic areas to participate in quality assurance programs. In fact, the increasing amplitude of this disease in Africa necessitates the rapid recognition of RVF outbreaks and implementation of effective control measures in order to prevent uncontrolled and wider spread of the virus.Most of the laboratories  reported the use of qRT-PCR techniques allowing a rapid detection as well as quantification of the virus genome. This confirms that the use of qRT-PCR has remarkably expanded although it requires expensive equipment. All datasets obtained by qRT-PCR only were scored with 13 or 14 points indicating an evenly high performance of all qRT-PCR procedures performed by the different laboratories.Protocols from Drosten et al, 2002 Information on the viral load of RVFV in human samples can be very useful to monitor the progress of clinical manifestations and to study the pathogenesis of RVFV. Interestingly, not all laboratories employing qRT-PCR techniques have reported quantified results and most of them (64%) reported the results as cycle thereshold (Ct) values and not the number of genome copies. This indicates that most laboratories do not resort to RVFV standards while performing qRT-PCR although such standards would allow them to quantify viral genome in each sample without performing any additional assay. Accordingly to the results of this EQA as well as previous EQA studies, there is still room for improvement concerning viral load determination The most widely used technique after qRT-PCR was nested RT-PCR with 5 laboratories which referred to 2 different protocols It is interesting to notice the appearance of newly developed techniques which are suitable for rapid field diagnostics such as RT-LAMP developed in 2009 On the other hand, RT-LAMP results indicated difficulties in detecting RVFV genome in the less concentrated samples of the panel . These results suggest some limitations in test sensitivity. However, very high test sensitivity is not essential for field diagnostics in an outbreak situation where most diagnosed patients are in the acute phase of the disease and are expected to present a high viremia.Three laboratories have provided different sets of results which referred to the same technique and protocol but using different instruments . These datasets provided all optimal results by using two different instruments except for dataset #14 which reported a slightly lower sensitivity using the SmartCycler System from Cepheid  compared to the 7500 Real-Time PCR System from Applied Biosystems . However, this difference cannot be attributed with certainty to the use of a different instrument as result variability can also arise from a lack of repeatability of the procedure.Only a few participants provided complete or partial information regarding strain typing . However, correct results without strain or genetic lineage specification are satisfactory in the context of laboratory diagnosis. Nonetheless, RVFV strain typing is relevant for surveillance activities in order to monitor which strains are circulating in RVFV-endemic areas and what type of clinical manifestations are associated with these strains.Comparing the results of this EQA panel to previous EQA studies Nevertheless, variations in performance between laboratories using the same method were noted. The reason for such variations is difficult to establish but can be minimized by standardizing procedures, including controls and testing conditions.In order to ensure optimal performances for RVFV molecular diagnosis in expert laboratories, we recommend conducting EQA studies on a regular basis. Future EQA studies should include a wide range of RVFV isolates with limiting concentrations to assess as precisely as possible the diagnostic performances of various molecular protocols in different reference laboratories.