High-consequence Pathogens Research Articles

Benefits of a European project on diagnostics of highly pathogenic agents and assessment of potential "dual use" issues.

Quality assurance exercises and networking on the detection of highly infectious pathogens (QUANDHIP) is a joint action initiative set up in 2011 that has successfully unified the primary objectives of the European Network on Highly Pathogenic Bacteria (ENHPB) and of P4-laboratories (ENP4-Lab) both of which aimed to improve the efficiency, effectiveness, and response capabilities of laboratories directed at protecting the health of European citizens against high consequence bacteria and viruses of significant public health concern. Both networks have established a common collaborative consortium of 37 nationally and internationally recognized institutions with laboratory facilities from 22 European countries. The specific objectives and achievements include the initiation and establishment of a recognized and acceptable quality assurance scheme, including practical external quality assurance exercises, comprising living agents, that aims to improve laboratory performance, accuracy, and detection capabilities in support of patient management and public health responses; recognized training schemes for diagnostics and handling of highly pathogenic agents; international repositories comprising highly pathogenic bacteria and viruses for the development of standardized reference material; a standardized and transparent Biosafety and Biosecurity strategy protecting healthcare personnel and the community in dealing with high consequence pathogens; the design and organization of response capabilities dealing with cross-border events with highly infectious pathogens including the consideration of diagnostic capabilities of individual European laboratories. The project tackled several sensitive issues regarding Biosafety, Biosecurity and “dual use” concerns. The article will give an overview of the project outcomes and discuss the assessment of potential “dual use” issues.

Systems kinomics for characterizing host responses to high-consequence pathogens at the NIH/NIAID Integrated Research Facility-Frederick.

Currently, there is a paucity of information regarding the molecular pathogenesis for many high‐consequence pathogens (HCPs) that pose threats to both national and international public health. In spite of this, investigations of the molecular pathogenesis for many HCPs have been limited to gross pathological changes in animal models or global analysis of gene expression. Further, questions remain regarding the ability of animal models of disease to recapitulate human molecular pathogenesis or act as predictors of therapeutic efficacy. Thus, it is likely that medical countermeasure development for HCPs will rely on identifying therapeutic targets that are uniquely modulated during HCP infection. It is also appreciated that many cellular processes can be regulated independently of changes in transcription or translation through phosphorylation events. Cellular kinases, individually or collectively (the kinome), play critical roles in regulating complex biology, underlie various malignancies, and represent high‐priority drug targets. The growing interest in kinases in both basic and translational research has driven efforts to develop technologies that enable characterization of phosphorylation‐mediated signal transduction. To this end, enhanced technical capabilities at the IRF‐Frederick provide the unique capability for characterizing host responses to HCP insult during the course of infection and identify novel targets for therapeutic intervention.

Biocontainment laboratory risk assessment: perspectives and considerations

The ability to respond to public health emergencies involving infectious diseases as well as our ability to adequately prepare for as yet unknown or unrecognized emerging infectious diseases requires suitable facilities within which scientific investigations can take place. To ensure the safe conduct of such investigations so that laboratory workers and the general public are protected from potential consequences of accidental or intentional release of high consequence pathogens, special containment facilities have been designed and constructed. Evaluation of the adequacy of containment for these types of investigations requires a risk assessment (RA) as part of the overall construction project for these types of laboratories. A discussion of the RA process along with considerations that impact the design of such studies and the overall results is presented.

Low-Incidence, High-Consequence Pathogens

Infectious diseases that have epidemic or pandemic potential and spread rapidly through a population within a short time are an ongoing public health concern in industrialized and developing countries. Frequent exposure to infectious sources (e.g., food, infected animals, and vectors) or a high rate of person-to-person spread facilitates spread of these diseases. Foodborne illnesses and seasonal influenza are notable examples. These diseases typically are associated with high rates of illness and substantial societal and economic cost but relatively low rates of death in otherwise healthy persons. Other infectious diseases, in contrast, may occur infrequently but are associated with high rates of death. The low incidence of some of these diseases reflects effective public health prevention measures, such as vaccinations. For a select group of zoonotic infectious diseases with high death rates, the low incidence reflects infrequent spillover from an animal reservoir into humans. Often, humans represent a dead-end host for these pathogens, and person-to-person transmission is rare if appropriate infection control practices are followed. Many of the pathogens highlighted in the current issue of Emerging Infectious Diseases can collectively be described as low-incidence, high-consequence pathogens. Selected diseases caused by these pathogens are described below.

Evaluating the impacts of stressors of Pseudomonas syringae pathovar tomato on the effectiveness of multi-locus variable number tandem repeat analysis and multi-locus sequence typing in microbial forensic investigations.

BackgroundCrops in the USA are vulnerable to natural and criminal threats because of their widespread cultivation and lack of surveillance, and because of implementation of growing practices such as monoculture. To prepare for investigation and attribution of such events, forensic assays, including determination of molecular profiles, are being adapted for use with plant pathogens. The use of multi-locus variable number tandem repeat (VNTR) analysis (MLVA) and multi-locus sequence typing (MLST) in investigations involving plant pathogens may be problematic because the long lag periods between pathogen introduction and discovery of associated disease may provide enough time for evolution to occur in the regions of the genome employed in each assay. Thus, more information on the stability of the loci employed in these methods is needed.ResultsThe MLVA fingerprints and MLST profiles were consistent throughout the experiment, indicating that, using a specific set of primers and conditions, MLVA and MLST typing systems reliably identify P.s. tomato DC3000. This information is essential to forensic investigators in interpreting comparisons between MLVA and MLST typing profiles observed in P.s. tomato isolates.ConclusionsOur results indicate that MLVA and MLST typing systems, utilizing the specified primers and conditions, could be employed successfully in forensics investigations involving P.s. tomato. Similar experiments should be conducted in the field and with other high-consequence plant pathogens to ensure that the assays are reliable for pathogens infecting plants in their natural environment and for organisms that may display faster rates of mutation.

Protein markers for identification of Yersinia pestis and their variation related to culture

The detection of high consequence pathogens, such as Yersinia pestis, is well established in biodefense laboratories for bioterror situations. Laboratory protocols are well established using specified culture media and a growth temperature of 37 °C for expression of specific antigens. Direct detection of Y. pestis protein markers, without prior culture, depends on their expression. Unfortunately protein expression can be impacted by the culture medium which cannot be predicted ahead of time. Furthermore, higher biomass yields are obtained at the optimal growth temperature (i.e. 28 °C–30 °C) and therefore are more likely to be used for bulk production. Analysis of Y. pestis grown on several types of media at 30 °C showed that several protein markers were found to be differentially detected in different media. Analysis of the identified proteins against a comprehensive database provided an additional level of organism identification. Peptides corresponding to variable regions of some proteins could separate large groups of strains and aid in organism identification. This work illustrates the need to understand variability of protein expression for detection targets. The potential for relating expression changes of known proteins to specific media factors, even in nutrient rich and chemically complex culture medium, may provide the opportunity to draw forensic information from protein profiles.

Retrospective Studies: Excellent Tools to Complement Surveillance

Emerging infectious diseases pose an important challenge to public health globally. The identification of an etiologic pathogen responsible for an emerging infection in humans can be a major task. The time required to isolate and characterize a new infectious agent sufficiently to adopt control measures and, when possible, to select and make available curative and preventive treatments can be considerable and take years. If the novel pathogen exhibits rapid pathogenesis, causing serious illness and death in a significant percentage of infected individuals, in addition to efficient transmission and spread, serious consequences for the human population can be readily observed. The race between the spread of a high-consequence pathogen and the deployment of adequate public health measures can hardly be satisfactory from a public perspective, because it is a reactive chain of events to the initial spread of the infectious agent. Predicting the emergence of infectious diseases months or years in advance would be a perfect solution but for now has remained a theoretical concept. Improved surveillance systems to detect emerging infectious diseases and new technologies using algorithms specifically developed to isolate and identify causative infectious agents have been emphasized to minimize public health emergencies related to emerging infections. Enhanced surveillance and detection is one of the most significant improvements to regional and global public health of the past several decades. For example, several regional or national networks, such as the Global Public Health Intelligence Network in Canada and the Global Disease Detection program of the Centers for Disease Control and Prevention network in the United States, are informing and cooperating with international networks, such as the Global Outbreak Alert and Response Network developed by the World Health Organization [1–3]. Unfortunately, surveillance and detection methods are region specific, and many regions are currently not being actively monitored. Regions under surveillance oftenmake use of different technologies and approaches that are not yet standardized, thus creating many potential holes in the global network intended to detect new pathogens before they cause damage. More work will be required before these newly developed systems are ideal, but the recognition of their importance and usefulness is a critical step toward enhanced preparedness and response and ultimately prevention. Viruses are responsible for a significant proportion of emerging infectious diseases, the numbers of which may be on an increasing trend overall [4, 5]. It is clear that surveillance and identification of unknown pathogens has greatly improved in the past decade, which has resulted in the concomitant rise in the number of new pathogens being discovered each year. Again, this increase has occurred despite the fact that the detection and identification of previously unknown pathogens currently depends on heterogeneous surveillance systems of variable sensitivity and precision. Regardless, heightened surveillance, detection, and identification of emerging pathogens are currently active and prolific fields of research. Whether there exists a real or apparent increase in the number of viruses emerging in the past 50 years, compared with the number that emerged 50 or 100 years earlier, there is certainly an increasing number of viruses being detected and identified in recent years. A PubMed search using the term “emerging viruses” revealed that the number of scientific articles published on the subject of emerging viruses has jumped from <10 per year in the early 1990s to nearly 70 in the early 2000s and up to 220 per year during 2011–2012, reflecting a Received and accepted 6 November 2013; electronically published 14 November 2013. Correspondence: Gary Kobinger, Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St, Winnipeg, Manitoba, R3E 3R2 (gary.kobinger@phac-aspc.gc.ca). The Journal of Infectious Diseases 2014;209:811–2 © Crown copyright 2013. DOI: 10.1093/infdis/jit604

News from the CDC: chronic fatigue syndrome (CFS) and standardized patient videos — a novel approach to educating medical students about CFS

News from the CDC: chronic fatigue syndrome (CFS) and standardized patient videos — a novel approach to educating medical students about CFS

The Microbial Rosetta Stone Central Agricultural Database: An Information Resource on High-Consequence Plant Pathogens.

Microbial pathogens of humans, animals, and plants can serve as potential agents of biowarfare, bioterrorism, and biocrime. Previously, the Microbial Rosetta Stone (MRS) Central database, an easily accessible informational resource tool, was developed to assist law enforcement personnel in the event of a disease investigation by providing key information on pathogens of concern. Although the database already contained information on a few high-profile plant pathogens, the coverage was insufficient considering the large number of plant pathogens that pose a threat, not only to agricultural production but also to natural plant resources such as forests and rangelands. In this project, 100 plant pathogens of high consequence were selected for study, existing literature on these agents was reviewed, and both the sources and key pathogen information provided therein were curated in the new Agricultural Database (AgDB), an accessory to the existing MRS Central Database. Chosen for inclusion in the MRS Central AgDB were plant pathogens having significant potential for damage to U.S. agricultural and natural ecosystems. The selection process included review of several previously developed plant-pathogen threat lists and recommendations from experts within the U.S. plant biosecurity community. Pathogen information was collected by searching a number of relevant literature databases, sites on the World Wide Web, and other resources. For inclusion in the MRS, the information was curated into categories: pathogen taxonomy, nomenclature synonyms, disease symptoms and geographic distribution, plant hosts, insect vectors, detection and diagnostic methods, laboratory and field protocols, sample collection, and epidemiology. The resulting AgDB enhances the MRS Central Database by summarizing and linking key information on high-threat plant diseases and their causal agents to relevant scientific literature and internet resources. The AgDB contains critical, key information on high-consequence plant pathogens, curated in a format that is readily accessible and easily searched. The resource enhances the existing MRS Central Database and provides law enforcement, forensic, and investigative personnel with an additional tool with which to respond to microbial emergencies, particularly those affecting the agricultural and environmental sectors.

Bioterrorism: Health sector alertness

The global events of the last two decades indicate that the threat of biological warfare is not a myth, but a harsh reality. The successive outbreaks caused by newly recognized and resurgent pathogens and the risk that high-consequence pathogens might be used as bioterrorism agents amply demonstrate the need to enhance capacity in clinical and public health management of highly infectious diseases. This review article provides a concise overview of bioterrorism, the agents used, and measures to counteract it, with a relevant note on India's current scenario of surveillance systems, laboratory response network, and the need for preparedness.

Probability of introducing foot and mouth disease into the United States via live animal importation

Foot and mouth disease (FMD) continues to be a disease of major concern for the United States Department of Agriculture (USDA) and livestock industries. Foot and mouth disease virus is a high-consequence pathogen for the United States (USA). Live animal trade is a major risk factor for introduction of FMD into a country. This research estimates the probability of FMD being introduced into the USA via the legal importation of livestock. This probability is calculated by considering the potential introduction of FMD from each country from which the USA imports live animals. The total probability of introduction into the USA of FMD from imported livestock is estimated to be 0.415% per year, which is equivalent to one introduction every 241 years. In addition, to provide a basis for evaluating the significance of risk management techniques and expenditures, the sensitivity of the above result to changes in various risk parameter assumptions is determined.

Complete Genome Sequences of Mycoplasma leachii Strain PG50 T and the Pathogenic Mycoplasma mycoides subsp. mycoides Small Colony Biotype Strain Gladysdale

Mycoplasma mycoides subsp. mycoides small colony biotype (SC) is the high-consequence animal pathogen causing contagious bovine pleuropneumonia. We report the complete genome sequences of the pathogenic strain M. mycoides subsp. mycoides SC Gladysdale and a close phylogenetic relative, Mycoplasma leachii PG50(T), another bovine pathogen of the M. mycoides phylogenetic clade.

Assessment of High-Throughput Screening (HTS) Methods for High-Consequence Pathogens

Currently, there are no Food and Drug Administration (FDA)-approved antiviral drugs or therapeutics for many of the biosafety level three (BSL-3) and four (BSL-4) pathogens. Many of these high-consequence pathogens, including Venezuelan equine encephalitis virus (VEEV), Ebola virus (EBOV), Marburg virus (MARV), and Lassa virus (LASV), are classified as biothreat agents and the development of therapeutic treatments for these diseases is an important area of research. In recent years, high-throughput screening (HTS) assays have become an effective and robust tool used for drug and therapeutic discovery. There are several types of HTS methods available, including targeted screening, diversity and high-content screening, and RNA interference (RNAi). These screens have been used effectively with a number of BSL-2 pathogens, but present unique challenges for the BSL-3/4 pathogens due to the requirement for higher level biocontainment facilities as well as biosurety requirements. Addressing and overcoming these challenges is essential for the proper adaptation of HTS into higher biocontainment facilities. In this article, we will discuss the advantages and disadvantages of each of the aforementioned HTS methods in the context of BSL-3/4 containment.

Ralstonia solanacearum Race 3 Biovar 2 Causes Tropical Losses and Temperate Anxieties

R. solanacearum Race 3 biovar 2, which causes potato brown rot and Southern wilt of geranium, is a high-consequence quarantine pathogen in Europe and North America and causes large crop losses in tropical highlands. This practical review emphasizes prevention, detection, diagnosis, and effective management of diseases caused by this destructive pathogen. Accepted for publication 17 January 2009. Published 13 March 2009.

Rapid microchip-based electrophoretic immunoassays for the detection of swine influenza virus

Towards developing rapid and portable diagnostics for detecting zoonotic diseases, we have developed microchip-based electrophoretic immunoassays for sensitive and rapid detection of viruses. Two types of microchip-based electrophoretic immunoassays were developed. The initial assay used open channel electrophoresis and laser-induced fluorescence detection with a labeled antibody to detect influenza virus. However, this assay did not have adequate sensitivity to detect viruses at relevant concentrations for diagnostic applications. Hence, a novel assay was developed that allows simultaneous concentration and detection of viruses using a microfluidic chip with an integrated nanoporous membrane. The size-exclusion properties of the in situ polymerized polyacrylamide membrane are exploited to simultaneously concentrate viral particles and separate the virus/fluorescent antibody complex from the unbound antibody. The assay is performed in two simple steps--addition of fluorescently labeled antibodies to the sample, followed by concentration of antibody-virus complexes on a porous membrane. Excess antibodies are removed by electrophoresis through the membrane and the complex is then detected downstream of the membrane. This new assay detected inactivated swine influenza virus at a concentration four times lower than that of the open-channel electrophoresis assay. The total assay time, including device regeneration, is six minutes and requires <50 microl of sample. The filtration effect of the polymer membrane eliminates the need for washing, commonly required with surface-based immunoassays, increasing the speed of the assay. This assay is intended to form the core of a portable device for the diagnosis of high-consequence animal pathogens such as foot-and-mouth disease. The electrophoretic immunoassay format is rapid and simple while providing the necessary sensitivity for diagnosis of the illness state. This would allow the development of a portable, cost-effective, on-site diagnostic system for rapid screening of large populations of livestock, including sheep, pigs, cattle, and potentially birds.

Spatio-Temporal Processing for Multichannel Biosensors Using Support Vector Machines

Rapid-response biosensing systems are necessary to counteract threats due to foreign and high-consequence pathogens. A yes/no multichannel biosensor is an important tool that enables simultaneous detection of different pathogens, independent of their relative concentration level. This paper proposes a novel multichannel biosensing technique, which combines multiclass support vector machines (SVMs) with multichannel immunosensors. The method combines spatial and temporal information generated by the multichannel immunosensor for rapid and reliable discrimination between pathogens of interest. This paper demonstrates that by including temporal and cross-reactive spatial signatures, the accuracy of the system can be improved at low pathogen concentration levels and for discrimination between closely related strains of pathogens. Compensation of systematic and biosensor fabrication errors is achieved by the use of a supervised SVM training which is also used in system calibration. Experimental results, with a prototype multichannel biosensor used for discriminating strains of E. coli (K12 and O157 : H7) and Salmonella enterica serovar Thompson, show an accuracy of 98% for concentration levels, 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">0</sup> -10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">8 </sup> colony forming units per milliliter, and total detection time of less than 6 min

Biological weapons

.In the last 2 decades, successive outbreaks caused by new, newly recognised and resurgent pathogens, and the risk that high-consequence pathogens might be used as bioterrorism agents amply demonstrated the need to enhance capacity in clinical and public health management of highly infectious diseases. In this article we review these recent and current threats to public health, whether naturally occurring or caused by accidental or intentional release. Moreover, we discuss some components of hospital preparedness for, and response to, infectious disease of the emergencies in developed countries. The issues of clinical awareness and education, initial investigation and management, surge capacity, communication, and caring for staff and others affected by the emergency are discussed. We also emphasise the importance of improving the everyday practice of infection control by healthcare professionals.

Armored RNA as Virus Surrogate in a Real-Time Reverse Transcriptase PCR Assay Proficiency Panel

In recent years testing responsibilities for high-consequence pathogens have been expanded from national reference laboratories into networks of local and regional laboratories in order to support enhanced disease surveillance and to test for surge capacity. This movement of testing of select agents and high-consequence pathogens beyond reference laboratories introduces a critical need for standardized, noninfectious surrogates of disease agents for use as training and proficiency test samples. In this study, reverse transcription-PCR assay RNA targets were developed and packaged as armored RNA for use as a noninfectious, quantifiable synthetic substitute for four high-consequence animal pathogens: classical swine fever virus; foot-and-mouth disease virus; vesicular stomatitis virus, New Jersey serogroup; and vesicular stomatitis virus, Indiana serogroup. Armored RNA spiked into oral swab fluid specimens mimicked virus-positive clinical material through all stages of the reverse transcription-PCR testing process, including RNA recovery by four different commercial extraction procedures, reverse transcription, PCR amplification, and real-time detection at target concentrations consistent with the dynamic ranges of the existing real-time PCR assays. The armored RNA concentrations spiked into the oral swab fluid specimens were stable under storage conditions selected to approximate the extremes of time and temperature expected for shipping and handling of proficiency panel samples, including 24 h at 37 degrees C and 2 weeks at temperatures ranging from ambient room temperature to -70 degrees C. The analytic test performance, including the reproducibility over the dynamic range of the assays, indicates that armored RNA can provide a noninfectious, quantifiable, and stable virus surrogate for specific assay training and proficiency test purposes.

Encyclopedia of bioterrorism defense

Preface. Contributors. Acknowledgment. Abu Sayyaf Group. Aerosol. Agricultural Bioterrorism. Aliens of America: A Case Study. Al-Quida. Animal Aid Association: A Case Study. Anthrax (Bacillus anthracis). Anthrax Hoaxes: Case Studies and Discussion. Anti-Material Agents. Armed Islamic Group: A Case Study. Army of God. Army Technical Escort Unit. Assassinations. Aum Shinrikyo and the Aleph. Baader-Meinhof Gang. Biological Simulants. Bioregulators. Biosecurity: Protecting High Consequence Pathogens and Toxins Against Theft and Diversion. Biotechnology and Bioterrorism. Bioterrorist Attack, Stages, and Aftermath. Botulinum Toxin. Breeders: A Case Study. Brucellosis (Brucella spp.). Camelpox. CDC Category C Agents. Centers for Disease Control and Prevention's Bioterrorism Preparedness Program. Central Intelligence Agency. Characteristics of Future Bioterrorists. Chechen Separatists. Christian Identity. Consequence Management. Cost-Effectiveness of Biological Weapons. Crisis Management. Cuba. Dark Harvest. Dark Winter. Defense Threat Reduction Agency. Defense Research and Development Canada-Suffield. Delivery Methodologies. Department of Defense. Department of Health and Human Services. Department of Homeland Security. Department of Justice. Department of State. Detection of Biological Agents. Diane Thompson: A case Study. Director of Central Intelligence Counterterrorist Center. DoD Policies on Force Health Protection: Medical Defense Against Biological Warfare Agents. Dual-Use Equipment and Technology. Dugway Proving Ground. Edgewood Chemical Biological Center (Formerly Edgewood Arsenal), Aberdeen Proving Ground. Education for Biodefense. Epidemiology in Bioterrorism. Equine Encephalitis, Venezuelen, and Related Alphaviruses. Ethnic Weapons. Fatality Management. Federal Bureau of Investigation. Food and Beverage Sabotage. Food and Drug Administration. Food and Waterborne Pathogens. Fort Detrick and USAMRIID. Glanders (Burkholderia mallei). HAMAS. Hemorrhagic Fever Viruses. Hizballah. Homeland Defense. Human Immunodeficiency Virus. Influenza. Intelligence Collection and Analysis. International Cooperation and Bioterrorism Preparedness. International Regulations and Agreements Pertaining to Bioterrorism. Iran. Iraq. Islam, Shi'a and Sunna. Islamism. Israel. Joint Task Force Civil Support. Korea, Democratic People's Republic of Kurdistan. Laboratory Response to Bioterrorism. Larry Wayne Harris. Lawrence Livermore National Laboratory. Liberation Tigers of Tamil Eelam. Libya. Los Alamos National Laboratory. Marine Algal Toxins. Marine Corps Chemical and Biological Incident Response Force. Mau-Mau. Media and Bioterrorism. Metropolitan Medical Response System. Minnesota Patriots Council. Minutemen: Case Studies. Modeling the Public Health Response to Bioterrorism. National Institutes of Health and National Institute of Allergy and Infectious Diseases. NATO and Bioterrorism Defense. North American Militia. NORTHCOM (U.S. Northern Command). Office International des Epizooties: World Organization for Animal Health. Orange October: A Case Study. Palestine Liberation Organization. Palestinian Islamic Jihad. Pharmaceutical Industry. Pine Bluff Arsenal. Plague (Yersinia pestis). POLISARIO. Prevention and Treatment of Biological Weapons-Related Infection and Disease. Prion Diseases. Psychological and Social Sequelae of Bioterrorism. Public Health Preparedness in the United States. Rajneesjees. Republic of Texas: A Case Study. Ricin and Abrin. RISE: A Case Study. Risk Assessment in Bioterrorism. Sandia National Alboratories. Scientists, Societies, and Bioterrorism Defense in the United States. Smallpox. Staphylococcal Enterotoxins. Sudan, Republic of. Suicide Terrorism. Syndromic Surveillance. Syria. Terrorist Group Identification. Threat Reduction in the Former Soviet Union. TOPOFF. TOPOFF 2. Toxins: Overview and General Principles. Tularemia (Francisella tularensis). Typhus, Epidemic (Rickettsia prowazekii). United Kingdom: Bioterrorism Defense. United States Department of Agriculture. United States Legislation and Presidential Directives. Water Supply: Vulnerability and Attack Specifics. Weapons of Mass Destruction Civil Support Teams. Weather Underground: A Case Study. Index.

Use of a Novel Virus Inactivation Method for a Multicenter Avian Influenza Real-Time Reverse Transcriptase—Polymerase Chain Reaction Proficiency Study

Proficiency assessments are important elements in quality control for diagnostic laboratories. Traditionally, proficiency testing for polymerase chain reaction (PCR)-based assays has involved the use of clinical samples, samples "spiked" with live agents or DNA plasmids. Because of government regulations and biosecurity concerns, distribution of live high-consequence pathogens of livestock and poultry, such as avian influenza, is not possible, and DNA plasmids are not technically suitable for evaluating RNA virus detection. Therefore, a proficiency testing panel using whole avian influenza in a diluent containing a phenolic disinfectant that inactivates the virus while preserving the RNA for at least 8 weeks at -70 C was developed and used in a multicenter proficiency assessment for a type A influenza real-time reverse transcriptase (RT)-PCR test. The test, which was highly standardized, except for variation in the real-time RT-PCR equipment used, was shown to be highly reproducible by proficiency testing in 12 laboratories in the United States, Canada, and Hong Kong. Variation in cycle threshold values among 35 data sets and 490 samples was minimal (CV = 5.19%), and sample identifications were highly accurate (96.7% correct identifications) regardless of real-time PCR instrumentation.