WAVLD Symposium Handbook_V4.indd - csiro

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World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 1030 - 1230 Concurrent Session 2.4 - Quality Assurance LABS OF THE FUTURE A CULTURAL SHIFT IN CANADA FOR FOREIGN ANIMAL DISEASE (FAD) DETECTION D. Ridd & A. Copps, National Centre for Foreign Animal Disease, Canadian Food Inspection Agency (CFIA) Presenter: Dr. R.P. Kitching, Director, National Centre for Foreign Animal Disease, CFIA The success of a country’s capability for rapid detection of potentially epidemic animal diseases depends on many factors, one of which is laboratory diagnostic capacity and capability. Canada’s lab strategy under the Avian Influenza (AI) Pandemic Preparedness Plan brought forward the priority in 2006 to accelerate the plans and timelines for laboratory interoperability and to establish a national early warning system for perceived risks of Avian Influenza. Within 6 months, 4 federal and 7 provincial laboratories in the Canadian Animal Health Surveillance Network (CAHSN) were trained, equipped and approved for testing the presence of AI virus. The CAHSN was developed to consolidate the efforts across provincial, territorial and federal jurisdictions to respond to animal disease threats that could affect animal health, the food supply, or public health. Laboratory interoperability is a means towards meeting Canada’s requirements of an early warning system for outbreak management and emergency preparedness, planning for breaches of biosecurity and threats of bioterrorism. Quality Assurance (QA) Support and Laboratory Support although separate programs within CAHSN, are working together in achieving these goals and objectives. These programs’ are designed to deliver and harmonize quality assurance and laboratory systems through collaborations between federal/provincial animal health laboratories across Canada. The provincial laboratories as first responder laboratories will have the capacity and capability to rapidly diagnose foreign animal diseases, having been trained in FAD test methods and receiving the necessary supports towards lab infrastructure. The CAHSN Quality Assurance Support Program delivers support to the development of internal quality systems for FAD within the network laboratories with the goal of the labs achieving ISO/IEC 17025 accreditation. Based on site evaluations, funding has been allocated to establish and aid the implementation and maintenance of a standardized quality management program and to support the achievement of ISO/IEC 17025 accreditation. Resources were provided for QA Officers, ISO training, and ongoing accreditation costs. The CAHSN QA Support Program Manager liaises with the Network Laboratory Directors, Laboratory Managers/Supervisors and Quality Assurance Officers in a consulting, advisory capacity and provides oversight regarding all QA aspects in network laboratories relating to FAD testing. The CAHSN Laboratory Support Program ensures the animal health laboratories sharebest diagnostic practice, particularly for the major animal diseases such as Foot-and-Mouth Disease, Classical Swine Fever, Avian Influenza and Newcastle Disease, and establishes common diagnostic protocols and reagents, while ensuring diagnostic competence is maintained by the regular distribution and evaluation of proficiency panels. Where necessary, new equipment is provided to allow all network laboratories to make use of the most sensitive and specific diagnostic tests available. The CAHSN Laboratory Support Manager liaises with the Network Laboratory Directors and Laboratory Managers/Supervisors in a consulting, advisory capacity regarding all aspects of training/certification for network labs, encompassing the areas of equipment advice and purchases, reagents/supplies, surge capacity issues and is directly involved in training and certification of analysts. Conclusion: Canada’s jurisdictional requirements for diagnostic surge capacity are being achieved through harmonization and standardization of quality assurance and lab interoperability. The CAHSN Quality Assurance Support Program and the Laboratory Support Program are integral in aiding the network laboratories in the detection of emerging animal disease threats which could have zoonotic potential. This capability of early diagnosis and control is essential to minimizing the human health and economic consequences to the country. Ann Copps, CAHSN QA Support Manager coppsa@inspection.gc.ca Deidre Ridd, CAHSN Lab Support Manager dridd@inspection.gc.ca Wed 14 November Wed 14 November World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 LABORATORY-BASED EARLY ANIMAL DISEASE DETECTION UTILIZING A PROSPECTIVE SPACE-TIME PERMUTATION SCAN STATISTIC *C.N. Carter, University of Kentucky; A. Odoi, University of Tennessee; J. Riley, Hensley and Associates, Lexington, Kentucky; J. Smith, University of Kentucky; R. Stepusin, University of Kentucky; T. Cattoi, University of Kentucky; S. McCollum, University of Kentucky Introduction Veterinary diagnostic laboratories are in a unique position to analyze data from large numbers of clinical cases and to help with the early detection of health problems. The aim of this study is to develop a system for early detection of clusters of health events in animal populations using diagnostic laboratory data. The system provides near-real-time cluster alarms/alerts and medical situational awareness. Thus, it aids in the decision-making process related to conducting field investigations and/or mounting appropriate and timely medical responses. The system could, as well, be used to detect clusters of animal health events of public health significance (e.g. bioterrorist attacks). Material & methods Kulldorff’s prospective space-time permutation scan statistic was utilized in this project. The method uses a cylindrical window of varying size to scan for potential clusters in space and time. The statistic only requires case health events (e.g. confirmed diagnoses, deaths), thereby eliminating the need for population-at-risk data. We developed an engine that automatically performs statistical analysis at the close of business each day. A likelihood ratio test is used to test statistical significance of potential clusters. Identified clusters are automatically mapped for daily epidemiological decision-making. Here we present the methodology that was used to detect outbreaks of bovine blackleg and equine leptospirosis which occurred in 2006-2007. Results The system successfully detected significant clusters of bovine blackleg and equine leptospirosis cases during the 2006-2007 outbreaks. The detection of these clusters allowed the laboratory epidemiology section to mount a timely awareness campaign that included email alerts, field investigations, and direct consults with practicing veterinarians. Although not verifiable, this campaign likely led to enhanced vaccination efforts against bovine blackleg and aggressive MAT titer screening of horse farms for possible prophylactic treatment of mares. Blackleg losses for the 2006-2007 season are estimated at over $500,000 USD for eastern Kentucky alone. In addition, economic losses reported by 13 horse farms that experienced equine leptospiral abortion amounted to over $3 million USD for the 2006-2007 season alone. Earlier medical responses to outbreaks should result in reducing economic losses. Discussions & conclusions The appropriate analysis of laboratory-detected animal health events can be successfully used to generate medical alerts which can lead to timely and appropriate responses that can improve overall health outcomes and reduced economic losses. Further studies will be conducted to assess the sensitivity and specificity of medical alerts generated by this system. References Carter CN, Odoi A: Diagnostic Laboratory Surveillance and Epidemiology: Serving Agriculture and Public Health, Proceed 143 rd AVMA Convention, CD-ROM, July, 2006. Carter CN: Equine Disease Surveillance in Kentucky, Equine Disease Quarterly, Oct, 2005, Vol. 14, No. 4, pp 5-6. Bradley CA, Rolka H, et al. BioSense: implementation of a national early event detection and situational awareness system. MMWR Morb Mortal Wkly Rep. 2005 Aug 26; 54 Suppl: 11-9. Kulldorff M, Heffernan R, Hartman J, Assuncao R, Mostashari F. A space-time permutation scan statistic for disease outbreak detection. PLoS Medicine 2005 March; 2(3): 216-224. Norström M, Pfeiffer DU, Jarp J. A space-time cluster investigation of an outbreak of acute respiratory disease in Norwegian cattle herds. Preventive Veterinary Medicine, 47: 107-119, 2000. Perez AM, Ward MP, Torres P, Ritacco V. Use of spatial statistics and monitoring data to identify clustering of bovine tuberculosis in Argentina. Preventive Veterinary Medicine, 56: 63-74, 2002.
World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 DEVELOPMENT, VALIDATION AND IMPLEMENTATION OF MOLECULAR DIAGNOSTIC TESTS FOR IMPORTANT PATHOGENS OF FARMED ANIMALS AND WILDLIFE – A PRAGMATIC APPROACH *P.R. Wakeley, J. Errington, N. Johnson, C. Fearnley, M. J. Slomka, F. Yong and J. Sawyer Veterinary Laboratories Agency – United Kingdom Introduction We have developed a panel of molecular diagnostic tests for the detection of pathogens of farmed animals and wildlife to support diagnosis and surveillance activities. The tests developed include those to detect pathogens of farmed food animals such as a bovine viral diarrhoea virus/border disease virus (BVDV/BDV) and an assay to detect pathogenic leptospires. We have also developed and validated assays for the detection of pathogens of wildlife with zoonotic potential such as lyssavirus 1 and avian influenza, as well as assays to support the horse industry e.g. for the detection of Taylorella equigenitalis 2 the causative agent of contagious equine metritis (CEM). Molecular diagnostics offer the advantage over traditional “gold standard” technologies of viral or bacterial culture in that they are rapid, allow differentiation of closely related species and are not reliant on the presence of viable pathogen. Material & methods The tests developed make use of real-time polymerase chain reactions using fluorescence detection, either TaqMan or FRET. Where possible the assays have been validated against the accepted “gold standard” test which generally involves culture methods. However, we have found that culture techniques are either expensive to perform and can be substituted by inexpensive tests such as antigen ELISA e.g. for BVDV, or the confirmatory test is extremely lengthy to perform such as culture of leptospires (36 weeks) requiring other confirmatory tests of positive PCRs e.g. nucleic acid sequencing. Assays have been characterised with respect to analytical and diagnostic sensitivity and specificity as well as repeatability and robustness in comparison to the standard assays and have also been field trialled. In most instances we have taken a pragmatic approach to field validation as sufficient positive samples have not been available. One of the key limitations to successful performance of a PCR test is the efficient extraction of nucleic acids. Control assays have been developed to detect either RNA transcripts from the host, based on β-actin expression or for bacterial pathogens, such as leptospires and T.equigenitalis, real time detection assays based on the generic 16S rDNA of eubacteria. These controls are crucial for assays where the specimen cannot be resampled e.g. cloacal swabs from live captured wild birds for avian influenza surveillance, and provide confidence that the extraction component of the assay is performing optimally. Automated extraction of nucleic acids has been implemented to reduce contamination and “hands on time” for both the BVDV/BDV assay where we have used the lower throughput MagNApure (Roche) and avian influenza virus assays where we have used the higher throughput BioRobot 96 (QIAGEN). Results Field validation of several assays has led to novel and interesting findings. The first BVDV genotype 2 in the UK, BVDV in alpaca in the UK and the first border disease virus in cattle were discovered during the validation process of the BVDV/BDV TaqMan assay. Using the lyssavirus assay a number of European bat lyssavirus genotype 2 have been found in bats in the UK, including in an urban area close to the VLA. Implementation of the CEM assay has not only allowed detection of the reportable bacteria in infected horses in transit but due to the ability of the assay to discriminate T.equigenitalis from a closely related Taylorella species has led to changes in the controls used for culture purposes in our laboratories. The leptospire PCR has been successfully used to detect and trace pathogenic leptospire in a human case of leptospirosis and has also led to discussion of the impact of these bacteria on bovine abortion in the UK due to the low numbers of positives detected using the PCR. Discussions & conclusions The design and development of molecular tests for animal pathogens is not time consuming and costly; the validation of such tests most definitely is. Due to time constraints, the non-availability of appropriate confirmatory tests and the lack of suitable field samples, a pragmatic approach must be adopted to validation. Wherever possible internal controls should be implemented for not only the control of the amplification process but also the extraction. Validation processes in our experience can frequently lead to the discovery of novel scientific findings. References (1) Wakeley et al. J. Clin. Microbiol. 2005. 43 (6):2786-2792 (2) Wakeley et al. Vet. Microbiol. 2006. 118 (3-4):247-254 Wed 14 November Wed 14 November World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 QUALITY ASSURANCE AND CONTROL ISSUES WITH PCR AS A DIAGNOSTIC TOOL J. Oakey, Biosecurity Queensland, Tropical and Aquatic Animal Health Laboratory, Department of Primary Industries and Fisheries This aims to be a discussion of the quality assurance principles and current issues with the use of molecular biology techniques as tools for veterinary diagnosis. Detection of identifiable nucleic acid rapidly has become commonplace in veterinary diagnostic laboratories, presenting a number of significant advantages over conventional techniques. These methods are often faster, and more sensitive and specific. However, the routine use of what were so recently considered as research tools may be prone to over-reliance and lack of adequate quality management, quality control and overall quality assurance. It is essential that such techniques are demonstrated to be at least as reliable and “fit-for-purpose” as conventional methods if the test results are to be universally accepted. Standard regulations, manuals and official guidelines are increasingly being implemented. These will greatly assist in ensuring the reliability of the test results through standardisation of procedures, techniques, level of facilities and control of this powerful technology. It is thought that these guidelines will continue to form the basis for accreditation, validation and demonstration of proficiency of testing laboratories. This presentation will describe some of the current and draft guidelines in the context of using nucleic acid amplification (in the form of polymerase chain reaction) as diagnostic tools in veterinary laboratories and as prescribed tests for international trade. Practical issues that may affect quality assurance, application and standardisation of these techniques, and are yet to be addressed by standard documentation, will be identified from a laboratory implementation perspective. These concerns include availability of reagents and equipment on an international basis, inter-changeability of reagents and equipment, interpretation of methodology, geographical variations in target organisms and reactivity, and variations in accreditation standards. The processes of PCR will be discussed with application of a HACCP-like approach to identifying critical QA points affecting accuracy, reproducibility, and contamination control. Critical control points will be identified that may contribute to problematic standardisation between laboratories. In this context PCR will be split into pre-processing; sample processing / nucleic acid extraction; reaction mix preparation; adding template material to reaction mix; running a reaction; post-amplification processes; and post-amplification analyses. Each of these stages will be discussed with identification of perceived critical control points that may contribute towards reproducibility and standardisation concerns between laboratories.
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