Laboratory Diagnostics for Plant Pathogens of Regulatory Concern

1. Laboratory Diagnostics for Plant Pathogens of Regulatory Concern A National Laboratory Accreditation and Certification System Phil Berger National Science Program Leader - Molecular Diagnostics & Biotechnology USDA APHIS PPQ Center for Plant Health Science & Technology

5. NPDN Critical System Requirements Validated Diagnostics Plant Diagnostic Information System Information Management (Alerts) Data Security, Analysis, and Flow Training & Scenario Exercises Multidisciplinary Scientific Expertise Cross-ties to Other Networks Linkage with APHIS VS National Seed Health System (ASTA) FDA (ISO 17025) International Regulatory Agency Contacts EPPO, NAPPO, CFIA, DEFRA, IPPC Other (NBIS) We will learn from the APHIS VS advanced levels of experience on diagnostic network issues…they are more advanced than the systems for the NPDN (the whole system…not just CSREES) Diagnostic programs and accreditation systems in place to meet phytosanitary requirements for seed export, etc.We will learn from the APHIS VS advanced levels of experience on diagnostic network issues…they are more advanced than the systems for the NPDN (the whole system…not just CSREES) Diagnostic programs and accreditation systems in place to meet phytosanitary requirements for seed export, etc.

6. Challenges to the NPDN Diversity and Numbers of Crops, Pests, Pathogens, & Production Systems Over 1 billion Acres of crops, forage and forest Biology, Epidemiology Response and Recovery Threat Awareness Technology…“State of the Art” We are dealing with the equivalent of thousands of different pathogen-host interactions….and with tens of thousands of different pests and pathogens….. Response and Recovery are complex and strategies are not all in place nor are they likely to be adequate to maintain agricultural productivity. Threat Awarenss: tip off to something that might happen…OPIS and GPDD in APHIS. Networks within CDC, FBI, CIA etc. receive information…how can we get that information…..We are dealing with the equivalent of thousands of different pathogen-host interactions….and with tens of thousands of different pests and pathogens….. Response and Recovery are complex and strategies are not all in place nor are they likely to be adequate to maintain agricultural productivity. Threat Awarenss: tip off to something that might happen…OPIS and GPDD in APHIS. Networks within CDC, FBI, CIA etc. receive information…how can we get that information…..

7. ID/Diagnostic Challenges Symptoms frequently not definitive Morphology of pathogens Immunology (ELISA) Molecular diagnostics (PCR) Biochemical analyses Culture Knowledge base (something new & different) The numbers of targets pathogens is so large that we are likely to encounter situations where practical validated diagnostics will not be ready and available when we need them.The numbers of targets pathogens is so large that we are likely to encounter situations where practical validated diagnostics will not be ready and available when we need them.

8. Meeting the Challenges Validated Diagnostics Laboratory Accreditation Certification, Proficiency Testing Permits Harnessing New Technology Roles & Responsibilities (NPDN ? Regulatory Agencies) Offshore Pest Information Systems Information Technologies Accreditation = the laboratory has the competency and capacity to perform the validated tests. Certification = The accredited lab is certified to perform specific tests. Accreditation = the laboratory has the competency and capacity to perform the validated tests. Certification = The accredited lab is certified to perform specific tests.

11. National Plant Diagnostic Laboratory Certification WorkshopOctober 12-13, 2004 Organized by CSREES, APHIS & ARS Attendees from NPDN, NPB, ESCOP, private industry, and USDA (CSREES, APHIS, ARS) Presentations: Validation procedures, Peter Emanuel, DOD Accreditation procedures, Leon Thacker, AAVLD [Certification procedures, Barbara Martin, NAHLN] Certification and Accreditation, Denis McGee, NSHA Accreditation procedures, Cathy Burns, FDA Canadian Food Inspection Agency & NAPPO, John McDonald

12. National Plant Diagnostic Laboratory Certification WorkshopDesired outcomes Understanding other laboratory accreditation and certification systems What worked & what didn’t - what can we do to avoid the same mistake(s); what are the pitfalls and how can they be addressed Clarification of: Guiding principles for the system Physical and technical requirements for labs Procedures for accreditation and certification Identification of resources needed to implement the system and major milestones in implementation How much will it cost to implement and maintain?

13. Outcomes: APHIS and CSREES to collaboratively organize a Planning/Coordinating Committee (with APHIS, CSREES, ARS, NPDN, NPB representation) determine how to get the accreditation process underway and keep things moving provide oversight and set standards Hire ‘Accreditation Manager’ in APHIS to develop criteria Hire Accreditation Developer and Implementer in CSREES to set up and implement process, based on specifications provided by APHIS other … National Plant Diagnostic Laboratory Certification WorkshopOctober 12-13, 2004

14. Desirable/optimal Characteristics of a System for Laboratory Accreditation for Plant Pathogen Diagnostics All labs are ISO registered process for documentation of non-conformances process for continual improvement performance Minimum competency standards would be established Labs would meet regulatory requirements There would be assurance of acceptance of laboratory data Interstate assurances International assurances

15. ISO/IEC 17025-1999 Accreditation - a framework for operating procedures, QA, etc. … This standard requires documentation of quality policies, procedures, and test methods in a Quality System Manual and is geared toward laboratories conducting routine testing. One of the challenges in applying ISO/IEC 17025 to the regulatory diagnostic mandate is that the standard was designed to suit laboratories working in the physical sciences. The process of pest identification goes far beyond determining one or more characteristics by the act of separating an organism from all others, based on a complex array of recognized taxonomic characteristics, e.g. morphological, biochemical and DNA sequence.

16. ISO/IEC 17025 and Plant Pest Identification ISO/IEC 17025 requires that all tests either be standard methods, that contain the following information, or be non-standard methods, that shall be subject to agreement with the client. The laboratory shall also have a clear specification of the client’s requirements and use tests that have been validated and documented appropriately. Documentation should include at least the following: a) appropriate identification; b) scope; c) description of the type of item to be tested; d) parameters or quantities to be determined; e) apparatus and equipment, including technical performance requirements; f) reference standards and reference materials required; g) environmental conditions required and any stabilization period needed;

17. ISO/IEC 17025 and Plant Pest Identification (con’t) h) description of the procedure, including: affixing of identification marks, handling transporting, storing and preparation of items, checks to be made before the work is started, checks to be made that the equipment is working properly and, where required, calibration and adjustment of the equipment before each use, the method of recording and the observations and results, any safety measures to be observed; i) criteria and/or requirements for approval/rejection; j) data to be recorded and method of analysis and presentation; k) the uncertainty or the procedure for estimating uncertainty.

18. Accreditation of a Diagnostic Laboratory To be accredited to perform APHIS-PPQ validated diagnostic tests, a laboratory must have: Appropriate facilities and infrastructure Adequate and sustained institutional financial commitment Appropriate and properly maintained instrumentation Personnel (preferably permanent) with appropriate experience and training These parameters will need to be defined by APHIS…These parameters will need to be defined by APHIS…

19. Training Programsknowledge vs. skill An effective technician understands the method (and the science behind it) and has skill in using it. S/he can recognize when the method is out of specification. requires an ‘RO’ with appropriate background notification/review if RO changes --> technicians need appropriate skill and supervision Designed to provide practitioners with relevant background information (e.g., basic biology of P. ramorum) Basic background on the science underlying the method(s) used for assay Detailed knowledge of the specific method Troubleshooting Proficiency

20. Proficiency Demonstration that the laboratory can perform the method within specification Continued proficiency: what works today needs to work tomorrow, and the next day, and the next … Developing proficiency panels that demonstrate skill in performing the method can be challenging, e.g., Laboratory prepared samples vs. ‘real world’ samples Continuous verification of panel integrity Internal verification and auditing of panel samples Experimental material cannot be used

21. Issues to be addressed/resolved Who is to be accrediting body? At present not identified, but could be a joint function of USDA-APHIS and USDA-CSREES. Who will provide initial and annual funding to develop and sustain system? Administrative unit, regional centers, diagnostic labs… Is rule making required? May be necessary if regulatory action is required by USDA-APHIS on the basis of diagnosis made in the NPDN. Would significantly increase the time needed to implement the system. Where and how does ISO fit in the plan? The system, itself, must first be put in place. To comply with an ISO Standard, extensive resources of time and labor are required in document preparation. Significant fees and auditing cost are also required.

22. What steps should be taken to begin the planning process Approval of a plan by the potential Accrediting Bodies (CSRESS and USDA-APHIS). Formation of a Planning Committee. This could be a group that evolves into the Stakeholder’s Committee. Appoint a Planning Director with appropriate resources and a mandate to manage development of the system within a certain timeframe.

23. Identify labs to perform Phytophthora ramorum diagnostics Spring 2005 December ‘04! Goal: Up to ca. 10 labs to perform PPQ Pr diagnostics APHIS will establish criteria to identify candidate laboratories: - Laboratory infrastructure (human, physical, & fiscal commitment - Conduct site visits - Identify RO for each location - Provide training - Verify diagnostic proficiency

27. Common misconceptions Assay Validation ? Assay Optimization ? Assay Qualification A Validated Method is NOT necessarily a “tight” method Repeating an assay a number of time does not constitute validation source: FDA

28. Assay Validation Parameters:

29. Accuracy Accuracy is the measure of exactness of an analytical method, or the closeness of agreement between the measured value and the value that is accepted as a conventional true value or an accepted reference value. The determination of Accuracy usually requires a “gold standard” or an accepted method to which a new method can be compared - no gold standard available - no accepted method available

30. Future Diagnostics Real-Time PCR Multiplex Field deployable instrumentation & kits Microarray hybridization e.g., diagnostics for mollusks; pathogen arrays Immunological methods Biosensors volatile organic compounds Gene fragment analyses Improved diagnostics Biogeographic analyses

31. Photos (clockwise from top): -Gypsy moth on leaf -Dr. Nada Carruthers, Western Regional Research Center -Potato Mop-top virus symptoms in tuberPhotos (clockwise from top): -Gypsy moth on leaf -Dr. Nada Carruthers, Western Regional Research Center -Potato Mop-top virus symptoms in tuber

32. The concept of the NPDN was conceived in 2001 under the leadership of CSREES to harness the expertise and capacity of Land Grant Universities to develop a system based on existing resources to improve US ability to detect crop diseases of high consequence. Within the LGU’s are diagnostic labs, plant pathology & other pertinent departments, and Cooperative Extension that play significant roles in the NPDN. The NPDN has multiple interfaces with state and federal regulatory agencies (National Plant Board and APHIS PPQ). State and PPQ Regulatory Laboratories…all doing similar things…although NPDN is servicing the domestic agricultural production needs as well….The concept of the NPDN was conceived in 2001 under the leadership of CSREES to harness the expertise and capacity of Land Grant Universities to develop a system based on existing resources to improve US ability to detect crop diseases of high consequence. Within the LGU’s are diagnostic labs, plant pathology & other pertinent departments, and Cooperative Extension that play significant roles in the NPDN. The NPDN has multiple interfaces with state and federal regulatory agencies (National Plant Board and APHIS PPQ). State and PPQ Regulatory Laboratories…all doing similar things…although NPDN is servicing the domestic agricultural production needs as well….

33. End Game: Protect US Ag Production, Trade, Natural Resources and Food Supply Early Detection ? Low Impact System requirements: Technology: Secure Communication Data Management Validated Diagnostics Laboratory Certification High Level of Collaboration Clarity of Roles, Responsibilities, and Authorities Capacity and Capabilities Technical Working Groups Training (Develop & Deliver) Bioterror: Interaction with DHS; Regulatory Bioterror: Interaction with DHS; Regulatory

34. Plant Disease & Pest Pathways Intentional Introduction (Bioterror: Select Agents) Smuggling Natural Spread (Asiatic Soybean Rust) Trade (e.g., Rsr3b2, ALB, EAB) Tourism and Hurricanes (Citrus canker) Trade and Unknown (Sudden Oak Death) Propagative Material (Plum Pox)

35. Safeguarding of US Agriculture and Natural Resources A Systems Approach: NPDN: Domestic Surveillance, Detection, Identification, Surge Capacity & Reporting System Offshore Pest Detection Initiatives & Surveillance Systems CAPS: Systematic survey and delineation of high consequence or pests and pathogens in the US Emergency Preparedness (ICS) Detection, Response, Recovery

36. NPDN Summary Stewardship resides with USDA CSREES There is a high level of collaboration Development of NPDN is on-going Challenges to the program are significant System Multidisciplinary solutions required Diversity of target Technology Future Threat awareness Linkages to other networks Technology