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

4. NPDN Linkages APHIS-PPQ Cooperative Ext Industry NPDN State Dept of Ag (NPB) APHIS PPQ Databases CAPS GPDD NAPIS OPIS LGU NBIS

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)

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”

7. ID/Diagnostic Challenges • Symptoms frequently not definitive • Morphology of pathogens • Immunology (ELISA) • Molecular diagnostics (PCR) • Biochemical analyses • Culture • Knowledge base (something new & different)

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

9. Working Definitions ➢Accreditation: The determination that a laboratory is capable of performing competent diagnoses. ➢Certification: The determination that a laboratory has undergone training, method validation, and quality assurance procedures for a specific diagnostic method. ➢Assay: a quantitative or qualitative test of a substance (especially an ore or a drug) to determine its components; frequently used to test for the presence or concentration of infectious agents or antibodies etc. ➢Proficiency: competence in something, or knowledge of it. . ➢Validation: Assay validation is a process of determining the suitability of a given laboratory method for providing useful analytical data. It describes in mathematically quantifiable terms the performance characteristics of an assay, identifying the source of potential errors and the likelihood of their occurrence. (NIAID) ➢Registration: Evaluation and recognition of a quality system that defines how to produce the outputs (products or services) of the organization. Does not certify or guarantee the quality of products or service for compliance with specific specifications.

10. Purpose of Accreditation To accredit laboratories in the National Plant Disease Network (NPDN), State Dept. of Ag., and private or commercial sector, to carry out diagnostic tests on plant pathogens under defined standards for facilities, equipment, personnel training, and certified methods.

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. National Plant Diagnostic Laboratory Certification WorkshopOctober 12-13, 2004 • 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 …

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. Courtesy J.G. McDonald, CFIA

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; Courtesy J.G. McDonald, CFIA

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. Courtesy J.G. McDonald, CFIA

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

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 performPhytophthora 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

24. Questions?

26. Assay Validation • Assay validation is a process of determining the suitability of a given laboratory method for providing useful analytical data. It describes in mathematically quantifiable terms the performance characteristics of an assay, identifying the source of potential errors and the likelihood of their occurrence. (NIAID)

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: Accuracy Precision Limit of Detection Method Validation Limit of Quantitation Specificity Linearity and Range Ruggedness/Robustness System Suitability source: FDA

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 source: FDA

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. Pink Bollworm eradication Sudden Oak Death Pink Hibiscus Mealybug Ralstonia solanaceraum r3b2 Citrus Canker detection Saltcedar Gypsy Moth Plum pox Asian longhorn beetle Genetically modified Fluorescent Green PBW Larva Silverleaf whitefly PMTV Asian soybean rust Rearing of sterile medflies Safeguarding US Agriculture

32. USDA: National Plant Diagnostic Network * * * * * David T. Kaplan & Philip H. Berger USDA APHIS PPQ Kitty F. Cardwell USDA CSREES November 5, 2004

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)

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