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Impact, validation and regulatory implication of rapid methods

Impact, validation and regulatory implication of rapid methods. Peter Feng, Research Microbiologist FDA Center for Food Safety and Applied Nutrition (CFSAN) Wednesday, May 28, 2008. Impact, validation and regulatory implication of rapid methods Peter Feng, Ph.D. Food safety regulations

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Impact, validation and regulatory implication of rapid methods

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  1. Impact, validation and regulatory implication of rapid methods Peter Feng, Research Microbiologist FDA Center for Food Safety and Applied Nutrition (CFSAN) Wednesday, May 28, 2008

  2. Impact, validation and regulatory implication of rapid methods Peter Feng, Ph.D. • Food safety regulations • impact of food safety laws vs method development • Impact of changes • society/lifestyle - new problems, regulations, methods • Problems in Food Analysis • Rapid Methods - definitions and origin • Next generation technologies • Advantages and limitations • Validation • Impact on regulations

  3. Food Safety Law – impact on method development • 1785 - State of MA: 1st law on food adulteration • 1787 – U.S. Constitution – Federal Government • Society - Agricultural to industrial (mid to late 1800’s) • 1902 - “poison squad” Harvey Wiley, USDA • 1906 - Food and Drug Act, USDA • prohibit manufacture/interstate commerce of adulterated and • misbranded food, drinks & drugs (burden of proof - Fed Government) • 1938: Federal Food, Drug & Cosmetic Act • safety of cosmetic and medical devices • tolerance of unavoidable poison in food • standard of product identity, quality and fill • factory inspection authority • producer must show product safety (burden of proof – industry)

  4. Impact of Changes • Population – advances in medicine • Longevity - > immunosusceptible population • Lifestyle – • Healthier diet – fruits and vegetables; > imports • Dual income family – changes in eating habits. > restaurants, fast food & carry out. • Consequences • Convenience industry – RTE; ie: bagged produce. • Mass production & Broad distribution – large outbreaks • Bottomline: new food safety problems, regs - new methods

  5. Microbiological Analysis of Foods • Pathogens – • Presence/absence • Indicators – TVC, coliforms, E. coli,enterobacteriaceae • Enumeration - Direct: plate counts, membrane filtration (MF) - Indirect: Most Probably Number (MPN); enzymatic activity on specific substrate to estimate cell density

  6. Pathogen & Toxins in Foods – presence/absence Solutions Problems Bacteria Toxins • Complex Matrices enrichment (dil.) extraction • Normal flora Sel. enrichment little effect • Low target levels Post-enrichment concentration • Processing (injury) Pre-enrichment renaturation Indicators in Foods - enumeration • Problems Solutions • Complex Matrices pre-filtration, dilutions • High Cell density serial dilution

  7. Conventional microbiological analysis of foods toxins pathogens Food Conc. extract Non-selective media Pre-enrichment resuscitation Test + Buffer Incubate indicators Sub culture Serial dilutions Selective media Selective enrichment selection Enumeration TVC, MPN, MF Incubate Presumptive identification Sub culture Post-enrichment > cell # Differential/ Selective agar medium Incubate growth media plate Pick colonies Serotyping and/or PFGE Characterization Biochemical testing isolates Identification

  8. Commercial kits (rapid methods) – food borne pathogens 1990 1998 2006 TargetAbAbDNAAbDNA Campylobacter03 317 4 C. botulinum toxin 0 1 0 5 1 E. sakazakii 00 0 1 1 EHEC (non-O157) 0 0 0 16 0 O157015141 9 Shiga toxin 0 3 0 10 0 Listeria & Lm183 22 19 Salmonella421 3 42 10 S. enteritidis 00 0 4 0 S. aureus03 1 9 4 enterotoxin25 011 0

  9. “Rapid”Methods • “..determines the presence of dangerous strain of E. coli in 5 to 10 min instead of 48 hr…” WA Post (1997) • Definition • “Rapid”? • Origin? biotechnology 1950 1960 1970 1980 1990 2000 1940 pre-API “rapid method”

  10. History of Biotechnology 1950’s Fundamental Information 1960’s DNA Genetic Engineering Monoclonal Antibody Tissue Culture Technology Fermentation Technology 1970’s Basic Technology Development 1980’s Future Applications Human & animal health care products Custom-design of raw agricultural commodities Engineered enzymes & proteins Biosensor applications Natural ingredients & chemicals by fermentation or cell culture DNA probe & monoclonal antibody-based detection systems Bioreactor design, down-stream processing & product purification 1990’s and beyond Harlander (1989)

  11. Delphi Forecast on Rapid Methods (1981) • Microbial enumeration: • automated colony counting (1991) • by microbial metabolites (2001) • by microbial activity (2001) • Microbial identification: • miniaturized biochemical (1986) • selective media/automation (1997) • specific substrates/metabolites (1996) Adapted from Gutteridge & Arnott (1989)

  12. Rapid Methods - Foodborne Pathogens 1. Miniaturized Biochemical Identification • (API 20E, MicroID, Crystal, RapidID 32E, etc) • pure cultures; incubations 2. Modification - Conventional methods • (ATP & Chromogenic, fluorogenic substrates, etc.) • ATP – fast; total counts only – no ID • substrates – incubations & presumptive data, cost 3. Automated systems • (various technol: eg. biochemical, FA, C oxidation) • identification – pure culture • detection – few manipulation; need enrichment

  13. Rapid Methods – Food borne Pathogens 4. Antibody-based Assays • Latex Agglutination: low sensitivity (log 7); pure culture • ELISA: manipulations: need enrichment. • IMS:= selective enrichment; binding efficiency?; not all foods; culture not pure; notstand alone. • Immunoprecipitation: fast (min), but need enrichment 5. Nucleic acid-based Assays • DNA probes: manipulations; need enrichment • Phage:lux or ina; few on market; need enrichment • PCR: inhibitors in foods; need enrichment

  14. Food Analysis - timeline “Ideal” Rapid Methods Conventional Methods < 8 hr 3 days 0 1 day 2 days existing rapid methods confirmation Enrichment/ isolation - antibody assays - DNA assays - Automated assays - Miniaturized biochemical ID key problemsin food testing – food complexity; speed

  15. History of Biotechnology.2 Basic information 1950’s & 60’s DNA Genetic Engineering Monoclonal Antibody Fermentation & Tissue Culture Technology, PCR Basic Technology Development 1970’s & 80’s Health care products Engineered enzymes & proteins DNA probe & monoclonal antibody assays Custom-designed agricultural commodities bioreactors, down-stream processing & purification ingredients & chemicals by fermentation or cell culture Applications 1990’s Automated sequencing, bioinfomatics (BLAST), microfluidics, MALDI-TOF, nanotechnology, rt-time PCR, DNA and phenotypic microarrays, biosensors. Tech. Dev. 2000’s Modified fromFeng (2001)

  16. “…7 technologies with greatest impact on life science research…”(Scientist 19:6, 2005) 1. Automated DNA sequencing 2. Basic Local Alignment Search Tool (BLAST) 3. DNA microarray (DNA Chip) 4. Yeast two-hybrid assay – novel protein-protein interactions 5. MALDI-TOF - matrix-assisted laser desorption ionization-time of flight 6. Microfluidics 7. Optical trap/tweezers – use of laser to move cells, organelle, atoms without cell disruption

  17. Next Generation Technologies • DNA Microarrays (DNA Chips) • Simultaneous detection/characterization of multiple genotype • Capable of 400,000 tests per chip • Ie: MWG; Panorama – K-12 genomic array (> 4000 ORF) • Phenotypic Microarray (phenotypes) • Simultaneous detection/characterization of multiple phenotype • Ie: Omnilog – Phenotype microarray (2000 phenotypic growth curves) • real-time PCR • Rapid amplification & real-time results (SYBRgreen, Taqman, FRET hybridization probes, molecular beacons) Ie: LightCycler & iQ-Check – Salmonella and L. monocytogenes • Biosensors – • Biological component (antibodies, ligands, etc) – specificity. • Physical component (fluorescence, light, electromagnetic wave, etc) – detection. eg: flow cytometry, SPR, etc. • Rapid detection in minutes. in-line monitoring?

  18. rt-PCR and biosensor assays for food borne pathogens Pathogenrt-PCRbiosensors B. cereus 1 0 Campylobacter 2 3 C. bot./perf. 2 0 E. coli O157:H7 6 5 E. sakazakii 1 0 Listeria spp. 4 4 L. monocytogenes 6 2 Salmonella 6 4 Shigella 2 0 S. aureus 2 0

  19. Logistics to consider • Practicality – • Technology - fascination or publicity? • Many “solutions looking for problems” • Applicability – ample formats to meet all needs • User’s needs – characterization or screening • Screening - amount of data/discrimination vs cost? • volume of testing - instrument capacity; disposible; cost • Advantages and Limitations • Fast – DNA chips, biosensor, rt-PCR - < 1hr assay time • Food matrix interference? • Validation?

  20. Rapid Methods – Pros and Cons • Reliance on cultural enrichment • Assay in min, but enrichment takes days • limits speed but offers other benefits • Screening application • (-) accepted; (+) Is presumptive, need to be confirmed • Food dependant performance and interference • Need for comparative analysis • DNA assays • not detect gene expression • Can’t detect pre-formed toxins • Single target format • Ideal for screening application • Over abundance of assays • Lack of comparative evaluation or validation

  21. Sample Analysis Time of Various Assays • ELISA:45 min - 2 henrichment: 16 - 72 h • Immunoprecipitation:10 minenrichment: 18 - 24 h • DNA Probes:2 - 2.5 henrichment: 48 h • PCR:1 - 4 henrichment: 16 - 72 h • Bacteriophage:3 h enrichment: 22 h

  22. Rapid Methods – Pros and Cons • Reliance on cultural enrichment • Assay in min, but enrichment takes days • limits speed but offers other benefits • Screening application • (-) accepted; (+) is presumptive, need to be confirmed • Food dependant performance and interference • Varying sensitivities • Need for comparative analysis • DNA assays • not detect gene expression • Can’t detect pre-formed toxins • Single target format • Ideal for screening application • Over abundance of assays • Lack of comparative evaluation or validation

  23. Detection Sensitivity of Assays AssayBacteria(cfu/g)Toxins (ng/ml) Adenosine triphosphate (ATP) 104 NA Latex Agglutination 107 NA Reverse Passive LA NA 0.5 - 4.0 ELISA 104 - 105 0.01 - 1 Immunomagnetic Separation < 103 NA Immunodiffusion (1-2 Test) 105 - 106 NA Immunoprecipitation 104 - 105 NA for stx 107 - 108 6 - 20 DNA Probe 104 - 106 NA phage (INA or bioluminescence) 25 - 100 NA PCR 101 - 102 NA Biosensor 101 - 102 NA NA - information not available or applicable

  24. Rapid Methods – Pros and Cons • Reliance on cultural enrichment • Assay in min, but enrichment takes days • limits speed but offers other benefits • Screening application • (-) accepted; (+) Is presumptive, need to be confirmed • Food dependant performance and interference • Varying sensitivities • Need for comparative analysis • DNA assays • not detect gene expression • Can’t detect pre-formed toxins • Single target format • Ideal for screening application • Over abundance of assays • Lack of comparative evaluationor validation

  25. Rapid Methods in food Testing – validations claims • “….our assay is approved by the FDA for testing X pathogen in foods…..” • “..FDA-approved method X quickly determines the disease causing toxins produced by EHEC in foods, especially ground beef and milk.” • “.. Company X received FDA clearance to market a rapid test for E. coli O157:H7, which is often transmitted via ground beef” • “…the FDA is currently using this assay in their labs.”

  26. Test Kit Validations in the U.S. • Clinical: FDA (Center for Devices and Radiological Health) • Food: • U.S. USDA Microbiology Laboratory Guidebook • Food Safety Inspection Service(internal validation) • U.S. FDA Bacteriological Analytical Manual • Center for Food Safety & Applied Nutrition(internal validation) • AOAC Int. - Official Methods (Collaborative Study) - Peer-Verified Methods (non-propriety) • AOAC-RI -Test Kit Performance Tested Methods (proprietary) (AOAC Research Institute)

  27. Administrator: AOAC Application fee: 35K Reviewers:General Referee, Methods Committee (volunteers) Studies:in-house, precollab, collab Lab Requirements:15 (academia, industry, government) Sample Requirements:5 types Seed Levels:uninoculated, lowhigh (5 replicates each). Status:Official Standard Method Official 1st action - 2 yrs Official Final action - indefinite Administrator: AOAC RI Application fee:21K Reviewers:2 Experts selected by AOAC RI (retainer) Studies:proposed protocol Lab Requirements:2 (Sponsor & RI contractor) Sample Requirements:none Seed Levels: specified by study protocol Status:Not official Validated status - renewed annually + 10K AOAC: Official vs Performance Tested

  28. AOAC Method Designations Official-AOAC Official Method 996.09 Research Institute - PERFORMANCE TESTED AOAC RESEARCH INSTITUTE LICENSE NUMBER 930706

  29. AOAC – eCAM Validation Program • REG – Regulatory Methods • Specified in regs by National & International regulatory agencies • HCV – Harmonized Collaboratively Validated Methods • Meets ISO and AOAC standards – full collaborative • MLV – Multi-Laboratory Validated Methods • Same as above, but only 2-8 lab collaborative • SLV – Single Laboratory Validated Methods • Single lab validation • DNV – Developmental Non-Validated Methods • Provide information only on potentially useful methods; not optimized or validated

  30. Validation Programs - Key Points • FDA (CDRH) - reviews and approves assays used in clinical testing. • FDA (CFSAN) - does notreview or approve assays used for pathogens & toxin testing in foods • Performance Tested Methods by AOAC Research Institute - not official • Collaborative Study Methods of AOAC International - Official (Standard) Methods (must be performed as specified)

  31. Rapid Methods in food Testing – claims • “..determines the presence of dangerous strain of E. coli in 5 to 10 min instead of 48 hr…” Newspaper • “….our assay is approved by the FDA for testing pathogen X in foods…..” kit Company Sales Reps • “..FDA-approved X method quickly determines the disease causing toxins produced by EHEC in foods, especially ground beef and milk.” kit Company Newsletter • “.. Company X received FDA clearance to market a rapid test for E. coli O157:H7, which is often transmitted via ground beef.” kit Company Press Release • “…the FDA is currently using this assay in their labs” kitCompany Brochure

  32. Validation – Misconceptions? Importance of Validation • complexity of foods • food-dependant assay efficiency • different sensitivities • abundance of assays • regulatory testing • Validated methods are good? • Compared vs standard or official method • Tested with seeded foods • Validated methods are applicable to all foods? • Exotic imports

  33. Rapid Methods - Impact on Regulation • Increased Sensitivity • “zero tolerance” pathogens • Health Risk? • Upgrade in Quality Control Programs? • Cost? • Assay-dependent Regulations? • False-negative reactions? • Applicability: ie: All Foods? • Endorsement/conflict of Interest?

  34. Food Microbiology: Fundamentals & Frontiers, 2nd ed.2001 ASM Press, Washington DC Feng, P. Chap. 38. Development and Impact of Rapid Methods for Detection of Foodborne Pathogens, pp.775-796. Food Microbiology: Fundamentals & Frontiers, 3rd ed. 2007 ASM Press, Washington DC. Feng, P. Chap. 43. Rapid Methods for the Detection of Foodborne Pathogens: Current and next-generation technologies, pp.911-934. peter.feng@fda.hhs.gov

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