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Rapid Pathogen Detection using Phage Technology

Rapid Pathogen Detection using Phage Technology. Dubai International Food Safety Conference Workshop: Advancements in Microbiological Testing Fabrice LESAULT – METERA Regional Business Director February 28, 2011 – Dubai UAE. Foodborne Pathogens risks International regulations

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Rapid Pathogen Detection using Phage Technology

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  1. Rapid Pathogen Detection using Phage Technology Dubai International Food Safety Conference Workshop: Advancements in Microbiological Testing Fabrice LESAULT – METERA Regional Business Director February 28, 2011 – Dubai UAE

  2. Foodborne Pathogens risks • International regulations • International validations • Evolving solutions • Perspectives • Foodborne Pathogens risks • International regulations • International validations • Evolving solutions • Perspectives

  3. Microbiological risk(1/2) Any food product is favourable to a microorganism growth. Sanitary risk for the consumer or the patient Risk of commercial quality deterioration by microorganisms • Salmonella in cooked meal • No taste or smell modification • Foodborne outbreak • Yeasts in fruit juice • Turbidity, gaz, different taste • No danger for the consumer

  4. Microbiological risk(2/2) • More ready-to-eat foods • Worldwide distribution • Mass production • Customers concentration Increase in the number of food poisoning cases Risk of larger outbreaks

  5. Pathogens incidence worldwide WHO data (per year and worldwide), 2006 data • 2 billion of illnesses • 1.8 million of deaths.

  6. Pathogens incidence USA CDC data (per year and in USA), 2006 data • 76 millions of foodborne illnesses, • 300 000 hospitalizations, • 5 000 deaths, • 1 200 outbreaks.

  7. Pathogens incidence USA (2006)

  8. Pathogens incidence Europe (2007)

  9. FoodbornePathogens risks • International regulations • International validations • Evolving solutions • Perspectives

  10. Due Diligence From stable To table “All food business operators are involved in food safety”

  11. International regulation North America • USA • USDA-FSIS • FDA-CFSAN • AOAC for Rapid Methods

  12. FDA Guidance document Bacteriological Analytical Manual 7th Edition (1992) • Provides quantitative and qualitative bacteriological testing procedures for detecting microbiological contamination. • Chapter 4a : Diarrheagenic Escherichia coli • Chapter 5 : Salmonella • Chapter 10 : Listeria monocytogenes Source : http://www.cfsan.fda.gov/~dms/guidance.html#proc

  13. International regulation Europe • Commission regulation 2073/2005 • 12 articles • Based on HACCP program • Food safety criteria • Process hygiene criteria

  14. Salmonella1/2

  15. Salmonella2/2

  16. Listeria monocytogenes

  17. Conventional Methods1/3 Conventional Methods = Reference Methods SalmonellaISO 6579 MLG 4.04 BAM Chap.5 ListeriaISO 11290 MLG 8.06 BAM Chap.10 E. Coli O157:H7 ISO 16649 MLG 5.04 BAM Chap.4a

  18. Conventional Methods2/3 Slow Results • Delays release finished products and ingredients • Delays response to data from environmental monitoring programs • Aerobic Count =72 hours • MPN of Coliform Bacteria =72 hours • Listeria Neg=4-5 daysPos=5-7 days

  19. Conventional Methods3/3 Inefficient • Laborious. • Numerous supplies. • High human cost. • Necessity for well-trained operators. • Very subjective results, depending on each operator’s competence. • Many yield false positive and false negative results • Large measurement uncertainty

  20. Why Rapid Methods? • Large volume of product produced • Lack of space in warehouse • Short shelf-life • Fast sorting of raw material • Reliability and reproducibility of results

  21. Room for Alternative Methods Article 5: Specific rules for testing and sampling “The use of alternative method analytical methods is acceptablewhen the methods are validated again the reference method in Annex1 and if a proprietary method, certified by a third party in accordance with the protocol set out in EN/ISO standard 16140 or other internationally accepted similar protocols, is used.” EN ISO 16140-2003: Food Microbiology : Protocol for validation of alternative method

  22. USDA-FSIS MLG Commercially available test kits • Salmonella : Any screening method under considerationfor Salmonella testingmust meet or exceed the following performance characteristics: sensitivity > 97%, specificity > 90%, false negative rate < 3% and false-positive rate < 10%. • E. coli O157:H7: The screening test for the detection of E. coli O157:H7/NM shouldmeet or exceed the following performance characteristics: sensitivity > 98%, specificity > 90%, false negative rate < 2% and false-positive rate < 10%. • L. monocytogenes : Any screening method under considerationfor L. monocytogenes testing must be validated for the intended use and must be at least as sensitive as the culture method described in this procedure Source : http://www.fsis.usda.gov/Science/Microbiological_Lab_Guidebook/index.asp

  23. Detection on selective media Selective Enrichment Identification Pre-enrichment Genotyping 24 HRS 24 HRS 24 – 48 HRS 24 HRS Few hrs Selective Enrichment (optionnal) Automated Detection Automated Identification Pre-enrichment Genotyping 24 HRS 24 HRS 1 HR Few hrs Few hrs Negative samples Positive samples Mains steps for Pathogen detection

  24. Conventionnal Methods vs Rapid Methods Salmonella Testing ISO 6579 Rapid Method 5 days 1 day 2 days

  25. Alternative Methods1/2 Objectives • Shorter Time to results • Increase Lab Efficiency/productivity • Increase Reliability by • Objective results by automation • Limited steps in protocols Characteristics • Ready to use • Fast: 1 or 2 days results • Validated • Could be automated

  26. Alternative Methods2/2 But also… • Higher risk for Interference / inhibition matrix. • Need for International Validation • Request for Internal Evaluation • Screening Method in case of Qualitative Method (necessity for confirming positive presumptive) No Method is Perfect or Absolute !!

  27. Foodborne Pathogens risks • International regulations • International validations • Evolving solutions • Perspectives

  28. How to choose? • Interest for faster and/or more practical (“alternative”) methods. • Offer of important, steady-developed, alternative methods • Field evaluations are costly, require high scientific competence and a lot of time • How to choose the suitable method? • Do all of them work well?

  29. Complete validated solution By working at all steps of the analysis • Enrichment:balance of selectivity and fertility : media optimised for a full solution • Proprietary media, standard media used as enrichment broths • Screening step:A balance between the enrichment and the detection. Sensitivity and specificity • Immuno-assay • Chromogenic media • Molecular biology • Confirmation:selective media, latex, identification

  30. International Validations North America • USA • AOAC Official Method • AOAC Performance tested Method • Canada • Health Canada

  31. Official Method of Analysis Two Phases Validation • Pre-Collaborative Study • Inclusivity and Exclusivity • Method Comparison • 20 foods • USDA or FDA reference methods • Collaborative Study • Method Comparison • 20 foods • USDA or FDA reference methods • Quantitative method: 8 laboratories minimum • Qualitative method: 10 laboratories minimum

  32. Adopted as… First Action • Successful Collaborative Study • In accordance with AOAC specifications • Recommended by General Referee • Approved by Methods Committee • Published in Journal of AOAC • Compiled in OMA Final Action • Approved methods eligible for final action after 2 years of availability to public

  33. OMA Approval Process - Overview

  34. Performance Tested Method (PTM) Monitored by the Research Institute PTM-Approval has gained wide acceptance in the US, Europe, and globally. • Third Party validation • Independent “single” Lab Validation • Certification Mark • Annual Review

  35. Performance Tested Method (PTM) • Two-part Validation – Internal Studies and Independent Study • One Independent Laboratory required – contracted by AOAC RI • Use AOAC, FDA, USDA, ISO, AFNOR or other official reference methods • Data review by two Expert Reviewers and General Referee • Validation Time: can be less than 6 months

  36. Internal Study Inclusivity Exclusivity Method Comparison 10 foods for “Variety of Foods” choice of reference methods Ruggedness Stability Lot-to-Lot Variation Independent Study Method Comparison 1 laboratory 1-2 foods Performance Tested Method (PTM)

  37. List of PTMSM Approved Methods

  38. AOAC RI Certificate

  39. International Validations Europe Article 5: Specific rules for testing and sampling “The use of alternative method analytical methods is acceptable when the methods are validated again the reference method in Annex1 and if a proprietary method, certified by a third party in accordance with the protocol set out in EN/ISO standard 16140 or other internationally accepted similar protocols, is used.”

  40. EN ISO 161401/2 AFNOR (French Association of Normalization)MICROVAL (European Validation Association), and other European bodies participated in the development of the first ISO international standard for the validation of alternative microbiological methods. EN/ISO 16140 : 2003 “Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods”

  41. EN ISO 161402/2 Publication date:May, 2003 Objective:Protocol for the validation of alternative methods applicable to food microbiology AFNORapplies ISO 16140 since 2004 MicroValapplies ISO 16140 since 2006

  42. Most Frequent Reference Methods used It depends on the organism tested: Salmonella • EN ISO 6579: Microbiology of food and animal feeding stuffs – Horizontal Method for the detection of Salmonella spp. Listeria monocytogenes • EN ISO 11290-1: Microbiology of food and animal feeding stuffs – Horizontal Method for the detection of Listeria monocytogenes • EN ISO 11290-2: Microbiology of food and animal feeding stuffs – Horizontal Method for the enumeration of Listeria monocytogenes E. coli O157 • ISO 16654:2001: Microbiology of food and animal feeding stuffs – Horizontal Method for the detection of Escherichia coli O157

  43. Two Phases Validation Preliminary Study • Inclusivity and Exclusivity • LOD50 (Relative detection limit) • Method Comparison • Ease of Use Collaborative Study • Method Comparison • 1 food, 8 replicates • 1 strain at 3 levels • 10 laboratories minimum

  44. Qualitative methods

  45. Validation Process Expert Lab - Organizer Protocol Study Expertise and comparison of method study + report Analysis inter-lab study + report 5 weeks C 6 months C 2-3 months C General Committee

  46. ISO 16140 • All key performance criteria are specified • Renewed every 4 years • Available on the AFNOR website

  47. ISO 16140 • All key detailled performance criteria are specified • Renewed once there is a change in the protocol • Available on the AFNOR website

  48. Summary USA regulation • Meat, Poultry, Eggs = USDA • Traditional Methods = MLG • Rapid Methods = AOAC RI or OMA (comparing with MLG) • Others = FDA • Traditional Methods = BAM • Rapid Methods = AOAC RI or OMA (comparing with BAM) • Ease of Use EU regulation • Food Micro criteria = 2073/2005 • Traditional Methods = ISO Methods • Rapid Methods = ISO 16140

  49. Foodborne Pathogens risks • International regulations • International validations • Evolving solutions • Perspectives

  50. VIDAS…always evolving Listeria LMO2 SET2 LDUO SLMX LMX VIDAS Heat & Go ECPT ICS+ SLM ELFA Immuno Concentration Fab Fragment Recombinant phage protein 2002 2003 2004 2006 1992 2007 2008 1996 1993 2009 LSX NextDay ECO to improve the reliability and the TTR of the solution

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