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), data
2 billion of illnesses
1.8 million of deaths.

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

7. Pathogens incidence USA (2006)

8. Pathogens incidence Europe (2007)

9. Foodborne Pathogens 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 2

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 : 2

13. International regulation
Europe
Commission regulation 2073/2005
12 articles
Based on HACCP program
Food safety criteria
Process hygiene criteria 2

14. Salmonella 1/2

15. Salmonella 2/2

16. Listeria monocytogenes

17. Conventional Methods 1/3
Conventional Methods = Reference Methods
Salmonella ISO 6579 MLG 4.04 BAM Chap.5
Listeria ISO MLG 8.06 BAM Chap.10
E. Coli O157:H7 ISO MLG 5.04 BAM Chap.4a

18. Conventional Methods 2/3
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 days Pos= 5-7 days

19. Conventional Methods 3/3
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 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 or other internationally accepted similar protocols, is used.”
EN ISO :
Food Microbiology : Protocol for validation of alternative method

22. USDA-FSIS MLG Commercially available test kits
Salmonella : Any screening method under consideration for Salmonella testing must 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 should meet 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 consideration for 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 : 2

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

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

25. Alternative Methods 1/2 Objectives Characteristics
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 Methods 2/2 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 2

30. International Validations
North America
USA
AOAC Official Method
AOAC Performance tested Method
Canada
Health Canada 2

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
Quantitative method: 8 laboratories minimum
Qualitative method: 10 laboratories minimum

32. Adopted as… First Action Final 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
Order of steps to be followed for method adoption.

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
Differs from PVM
1. Independent evaluation done
2. Proprietary methods
3. Package insert is reviewed
4. Lab must have a QA system in place
5. Method reviewed annually
Data submission - data supporting claims of product
Independent testing lab - verify performance of kit, evaluation package insert, GR and reviewers go over data and make final decision
Certification mark - recognized my variety of organizations
Reviews - verify no changes have been made

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. Performance Tested Method (PTM)
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

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 or other internationally accepted similar protocols, is used.” 2

40. EN ISO /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 : 2003
“Microbiology of food and animal feeding stuffs -
Protocol for the validation of alternative methods”

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

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 : Microbiology of food and animal feeding stuffs – Horizontal Method for the detection of Listeria monocytogenes
EN ISO : 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 2

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

44. Qualitative methods Study Protocol Methods Inclusivity study
50 pure positive strains
Alternative method
Exclusivity study
30 pure negative strains
Relative detection level
5 food products
5 positive strains
4 level of contamination
6 replicates/level +
Reference Method
Comparative study
5 food categories
60 products per category
~ 50% of positive results
Interlaboratory study
10 labs without outliers
1 food products
1 positive strain
3 levels of contamination
8 replicates per level
Alternative method +
Reference Method 2

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

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 EU regulation Food Micro criteria = 2073/2005
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
ELFA
Immuno
Concentration
Fab Fragment
Recombinant phage
protein
LMO2
SET2
ECPT
Listeria
SLMX
LMX
ICS + SLM
LDUO
VIDAS
Heat & Go
1992
1993
1996
2002
2003
2004
2006
2007
2008
2009
Cette slide est très importante car elle résume les principales innovations de ces seize dernières années.
Toutes ces innovations sont liées à l’évolution de vos besoins en terme de rapidité, praticabilité, performance et du workflow.
LSX
Next Day
ECO
to improve the reliability and the TTR of the solution

51. VIDAS Principle CAPTURE OF ANTIGENS The antibody captures
the target pathogens
SANDWICH TEST
A second antibody conjugated with an enzyme binds to specific antigens
DETECTION
The intensity of the reaction is interpreted by the system
370 nm
450 nm

52. Portfolio Salmonella Listeria spp Listeria monocytogenes
Escherichia coli 0157 (including H7)
Campylobacter
Staphyloccocal enterotoxins

53. An objective result with Ready to Use reagents
Automated ELFA
1992
1993
1996
2002
2003
2004
2006
2007
2008
2009
Listeria
Cette slide est très importante car elle résume les principales innovations de ces seize dernières années.
Toutes ces innovations sont liées à l’évolution de vos besoins en terme de rapidité, praticabilité, performance et du workflow.
ECO
An objective result with Ready to Use reagents

54. A faster result within 24 hours
Immuno Concentration
1992
1993
1996
2002
2003
2004
2006
2007
2008
2009
Listeria
Cette slide est très importante car elle résume les principales innovations de ces seize dernières années.
Toutes ces innovations sont liées à l’évolution de vos besoins en terme de rapidité, praticabilité, performance et du workflow.
ECO
ICS + SLM
A faster result within 24 hours

55. Antibodies fragment Improved performanes
Fab Fragment
1992
1993
1996
2002
2003
2004
2006
2007
2008
2009
Listeria
Cette slide est très importante car elle résume les principales innovations de ces seize dernières années.
Toutes ces innovations sont liées à l’évolution de vos besoins en terme de rapidité, praticabilité, performance et du workflow.
ECO
ICS + SLM
LMO2
SET2
Improved performanes

56. A new technology on VIDAS
Recombinant phage
protein
1992
1993
1996
2002
2003
2004
2006
2007
2008
2009
Listeria
Cette slide est très importante car elle résume les principales innovations de ces seize dernières années.
Toutes ces innovations sont liées à l’évolution de vos besoins en terme de rapidité, praticabilité, performance et du workflow.
LSX
Next Day
ECO
ICS + SLM
LMO2
SET2
LDUO
VIDAS
Heat & Go
ECPT
Most advanced technology for high performances

57. What’s a bacteriophage ?
From phage to VIDAS
What’s a bacteriophage ?
Virus: A virus that only infects bacteria.
Very common: The most abundant form of life on earth.
Optimized by nature: Need for a specific host for its reproduction

58. A new technology on VIDAS

59. A simple and rapid protocol…
25 gr.
in BPW
375 g
in BPW +/- vancomycin
Raw beef
Raw beef
7-24 hrs
41.5 +/- 1 °C
8-24 hrs
41.5 +/- 1 °C
Heat in boiling bath 5 min
Heat & Go 5 min
50 min.
50 min.
VIDAS UP E. coli O157 including H7
VIDAS UP E. coli O157 including H7

60. Internationally recognized and validated…
ISO 16140/AFNOR Certification in May 2009 for raw ground beef.
ISO 16140/AFNOR Certification in December 2009 for all food and environmental samples
AOAC RI validated in July 2009 for raw ground beef, beef trim, produce and irrigation water.

62. One step protocol for Next Day results
…always evolving
1992
1993
1996
2002
2003
2004
2006
2007
2008
2009
Listeria
Cette slide est très importante car elle résume les principales innovations de ces seize dernières années.
Toutes ces innovations sont liées à l’évolution de vos besoins en terme de rapidité, praticabilité, performance et du workflow.
LSX
Next Day
ECO
ICS + SLM
LMO2
SET2
LDUO
VIDAS
Heat & Go
ECPT
SLMX
LMX
One step protocol for Next Day results

63. *Except for pasteurized eggs
VIDAS SLMX protocol
Raw beef and veal
Pasteurized eggs (liquid, powder…)
225 ml of BPW
Pre-warmed at 41.5 °C
16-24 hr
Validated according to ISO on raw beef & veal and pasteurized egg products. (BIO 12/26-07/09)
41.5°C +/- 1°C
Heating Step 5 min
*Except for pasteurized eggs
45 min.
VIDAS SLMX

64. VIDAS SLMX performances
VIDAS SLMX is certified by AFNOR Validation according to the ISO norm on raw beef & veal and pasteurized egg products. (BIO 12/26-07/09)
Results from ISO preliminary study
                                  
Reference Method
(ISO 6579)
Positive
Negative
VIDAS SLMX 63 61

65. VIDAS Listeria monocytogenes Xpress
225 ml LMX broth
+ 0.5 ml of LMX supplement
Validated according to ISO on human food and production environmental samples. (BIO 12/27-02/10)
26-30 h
37°C +/- 1°C
5 min
80 min.
VIDAS LMX

66. VIDAS LMX PRINCIPLE Next Day Results = P60 protein biotin
ALP
ALP S
ALP
Streptavidin-PAL
Fab’
AcM
Specificity
Sensitivity + =
Next Day Results

67. Foodborne Pathogens risks
International regulations
International validations
Evolving solutions
Perspectives

68. Keep extending Phage technology
VIDAS UP Salmonella
VIDAS UP Listeria
Simple and Rapid

69. VIDAS UP SALMONELLA
Simplified protocol for Next Day detection of Salmonella
Detection of both motile and non motile strains

70. VIDAS UP SALMONELLA Heat & Go 5 min VIDAS UP Salmonella
Food & environment.
BPW + supplement
41.5 +/- 1 °C
18-24 hrs
Heat & Go 5 min
VIDAS UP Salmonella

71. VIDAS UP SALMONELLA External study
757 food products : Meat, poultry, vegetables, seafood, dairy products, egg products, confectionary, environmental samples from production area, feeds and pet foods.
657 negative products to evaluate the specificity of the method
100 positive products, 85 naturally contaminated and 15 artificially contaminated with stressed Salmonella (less than 10 cells/25g)
ISO 6579:2002 as the reference method 2

72. Specificity of 98.8% Sensitivity 100%
VIDAS UP SALMONELLA
External study
ISO 6579 + -
VIDAS SPT
100
657 *
1 seule matrice – lait . Même souche qui croisait  pdt a été retravaillé depuis
* 6 presumptive positive samples by the alternative method were negative after confirmation
Specificity of 98.8% Sensitivity 100%

73. Listeria
Simplified protocol for Next Day detection of Listeria species

74. VIDAS UP LISTERIA SPP
Simplified protocol for Next Day detection of Listeria species
Detection protocols for Food samples and environmental samples

75. Environmental samples
VIDAS UP LISTERIA SPP
Environmental samples
Food
Listeria broth + supplement
Listeria broth + supplement
24-26 hrs
30 +/- 1 °C
26-30 hrs
30 +/- 1 °C
Heat & Go 5 min
Heat & Go 5 min
VIDAS UP Listeria
VIDAS UP Listeria

76. TAKE AVWAY
Reminder
International regulations keep room for Rapid Method
International validations have strong process to assess rapid methods
Time to result can be shortened to less than 24 hours for Salmonella
Beyond TTR, Rapid Methods bring Ease of use w/ limited steps
Automation limits risk of error
Rapid Methods are evolving with state of the art technology

77. THANK YOU
FOR YOUR ATTENTION