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

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

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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

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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
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Pathogens incidence worldwide
WHO data (per year and worldwide), 2006 data

• 2 billion of illnesses
• 1.8 million of deaths.

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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.

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Pathogens incidence USA (2006)
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Pathogens incidence Europe (2007)
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• Foodborne Pathogens risks
• International regulations
• International validations
• Evolving solutions
• Perspectives

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Due Diligence
From stable
To table
All food business operators are involved in food
safety
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International regulation

• North America
• USA
• USDA-FSIS
• FDA-CFSAN
• AOAC for Rapid Methods

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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.ht
mlproc
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International regulation

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

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Salmonella 1/2
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Salmonella 2/2
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Listeria monocytogenes
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Conventional Methods 1/3
Conventional Methods Reference
Methods Salmonella ISO 6579 MLG 4.04 BAM
Chap.5 Listeria ISO 11290 MLG 8.06 BAM
Chap.10 E. Coli O157H7 ISO 16649 MLG 5.04 BAM
Chap.4a
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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

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Conventional Methods 3/3

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

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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

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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 16140 or other
internationally accepted similar protocols, is
used.
EN ISO 16140-2003 Food Microbiology Protocol
for validation of alternative method
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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 gt 97, specificity gt 90, false
  negative rate lt 3 and false-positive rate lt 10.
• E. coli O157H7 The screening test for the
  detection of E. coli O157H7/NM should meet or
  exceed the following performance characteristics
  sensitivity gt 98, specificity gt 90, false
  negative rate lt 2 and false-positive rate lt 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 http//www.fsis.usda.gov/Science/Microbio
logical_Lab_Guidebook/index.asp
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Mains steps for Pathogen detection
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Conventionnal Methods vs Rapid Methods
Salmonella Testing
ISO 6579
Rapid Method
5 days
2 days
1 day
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Alternative Methods 1/2

• 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

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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 !!

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

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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?

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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

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International Validations

• North America
• USA
• AOAC Official Method
• AOAC Performance tested Method
• Canada
• Health Canada

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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

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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

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OMA Approval Process - Overview
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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

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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

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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

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List of PTMSM Approved Methods
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AOAC RI Certificate
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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.
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EN ISO 16140 1/2
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
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EN ISO 16140 2/2
Publication date May, 2003
Objective Protocol for the validation of
alternative methods applicable to food
microbiology
AFNOR applies ISO 16140 since 2004
MicroVal applies ISO 16140 since 2006
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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 166542001 Microbiology of food and animal
  feeding stuffs Horizontal Method for the
  detection of Escherichia coli O157

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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

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Qualitative methods
Study Protocol Methods
Inclusivity study 50 pure positive strains Alternative method
Exclusivity study 30 pure negative strains Alternative method
Relative detection level 5 food products 5 positive strains 4 level of contamination 6 replicates/level Alternative method Reference Method
Comparative study 5 food categories 60 products per category 50 of positive results Alternative method Reference Method
Interlaboratory study 10 labs without outliers 1 food products 1 positive strain 3 levels of contamination 8 replicates per level Alternative method Reference Method
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Validation Process
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ISO 16140

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

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ISO 16140

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

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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

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

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VIDASalways evolving
Fab Fragment
ELFA
Immuno Concentration
Recombinant phage protein
2002
2003
2004
2006
1992
2008
2007
1996
2009
1993
LSX NextDay
ECO
to improve the reliability and the TTR of the
solution
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VIDAS Principle
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Portfolio

• Salmonella
• Listeria spp
• Listeria monocytogenes
• Escherichia coli 0157 (including H7)
• Campylobacter
• Staphyloccocal enterotoxins

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Automated ELFA
2002
2003
2004
2006
1992
1996
2008
2007
2009
1993
Listeria
ECO
An objective result with Ready to Use reagents
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Immuno Concentration
2002
2003
2004
2006
1992
1996
2008
2007
2009
1993
Listeria
ECO
ICS SLM
A faster result within 24 hours
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Antibodies fragment
2002
2003
2004
2006
1992
2008
2007
1996
2009
1993
Listeria
LMO2
SET2
ECO
ICS SLM
Improved performanes
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A new technology on VIDAS
2002
2003
2004
2006
1992
1996
2008
2007
2009
1993
LSX NextDay
Listeria
LMO2
SET2
LDUO
ECO
ECPT
VIDAS Heat Go
ICS SLM
Most advanced technology for high performances
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From phage to VIDAS

• Whats 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

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A new technology on VIDAS
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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
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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.

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always evolving
2002
2003
2004
2006
1992
2008
2007
1996
2009
1993
LSX NextDay
Listeria
LMO2
SET2
LDUO
SLMX LMX
ECO
ECPT
VIDAS Heat Go
ICS SLM
One step protocol for Next Day results
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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 16 140 on raw beef
  veal and pasteurized egg products. (BIO
  12/26-07/09)

41.5C /- 1C
Heating Step 5 min Except for pasteurized eggs
45 min.
VIDAS SLMX
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VIDAS SLMX performances

• VIDAS SLMX is certified by AFNOR Validation
  according to the ISO 16 140 norm on raw beef
  veal and pasteurized egg products. (BIO
  12/26-07/09)
• Results from ISO 16 140 preliminary study

Reference Method (ISO 6579) Reference Method (ISO 6579)
Positive Negative
VIDAS SLMX Positive 63 0
VIDAS SLMX Negative 0 61
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VIDAS Listeria monocytogenes Xpress
225 ml LMX broth 0.5 ml of LMX supplement

• Validated according to ISO 16 140 on human food
  and production environmental samples. (BIO
  12/27-02/10)

26-30 h
37C /- 1C
5 min
VIDAS LMX
80 min.
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VIDAS LMX PRINCIPLE
biotin
P60 protein
Streptavidin-PAL
Fab
AcM


Next Day Results
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• Foodborne Pathogens risks
• International regulations
• International validations
• Evolving solutions
• Perspectives

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Keep extending Phage technology

• VIDAS UP Salmonella
• VIDAS UP Listeria

Simple and Rapid
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VIDAS UP SALMONELLA

• Simplified protocol for Next Day detection of
  Salmonella
• Detection of both motile and non motile strains

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VIDAS UP SALMONELLA
Food environment.
BPW supplement
41.5 /- 1 C 18-24 hrs
Heat Go 5 min
VIDAS UP Salmonella
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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 65792002 as the reference method

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VIDAS UP SALMONELLA
External study
ISO 6579 ISO 6579
-
VIDAS SPT 100 0
VIDAS SPT - 0 657
6 presumptive positive samples by the
alternative method were negative after
confirmation

• Specificity of 98.8 Sensitivity 100

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Simplified protocol for Next Day detection of
Listeria species
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VIDAS UP LISTERIA SPP

• Simplified protocol for Next Day detection of
  Listeria species
• Detection protocols for Food samples and
  environmental samples

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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
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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

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THANK YOU FOR YOUR ATTENTION