Title: Trame de p
1Rapid 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
3Microbiological 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
4Microbiological 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
5Pathogens incidence worldwide
WHO data (per year and worldwide), 2006 data
- 2 billion of illnesses
- 1.8 million of deaths.
6Pathogens 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.
7Pathogens incidence USA (2006)
8Pathogens incidence Europe (2007)
9- Foodborne Pathogens risks
- International regulations
- International validations
- Evolving solutions
- Perspectives
10Due Diligence
From stable
To table
All food business operators are involved in food
safety
11International regulation
- North America
- USA
- USDA-FSIS
- FDA-CFSAN
- AOAC for Rapid Methods
12FDA 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
13International regulation
- Europe
- Commission regulation 2073/2005
- 12 articles
- Based on HACCP program
- Food safety criteria
- Process hygiene criteria
14Salmonella 1/2
15Salmonella 2/2
16Listeria monocytogenes
17Conventional 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
18Conventional 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
19Conventional 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
20Why 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
21Room 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
22USDA-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
23Mains steps for Pathogen detection
24Conventionnal Methods vs Rapid Methods
Salmonella Testing
ISO 6579
Rapid Method
5 days
2 days
1 day
25Alternative 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
26Alternative 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
28How 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?
29Complete 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
30International Validations
- North America
- USA
- AOAC Official Method
- AOAC Performance tested Method
- Canada
- Health Canada
31Official 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
32Adopted 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
33OMA Approval Process - Overview
34Performance 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
35Performance 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
36Performance 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
37List of PTMSM Approved Methods
38AOAC RI Certificate
39International 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.
40EN 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
41EN 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
42Most 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
43Two 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
44Qualitative 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
45Validation Process
46ISO 16140
- All key performance criteria are specified
- Renewed every 4 years
- Available on the AFNOR website
47ISO 16140
- All key detailled performance criteria are
specified - Renewed once there is a change in the protocol
- Available on the AFNOR website
48Summary
- 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
50VIDASalways 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
51VIDAS Principle
52Portfolio
- Salmonella
- Listeria spp
- Listeria monocytogenes
- Escherichia coli 0157 (including H7)
- Campylobacter
- Staphyloccocal enterotoxins
53Automated ELFA
2002
2003
2004
2006
1992
1996
2008
2007
2009
1993
Listeria
ECO
An objective result with Ready to Use reagents
54Immuno Concentration
2002
2003
2004
2006
1992
1996
2008
2007
2009
1993
Listeria
ECO
ICS SLM
A faster result within 24 hours
55Antibodies fragment
2002
2003
2004
2006
1992
2008
2007
1996
2009
1993
Listeria
LMO2
SET2
ECO
ICS SLM
Improved performanes
56A 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
57From 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
58A new technology on VIDAS
59A 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
60Internationally 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.
61(No Transcript)
62always 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
63VIDAS 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
64VIDAS 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
65VIDAS 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.
66VIDAS LMX PRINCIPLE
biotin
P60 protein
Streptavidin-PAL
Fab
AcM
Next Day Results
67- Foodborne Pathogens risks
- International regulations
- International validations
- Evolving solutions
- Perspectives
68Keep extending Phage technology
- VIDAS UP Salmonella
- VIDAS UP Listeria
Simple and Rapid
69VIDAS UP SALMONELLA
- Simplified protocol for Next Day detection of
Salmonella - Detection of both motile and non motile strains
70VIDAS UP SALMONELLA
Food environment.
BPW supplement
41.5 /- 1 C 18-24 hrs
Heat Go 5 min
VIDAS UP Salmonella
71VIDAS 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
72VIDAS 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
73Simplified protocol for Next Day detection of
Listeria species
74VIDAS UP LISTERIA SPP
- Simplified protocol for Next Day detection of
Listeria species - Detection protocols for Food samples and
environmental samples
75VIDAS 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
76TAKE 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
77THANK YOU FOR YOUR ATTENTION