Rapid Pathogen Detection
using Phage Technology
Dubai International Food Safety Conference
Workshop: Advancements in Microbiological Testing
1
Fabrice LESAULT – METERA Regional Business Director
February 28, 2011 – Dubai UAE
 Foodborne Pathogens risks
 International regulations
 International validations
 Evolving solutions
 Perspectives
2
Microbiological risk
(1/2)
Any food product is favourable to
a microorganism growth.
Sanitary risk for the
consumer or the
patient
 Salmonella in cooked meal
 No taste or smell modification
 Foodborne
3
outbreak
Risk of commercial
quality deterioration by
microorganisms
 Yeasts in fruit juice
 Turbidity, gaz, different taste
 No
danger for the consumer
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
4
Pathogens incidence worldwide
WHO data (per year and worldwide), 2006 data
 2 billion of illnesses
 1.8 million of deaths.
5
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.
6
Pathogens incidence USA (2006)
10 000 000
1 000 000
100 000
10 000
nb of cases
nb of death
1 000
100
10
7
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15
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7
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1
Pathogens incidence Europe (2007)
1 000 000
100 000
10 000
nb of cases
nb of deaths
1 000
100
10
1
lm
a
s
8
on
la
l
e
7
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7
p
5
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1
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to
 Foodborne Pathogens risks
 International regulations
 International validations
 Evolving solutions
 Perspectives
9
Due Diligence
From stable
To table
“All food business operators are involved in food safety”
10
International regulation
North America

USA
 USDA-FSIS
 FDA-CFSAN
 AOAC for Rapid Methods
11
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
12
Source : http://www.cfsan.fda.gov/~dms/guidance.html#proc
International regulation
Europe

Commission regulation 2073/2005
 12 articles
 Based on HACCP program
 Food safety criteria
 Process hygiene criteria
13
Salmonella
Fo o d catego ry
M icro -o rganisms
1.4 M inced meat and meat
preparatio ns intended to be eaten
raw
Salmo nella
1.5 M inced meat and meat
preparatio ns made fro m poultry
meat intended to be eaten co o ked
Salmo nella
1.6 M inced meat and meat
preparatio ns made fro m o ther
species than poultry meat intended
to be eaten co o ked
Salmo nella
1.7 M echanically separated M eat
(M SM )
Salmo nella
Sampling P lan
n
5
Limits
c
m
M
0
A bsence in 25 g
A nalytical reference
method
EN/ISO 6579
A bsence in 10 g (2006)
5
0
EN/ISO 6579
Stage where the criterio n applies
P ro ducts placed o n the market
during their shelf-life
P ro ducts placed o n the market
during their shelf-life
A bsence in 25 g (2010)
1.8 M eat pro ducts intended to be
Salmo nella
eaten raw, excluding pro ducts where
the manufacturing pro cess o r the
co mpositio n o f the pro duct will
eliminate the Salmo nella risk
14
1/2
1.9 M eat pro ducts made fro m
poultry meat intended to be eaten
co o ked
Salmo nella
1.10 Gelatin and co llagen
Salmo nella
P ro ducts placed o n the market
during their shelf-life
5
0
A bsence in 10 g
EN/ISO 6579
5
0
A bsence in 10 g
EN/ISO 6579
P ro ducts placed o n the market
during their shelf-life
P ro ducts placed o n the market
during their shelf-life
5
0
5
0
A bsence in 25 g
EN/ISO 6579
A bsence in 10 g (2006)
EN/ISO 6579
P ro ducts placed o n the market
during their shelf-life
A bsence in 25 g (2010)
1.11Cheeses, butter and cream
Salmo nella
made fro m raw milk o r milk that has
undergo ne a lo wer heat treatment
than pasteurizatio n
5
0
A bsence in 25 g
EN/ISO 6579
5
0
A bsence in 10 g
EN/ISO 6579
P ro ducts placed o n the market
during their shelf-life
P ro ducts placed o n the market
during their shelf-life
Salmonella
1.12 M ilk powder and whey powder
Salmo nella
5
1.13 Ice cream, excluding pro ducts
Salmo nella
where the manufacturing pro cess o r
the co mposition o f the pro duct will
eliminate the Salmo nella risk
1.14 Egg pro ducts, excluding
pro ducts where the manufacturing
pro cess o r the co mposition o f the
pro duct will eliminate the
Salmo nella risk
15
5
0
0
A bsence in 25 g
A bsence in 10 g
EN/ISO 6579
EN/ISO 6579
Salmo nella
Salmo nella
0
A bsence in 25 g
1.19 P re-cut fruit and vegetables
Salmo nella
(ready to eat)
1.20 Unpasteurised fruit and
Salmo nella
vegetables juices (ready-to -eat)
1.21Dried Infant fo rmula and dried
Salmo nella
dietary fo o ds fo r special medical
purpo ses intended fo r infants below
6 mo nths o f age.
P roducts placed o n the market
during their shelf-life
EN/ISO 6579
P roducts placed o n the market
during their shelf-life
5
0
A bsence in 10 g
EN/ISO 6579
5
0
A bsence in 25 g
EN/ISO 6579
5
0
A bsence in 10 g
EN/ISO 6579
5
0
A bsence in 25 g
EN/ISO 6579
5
0
A bsence in 10 g
EN/ISO 6579
5
0
A bsence in 25 g
EN/ISO 6579
30
0
A bsence in 10 g
EN/ISO 6579
Salmo nella
Salmo nella
P roducts placed o n the market
during their shelf-life
P roducts placed o n the market
during their shelf-life
5
1.15 Ready-to -eat fo o ds co ntaining Salmo nella
raw eggs, excluding pro ducts where
the manufacturing pro cess o r the
co mposition o f the pro duct will
eliminate the Salmo nella risk
1.16 Co o ked crustaceans and
mo lluscan shellfish
1.17 Live bivalve mo lluscs and live
echinoderms, tunicates and
gastropods
1.18 Sprouted seeds (ready-to -eat)
2/2
P roducts placed o n the market
during their shelf-life
P roducts placed o n the market
during their shelf-life
P roducts placed o n the market
during their shelf-life
P roducts placed o n the market
during their shelf-life
P roducts placed o n the market
during their shelf-life
P roducts placed o n the market
during their shelf-life
Listeria monocytogenes
Food category
Micro-organisms
Sampling Plan
n
1.1 Ready-to-eat foods intended for Listeria
infants and ready-to-eat foods for
monocytogenes
special medical purposes
1.1 Ready-to-eat foods able to
support the grow th of L.
monocytogenes , other than those
intended for infants and for special
medical purposes
Listeria
monocytogenes
10
5
5
1.1 Ready-to-eat foods unable to
support the grow th of L.
monocytogenes , other than those
intended for infants and for special
medical purposes
16
c
0
0
0
Limits
m
Analytical
Stage w here the criterion
reference method applies
M
Absence in 25 g
100 CFU/g
Absence in 25 g
EN/ISO 11290-1
EN/ISO 11290-2
EN/ISO 11290-1
Listeria
monocytogenes
Products placed on the
market during their shelf-life
Products placed on the
market during their shelf-life
Before the food has left the
immediate control of the food
business operator, w ho has
produced it
Products placed on the
market during their shelf-life
5
0
100 CFU/g
EN/ISO 11290-2
Conventional Methods
1/3
Conventional Methods = Reference Methods
17
Salmonella
ISO 6579
MLG 4.04
BAM Chap.5
Listeria
ISO 11290
MLG 8.06
BAM Chap.10
E. Coli O157:H7
ISO 16649
MLG 5.04
BAM Chap.4a
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
18
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
19
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
20
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
21
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
22
Source : http://www.fsis.usda.gov/Science/Microbiological_Lab_Guidebook/index.asp
Mains steps for Pathogen detection
Pre-enrichment
24 HRS
Pre-enrichment
Selective
Detection
Enrichment
on selective media
24 HRS
24 – 48 HRS
Selective
Automated
Enrichment
Detection
Identification
Genotyping
24 HRS
Few hrs
Automated
Identification
Genotyping
Few hrs
Few hrs
(optionnal)
24 HRS
23
24 HRS
1 HR
Negative samples
Positive samples
Conventionnal Methods vs
Rapid Methods
Salmonella Testing
ISO 6579
Rapid Method
BPW BPW
Supplemented
X g into 10X mL BPW
24hrs @ overnight
37±1°C @ 37±1°C
16-20 hrs @ 37±1°C
1 mL into 10 mL
MKTTn broth
0.1 mL into 10 mL
Rappaport Vassiliadis Soya broth
21-27hrs @ 41,5 ±1 °C
21-27 hrs @ 37±1 °C
+
Other selective media
Manufacturer’s recommendations
Selective Enrichment
22-26 hrs @ 41.5±1°C
heating
+
XLD
XLD
Other selective media
21-27 hrs @ 37±1 °C
Manufacturer’s recommendations
reading
heating
VIDAS
Pick up 1 to 5 colonies
Nutrient agar
VIDAS
21-27 hrs @ 37±1 °C
Biochemical confirmation
24
Serological confirmation
5 days
1 day
2 days
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
25
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 !!
26
 Foodborne Pathogens risks
 International regulations
 International validations
 Evolving solutions
 Perspectives
27
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?
28
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

29
Confirmation: selective media, latex, identification
International Validations
North America
 USA
 AOAC Official Method
 AOAC Performance tested Method
 Canada
 Health Canada
30
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
31
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
32
OMA Approval Process - Overview
Study Director
Approved
Precollaborative
Protocol
Precollaborative
Study
General Referee
Precollaborative Final
Report
Approved
Collaborative
Protocol
Collaborative
Study
Collaborative
Final
Report
First Action
Final Action
33
Performance Tested Method (PTM)
Monitored by the Research Institute
PTM-Approval has gained wide acceptance in the
US, Europe, and globally.




34
Third Party validation
Independent “single” Lab Validation
Certification Mark
Annual Review
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
35
Performance Tested Method (PTM)
Internal Study
Independent Study
 Inclusivity
 Exclusivity
 Method Comparison
 10 foods for “Variety of Foods”
 choice of reference methods
 Ruggedness
 Stability
 Lot-to-Lot Variation
36
 Method Comparison
 1 laboratory
 1-2 foods
List of PTM Approved Methods
SM
37
AOAC RI Certificate
38
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.”
39
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”
40
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
41
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

42
ISO 16654:2001: Microbiology of food and animal feeding stuffs – Horizontal
Method for the detection of Escherichia coli O157
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
43
Qualitative methods
Study
44
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
Validation Process
Expert Lab - Organizer
Expertise and comparison
of method study + report
Protocol Study
5 weeks
C
6 months
General
Committee
45
Analysis inter-lab
study + report
C
2-3 months
C
ISO 16140
 All key performance criteria
are specified
 Renewed every 4 years
 Available on the AFNOR
website
46
ISO 16140
 All key detailled
performance criteria are
specified
 Renewed once there is a
change in the protocol
 Available on the AFNOR
website
47
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
48
 Foodborne Pathogens risks
 International regulations
 International validations
 Evolving solutions
 Perspectives
49
VIDAS…always evolving
ELFA
1992
Immuno
Concentration
1993
Listeria ECO
Fab Fragment
Recombinant phage
protein
1996
2002
2003
2004
2006
2007
2008
2009
ICS
+ SLM
LMO2
SET2
LSX
Next
Day
LDUO
VIDAS
Heat & Go
ECPT
SLMX
LMX
to improve the reliability and the TTR of the solution
50
VIDAS Principle
CAPTURE OF ANTIGENS
SANDWICH TEST
DETECTION
370 nm
The antibody captures
the target pathogens
51
A second antibody
conjugated with an
enzyme
binds
to
specific antigens
450 nm
The intensity of
the reaction is
interpreted by the
system
Portfolio
 Salmonella
 Listeria spp
 Listeria monocytogenes
 Escherichia coli 0157 (including H7)
 Campylobacter
 Staphyloccocal enterotoxins
52
Automated ELFA
1992
1993
1996
2002
2003
2004
2006
2007
2008
Listeria ECO
An objective result with Ready to Use reagents
53
2009
Immuno Concentration
1992
1993
Listeria ECO
1996
2002
2003
2004
2006
2007
ICS
+ SLM
A faster result within 24 hours
54
2008
2009
Antibodies fragment
Fab Fragment
1992
1993
Listeria ECO
1996
2002
2003
ICS
+ SLM
LMO2
SET2
2004
2006
Improved performanes
55
2007
2008
2009
A new technology on VIDAS
Recombinant phage
protein
1992
1993
Listeria ECO
1996
2002
2003
2004
2006
2007
2008
ICS
+ SLM
LMO2
SET2
LSX
Next
Day
LDUO
VIDAS
Heat & Go
ECPT
Most advanced technology for high performances
56
2009
From phage to VIDAS
What’s a bacteriophage ?
57
 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
A new technology on VIDAS
58
A simple and rapid protocol…
Raw beef
25 gr.
Raw beef
in BPW
in BPW +/- vancomycin
7-24 hrs
8-24 hrs
41.5 +/- 1 °C
41.5 +/- 1 °C
Heat & Go 5 min
50 min.
VIDAS UP E. coli O157
including H7
59
375 g
Heat in boiling bath
5 min
50 min.
VIDAS UP E. coli O157
including H7
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.
60
61
…always evolving
1992
1993
Listeria ECO
1996
2002
2003
2004
2006
2007
2008
2009
ICS
+ SLM
LMO2
SET2
LSX
Next
Day
LDUO
VIDAS
Heat & Go
ECPT
SLMX
LMX
One step protocol for Next Day results
62
VIDAS SLMX protocol
Raw beef and veal
Pasteurized eggs (liquid, powder…)
225 ml of BPW
Pre-warmed at 41.5 °C
16-24 hr
41.5°C +/- 1°C
Heating Step 5 min
*Except for pasteurized eggs
45 min.
VIDAS SLMX
63
Validated according to ISO 16 140 on
raw beef & veal and pasteurized
egg products. (BIO 12/26-07/09)
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)
Positive
Negative
Positive
63
0
Negative
0
61
VIDAS
SLMX
64
VIDAS Listeria monocytogenes Xpress
225 ml LMX broth
+ 0.5 ml of LMX supplement
26-30 h
37°C +/- 1°C
5 min
80 min.
65
VIDAS LMX
Validated according to ISO 16 140 on human
food and production environmental samples.
(BIO 12/27-02/10)
VIDAS LMX PRINCIPLE
P60 protein biotin
ALP
S
ALP
ALP
Streptavidin-PAL
Fab’
AcM
Specificity
+
Sensitivity
=
Next Day Results
66
 Foodborne Pathogens risks
 International regulations
 International validations
 Evolving solutions
 Perspectives
67
Keep extending Phage technology
 VIDAS UP Salmonella
 VIDAS UP Listeria
Simple and Rapid
68
VIDAS UP SALMONELLA
 Simplified protocol for Next Day detection of Salmonella
 Detection of both motile and non motile strains
69
VIDAS UP SALMONELLA
Food & environment.
BPW + supplement
41.5 +/- 1 °C
18-24 hrs
Heat & Go 5 min
VIDAS UP Salmonella
70
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
71
VIDAS UP SALMONELLA
External study
ISO 6579
+
-
+
100
0
-
0
657 *
VIDAS SPT
* 6 presumptive positive samples by the alternative method were negative after confirmation
 Specificity of 98.8%
72
Sensitivity 100%
Listeria
Simplified protocol for Next Day detection of Listeria species
73
VIDAS UP LISTERIA SPP
 Simplified protocol for Next Day detection of Listeria species
 Detection protocols for Food samples and environmental
samples
74
VIDAS UP LISTERIA SPP
Environmental
samples
Food
Listeria broth + supplement
Listeria broth + supplement
24-26 hrs
26-30 hrs
30 +/- 1 °C
30 +/- 1 °C
Heat & Go 5 min
VIDAS UP Listeria
75
Heat & Go 5 min
VIDAS UP Listeria
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
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