AFNOR Validation2008

AFNOR VALIDATION SYMPOSIUM, BRUSSELS 2008
RVA J0958
AFNOR VALIDATION
EUROPEAN CERTIFICATION
FOR MICROBIOLOGICAL TEST KITS
19 JUNE 2008 - BRUSSELS
***
Many thanks to :
AFSSA-LERQAP, SANCO E2, AFNOR Certification, Insitut Pasteur,
ADRIA Dveloppement, Deutsches Institut fr Lebensmitteltechnik,
3M Microbiology, bioMrieux Industry, Nestl Research Center,
University of Lige, Genesystems, Bio-Rad.
AFNOR all rights reserved. RVA J0958-2008
9:30 Registration
10:00 Welcome and introduction
Speaker : Bertrand Lombard, AFSSA-LERQAP
10:10 European regulation - Specific requirements for the use of alternative
methods for microbiological criteria
Speaker : Ari Hrman, DVM PhD, MPH Legislative officer, END, SAMCO E2
10:30 AFNOR VALIDATION : European status and Certification Scheme
Speaker : Valentine Digonnet, AFNOR VALIDATION Scheme Manager
10:45 Methods performance assessment according to the EN ISO 16140
standard and the AFNOR Validation technical rules : Qualitative methods &
Discussion
Speaker : Virginie EWE, Institut Pasteur Lille, Food microbiology
laboratotory Technical Manager
11:10-11:30 : Coffee Break
11:30 Methods performance assessment according to the EN ISO 16140
standard and the AFNOR Validation technical rules : Quantitative methods &
Discussion
Speaker : Danile Sohier, ADRIA Dveloppement, IDEA Unit Manager
12:00 More information on Inter-laboratory studies
Speaker : Dr. Steinkamp, Deutsches Institut fr Lebensmitteltechnik
12h15-13/30h15 : Lunch Breakoffered
13:30 Two kit manufacturers validation experiences& Discussion
* 3M Microbiology: Marie-Pierre COPIN; New products and Regulatory
Manager
* bioMrieux I ndustry : Raffaella Giardino, Global Marketing Manager,
Program Director QI
14:20 Experience from a perspective of a Food Manufacturer
Speaker : J ohn Marugg, Nestl Research Center, Microbiological Safety
14:45-15:15 :Coffee Break
15:15 Alternative validated methods in Belgium, an example of
application of the European Regulation EC n2073/2005
Speaker : Caroline De Backer, University of Liege, National Reference
Laboratory for food microbiology
15:40 Revision of EN ISO 16140 : current status and foreseen changes
Speaker : Paul in 't Veld, Chairmanof ISO/TC 34/SC 9/WG3
16:00 PCR Legionella : the interest of an AFNOR Validation with 2
examples & Discussion
Speaker : Bertrand Coissac, Genesystems-Sales Manager and
Frederic Martinez, Bio-Rad - Food Science Division / Group Product
Manager
16:30 Debate & Conclusion
17:00 Closure of the Symposium
RVA J0958
RVA J0958
Welcome to Afnor Validation Symposium !
***
Convenor
Bertrand LOMBARD
Co-ordinator Scientific & Technical Advice, Community Reference
Laboratories, International Relations ;
AFSSA-LERQAP (French Agency for Food Safety - Laboratory for Study &
Research on Food Quality & Food Processes)
23 avenue du Gnral de Gaulle - 94706 Maisons-Alfort, France
Tl: 00 33 1 49 77 26 96 - Fax: 00 33 1 49 77 26 95 - e-mail: b.lombard@afssa.fr
web: www.afssa.fr
Regulation (EC) No 2073/2005 on
microbiological criteria for foodstuffs
The use of rapid methods
Ari Hrman
European Commission
DG SANCO
AFNOR Validation Symposium, June 2008, Brussels
AFNOR Validation Symposium, 19th of June 2008 Brussels
The aim of the Regulation
To ensure a high level of human health
protection
Reduction of human salmonellosis and
listeriosis cases
To harmonise microbiological criteria in
the EU
Uniform rules for food business operators
and competent authorities within the EU as
well as for third countries importing to the
EU
Testing against criteria
Food business operators (Reg
2073/2005)
For validation and verification of procedures
based on HACCP and GHP
For batch acceptability testing
Competent authorities (Reg 882/2004 on
official controls)
To verify that food is in comliance with the
Community criteria
Regulation
Latest scientific advice
Broad consensus
Flexible
Sampling procedures
Analytical methods
Sampling frequencies (risk-based)
Process hygiene criteria
Trends
Components of a microbiological
criterion (Codex)
!Micro-organism of concern
!Analytical method
!Sampling plan
!Microbiological limits
!The foodstuff
!The point of the food chain where the limit
applies
!Actions to be taken when unsatisfactory
results
Analytical methods
Reference methods in the legislation
Preference to horizontal methods
Methods of international standardisation
organisations
For in-house control
Alternative methods can be used under
certain conditions (Regulation 2073/2005)
For official controls
The use of EN/ISO methods recommended
(Guidance document on official controls)
The use of rapid methods
An advantage for food business
operators
Results available more rapidly than by
using traditional methods
Rapid corrective measures and actions
possible
Alternative methods
The use of alternative analytical
methods is acceptable when the
methods are:
validated against the reference method in
Annex I
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 protocols, is used.
Alternative methods
If the food business operator
wishes to use analytical methods
other than those validated and
certified as described in paragraph
3 the methods shall be validated
according to internationally
accepted protocols and their use
authorised by the competent
authority
Thank you
ari.horman@ec.europa.eu
AFNOR Validation :
Certifying analytical performances
of commercial methods
Valentine Digonnet
AFNOR Validation Symposium, June 2008, Brussels 2
AFNOR CERTIFICATION AFNOR CERTIFICATION
An AFNOR owned commercial subsidiary offering
a complete range of certification services with
COFRAC accreditation (French signatory of EA
and IAF Mutual Agreements)
Staff : 250 employees
Turnover : 60 million euros
AFNOR CERTIFICATION SERVICES
Conformity assessment
Attestation of
conformity to EC
regulations
Product Certification
Competence
certification
Services certification
Systems certification
(ISO 9001, ISO
14001,)
Purpose of AFNOR Validation
!Conformity assessment by a third party body
that test results obtained with commercial kits
are comparable to results obtained using the
corresponding reference methods
" individual approach (proprietary methods)
Who needs AFNOR Validation?
Kit manufacturers
#to highlight the performance of their methods
Food industry, private and official labs
#for internal quality control or for services
Users of analytical results
#because they need safeguards
What is an alternative method?
For a given given scope scope, it shall estimate the same same
analyte analyte than the one measured by the
corresponding reference method,
and shall meet the following requirements:
rapid rapid analysis and/or response
easy easy carrying and/or automation
analytical performances performances (accuracy, sensitivity..)
What is included in the alternative method ?
" The term "alternative alternative method method" refers to the
combination of product, equipment and
operating instructions
" It includes all elements necessary for
implementing the alternative method
(equipment, reagents, culture media, software for analysis
of results,)
What is a reference method?
" A reference method reference method is automatically a
standardised method (CEN, ISO).
If no CEN or ISO method : an official method (if
existing), or in certain (rare) cases a well known
and widely practised method will serve as a
reference.
List of main reference methods used for
AFNOR Validation
EN ISO 6579 : Salmonella
EN ISO 11290-1 : Listeria (detection)
EN ISO 11290-2 : Listeria (enumeration)
EN ISO 6888-1 : S. aureus
ISO 21528-1 : Enterobacteriaceae
ISO 4831 & 4832 : Coliforms
ISO 16649-2 : E. coli
ISO 4833 : Total count
Who acts in AFNOR Validation?
AFNOR CERTIFICATION
administrative management
responsible for implementation
Expert laboratories
independant & qualified
achieve the validation studies
make presentations of the results
Validation Commission
advises on :
general operating rules, policies
collective advertising
Auditors
independant & qualified
perform the quality audits
Technical Board
advises on :
technical validation and renewal
specific technical rules.
20 members
5 meetings (x 2 days) / year
AFNOR Validation
Certification procedure
Based upon 3 3 principles principles:
1- A standardized validation protocol (EN ISO
16140)
2- Quality control of the production (audits)
3- Implementation of certification rules (including
regular control of the validated methods)
AFNOR Validation
technical protocol
Since Since July 2003 July 2003 :
standard EN ISO 16140
specific requirements and interpretation set up
by the Technical Board
The preliminary study
based on EN ISO 16140 requirements
to characterize characterize the alternative method
(selectivity, practicability)
to compare compare the performances of the alternative
method against the performances of the reference
method (relative accuracy, sensitivity, specificity,
relative detection level,)
Mandatory third party expert lab (no internal data)
The inter-laboratory study
To define variability variability of of results obtained results obtained in in different different
laboratories using the same samples laboratories using the same samples
According to EN ISO 16140:
at least 10 collaborative laboratories giving non
outlier results (or 8 labs for quantitative methods)
implementing both alternative and reference
methods
Quality requirements
for the manufacturer
Adoption of audit requirements based upon
EN ISO 13485 (medical devices)
Additional specific requirements concerning the
control of control of products products
AFNOR Validation :
Control of alternative methods
Duration Duration of of certificate certificate : 4 years
Quality Quality control on site control on site : according to the certification
already granted to the supplier
one audit every 2 years (if not certified)
one audit every 4 years (if certified ISO 9001 or 13485)
Renewal Renewal every 4 years : complete review of changes
in the alternative method, reference method and
Validation protocol
#complementary assays if needed
AFNOR Validation Certificate
Contents Contents :
Administrative Administrative details details:
" Date of validation / end of
validity
" Certificate reference
" Address of manufacturer
and retailer
" Reference of the kit users
manual / technical sheet
Technical Technical details details:
" Principle of the method
" Scope of validation
" Restrictions for use
" Reference method used
" Analytical performances
(Resume of main results
obtained)
AFNOR Validation
Reference documents
Operating Operating Rules Rules (general part + appendices)
#to define the general policies
Technical protocol Technical protocol for the validation studies
(based upon EN ISO 16140 + interpretation)
#to be used by the expert laboratories
EU Regulation on microbiological Criteria,
DG SANCO
Article 5 (published in January 2006) :
use of alternative proprietary methods when
certified by a third party in accordance with the
protocol set in EN ISO 16140 standard (or similar)
and validated against the reference method set in
Annex I (mainly EN ISO methods)
AFNOR Validation complies with EU Regulation
AFNOR VALIDATION scheme was developed in
order to comply with EU requirements defined in
regulation, granting certificates to alternative
methods which are:
Validated against EN/ISO reference methods
Certified by a third party (AFNOR CERTIFICATION)
Tested against EN ISO 16140 standardized
protocol
AFNOR Validation :
from 1989 to 2008
!1989 : first kit certified AFNOR Validation
!2008 : 73 validated methods from 15 companies
#70% pathogen detection
#Methods based on several different principles such as :
Chromogenic media, Immunoassay, Real time PCR,
International and European suppliers
with AFNOR validated methods
3M Healthcare (USA)
AES Chemunex (F)
BIO-ART (BE)
BIOCONTROL Systems (USA)
BIOLINE (DK)
BioMrieux (F)
BIO-RAD (USA, F)
ChromAGAR (F)
CHR Hansen (F)
DUPONT QUALICON (USA)
EUROPROBE (Austria, F)
GENEPROBE (USA)
GENESYSTEMS (F)
OXOID Thermofisher (UK)
SOLABIA BIOKAR (F)
SY-LAB (Austria)
TECRA (Australia)
Implementation of EN ISO 16140
in AFNOR VALIDATION
5 years work on implementation of EN ISO 16140
$Working groups and Technical Board meetings
" 63 method validations or renewals according to
EN ISO 16140 protocol
" Contribution to ISO/CEN for standard revision since 2005
based on real experience according to validation studies on
qualitative and quantitative methods based on several principles
Recent development of AFNOR Validation
% Creation of the logo and registration of the
collective European mark (2005)
% Current contribution to revision of technical
protocol EN ISO 16140
% Extension of AFNOR Validation scope to water
microbiological analysis in 2007
Development of AFNOR Validation
Objectives in the near future
To make a request for accreditation scope extension of AFNOR
Certification for AFNOR Validation scheme
To work on mutual agreement with other Validation schemes:
% 1- Accepting test results when possible
% 2- Working on current obstacles:
- use of same reference methods (ISO CEN standards),
- use of same validation protocol (standard EN ISO 16140)
- use of a significant part of naturally contaminated samples from
various matrices in the comparative study
- 3
rd
party certification with a regular survey of the product quality
and the production site
To extend AFNOR Validation scope (GMO,)
19/06/2008 Virginie Ewe - Institut Pasteur Lille 1
Methods performance assessment
according to
the EN ISO 16140 standard and
the AFNOR Validation technical rules
- Qualitative methods
Processus of validation
Performance assesment =
Study in 2 steps :
Preliminary study
Interlaboratory study
Comparison with an EN ISO standard method:
in relation with the european regulation
Preliminary study
Aim: to verify if results obtained with the
alternative method are equivalent to those
obtained with the reference method
Determination of Relative ACcuracy, Relative
SPEcificity and Relative SEnsitivity (%)
Determination of Relative Detection Level (LOD
50
)
Inclusivity and exclusivity (pure culture strains)
Practicability
(AFNOR certification technical rules)
Relative AC, SPE & SE
Analyses on different samples with:
Alternative method
Reference method
Comparison of results obtained for the same
samples
confirmation of positive tests
Relative AC, SPE & SE
Number of samples:
5 categories: meat products, dairy products,
choice of products depending on target
microorganism
60 results per category (around 50% positive
results and 50% negative results)
At least 30 positive results in AFNOR
Certification rules
Samples
Natural or Artificial contamination?
Natural contamination if possible, to consider:
Effect of background flora
Physiologic status of the strains
Minimal % of naturally contaminated samples in
AFNOR Certification rules :
50% for Listeria monocytogenes
25% for Salmonella
Examples of natural
contamination rates
Listeria studies:
! 55% in lot of studies to 80% in some studies
! Smoked salmons, raw meats, delicatessen,
environmental samples
! Dairy products
Salmonella studies:
! More difficult to find naturally contaminated
samples : meat and egg products
! 26 25 %
Samples
Rules for artificial contamination:
ISO 16140: use stressed microorganisms and
determine the stress and level of contamination
Respect of origin / samples
Different types of stress
log (non selective selective medium) > 0.5
Level of contamination: < 30 cells/25g
Analysis protocol
homogenization
Same first step
incubation
Reference method Alternative method
Analysis protocol
homogenization
Same first step
incubation
BPW or Half Fraser
Analysis protocol
Product Homogenisation
Different first step
Calculation and interpretation
PD PA (A+)
NA ND (A-)
(R-) (R+) Results
PA = positive accordance (R+ / A+)
NA = negative accordance (R- / A-)
PD = positive deviation (R- / A+) = additional positive
ND = negative deviation (R+ / A-) = false negative
(R+) (R-)
(A+)
A+ / R+
PA = 155
A+ / R-
PD = 2
(A-)
A- / R+
ND = 1
A- / R-
NA = 169
Same first step between
alternative and
reference protocols
Examples
(R+) (R-)
(A+)
A+ / R+
PA = 155
A+ / R-
PD = 16
(A-)
A- / R+
ND = 7
A- / R-
NA = 186
Different first step
between alternative and
reference protocols
Examples
Relative Accuracy (AC)
Degree of correspondence between the
response obtained by the reference method
and the response obtained by the alternative
method on identical samples
AC = (NA + PA) / N
Relative Specificity (SP)
Ability of the method not to detect the target
organism if the reference method doesnt detect it
SP = NA / (NA + PD)
i.e. additionnal positive (PD) are considered as
false positive, although they were confirmed as true
positive
# the target organism is not present
Relative Sensitivity (SE)
Ability of the method to detect the target organism
when the reference method detects it
SE = PA / (PA + ND)
Sensitivity
SE = POS x 100% in AFNOR Certification rules
N+
with N+ = PA + ND + PD
et POS = confirmed positive results of the
considered method
99.4 %
98.8 %
99.1%
95.7% Relative sensitivity : SE%
92.1% Relative specificity : SP%
93.7% Relative accuracy : AC%
Examples
3 discordances:
1 ND and 2 PD
23 discordances:
7 ND and 16 PD
99.4 % 95.7 % Relative sensitivity : SE%
98.7 % 99.4 %
(PA + ND) / (PA + PD + ND) (PA + PD) / (PA + PD + ND)
91.0 % 96.1 %
Reference method (SE%) Alternative method (SE%)
Examples
SE = PA / (PA + ND)
Calculation and interpretation
No criteria of acceptance in the standard
Analysis of discordances
Statistical test to determine if the methods
are equivalent or not
Ccl d min
~
16 7 = 9 10 23
d = |PD ND| discordances
Examples
Less than 6 discordances:
no statistical test
Relative detection level
= the smallest number of culturable
organisms that can be detected in the
sample in 50% of occasions
Measurement protocol
5 products (one per category)
5 target microorganisms
3 to 5 levels of contamination
6 replicates per level
Interpretation
The relative detection level lies between the
two contamination levels:
less than 50 % positive results
more than 50 % positive results
Interpretation
"Hitchins A. Proposed Use of a 50 % Limit of
Detection Value in Defining Uncertainty
Limits in the Validation of Presence-Absence
Microbial Detection Methods, Draft 10th
December, 2003".
LOD 50
Example
Matrix Strain Reference
method
(CFU / 25 g)
Alternative
method
(CFU / 25 g)
Raw milk L.mono 1/2b 0,4 [0,3 0,6] 0,4 [0,3 0,6]
Pt L.mono 1/2c 0,4 [0,3 0,7] 0,4 [0,3 0,7]
Red cabbage L.mono 4b 0,6 [0,4 1,1] 0,6 [0,4 1,1]
Smoked salmon L.mono 1/2a 0,4 [0,3 0,6] 0,4 [0,3 0,6]
Process water L.mono 1/2c 0,8 [0,4 1,8] 0,8 [0,4 1,8]
Inclusivity
detection of the target microorganism from a
wide range of strains
50 strains
Application of the complete protocol of the
alternative method
Exclusivity
= the lack of interference from a relevant
range of non-target microorganisms
30 strains
Use of non selective broths
Practicability
13 criteria defined in AFNOR Certification
rules like :
Qualification of technical staff
Environment of analyses
Materials
Real time handling
Time to result
Aim:
to determine the variability of the results
obtained in different laboratories using
identical samples
organisation
At least valid data from 10 laboratories
3 levels of contamination
8 replicates per level
Analyses
Analyses with both methods, alternative and
reference
48 results: 24 with each method
Interpretation
Comparison of alternate method and
reference method results against expected
results
FP : false positive of L
0
level
TP : true positive of L
1
et L
2
levels
Interpretation
TP TP FP Total
/8 /8 /8 etc
/8 /8 /8 Lab 2
/8 /8 /8 Lab 1
L2 L1 L0
Contamination level
Lab
Pour each method : number of
positive results
Interpretation
L
0
Level
SP = (1 FP ) X 100%
N-
FP = number of false positive
N- = total number of samples for L
0
level
Interpretation
L
1
et L
2
Levels
SE = TP X 100%
N+
TP = number of true positive
N- = total number of samples for each level
Interpretation
Accuracy : AC = PA + NA x 100%
N
Accordance, concordance,
odds ratio
Notions of repeatability, reproducibility and
odds ratio (informative annex)
Conclusion
Preliminary study: informations of method
performances
Interlaboratory study:
practicability
lot of work for the participant laboratories
Certificate
Edition of a certificate
Summary report
www.afnor-validation.com
08.19.06 daniele.sohier@adria.tm.fr
Methods performance assessment
accordingto
the EN ISO 16140 standard
and the AFNOR
Validation technical rules
QUANTITATIVE METHODS
08.19.06
ADRIA
Developpement
! Agro-Food Application and Technologies Centre
focuses on expert courses and training programs
management
coordinates innovation programs in close collaboration
with food and diagnosis industrial partners
" predictive tools conception for food process and food
products shelf-life control and monitoring
# www.symprevius.org
" nano-scales devices development for food microbial
ecosystems analysis based on OMIC knowledge
" expert lab which provides daily validation of
microbiological alternative methods accordingto
theISO 16140 standard
08.19.06
Alternative method?
! The European regulation (Regulation (EC) No 852)
specified
Alternative methods are validated against the reference
method; the results are certified by a third party in
accordance with the protocol set out in EN/ISO standard
16140
08.19.06
EN I SO 16140
standard
! Microbiology of food and animal feeding
stuffs Protocol for the validation of
alternative method
Qualitative methods
Quantitative methods
08.19.06
EN I SO 16140
standard
alternative method
Qualitative methods
$ Microbial Indicators of food quality & safety
08.19.06
EN I SO 16140
standard
alternative method
Qualitative methods
And according to the AFNOR validation technical rules
08.19.06
I SO 16140
standard
alternative method
Qualitative methods
Or the need of a revised standard
08.19.06
"Methods comparison study
Specificity Specificity & & selectivity selectivity
Linearity Linearity
Relative Relative accuracy accuracy
Methods precision
Detection & quantification limits
"Inter-laboratory study
Bias Bias
Repeatability Repeatability & & Reproducibility limits Reproducibility limits
Dispersion between laboratories
Validation study
process
!
08.19.06
! Food categories : 5 categories to test (minimum)
Dairy products
Meat products
Fish & seafood products
Fuits and vegetables based products
Egg based products
Animal feeds
Environmental samples
Methods comparison
study
08.19.06
Specificity /
Selectivity
! Pure cultures
30 target strains
20 non-target strains
Enumeration by the reference and the alternative methods
Interpretation : the alternative method is conformed
to the manufacturers specifications and to the specific
requirements for its reliable use.
08.19.06
Linearity
! 5 contamination levels, 4 log range
! 1 matrix / category, x 5 categories
! Lack-of-fit test
0
1
2
3
4
5
6
0 1 2 3 4 5 6
Log (Reference method)
L
o
g

(
A
l
t
e
r
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a
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i
v
e

m
e
t
h
o
d
)
Matrix 1
Sample Regression P% Correlation
coefficient
Regression equation
Matrix 1 GMFR 100 0,999 log alt =0,975 logRf. + 0,101

Ratio : global repeatability standard deviations
GMFR : Orthogonal regression
OLS : Ordinary least squares linear regression
F-table (Snedecor)
08.19.06
Linearity
! Lack-of-fit test
coefficient
Regression equation

0
1
2
3
4
5
6
0 1 2 3 4 5 6
L
o
g

(
A
l
t
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r
n
a
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i
v
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m
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t
h
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d
)
Matrix 1
Correlation coefficient = 0,999, P > 5%
The linearity is accepted
08.19.06
coefficient
Regression equation
Matrix 2 GMFR 2 0,999 log alt =0,975 log Rf. + 0,093

0
1
2
3
4
5
6
0 1 2 3 4 5 6
L
o
g

(
A
l
t
e
r
n
a
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i
v
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m
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t
h
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d
)
Linearity
! Lack-of-fit test
Correlation coefficient = 0,999, but P < 5%
Linearity test robustness?
Matrix 2
08.19.06
Relative accuracy
Method 1
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7
L
o
g

(
A
l
t
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r
n
a
t
i
v
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m
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t
h
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)
P%
All
products
n Regression intercept slope
Intercept
= 0
Slope
= 1
Method 1 73 GMFR 0,035 0,991 37 27

! Minimum 10 samples/category, x 5 categories
! Priority to naturally contaminated samples
! Slope = 1, Intercept = 0 (linear model)
Discrete cluster
Intercept = 0 with P > 5%
Slope = 1 with P > 5%
No systematic bias, ideal accuracy
08.19.06
Relative accuracy
P%
All
products
Intercept
= 0
Slope
= 1
Method 1 73 GMFR 0,035 0,991 37 27
Method 2 73 GMFR -0,135 1,013 1 39

Method 2
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7
L
o
g

(
A
l
t
e
r
n
a
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i
v
e

m
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t
h
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d
)
! Minimum 10 samples, x 5 categories
! Slope = 1, Intercept = 0
Discrete cluster, no outlier
Intercept 0 with P < 5%
08.19.06
Relative accuracy
! Minimum 10 samples, x 5 categories
! Slope = 1, Intercept = 0
P%
All
products
Intercept
= 0
Slope
= 1
Method 1 73 GMFR + 0,035 0,991 37 27
Method 2 69 GMFR - 0,135 1,013 1 39
Method 3 61 GMFR - 0,895 1,044 10 48

Method 3
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7
Log (reference method)
L
o
g

(
A
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a
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i
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m
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h
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)
Intercept =0 with P > 5% ?
Systematic bias ?
Tests robustness ?
08.19.06
Methods comparison
study
! Current EN ISO 16140 standard
Selectivity & specificity
Linearity & relative accuracy
"The alternative method gives results
comparable to the reference method
"reliability of the tests?
! Reports & certificates : Deep informations
& explanations!
08.19.06
"Methods comparison study
Specificity Specificity & & selectivity selectivity
Linearity Linearity
Relative Relative accuracy accuracy
Methods precision
Detection & quantification limits
"Inter-laboratory study
Bias Bias
Repeatability Repeatability & & Reproducibility limits Reproducibility limits
Dispersion between laboratories
Validation study
process
#
!
08.19.06
! Usually pasteurized milk samples with background microflora
! 3 different levels of contamination, + non inoculated
samples, i.e. 1< to 3 Log CFU/ml
! 2 replicates / contamination level
! samples analysed by the reference and the alternative
methods
! 8 laboratories (minimum)
Inter-laboratory
study
08.19.06
Robust estimators
Student t table
Contamination
level
Log CFU/ml
Number of labs
Bias D
Log CFU/ml
Conclusion
1 n = 12 - 0,05 non significant
2 n = 12 + 0,08 significant
M
e
t
h
o
d

2

3 n = 12 + 0,16 significant
Bias
Contamination
level
Log CFU/ml
Number of labs
Bias D
Log CFU/ml
Conclusion
M
e
t
h
o
d

1

3 n = 12 + 0,01 non significant
A non significant bias for each analyte level is expected.
Bias = 0,16 Log CFU/gsignificant?
08.19.06
Reproducibility limits Contamination
level
Log CFU/ml
Number of labs
Reference
method
Alternative
method
P %
1 n = 10 0,522 0,277 4
2 n = 10 0,427 0,227 4
3 n = 10 0,247 0,187 21

Repeatability &
reproducibility limits
Repeatability limits Contamination
level
Log CFU/ml
Number of labs
Reference
method
Alternative
method
P %
1 n = 10 0,542 0,601 63
2 n = 10 0,141 0,094 11
3 n = 10 0,106 0,249 1

Ideal comparison, P > 5%
According to the current EN ISO 16140 standard : at present no
criteria are available for the rejection of an alternative method
F-table
08.19.06
Inter-laboratory
study
! Current EN ISO 16140 standard
Bias
Repeatability & reproducibility limits
"The alternative method gives results
comparable to the reference method
"reliability of the tests
! Reports & certificates : Deep informations
& explanations!
08.19.06
Conclusions
"Alternative methods validated according to the EN
ISO 16140 standard (experimental design)
Give comparable results to the reference methods
Are quicker and/or easier than the reference methods
Offer a rapid & easy assessment of the microbiological
quality of raw materials & finished products
Are internationally recognised.
08.19.06
Conclusions
"reliable tests?
friendly tools needed for users,
i.e. food microbiology laboratories !
The revision is now in progess
08.19.06 daniele.sohier@adria.tm.fr
Thanks for your attention!
Deutsches Institut fr Lebensmitteltechnik e. V.
AFNOR VALIDATION
European certification for microbiological test kits
19th June 2008, Brussels
More Information on Inter-laboratory-studies
German Institute of Food Technology (DIL)
Dr.-Ing. Helmut Steinkamp
Business Unit Manager
Food Safety Division
Quakenbrck, Germany
h.steinkamp@dil-ev.de
+49 (0)5431 / 183 - 135
Competences of DIL
Applied Research
(product and process
development)
Food Analysis
(chemical, microbiological,
physical)
Quality & Safety Management
Technology Transfer
Apparatus / Prototype Design
and Construction,
3
DIL works in partnership with small and medium sized food companies
and supports technology transfer from science to business.
Concept of DIL
Stuff at DIL:
- 30 scientists
- 60 technician
TTZ
DI L
A& F
NI ZO
FHL
I GV
I VV
KI N
Bf EL
ATB
LUFA
MLUA
TUM
UH
UK
FHT
FHW
UB
FHF
FHK
TUD
FHH
FHB
FHN
TUB
TFH
TNO
Bf R
UM
TUHH
DIL in Food Innovation Process
Basic
research
Applied
research
Development
of products/
processes
Distribution
Way to
market
Food industry
Companies
Service of DIL
Universities /
Research Institutions
Customer
Food Safety Division
Analytics
Microbiological and
chemical analytics
Service for food and
feed industry
Marketebility
certificate
Implementation of
monitoring systems
Quality Management
Implementation of
Quality management
systems (HACCP, BRC,
IFS)
Cleaning control
Cross contamination
control
Training of internal
auditors and staff
Research/Consulting
Detection of R&D
support in industry
Food Supply Chain
Management
Cross contamination of
allergens
Materials in contact with
food
Microbiological Analyis for Food Industry
Analysis of food products
Specific detection of pathogenic microorganisms
Detection of risk material, animal protein of unwanted
species, genetically modified organisms (GMOs) and
allergens
Cultivation and PCR technology
Identification of microorganisms
Shelf life testing
Sample Range
Meat and Meat products
Eggs and Egg products
Baked goods and their raw materials / ingredients
Salads and Dressings
Potato Products
Canned Food
Water (human /animal), Beverages, Ice Cream
Feed
Packing materials

1
Microbiological Testing
New EU-Regulations on Food Hygiene
Improved control systems are needed in
Food Industry to improve safety
Delays in Food Production Chain are getting
more important financial aspects
New analytical techniques needed for rapid
conformation of safety
Why participating in
Inter-laboratory Studies?
Requirements
DIL runs accreditated laboratories
Its a Question of Quality of our analysis
Challenge
Comparision in these studies shows us the accuracy
of our service for industry
Co-operation with Bio-Rad
Food Industry wants State-of-the-Art
Inter-Laboratory Studies at DIL
50% of AFNOR tested micro-organisms
Time Micro-organism detected Reference Methode AFNOR-Validation MethodeSuccess
Mai 04 Salmonella spp. ISO 6579 Bio-Rad iQ Check Salmonella PCR ja
Okt 04 Coliforme/E.coli NF ISO 4832 / NF ISO Bio-Rad RAPIDE.coli 2 cultivation ja
Dez 04 Koagulase-positive Staphyloko NF EN ISO 6888-1 Bio-Rad RAPIDStaph cultivation ?
Feb 05 Listeria monocytogenes ISO 11290-1 Bio-Rad iQ Check Listeria monoPCR ?
Okt 05 Listeria monocytogenes ISO 11290-2 Bio-Rad RAPIDL.mono cultivation ja
Okt 05 Salmonella spp. ISO 6579 Bio-Rad RAPIDSalmonella cultivation tw.
Jul 06 Listeria monocytogenes ISO 11290-1 Bio-Rad RAPIDL.mono cultivation ja
Nov 06 Listeria spp. ISO 11290-1 Bio-Rad RAPIDListeria spp. cultivation ja
Apr 07 Listeria spp. ISO 11290-1 Bio-Rad iQ Check Listeria spp. PCR ?
Jun 07 E. coli O157:H7 ISO 16654 Bio-Rad RAPIDE.coli O157:H7cultivation ja
Mai 08 E. coli O157:H7 ISO 16654 Bio-Rad iQ Check E. coli O157 PCR ?
by Bio-Rad
What does DIL get out of
Inter-laboratory Studies
Fulfil requirement of accreditation
Control of our analytical work
Challenge for the laboratory stuff
Wide range of experience by detecting in
different food systems/matrices
Implementing new methods
No internal validation needed
External testing helps improving
Cultivation Techniques
Well-known and easy to handle
Nearly no equipment needed
Less expensive
Time consuming
Results after three days
Enrichment in
Pepton Water
21h
Enrichment in
selektive
bouillons
24h
Selektive
Media
24h
Cultivation
Techniques
(ASU L00.00-20)
PCR methods
ASU L00.00-52
Enrichment in
Peptone Water
21h
PCR
3h
Proben-
Vorbereitung
results
1
PCR vs Cultivation Technology
Samples from
rapeseed
meat: pork, poultry
milk powder
eggs and egg produkts
In total 828 samples tested on Salmonella spp.
False-negative results:
Very few samples were tested positive by
Cultivation but not by PCR
False-positive results:
Due to remained DNA of cells especially in
processed eggs
PCR vs Cultivation Technology
Results of Internal Testing
Nearly 5% more positive by PCR technology
Intensive internal testing
Nearly all false-positive results were detected
as positive for Salmonella spp.
Close relation to
food matix (i.e. egg products, rapeseed)
stress on micro-organisms
cultivation method
1 day vs 3 5 days for results of analysis
More equipment needed
More expensive
Higher accuracy
PCR Technique
1
3M Microbiology validation experiences
SYMPOSIUM BRUSSELS JUNE 19th 2008
EUROPEAN VALIDATION
3M MicrobiologyValidation experience
Marie-Pierre Copin (Laboratoires 3M Sant : Cergy, France)
2
Summary
! Introduction to 3M Company
! 3M Microbiologyproducts
! 3M Microbiologyproductsvalidations and recognitions
! Somemilestones on microbiological methodvalidation in Europe
! ISO 16140 in the Europeanregulationon microbiological criteria
! Afnor certificatesandISO 16140
! Conclusion
3
Six Market-Leading Businesses
4
Consumer and Office Business
! Sales: $3.4 billion
! Operating income: $688 million
Simplifying life at home and at work
5
Display and Graphics Business
! Operating income: $1.2 billion
The products of choice in four large and growing industries
6
Electro and Communications Business
Turning 3M technology into solutions in electrical, electronics and
telecommunications markets worldwide
7
Industrial and Transportation Business
Helping industrial customers solve their problems
8
Safety, Security and Protection Services Business
Responding to growing demand for safety, security and protection
9
Health Care Business
Reinvesting in core businesses, pursuing strategic synergies
10
Solving Problems Everywhere
! Operating companies in more than 60 countries and
selling products in more than 200
! 63 % of sales are outside the United States
! More than 42,000 employees internationally
! We provide borderless customer success
3M MicrobiologyMission
3MMICROBIOLOGY MISSION: Providea broadrange of innovative
solutions thathelp improvesafety, qualityandbusiness resultsfor food&
beverageprocessors, foodpreparationsites and referencelaboratories
globally. Our offeringwill have relevant regulatoryacceptanceand will be
backedwithexceptional customer support.
12
Plastic film
Adhesive +
indicator
Adhesive
Plastic coated
paper printed
with a grid
Multi-layer coating technology
Cold water
soluble gel
Standard methods
nutrients
Until November 2006: Main products range was
3MPetrifilmplates:
alternative methods for Hygiene indicators
13
Total Count Enterobacteriaceae
E. coli/Coliform
Yeast and Mold
Rapid
Coliform
Coliform
High-Sensitivity Coliform
Staph Express Select E. coli
3MPetrifilmplates: Range of enumeration tests
Environmental
Listeria
14
Using 3MPetrifilmplates for product analysis
1 Inoculation
2 Incubation
3 - Interpretation
3 steps only ..
15
November 2006:
3M company acquires Biotrace Ltd.
I. Hygiene monitoring control
3M Clean-Trace
3M Clean-Trace Water
3M NG
3M Protect
II. Environmental sampling products
III. Pathogen detection range
16
3M TECRA Pathogen test range
Alternative method for Pathogen tests
3M TECRA VIA and ULTIMA
Salmonella VIA
Salmonella ULTIMA
Listeria VIA
Campylobacter VIA
E. coli O157 VIA
Pseudomonas VIA
Staphylococcus aureus VIA
3M TECRA Toxin VIA
Bacillus Diarrhoeal Enterotoxin VIA
Staphylococcal Enterotoxins VIA
3M TECRA Unique Salmonella
ELISA Tests Immunocapture Tests
17
Analytical methods
Standardized methods
Standardized methods Commercial/Alternative
methods
Commercial/Alternative
methods
References (ISO)
References (ISO) Appropriate
for routine use
Appropriate
for routine use
OUR Customers need:
To get evidence that choosen alternative method will give results
equivalent to reference method
?
18
Somestepsin the methodapproval organisation in Europe
! 1987: AFNOR validation committeeimplementation
( 3M representatedin thekit manufacturerscollege)
! 1989: First certificatesissued
( 3M Petrifilmplates)
! 1993-1998: Eureka Microval project foundedby Europe to get Europeanscheme
and rulesin Europe
(3M Participation in workinggroups)
! 1998- 2003:CEN group wroteEN 16140 Standard, becamethenISO standard
! 1998 Microval thirdpartybody implementation
(3M representativememberof steeringcommittee)
! 1999: Nordval system isset-up
! 2006: New EuropeanRegulationon microbiological criteria, referingto ISO 16140
standard
! 2007 First Microval certificate
19
3M Microbiology:
Pioneer in 3rd party methodapprovalsActions
! 1986 :Petrifilm Aerobiccount plate and Petrifilm
coliformcount plate in milk: First alternative methods
recognizedby AOAC int
! 1989: Petrifilm Aerobiccount plate and Petrifilm
coliformcount plate: First methodsvalidatedby
AFNOR
! 1993: SomePetrifilmplates recognizedas NMKL,
thenswitchedto NORDVAL validation.
20
Alternative methodin the EuropeanRegulation
on microbiological criteria(EN 2073/2005)
Article 5
The use of alternative analytical methods is
acceptable, when the methods arevalidatedagainstthe
reference method set down in Annex I *and certifiedby
a third party in accordance with the protocol set in
EN/ISO standard 16140or other internationally accepted
similar protocols
21
Analytical methods
Equivalence of methods
methods
methods
Validation according to ISO 16140
by an independent third party
Validation according to ISO 16140
by an independent third party
"
Microval
ISO, CEN
22
Ex Food SafetyCriteria(in EN 2073/2005 )
Salmonella in fruit and vegetable
23
Examples of Process hygiene criteria (in EN 2073/2005 )
Aerobic count and E .coli inMeat and Meat products
24
! Ex of afnor certificate
! standard method
25
Referenceto standard methodisgivenin theAFNOR
certificate
26
Validations and recognitions for 3M Petrifilm plates
+ Environmental Listeriaplate
+ + + Yeast and Mould Count plates
+ + + + StaphExpress Count plates
+ + Select E. coli Count plates
+ + + E. coli and ColiformCount plates
+ + High Sensitivity ColiformCount plates
+ + Rapid ColiformCount plates
+ + + ColiformCount plates
+ + + + Enterobacteriaceae Count plates
+ + +
Aerobic Count plates
RI OMA
CMMAS
ISO 16140
27
3M TECRA range productsvalidation
AFNOR
Salmonella UniqueTEC-24/2-04/03 ( negative results in 24 hours)
Salmonella UltimaTEC-24/3-12/03 ( negative results in 48 hours)
Listeria Ultima TEC-24/4-09/04 ( negative results in 48 hours)
AOACINTERNATIONAL Official Methodof Analysis
Salmonella VIA Method 989.14
Salmonella UltimaMethod 998.09:
ListeriaVIA Method 995.22:
28
Conclusion
Validation scheme:
! Bigprogresses to get a unique schemebasedon an international standard: ISO
16140
In reality: Recognition ismore a Europeanone.
What about a more commonschemewithAOAC?
ThirdParties
! AFNOR has alreadycertified63 methodsfollowingISO 16140
MICROVAL issued 5 certificates
As a manufacturer, weappreciatehavingthe possibilityto choosea thirdparty
expert laboratoryto get our methodsvalidated( system improvement, speed )
Methods validation:
A kit manufacturer perspective
Symposium AFNOR VALIDATION
19 June 2008
Dr. Raffaella Giardino
Global Marketing Manager
Program Director Quality indicators
Agenda Agenda Agenda Agenda
1. Introduction
2. AFNOR Validation
3. Example EN ISO 16140 for TEMPO
!
! Part A: Method comparison study
! Part B: Inter-laboratory study
4. Conclusions
Chromogenic Pathogens Quality Indicators Identification
VITEK
2
LIMS connection
DiversiLab
Typing
A Comprehensive Offer for A Comprehensive Offer for A Comprehensive Offer for A Comprehensive Offer for
the the the the
Food Microbiology Lab Food Microbiology Lab Food Microbiology Lab Food Microbiology Lab
API
Traditional methods
ChromID
TM
VIDAS
TEMPO
Count-Tact
TM
air IDEAL
BacT/ALERT
3D
PPM BioBall
ID32

Validation in Food Validation in Food Validation in Food Validation in Food
Microbiology Microbiology Microbiology Microbiology
" Third party assessment of method
performances
" Official Authority requirement
" Public Health issue
" Customers need (external laboratories
and food industry)
bioMerieux bioMerieux bioMerieux bioMerieux
Development Process Development Process Development Process Development Process
1. Feasibility
2. Optimisation
3. Internal Verification
4. External Verification
Design Control Phases (controlled by FDA): Design Control Phases (controlled by FDA): Design Control Phases (controlled by FDA): Design Control Phases (controlled by FDA):
Regulatory
Affairs Dept.
Clinical
Affairs Dept.
4 independents
laboratory WW
Food Micro Validation Food Micro Validation Food Micro Validation Food Micro Validation
5. Industrialisation
6. External Validation
7. Launch authorisation
Food Validations Food Validations Food Validations Food Validations
of of of of bMx bMx bMx bMx products products products products
" NordVal, vs. NMKL (Nordic Countries)
" AFNOR Validation, EN ISO 16140, vs ISO-CEN
" AOAC validation, USA, vs USDA or FDA
" EMMAS Assessment (UK)
" Health Protection Branch of Canada
" DIN Committee (Germany)
" Chinese Food Safety Authority
" Japanese Government
" Thailand Veterinary Authority
" "
List of Validations obtained in Food Micro for bioM#rieux
bioM bioM bioM bioM# ## #rieux rieux rieux rieux
AOAC Approvals AOAC Approvals AOAC Approvals AOAC Approvals
VIDAS Salmonella SLM (SC + TT) (May 1996 - Official Method N 996.08)
VIDAS Salmonella SLM (RV + TT) (Jan 2004 - Official Method N 2004.03)
VIDAS Salmonella ICS / plate (April 2001 - Official Method N 2001.07)
VIDAS Salmonella ICS/ plate (April 2001 - Official Method N 2001.08)
VIDAS Salmonella ICS/ SLM (April 2001 - Official Method N 2001.09)
VIDAS Listeria LIS (December 1998 - Certificate N 981202)
VIDAS Listeria LIS (harmonized protocol) (May 1999 - Official Method N 999.06)
VIDAS Listeria LIS (harmonized protocol) (June 2004 - Official Method N 2004.06)
VIDAS Listeria monocytogenes II (January 2004 - Official Method N 2004.02)
VIDAS Listeria Species Xpress (LSX) (October 2005 - Certificate N 100501)
with Ottaviani Agosti Agar (OAA)
VIDAS Listeria spp & mono(LDUO) (October 2007- Certificate N 100702 )
VIDAS Staph enterotoxin SET2 (September 2004 - Certificate N 070404)
VIDAS Staph enterotoxin SET2 (October 2007 - Official Method N 2007.06)
VIDAS ECO + O157:H7 ID plate (January 2005 - Certificate N 010502)
8 and 24 hour ground beef protocols
Salmonella
Listeria
Staph
enterotoxin
E.coli
O157:H7
bioM bioM bioM bioM# ## #rieux rieux rieux rieux
AOAC Approvals AOAC Approvals AOAC Approvals AOAC Approvals
E. coli TEMPO EC (Escherichia coli ) August 2006 - Certificate N 080603; OMA on going
Total Flora TEMPO TVC (Total Viable Count) December 2006- Certificate N 120602; OMA on going
Coliform TEMPO CC (Coliform Count) June 2007 - Certificate N 060702; OMA on going
Enterobacteriacea TEMPO EB (Enterobacteriaceae) May 2008 - Certificate N 050801
VITEK Identification Listeria (GPI and GNI+) (1992 - Official Method N 992.19)
VITEK Identification of Salmonella, E. coli (1991 - Official Method N 991.13)
and Other Enterobacteriaceae (GNI+)
API Biochemical Identification of Salmonella (1978 - Official Method N 978.24)
VITEK 2 GN - Identification of gram negative February 2008 - Certificate N 020802
VITEK 2 GP - Identification of gram positive December 2007 - Certificate N 120702
VITEK 2 BCL - Identification of Bacillus Pending AOAC RI approval
Identification
1. Introduction
2. AFNOR Validation
!
4. Conclusions
21 AFNOR validated methods, and all accordingly all accordingly all accordingly all accordingly
the EN ISO 16140 protocol the EN ISO 16140 protocol the EN ISO 16140 protocol the EN ISO 16140 protocol
AFNOR AFNOR AFNOR AFNOR
Approved Methods Approved Methods Approved Methods Approved Methods
0
5
10
15
20
25
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
N
b

o
f

I
S
O

1
6
1
4
0

a
p
p
r
o
v
e
d

m
e
t
h
o
d
s
13 Qualitative Methods (pathogens detection)
5 methods for Salmonella detection
7 methods for Listeria/Listeria monocytogenes detection
1 method for E. coli O157:H7 detection
Qualitative methods Qualitative methods Qualitative methods Qualitative methods
VIDAS SLM double voie BIO 12/1-04/94 09/06/2010
VIDAS SLM simple voie BIO 12/10-09/02 18/09/2010
VIDAS Easy Salmonella BIO 12/16-09/05 20/09/2009
VIDAS ICS2-SLM BIO 12/22-05/07 24/05/2011
VIDAS ICS2-boite BIO 12/23-05/07 24/05/2011
VIDAS Listeria BIO 12/2-06/94 09/06/2010
VIDAS LSX BIO 12/12-07/04 01/07/2008
VIDAS LMO2 (0.1ml-37C) BIO 12/11-03/04 12/03/2012
VIDAS LMO2 (1ml-30C) BIO 12/9-07/02 03/07/2010
Accuprobe LMO BIO 12/4-02/95 07/02/2011
VIDAS LDUO BIO 12/18-03/06 09/03/2010
OAA dtection BIO 12/14-04/05 07/04/2009
E. coli O157 VIDAS ECO BIO 12/8-07/04 05/07/2012
Mthodes qualitatives
Salmonelles
Listeria
Approved Methods Approved Methods Approved Methods Approved Methods
8 Quantitative methods:
1 for Total Count enumeration
3 for E. coli enumeration
2 for coliforms enumeration
1 for Enterobacteriaceae enumeration
1 for Listeria monocytogenes enumeration
Quantitative methods Quantitative methods Quantitative methods Quantitative methods
Flore totale TEMPO TVC BIO 12/15-09/05 19/09/2009
TEMPO EC BIO 12/13-02/05 04/02/2009
Coli ID E. coli 37C BIO 12/19-12/06 14/12/2010
Coli ID E. coli 44C BIO 12/5-03/99 19/01/2011
TEMPO TC BIO 12/17-12/05 08/12/2009
Coli ID coliformes 37C BIO 12/20-12/06 14/12/2010
Entrobactries TEMPO EB BIO 12/2112/06 14/12/2010
Listeria monocytogenes OAA dnombrement BIO 12/24-03/08 28/03/2012
Mthodes quantitatives
Escherichia coli
Coliformes
approved Methods approved Methods approved Methods approved Methods
Classified by technology:
11 VIDAS methods
4 TEMPO methods
5 media methods
1 molecular hybridation method
approved Methods approved Methods approved Methods approved Methods
1. Introduction
2. AFNOR Validation
!
4. Conclusions
Validation Protocol Validation Protocol Validation Protocol Validation Protocol
ISO 16140 ISO 16140 ISO 16140 ISO 16140
" A. Method comparison study
1. Linearity
2. Relative Accuracy
3. Detection & Quantification limits
4. Relative sensitivity
5. Inclusivity and Exclusivity
6. Practicability
1. Linearity 1. Linearity 1. Linearity 1. Linearity - -- - TVC TVC TVC TVC
" TVC: in double at 40h and in double at 48h = 250 results
2.08 $ 4.66 trout Listeria seeligeri BR1 RTE Salmon
with pates
2.41 $ 5.28 raw milk Staphylococcus
aureus 501
Custard
cream
2.41 $ 4.93 plant Xanthomonas
maltophilia 11.2
Green beans
2.32 $ 4.95 mechanically
separated meat
Bacillus cereus 29 Wet cat food
2.30 - 5.04 buf
bourguignon
Enterobacter
agglomerans 72
Porc pt
Range (log) Range (log) Range (log) Range (log) Isolated from Isolated from Isolated from Isolated from Strain Strain Strain Strain Food Food Food Food
"5 food categories + 5 strains
"5 contamination levels $ to cover the whole range
"2 repetitions with TEMPO and Ref method per sample
1. Linearity 1. Linearity 1. Linearity 1. Linearity - -- - TC TC TC TC
" Graphs for all foods/parameters + non-linearity tests
" (example TC, dilution 1)
" The correlation coefficients are all greater than 0.98
" The non-linearity test is not significant, therefore the linearity linearity linearity linearity
of TEMPO TVC, TC, EC and EB is good of TEMPO TVC, TC, EC and EB is good of TEMPO TVC, TC, EC and EB is good of TEMPO TVC, TC, EC and EB is good.
2. Relative accuracy 2. Relative accuracy 2. Relative accuracy 2. Relative accuracy
! 5 food categories Ex. TC range (log)
o Meat, poultry products 1.00 $ 4.63
o Pet-food 1.00 $ 5.43
o Dairy products 1.52 $ 5.48
o Fruit and vegetables 1.85 $ 6.15
o Sea-food 1.40 $ 5.04
! As much as possible naturally contaminated sample,
artificial with strain stress
! TVC: in double at 40h and in double at 48h
" Protocol
2. Relative accuracy 2. Relative accuracy 2. Relative accuracy 2. Relative accuracy - -- - TVC TVC TVC TVC
" 157 different naturally contaminated samples x (2 Ref. +
4 TEMPO) = 942 tests
3. Detection and quantification 3. Detection and quantification 3. Detection and quantification 3. Detection and quantification
limits limits limits limits
" Critical Level CL
" Detection Limit LOD
" Quantification Limit
Data from statistical calculations; they are not
included in the validation certificate, because not
necessary. They will be probably change in the
next revision of the EN ISO standard
4. Relative sensitivity 4. Relative sensitivity 4. Relative sensitivity 4. Relative sensitivity
! Example plot from TC
! Use data from the linearity study
! Plot the precision profile CV (<x(y)>) vs. x(y)
5. 5. 5. 5. Inclusivity Inclusivity Inclusivity Inclusivity and exclusivity and exclusivity and exclusivity and exclusivity
" Protocol Protocol Protocol Protocol
1. Inclusivity: 30 positive pure strains (collection or food isolates)
! In double with TEMPO, VRBG, VRBL/TBX and PCA
! 3 Lactose
neg
strains (TC), 2 -glucuronidase
neg
strains (EC)
! 180 tests per each parameter
2. Exclusivity: 20 negative strains (collection of food isolates)
! In double with TEMPO, VRBG, VRBL/TBX and PCA
! 120 tests per each parameter
3. Not applicable to TVC
5. 5. 5. 5. Inclusivity Inclusivity Inclusivity Inclusivity : EC : EC : EC : EC
Conclusion: Conclusion: Conclusion: Conclusion:
Perfect
correspondence
between TEMPO EC
and ISO 16649-2
standard
5. Exclusivity : EC 5. Exclusivity : EC 5. Exclusivity : EC 5. Exclusivity : EC
Conclusion: Conclusion: Conclusion: Conclusion:
No interference
by non target
strains
6. Practicability 6. Practicability 6. Practicability 6. Practicability
" 13 evaluation criteria (only AFNOR)
! Training duration of an operator
A technician can be fully trained in 2 days
! Traceability of results
A complete traceability for TEMPO Filler and Reader
! Delay to the results
TEMPO
TVC gives results in 40h compared to 72h of

the ref. method, with a saving of more than one day
! QC check and lab maintenance
Monthly use of the kit QC to validate the TEMPO Filler
and Reader performances
6. Practicability 6. Practicability 6. Practicability 6. Practicability
" Real hands-on time (work-flow study) in relation to
the number of samples to analyse*
4.1 4.1 4.1 4.1 13.9 13.9 13.9 13.9 Time for 1 sample Time for 1 sample Time for 1 sample Time for 1 sample
82 278 Total time
2 90 Reading
20 107 Dilution and inoculation
- 21 Petri dishes preparation
15 15 Dilution and stomaching
45 45 Sample Weighing
Method TEMPO TVC Method TEMPO TVC Method TEMPO TVC Method TEMPO TVC Method ISO 4833 Method ISO 4833 Method ISO 4833 Method ISO 4833 20 samples 20 samples 20 samples 20 samples
* Media and tubes preparation time non included
Validation Protocol Validation Protocol Validation Protocol Validation Protocol
ISO 16140 ISO 16140 ISO 16140 ISO 16140
" B. Inter-laboratory study
1. Relative Accuracy
2. Repeatability & Reproducibility limits
3. Ratio R/r
4. Dispersion between laboratories
Inter Inter Inter Inter- -- -laboratory study laboratory study laboratory study laboratory study
" Protocol Protocol Protocol Protocol
! 12-13 European labs participating to each
study + organising lab
! 8 Pasteurized milk samples with natural flora
for TC, EB and EC, UHT for TVC
! Samples at 4 levels of artificial contamination
over the whole range
! Analysed with TEMPO x 2 and ISO method at
day 1
Example TEMPO TC Example TEMPO TC Example TEMPO TC Example TEMPO TC
Example TEMPO TC Example TEMPO TC Example TEMPO TC Example TEMPO TC
Alternative ISO Alternative ISO
1.97 0.46 0.23 0.5 0.27
2.9 0.28 0.16 0.39 0.18
3.92 0.28 0.14 0.31 0.16
Contamination
Level (log cfu/g)
Repeatibility Reproducibility
TVC
Alternative ISO
0.78 0.88
0.91 1.24
1.27 1.78
Dispersion between
laboratories
EC
Example TEMPO EC Example TEMPO EC Example TEMPO EC Example TEMPO EC
1. Introduction
2. AFNOR Validation
!
4. Conclusions
AFNOR Validation AFNOR Validation AFNOR Validation AFNOR Validation
1. A clear set of rules
" Which makes it possible to organise the validation process without major
unexpected events
" The different parameters undergo similar procedures, allowing a general
coherence in the validation activities:
! submission of the data file
! content of the validation certificate
! Content of the Summary Reports
! post validation management (I.e. new software versions, etc.),
! in the audits and in the overall costs
2. Good Representativity in the AFNOR Technical
Committee
" Presence of Official Bodies, alternative method users, Kit manufacturers,
etc., assuring an equilibrated evaluation of the data files
" the practical side (users point of view) is also taken into account, and
sometimes the good sense prevails on purely statistical considerations
5. Professional excellence of expert laboratories
" Very good knowledge of the ISO 16140 protocol which allows the
production of clear reports and presentations
3. Different expertise in the AFNOR Technical
Committee
" Good associations of scientific, regulation and technical knowledge; a
group of people $ not only one person taking decision
4. Frequency of the Technical Committee meetings
" Approximately every two months. The validation delays are easily
foreseeable
6. Public web-site
" Where is possible to download not only the Validation Certificate, but the
Summary Report too.
AFNOR Validation AFNOR Validation AFNOR Validation AFNOR Validation
Nestl Research Center 2007-03-20 NRC/QS JMr 1
- Rapid method validation -
Experiences from a perspective of a food
manufacturer
AFNOR Validation - 19 June 2008
John D. Marugg,
Nestl Research Centre, Lausanne, Switzerland
Nestl Research Center 2007-03-20 NRC/QS JMr
2
This presentation
1 Does the food industry need rapid methods?
2 Requirements and selection process
3 Nestl validation procedure
4 Examples of Nestl/AFNOR approved alternative methods
5 Harmonisation and challenges
3
Nestl, the biggest Food Company
276'000 employees
Sales of CHF 107.6 billion in 2007
93% of sales is food and beverages
480 factories in 86 countries
Prepared dishes &
Cooking aids
17%
Pharmaceutical
products
7%
Milk products & Ice cream
19%
Powdered & liquid
beverages
17%
Nestl Waters
10%
Confectionery
11%
PetCare
11%
Nestl Nutrition
8%
4
Nestl's Billionaire Brands
5
Many products. many analyses needed
! > 250 Labs involved in microbiological testing
finished products, ingredients, environmental samples
! ~ 50 Salmonella labs
! ~ 1.000.000 Salmonella analyses per year
> 99% negative results
! ~ 0 Salmonella outbreaks per year
6
Common limitations of traditional
(cultural) methods
! Slow
Delays release finished products and
ingredients
Delays response to data from environmental
monitoring programs
! Many yield false negative and false
positive results/large measurement
uncertainty
! Labour intensive
Conclusion: Food industry indeed needs
rapid methods
Choice is enormous
How to decide which method is best?
7
Rapid Methods: huge diversity in
principles + strategies
! metabolic properties
Miniaturisation and Diagnostic Kits,
Biochemical Identification Techniques
chromogenic agar broths
! antibody-based
Elisa, dipstick, lateral flow...
Immunoconcentration
Immunomagnetic separation
! Nucleic acid-based
Hybridisation
PCR
riboprinting, AFLP,PFGE, DNA chips.
8
Minimum requirements for the ideal
rapid method
Flexibility
multi-purpose, multi-target, detection + identification, non-destructive
Future Nestl microbiologist in search of Salmonella
Sensitivity
100%
Specificity
100%
Speed
fast "on-line
Ease-of-use
fully automated
Costs
zero
9
Rapid Methods: Reality
Challenges:
Foods represent a wide variety of complex matrices
Target organisms often present in low numbers
Intrinsic bacterial flora may hinder identification
Processing of foods results in stressed or injured
cells
Lengthy enrichment procedures required to detect
viable cells
10
Requirements Rapid Methods
! Validated
preferably ISO 16140, AOAC, Nordval etc.
! Reliable
Also suited for Nestl specific matrices
Composite samples (pooling)
! Recognized by authorities and trading partners
! Fast result
! Low costs
Equipment
Reagents
Personnel
Laboratory space
costs of waste disposal
11
Requirements rapid methods (cont)
! No extra requirements for technicians
easy training
! Compatible with normal lab organisation
! Sufficient capacity/throughput
! Multifunctional (adapted for Salmonella, Listeria, etc)
! Automation
limit possibilities human errors
! Compatible LIMS
! Guaranteed availability/delivery
reliable supplier
world wide available (also in the future)
quick repair/back up equipment in case of problems
! Easy exit strategy
12
Collect information candidate methods
- Supplier
- Validation organisations
- Colleague microbiologists
- Publications
- Other suppliers
13
Pragmatic solution
Procedure:
Review literature, data from supplier and external independent
validation data
Comparison with other methods
Test pure cultures of target and non-target microorganisms
Analysis of different matrices (spiked and naturally contaminated)
Repeatability & reproducibility
Compile results
Decision taken by an expert panel
14
Development of a coordinated
approach for method selection
! Method evaluation and approval
Expensive
Requires specific competence
Should be coordinated
! 1998, implementation NesVal system: a Common
framework for internal evaluation and validation of new and
alternative methods
15
NesVal, main features
! Centrally coordinated evaluation of rapid methods
microbiology
allergens, chemical contaminants (mycotoxins, antibiotics)
Modifications of existing methods, scope extensions (new matrices)
! Systematic choice of candidate methods
Determined by needs of laboratories & offers from method suppliers
! Fixed procedure, complementary to ISO 16140
Special attention to Nestl specific matrices
Comprises usually an first evaluation by NRC followed by inter-
laboratory studies
Judges methods also with respect to other requirements
16
NesVal, main features (cont)
! Technical Expert Committee
Technical evaluation at the Nestl Research Centre Switzerland
- Assessment external evaluation data (suppliers, external validation)
- Preliminary screening trials
- Coordination of trials
Maintain contacts with suppliers
- Feedback on results of trials
- Explain what our needs are
! Advisory committee
Ca 20 members, representing markets, R&D, Corporate QM,
business units
Define needs and priorities
Ensure communication and implementation
17
Validation status rapid methods
NesVal May 2008
Method Target Throughput Approval/ LI number
(s/series) Status
ELISA-based (<50 h)
! Vidas S, Lm, L 1230 AFNOR/AOAC LI-00.742; -745;
! (Tecra Via S, L 96 AFNOR/AOAC LI-00.747; -749)
! Transia S, L 186-372 AFNOR LI-00.746; -754
PCR-based (<30 h)
! BAX S, Es 96 AFNOR/AOAC LI-00.744; -748;
Diverse (chromogenic/petrifilm)
! Petrifilm TVC, EB, -- AFNOR/AOAC LI-00.701; -709;
CC/EC/HSCC, STX LI-00.738; -750
! EB shortened EB -- NesVal/ISO21528 LI-00.709
! ALOA Lm -- AFNOR/ISO11290 LI-00.716; -727
! ESIA Es -- ISO/TS22694 LI-00.743
! RAPIDLmono Lm -- AFNOR/AOAC LI-00.755
Under evaluation
! Simplate TVC, Y&M -- AOAC LI-00.7xx
! Tempo EB, TVC -- AFNOR/AOAC LI-00.7xx
! RT-PCR (BD/LC2) EB + Es 32 Microval/AOAC LI-00.756
18
VIDAS system (BioMrieux)
! The VIDAS is an Enzyme Linked Fluorescent
Assay (ELFA) for detection of Salmonella
antigens carried out on a multi-parameter
automated immuno analyser.
! Each test consists of:
! Solid Phase Receptacle (SPR)
solid phase coated with anti-S antibodies
pipetting device
! Ready-to-use reagent strips
Tests available: SLM, easy SLM, LIS, LMO2, LSX,
enterotoxins, etc.
0.1 ml
Pre-enrichment 18h
RVS 7h
1 ml
1 ml
M-broth 16h
15 min 100C
Elisa 1h
0.2 ml
19
Transia system (Biocontrol)
Transia Plate Salmonella Gold
is a sandwich type Elisa assay on a
microtiter plate format
Each test consists of:
microtiter plate with
solid phase (strips) coated with
anti-Salmonella antibodies
polyclonal Ab as capture
specific monoclonal Ab as tracer
Other assays:
Listeria,
enterotoxins,
allergens
1 ml
0.1 ml
1 ml
Pre-enrichment 18h
RVS 20h
15 min 100C
Elisa 1h
20
PCR method dedicated to use in food industry
Automated DNA amplification with fluorescent
detection
# Tablets contain all reagents necessary for PCR
- Taq polymerase
- Primers
- Nucleotides
- Control reaction
- Fluorescent dye
# Assays
Salmonella, Listeria genus, Listeria
monocytogenes, E. coli O157 and E.sakazakii
BAX system (Dupont Qualicon)
50 l
10 l
5 l
Pre-enrichment 18h
BHI 3h
Lysis reagent
20 min 37C
5min 100C
PCR 3+1h
2007-03-20NQAC
Cergy/OGG
NRC/QS JMr 21
Day 1 Day 1 Day 1 Day 1
Rapid! Rapid! Rapid! Rapid!L. mono L. mono L. mono L. mono
37"C, 24 37"C, 24 37"C, 24 37"C, 24 2 h hh h
Suspend 25g or 25 ml
sample 1:10
1/2 Fraser broth Fraser broth Fraser broth Fraser broth
30"C, 24 30"C, 24 30"C, 24 30"C, 24 # ## # 2 22 2 h hh h
Confirmation(s) Confirmation(s) Confirmation(s) Confirmation(s)
Spot on Ottaviani & Agosti Spot on Ottaviani & Agosti Spot on Ottaviani & Agosti Spot on Ottaviani & Agosti
or Gen Probe or Gen Probe or Gen Probe or Gen Probe
or others or others or others or others
0.1 ml
Palcam + O&A Palcam + O&A Palcam + O&A Palcam + O&A
0.1 ml in 10 ml 0.1 ml in 10 ml 0.1 ml in 10 ml 0.1 ml in 10 ml
Fraser Fraser Fraser Fraser
37"C, 24 37"C, 24 37"C, 24 37"C, 24 2 h hh h
37"C, 48 37"C, 48 37"C, 48 37"C, 48 2 h hh h
Palcam + O&A Palcam + O&A Palcam + O&A Palcam + O&A
37"C, 24 37"C, 24 37"C, 24 37"C, 24 2 h hh h
Confirmation Confirmation Confirmation Confirmation
Confirmation Confirmation Confirmation Confirmation
EN ISO 11290-1 RLM
Nestl Research Center
2007-03-20 NRC/QS JMr 22
Hygiene indicators: Need for rapid validated
methods
! Hygiene indicators are used for product release
TVC, Enterobacteriaceae, Coliforms, E. coli
! Main available methods:
Lab efficiency TAT Calibration ISO16140
Petrifilm + No Yes
MicroFoss + ++ Required No
/Bactrac /Yes
Simplate ++ ++ No No
(AOAC/OMA)
TEMPO
++ + No Yes
Nestl Research Center
2007-03-20 NRC/QS JMr 23
Release of starter infant formula
products
! Per 2008:
Absence of EB (10 x 10 g) and absence of Es (30 x 10 g)
! Current Methodology Duration
EB:
cultural ISO21528 (2004);
(shortened ISO procedure) 2 3 days
Es:
cultural ISO/TS 22964 (2006) 3 5 days
BAX-PCR Es; 30 hours
2007-03-20 NRC/QS JMr 24
LightCycler-System 2.0
2007-03-20 NRC/QS JMr 25
Detection of Enterobacteriaceae/E.
sakazakii/Salmonella
PCR 1
Enterobacteriaceae/E. sakazakii
Enrichment
20-24 h
DNA Extraction
CH 610
IPC
CH 640
Enterobacteriaceae
CH 670
E. sakazakii
Positive Result
PCR 2
Salmonella
Negative Result
Enterobacteriaceae neg.
E. sakazakii neg.
Salmonella neg.
26
NesVal, Official Recognition
! NesVal approved methods accredited by Swiss authorities
Based on NesVal validation files
May facilitate discussions with local accreditors
! NesVal approved methods have official validation status
Majority based on AFNOR/ISO 16140 (or are in the process to obtain this)
AOAC (RI/Official Method)
ISO 16140 validation requirement for new alternative methods
! Official validation for key conventional methods (in-house methods)
Nestl S Method (easier variant of ISO6579) under validation (MicroVal/ISO
16140)
EB detection (ISO21528-1): shortened protocol (- EE selective enrichment
step) proposed and accepted for next ISO 21528 revision
27
Harmonisation is required:
! Reference methods (ISO/CEN versus AOAC/BAM)
To avoid disputes on release/acceptance of ingredients or products
- example: TVC: ISO4833 (30C/72 h) is not equivalent to BAM (35C/48 h), and do
not provide the same results!
- example: ISO 6888 (Coag + Staphylocci) <-> BAM (S. aureus)
To facilitate international trade and acceptance by authorities
To facilitate the validation of alternative methods
- In principle, a single validation based on a single reference method would be
sufficient...
! International validation procedures
To avoid unnecessary duplication of work
To speed up the process and reduce costs
To avoid different protocols for the same method
- example, VIDAS SLM (one way <-> two way)
To facilitate acceptance by authorities/accredition bodies.
28
Challenges for method developers
! Consider the challenges of food matrices
! Consider the challenges of high throughput laboratories
! Include challenging matrices in evaluation studies
! Include stressed microorganisms in evaluation studies
! Independently approved methods are preferred
EN/ISO 16140:2003
Microval/AFNOR
AOAC/Nordval
! Wide acceptance of methods is necessary in most cases
29
Challenges for AFNOR
! Include relevant and challenging industrial food matrices in
studies
shift from local/artisanal products to industrial foods
! Include pooling protocols in validation (composite samples)
many analyses in industrial labs are done on composite samples
! Include relevant environmental samples
powders, vacuum cleaner contents, line scrapings, etc.
! Include stressed microorganisms in evaluation studies
consider artificially spiking with low levels of stressed m.o., rather than insisting on
naturally contaminated samples (in non-relevant matrices!)
! Include lyophilisates in collaborative trials
to facilitate the organisation of international shipments of samples.
! AFNOR Validation as organisation:
Is perceived as French rather than European (composition Technical Board.)
Involve international, independent experts
Consider closer cooperation with Microval - mutual recognition agreement
Actively work on harmonisation of international validation procedures
30
Conclusions
! The food industry still leans heavily on traditional
methods
! Rapid and reliable low cost alternatives are needed
! Offer is enormous, the choice difficult
! Evaluation is expensive. It requires scientific
competence and understanding of needs
! NesVal: successful coordinated and systematic
approach for method evaluation, approval and
implementation.
! Official validation is essential Harmonisation of official
procedures is a must.
Nestl Research Center 2007-03-20 NRC/QS JMr 31
Many thanks to:
Han Joosten, Patrick Deberghes, NRC/QS
Tim Jackson (CO-QM)
Alternative validated methods in Belgium, an
example of application of the European
Regulation (EC) n2073/2005
C. De Backer
National Reference Laboratory for food microbiology
Faculty of Veterinary Medicine - University of Liege
Boulevard de Colonster 20 bt. B43bis
4000 Liege
Tl +32 (0)4 366 42 26
Fax +32 (0)4 366 40 44
caroline.debacker@ulg.ac.be
Agence fdrale
pour la Scurit
de la Chane alimentaire
In collaboration with the Federal Agency for the Safety of the Food Chain
AFNOR Validation Symposium, Brussels 19
th
of June 2008
Belgian National Reference Laboratory for food microbiology 2
Plan of the lecture
! Legislative context
! Analytical context
! Methods categories
! Available alternative methodologies
! Illustration
! Conclusion
! References
Legislative context
Commission Regulation (EC) n 2073/2005
of 15 November 2005
on microbiological criteria for foodstuffs
" Reference methods (mainly ISO standard methods)
" Alternative validated methods
! Reference methods issued from the regulation
! Protocol set out in the standard EN ISO 16140 or internationally
accepted similar protocols
! Certified by a third party
Analytical context
! FASFC requirements for the choice of methods
according to the analytical context
analytical context FASFC requirements in practice date of
application
R & D no requirement NA
private analyses methods consistent with the
Commission Regulation (EC) n
2073/2005
any method among :
- reference methods
- alternative validated methods
01/01/08
FASFC analyses and
all counter-analyses
methods imposed by FASFC among
methods consistent with the
Commission Regulation (EC) n
2073/2005
imposed methods among :
- reference methods
- alternative validated methods
01/01/08
Methods categories
! Standard methods
! Alternative validated methods
! Belgian official methods
Methods categories - 2
! Standardized methods
" Characterized performances
" Entire protocol, no simplification
" French routine standard methods and some other standards :
progressive abolition (from October 2006 until January 1, 2008)
method category examples
standard international ISO, IDF
European EN
national NBN, NF, NEN, DIN, NMKL
! Alternative validated methods
" Commercial methods: simpler, faster and/or more economic than the reference
methods
" Standard EN ISO 16140 : 2003 Protocol for the validation of the alternative
methods
! International recognition
" FASFC considers that the alternative methods validated by AFNOR Certification
and MicroVal are in conformity with the provisions of article 5 of the Commission
Regulation (EC) n 2073/2005
" The alternative methods validated by other organizations or laboratories will be
also recognized in Belgium if they fulfil the requirements of article 5 of the
Commission Regulation (EC) n 2073/2005 and if they provide at least the same
guarantees as those described in the protocol of the standard EN ISO 16140
" Validation file to provide to FASFC and National Reference Laboratory in food
microbiology for evaluation and possibly recognition
! Belgian official methods
" Worked out to meet a need
" Written by the National Reference Laboratory in food
microbiology and recognized by FASFC
" Since January 1, 2008 only valid the ones where no reference
method exists
Methods categories - end
! Methods : summary
methode category examples
standardized international ISO, IDF
European EN
national NBN, NF, NEN, DIN, NMKL
alternative validated EN ISO 16140 AFNOR
Belgian official SP-VG
AOAC?, NordVal?
MicroVal EN ISO 16140
Available alternative methodologies
! Principal available methodologies within the alternative validated
methods and some examples
" Important : step of confirmation of the positive results
Tempo MPN
Lumiprobe, Accuprobe Molecular hybridization
BAX, IQ check, TaqMan, GeneDisc Genetic test
Vidas, Transia, TAG, Tecra, Bioline, Rapidyme, Oxoid
Rapid test
Immunoassay
SMS, Rapid, SESAME, ALOA, CHROMagar, Compass,
Transia, Precis,OAA
Culture media detection
BACTRAC Impedance
Rapidmedia, media ID, Compass, CHROMagar,ALOA,
REBECCA, OAA
Chromogenic media
Petrifilm, Compact dry Dehydrated media counting
Example of methods Per parameter
Practical aspects of the list
! Two times a year
" Scientific evaluation
! Update of the dates of standards and alternative methods review
! suppression of the repealed methods
! evaluation of innovations
! follow-up of the modifications compared to the former version
" Proposition of list to the Food Agency
" Comments and agreement of the Food Agency
" Official circular of the Food Agency to the laboratories and list
available on the website
! www.afsca.be> business sectors > Laboratories Administration >
Approvals > Approved laboratories: Office circular
" Next version scheduled for July 2008
Illustration
Example coming from the list of recognised methods before application of
the Regulation EC n 2073/2005 (version April 2007)
Germes Matrices M thodes Intitul
Limite de
validit
AFNOR
Validation
ISO 16140
Ann e de
publication
remarques
14
Recherche de
Salmonella spp
(6)
toutes denr es ISO 6579 (1)
M thode horizontale pour la recherche des
Salmonella spp
2002 Cor 1:2004
denr es d'origines animales,
mati res f cales des animaux,
chantillons au stade de la
production primaire
ISO 6579/FDAmd 1
Recherche de Salmonella spp. dans les
mati res f cales des animaux et dans des
chantillons au stade de la production
primaire
toutes denr es NMKL 71 Salmonella.Detectioninfoods. 1999 accept e jusqu'au 31/12/07
viandes et produits base de viande SP-VG M002 (3)
M thode pour la recherche de Salmonella
(m thode de routine par enrichissement
en milieu semi-solide Diassalm)
1998 accept e jusqu'au 31/12/07
lait et produits laitiers ISO 6785 = IDF 93 Recherche de Salmonella spp. 2001 accept e jusqu'au 31/12/07
eau ISO/CD 19250
Recherche et d nombrement d'esp ces
Salmonella
paratre
eau ISO 6340 Recherche et d nombrement de Salmonella 1995 accept e jusqu'au 31/12/07
toutes denr es et pr l vements de
l'environnement (hors levage)
AFNOR AES-10/4-05/04 (2) Simple Method for Salmonella (SMS) 7/05/08 oui
toutes denr es AFNOR ART-27/01-12/05 (2) Rapidyme Salmonella 9/12/09 oui
toutes denr es AFNOR BIO-12/1-04/94 (2) VIDAS Salmonella (double voie) 9/06/10 oui
toutes denr es AFNOR BIO-12/6-03/99 VIDAS ICS SLM 30/09/07
toutes denr es AFNOR BIO-12/7-03/99 VIDAS ICS-Bote 30/09/07
toutes denr es AFNOR BIO-12/10-09/02 (2) VIDAS Salmonella (simple voie) 18/09/10 oui
AFNOR BIO-12/16-09/05 (2) VIDAS Easy Salmonella 20/09/09 oui
toutes denr es AFNOR BLN-26/01-03/04
Bioline Salmonella Elisa Test SELECTA
(enrichissement 24h )
12/03/08
toutes denr es AFNOR BLN-26/02-03/04
Bioline Salmonella Elisa Test Optima
(enrichissement 40h)
12/03/08
AFNOR BRD-07/6-07/04 (2) IQ-Check Salmonella 1/07/08 oui
toutes denr es AFNOR BRD-07/11-12/05 (2) Rapid'Salmonella 9/12/09 oui
toutes denr es AFNOR EUR-15/2-11/00 Lumiprobe 24 Salmonella 29/11/08 en cours
toutes denr es AFNOR GEN-25/01-12/03 GeneDisc Cycler Salmonella 12/12/07
AFNOR QUA-18/3-11/02 (2) BAX Salmonella (automatis ) 28/11/10 oui
toutes denr es AFNOR TEC-24/2-04/03 Tecra Ultima Salmonella 12/10/07
toutes denr es AFNOR TEC-24/3-12/03 Tecra Unique Salmonella 12/12/07
AFNOR TRA-02/8-03/01 (2) Transia Plate Salmonella Gold 3/02/09 oui
toutes denr es AFNOR UNI-03/1-05/91 Salmonella Rapid Test 7/09/07
Illustration
Salmonella spp detection
from January 1, 2008
standard alternative validated Belgian official
ISO 6579 AFNOR AES-10/4-05/04 (2) SP-VG M002
NMKL 71 AFNOR ART-27/01-12/05 (2)
ISO 6785 = IDF 93 AFNOR BIO-12/1-04/94 (2)
ISO 6340 AFNOR BIO-12/6-03/99
ISO/CD 19250 AFNOR BIO-12/7-03/99
AFNOR BIO-12/10-09/02 (2)
AFNOR BIO-12/16-09/05 (2)
AFNOR BLN-26/01-03/04
AFNOR BLN-26/02-03/04
AFNOR BRD-07/6-07/04 (2)
AFNOR BRD-07/11-12/05 (2)
AFNOR EUR-15/2-11/00
AFNOR GEN-25/01-12/03
AFNOR QUA-18/3-11/02 (2)
AFNOR TEC-24/2-04/03
AFNOR TEC-24/3-12/03
AFNOR TRA-02/8-03/01 (2)
AFNOR UNI-03/1-05/91
AFNOR UNI-03/1-05/91
Note
! For some parameters, it doesnt exist recognised
methods anymore since January 2008
" No requirement on the European level, and not included in
the national monitoring
" To work with other relevant parameters
" Or to keep the same parameters using a method without
any preference (but no official recognition of FASFC
anymore)
! Example: enumeration of fecal coliforms
Conclusion-1
! List of the analytical methods recognised by FASFC for the food
microbiological analyses in Belgium since about 12 years
! List strongly modified by the Commission Regulation (EC) n
2073/2005
! Reference methods and validated alternative methods
! Obligation for private analytical controls to also align on Commission
Regulation (EC) n 2073/2005, since January 1, 2008
! Imposition of specific methods for the analyses for FASFC since
January 1, 2008
Conclusion-2
! Alternative validated methods recognized by FASFC if they fulfil
requirements of the Commission Regulation (EC) n 2073/2005 and
if they provide at least the same guarantees as those described in
the protocol of the standard EN ISO 16140
! Validation file to provide to FASFC for evaluation and possibly
recognition
! List updated twice a year and available on the website of FASFC
(www.afsca.be > business sectors > Laboratories Administration >
Approvals > Approved laboratories: Office circular
! Constant evolution, must anticipate changes
References and useful links
" Standard ISO 16140:2003, Microbiology of food and animal
feeding stuffs Protocol for the validation of alternative methods.
" Commission Regulation (EC) n 2073/2005 of 15 November 2005
on microbiological criteria for foodstuffs. Official Journal of the
European Union, December 22, 2005, L 338/1 L 338/26.
" www.afsca.be: website of the Federal Agency for the Safety of
the Food Chain (FASFC)
" www.afnor-validation.org: website of AFNOR Validation
" www.microval.org: website of MicroVal
" www.wssn.net : World Standards Services Network : network
gathering websites of the international, regional or national
organizations of standardization.
19 June 2008
Revision of EN ISO 16140:
Current status and foreseen changes
AFNORValidation Symposium : European certification for microbiological test kits
19 June 2008
Revision of EN ISO 16140 | Paul in 't Veld 2
Revision of EN ISO 16140
- History
- ISO/CEN resolutions
- ISO TC 34/SC 9/WG 3
- Division into project groups
- Discussions in PG2
- Likely changes
- Time frame
19 June 2008
History
1993: start of Eureka project called Microval
June 1996: Acceptance of new work item proposal for Protocol for
the validation of alternative microbiological methods. Start of TAG2
December 1999: ISO/DIS 16140 published for comments
May 2003: First edition of ISO 16140
June 2005: Resolutions from CEN and ISO to revise the ISO 16140
Mainly based upon document of AFAQ AFNOR Certification
describing proposed corrections, modifications and interpretations.
19 June 2008
ISO/CEN resolution
The ISO TC 34/ SC 9 resolution 265 decided:
Setting up of WG 3 Validation of microbiological methods
Objectives of WG 3:
Establish terminology
Revision of ISO 16140 (full validation of proprietary methods)
and taking into account the inclusion of IDF 161 (Quantitative
determination of bacteriological quality)
Development of standard for in house validation
19 June 2008
ISO/CEN resolution (ctnd)
Laboratory verification of methods
Intermediate validation based on AOAC classification
Minimum requirements for establishing/revising reference
methods
Inquiry for nomination of members for WG 3
19 June 2008
ISO TC 34/SC 9/WG 3
- First meeting January 2006: nomination of convenor and divisionof
tasks
- so far two meetings per year
- ca 25 participants in WG from 15 different countries/organisations
- division of the work into project groups, project group maximumof 6
participants
19 June 2008
Division into project groups
PG 1: Terminology
PG 2: Proprietary methods
PG 3: Intermediate validation
PG 4: Method verification
PG 5: In house method validation
PG 6: Technical requirements for the establishment/revision of
standardised methods
19 June 2008
PG 1: Terminology
- Project leader: Mieke Uyttendaele(RUG, Belgium)
- Terminology will be a separate part of the revised 16140
- Current status: document has been prepared taking into account the
definitions used by other organisations such as for example EU,
AOAC, ILAC. Draft has been discussed at WG3 level.
19 June 2008
PG 3: Intermediate validation
- Project leader: Rachel Rangdale(CEFAS,UK)
- Intermediate validation is a validation study including in inter-
laboratory study with a limited number of participating laboratories
- Intermediate validation will be a separate part of the revised 16140
- Current status: Difficult to use less laboratories and still produce
statistically valid results. Proposal from Germany still under
discussion, also in WG2 (statistics)
19 June 2008
PG 4: Method verification
- Project leader: Henk Stegeman(NL)
- Verification: Demonstration by experiment that an established
method functions in the users hands according to the methods
specifications determined in the validation study.
- Method verification will be a separate part of the revised 16140
- Current status: Still under discussion in WG3
19 June 2008
PG 5: In house method validation
- Project leader: Thorsten Trumpf (LVUA, Germany)
- In house validation: a validation study without an inter-laboratory
study
- In house validation will be a separate part of the revised 16140
19 June 2008
PG 6: Technical requirements for the establish-
ment/revision of standardised methods
- Project leader: Olli Pensala(Finland)
- Will become a separate ISO publication as a Technical Report (TR)
19 June 2008
PG 2: Proprietary methods
- Project leaders: MaxFeinberg(INRA, France) and Paul in t Veld
(VWA, Netherlands)
- Topics discussed so far:
- Inclusion IDF 161 (Bactoscan)
- Natural versus artificially contaminated samples
- Outline collaborative studies
- Statistics for the evaluation for qualitative data
- Statistics for the evaluation for quantitative data
19 June 2008
PG2: Inclusion of IDF 161 (Bactoscan)
IDF validates alternative methods for the hygienic status of milk.
Farmers are being paid on that basis. Currently the Bactoscanis
used as the alternative method and has been extensively validated.
WG 3 noted that the scope for this validation was very limited and
specific. ISO TC 34 SC9 confirmed this opinion.
In the revised standard a reference will be made to this specific IDF
standard.
19 June 2008
PG 2: Natural versus artificially
contaminated samples
Annex C: current order of preference will remain the same:
1) natural contamination is better than
2) dilution of contaminated product which is better than
3) spiking/inoculation.
PG 2 proposed to modify the procedure for inoculation of samples
(seeding protocol AOAC)
Advantage: better mimic natural contaminated samples
Disadvantage: not possible to count the level of contamination
(p/a testing)
19 June 2008
PG 2: Outline collaborative studies
PG confirmed the use of both alternative and reference method in
collaborative study
One matrix for evaluation in collaborative study is sufficient but
when more protocols for the examination of different samples for the
proprietary method exist more matrices are needed
19 June 2008
PG 2: Statistics for the evaluation of
qualitative data
Current evaluation does not give clear criteria for the acceptance of
results
Current parameters (concordance and accordance) depend on the
level of contamination of the samples used. Alternative approachis
the comparison of the (ratio of the) limit of detection (LOD).
This can work for both the collaborative study data as well as for the
method comparison study data.
19 June 2008
PG 2: Statistics for the evaluation of
quantitative data
Current evaluation does not give clear criteria for the acceptance of
results
Current analysis of data is dependant of the observed variation of the
methods and not on an (from a microbiological point of view)
acceptable difference between the reference and alternative method
Proposal from MaxFeinbergfor alternative approach, called
accuracy profile is being worked out.
19 June 2008
Small
uncertainty
Target value
Method A
(valid)
+
Acceptance
limits
High
uncertainty
Target value
Method B
(not valid)
+
Acceptance
limits
Acceptance limits
represent the
intended use of the
method
19 June 2008
Future prospective
In last 3 years significant progress has been made especially within
PG 2 which will be the basis for the other PGs.
It is intended to have the first draft from PG2 for discussion in WG 3
in September 2008. Depending on the discussion the draft can be
discussed at the next ISO meeting in May/June 2009.
So be patient.
AFNOR VALIDATION
19 June 2008 Brussels
1
PCR PCR PCR PCR Legi onel l a Legi onel l a Legi onel l a Legi onel l a : :: : t he i nt er est t he i nt er est t he i nt er est t he i nt er est of an of an of an of an
AFNOR Val i dat i on AFNOR Val i dat i on AFNOR Val i dat i on AFNOR Val i dat i on w i t h w i t h w i t h w i t h 2 22 2
ex ampl es ex ampl es ex ampl es ex ampl es
Frederic Martinez Bio-Rad
Bertrand Coissac - GeneSystems
AFNOR VALIDATION
2
Out l i ne
Legionella monitoring
PCR Legionella standard context
The PCR standard: XP T90-471
Performance assessment
An example of validation results
The interest of an AFNOR validation
AFNOR VALIDATION
3
Cont r ol of Legionella
Type of water concerned by control
Sanitary hot and cold water
Cooling tower water
Thermal water
Installations concerned by control
Travel associated buildings and installations
(Hotels, camping's, swimming pools, )
Hospitals and Health Care Establishments
All systems producing aerosols
(Public fountains, air conditioning, )
AFNOR VALIDATION
4
Official analysis
Regular control
Threshold requiring actions
Autocontrol monitoring
Without regulation
To manage disinfection processes
To secure people
Epidemiological inquiry
Importance to have a fast time to
results method for Legionella monitoring
Legionella Moni t or i ng
AFNOR VALIDATION
5
Rec ommendat i ons
The PCR method is useful :
for fast monitoring and assessing the risk of
Legionella growth in water circuits
the evolution of results over time enables driving
the installation and the necessary treatments ->
measuring the effectiveness of a biocide
treatment
in the context of an epidemiological inquiry,
research for contaminated sites can be carried
out in a short time
AFNOR VALIDATION
6
PCR st andar d c ont ex t
To meet expectations from customers and
governments faced with Legionella risk
management, especially in terms of time
To allow the use of new technologies while
overseeing the development work of various actors
in the sector
Importance to standardize
AFNOR VALIDATION
7
Agenda
APRIL 2006 : XPT 90 471 AFNOR STANDARD
JUNE 2006: PCR LEGIONELLA AFNOR VALIDATION
PROTOCOL
DECEMBER 2007 : GENESYSTEMS & BIORAD
LEGIONELLA METHOD AFNOR CERTICIFIED
Today in France,
about 50 laboratories use the XP T90-471 Standard
about 10 laboratories are accredited according to the
Standard
AFNOR VALIDATION
8
XP T90-471
The XP T90-471 standard is a rapid method (results within
few hours versus 10 to 12 days with culture method)
The method is applicable to all types of water
The method allows direct quantification of total Legionella
and L.pneumophila (without any culture stage and any
colonies count)
!Quantification of cultivable bacteria and viable but non
cultivable bacteria
!Insensitivity to background flora
AFNOR VALIDATION
9
XP T90-471
The standard allows the use of different technologies for
bacteria concentration, DNA extraction and purification
Different specific target sequences (primers and probes)
may be used for amplification and quantification
!Therefore, a performance approach (LD, LQ, linearity,
recovery, specificity) is used and need to be evaluated.
The submission of the results to a third party
certification is better.
Importance to validate the
performances
AFNOR VALIDATION
10
AFNOR val i dat i on
pr ot oc ol
Step 1 ! !! ! Preliminary study (Expert laboratory)
PCR performances :
Limit of detection (LOD)
Limit of quantification (LOQ)
Linearity
PCR specificity
Optimal recovery of the entire method
Practicability (18 criteria)
Step 2 ! !! ! Ring trial including at least 8 laboratories
AFNOR VALIDATION
11
Preliminary study
(Expert laboratory)
AFNOR val i dat i on
pr ot oc ol
AFNOR VALIDATION
12
PCR per f or manc es (1)
" Limit of detection (LOD): the most little GU quantity giving a PCR positive result with
90% confidence
#PCR analysis of 30 L. pneumophila ATCC 33152 DNA solutions calibrated at 5 GU/PCR well
100 % positive PCR results
" Limit of quantification (LOQ): the most little GU quantity giving a quantitative PCR result
with a measurement uncertainty (E) inferior to 0.15Log
#30 PCR analysis of a L. pneumophila ATCC 33152 DNA solutions calibrated at 25 GU/PCR well
E = !(bias
2
+ (SD
2
) = 0.15
Method Result
Validation: AFNOR Certification
Objective: AFNOR XP T90-471
AFNOR VALIDATION
13
" Linearity:
! validation of the linear regression model by a Fisher test ( risk = 5%)
! E < 0.15 for each concentration level of the L. pneumophila DNA
! 75% < PCR efficacy < 125%
# PCR analysis of 5 L. pneumophila ATCC 33152 DNA ranges
F = 0.24 (< 3.10) ! !! !OK
E max = 0.09 (< 0.15) ! !! !OK
PCR efficacy = 93.7% ! !! !OK
" Specificity: minimal tests required
! 15 listed L. pneumophila strains ! !! !inclusivity: 10
2
GU/PCR
! 21 listed Legionella spp strains
! 17 listed strains others than Legionella ! !! !exclusivity: 10
4
GU/PCR
# PCR analysis with Legionella spp & L. pneumophila GeneDiscs Pack Premium
100 % specific
PCR per f or manc es (2)
AFNOR VALIDATION
14
" 3 matrix: ! mineral water
! hot sanitary water (HSW)
! cooling tower water (CTW)
" 3 contamination level of L. pneumophila ATCC 33152 / 250mL :
! 10
3
GU
! 10
4
GU
! 10
5
GU
" 6 independent samples / contamination level / matrix
=> 54 samples
Objective: Recovery > 25% or -0.6 Log < Bias (Log) < 0.6 Log
Opt i mal r ec over y (1)
AFNOR VALIDATION
15
Opt i mal r ec over y (2)
Contami
-nation
level
Recovery
Mean (%)
Bias Mean
(Log)
Bias SD
Mineral
water
10
3
75 -0.19 0.25 10
4
10
5
HSW
10
3
64 -0.27 0.28 10
4
10
5
CTW
10
3
66 -0.24 0.27 10
4
10
5
No
matrix
effect
AFNOR VALIDATION
16
Pr ac t i c abi l i t y
18 criteria inherent to kits and systems such as:
Quality control
Traceability
External quantitative control
Inhibition control
Ease of Use & Performances
Minimal volume to handle
All inclusive reagents / need to prepare reagent
Qualification of operator requirement
Hands on time
Time to results
Storage condition / stability of reagent / Maintenance
Rooms needed for analysis
AFNOR VALIDATION
17
AFNOR val i dat i on pr ot oc ol
Ring Trial
(minimum 8 laboratories)
AFNOR VALIDATION
18
AFNOR val i dat i on pr ot oc ol
" 3 steps:
! Calibrated DNA analysis (2 different tubes)
! !! !PCR reproducibility
! Artificially contaminated sanitary water (1 sample analyzed in duplicate)
! !! !DNA extraction + PCR reproducibility
! Naturally contaminated hot sanitary water (1 sample)
! !! !DNA extraction + PCR reproducibility
AFNOR VALIDATION
19
Ri ng t r i al
Calibrated DNA
Artificially contaminated
sanitary water
Naturally
contaminat
ed HSW
Sample DNA 1 DNA 2
Sanitary
water 3
Sanitary
water 3
HSW 4
N Labs 14 14 15 15 11
Bias (Log) 0.246 0.200 0.185 0.124 0.157
SD
r
repeatab.
0.051 0.035 0.113 0.084 0.098
SD
R
reproduc.
0.130 0.104 0.445 0.440 0.425
< 0,15 < 0,5
AFNOR VALIDATION
20
AFNOR Cer t i f i c at i on
AFNOR has created a standardized method &
validation protocol:
Guaranteed performances
Third party validation
Easier for labs to be accredited
iQ-Check Legionella GeneSystems Legionella
AFNOR VALIDATION
21
Thank you for attention
www.genesystems.fr
www.bio-rad.com
RVA J0958
AFNOR VALIDATION
EUROPEAN CERTIFICATION
19 JUNE 2008 - BRUSSELS
***
Many thanks to :
AFSSA-LERQAP, SANCO E2, AFNOR Certification, Insitut Pasteur,
ADRIA Dveloppement, Deutsches Institut fr Lebensmitteltechnik,
3M Microbiology, bioMrieux Industry, Nestl Research Center,
University of Lige, Genesystems, Bio-Rad.

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AFNOR VALIDATION SYMPOSIUM, BRUSSELS 2008

AFNOR Validation Symposium, June 2008, Brussels

TVC gives results in 40h compared to 72h of

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