afnor-validation2008
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AFNOR VALIDATION SYMPOSIUM, BRUSSELS 2008RVA J0958
AFNOR VALIDATION
EUROPEAN CERTIFICATION
FOR MICROBIOLOGICAL TEST KITS19 JUNE 2008 - BRUSSELS
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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
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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 criteriaSpeaker : 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 Break offered
13:30 Two kit manufacturers validation experiences& Discussion
* 3M Microbiology : Marie-Pierre COPIN; New products and Regulatory Manager
* bioMrieux Industry : Raffaella Giardino, Global Marketing Manager, Program Director QI
14:20 Experience from a perspective of a Food Manufacturer
Speaker : John 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, Chairman of 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
AFNOR all rights reserved. RVA J0958-2008
AFNOR VALIDATION SYMPOSIUM, BRUSSELS 2008RVA J0958
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AFNOR VALIDATION SYMPOSIUM, BRUSSELS 2008RVA 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, FranceTl: 00 33 1 49 77 26 96 - Fax: 00 33 1 49 77 26 95 - e-mail: [email protected]: www.afssa.fr
AFNOR all rights reserved. RVA J0958-2008
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Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffsThe use of rapid methods
Ari HrmanEuropean Commission
DG SANCO
AFNOR Validation Symposium, June 2008, Brussels
AFNOR Validation Symposium, 19th of June 2008 Brussels
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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
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Testing against criteriaFood business operators (Reg 2073/2005)
For validation and verification of procedures based on HACCP and GHPFor batch acceptability testing
Competent authorities (Reg 882/2004 on official controls)
To verify that food is in comliance with the Community criteria
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RegulationLatest scientific adviceBroad consensus Flexible
Sampling proceduresAnalytical methodsSampling frequencies (risk-based)Process hygiene criteria Trends
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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
resultsAFNOR Validation Symposium, June 2008, Brussels
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Analytical methodsReference methods in the legislation
Preference to horizontal methodsMethods of international standardisation organisations
For in-house control Alternative methods can be used under certain conditions (Regulation 2073/2005)
For official controlsThe use of EN/ISO methods recommended (Guidance document on official controls)
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The use of rapid methods
An advantage for food business operators
Results available more rapidly than by using traditional methodsRapid corrective measures and actions possible
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Alternative methodsThe 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.
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Alternative methodsIf 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
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Thank you
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AFNOR Validation :Certifying analytical performances
of commercial methods
Valentine Digonnet
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AFNOR CERTIFICATIONAFNOR 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
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AFNOR CERTIFICATION SERVICES
Conformity assessmentAttestation of
conformity to ECregulations
Product CertificationCompetencecertification
Services certification
Systems certification (ISO 9001, ISO
14001,)
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Purpose of AFNOR Validation
!Conformity assessment by a third party bodythat test results obtained with commercial kits
are comparable to results obtained using the corresponding reference methods
" individual approach (proprietary methods)
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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
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What is an alternative method?
For a givengiven scopescope, it shall estimate the same same analyteanalyte than the one measured by the corresponding reference method,
and shall meet the following requirements:
rapidrapid analysis and/or response
easyeasy carrying and/or automation
analytical performancesperformances (accuracy, sensitivity..)
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What is included in the alternative method ?
" The term "alternativealternative methodmethod" refers to the combination of product, equipment andoperating instructions
" It includes all elements necessary forimplementing the alternative method
(equipment, reagents, culture media, software for analysisof results,)
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What is a reference method?
" A reference methodreference 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.
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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
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Who acts in AFNOR Validation?
AFNOR CERTIFICATIONadministrative management
responsible for implementation
Expert laboratoriesindependant & qualified
achieve the validation studiesmake presentations of the results
Validation Commissionadvises on :
general operating rules, policies collective advertising
Auditorsindependant & qualified perform the quality audits
Technical Boardadvises on :
technical validation and renewalspecific technical rules.
20 members5 meetings (x 2 days) / year
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AFNOR ValidationCertification procedure
Based upon 33 principlesprinciples:
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)
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AFNOR Validationtechnical protocol
SinceSince July 2003July 2003 :
standard EN ISO 16140
specific requirements and interpretation set up by the Technical Board
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The preliminary study
based on EN ISO 16140 requirements
to characterizecharacterize the alternative method(selectivity, practicability)
to comparecompare the performances of the alternativemethod against the performances of the reference method (relative accuracy, sensitivity, specificity, relative detection level,)
Mandatory third party expert lab (no internal data)
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The inter-laboratory study
To define variabilityvariability ofof results obtainedresults obtained inin different different laboratories using the same sampleslaboratories using the same samples
According to EN ISO 16140:
at least 10 collaborative laboratories giving nonoutlier results (or 8 labs for quantitative methods)
implementing both alternative and reference methods
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Quality requirementsfor the manufacturer
Adoption of audit requirements based uponEN ISO 13485 (medical devices)
Additional specific requirements concerning thecontrol ofcontrol of productsproducts
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AFNOR Validation :Control of alternative methods
DurationDuration ofof certificatecertificate : 4 years
QualityQuality control on sitecontrol on site : according to the certificationalready granted to the supplier
one audit every 2 years (if not certified)
one audit every 4 years (if certified ISO 9001 or 13485)
RenewalRenewal every 4 years : complete review of changes in the alternative method, reference method andValidation protocol
#complementary assays if needed
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AFNOR Validation CertificateContentsContents :
AdministrativeAdministrative detailsdetails:
" Date of validation / end ofvalidity
" Certificate reference
" Address of manufacturerand retailer
" Reference of the kit users manual / technical sheet
TechnicalTechnical detailsdetails:
" Principle of the method
" Scope of validation
" Restrictions for use
" Reference method used
" Analytical performances (Resume of main results obtained)
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AFNOR ValidationReference documents
OperatingOperating RulesRules (general part + appendices)# to define the general policies
Technical protocolTechnical protocol for the validation studies(based upon EN ISO 16140 + interpretation)# to be used by the expert laboratories
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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 inAnnex I (mainly EN ISO methods)
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AFNOR Validation complies with EU Regulation
AFNOR VALIDATION scheme was developed inorder to comply with EU requirements defined inregulation, granting certificates to alternativemethods which are:
Validated against EN/ISO reference methodsCertified by a third party (AFNOR CERTIFICATION)Tested against EN ISO 16140 standardized protocol
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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,
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International and European supplierswith 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)
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Implementation of EN ISO 16140in AFNOR VALIDATION
5 years work on implementation of EN ISO 16140
$Working groups and Technical Board meetings
" 63 method validations or renewals according toEN 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
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Recent development of AFNOR Validation
% Creation of the logo and registration of thecollective European mark (2005)
% Current contribution to revision of technical protocol EN ISO 16140
% Extension of AFNOR Validation scope to watermicrobiological analysis in 2007
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Development of AFNOR ValidationObjectives 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- 3rd party certification with a regular survey of the product quality
and the production site
To extend AFNOR Validation scope (GMO,)
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Methods performance assessment according tothe EN ISO 16140 standard and the AFNOR Validation technical rules
- Qualitative methods
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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
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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 (LOD50) Inclusivity and exclusivity (pure culture strains)
Practicability (AFNOR certification technical rules)
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Relative AC, SPE & SE
Analyses on different samples with: Alternative method Reference method
Comparison of results obtained for the same samplesconfirmation of positive tests
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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
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SamplesNatural or Artificial contamination?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 monocytogenes25% for Salmonella
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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 %
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SamplesNatural or Artificial contamination?Natural or Artificial contamination?
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
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Analysis protocol
homogenization
Same first step
incubation
Reference method Alternative method
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Analysis protocol
homogenization
Same first step
incubation
Reference method Alternative method
BPW or Half Fraser
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Analysis protocol
Product Homogenisation
Reference method Alternative method
Different first step
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Calculation and interpretation
PDPA(A+)
NAND(A-)
(R-)(R+)Results
PA = positive accordance (R+ / A+)NA = negative accordance (R- / A-)PD = positive deviation (R- / A+) = additional positiveND = negative deviation (R+ / A-) = false negative
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(R+) (R-)
(A+) A+ / R+PA = 155A+ / R-PD = 2
(A-) A- / R+ND = 1A- / R-
NA = 169
Same first step between alternative and reference protocols
Examples
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(R+) (R-)
(A+) A+ / R+PA = 155A+ / R-PD = 16
(A-) A- / R+ND = 7A- / R-
NA = 186
Different first stepbetween alternative and reference protocols
Examples
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Relative Accuracy (AC)
Degree of correspondence between the response obtained by the reference method and the response obtained by the alternative method on identical samplesAC = (NA + PA) / N
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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
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Relative Sensitivity (SE)
Ability of the method to detect the target organism when the reference method detects it
SE = PA / (PA + ND)
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Sensitivity
SE = POS x 100% in AFNOR Certification rulesN+
with N+ = PA + ND + PD
et POS = confirmed positive results of the considered method
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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
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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)
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Calculation and interpretation
No criteria of acceptance in the standard
Analysis of discordances
Statistical test to determine if the methods are equivalent or not
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Ccld min
~16 7 = 91023
d = |PD ND|discordances
Examples
Less than 6 discordances: no statistical test
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Relative detection level
= the smallest number of culturableorganisms that can be detected in the sample in 50% of occasions
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Measurement protocol
5 products (one per category) 5 target microorganisms 3 to 5 levels of contamination 6 replicates per level
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Interpretation
The relative detection level lies between the two contamination levels: less than 50 % positive results more than 50 % positive results
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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
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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]
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Inclusivity
detection of the target microorganism from a wide range of strains
50 strains
Application of the complete protocol of the alternative method
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Exclusivity
= the lack of interference from a relevant range of non-target microorganisms
30 strains
Use of non selective broths
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Practicability
13 criteria defined in AFNOR Certification rules like : Qualification of technical staff Environment of analyses Materials Real time handling Time to result
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Interlaboratory study
Aim:to determine the variability of the results obtained in different laboratories using identical samples
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Interlaboratory study organisation
At least valid data from 10 laboratories 3 levels of contamination 8 replicates per level
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Analyses
Analyses with both methods, alternative and reference
48 results: 24 with each method
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Interpretation
Comparison of alternate method and reference method results against expected results
FP : false positive of L0 level TP : true positive of L1 et L2 levels
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Interpretation
TPTPFPTotal
/8/8/8etc
/8/8/8Lab 2
/8/8/8Lab 1
L2L1L0Contamination level
Lab
Pour each method : number of positive results
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Interpretation
L0 LevelSP = (1 FP ) X 100%
N-
FP = number of false positive
N- = total number of samples for L0 level
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Interpretation
L1 et L2 Levels
SE = TP X 100%N+
TP = number of true positive
N- = total number of samples for each level
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Interpretation
Accuracy : AC = PA + NA x 100%N
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Accordance, concordance, odds ratio
Notions of repeatability, reproducibility and odds ratio (informative annex)
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Conclusion
Preliminary study: informations of method performances
Interlaboratory study: practicabilitylot of work for the participant laboratories
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Certificate
Edition of a certificate
Summary report
www.afnor-validation.com
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Methods performance assessment according to
the EN ISO 16140 standard and the AFNOR
Validation technical rules
QUANTITATIVE METHODS
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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 ofmicrobiological alternative methods according tothe ISO 16140 standard
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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 inaccordance with the protocol set out in EN/ISO standard 16140
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EN ISO 16140 standard
! Microbiology of food and animal feeding stuffs Protocol for the validation of alternative method Qualitative methods Quantitative methods
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EN ISO 16140 standard
! Microbiology of food and animal feeding stuffs Protocol for the validation of alternative method Qualitative methods Quantitative methods
$ Microbial Indicators of food quality & safety
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EN ISO 16140 standard
! Microbiology of food and animal feeding stuffs Protocol for the validation of alternative method Qualitative methods Quantitative methods
And according to the AFNOR validation technical rules
$ Microbial Indicators of food quality & safety
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ISO 16140 standard
! Microbiology of food and animal feeding stuffs Protocol for the validation of alternative method Qualitative methods Quantitative methods
Or the need of a revised standard
$ Microbial Indicators of food quality & safety
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"Methods comparison study
SpecificitySpecificity && selectivityselectivity
Linearity Linearity
Relative Relative accuracyaccuracy Methods precision
Detection & quantification limits
" Inter-laboratory study
BiasBias
RepeatabilityRepeatability & & Reproducibility limitsReproducibility limits Dispersion between laboratories
Validation study process
!
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! 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
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Specificity /Selectivity
! Pure cultures
30 target strains
20 non-target strains
Enumeration by the reference and the alternative methods
Interpretation : the alternative method is conformedto the manufacturers specifications and to the specific
requirements for its reliable use.
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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
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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 deviationsGMFR : Orthogonal regressionOLS : Ordinary least squares linear regressionF-table (Snedecor)
AFNOR Validation Symposium, June 2008, Brussels
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08.19.06
Linearity
! 5 contamination levels, 4 log range
! 1 matrix / category, x 5 categories
! Lack-of-fit test
Sample Regression P% Correlation coefficient
Regression equation
Matrix 1 GMFR 100 0,999 log alt =0,975 logRf. + 0,101
0
1
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Log (Reference method)
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Matrix 1
Correlation coefficient = 0,999, P > 5% The linearity is accepted
AFNOR Validation Symposium, June 2008, Brussels
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08.19.06
Sample Regression P% Correlation coefficient
Regression equation
Matrix 1 GMFR 100 0,999 log alt =0,975 logRf. + 0,101
Matrix 2 GMFR 2 0,999 log alt =0,975 log Rf. + 0,093
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Linearity
! 5 contamination levels, 4 log range
! 1 matrix / category, x 5 categories
! Lack-of-fit test
Correlation coefficient = 0,999, but P < 5%Linearity test robustness?
Matrix 2
AFNOR Validation Symposium, June 2008, Brussels
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08.19.06
Relative accuracy
Method 1
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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 clusterIntercept = 0 with P > 5%Slope = 1 with P > 5% No systematic bias, ideal accuracy
AFNOR Validation Symposium, June 2008, Brussels
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08.19.06
Relative accuracy
P% All products n Regression intercept slope 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
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! Minimum 10 samples, x 5 categories
! Priority to naturally contaminated samples
! Slope = 1, Intercept = 0
Discrete cluster, no outlierSlope = 1 with P > 5% Intercept 0 with P < 5%
AFNOR Validation Symposium, June 2008, Brussels
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08.19.06
Relative accuracy
! Minimum 10 samples, x 5 categories
! Priority to naturally contaminated samples
! Slope = 1, Intercept = 0P% All
products n Regression intercept slope 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
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Intercept = 0 with P > 5% ?Slope = 1 with P > 5%Systematic bias ? Tests robustness ?
AFNOR Validation Symposium, June 2008, Brussels
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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!
AFNOR Validation Symposium, June 2008, Brussels
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08.19.06
"Methods comparison study
SpecificitySpecificity && selectivityselectivity
Linearity Linearity
Relative Relative accuracyaccuracy Methods precision
Detection & quantification limits
" Inter-laboratory study
BiasBias
RepeatabilityRepeatability & & Reproducibility limitsReproducibility limits Dispersion between laboratories
Validation study process
#
!
AFNOR Validation Symposium, June 2008, Brussels
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! 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
AFNOR Validation Symposium, June 2008, Brussels
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08.19.06
Robust estimatorsStudent 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 Me
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3 n = 12 + 0,16 significant
Bias
Contamination level
Log CFU/ml Number of labs Bias D Log CFU/ml Conclusion
1 n = 12 - 0,19 non significant
2 n = 12 - 0,02 non significant Me
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3 n = 12 + 0,01 non significant
A non significant bias for each analyte level is expected.Bias = 0,16 Log CFU/gsignificant?
AFNOR Validation Symposium, June 2008, Brussels
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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 nocriteria are available for the rejection of an alternative method
F-table
AFNOR Validation Symposium, June 2008, Brussels
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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!
AFNOR Validation Symposium, June 2008, Brussels
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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.
AFNOR Validation Symposium, June 2008, Brussels
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08.19.06
Conclusions
"reliable tests ?friendly tools needed for users,
i.e. food microbiology laboratories !
The revision is now in progess
AFNOR Validation Symposium, June 2008, Brussels
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08.19.06 [email protected]
Thanks for your attention!
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
AFNOR VALIDATIONEuropean certification for microbiological test kits
19th June 2008, Brussels
More Information on Inter-laboratory-studies
German Institute of Food Technology (DIL)
Dr.-Ing. Helmut SteinkampBusiness Unit Manager
Food Safety Division
Quakenbrck, [email protected]+49 (0)5431 / 183 - 135
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
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 companiesand supports technology transfer from science to business.
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
Concept of DIL
Stuff at DIL:
- 30 scientists
- 60 technician
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
TTZ
DIL
A&FNIZO FHL
IGV
IVV
KIN
BfEL
ATB
LUFAMLUA
TUMUH
UK
FHTFHW
UBFHF
FHK
TUD
FHH
FHBFHN
TUBTFH
TNO
BfR
UM
TUHH
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
DIL in Food Innovation Process
Basic research
Applied research
Development of products/ processes
DistributionWay to market
Food industry
Companies
Service of DIL
Universities / Research Institutions Customer
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
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 managementsystems (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 withfood
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
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
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
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
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
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
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
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
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
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 ?
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
by Bio-Rad
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
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
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
Cultivation Techniques
Well-known and easy to handle Nearly no equipment needed Less expensive Time consuming Results after three days
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
Enrichment in Pepton Water
21h
Enrichment in selektive bouillons
24h
SelektiveMedia
24h
CultivationTechniques(ASU L00.00-20)
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
PCR methodsASU L00.00-52
Enrichment in Peptone Water
21h PCR
3h
Proben-Vorbereitung
results
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
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
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
PCR vs Cultivation Technology
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
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
AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.AFNOR Validation Symposium, June 2008, Brussels
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Deutsches Institut fr Lebensmitteltechnik e. V.
More equipment needed More expensive Higher accuracy
PCR Technique
AFNOR Validation Symposium, June 2008, Brussels
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13M Microbiology validation experiencesSYMPOSIUM BRUSSELS JUNE 19th 2008
EUROPEAN VALIDATION FOR MICROBIOLOGICAL TEST KITS
3M Microbiology Validation experience
Marie-Pierre Copin (Laboratoires 3M Sant : Cergy, France)
AFNOR Validation Symposium, June 2008, Brussels
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2Summary
! Introduction to 3M Company! 3M Microbiology products! 3M Microbiology products validations and recognitions! Some milestones on microbiological method validation in Europe! ISO 16140 in the European regulation on microbiological criteria! Afnor certificates and ISO 16140! Conclusion
AFNOR Validation Symposium, June 2008, Brussels
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3Six Market-Leading Businesses
AFNOR Validation Symposium, June 2008, Brussels
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4Consumer and Office Business
! Sales: $3.4 billion! Operating income: $688 million
Simplifying life at home and at work
AFNOR Validation Symposium, June 2008, Brussels
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5Display and Graphics Business
! Sales: $3.9 billion! Operating income: $1.2 billion
The products of choice in four large and growing industries
AFNOR Validation Symposium, June 2008, Brussels
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6Electro and Communications Business
! Sales: $2.8 billion! Operating income: $481 million
Turning 3M technology into solutions in electrical, electronics and telecommunications markets worldwide
AFNOR Validation Symposium, June 2008, Brussels
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7Industrial and Transportation Business
! Sales: $7.3 billion! Operating income: $1.5 billion
Helping industrial customers solve their problems
AFNOR Validation Symposium, June 2008, Brussels
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8Safety, Security and Protection Services Business
! Sales: $3.1 billion! Operating income: $611 million
Responding to growing demand for safety, security and protection
AFNOR Validation Symposium, June 2008, Brussels
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9Health Care Business
! Sales: $4.0 billion! Operating income: $1.9 billion
Reinvesting in core businesses, pursuing strategic synergies
AFNOR Validation Symposium, June 2008, Brussels
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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
AFNOR Validation Symposium, June 2008, Brussels
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3M Microbiology Mission3M MICROBIOLOGY MISSION: Provide a broad range of innovativesolutions that help improve safety, quality and business results for food & beverage processors, food preparation sites and reference laboratoriesglobally. Our offering will have relevant regulatory acceptance and will bebacked with exceptional customer support.
AFNOR Validation Symposium, June 2008, Brussels
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12
Plastic film
Adhesive + indicator
AdhesivePlastic coated paper printed with a grid
Multi-layer coating technology
Cold water soluble gel
Standard methods nutrients
Until November 2006: Main products range was
3M Petrifilm plates:alternative methods for Hygiene indicators
AFNOR Validation Symposium, June 2008, Brussels
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13
Total Count Enterobacteriaceae
E. coli/Coliform
Yeast and Mold RapidColiform
Coliform
High-Sensitivity Coliform
Staph Express Select E. coli
3M Petrifilm plates: Range of enumeration tests
EnvironmentalListeria
AFNOR Validation Symposium, June 2008, Brussels
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14
Using 3MPetrifilm plates for product analysis
1 Inoculation
2 Incubation
3 - Interpretation
3 steps only ..
AFNOR Validation Symposium, June 2008, Brussels
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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
AFNOR Validation Symposium, June 2008, Brussels
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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
AFNOR Validation Symposium, June 2008, Brussels
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17
Analytical methods
Standardized methodsStandardized 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 resultsequivalent to reference method
?
AFNOR Validation Symposium, June 2008, Brussels
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18
Some steps in the method approval organisation in Europe
! 1987: AFNOR validation committee implementation( 3M representated in the kit manufacturers college)
! 1989: First certificates issued ( 3M Petrifilm plates)
! 1993-1998: Eureka Microval project founded by Europe to get European schemeand rules in Europe(3M Participation in working groups)
! 1998- 2003:CEN group wrote EN 16140 Standard, became then ISO standard! 1998 Microval third party body implementation
(3M representative member of steering committee)! 1999: Nordval system is set-up! 2006: New European Regulation on microbiological criteria, refering to ISO 16140
standard! 2007 First Microval certificate
AFNOR Validation Symposium, June 2008, Brussels
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19
3M Microbiology: Pioneer in 3rd party method approvals Actions
! 1986 :Petrifilm Aerobic count plate and Petrifilm coliform count plate in milk: First alternative methodsrecognized by AOAC int
! 1989: Petrifilm Aerobic count plate and Petrifilm coliform count plate: First methods validated by AFNOR
! 1993: Some Petrifilm plates recognized as NMKL, then switched to NORDVAL validation.
AFNOR Validation Symposium, June 2008, Brussels
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20
Alternative method in the European Regulationon microbiological criteria (EN 2073/2005)
Article 5The use of alternative analytical methods is
acceptable, when the methods are validated against the reference method set down in Annex I * and certified by a third party in accordance with the protocol set in EN/ISO standard 16140 or other internationally accepted similar protocols
AFNOR Validation Symposium, June 2008, Brussels
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21
Analytical methodsEquivalence of methods
Standardized methodsStandardized methods Commercial/Alternative methodsCommercial/Alternative
methods
Validation according to ISO 16140by an independent third party
Validation according to ISO 16140by an independent third party
"
Microval
ISO, CEN
AFNOR Validation Symposium, June 2008, Brussels
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22
Ex Food Safety Criteria (in EN 2073/2005 )Salmonella in fruit and vegetable
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23
Examples of Process hygiene criteria (in EN 2073/2005 )
Aerobic count and E .coli in Meat and Meat products
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24
! Ex of afnor certificate! standard method
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25
Reference to standard method is given in the AFNOR certificate
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26
Validations and recognitions for 3M Petrifilm plates
+Environmental Listeria plate+++Yeast and Mould Count plates++++Staph Express Count plates
++Select E. coli Count plates+++E. coli and Coliform Count plates++High Sensitivity Coliform Count plates++Rapid Coliform Count plates+++Coliform Count plates++++Enterobacteriaceae Count plates+++Aerobic Count plates
RIOMACMMAS
ISO 16140
AFNOR Validation Symposium, June 2008, Brussels
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27
3M TECRA range products validation
AFNORSalmonella 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)
AOAC INTERNATIONAL Official Method of AnalysisSalmonella VIA Method 989.14Salmonella Ultima Method 998.09: Listeria VIA Method 995.22:
AFNOR Validation Symposium, June 2008, Brussels
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28
Conclusion
Validation scheme:! Big progresses to get a unique scheme based on an international standard: ISO
16140In reality: Recognition is more a European one.What about a more common scheme with AOAC?
Third Parties! AFNOR has already certified 63 methods following ISO 16140
MICROVAL issued 5 certificatesAs a manufacturer, we appreciate having the possibility to choose a third partyexpert laboratory to get our methods validated ( system improvement, speed )
AFNOR Validation Symposium, June 2008, Brussels
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Methods validation:A kit manufacturer perspective
Symposium AFNOR VALIDATION 19 June 2008
Dr. Raffaella GiardinoGlobal Marketing ManagerProgram Director Quality indicators
-
AgendaAgendaAgendaAgenda
1. Introduction
2. AFNOR Validation
3. Example EN ISO 16140 for TEMPO! Part A: Method comparison study! Part B: Inter-laboratory study
4. Conclusions
AFNOR Validation Symposium, June 2008, Brussels
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Chromogenic Pathogens Quality Indicators Identification
VITEK2
LIMS connection
DiversiLab
Typing
A Comprehensive Offer for A Comprehensive Offer for A Comprehensive Offer for A Comprehensive Offer for thethethethe
Food Microbiology LabFood Microbiology LabFood Microbiology LabFood Microbiology LabAPI
Traditional methods
ChromIDTM
VIDAS TEMPO
Count-TactTM
air IDEAL
BacT/ALERT 3D
PPM BioBall
ID32
AFNOR Validation Symposium, June 2008, Brussels
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Validation in Food Validation in Food Validation in Food Validation in Food MicrobiologyMicrobiologyMicrobiologyMicrobiology
" Third party assessment of method performances
" Official Authority requirement" Public Health issue" Customers need (external laboratories
and food industry)
AFNOR Validation Symposium, June 2008, Brussels
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bioMerieuxbioMerieuxbioMerieuxbioMerieuxDevelopment ProcessDevelopment ProcessDevelopment ProcessDevelopment Process
1. Feasibility2. Optimisation
3. Internal Verification4. 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 ValidationFood Micro ValidationFood Micro ValidationFood Micro Validation
5. Industrialisation6. External Validation7. Launch authorisation
AFNOR Validation Symposium, June 2008, Brussels
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Food Validations Food Validations Food Validations Food Validations of of of of bMx bMx bMx bMx productsproductsproductsproducts
" 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 bioMrieux
AFNOR Validation Symposium, June 2008, Brussels
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bioMbioMbioMbioMrieux 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
StaphenterotoxinE.coliO157:H7
AFNOR Validation Symposium, June 2008, Brussels
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bioMbioMbioMbioMrieux 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
AFNOR Validation Symposium, June 2008, Brussels
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AgendaAgendaAgendaAgenda
1. Introduction
2. AFNOR Validation
3. Example EN ISO 16140 for TEMPO! Part A: Method comparison study! Part B: Inter-laboratory study
4. Conclusions
AFNOR Validation Symposium, June 2008, Brussels
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21 AFNOR validated methods, and all accordingly all accordingly all accordingly all accordingly the EN ISO 16140 protocolthe EN ISO 16140 protocolthe EN ISO 16140 protocolthe EN ISO 16140 protocol
AFNOR AFNOR AFNOR AFNOR Approved MethodsApproved MethodsApproved MethodsApproved Methods
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1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
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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 methodsQualitative methodsQualitative methodsQualitative methods
AFNOR Validation Symposium, June 2008, Brussels
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VIDAS SLM double voie BIO 12/1-04/94 09/06/2010VIDAS SLM simple voie BIO 12/10-09/02 18/09/2010VIDAS Easy Salmonella BIO 12/16-09/05 20/09/2009VIDAS ICS2-SLM BIO 12/22-05/07 24/05/2011VIDAS ICS2-boite BIO 12/23-05/07 24/05/2011VIDAS Listeria BIO 12/2-06/94 09/06/2010VIDAS LSX BIO 12/12-07/04 01/07/2008VIDAS LMO2 (0.1ml-37C) BIO 12/11-03/04 12/03/2012VIDAS LMO2 (1ml-30C) BIO 12/9-07/02 03/07/2010Accuprobe LMO BIO 12/4-02/95 07/02/2011VIDAS LDUO BIO 12/18-03/06 09/03/2010OAA 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
AFNOR AFNOR AFNOR AFNOR Approved MethodsApproved MethodsApproved MethodsApproved Methods
AFNOR Validation Symposium, June 2008, Brussels
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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 methodsQuantitative methodsQuantitative methodsQuantitative methods
AFNOR Validation Symposium, June 2008, Brussels
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Flore totale TEMPO TVC BIO 12/15-09/05 19/09/2009TEMPO EC BIO 12/13-02/05 04/02/2009Coli ID E. coli 37C BIO 12/19-12/06 14/12/2010Coli ID E. coli 44C BIO 12/5-03/99 19/01/2011TEMPO TC BIO 12/17-12/05 08/12/2009Coli ID coliformes 37C BIO 12/20-12/06 14/12/2010
Entrobactries TEMPO EB BIO 12/2112/06 14/12/2010Listeria monocytogenes OAA dnombrement BIO 12/24-03/08 28/03/2012
Mthodes quantitatives
Escherichia coli
Coliformes
AFNOR AFNOR AFNOR AFNOR approved Methodsapproved Methodsapproved Methodsapproved Methods
AFNOR Validation Symposium, June 2008, Brussels
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Classified by technology:11 VIDAS methods
4 TEMPO methods
5 media methods
1 molecular hybridation method
AFNOR AFNOR AFNOR AFNOR approved Methodsapproved Methodsapproved Methodsapproved Methods
AFNOR Validation Symposium, June 2008, Brussels
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AgendaAgendaAgendaAgenda
1. Introduction
2. AFNOR Validation
3. Example EN ISO 16140 for TEMPO! Part A: Method comparison study! Part B: Inter-laboratory study
4. Conclusions
AFNOR Validation Symposium, June 2008, Brussels
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Validation Protocol Validation Protocol Validation Protocol Validation Protocol ISO 16140ISO 16140ISO 16140ISO 16140
" A. Method comparison study1. Linearity2. Relative Accuracy3. Detection & Quantification limits4. Relative sensitivity5. Inclusivity and Exclusivity6. Practicability
AFNOR Validation Symposium, June 2008, Brussels
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1. Linearity 1. Linearity 1. Linearity 1. Linearity ---- TVCTVCTVCTVC
" TVC: in double at 40h and in double at 48h = 250 results
2.08 4.66troutListeria seeligeri BR1RTE Salmon with pates
2.41 5.28raw milkStaphylococcusaureus 501
Custard cream
2.41 4.93plantXanthomonas maltophilia 11.2
Green beans
2.32 4.95 mechanically separated meat
Bacillus cereus 29Wet cat food
2.30 - 5.04bufbourguignon
Enterobacter agglomerans 72
Porc pt
Range (log)Range (log)Range (log)Range (log)Isolated fromIsolated fromIsolated fromIsolated fromStrainStrainStrainStrainFoodFoodFoodFood
"5 food categories + 5 strains"5 contamination levels to cover the whole range"2 repetitions with TEMPO and Ref method per sample
AFNOR Validation Symposium, June 2008, Brussels
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1. Linearity 1. Linearity 1. Linearity 1. Linearity ---- TCTCTCTC
" 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 goodof TEMPO TVC, TC, EC and EB is goodof TEMPO TVC, TC, EC and EB is goodof TEMPO TVC, TC, EC and EB is good.
AFNOR Validation Symposium, June 2008, Brussels
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2. Relative accuracy2. Relative accuracy2. Relative accuracy2. Relative accuracy
! 5 food categories Ex. TC range (log)o Meat, poultry products 1.00 4.63o Pet-food 1.00 5.43o Dairy products 1.52 5.48o Fruit and vegetables 1.85 6.15o 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
AFNOR Validation Symposium, June 2008, Brussels
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2. Relative accuracy 2. Relative accuracy 2. Relative accuracy 2. Relative accuracy ---- TVCTVCTVCTVC
" 157 different naturally contaminated samples x (2 Ref. + 4 TEMPO) = 942 tests
AFNOR Validation Symposium, June 2008, Brussels
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3. Detection and quantification3. Detection and quantification3. Detection and quantification3. Detection and quantificationlimitslimitslimitslimits
" 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
AFNOR Validation Symposium, June 2008, Brussels
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4. Relative sensitivity4. Relative sensitivity4. Relative sensitivity4. Relative sensitivity
! Example plot from TC
! Use data from the linearity study! Plot the precision profile CV () vs. x(y)
AFNOR Validation Symposium, June 2008, Brussels
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5. 5. 5. 5. Inclusivity Inclusivity Inclusivity Inclusivity and exclusivityand exclusivityand exclusivityand 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 Lactoseneg strains (TC), 2 -glucuronidaseneg 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
AFNOR Validation Symposium, June 2008, Brussels
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5. 5. 5. 5. InclusivityInclusivityInclusivityInclusivity : EC: EC: EC: EC
Conclusion:Conclusion:Conclusion:Conclusion:
Perfect correspondence between TEMPO EC and ISO 16649-2 standard
AFNOR Validation Symposium, June 2008, Brussels
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5. Exclusivity : EC5. Exclusivity : EC5. Exclusivity : EC5. Exclusivity : EC
Conclusion:Conclusion:Conclusion:Conclusion:
No interference by non target strains
AFNOR Validation Symposium, June 2008, Brussels
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6. Practicability6. Practicability6. Practicability6. Practicability
" 13 evaluation criteria (only AFNOR)
! Training duration of an operatorA technician can be fully trained in 2 days
! Traceability of resultsA complete traceability for TEMPO Filler and Reader
! Delay to the resultsTEMPO TVC gives results in 40h compared to 72h of the ref. method, with a saving of more than one day
! QC check and lab maintenanceMonthly use of the kit QC to validate the TEMPO Filler and Reader performances
AFNOR Validation Symposium, June 2008, Brussels
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6. Practicability6. Practicability6. Practicability6. Practicability
" Real hands-on time (work-flow study) in relation to the number of samples to analyse*
4.14.14.14.113.913.913.913.9Time for 1 sampleTime for 1 sampleTime for 1 sampleTime for 1 sample
82278Total time
290Reading
20107Dilution and inoculation
-21Petri dishes preparation
1515Dilution and stomaching
4545Sample Weighing
Method TEMPO TVCMethod TEMPO TVCMethod TEMPO TVCMethod TEMPO TVCMethod ISO 4833Method ISO 4833Method ISO 4833Method ISO 483320 samples20 samples20 samples20 samples
* Media and tubes preparation time non included
AFNOR Validation Symposium, June 2008, Brussels
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Validation Protocol Validation Protocol Validation Protocol Validation Protocol ISO 16140ISO 16140ISO 16140ISO 16140
" B. Inter-laboratory study1. Relative Accuracy2. Repeatability & Reproducibility limits3. Ratio R/r4. Dispersion between laboratories
AFNOR Validation Symposium, June 2008, Brussels
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InterInterInterInter----laboratory studylaboratory studylaboratory studylaboratory study
" ProtocolProtocolProtocolProtocol
! 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
AFNOR Validation Symposium, June 2008, Brussels
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Example TEMPO TCExample TEMPO TCExample TEMPO TCExample TEMPO TC
InterInterInterInter----laboratory studylaboratory studylaboratory studylaboratory study
AFNOR Validation Symposium, June 2008, Brussels
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Example TEMPO TCExample TEMPO TCExample TEMPO TCExample TEMPO TC
InterInterInterInter----laboratory studylaboratory studylaboratory studylaboratory study
Alternative ISO Alternative ISO1.97 0.46 0.23 0.5 0.272.9 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 ISO0.78 0.880.91 1.241.27 1.78
Dispersion between laboratories
EC
Example TEMPO ECExample TEMPO ECExample TEMPO ECExample TEMPO EC
AFNOR Validation Symposium, June 2008, Brussels
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AgendaAgendaAgendaAgenda
1. Introduction
2. AFNOR Validation
3. Example EN ISO 16140 for TEMPO! Part A: Method comparison study! Part B: Inter-laboratory study
4. Conclusions
AFNOR Validation Symposium, June 2008, Brussels
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AFNOR ValidationAFNOR ValidationAFNOR ValidationAFNOR 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
AFNOR Validation Symposium, June 2008, Brussels
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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 ValidationAFNOR ValidationAFNOR ValidationAFNOR Validation
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr1
- Rapid method validation -Experiences from a perspective of a food
manufacturer
AFNOR Validation - 19 June 2008 John D. Marugg,
Nestl Research Centre, Lausanne, Switzerland
AFNOR Validation Symposium, June 2008, Brussels
AFNOR Validation Symposium, 19th of June 2008 Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr2
This presentation
1 Does the food industry need rapid methods?2 Requirements and selection process 3 Nestl validation procedure4 Examples of Nestl/AFNOR approved alternative methods5 Harmonisation and challenges
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr3
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%
Pharmaceuticalproducts
7%
Milk products & Ice cream19%
Powdered & liquid beverages
17%
Nestl Waters10%
Confectionery 11%
PetCare
11%
Nestl Nutrition 8%
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr4
Nestl's Billionaire Brands
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr5
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
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr6
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?
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr7
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.
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr8
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
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr9
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
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr10
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
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr11
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
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr12
Collect information candidate methods
- Supplier- Validation organisations
- Colleague microbiologists- Publications- Other suppliers
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr13
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
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr14
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
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr15
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
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr16
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
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr17
Validation status rapid methods NesVal May 2008
Method Target Throughput Approval/ LI number(s/series) Status
ELISA-based (
-
Nestl Research Center2007-03-20 NRC/QS JMr18
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
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr19
Transia system (Biocontrol)
Transia Plate Salmonella Goldis 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
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr20
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
# AssaysSalmonella, Listeria genus, Listeriamonocytogenes, 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
AFNOR Validation Symposium, June 2008, Brussels
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2007-03-20NQAC Cergy/OGG
NRC/QS JMr 21
Day 1Day 1Day 1Day 1
RapidRapidRapidRapidL. monoL. monoL. monoL. mono
37C, 24 37C, 24 37C, 24 37C, 24 2 hhhh
Suspend 25g or 25 ml sample 1:10
1/2 Fraser brothFraser brothFraser brothFraser broth
30C, 24 30C, 24 30C, 24 30C, 24 2222 hhhh
Day 2Day 2Day 2Day 2
Confirmation(s)Confirmation(s)Confirmation(s)Confirmation(s)Spot on Ottaviani & AgostiSpot on Ottaviani & AgostiSpot on Ottaviani & AgostiSpot on Ottaviani & Agosti
or Gen Probeor Gen Probeor Gen Probeor Gen Probeor othersor othersor othersor others
Day 3Day 3Day 3Day 3
0.1 ml
Palcam + O&APalcam + O&APalcam + O&APalcam + 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 FraserFraserFraserFraser
37C, 24 37C, 24 37C, 24 37C, 24 2 hhhh
Day 4Day 4Day 4Day 4
37C, 48 37C, 48 37C, 48 37C, 48 2 hhhh
Palcam + O&APalcam + O&APalcam + O&APalcam + O&A37C, 24 37C, 24 37C, 24 37C, 24 2 hhhh
ConfirmationConfirmationConfirmationConfirmation
ConfirmationConfirmationConfirmationConfirmationDay 5Day 5Day 5Day 5
EN ISO 11290-1RLM
AFNOR Validation Symposium, June 2008, Brussels
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Nestl Research Center2007-03-20 NRC/QS JMr22
Hygiene indicators: Need for rapid validated methods
! Hygiene indicators are used for product releaseTVC, Enterobacteriaceae, Coliforms, E. coli
! Main available methods:Lab efficiency TAT Calibration ISO16140
Petrifilm + No Yes
MicroFoss + ++ Required No/Bactrac /Yes
Simplate ++ ++ No