NF VALIDATION Validation of alternative analysis methods ... 5.3 C 3.9 C / As a result of transport conditions, 15 laboratories carried out the tests. Two laboratories were not included

NFVALIDATIONValidationofalternativeanalysismethodsApplicationtothefoodindustry

SummaryreportaccordingtotheENISO161402:2016standard

Qualitativemethod

VIDASListeriaDuoforthedetectionofListeriasppandListeriamonocytogenesin

humanfoodproductsandinenvironmentalsamplesCertificate#BIO12/1803/06

Expertlaboratory: For:LaboratoireMICROSEPT bioMrieuxZAdelaSablonnire ChemindelOrme15rueDenisPapin 69280MARCYLETOILE49220LELIONDANGERS FRANCEFRANCE

Thisreportcontains107pages,including72pagesofappendices.Thereproductionofthisdocumentisonlyauthorizedinitsentirety.The accreditation of the COFRAC (Section Laboratory) gives evidence of the expertise of thelaboratoryfortheonlytestscoveredbytheaccreditationthatarespecifiedbythesymbol().

Version0 April20,2018

Preamble

Protocolsofvalidation:

ENISO161401andNFENISO161402(September2016):MicrobiologyofthefoodchainMethodvalidationPart1:Vocabulary.Part 2: Protocol for the validation of alternative (proprietary)methods against areferencemethod.

Requirementsregardingcomparisonandinterlaboratorystudiesforimplementation

ofthestandardENISO161402(version6).

Referencemethod:

ENISO112901(July2017):HorizontalmethodforthedetectionandenumerationofListeriamonocytogenesandofListeriasppPart1:Detectionmethod.

Applicationscope:

Allhumanfoodproductsbyavalidationtestingofabroadrangeoffoods,including:meatproducts,dairyproducts,seafoodproducts,vegetalproducts,compositefoods,

Environmentalsamples.

Certificationbody:

AFNORCertification(https://nfvalidation.afnor.org/).

Microsept Summary report - v0 BIOMERIEUX - VIDAS LDUO

04/2018

2/107

Definitions

MethodcomparisonstudyThemethodcomparisonstudyisthepartofthevalidationprocessthatisperformedintheorganizinglaboratory.Itconsistsofthreepartsnamelythefollowing:Acomparativestudyoftheresultsofthereferencemethodtotheresultsofthealternativemethodin(naturallyand/orartificially)contaminatedsamples(socalledsensitivitystudy);Acomparativestudytodeterminetherelativelevelofdetection(RLOD)inartificiallycontaminatedsamples(socalledRLODstudy);Aninclusivity/exclusivitystudyofthealternativemethod.

Sensitivitystudy

Thesensitivitystudyaimstodeterminethedifferenceinsensitivitybetweenthereferenceandthealternativemethod.Thesensitivityistheabilityofthereferencemethodoralternativemethodtodetecttheanalyte.

Relativelevelofdetectionstudy

Acomparativestudyisconductedtoevaluatethelevelofdetection(LOD)ofthealternativemethodagainstthereferencemethod.Theevaluationisbasedonthecalculationoftherelativelevelofdetection(RLOD).Thelevelofdetectionat50%(LOD50)isthemeasuredanalyteconcentration,obtainedbyagivenmeasurementprocedure,forwhichtheprobabilityofdetectionis50%.TherelativelevelofdetectionlevelofdetectionatP=0,50(LOD50)ofthealternativemethoddividedbythelevelofdetectionatP=0,50(LOD50)ofthereferencemethod.

Inclusivityandexclusivitystudy

Theinclusivitystudyisastudyinvolvingpuretargetstrainstobedetectedorenumeratedbythealternativemethod.Theexclusivitystudyisastudyinvolvingpurenontargetstrains,whichcanbepotentiallycrossreactive,butarenotexpectedtobedetectedorenumeratedbythealternativemethod.

Interlaboratorystudy

Theinterlaboratorystudyisastudyperformedbymultiplelaboratoriestestingidenticalsamplesatthesametime,theresultsofwhichareusedtoestimatealternativemethodperformanceparameters.Theaimoftheinterlaboratorystudyistodeterminethedifferenceinsensitivitybetweenthereferenceandthealternativemethodwhentestedbydifferentcollaboratorsusingidenticalsamples(reproducibilityconditions).


04/2018

3/107

Tableofcontents1. Introduction.................................................................................................................................................6

2. Protocolsofthemethods............................................................................................................................6

2.1. Alternativemethod.............................................................................................................................6

2.1.1. Principleofthemethod...............................................................................................................6

2.1.2. Principleofthemethod...............................................................................................................8

2.1.3. Restrictions..................................................................................................................................8

2.2. Referencemethod...............................................................................................................................8

2.3. Studydesign........................................................................................................................................9

3. Methodscomparisonstudy.......................................................................................................................10

3.1. Sensitivitystudy.................................................................................................................................10

3.1.1. Protocolsappliedduringthevalidationstudy...........................................................................10

3.1.2. Artificialcontamination.............................................................................................................10

3.1.3. DetectionofListeriaspp............................................................................................................11

3.1.3.1. Numberandnatureofthesamples......................................................................................11

3.1.3.2. Results...................................................................................................................................12

3.1.3.3. Calculationofrelativetrueness(RT),sensitivity(SE)andfalsepositiveratio(PFR).............13

3.1.3.4. Analysisofdiscordantresults..................................................................................................1

3.1.3.5. Commentsontestsperformedafter72hoursofstorage......................................................3

3.1.4. DetectionofListeriamonocytogenes..........................................................................................5

3.1.4.1. Numberandnatureofthesamples........................................................................................5

3.1.4.2. Results.....................................................................................................................................5

3.1.4.3. Calculationofrelativetrueness(RT),sensitivity(SE)andfalsepositiveratio(PFR)...............6

3.1.4.4. Analysisofdiscordantresults..................................................................................................1

3.1.4.5. Commentsontestsperformedafter72hoursofstorage......................................................2

3.1.5. Conclusionofthesensitivitystudy..............................................................................................4

3.2. Relativedetectionlevelstudy.............................................................................................................4

3.2.1. Matricesused..............................................................................................................................4

3.2.2. Contaminationprotocol..............................................................................................................4

3.2.2.1. Initialvalidationstudy.............................................................................................................4

3.2.2.2. Thirdrenewalstudy.................................................................................................................5

3.2.3. Results.........................................................................................................................................5

3.2.4. Interpretationandconclusion.....................................................................................................5

3.3. Inclusivityandexclusivitystudy...........................................................................................................6

3.3.1.1. Testprotocols..........................................................................................................................6

3.3.1.2. Resultsandconclusion............................................................................................................6


04/2018

4/107

3.4. Practicability........................................................................................................................................6

3.5. Conclusion...........................................................................................................................................7

4. Interlaboratorystudy..................................................................................................................................8

4.1. Studyorganization...............................................................................................................................8

4.2. Controloftheexperimentalparameters............................................................................................8

4.2.1. Contaminationlevel....................................................................................................................8

4.2.2. Shippingconditions(temperatureandstateofthesamples).....................................................8

4.3. Testresults...........................................................................................................................................9

4.3.1. Expertlaboratoryresults.............................................................................................................9

4.3.2. Collaboratorsresults.................................................................................................................10

4.3.3. Resultsofthecollaboratorsusedforthestatisticalanalysis....................................................11

4.4. Calculationsandinterpretation.........................................................................................................11

4.4.1. Calculationofthespecificity......................................................................................................11

4.4.2. Summaryoftheresults.............................................................................................................11

4.4.3. Calculationofthesensitivityofthemethods,relativetruenessandfalsepositiveratio.........12

4.4.4. Determinationoftheacceptabilitylimitandconclusion..........................................................12

4.4.5. Determinationoftherelativelevelofdetection.......................................................................13

4.5. Conclusion.........................................................................................................................................13

5. Generalconclusion....................................................................................................................................14

AppendicesAppendixA:ProtocolofthealternativemethodAppendixB:ProtocolofthereferencemethodAppendixC:ArtificialcontaminationsAppendixD1:ResultsofthesensitivitystudyinitialvalidationAppendixD2:ResultsofthesensitivitystudyaftercoldstorageofthebrothsinitialvalidationAppendixD3:ResultsofthesensitivitystudythirdrenewalstudyAppendixE1:ResultsoftherelativelevelofdetectionstudyinitialvalidationAppendixE2:ResultsoftherelativelevelofdetectionstudythirdrenewalstudyAppendixF:ResultsoftheselectivitystudyAppendixG:Resultsoftheinterlaboratorystudy


04/2018

5/107

1. IntroductionTheVIDASListeriaDuomethod(VIDASLDUO) isvalidatedbyAFNORCertificationunderthemarkNFVALIDATIONwiththecertificationnumberBIO12/1803/06accordingtotheISO161402:2016standard.Themethodisintendedforallhumanfoodproductsandenvironmentalsamples(exceptprimaryproductionsamples)sinceitsinitialvalidation.Table1summarizesthedifferentstepsofthevalidationthatoccurredsincetheinitialvalidation.Table1:StepsofthevalidationAFNORcertification

Study Date Standard ExpertLaboratory Observation

Initialvalidation March2006 ISO16140:2003 SERMHAIPLNord /

Firstrenewal December2009 ISO16140:2003 EurofinsIPLNord Noadditionaltests

Secondrenewal January2014 ISO16140/A1:2011

InstitutScientifiquedHygineetdAnalyse Noadditionaltests

Thirdrenewal March2018 ISO161402:2016 Microsept

Additionalteststofulfillthe

requirementsofthevalidationstandard

Thepresentdocument introducestheresultsfortheAFNORCertificationvalidationstudiesofthemethodVIDASLDUOaccordingtothestandardISO161402:2016forabroadrangeoffoods.Apartof the results setout in this reportwereproducedduring validation tests carriedoutbySERMHAIPLNordaspartofNFValidation,inaccordancewithprevailingrequirements.The remaining part of the results is constituted by the analyses performed by the LaboratoryMicroseptaspartoftherequirementsoftheupdatedvalidationstandard.

2. Protocolsofthemethods

2.1. Alternativemethod

2.1.1. PrincipleofthemethodVIDASListeriaDuoisanimmunoenzymetestenablingthedetectionoftheListeriamonocytogenesand Listeria antigensusing the ELFA (Enzyme Linked FluorescentAssay)methodwith theVIDASautomatedsystem.Eachtestisbrokendownintotwocomponents:


04/2018

6/107

ThesingleuseSPRusedbothforthesolidphaseandasapipettingsystemforthetest.Theinside of the SPR is coatedwith antiListeriamonocytogenes antibodies and antiListeriaantibodiesadsorbedonitssurface.

Thestrip(seefigure1below)whichcontainsallofthereadytousereagentsrequiredforthetest:washing solution,antiListeriamonocytogenesantibodiesandantiListeriaantibodiesconjugatedwithalkalinephosphataseandsubstrate.

Figure1:ComponentsofaVIDASstrip

AllstepsareperformedautomaticallybytheVIDASanalyticalmodule.Analiquotofenrichmentbrothisplacedinthestripandundergoesasuction/dischargecyclewhosedurationisspecificallycalculatedtoactivatethereaction.Thus,thesystemperformstworevelationstagesandtworeadings:

thefirstreadingprovidesaresponseforListeriamonocytogenes(DLMO), thesecondreadingprovidesaresponseforListeriaspp(DLIS).

ThefluorescenceintensityismeasuredbytheVIDASopticalsystemat450nmandexpressedasarelativefluorescencevalue(RFV),interpretedbytheVIDASsystemasfollows.ThetestvaluescalculatedusingthefollowingformulaaresubsequentlycomparedtothethresholdvaluesdefinedforListeriamonocytogenesandforListeriaspp.

Testvalue(TV)=sampleRFV/standardRFV.ResponseforListeriamonocytogenes(DLMO) IfTV

Table2:TypesofresponseoftheVIDASLDUOtest

Case DLMO DLMOtestvalue DLISDLIStestvalue

Response(subjecttoconfirmation)

Case1 + Available +bydefaultNot

availablePresenceofListeriamonocytogenes

andbydefault,presenceofListeriaspp

Case2 Available + AvailableAbsenceofListeriamonocytogenes&PresenceofListeriaspp.otherthan

Listeriamonocytogenes

Case3 Available Available AbsenceofListeriaspp.,includingAbsenceofListeriamonocytogenes

2.1.2. PrincipleofthemethodTheprotocolisasfollows:

enrichmentinLXbroth,incubatedfor22to26hoursat30C1C, subcultureinLXbroth(0.1mlin6ml),incubatedfor22to26hoursat30C1C

TheworkflowofthemethodissetoutinAppendixA.TheVIDASLDUOtestisthenperformedusinganaliquotofLXheatedfor5minutes1minuteat95100C.Two responsesareprovided:presenceorabsenceofListeriamonocytogenes (DLMO)andpresenceorabsenceofListeriaspp.(DLIS).ThesamplesfoundtobepositiveusingtheVIDASLDUOtestareconfirmedbyisolatingthetubeofunheatedLXbrothonselectiveagarenablingthedevelopmentofallListeriaspecies(Palcam,Oxfordorchromogenicagar).The colonies characteristic of Listeria spp. are subsequently confirmed using conventionalbiochemicaltests.Inaddition,

intheeventofapositiveresponseforListeriamonocytogenes,asecondconfirmationoptionisoffered: in theevent that characteristic coloniesarepresenton chromogenicagar, theVIDASDLMOresultforListeriamonocytogenesisconsideredasconfirmed,

intheeventofapositiveresponseforListeriaspp.otherthanListeriamonocytogenes,thecoloniescharacteristicofListeriaareconfirmedusingconventionalbiochemicaltestsorusinganAPIteststripwithoutpriorpurificationifthecolonyissufficientlyisolated.

2.1.3. Restrictions

TherearenorestrictionsonuseforthemethodVIDASListeriaDuo.

2.2. ReferencemethodThe standard EN ISO 112901/A1:2005wasused for the initial validation study and for the twofollowingrenewalstudies.This standard was revised in 2017. Themain changes introduced in the ISO112901:2017 areconsideredasmajor.Thetechnicalchangeswereassessedandwereconsideredtohavenosignificanteffectonthemethodperformancecharacteristicsortestresults.


04/2018

8/107

ThatswhythemethoddescribedinthenewstandardENISO112901:2017HorizontalmethodforthedetectionandenumerationofListeriamonocytogenesandofListeriasppPart1:detectionmethodwasusedasreferencemethodforthesupplementarytestsperformedbytheLaboratoryMicrosept.TheworkflowofthereferencemethodispresentedinAppendixB.

2.3. StudydesignAsthereisnosharedenrichmentstepforboththealternativeandthereferencemethods,differenttestportionscomingfromthesamebatchorlotofproducthavetobeusedforthetwomethods.Thestudythusprovidesunpaireddataandthewordunpairedstudyisusedtodescribethestudydesign.


04/2018

9/107

3. MethodscomparisonstudyThestudywasconductedonavarietyofsamplesandstrainsrepresentativeoffoodproducts.Thisisnotanexhaustivelistofthevariousmatricesincludedintheapplicationscope.Foranyremarkonthealternativemethod,youcancontactAFNORCertificationbyconnecting to the Internetpagehttp://nfvalidation.afnor.org/contact2/.

3.1. SensitivitystudyThepurposeofthisstudyistocomparethetwomethodsthereferencemethodISO112901andthe VIDAS LDUO method on samples contaminated or not contaminated with ListeriamonocytogenesandListeriasppotherthanL.monocytogenes.

3.1.1. Protocolsappliedduringthevalidationstudy Incubation time: theminimum incubation times were tested, namely 22 hours for the

enrichmentinLXbrothand22hoursforthesubcultureinLXbroth Confirmation:presumptivepositiveresultswereconfirmedbystreaking10lofthesecond

enrichedLXbrothonanOttaviani&AgostiandonaPALCAMplate.Typicalcolonieswereconfirmed by the realization of a biochemical gallery without purification and by theimplementationofthetestsdescribedinthereferencemethodafterpurification.

ThetubesofLXbrothwerekeptfor72hoursat2C8CandthentestedagainusingVIDASandconfirmediftheVIDASLDUOtestwaspositive,inordertodocumenttheimpactofstoringthebrothsforupto72hoursat2C8C.

3.1.2. Artificialcontamination

ArtificialcontaminationwascarriedoutusingstressedstrainsinaccordancewiththerequirementsofthevalidationstandardandtheAFNORValidationTechnicalBureau(seeAppendixC).Table3givesthedistributionofthepositivesamplespertypeandlevelofcontamination.Table3:distributionofthepositivesamplespercontaminationtypeandlevel(LIS:parameterListeriaspp,LMO:parameterListeriamonocytogenes,cl:contaminationlevel)

Positivesamples

Naturallycontaminated

samples

ArtificiallycontaminatedsamplesTotalSpiking Seeding

cl5 5

Theyconcern:forListeriaspp: forL.monocytogenes:5samplescontaminatedatanundeterminedlevel 2samplescontaminatedabove

10CFU/testportion5samplescontaminatedbetween5and10CFU/testportion18samplescontaminatedabove10CFU/testportion

3.1.3. DetectionofListeriaspp

3.1.3.1. NumberandnatureofthesamplesThe sensitivity study for all categories concerned 530 samples: 438 analyzed during the initialvalidationstudyand92analyzedduringthisthirdrenewalstudy.Samplesanalyzedbycategoryandtypearepresentedintable4.Table4:Distributionofthesamplespercategoryandtype

Categories TypesPositiveresults Negative

results TotalLMO LIS LMO+LIS All

Meatproducts

a Rawmeatproducts 7 11 10 28 10 38

bRTRHproductsandmeatbasedproducts 7 1 4 12 10 22

c Delicatessen 7 8 16 31 15 46Total 21 20 30 71 35 106

Dairyproducts

a Cowrawmilkcheese 12 6 2 20 15 35bGoatorewerawmilkcheese 4 1 5 10 11 21c Othermilkproducts 6 7 3 16 15 31

Total 22 14 10 46 41 87

Seafoodproducts

a Rawproducts 9 11 3 23 18 41bSmoked,marinatedproducts 16 2 0 18 10 28

c Readytoeatandreadytoreheatproducts 10 1 0 11 10 21

Total 35 14 3 52 38 90

Vegetalproducts

a Rawvegetalproducts 8 4 0 12 22 34bPrecutfruitsandvegetables 8 2 6 16 10 26c Plantproductbasedfood 8 8 3 19 10 29

Total 24 14 9 47 42 89

Compositefoods

a Readytoeatproducts 7 3 2 12 10 22bReadytoreheatproducts 9 4 2 15 10 25c Pastriesandderivated,eggproducts 3 9 2 14 10 24

Total 19 16 6 41 30 71

Environmentalsamples

a Processandcleaningwaters 5 3 2 10 17 27bSurfacesamples 8 8 4 20 18 38c Residues 10 1 1 12 10 22

Total 23 12 7 42 45 87Total 144 90 65 299 231 530


04/2018

11/107

For the parameter Listeria spp, the proportions of Listeria spp (only or mixed with L.monocytogenes) and of L. monocytogenes among the positive samples for all categories arepresentedbelow:Table5:proportionsofListeriasppandofListeriamonocytogenesamongthepositivesamples

CategoryListeriaspponly(A)

Listeriaspp+L.monocytogenes(B)

TotalA+B L.monocytogenesonlyTotal

positivesamples# % # % # % # %

Meatproducts 20 28,2% 30 42,3% 50 70,4% 21 29,6% 71Dairyproducts 14 30,4% 10 21,7% 24 52,2% 22 47,8% 46

Seafoodproducts 14 26,9% 3 5,8% 17 32,7% 35 67,3% 52

Vegetalproducts 14 29,8% 9 19,1% 23 48,9% 24 51,1% 47Compositefoods 16 39,0% 6 14,6% 22 53,7% 19 46,3% 41Environmental

samples 12 28,6% 7 16,7% 19 45,2% 23 54,8% 42

All 90 30,1% 65 21,7% 155 51,8% 144 48,2% 299AccordingtotheRequirementsregardingcomparisonandinterlaboratorystudiesforimplementationof the standard EN ISO161402, v6, for Listeria genus studies, complianceper categorywith aproportionofatleast15to25Listeriasppcontaminatedsamples(aloneorcombinedwithListeriamonocytogenes)isrequested.Thisrequirementisfulfilledforeachcategory.

3.1.3.2. ResultsRawdataareshowninappendicesD1andD2fortheinitialvalidationstudyandinappendixD3fortherenewalstudy.Table6showstheresultsofthesensitivitystudyforallcategories.Table6:Distributionof thesamplespercategoryand type (PA:positiveagreement,NA:negativeagreement, ND: negative deviation, PD: positive deviation, PP: presumptive positive beforeconfirmation)

Category PA NA PD ND PPND PPNA Total1 Meatproducts 64 35 4 3 0 0 1062 Dairyproducts 37 41 7 2 0 0 873 Seafoodproducts 42 38 6 4 0 0 904 Vegetalproducts 40 42 3 4 0 0 895 Compositefoods 31 30 5 5 0 0 716 Environmentalsamples 35 45 6 1 0 0 87

Total 249 231 31 19 0 0 530


04/2018

12/107

3.1.3.3. Calculationofrelativetrueness(RT),sensitivity(SE)andfalsepositiveratio(PFR)

Thesetofresultsobtainedwereusedtocalculatetherelativetrueness,thesensitivityandthefalsepositiveratioforeachofthecategoriesandforallthecategories,accordingtotheformulassetoutintheENISO161402:2016standard(table7).


04/2018

13/107

Table7:valuesin%ofsensitivityforthetwomethods,relativetruenessandfalsepositiveratioforthealternativemethod(SEalt:sensitivityforthealternativemethod,SEref:sensitivityforthereferencemethod,RT:relativetrueness,FPR:falsepositiveratioforthealternativemethod)

Category Type PA NA ND PD N PPND PPNA SEalt SEref RT FPR

Meatproducts

a 26 10 0 2 38 0 0 100% 92,9% 94,7% 0%b 10 10 2 0 22 0 0 83,3% 100% 90,9% 0%c 28 15 1 2 46 0 0 96,8% 93,5% 93,5% 0%

Total 64 35 3 4 106 0 0 95,8% 94,4% 93,4% 0%

Dairyproducts

a 16 15 1 3 35 0 0 95,0% 85,0% 88,6% 0%b 9 11 0 1 21 0 0 100% 90,0% 95,2% 0%c 12 15 1 3 31 0 0 93,8% 81,3% 87,1% 0%

Total 37 41 2 7 87 0 0 95,7% 84,8% 89,7% 0%

Seafoodproducts

a 17 18 3 3 41 0 0 87,0% 87,0% 85,4% 0%b 14 10 1 3 28 0 0 94,4% 83,3% 85,7% 0%c 11 10 0 0 21 0 0 100% 100% 100% 0%

Total 42 38 4 6 90 0 0 92,3% 88,5% 88,9% 0%

Vegetalproducts

a 7 22 3 2 34 0 0 75,0% 83,3% 85,3% 0%b 16 10 0 0 26 0 0 100% 100% 100% 0%c 17 10 1 1 29 0 0 94,7% 94,7% 93,1% 0%

Total 40 42 4 3 89 0 0 91,5% 93,6% 92,1% 0%

Compositefoods

a 9 10 2 1 22 0 0 83,3% 91,7% 86,4% 0%b 11 10 1 3 25 0 0 93,3% 80,0% 84,0% 0%c 11 10 2 1 24 0 0 85,7% 92,9% 87,5% 0%

Total 31 30 5 5 71 0 0 87,8% 87,8% 85,9% 0%


a 8 17 0 2 27 0 0 100% 80,0% 92,6% 0%b 17 18 1 2 38 0 0 95,0% 90,0% 92,1% 0%c 10 10 0 2 22 0 0 100% 83,3% 90,9% 0%

Total 35 45 1 6 87 0 0 97,6% 85,7% 92,0% 0%

Allcategories Total 249 231 19 31 530 0 0 93,6% 89,6% 90,6% 0%


04/2018

14/107

Theresultsforallcategoriesaresummarizedinthetable8below.Table8:summaryoftheresultsforallcategories

Parameter FormulaENISO161402:2016 Resultsforallcategories

Sensitivityofthealternativemethod(SEalt)

93.6%

Sensitivityofthereferencemethod(SEref)

89.6%

Relativetrueness(RT)

90.6%

Falsepositiveratio(FPR)

0%

3.1.3.4. Analysisofdiscordantresults

DiscordantresultsareexaminedaccordingtothestandardISO161402:2016.Table9:summaryofthediscordantresults

Categories TypesInitialvalidation Thirdrenewal

Positivedeviations

Negativedeviations

Positivedeviations Negativedeviations

Meatproducts

abc

D7,T15

V10,C19

I10,L1L3

Dairyproducts

abc

C7,D14,H1,C10,

J19,J14,J17

L6

J20

Fishproductabc

G8,I36,S5,I37,I39,U3,

M13,M15,M16,S3

Vegetalproducts

abc

L1251

Q18,S8,S12

U9

1372306

1398372

Compositefoods

abc

Q10J28

C13

13723031370081/1372307

1372305/ 140926513983621398357

Environmentabc

H7,H13,G27,J1,I32,I43

O3

All deviations come from the nature of the study design. In an unpaired study, because of thedifferenceofsamplingbetweenbothmethods,andtheuseofnaturallycontaminatedsamplesorseededsampleswithlowlevelsofcontamination,nocellofL.monocytogenesmayhavebeenpresentinthesamplingofoneofthetwomethods.


04/2018

15/107

Forsample1398368,artificiallycontaminatedwithastrainofListeria ivanovii,anegativeresult isobtained by the alternativemethod.However the confirmation protocol allowed finding typicalcolonieswhichwereconfirmedasListeriaivanovii.Theresultofthereferencemethodisnegativeforthissample.Thecomparisonofthetwomethodsleadsthereforetoanegativeagreement.Its important tonote that the same strainofL. ivanoviiwasusedat the sameconcentration tocontaminatethesample1398355andthattheresultofthealternativemethodwasclearlypositiveandconfirmed.AnotherstrainofL.ivanoviiwasusedcombinedwithastrainofL.monocytogenestocontaminatethesample1409270.Theresultofthealternativewaspositiveandthetwospecieswerefoundduringtheconfirmation.Table 10 shows the difference between negative deviations and positive deviations and theacceptabilitylimits.Table10:acceptabilitylimits

Category Type ND PD (NDPD) Acceptabilitylimit(AL) Observation

Meatproducts

a 0 2/ /

(NDPD)AL:

b 2 0c 1 2

Total 3 4 1 3

Dairyproducts

a 1 3/ /b 0 1

c 1 3Total 2 7 5 3

Seafoodproducts

a 3 3/ /b 1 3

c 0 0Total 4 6 2 3

Vegetalproducts

a 3 2/ /b 0 0

c 1 1Total 4 3 1 3

Compositefoods

a 2 1/ /b 1 3

c 2 1Total 5 5 0 3


a 0 2/ /b 1 2

c 0 2Total 1 6 5 3

Allcategories Total 19 31 12 6

The observed values (ND PD) are below the acceptability limit for each category and for allcategories.Thealternativemethodproducesresultscomparabletothereferencemethod.


04/2018

16/107

3.1.3.5. Commentsontestsperformedafter72hoursofstorageTheLXbrothsweretestedusingtheVIDASLDUOTEST,immediatelyafterincubation,thenstoredfor3daysat2C8Candretested.Inaddition,LXbrothwasisolatedonselectiveagarandconfirmationtestswereperformed.SomediscordancesbetweenthetworesultsappearedduringtheVIDASLDUOtestperformedusingLXbrothstoredfor72hoursat2C8C:

7 samples: L11 (ice cream), H14 (residues), I19 (beefsteak), K6 (porc ribs), T15 (tomatoburger),V2 (catalanpoellee),V16 (tomatoburger,presented adiscordancebetween thespecifictestresultandtheidentificationresult(positiveDLMOtestresultwithidentificationofaListeriastrainotherthanL.monocytogenes).

3 samples:C11 rawmilk cheese, E4 soft cheesewith chocolate,R5Herring fillet, gave apositive result with the DLIS test but no Listeria was observed on selective agars. Thereferencemethodgaveanegativeresultforthese3samples.Thereareconsequentlyfalsepositiveresultsafterthestorageofthebroths.

a samplewhich gave a negative deviationwith the initial test (M13, fish fillet) becameconcordantfollowingstorageoftheLXbrothat2C8C.

Theseresultsaresummarizedbelow:

Samples Resultsattheendofincubation Resultsafterstorageat28CL11,H14,I19,K6,

V2,V16DLIS:+

Confirmation:+(PA)DLMO:+

Confirmation:presenceofListeriasppotherthanL.monocytogenes(PA)

T15 DLIS:+Confirmation:+(PD)

DLMO:+Confirmation:presenceofListeriasppotherthanL.monocytogenes(PA))

C11,E4,R5 DLIS:Confirmation:(NA)

DLIS:+Confirmation:(NA/FP)

M13 DLIS:(ND) DLIS:+Confirmation:+(PA)

TheresultsoftheVIDASLDUOtestsperformedontheLXbrothsstoredfor72hoursat2C8CarethereforegloballyequivalenttothoseobtainedwhentheVIDASLDUOtestiscarriedoutdirectlyafterincubation.Table 11 shows the difference between negative deviations and positive deviations and theacceptabilitylimits.


04/2018

17/107

Table11:acceptabilitylimits


Meatproducts

a 0 2/ /

(NDPD)AL:

b 2 0c 1 2

Total 3 4 1 3

Dairyproducts

a 1 3/ /b 0 1

c 1 3Total 2 7 5 3

Seafoodproducts

a 2 3/ /b 1 3

c 0 0Total 3 6 3 3

Vegetalproducts

a 3 2/ /b 0 0

c 1 1Total 4 3 1 3

Compositefoods

a 2 1/ /b 1 3

c 2 1Total 5 5 0 3


a 0 2/ /b 1 2

c 0 2Total 1 6 5 3

Allcategories Total 18 31 13 6

The observed values (ND PD) are below the acceptability limit for each category and for allcategoriesafterstorageofthebroths.Thealternativemethodproducesresultscomparabletothereferencemethod.


04/2018

18/107

3.1.4. DetectionofListeriamonocytogenes

3.1.4.1. NumberandnatureofthesamplesThe sensitivity study for all categories concerned 543 samples: 464 analyzed during the initialvalidationstudyand79analyzedduringthisthirdrenewalstudy.Samplesanalyzedbycategoryandtypearepresentedintable12.Table12:Distributionofthesamplespercategoryandtype

Categories Types PositiveresultsNegativeresults Total

Meatproducts

a Rawmeatproducts 17 21 38b RTRHproductsandmeatbasedproducts 10 12 22c Delicatessen 23 24 47

Total 50 57 107

Dairyproducts

a Cowrawmilkcheese 14 24 38b Goatorewerawmilkcheese 10 12 22c Othermilkproducts 10 21 31

Total 34 57 91

Seafoodproducts

a Rawproducts 12 29 41b Smoked,marinatedproducts 16 10 26c Readytoeatandreadytoreheatproducts 10 10 20

Total 38 49 87

Vegetalproducts

a Rawvegetalproducts 10 28 38b Precutfruitsandvegetables 15 13 28c Plantproductbasedfood 11 18 29

Total 36 59 95

Compositefoods

a Readytoeatproducts 10 10 20b Readytoreheatproducts 11 10 21c Pastriesandderivated,eggproducts 10 13 23

Total 31 33 64


a Processandcleaningwaters 10 21 31b Surfacesamples 14 32 46c Residues 12 10 22

Total 36 63 99Total 225 318 543

3.1.4.2. Results

RawdataareshowninappendicesD1andD2fortheinitialvalidationstudyandinappendixD3fortherenewalstudy.Table13showstheresultsofthesensitivitystudyforallcategories.


04/2018

19/107

Table13:Distributionofthesamplespercategoryandtype(PA:positiveagreement,NA:negativeagreement, ND: negative deviation, PD: positive deviation, PP: presumptive positive beforeconfirmation)

Category PA NA PD ND PPND PPNA Total1 Meatproducts 44 57 5 1 0 0 1072 Dairyproducts 30 57 4 0 0 0 913 Seafoodproducts 33 49 5 0 0 0 874 Vegetalproducts 29 59 4 3 0 0 955 Compositefoods 24 33 5 2 0 0 646 Environmentalsamples 34 63 1 1 0 0 99

Total 194 318 24 7 0 0 543

3.1.4.3. Calculationofrelativetrueness(RT),sensitivity(SE)andfalsepositiveratio(PFR)

Thesetofresultsobtainedwereusedtocalculatetherelativetrueness,thesensitivityandthefalsepositiveratioforeachofthecategoriesandforallthecategories,accordingtotheformulassetoutintheENISO161402:2016standard(table14).


04/2018

20/107

Table14:valuesin%ofsensitivityforthetwomethods,relativetruenessandfalsepositiveratioforthealternativemethod(SEalt:sensitivityforthealternativemethod,SEref:sensitivityforthereferencemethod,RT:relativetrueness,FPR:falsepositiveratioforthealternativemethod)

Category Type PA NA ND PD N PPND PPNA SEalt SEref RT FPR

Meatproducts

a 15 21 0 2 38 0 0 100% 88,2% 94,7% 0,0%b 9 12 1 0 22 0 0 90,0% 100% 95,5% 0,0%c 20 24 0 3 47 0 0 100% 87,0% 93,6% 0,0%

Total 44 57 1 5 107 0 0 98,0% 90,0% 94,4% 0,0%

Dairyproducts

a 14 24 0 0 38 0 0 100% 100% 100% 0,0%b 8 12 0 2 22 0 0 100% 80,0% 90,9% 0,0%c 8 21 0 2 31 0 0 100% 80,0% 93,5% 0,0%

Total 30 57 0 4 91 0 0 100% 88,2% 95,6% 0,0%

Seafoodproducts

a 9 29 0 3 41 0 0 100% 75,0% 92,7% 0,0%b 14 10 0 2 26 0 0 100% 87,5% 92,3% 0,0%c 10 10 0 0 20 0 0 100% 100% 100% 0,0%

Total 33 49 0 5 87 0 0 100% 86,8% 94,3% 0,0%

Vegetalproducts

a 5 28 3 2 38 0 0 70,0% 80,0% 86,8% 0,0%b 14 13 0 1 28 0 0 100% 93,3% 96,4% 0,0%c 10 18 0 1 29 0 0 100% 90,9% 96,6% 0,0%

Total 29 59 3 4 95 0 0 91,7% 88,9% 92,6% 0,0%

Compositefoods

a 6 10 1 3 20 0 0 90,0% 70,0% 80,0% 0,0%b 8 10 1 2 21 0 0 90,9% 81,8% 85,7% 0,0%c 10 13 0 0 23 0 0 100% 100% 100% 0,0%

Total 24 33 2 5 64 0 0 93,5% 83,9% 89,1% 0,0%


a 10 21 0 0 31 0 0 100% 100% 100% 0,0%b 13 32 1 0 46 0 0 93% 100% 98% 0,0%c 11 10 0 1 22 0 0 100% 91,7% 95,5% 0,0%

Total 34 63 1 1 99 0 0 97% 97,2% 98,0% 0,0%Allcategories Total 194 318 7 24 543 0 0 96,9% 89,3% 94,3% 0,0%


04/2018

21/107

Theresultsforallcategoriesaresummarizedinthetable15below.Table15:summaryoftheresultsforallcategories

Parameter FormulaENISO161402:2016 Resultsforallcategories

Sensitivityofthealternativemethod(SEalt)

96.9%

Sensitivityofthereferencemethod(SEref)

89.3%

Relativetrueness(RT)

94.3%

Falsepositiveratio(FPR)

0%

3.1.4.4. Analysisofdiscordantresults

DiscordantresultsareexaminedaccordingtothestandardISO161402:2016.Table16:summaryofthediscordantresults

Categories TypesInitialvalidation Thirdrenewal

Positivedeviations

Negativedeviations

Positivedeviations Negativedeviations

Meatproducts

abc

D7,V14

C19,V10,V11,

I10

Dairyproducts

abc

B16,C10J14,J17

/

Fishproductabc

G8,I36,U1I37,I39

/

Vegetalproducts

abc

L1251C1

Q18,S8,S12

1372306

1398372

Compositefoods

abc

1372303/14092711409275

1370081/1372307

1372305

1398362

Environmentabc

I43

O3

All deviations come from the nature of the study design. In an unpaired study, because of thedifferenceofsamplingbetweenbothmethods,andtheuseofnaturallycontaminatedsamplesor


04/2018

22/107

seededsampleswithlowlevelsofcontamination,nocellofL.monocytogenesmayhavebeenpresentinthesamplingofoneofthetwomethods.Table 17 shows the difference between negative deviations and positive deviations and theacceptabilitylimits.Table17:acceptabilitylimits


Meatproducts

a 0 2/ /

(NDPD)AL:

b 1 0c 0 3

Total 1 5 4 3

Dairyproducts

a 0 0/ /b 0 2

c 0 2Total 0 4 4 3

Seafoodproducts

a 0 3/ /b 0 2

c 0 0Total 0 5 5 3

Vegetalproducts

a 3 2/ /b 0 1

c 0 1Total 3 4 1 3

Compositefoods

a 1 3/ /b 1 2

c 0 0Total 2 5 3 3


a 0 0/ /b 1 0

c 0 1Total 1 1 0 3

Allcategories Total 7 24 17 6The observed values (ND PD) are below the acceptability limit for each category and for allcategories.Thealternativemethodproducesresultscomparabletothereferencemethod.

3.1.4.5. Commentsontestsperformedafter72hoursofstorageTheLXbrothsweretestedusingtheVIDASLDUOTEST,immediatelyafterincubation,thenstoredfor3daysat2C8Candretested.Inaddition,LXbrothwasisolatedonselectiveagarandconfirmationtestswereperformed.SomediscordancesbetweenthetworesultsappearedduringtheVIDASLDUOtestperformedusingLXbrothstoredfor72hoursat2C8C:


04/2018

23/107

7 samples: L11 (ice cream), H14 (residues), I19 (beefsteak), K6 (porc ribs), T15 (tomatoburger),V2 (catalanpoellee),V16 (tomatoburger,presented adiscordancebetween thespecifictestresult(positiveDLIStestresultwithidentificationofaListeriastrainotherthanL.monocytogenes)andtheidentificationresult(positiveDLMOtestresultwithidentificationofaListeriastrainotherthanL.monocytogenes).FortheresponseListeriamonocytogenes,theyarestillnegativedeviationsagreementsbuttheyareconsideredasfalsepositiveresults.

TheresultsoftheVIDASLDUOtestsperformedontheLXbrothsstoredfor72hoursat2C8CarethereforegloballyequivalenttothoseobtainedwhentheVIDASLDUOtestiscarriedoutdirectlyafterincubation.Table 18 shows the difference between negative deviations and positive deviations and theacceptabilitylimits.Table18:acceptabilitylimits


Meatproducts

a 0 2/ /

(NDPD)AL:

b 1 0c 0 3

Total 1 5 4 3

Dairyproducts

a 0 0/ /b 0 2

c 0 2Total 0 4 4 3

Seafoodproducts

a 0 3/ /b 0 2

c 0 0Total 0 5 5 3

Vegetalproducts

a 3 2/ /b 0 1

c 0 1Total 3 4 1 3

Compositefoods

a 1 3/ /b 1 2

c 0 0Total 2 5 3 3


a 0 0/ /b 1 0

c 0 1Total 1 1 0 3

Allcategories Total 7 24 17 6TheacceptabilitylimitsarenotmodifiedfortheresponseListeriamonocytogenes.Thealternativemethodproducesresultscomparabletothereferencemethodafterstorageofthebrothsfor3daysat53C.


04/2018

24/107

3.1.5. ConclusionofthesensitivitystudyThe statistical tests of the EN ISO 161402:2016 standard conclude that the alternativemethodproducescomparableresultstothereferencemethod.However,thesensitivityresultsshowedthatthealternativemethodbrothallowsabetterrecoveryofListeriaspp(includingListeriamonocytogenes)thanthereferencemethodbroth,thedifferenceofthedeviationsbeingclearlyinfavorofthealternativemethod.

3.2. Relativedetectionlevelstudy

3.2.1. MatricesusedVarious"foodmatrixstrain"pairswerestudiedinparallelusingthereferencemethodandtheVIDASLDUOmethod,forthecategoriesinquestion(cf.table19).Table19:couplesmatrixstrainforeachcategory

Category Couplematrixstrain OriginofthestrainStepofthevalidation

Meatproducts Rillettes/L.welshimeri Mincedmeat

Initialvalidationstudyaccording

toISO16140:2003standard

Dairyproducts Rawmilk/L.monocytogenes1/2b RawmilkcheeseRawmilk/L.innocua RawmilkcheeseSeafoodproducts

Smoked salmon / L. monocytogenes1/2a

Smokedsalmonoffcuts

Vegetalproducts

Redcabbage/L.monocytogenes4b Salad


Processwater/L.monocytogenes1/2c Surfacesample

Compositefoods Pastrycream/L.seeligeri

Pastrycreamfilledcake

3rdrenewalstudyaccordingto

ISO161402:2016standard

Thetotalfloraofthematrixwasdeterminedand issetout intheresultstables inappendicesE1andE2.

3.2.2. Contaminationprotocol

3.2.2.1. InitialvalidationstudyAt least four contamination levels, including the negative control,were performed. Each of the"matrixstrain level"combinationswas replicatedsix timesusing theVIDASLDUOalternativemethodandtheISO112901/A1referencemethod.As the firstenrichment stage isnotcommon, twelve25gbagsof foodproductsweremadeup,diluted to 1/10 in the appropriate diluent, then individually contaminated using a bacterialsuspensionwiththedeterminedtiter.Eachcontaminantsuspensionwasenumeratedon30platesofTSAYEagar.


04/2018

25/107

3.2.2.2. ThirdrenewalstudyThreelevelsofcontaminationwerepreparedconsistingofanegativecontrollevel,alowlevel,andahigherlevel.Thenegativecontrollevelshallnotproducepositiveresults.Fivereplicatesweretestedforthislevel.The low levelshallbethetheoreticaldetection level, itwascontaminatedat0.7 1CFUpertestportiontoobtainfractionalrecoveryresults.Twentyreplicatesweretestedforthislevel.Thehigherlevelshallbejustabovethetheoreticaldetectionlevel,itwascontaminatedat23CFUpertestportion.Fivereplicatesweretestedforthislevel.The pastry cream was contaminated using the seeding protocol. Bulk contaminations wereperformedonthematrix forthedifferent levelsofcontamination,thenthematrixwasstoredat53Cfortwodaysbeforeanalysis.Sampleswerethenanalyzedbythereferenceandthealternativemethod.

3.2.3. ResultsThedetailedresultstablesaresetoutinAppendicesE1andE2.TheRLODisdefinedastheratiooftheLODsofthealternativemethodandthereferencemethod:RLOD=LODalt/LODref.TheRLODscalculationswereperformedaccordingtothestandardISO161402:2016usingtheExcelspreadsheet available for download at http://standards.iso.org/iso/16140, with unknownconcentrations.ValuesoftheRLODsaresetoutintable20.Table 20:RLODs values for all categories (RLOD: the estimated relative level of detection value,RLODU:theupperlimitofthe95%confidenceintervalforRLOD,RLODL:thelowerlimitofthe95%confidenceintervalforRLOD,b=ln(RLOD):logarithmoftheRLODvalue,sd(b):standarddeviationofb,zTeststatistic:absolutevalueoftheteststatisticofthezTestwiththenullhypothesisH0:b=0,pvalue:pvalueofthezTest)

Name RLOD RLODL RLODU b=ln(RLOD) sd(b) zTeststatistic pvalue

Meatproducts 1.000 0.504 1.982 0.000 0.342 0.000 1.000DairyproductsL.monocytogenes 1.363 0.541 3.433 0.309 0.462 0.670 0.503

DairyproductsListeriaspp 1.000 0.368 2.718 0.000 0.500 0.000 1.000

Seafoodproducts 1.000 0.342 2.920 0.000 0.536 0.000 1.000Vegetalproducts 0.710 0.327 1.541 0.342 0.387 0.884 1.623Environmental

samples 1.380 0.524 3.632 0.322 0.484 0.665 0.506

Compositefoods 0.868 0.355 2.123 0.142 0.447 0.316 1.248Combined 1.000 0.712 1.405 0.000 0.170 0.000 1.000

3.2.4. Interpretationandconclusion

TheRLODsvaluesarebelowtheacceptabilitylimitsetat2.5,meaningthat,asstatedinISO161402: 2016, themaximum increase in LOD of the alternative versus the referencemethod is notconsideredasrelevantinconsiderationofthefitnessforpurposeofthemethod.


04/2018

26/107

In conclusion, alternative and referencemethods show similar LODs values for the detection ofListeriasppandofListeriamonocytogenesinthecategoriestested.

3.3. InclusivityandexclusivitystudyTheinclusivityandexclusivityofthemethodaredefinedbyanalyzing,respectively,50positivestrainsand30negativestrains.AstheVIDASLDUOmethodprovidesasimultaneousresponseforbothL.monocytogenesandListeriaspp.,50L.monocytogenesstrainsand30Listeriaspp.strains(otherthanL.monocytogenes)weretested.ThirtyonestrainsnotbelongingtotheListeriagenuswerealsotested.

3.3.1.1. Testprotocols

ProtocolforinclusivityForeachoftheListeriastrains,acultureinnutrientbrothwasperformedfor24hoursat30C.AnLXbrothwas inoculatedwithapproximately10Listeriaperml,thenthecompleteenrichmentprotocolforthemethodwasfollowedpriortoperformingtheVIDASLDUOtest.

ProtocolforexclusivityThevariousnegativestrainswereculturedinnutrientbrothfor24hoursat30C,inoculatedin10mlofnutrientbrothinordertoobtainlevelsofaround105cellsperml,thenincubatedfor24hoursat30CpriortoperformingtheVIDASLDUOtest.

3.3.1.2. ResultsandconclusionTheresultsaresetoutinAppendixF.The50L.monocytogenesstrainswerealldetectedusingtheLDUOtest(positiveDLMOresponse).The30ListeriastrainsotherthanListeriamonocytogeneswerealsodetectedusingtheLDUOtest(positiveDLISresponse).Nocrossreactionswereobtainedwiththe31nonListeriastrains.

3.4. PracticabilityThepracticabilityofthealternativemethodwas informedaccordingtothecriteriadefinedbytheTechnicalCommittee.1. Storageconditions,shelflifeandmodalitiesofutilizationafterfirstuseThekitsarepackagedin60testkitscontaining:

theLDUOstrips,madeofpolypropylene,consistingof10wellscoveredwithaluminumfoil, theLDUOSPRs,inaluminumpouchescontaining30units,withadesiccant, thevialofLDUOS1standard(Listeriamonocytogenes)(6ml), thevialofLDUOS2standard(Listeria)(6ml), thevialsofLDUOC1andC3positivecontrols(ListeriamonocytogenesandListeria)(6ml), thevialofC2negativecontrol(6ml).

Theteststoragetemperatureis28C.Theshelflifeoftestsisindicatedonthekits.Eachreagentshouldbestoredbetween+2Cand+8C.


04/2018

27/107

2. TimetoresultNegativeresultsareobtainedintwodays.Positiveresultsareobtainedin:

threetofourdaysusingaconfirmationbystreakingonachromogenicagarmedia, fourtofivedaysusingaAPIListeriagallerywithoutpurification, uptotendaysusingthetestsofthereferencemethod.

3. CommonstepwiththereferencemethodThealternativemethodhasnocommonstepwiththereferencemethod.

3.5. ConclusionThe comparative study of themethodswas performed according to the EN ISO 161402:2016standard.

SensitivitystudyThe performance of the VIDAS Listeria Duo (LDUO) method was compared to that of theENISO112901:2017referencemethodbyanalyzing543samplesdividedintosixproductcategories.For the response Listeria spp, the sensitivity of the alternative method was 93.6% and thesensitivityofthereferencemethodwas89.6%.FortheresponseListeriamonocytogenes,thesensitivityofthealternativemethodwas96.9%andthesensitivityofthereferencemethodwas89.3%.Theobservedvalues(NDPD)werebelowtotheacceptability limitforeachcategoryandforallcategoriesaftertheinitialtestandafterthreedaysofconservationat53C.Statistically,thealternativemethodproducesresultscomparabletothatofthereferencemethod.Thedifferenceofthedeviationsishoweverclearlyinfavorofthealternativemethod.

RelativelevelofdetectionstudyThe relativedetection levelof theVIDASLDUOmethodand referencemethodwasevaluatedbyartificiallycontaminatingsixdifferentproducts.Therelativelevelofdetectionofthealternativemethodwasbetween0.710and1.380cellspertestportion.TheVIDASLDUOmethodandthereferencemethodshowedsimilarLODsvaluesforthedetectionofListeriasppandListeriamonocytogenesinthecategoriestested.

InclusivityandexclusivitystudyThespecificityofthemethod issatisfactory,asallListeriamonocytogenesandListeriasppstrainsweredetected(inclusivity)andnocrossreactionswereobservedamongnontargetedtestedstrainswhenthecompleteprotocolforthealternativemethodwasimplemented(exclusivity).


04/2018

28/107

4. Interlaboratorystudy

4.1. Studyorganization- Numberofparticipatinglaboratories:seventeencollaboratorsreceivedsamples.

- Matrixused:pasteurizedmilkwasusedasmatrixfortheinterlaboratorystudy.- Strainused:thestrainusedforcontaminationwasastrainofListeriamonocytogenes(codedL37

bytheExpertLaboratory)isolatedfromdairyproducts.- Numberofsamplesperlaboratory:24samplespercollaboratorwerepreparedforthereference

methodand24samplesforthealternativemethod,brokendowninto3levels,with8samplesperlevel.Oneadditionalsample,notartificiallycontaminated,wasprovidedtothecollaboratorsfortheenumerationofthemicroorganismsofthematrix.

4.2. Controloftheexperimentalparameters

4.2.1. Contaminationlevel

Thecontaminationratesobtainedinthematrixaresetoutinthetablebelow:Table21:theoreticalandactualcontaminationlevels

Level Samples Theoreticaltargetlevel(CFU/25ml) Reallevel(CFU/25ml)

L0:Level0 3491015162122 0 0L1:Lowlevel 127813141920 3 3.2L2:Highlevel 56111217182324 30 33.0

4.2.2. Shippingconditions(temperatureandstateofthesamples)Thetemperaturesofthesamplesatreceptionforallthecollaboratorsaregivenintable22.


04/2018

29/107

Table22:temperatureandshippingconditions

Collaborator Temperaturesatreception CommentsGivenbythecollaborator IndicatedbytheprobeA 3.0C 2.7C /B 2.7C 2.7C /C 4.0C 3.2C /D 3.8C / AnalysesnotrealizedE 4.0C 2.2C /F 4.1C 2.7C /G 4.0C 2.7C /H 4.3C 3.7C /I / / DeliveryatD+2J 5.7C 2.2C /K 4.0C 2.7C /L 4.5C 5.7C /M 4.6C 4.7C /N 5.7C 3.2C /O 4.0C 2.7C /P 6.4C 3.7C /Q 5.3C 3.9C /

Asaresultoftransportconditions,15laboratoriescarriedoutthetests.Twolaboratorieswerenotincludedinthefinallistoflaboratories:

collaboratorIreceivedthesamplesafterthedeadline, forthecollaboratorD,theVIDASsystemhadnotbeenupdated.

4.3. Testresults

Thepostconfirmationpositiveresultsobtainedbythecollaboratorsandbytheexpertlaboratoryareset out in the following tables and in appendix G. The results of the enumeration of themicroorganismsofthematrixarealsoprovidedforeachlabinthisappendix.Forallcollaborators,resultsrangedbetween

4.3.2. CollaboratorsresultsTable24:Positiveresultsobtainedwiththereferencemethod

CollaboratorsContaminationlevels

L0 L1 L2CollaboratorA 0/8 7/8 8/8CollaboratorB 0/8 8/8 8/8CollaboratorC 0/8 7/8 8/8CollaboratorE 0/8 8/8 8/8CollaboratorF 0/8 8/8 8/8CollaboratorG 0/8 8/8 8/8CollaboratorH 0/8 8/8 8/8CollaboratorJ 0/8 7/8 8/8CollaboratorK 0/8 8/8 8/8CollaboratorL 0/8 7/8 8/8CollaboratorM 0/8 8/8 8/8CollaboratorN 0/8 7/8 8/8CollaboratorO 0/8 8/8 8/8CollaboratorP 0/8 8/8 8/8CollaboratorQ 0/8 8/8 8/8

Total 0/120 115/120 120/120Table25:Positiveresultsobtainedwiththealternativemethod

CollaboratorsContaminationlevels

L0 L1 L2CollaboratorA 0/8 8/8 8/8CollaboratorB 0/8 8/8 8/8CollaboratorC 0/8 8/8 8/8CollaboratorE 0/8 7/8 8/8CollaboratorF 0/8 8/8 8/8CollaboratorG 0/8 8/8 8/8CollaboratorH 0/8 7/8 8/8CollaboratorJ 0/8 8/8 8/8CollaboratorK 0/8 8/8 8/8CollaboratorL 0/8 8/8 8/8CollaboratorM 0/8 7/8 8/8CollaboratorN 0/8 8/8 8/8CollaboratorO 0/8 7/8 8/8CollaboratorP 0/8 8/8 8/8CollaboratorQ 0/8 8/8 8/8

Total 0/120 116/120 120/120Theresultsusingthereferencemethodandalternativemethodareconcordantforsixlaboratories:B,F,G,K,PandQ.


04/2018

31/107

ForcollaboratorF,oneofthenoncontaminatedsampleswasfoundtobepositiveusingtheVIDASLDUOtestatthethresholdvalue(VT=0.10)fortheDLISresponse.ThelaboratorystaffisolatedtheLXbrothanddidnotfindanycoloniesonselectiveagars.CollaboratorsA,C,J,LandNallfoundonesamplewithalowcontaminationlevelthatwasnegativeusingthereferencemethod.Likewise,collaboratorsE,H,MandOallfoundonesamplewithalowcontaminationratethatwasnegativeusingthealternativemethodfortheDLMOandDLISresponses.ItmustbenotedhoweverthatthecollaboratorsEandMappliedtheconfirmationprotocolfromtheLXbrothforthisnegativesampleofthelowcontaminationlevelandthatitwasfoundnegative.Thesedifferencesbetweenthetwomethodsmayhappen inanunpairedstudy.Twosetsofeightdistinctsampleshadbeenpreparedforthelowcontaminationlevel.Onesamplewasusedforonlyoneofthemethods(alternativeorreference),astheprimaryenrichmentbrothsweredifferent.Itislikelythatthesamplesfoundtobenegativewerenotcontaminated.

4.3.3. ResultsofthecollaboratorsusedforthestatisticalanalysisThe results of the 15 collaborators having realized the analyses are retained for the statisticalinterpretation.

4.4. Calculationsandinterpretation

4.4.1. CalculationofthespecificityThepercentagespecificity(SP)ofthereferencemethodandthealternativemethod iscalculated,usingthedataafterconfirmation,basedontheresultsoflevelL0asfollows:

Specificityofthereferencemethod: 1 100% Specificityofthealternativemethod: 1 100%where:NisthenumberofallL0tests,P0 is the total number of falsepositive results obtained with the blank samples beforeconfirmation,CP0isthetotalnumberoffalsepositiveresultsobtainedwithblanksamples.

Theresultsarethefollowing:

SPref=100% SPalt=100%

4.4.2. Summaryoftheresults

Asummaryofresultsobtainedatlevel1(L1),forwhichfractionalpositiveresultswereobtained,issetoutintable26.


04/2018

32/107

Table 25: tests results for the twomethods at level L1 (PA: positive agreement, NA: negativeagreement, ND: negative deviation, PD: positive deviation, PP: presumed positive beforeconfirmation,*:forthecollaboratorFonlywiththeDLISresponse)

Level AlternativemethodReferencemethod

Referencemethodpositive(R+)

Referencemethodnegative(R) Total

L1

Alternativemethodpositive(A+) PA=111 PD=5 116

Alternativemethodnegative(A)

ND=4including0PPND

NA=0including0PPNA 4

Total 115 5 120Thecauseofthepositiveandnegativedeviationsisexplainedin4.3.2(unpairedstudy).

4.4.3. Calculationofthesensitivityofthemethods,relativetruenessandfalsepositiveratio

Thesensitivityofthetwomethods,therelativetruenessandthefalsepositiveratioparametersarecalculatedwiththedataofthetable26,accordingtotheformulasbelow: Sensitivityforthealternativemethod: 100%

Sensitivityforthereferencemethod: 100% Relativetrueness: 100% Falsepositiveratioforthealternativemethod: 100%whereNisthetotalnumberofsamples(NA+PA+PD+ND)andFPisfalsepositiveresults.Theresultsarethefollowing:

SEalt=96.7% SEref=95.8% RT=92.5% FP=0%

4.4.4. Determinationoftheacceptabilitylimitandconclusion

The difference between (ND PD) for the levelwhere fractional recoverywas obtained (L1) iscalculated.Theobservedvaluefoundfor(NDPD)shallnotbehigherthantheacceptabilitylimit(AL).TheAL isdefinedas [(NDPD)max]and calculatedper levelwhere fractional recoverywasobtainedasdescribedbelowusingthefollowingthreeparameters: ,where

Px=numberofsampleswithapositiveresultobtainedwiththereferencemethodatlevelx,(L1orL2)foralllaboratories;Nx=numberofsamplestestedatlevelx(L1orL2)withthereferencemethodbyalllaboratories. ,where


04/2018

33/107

CPx=numberofsampleswithaconfirmedpositiveresultobtainedwiththealternativemethodatlevelx(L1orL2)foralllaboratories;Nx=numberofsamplestestedatlevelx(L1orL2)withthealternativemethodbyalllaboratories.

3 2 ,whereNx=thetotalnumberofsamplestestedforlevelx(L1orL2)byalllaboratories.The AL is notmet when the observed value is higher than the AL.When the AL is notmet,investigationsshouldbemade(e.g.rootcauseanalysis) inordertoprovideanexplanationoftheobservedresults.Basedon theALand theadditional information, it isdecidedwhether thealternativemethod isregardedasnotfitforpurpose.ThereasonsforacceptanceofthealternativemethodincasetheALisnotmetshallbestatedinthestudyreport.In this study, fractional positive results are observed at level L1 only. The different parametersobtainedbythecalculationaredetailedinthetablebelow:Table27:valuesobtainedforthedeterminationoftheacceptabilitylimit

Parameter Value(p+)ref 0.9583(p+)alt 0.9667

Acceptabilitylimit:AL=(NDPD)max 5.1Observedvalue:NDPD 1

The value (NDPD) is inferior to the acceptability limit, so the requirements of the standardISO161402:2016arefulfilled.

4.4.5. DeterminationoftherelativelevelofdetectionThis evaluation is performed according to Annex F of ISO 161402:2016 and using the Excelspreadsheetasdescribedinthisstandard.Asthereislimitedexperiencewiththeinterpretationofthisapproach,theresultsareusedonlyforinformation.Resultsareshowninthetablebelow:Table28:valuesobtainedforthedeterminationoftherelativelevelofdetection(RLOD:theestimatedrelative levelofdetectionvalue,RLODU: theupper limitof the95%confidence interval forRLOD,RLODL:thelowerlimitofthe95%confidenceintervalforRLOD,b=ln(RLOD):logarithmoftheRLODvalue,sd(b):standarddeviationofb,zTeststatistic:absolutevalueoftheteststatisticofthezTestwiththenullhypothesisH0:b=0,pvalue:pvalueofthezTest)Category RLOD RLODL RLODU b=ln(RLOD) sd(b) zTeststatistic pvalue

ILS 0,934 0,627 1,393 0,068 0,200 0,340 1,266

4.5. Conclusion

ThedataandtheirinterpretationmeettherequirementsofthestandardENISO161402:2016.Theperformanceofthealternativemethodandthereferencemethodcanbeconsideredasequivalent.


04/2018

34/107

5. GeneralconclusionThedataandtheinterpretationofthemethodscomparisonstudyandoftheinterlaboratorystudyfulfill the requirementsof the standardEN ISO161402:2016.TheVIDAS ListeriaDuomethod isconsideredasequivalenttothestandardENISO112901:2007. LeLiondAngers,April20,2018 FranoisLeNestour HeadoftheMethodsValidationstudiesandR&D


04/2018

35/107

APPENDICES


04/2018

36/107

APPENDIXAVIDASLISTERIADUO

Diagram oftheprocedure asdescribed intheuserguide

25gofsample +225mlofLXbroth

Incubationat301Cfor242h

Transferof0.1mlin6mlofLXbroth

Heating at95 100Cfor51min(1to2mlinatubeforuseofawaterbathor0.5mlinaVIDASstrip foruseofVIDASHeat&Go


Realization oftheVIDAStest

ConfirmationListeriamonocytogenes

Streaking oftheLXnonheated brothonAgarListeriaacc.toOttaviani and

Agosti orPALCAMorOxford.Incubationacc.touserguides

Identificationof1to5typicalcoloniesusingtheconventionaltestsdescribedinthemethodsstandardizedbytheCENorlSO

(includingthepurificationstep)

or

Useofachromogenicagar,thatispartofamethodcertifiedNFVALIDATION.The

presenceoftypicalcoloniesconfirmsthepresenceofListeriamonocytogenes

ConfirmationListeriaspp

Streaking oftheLXnonheated brothonAgarListeriaacc.toOttaviani and

Agosti orPALCAMorOxford.Incubationacc.touserguides

Identificationof1to5typicalcoloniesusingtheconventionaltestsdescribedinthemethodsstandardizedbytheCENorlSO

(includingthepurificationstep)

or

FromAgarListeriaacc.toOttaviani andAgosti,typical coloniesofListeria can be

confirmed bytheuseofbiochemical testsorbytheuseofanAPIgallery without

purification.


04/2018

37/107

APPENDIXAVIDASLISTERIADUO

Diagram oftheprocedure applied bytheExpertLaboratory

25gofsample +225mlofLXbroth


Transferof0.1mlin6mlofLXbroth

Heating of0,5mlinaVIDASstrip at95 100Cfor51minusing aVIDASHeat&Go


Realization oftheVIDAStest

Isolationofthenonheated LXbroth on

ALOAIncubationat371Cfor242hand482h

PALCAMIncubationat371Cfor24hand48h

Confirmationof1to5coloniesfrom 1selective medium:realization ofthebiochemical testsafter purificationand

realization ofanAPIgallery without purification


04/2018

38/107

APPENDIXBENISO112901:2017

Diagram oftheprocedure asdescribed inthestandard

25gofsample +225mlofhalfFraserbroth


Transferof0.1mlin10mlofFraserbroth

Plating outon: AgarListeria according toOttaviani andAgosti

Secondselective medium


ALacc.O&A:incubationat371Cfor242handfor242hOther selective medium:incubationasspecified

Confirmation:1typical colony on1selective medium,uptoamaximumof5coloniesoneach medium

Streaking onnonselective agarIncubationat37Cfor213horuntil sufficient growth

ListeriamonocytogenesMandatory tests:

Microscopic apect haemolysis Lrhamnose Dxylose

Optional tests: Catalase Motility at25C CAMPtest

Listeria sppMandatory tests:

Microscopic apect Catalase

Optional tests: VPtest Motility at25C


04/2018

39/107

APPENDIXC

ARTIFICIALCONTAMINATIONS


04/2018

40/107

ARTIFICIALCONTAMINATIONSINITIALVALIDATIONSTUDY

Code Matrix Artificial contamination

Result Strain Type of stress Evaluation of stress CFU / 25 g No. Name Origin

H1 Grated Gruyre L64 L.innocua poisses cheese 45 mins. at 55C, then 30 mins. at -80C 1.0 16.8 + H2 Vieux pn cheese L64 L.innocua poisses cheese 45 mins. at 55C, then 30 mins. at -80C 1.0 33.6 + H3 Ossau-Iraty sheep's milk cheese L142 L.seeligeri Cheese made with raw milk 45 mins. at 55C, then 30 mins. at -80C 3.9 28.0 - H4 Brie cheese L142 L.seeligeri Cheese made with raw milk 45 mins. at 55C, then 30 mins. at -80C 3.9 56.0 - H5 Reblochon cheese L64 L.innocua poisses cheese 45 mins. at 55C, then 30 mins. at -80C 1.0 50.4 + H6 Reblochon cheese L142 L.seeligeri Cheese made with raw milk 45 mins. at 55C, then 30 mins. at -80C 3.9 84.0 - H7 Water from final rinsing L115 L.seeligeri Standing water 45 mins. at 55C, then 30 mins. at -80C 1.6 8.0 + H8 Rinsing bath L115 L.seeligeri Standing water 45 mins. at 55C, then 30 mins. at -80C 1.6 16.0 + H9 Washing basin L115 L.seeligeri Standing water 45 mins. at 55C, then 30 mins. at -80C 1.6 24.0 -

H10 Residual water L132 L.innocua Cheese counter chopping board 45 mins. at 55C, then 30 mins. at -80C 0.2 0.1 - H11 Doser rinsing water L132 L.innocua Cheese counter chopping board 45 mins. at 55C, then 30 mins. at -80C 0.2 0.1 - H12 Standing water from storage room L132 L.innocua Cheese counter chopping board 45 mins. at 55C, then 30 mins. at -80C 0.2 0.2 -

H13 Water from light rinsing L115 L.seeligeri Standing water 45 mins. at 55C, then 30 mins. at -80C 1.6 4.0 + L132 L.innocua Cheese counter chopping board 45 mins. at 55C, then 30 mins. at -80C 0.2 0.0 H14 Residue from machine filter L115 L.seeligeri Standing water 45 mins. at 55C, then 30 mins. at -80C 1.6 16.0 + H15 Ground surface in storage room L115 L.seeligeri Standing water 45 mins. at 55C, then 30 mins. at -80C 1.6 24.0 + H16 Surface of dirty service lift L132 L.innocua Cheese counter chopping board 45 mins. at 55C, then 30 mins. at -80C 0.2 0.1 + I14 Rabbit terrine L77 L.innocua Toulouse sausage 45 mins. at 55C, then 30 mins. at -80C 1.1 1.2 - I15 Farmhouse liver pt L77 L.innocua Toulouse sausage 45 mins. at 55C, then 30 mins. at -80C 1.1 0.8 - I16 Country pt L87 L.welshimeri Minced beef burger 45 mins. at 55C, then 30 mins. at -80C ND



J14 Raw milk L37 L.monocytogenes Maroilles cheese 45 mins. at 55C, then 30 mins. at -80C 0.6 3.4 + L72 L.innocua Boulette dAvesnes cheese 45 mins. at 55C, then 30 mins. at -80C, then 24 hrs at 4C 0.6 5.0 J15 Raw milk L72 L.innocua Boulette dAvesnes cheese 45 mins. at 55C, then 30 mins. at -80C, then 24 hrs at 4C 0.6 7.5 + J16 Raw milk L72 L.innocua Boulette dAvesnes cheese 45 mins. at 55C, then 30 mins. at -80C, then 24 hrs at 4C 0.6 10.0 +

J17 Raw milk L37 L.monocytogenes Maroilles cheese 45 mins. at 55C, then 30 mins. at -80C 0.6 5.1 + L72 L.innocua Boulette dAvesnes cheese 45 mins. at 55C, then 30 mins. at -80C, then 24 hrs at 4C 0.6 7.5

J18 Powdered milk L37 L.monocytogenes Maroilles cheese 45 mins. at 55C, then 30 mins. at -80C 0.6 5.1 + L72 L.innocua Boulette dAvesnes cheese 45 mins. at 55C, then 30 mins. at -80C, then 24 hrs at 4C 0.6 7.5 J19 Powdered milk L72 L.innocua Boulette dAvesnes cheese 45 mins. at 55C, then 30 mins. at -80C, then 24 hrs at 4C 0.6 7.5 + J20 Powdered milk L72 L.innocua Boulette dAvesnes cheese 45 mins. at 55C, then 30 mins. at -80C, then 24 hrs at 4C 0.6 10.0 +

J21 Chantilly cream puff L37 L.monocytogenes Maroilles cheese 45 mins. at 55C, then 30 mins. at -80C 0.6 5.1 + L72 L.innocua Boulette dAvesnes cheese 45 mins. at 55C, then 30 mins. at -80C, then 24 hrs at 4C 0.6 7.5 J22 Strawberry melba L72 L.innocua Boulette dAvesnes cheese 45 mins. at 55C, then 30 mins. at -80C, then 24 hrs at 4C 0.6 10.0 + J28 Strawberry ice cream L72 L.innocua Boulette dAvesnes cheese 45 mins. at 55C, then 30 mins. at -80C, then 24 hrs at 4C 0.6 5.0 + J29 Vanilla ice cream L72 L.innocua Boulette dAvesnes cheese 45 mins. at 55C, then 30 mins. at -80C, then 24 hrs at 4C 0.6 7.5 +

J30 Surface of stainless steel table in butcher's facility L115 L.seeligeri Standing water 48 hrs at -20C, 45 min. 55C, then 30 mins. at -80C 1.0 0.3 -

K1 Minced pork loin L77 L.innocua Toulouse sausage 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 0.8 4.1 + K2 Chicken gizzard L77 L.innocua Toulouse sausage 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 0.8 5.5 + K3 Mortadella sausage L77 L.innocua Toulouse sausage 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 0.8 6.8 + K4 Beef L77 L.innocua Toulouse sausage 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 0.8 8.2 + K5 Ham L87 L.welshimeri Minced beef burger 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 1.3 9.6 + K6 Pork chop L87 L.welshimeri Minced beef burger 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 1.3 12.0 + K7 Jellied tongue L87 L.welshimeri Minced beef burger 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 1.3 14.4 + K8 Potjevlesch potted meat L87 L.welshimeri Minced beef burger 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 1.3 16.8 +

K9 Bayonne ham L77 L.innocua Toulouse sausage 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 0.8 6.8 + L87 L.welshimeri Minced beef burger 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 1.3 12.0

K10 Meat pt L77 L.innocua Toulouse sausage 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 0.8 5.5 + L87 L.welshimeri Minced beef burger 30 mins at 55C, 20 mins at -80C, 5 mins at 45C 1.3 9.6 L1 Tuscan minced pork L91 L.welshimeri Aoste rosette dried sausage 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.6 2.3 + L2 Strasbourg sausages L91 L.welshimeri Aoste rosette dried sausage 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.6 1.8 + L3 Rillettes L151 L.ivanovii Minced beef burger 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.2 0.3 + L4 Chicken rillettes L151 L.ivanovii Minced beef burger 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.2 0.6 - L5 Camembert L64 L.innocua poisses cheese 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.2 5.0 + L6 Munster cheese L111 L.innocua Munster cheese 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.5 2.4 +

L7 Double-cream cheese L64 L.innocua poisses cheese 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.2 10.0 + L111 L.innocua Munster cheese 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.5 8.7 L8 Leerdamer cheese L64 L.innocua poisses cheese 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.2 10.0 + L9 Normandy apple pie L111 L.innocua Munster cheese 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.5 8.7 +

L10 Egg custard pie L64 L.innocua poisses cheese 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.2 15.0 + L111 L.innocua Munster cheese 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.5 8.7 L11 Strawberry vanilla ice cream L64 L.innocua poisses cheese 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.2 4.5 + L12 Raw milk L111 L.innocua Munster cheese 30 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.5 2.7 + M2 Minced meat L2 L.innocua Minced beef burger 30 mins at 55C, 25 mins at -80C, 5 mins at 46C 0.9 5.7 +

M5 Rosette dried sausage L44 L.monocytogenes Dried sausage 30 mins at 55C, 25 mins at -80C, 5 mins at 46C 0.5 7.0

+ L2 L.innocua Minced beef burger 30 mins at 55C, 25 mins at -80C, 5 mins at 46C 0.9 3.8


04/2018

42/107



M13 Fish fillet L86 L.welshimeri ATCC 35897 30 mins at 55C, 25 mins at -80C, 5 mins at 46C 0.7 3.8 + M15 Dogfish L86 L.welshimeri ATCC 35897 30 mins at 55C, 25 mins at -80C, 5 mins at 46C 0.7 5.7 + M16 Fillet of scabbardfish L86 L.welshimeri ATCC 35897 30 mins at 55C, 25 mins at -80C, 5 mins at 46C 0.7 4.8 +

M27 Rinsing water L44 L.monocytogenes Dried sausage 30 mins at 55C, 25 mins at -80C, 5 mins at 46C 0.5 7.0 + L2 L.innocua Minced beef burger 30 mins at 55C, 25 mins at -80C, 5 mins at 46C 0.9 3.8

M28 Water from washing container L44 L.monocytogenes Dried sausage 30 mins at 55C, 25 mins at -80C, 5 mins at 46C 0.5 8.8 + L2 L.innocua Minced beef burger 30 mins at 55C, 25 mins at -80C, 5 mins at 46C 0.9 3.8 -

O10 Residue from cutting facility stainless steel table L149 L.monocytogenes Environmental sample 45 mins at 55C, 30 mins at -80C, 10 mins at 55C 1.0 13.0 + L144 L.innocua Bin surface 45 mins at 55C, 30 mins at -80C, 10 mins at 55C 0.7 10.0

O11 Surface in cooling room L149 L.monocytogenes Environmental sample 45 mins at 55C, 30 mins at -80C, 10 mins at 55C 1.0 19.5 + L144 L.innocua Bin surface 45 mins at 55C, 30 mins at -80C, 10 mins at 55C 0.7 7.5

O12 Floor of cold packaging room L149 L.monocytogenes Environmental sample 45 mins at 55C, 30 mins at -80C, 10 mins at 55C 1.0 9.7 + L144 L.innocua Bin surface 45 mins at 55C, 30 mins at -80C, 10 mins at 55C 0.7 5.0

O13 Stainless steel shelf surface in cold store L149 L.monocytogenes Environmental sample 45 mins at 55C, 30 mins at -80C, 10 mins at 55C 1.0 6.5 +

O14 Residue from sink in cutting facility L149 L.monocytogenes Environmental sample 45 mins at 55C, 30 mins at -80C, 10 mins at 55C 1.0 9.7 + O15 Residue from packaging container L153 L.ivanovii Environmental sample 45 mins at 55C, 30 mins at -80C, 10 mins at 55C ND



R16 Cucumbers L47 L.monocytogenes Fried potatoes 45 mins at 55C, 30 mins at -80C, 5 mins at 46C ND < 0.1 - R17 Broccoli L47 L.monocytogenes Fried potatoes 45 mins at 55C, 30 mins at -80C, 5 mins at 46C ND < 0.1 - R18 Carrots L47 L.monocytogenes Fried potatoes 45 mins at 55C, 30 mins at -80C, 5 mins at 46C ND < 0.1 - R19 Lamb's lettuce L47 L.monocytogenes Fried potatoes 45 mins at 55C, 30 mins at -80C, 5 mins at 46C ND < 0.1 - R20 White cabbage L47 L.monocytogenes Fried potatoes 45 mins at 55C, 30 mins at -80C, 5 mins at 46C ND < 0.1 - S2 Prawns L113 L.innocua Smoked halibut 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 1.6 - S3 Smoked Atlantic salmon L113 L.innocua Smoked halibut 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 2.5 + S4 Prawns L113 L.innocua Smoked halibut 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 2.5 + S5 Cod fillet L113 L.innocua Smoked halibut 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 2.5 + S6 Fillet of coalfish L113 L.innocua Smoked halibut 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 1.6 + S7 Fillet of pollock with baby vegetables L113 L.innocua Smoked halibut 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 1.6 + S8 Carrots L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 4.9 + S9 Mushrooms L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 4.9 +

S12 Red cabbage L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 20.0 + S13 Vegetable soup L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 10.0 + S14 Celery and beetroot salad L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 20.0 + S15 Ratatouille L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 10.0 + S16 Potato flakes L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 10.0 + S19 Frozen fries L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.7 20.0 + T3 Broccoli and cauliflower patties L58 L.monocytogenes Salad 45 mins at 55C, 30 mins at -80C 1.3 21.5 + T4 Celery cake L140 L.seeligeri Frozen fries 45 mins at 55C, 30 mins at -80C > 4.5 30.0 + T5 Frozen mushrooms L140 L.seeligeri Frozen fries 45 mins at 55C, 30 mins at -80C > 4.5 ND + T6 Mixed salad L58 L.monocytogenes Salad 45 mins at 55C, 30 mins at -80C 1.3 21.5 + T8 Smoked Atlantic salmon L85 L.seeligeri Collection 45 mins at 55C, 30 mins at -80C > 1.3 0.8 - T9 Smoked halibut L85 L.seeligeri Collection 45 mins at 55C, 30 mins at -80C > 1.3 0.4 - U2 Prawns L113 L.innocua Smoked halibut 45 mins at 55C, 30 mins at -80C 0.5 4.4 + U3 Smoked trout L113 L.innocua Smoked halibut 45 mins at 55C, 30 mins at -80C 0.5 2.7 + U9 Pan-fried courgettes L140 L.seeligeri Frozen fries 45 mins at 55C, 30 mins at -80C > 3 0.3 +

U10 Pan-fried Mediterranean vegetables L140 L.seeligeri Frozen fries 45 mins at 55C, 30 mins at -80C > 3 0.4 -

U11 Pan-fried mushrooms and vegetables L140 L.seeligeri Frozen fries 45 mins at 55C, 30 mins at -80C > 3 0.5 -

L125-1 Red cabbage L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C 0.6 2.7 + L125-2 Red cabbage L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C 0.6 2.7 - L125-3 Red cabbage L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C 0.6 2.7 - L125-4 Red cabbage L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C 0.6 2.7 - L125-5 Red cabbage L125 L.monocytogenes Pan-fried vegetables 45 mins at 55C, 30 mins at -80C 0.6 2.7 -

V3 Pan-fried Mediterranean vegetables L66 L.innocua Spinach 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.6 4.2 +

V4 Pan-fried mushrooms and vegetables L66 L.innocua Spinach 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.6 7.2 +

V5 Pan-fried vegetables L66 L.innocua Spinach 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.6 4.2 + V6 Vegetable pure L66 L.innocua Spinach 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.6 5.6 + V7 Cooked carrots L66 L.innocua Spinach 45 mins at 55C, 30 mins at -80C, 5 mins at 46C 0.6 7.2 +


04/2018

44/107

Category Strain Serovar Code Origin ContaminationContamination

levelConservation # Sample

3daysat5C 1398360 Spicedcookedchickenwings3daysat5C 1398361 Bologneseescalope

Listeriamonocytogenes 1/2b JAR249 Pasteurizedmilkcheese Seeding 2.4 3daysat5C 1370083 Slicedcheddar(pasteurizedmilk)3daysat5C 1398363 Rawcowmilk3daysat5C 1398364 Slicefourmed'Ambert(pasteurizedmilk)3daysat5C 1398367 Cheeseproductwithgarlicandherbs(pasteurizedmilk)3daysat5C 1398365 Ewesoftcheese(pasteurizedmilk)3daysat5C 1398366 Ewecheese,pressednotcooked(pasteurizedmilk)

Listeriaivanovii / / Creampastrycake Seeding 1.2 2weeksat24C 1398371 Coffeeicecreammwithhazelnutsauce3daysat5C 1420538 Goatrawmilkcheese13daysat5C 1420539 Goatrawmilkcheese23daysat5C 1398351 Scallopterrine3daysat5C 1398352 MincedAlaskapollockwithlemonandonion3daysat5C 1398353 Tunarillettes3daysat5C 1398354 Surimisticks

2.4 3daysat5C 1372306 Freshchive1.6 3daysat5C 1372308 Mixtureofaromaticherbs

2weeksat24C 1398372 Frozenvegetablesgyoza2weeksat24C 1398373 Frozenpuree:carot,sweetpotato,pumpkinandpotato2weeksat24C 1398374 Frozenstew:greenbeans,potatoes,mushrooms,carots3daysat5C 1409272 Fruitsalad3daysat5C 1409273 Appleredberriescompotewithwhippedcream3daysat5C 1420540 Strawberries2weeksat24C 1420541 Finebeans(frozen)

Listeriamonocytogenes 1/2a SCZ856 Stuffedmonkfish Seeding 1.0 3daysat5C 1370078 GambasandscallopmarinadewithlemonListeriamonocytogenes 1/2c TED200 Rilletttes Seeding 0.2 3daysat5C 1370081 SweetbreadboucheListeriamonocytogenes 1/2b JAR249 Pasteurizedmilkcheese Seeding 1.2 3daysat5C 1370082 GoatcheesesoufflListeriamonocytogenes 1/2a FCY076 Eggplantgratin Seeding 1.8 3daysat5C 1372303 MixedvegetableswithmayonnaiseListeriamonocytogenes 1/2c TED200 Rilletttes Seeding 0.2 3daysat5C 1372304 ChorizoslicesListeriamonocytogenes 1/2a MEF831 Celerypuree Seeding 3.8 3daysat5C 1372305 ColeslawsaladListeriamonocytogenes 1/2c TED200 Rilletttes Seeding 0.2 3daysat5C 1372307 Cookedturkeyaiguillettes

3daysat5C 1398355 Chocolatefondant3daysat5C 1398368 Chocolateandcoffee"religieuse"pastry3daysat5C 1398357 Custard3daysat5C 1398356 Ricottaspinachravioli3daysat5C 1398358 Cheesepie3daysat5C 1398359 Tortillawithonions3daysat5C 1398362 Porknems3daysat5C 1398369 Grilledchickenandmayonnaisesandwich3daysat5C 1398370 Chickencheddarhamburgerwithbarbecuesauce

Listeriawelshimeri / DJC260 Porkmeat Seeding 2.4 3daysat5C 1409265 SandwichhamchedddarsaladListeriamonocytogenes 1/2b JAR249 Pasteurizedmilkcheese Seeding 1.0 3daysat5C 1409266 Cheesebiteswithspicesandherbs

Listeriainnnocua / FEY823 Liverpt Seeding 2.4 3daysat5C 1409267 Wrapbacon,yoghurtsauce,egg,marinatedtomatoes3daysat5C 1409264 Cocktailbitesbacon/cheese3daysat5C 1409268 Sandwichbeef,cheesesauceandcheese3daysat5C 1409269 Rillauds(readytoeatcookedporkbites)

Listeriamonocytogenes 4b ALB748 Salmontagliatelle Seeding 1.0Listeriaivanovii / / Pork Seeding 1.6

Listeriamonocytogenes 1/2a XBB696 Peeledfrozenbeans Seeding 0.8Listeriainnocua / CLM641 Garlic Seeding 2.4

Listeriawelshimeri / DJC260 Porkmeat Seeding 2.0 3daysat5C 1409274 BeefmuzzlelalyonnaiseListeriamonocytogenes 1/2a EFV356 Bacon Seeding 2.0 3daysat5C 1409275 SaveloysaladwithvinaigretteListeriamonocytogenes 1/2a FCY076 Eggplantgratin Seeding 1.2 3daysat5C 1420542 Saladbulgur,quinoa,cranberries

3daysat5C 1420543 Liquidpasteurizedwholeeggs3daysat5C 1420544 Bchamelsauce3daysat5C 1420545 Liquidpasteurizedeggwhites3daysat5C 1420546 Pralinflavouredcreampastry3daysat5C 1420547 Fraisier(strawberryflavouredcreampastry)3daysat5C 1420548 Processwaterfishplant13daysat5C 1420549 Processwaterfishplant23daysat5C 1420550 Processwatervegetableprocessingarea3daysat5C 1420551 Processwaterdairyplant3daysat5C 1420552 Processwatereggprocessingplant

3a RAX819

SeedingCodeggsHCX5171/2a

Seeding

SeedingSpongebutchery

SwabwashingstationGEB6391/2a 1.8

2.4

Compositefoods


Fusilicarbonara14092703daysat5C

3daysat5C 1409271 Cucumberwithcottagecheeseandchives

Creampastrycake//Listeriaivanovii

Listeriawelshimeri


SAE286 Ham Seeding

SeedingMealwithfermentedporkKFT1541/2aListeriamonocytogenes

2.8

Vegetalproducts

2.4

2.2

2.8

1.8

1.4

0.6Listeriamonocytogenes 1/2c

0.8

SeedingBouchewithporkEGK088/

Seeding

SeedingSalmontagliatelleALB7484b

Seafoodproducts

1.0

0.8Listeriamonocytogenes

SeedingFishbrandadeVCD6384bListeriamonocytogenes

SeedingStuffedmonkfishSCZ8561/2aListeriamonocytogenes

Meatproducts

Dairyproducts

SeedingGoatrawmilkcheeseHBP6524bListeriamonocytogenes

SeedingRawmilkcheeseCLM6411/2bListeriamonocytogenes

Seeding

CelerypureeMEF8311/2a

SeedingEggplantgratinFCY0761/2aListeriamonocytogenes

3.0

2.2

2.6

3.0

2.6

Seeding

ARTIFICIALCONTAMINATIONSTHIRDRENEWALSTUDY






BuckwheatflourRCJ2804b

RawmilkcheeseGCQ4714dListeriamonocytogenes

SeedingRawchickenlegDHN2311/2cListeriamonocytogenes

SeedingPeeledfrozenbeansXBB6961/2a

Seeding


04/2018

45/107

APPENDIXD1

INITIALVALIDATIONSTUDY

SENSITIVITYRAWRESULTS


04/2018

46/107

MeatproductsListeriaspp APPENDIX D

P1 OA1 P2 OA2 IDENTIF. RESULT RFV LMO VTRESULT

TEST LMORFV LIS VT

RESULT TEST LIS PAL RLM OAA IDENTIF.

E12 Lamb kebab PC1 No -LE / - -3 0,00 - 17 0,00 - / / / / - NAF1 Tournedos PC1 No / - -5 0,00 - 22 0,00 - / / / / - NAF3 Horse meat fillet PC1 No / - 0 0,00 - 34 0,01 - / / / / - NAF4 pork chop PC1 No / - -3 0,00 - 19 0,00 - / / / / - NAF5 Rump steak PC1 No / - -5 0,00 - 22 0,00 - / / / / - NAF6 Rib steak PC1 No / - -3 0,00 - 24 0,00 - / / / / - NAM1 Lamb kidneys PC1 No -ME / - -3 0,00 - 21 0,00 - / / / / - NAM6 Chicken breasts PC1 No / - -3 0,00 - 21 0,00 - / / / / - NAM11 Kidneys PC1 No -LE -LE / - -3 0,00 - 25 0,01 - / / / / - NAW14 Minced horse meat PC1 No -LE / - 22 0,00 - 24 0,00 - / / / / - NAF2 Tomato & basil chipolata sausages PC2 No -LE -LE / - -4 0,00 - 39 0,01 - / / / / - NAM3 Blood sausage PC2 No / - -4 0,00 - 21 0,00 - / / / / - NAM12 Blood sausage PC2 No / - -3 0,00 - 16 0,00 - / / / / - NAV12 Tomato stuffing PC2 No -LE -LE / - -5 0,00 - 16 0,00 - / / / / - NAV18 Bolognese minced meat PC2 No -LE / - -5 0,00 - 33 0,01 - / / / / - NAM10 Pt with pastry crust PC2 No -LE / - -2 0,00 - 21 0,00 - / / / / - NAI14 Rabbit terrine PC2 Yes 1,17 / - -2 0,00 - 30 0,01 - / / / / - NAI17 Duck pt PC2 Yes



TEST LMORFV LIS VT


COMPARISONCODE MATRICES Cat. AC CFU/25gFRASER 1/2 FRASER CONFIRMATION VIDAS LDUO CONFIRMATION

FINAL RESULT

NF EN ISO 11290-1 METHOD VIDAS LDUO METHOD

T15 Tomato burger PC1 No / - 11 0,00 - 7048 3,13 + +HA +HA +MB L.innocua + PDD7 Beef minced meat PC1 No / - 199 0,05 + / / + par dfaut +LA +LA +LA L.monocytogenes + PDC23 Beef heart PC1 No +MA +MB +MA +MA L.monocytogenes + 7363 1,97 + / / + par dfaut +HA +HA +MA L.monocytogenes + PA

D1 Turkey kebab PC1 No +LA* +LA* +MA* +MA*L.monocytogenes

L.innocua L.welshimeri

+ 1474 0,37 + / / + par dfaut +HA* +HA* +MA*L.monocytogenes


+ PA

D2 Chicken wings PC1 No +MA* +MA* +HA* +MA* L.monocytogenes L.innocua + 10982 2,78 + / / + par dfaut +HA +HA +HAL.monocytogenes

L.innocua + PA

E2 Minced meat PC1 No +LA +LA(2) +MA +MA L.monocytogenes + 7515 1,90 + / / + par dfaut +HA +HB +MA L.monocytogenes + PAE3 Minced meat PC1 No +LA +LA +MA +MA L.monocytogenes + 8260 2,09 + / / + par dfaut +HA +HA +HA L.monocytogenes + PAE7 Minced meat PC1 No +HA +HA L.monocytogenes + 7980 2,02 + / / + par dfaut +HA +HA +HA L.monocytogenes + PAE10 Lean bourguignon beef PC1 No +LA +LA +MA +MA L.welshimeri + 8 0,00 - 7812 3,00 + +HA +HA +HA L.welshimeri + PA

I21 Pork kidneys PC1 No +LA* +LA* +HA* +LA* L.welshimeri L.innocua + 26 0,00 - 6924 2,96 + +MB +MA* +MA*L.welshimeri

L.innocua + PA

M4 Chicken thighs PC1 No +LA +LA* +MA* +MA*L.monocytogenes

L.welshimeri L.innocua

+ 6 0,00 - 8155 3,62 + +MA +HA +MAL.monocytogenes


+ PA

T11 Fillet of duck breast PC1 No +HA +HB L.monocytogenes L.innocua + 9390 2,39 + / / + par dfaut +HA +MA* +HAL.monocytogenes

L.innocua + PA

T16 Veal PC1 No +LA(1) -LE +HB +MB L.welshimeri + 9 0,00 - 8603 3,83 + +MA +MA* +MD L.welshimeri + PA

T19 Turkey kebab PC1 No +LA(4) +HD +MA +MA* L.monocytogenes L.innocua + 10155 2,59 + / / + par dfaut +HA +HA* +MAL.monocytogenes

L.innocua + PA

V8 Frozen minced beef burger PC1 No +LB +LB +HA +MA L.monocytogenes + 7871 2,03 + / / + par dfaut +HA +HB +MA L.monocytogenes L.innocua + PA

V9 Fillet of duck breast PC1 No +MA +MA L.monocytogenes + 9836 2,54 + / / + par dfaut +HA +HA +MA L.monocytogenes + PA

V14 Chicken fillet PC1 No +HA +MA L.welshimeri + 1934 0,50 + / / + par dfaut +HA +HA +MA* L.monocytogenes L.welshimeri + PA

D10 Minced meat PC1 No +LA(4) +LA*(2) +HA* +HA* L.monocytogenes L.innocua + 1339 0,33 + / / + par dfaut +HA +HA +MA*L.monocytogenes

L.innocua + PA

I13 Fillet of duck breast PC1 No +LA* +LA* +HA* +MA* L.monocytogenes L.innocua + 9599 2,44 + / / + par dfaut +HA +MA* +MA*L.monocytogenes

L.innocua + PA

T10 Minced beef burger with oignons PC1 No +LA(3) +LB +HB +MA L.innocua + 9 0,00 - 8008 3,56 + +HA +HA +MA L.innocua + PAI8 Hamburger PC1 No +LA +MA +HB +MA L.monocytogenes + 7818 1,99 + / / + par dfaut +MA +HB +MA L.monocytogenes + PA

V16 Tomato burger PC1 No +HA +MA L.welshimeri + 27 0,00 - 7573 2,65 + +HA +HA* +MA L.welshimeri + PAI19 Beef steak PC1 Yes



TEST LMORFV LIS VT


COMPARISONCODE MATRICES Cat. AC CFU/25gFRASER 1/2 FRASER CONFIRMATION VIDAS LDUO CONFIRMATION

FINAL RESULT

NF EN ISO 11290-1 METHOD VIDAS LDUO METHOD

K8 Potjevlesch potted meat PC2 Yes 16,8 +HA +HA +HA +MA L.welshimeri + -2 0,00 - 7565 3,23 + +MB +MB +MA L.welshimeri + PA

C19 Chipolata sausage PC3 No -LE -LE / - 8633 2,31 + / / + par dfaut +HA +HB +MB L.monocytogenes L.welshimeri + PD

B30 Sausage PC3 No +HA +HA +HA* +HA* L.monocytogenes L.innocua + 8627 2,31 + / / + par dfaut +HA +HA* +MA*L.monocytogenes

L.innocua + PA

D3 Merguez sausage PC3 No +MA* +MB* +HA* +MA*L.monocytogenes


+ 10263 2,60 + / / + par dfaut +HA +HA* +HBL.monocytogenes


+ PA

D4 Merguez sausage PC3 No +MA +MA L.monocytogenes + 592 0,15 + / / + par dfaut +HA* +MA* +MB* L.monocytogenes L.innocua + PA

D6 Chipolata sausage PC3 No +HC +MB L.monocytogenes + 9036 2,29 + / / + par dfaut +HA +HA* +MA L.monocytogenes L.innocua + PA

D11 Andouillette sausage PC3 No +LA +LA +HA +HA L.monocytogenes + 7809 1,98 + / / + par dfaut +HA +HA +MB L.monocytogenes + PA

E9 Chipolata sausage with olives PC3 No +LA +LB +LB +MA*L.monocytogenes


+ 452 0,11 + / / + par dfaut +MA* +MA* +MA*L.monocytogenes


+ PA

E11 Toulouse sausage PC3 No +LA* +LA* +HA +HA* L.monocytogenes L.innocua + 3134 0,79 + / / + par dfaut +HA +HA* +HA*L.monocytogenes

L.innocua + PA

F7 Chipolata sausage PC3 No +MA* +MA* +MA* +MA* L.monocytogenes L.innocua + 10480 2,80 + / / + par dfaut +HA* +MB* +MBL.monocytogenes

L.innocua + PA

F8 Merguez sausage PC3 No +MB +LA +HA +MA*L.monocytogenes


+ 9529 2,54 + / / + par dfaut +HB +HB +HAL.monocytogenes


+ PA

M8 Merguez sausage PC3 No +HB +HB +MB +MB L.monocytogenes L.innocua + 9253 2,33 + / / + par dfaut +HB +HA* +HBL.monocytogenes

L.innocua + PA

V10 Strasbourg sausages PC3 No -LE / - 7711 1,99 + / / + par dfaut +HA +HA +MA L.monocytogenes + PD

D5 Lardons PC3 No +MA* +MA* +HA* +MA*L.monocytogenes


+ 8706 2,20 + / / + par dfaut +HA +HA* +HB L.monocytogenes L.innocua + PA

D8 Cured ham PC3 No +MA +MA +MA +MA L.welshimeri + 61 0,01 - 7666 2,94 + +HA +HA +HA L.welshimeri + PAG3 Paris style cooked ham PC3 No +MA +MA +HA +HA L.monocytogenes + 7946 2,01 + / / + par dfaut +MA +MA +MA L.monocytogenes + PAI12 Knuckle of ham PC3 No +MA +MA +HA +HA L.monocytogenes + 8991 2,29 + / / + par dfaut +MA +HA +MA L.monocytogenes + PA

T12 Somked lardons PC3 No +LA(2) +MA +HA* +MA* L.monocytogenes L.welshimeri + 10064 2,57 + / / + par dfaut +HA +HA* +HAL.monocytogenes

L.welshimeri + PA

T13 Spreadable sausage PC3 No +MA* +MA* +HB +MA* L.monocytogenes L.welshimeri + 9739 2,48

Documents

NF VALIDATION Validation of alternative analysis methods ... 5.3 C 3.9 C / As a result of transport conditions, 15 laboratories carried out the tests. Two laboratories were not included