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文件.docx
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SUMMARYANDEXPLANATION
ThoATBFUNGUS3atrtpenablesthodotormlnauonofthesuscoptlbllltyofCandidaandCryptococcusnoolomnorrstonntlfungalagentinaseml-solidmodiumunderconditionssimilartothereferencemothodformicro-dilution(acoordingtoEUCAST(1.6,8)andCLSI(2,6)recommendations).
PRTNCIPLE
TheATBFUNGUS3stripconsistsof18pairsofcupules.Thefirstpairdoesnotcontainanyantifungalagentandisusedasapositivegrowthcontrol.Thenoxt15pairscontain5antifungalagentsatseveralconcentrations,enablingMICdeterminationand/orclinicalsusceptibilitycategorization.Asuspensionispreparedwiththeyeasttobetested,thentransferredintotheculturemediumandinoculatedintothestrip.Afterincubation.readingofthegrowthinthecupuleslsperformedeithervisuaily.orautomaticallybytheATBInstrumentorminiAPI.TheresultobtainedgivesaMIC(AmphotericinB[AMB].Fluconazole[FCA].itraconazole[LTR].Voriconazole[VRCJ]and/orclassifiesthestrainasSensiive.IntermediateorResistant(Flucytosine[5FC]).
CONTENTOFTHEKIT(Kitfor25susceptibilitytests):
-25ATBFUNGUS3stnpsindividuallywrapped
+deslccant
-25incubationlids
-25ampulesofATBF2Medium
-25resultsheets
-1packageinsert
COMPOSITION
Strips
ThecompositjonoftheATBFUNGUS3stripisindicated
inthe<
insert.
Medium
ATBF2Medium
YeastN;trogonBase6.7g
Glucose6.5g
Asparagine1.5g
Disodiumphosphate2.5g
Trisodlumcttroto2.5g
Potassiumnitrate5.5g
Agar1.5g
Demineraiuedwater1000ml
PH:
6.5-6.8
NOTE:
ThecloloroftheATBF2Mediummayvaryslightly
Fromoneampuletoanother.Thisdoesnotaffectproductperformanceinanyway.
REAGENTSANDMATERIALREQUIREDBUTNOTPROVIDED
ReagentsandInatrumonts
-APINaCl0.85%Medium(Ref.20070or20040or20230)or
APISuspensionMedium(Ref.70700or70640or20150)
-McfarlandStandard(Ref70900)or
DENSIMAT(Ref99234)orATBDensltometer
-ATBElectronicPipette(consultbioMerieux)or
ATBInoculatorandTips(Ref.15710)
-ATBInstrumentorminlAPIwithsoftware(consult
bioMerleux)
Matorlal
-Pipette
-Ampuleprotector
-Ampulerack
-Air-tightcontainer,e.g<
-Generalmicrobiologylaboratoryequipment
WARNINGSANDPRECAUTIONS
·ForInvitrodiagnosticuseonly.
·Forprofessionaluseonly.
·Allspecimens,yeastculturesandinoculatedproductsshouldbeconsideredinfectiousandhandledappropriatelyAseptictechniqueandusualprecautionsforhandlingyeastsshouldbeobservedthroughoutthisprocedure.RefertoCLSIM29-A,ProtectionofLaboratoryWorkersFromOccupationallyAcquiredInfecfions;ApprovedGuideline-Currentrevision.Foradditionalhandlingprecautions,referto'BiosafetyinMicrobiologicalandBiomedicalLaboratories-CDC/NIH-Latestedition,ortotheregulationscurrentlyinuseIneachcountry.
·Donotusereagentspasttheexpirydate.
·Beforeuse,checkthatthepackagingandcomponents
areintact.
·Donotusestripswhichhavebeendamaged:
cupules
deformed,desicantsachetopen,etc.
·Allowmediatocometoroomtemperaturebeforeuse.
•Openampulescarefullyasfollows
-Placetheampuleintheampuleprotector.
-Holdtheprotectedampuleinonehandinaverticalposition(whiteplasticcapuppermost)
-Pressthecapdownasfaraspossible.
Positionthethumbtiponthestriatedpartofthecapandpressforwardtosnapoffthetopoftheampule.
-TaketheampuleoutoftheampuleprotectorandputtheProcedureasideforsubsequentuse.
Carefullyremovothecap
·Theperformancedatapresentedwereobtainedusingthe
procedureindicatedinthispackageinsert.Anychangeor
modificationintheproceduremayaffecttheresults.
·Theinterpretationandvalidationoftheantifungalsusceplibilitytestresultsshouldbemadetakingintoconsiderationthepatient'shistory.Thesourceofthespecimen,theidenlificationofthestrain.And,ifnecessary,theresutltsofanysupplementarytestsperformedandanylocalapplicablerecommendations,UseoftheATBExpertsystemfacilitatesmterpretationandvalidation.
STORAGECONDITIONS
Thestripsaridmediashouldbestoredat2-8'CInthedarkuntiltheexpirydateindicatedonthebox.
SPECIMENS(COLLECTIONANDPREPARATION)
TheATBFUNGUS3stripisnotforusedirecuywithclinicalorothorspecimens.Themicroorganismstobeidentifiedmustfirstbeisolatedonasuitableculturemediumaccordingtostandardmicrobiologicaltechniques.ThefollowingbioMerieux
isolationmediacanbeused.
-Sabouraud2agar.
-Sabouraudagar+2%Glucose[EUCAST(1.8)andCLSI/NCCLS
(2)recommendedmedium]
-SabouraudGontamion-Chloramphenicol2agar.
-CPSID3agar
-Columbiaagar+5%sheepblood,
-TrypcaseSoyagar+5%horseblood.
-Chocolateagar+PolyVitex,
-BCPagar,
-CandidaID2agar
INSTERUCTIONSFORUSE
Preparationofstrip
·Removethestripfromitspackaging
·Recordtheidentificationnumberoftheyeaststraintobetestedontheelongatedflapofthestrip.
Preparationofthoinoculum
·OpenanampuleofAPINacl0.85%Medium(orAPISuspensionMedium)asindicatedintheparagraph"WarningsnndPrecautions"ofthepackageinsertforthesemedia.
·Usingapipette,takeupseveralcolonieswhicharenomorethan4daysold.Prepareasuspensionwithaturbtdityequiva+entto2McFarland.
·ComparetotheturbiditycontrolintheMcFarlandStandardkitorusetheATBDensitometerortheDENSIMAT(refertotheUser'sManual)Thissuspensionmustbeusedimmediatelyafterpreparation.
NOTE:
ittsrecommendedcontrolthepurityoftheinoculumandtore-isolateinthecaseofmixedcultures.
·Usingapipette,transfer20ulofthissuspension
intoanampuleofATBF2Medium.
inoculationofthestrip
·MATJUALinoculation:
-HomogenizeATBF2MediumwiththeATBElectronicPipette,avoidingthoformationofbubbles.
-Inoculatethestripbydispensing135ulofATBF2MediumintoeachcupuleusingtheATBElectronicPipette(approximately3×10yeasts/mlor4×10yeasts/cupule).
AUTOTAATICinoculation:
RefertotheATBinoculatorUser'sManual.
·PlacealidOnthestrip
·Putthestripinair-EightcontalperoraGENBox-typeJarcontainingmoistabsorbentpaper
·IncubateCandidafor24hours(2hours)andCryptococcusneorormonsfor48hours(6hours)at35'C(2'C)inaerobicconditions.
READINGANDINTERPRETATION
Checkthatgrowthinthecontrolcupulesissufficient.ForCandidastrains.ifgrowthinsufficientorifthereisnogrowthinthese2cupules.Makingitditftcultorimpossibletoreadthescripafter24hoursofincubation.Incubateforafurthe:
24hoursinthesameconditions.
Theabsenceofgrowthorinsuffioentgrowthinoneofthetwocontrolcupulesafter24or48hoursofincubation.invalidatesthetestwhichmustberepeated
1.MTCdotermtnation(AMB,FCA.ITR.VRC):
·ObserveforandquantjfygrowthineachcupulebyreadingvisuallyorautomaticallyusingtheATBinstrumentorminiAPI(refertotheUsersManuals).
·Beforeperformingvisualroading,itisrecommendedtoplacethestriponablackbackgrourld(ATBFUNGUS3visualreadingaidavailablefrombioMerieux)-Foreachantifungalagent.startwiththeIowstconcentrationandrecordthegrowthscoreontheresultsheetforeachofthecupulescomparedwiththecontrolcupules:
Debnition
Score
Noreductionmcvowth
4
Slightreductionjngrowth
3
Disljnctreductioningrowth
2
Veryweakgrowth
1
Nogrowth
0
-ForAmphotericinB(AMB).TheMICcorrespondstothelowestconcentrationenablingcompletegrowthinhrbition(score="0").
Note:
one(orseveral)isolatedcoloniesorsignsofgrowthattheperipheryofthecuputeshouldbeinterpretedas1.
-ForFluconazole(FCA),ltraconnzole(1TR)andVoriconazote(VRC).Asthepossibilityoftrallinggrowthexists,theMICcorrespondstothelowestConcentrationoftheantifungalagentwithwhichascoreof"2","1"or"0"isobtained,
Note:
signsofgrowthattheperipheryofthecupuleshouldbeinterpetedas"0"or"1".
2.Flucytoslno(5FC)intorprotation:
ObserveforandqunntifygrowthinbothcupulesbyreadingvisuallyorautomaticallyusingtheATBinstrumentorminiAPI(refertotheUser'sManuals).
ForFlucytosinetestedattwoConcentrations:
GrowthleoroRosuthThoatralnla
0/1/20/1/2--SENSTTIVE
3/40/1/2++INTERMEDTATE
3/43/4++RESISTANT
Notes:
Thedefinitionofthegrowthscoresisidenticaltothat
IndicatedaboveforMICdetermination.Signsofgrowthattheperipheryofthecupuleshouldbeinterpretedas"0"or"1".
3.Breakpointsused
CLSuNCCLSrecommendedbreahpohlts
(Inmgn)forCandidasp(2,3)
s
i
r
Flucytogjne
<=4
8-16
>=32
AmphotericinB
ND
ND
ND
Fluconazole
<=8
16-32
>=64
ltraconazole
<=0.125
0.25-0.5
>=1
Voriconazole
<=1
2
>=4
ND:
NotformallydelinedbytheCLSI/NCCLS
Notes:
-AsCandidakruseiisaspecieswithintrinsicresistancetoFruconazole,thetestshouldbesystematicallyinterpretedasR.
ForAmphotericinB,aMIC>=2mg/1suggests
resistance
(2)
DataProvidedforinformationpurpososonly(absenceofofficialrecommendattions):
Breakpoints(Inmg/I)for
Cryptococcusneoformans
s
i
r
Flucytosine(10)
<=4
8-16
>=32
AmphotericinB
ND
ND
ND
Ftuconazole(5)
<=4
8
>=16
ltraconazole
ND
ND
ND
Voriconazole
ND
ND
ND
ND:
NotDefined
():
Literaturereference
Note:
ForAmphotencinB,Amic>=2mg/lsuggestsresistance(theMICsusuallyobtainedforCrneofomansare0.5and1mg/l).
NOTES:
Whenporformingautornaticroadingofthestrip:
-checkthatthemiddlepartofthestripiscreansothatthereadercanrecognizethestrip
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