SHEA 2012 Diekema
Transcript of SHEA 2012 Diekema
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MolecularTes,ngand
Infec,onPreven,on
DanielJ.DiekemaMDD(ABMM)
UniversityofIowaCarverCollegeofMedicine
Disclosures:
ResearchfundingfrombioMerieuxCerexaMerckPfizerPurthread
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Objec,ves
• Knowwhichavailablemoleculardiagnos,c
testsholdpromiseforimprovingthediagnosis
andpreven,onofHAIs
• Understandthelimita,onsofmolecular
diagnos,csandhowthoselimita,onsmay
impactdiagnosisandsurveillanceac,vi,es
• Beabletodiscusswithyourlabdirectortheprosandconsofintroducingcertainmolecular
assays.
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Caveats:Intheinterestof,me…
• Willemphasizeapplica,ontohealthcareepi
– mostcommonlyencounteredissues
– MDROsC.difficilemoleculartyping• Willnotdiscussspecificproductsindetail
– Subjecttorapidchangeoenmorethanone
usingaspecificapproach
• Emphasizetechnologyavailabletomostlabs
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Majorusesofmoleculartes,ngfor
infec,onpreven,on• Detec,onofpathogensandresistance(MDRO)
– Allowearlierinterven,on
– Focusedtherapyisola,ondecoloniza,on
• Typingtoassessgene,crelatedness
– Inves,gatepathogenesistransmissionoutbreaks
– PFGEribotypingrep-PCRmul,locussequencetypingspatyping(S.aureus)
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Moleculardetec,onofpathogens
• Directfromsampleorfromculturegrowth
• Advantagesincomparisonwithculture:
– Fasteranaly,cturnaround,me(TAT)
– Increasedsensi,vity(some,mes)
– Bestalterna,vefordifficult-to-growpathogens
PCRornucleicacidamplifica,ontes,ng(NAAT)methods
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Moleculardetec,onofMRSAfromnares
Thisshouldbeeasyright?
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Butproblemsremain
• SingletargetmayremainaerlossofmecA
(“mecAdropouts”)
– Foundin5-10%ofall+samplesinsomecenters
• ArbefevilleSSetal.JClinMicrobiol2011;49:2996-9.
• SomeMR-CoNSmaytestposi,ve• Malhotra-Kumaretal.JClinMicrobiol2010;48:4598.
• Posi,vepredic,vevaluefortheseassayscan
thereforebelowerthanculture• Herdmanetal.JClinMicrobiol2009;47:4102.
Newtes;ngapproachesincludemecAprimers,toaddressbullet1,
butremainvulnerabletomixedpopula;onsorfalse+MR-CoNS
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Falseposi,vesaren’ttheonlyissue
• mecALGA251
– UndetectedbycurrentPCR
– WidespreadinUKamongLA-MRSA
– Mul,plespaandMLSTtypes
• I’msurethiswon’teverhappenagain…
Garcia-Alvarezetal.LancetInfectDis2011;11:595-603.
ShoreACetal.An,microbAgentsChemotherJune2011
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Themoralofthisstory:
Don’ttossthoseagarplates!• LessvulnerablethanPCRtogene,cvariability
– Emergingclones“emptycassee”variants
• ImprovedPPVhelpsdetectassayproblems• OrganismisavailableforASTtypingetc.
• Greaterflexibilityofuse(sitesample)
• Addingamoleculartesttocultureratherthan
replacingculture
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TheimportanceofMSSAdetec,on
• Focusperiopdecoloniza,onofSAcarriers
• Tailoran,microbialprophylaxisforMRSAcarriers
Bodeetal.NEnglJMed2010;362:9-17
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• Usesbacteriophagestoamplifysignal
(mustbespecies-specific)
• SimpleimmunoassaydetectsphageAg
• Tes,ng+/-anan,bio,ccanserveasamethodofsuscep,bilitytes,ng
• OnlycurrentFDA-approvedassayisfor
bloodcultures+forGramposi,vecocci
www.microphage.com
Anewtestmethodofinterestnarestestnotyetavailable:
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Rapiddetec,onofMRSA/MSSA
Frombloodculturesornaressamples
Method TAT Sens Spec Equipment Pertest
PCR(various)* 1-4 90-98 91-99 30-150K 25-50
Bacteriophage** 5-6 92 98 n/a ?
Chromogenicagar 18-48 60-90 90-100 n/a 5
*Commonlyusedcommercialassays:homebrewPCRisfarlessexpensive
**bloodcultureonlynarestestindevelopment
***TATisanaly=cTATonly!
Bhowmicketal.50thICAACBostonMA2010AbstractD-155.
CarrollKC.MolDiagnTher2008;12:15
Malhotra-Kumaretal.JClinMicrobiol2010;48:4598.
ReyesRCetal.DiagnMicrobiolInfectDis2008;60:225
PapeJetal.JClinMicrobiol2006;44:2575.
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Scenario
Yourlabdirectorcomestoyourmonthlyinfec,oncontrol
mee,ngtoaskforyoursupporttobringinanewreal-,me
PCRtestforMRSAdirectfromnasalswabs.Yourhospital
currentlyscreensalladmissionstoselectedunitshasa4%
admissionprevalenceandhasseena60%declineinMRSA
HAIrateoverthepast5years.Thecurrentscreeningmethod
ischromogenicagarwith~24hrTAT.
Thelabdirectorasks:willadop,ngthenewtestbe
cost-effec,ve?Willitresultinimprovedpreven,onofMRSAtransmissionandinfec,on?
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Bestwaytoanswertheques,on:
• Prospec,vetrialwithconcurrentcontrols!
• Culturevs.NAAT(PCRwithreducedTAT)
• OutcomestoincludeMRSAtransmission
(acquisi,on)andinfec,onevents• Atleast3suchtrialshavebeenpublishedall
useunit-basedcrossoverdesigns:
1. Jeyaratnametal.BMJ2008;336:927-930.
2. Aldeyabetal.JHospInfect2009;71:22-8.
3. Hardyetal.ClinMicrobiolInfect2010;16:333.
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Summaryofcontrolledtrials
• TwoshownodifferenceinMRSAoutcomes12
– ButoverallTATonlyreducedto19-22hours
• HardyetalshowedbenefitforPCR3:
– Ptsoncxunits1.5XmorelikelytoacquireMRSA
– Almost2-foldmoreunscreenedinculturearm
– Only17%ofcolonizedwereproperlyisolated
– 71%ofPCR+decolonized(vs.41%incx+arm)• Reallyatrialofdecoloniza,on(notof“ADI”)
1. Jeyaratnametal.BMJ2008;336:927-930.
2. Aldeyabetal.JHospInfect2009;71:22-8.
3. Hardyetal.ClinMicrobiolRev2010;16:333-39.
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Whatisyourtrueturnaround,me?
Pre-analy,c
• Collec,on
• Transport
Analy,c
• Processing
• Tes,ng
Post-analy,c
• Repor,ng
• Interven,on
Howlongiseachstep?Whatpor,onofTATisaributabletothetest?
ArePCRtestsbatched?
Isthe“efferentarm”ofyourprogramfunc,oning?
Itisn’tassimpleas2hoursversus24-72hours….
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MRSA/MDROpreven,on:
Somuchmorethanalabtest• Environmentaldisinfec,onprac,ces
• Handhygieneadherence(40%or90%?)
• Contactprecau,onsadherence• Hospitaldesign:%singlerooms
• AdherencetoCLABSI/VAP/SSIbundles
• Decoloniza,onprac,ces(CHGmupirocin)• Pa,entpopula,on(riskfactors)
• AdmissionMRSAprevalence
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Considertwounits
A. 40%adherencetoHHandCPpoor
implementa,onofDAIpreven,onbundles
B. 90%adherencetoHHandCPnearly100%
adherencetoDAIbundlemeasures
• ReduceTATtozeroinunitAyous,llhaveno
impactonMRSAdisease.
• InunitBMRSAdiseasefalls85-90%before
screeningcanbeimplemented.
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ClinMicroNetSurvey2011Hasyourlaboratoryadoptedarapidmoleculartestfordetec,on
ofMRSAand/orMSSAdirectfromclinicalsamples?
35 (92%) for nares
13 (34%) for BSI38 (54%)32 (46%)
24 (75%): cost
3 (9%): performance4 (13%): both
Diekema D, Peterson L. ClinMicroNet survey.
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Hasyourlaboratoryadoptedarapidtestand
thengonebacktoaculture-basedtest?• Thirteen(19%)ofthelabsreportedhaving
gonebacktoanagar-basedmethodfromarapiddetec,ontest
– Eightwentcompletelyback(alltes,ng)
– Fives,lldidsomerapidmoleculartes,ng• Physicianpreference
• Agar-basedforoneunitwithendemicemptycassee
• WentbackfornaresbutnotBSI
– Reasons:Cost(7)performance(3)cost+perf(3)• Emptycasseesfailuretodemonstratesavings/impact
Diekema D, Peterson L. ClinMicroNet survey.
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Quoteexcerptsfromsurveyresponses• MaygobacktoculturefromPCRb/cpromisedcostsavingshaven'tbeen
demonstrated
• DidacomparisonandPCRnotbeerathighercost
• AdoptedPCRbuthadnoimpactonMRSAratesinICU
• Concernsaboutsensi,vityandapplica,ontonon-naressites
• Costplusnotvalidatedfornon-naressamples
• NodiffinperformanceinourhandsTATnotdifferentenough
• ActualTATinourlabnotmuchdifferentforchromvsPCR
• Wouldcostover500KperyearnoproofthatfasterTATcausesfewer
MRSAinfxs
• Wesaveabout450Kperyearbyusingchromogenicagar
• Nostudyshowingreduc,oninMRSAtransmissionformolecularvs24hr
chromogenicculturedetec,on
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Lessonslearnedfrommolecular
tes,ngforS.aureus
• Gene,cvariabilityandevolu,onwillbeanongoingchallengeforNAATtests
• Cultureback-upremainsimportant• Analy,cTATmaydifferfromactualTAT
– Knowhowyourlabwillincorporatethetest
• Costsavinges,matesmustbeindividualized:NAATmaynotbetherightchoiceforall
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Vancomycin-resistantenterococci
• AnotherMDROwithwell-characterized
resistancegenes(vanA/B/C/D/E/G/L/M)
• vanAandvanBdetec,onmostimportant
– Mobilegene,celementsE.faeciumandE.faecalis
• Problemforrapiddetec,on:reservoir
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RapidVREdetec,on
• Moleculararac,vegivenTATofculture
• Someobstaclestokeepinmind:
– lowerlimitofdetec,on(varies~10-100cfu/ml)
• effectofan,microbialsondetec,on!
– vanBispresentinselectedgutanaerobes
• Whatdoesanega,veVREscreenmean?
– Beforeversusaeran,microbialexposure
• Whatdoesaposi,veVREscreenmean?
Ballardetal.An,microbAgentsChemother2005;49:77-81.
Gazinetal.EurJClinMicrobiolInfectDis2012;31:273-76.
Donskeyetal.NEnglJMed2000;343:1925-32.
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PerformanceofvanA/vanBscreenvs.broth
enrichedculture
XpertvanA/vanBpackageinsert(www.cepheid.com)
Gazinetal.EurJClinMicrobiolInfectDis2012;31:273-76.
Posi,vepredic,vevalueinmul,centerevalua,on~50%
ForvanBPPVinonestudywas<10%for2diffassaysDon’ttossthoseagarplates!
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Lessonslearnedfrommolecular
tes,ngforVRE• Tes,ngsamplesfromcomplexorganism
popula,ons(directfromsamplesofnon-
sterileenvironment)ischallenging• Otherspeciesshareresistancedeterminants
– mecA:foundinS.aureus+coagnega,vestaph
– vanB:foundinVRE+selectedanaerobes
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Peleg and Hooper. N Engl J Med 2010;362:1804-13.
Moleculardetec,onofMDR-GNR
Evenmorecomplicated!
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Rapidemergenceandspreadofcarbapenemases(NDMKPCVIMIMP)
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Obstaclestowidelyavailable
moleculardetec,onofMDR-GNR• Mul,plicityoftargetsemergingnewtargets
– Difficultto“keepup”withFDA-approvedmethods
• Genepresence≠geneexpression – e.g.chromosomalcephalosporinaseinE.coli
• Someimportantphenotypesarecomplex
– CarbapenemRfromstablyde-repressedampC
cephalosporinase+porinmuta,on…
• Therealmofreferralreferenceresearchlabs
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Currentapproaches
• PCRtarge,ngmostcommonproblems – e.g.“homebrew”KPCdetec,onassayscommon
– onisolatedcolonieslimitedtargets
• Forscreeningmicroarraymostpromising
Naasetal.JClinMicrobiol2011;Feb16(onlineaheadofprint)
SpecificnucleicacidprobesOnlyhybridizedprobesamplified
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MDR-GNRNAATtes,ng:Summary
• Complexity/varietyofresistancedeterminantsisachallengefortestdevelopment
• Fornow“screening”forcarriagebestdone
withselec,veculture(e.g.chromogenicagar) – Laborintensiveandidealscreeningsitesassay
performances,lluncertain
• Performanceofanynewassayinmixedand
complexsamples(e.g.stoolbloodculturebrothrespiratorysecre,ons)willbekey
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Moleculardetec,onofC.difficile
• Therighttargetformoleculartes,ng:
– Difficult/laborioustoculture
– Standardtests(EIA)performpoorly(60-70%sens)
– Toxingenepresencecorrelateswithpathogenicity
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ToxinEIAs:Poorsensi,vityandPPV
GoldenbergSDetal.JHospInfect2011;79:4-7.
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SeveralNAATtestsnowavailable
• MosttargettcdBgene(genenottoxin!)
• Importanttotestonlythosewithdisease
• Sensi,vityandspecificityinmid-90s• Repeattes;ngisnotnecessary
Tableadaptedfrom:CarrollKC.Anaerobe2011;17:170-174.
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Alessexpensiveapproach?
Twostep:Glutaminedehydrogenase→PCR/NAAT
Problem:GHDEIAisn’tsensi,veenough(?Mid-80%)
ChapinKetal.JMolDiagn2011;13:395-400.
Selvarajuetal.DiagnMicrobiolInfectDis2011;71:224-29.
CarrollKC.Anaerobe2011;17:170-174.
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Besttes,ngprac,cesareessen,al!
Diagnos,cs101• PPVdependsupondiseaseprevalence
• Prevalencedropsiftestisperformedonthose
withlowpre-testprobabilityofdisease – e.g.ata4-5%posi,vityratePPV~50%
– Usuallyreflectspoortes,ngprac,ces
• NOtes,ngofnon-diarrhealstool(C-T-C)
• NOrepeattes,ngwithin7days
• NOtestofcure
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Whatisyourprevalence?
Pooledsensi,vity=90%
Pooledspecificity=96%
Deshpandeetal.ClinInfectDis2011;53:e81-e90.
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HowaboutthoseC.difficilerates!
Theimpactof↑sensi,vity
EIA PCR
Nsamples 2579 2534
N(%)posi,ve 167(6.5) 382(15.1)
CDIrates* 4.9 10.3*cases/10000pa,entdays
FongKSetal.InfectContHospEpidemiol2011;32:932.
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C.difficiletes,ng:summary
• PCR/NAATseemsthebestsingleop,on
• PrepareforCDADrateincreaseofup>50%
• Strictenforcementoftestrejec,onpolicies
– NPV>99%allowsthispreservesgoodPPV
• Considerreviewofclinicaltes,ngapproach
– >3diarrheastoolsperdayan,bio,cusehistory
• Periodicallyassessposi,vityrate – If<15%assessrejec,onpolices/tes,ngprac,ce
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UseofMolecularTyping
• Studypathogenesisofinfec,on – Colonizingandinfec,ngstrains
– Contamina,onversuspathogen
• Assessextentandmodeofpathogentransmission – Effec,venessofinfec,oncontrolefforts
– Outbreakinves,ga,on
• Detectepidemiologicallyimportant
pathogen(e.g.NAP1/BIC.difficile)
Requiresmaintaininganorganismbank
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Methodsareallbasedupondetec,ng
differencesinnucleo,desequence• Bandingpaerns:sizeoffragmentsproducedby
amplifica,onand/orenzyma,cdiges,onof
genomicDNA
– PFGERFLPribotypingAFLPrep-PCRRAPDMLVAetc.
• DNAsequencing:polymorphismofDNAsequences
– Mul,locussequencetyping(MLST)spa(S.aureus)
– Wholegenomesequencing
• DNAhybridiza,on:usingnucleo,deprobes
– Spoligotyping(macroarray—M.tb)microarrays
LiRaoultandFornier.FEMSMicrobiolRev2009;33:892-916.
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CriteriaforEvalua,ngTypingSystems
• Typeability:Abilitytoobtainan
unambiguousresultforeachisolate
analyzed
• Reproducibility:Abilitytogivethesame
resulteach,meanisolateistested
• Discriminatorypower:Abilityto
differen,ateamongunrelatedstrains
• Cost($$laboreaseofuseetc.)
• Turnaround,me
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ChoosingaTypingMethod
• PFGE:excellentdiscrimina,onflexibility
– LaborintensivelongerTAT(2-3days)
• SeveralPCRmethodsprovidegood
discrimina,onw/fasterTAT(rep-PCRMLVA)
• Methoduseddeterminedby:
– Purpose(localoutbreak/clustervs.“library”)
– Organism(somespecificforbug(e.g.spatyping))• Combina,onofmethodsmayprovide
greatestdiscrimina,on(e.g.MRSA)
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Comparingcommontypingmethods
Method Reproducibility Discrimina,on Cost TAT
PFGE Intra(Y)/Inter(V) Excellent $$ 2d
rep-PCR Yes(autoonly) Good $$ 4h
MLVA Yes VeryGood $ 4h
Ribotype Yes Fair-Good $$$ 1d
MLST Yes Fair $$ 2-3d
WGS Yes Excellent $$$ ??
Whenwillrapidthroughputsequencingcometotheclinicallab?
Howwillwehandleallthedata?
Resistancedetec,ontypingvirulenceetc.etc.etc.
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Typingdatainformsgoodepidemiology
(itdoesn’treplaceit)
• “Indis,nguishable”≠immediatecommonsource
Same:
PFGE
spa
MLST
SCCmecrep-PCR
Zazakovaetal.NEnglJMed2005;352:468.
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Typingdatainformsgoodepidemiology
(itdoesn’treplaceit)
• “Different”(typeorspecies)doesnot“rule
out”transmission
– e.g.spreadofplasmid-mediatedESBLs
RegionalKPCspread
MostwereKPNInterspeciestoE.coli
onatleast2occasions
Wonetal.ClinInfectDis2011;53:532.
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Moleculartyping:Summary
• Manyacceptablemethodsavailable
• Discussper,nentissueswithyourlabdirector
– TAT:needforinhouseversussendouttes,ng
– Localuseforcluster/outbreaksonly?
– Needforlibrarymethod(e.g.MLSTspa)
• MostwellservedbyPFGErep-PCRMLVA
• Mostimportantpointistointerpretresultsincontext(buggene,csofRepidemiology)
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Proteomics
• Matrixassistedlaserdesorp,on/ioniza,on–,me
offlight(MALDI-TOF)
• Producesdetailedprotein
fingerprint
• AccuratespeciesID
– Rapidinexpensive
– Requiresisolatedcolony
• Otherpoten,alapplica,ons?
– Straintyping
– Resistancedetec,on
– Analysisofmixedsamples
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Summary
• Moleculartes,ngincreasinglyoffersfaster
moresensi,vedetec,onofHAIpathogens
• Notestisperfect:understandlimita,onsand
implica,ons(diagnosissurveillancecost)
• Closecollabora,onwithlabdirector
– Regularroundsinthemicrolab
– Labpar,cipa,ononinfec,oncontrolcommiee – Knowimplica,onsofICini,a,vesonthelab