shea 2012 diekema

8/2/2019 SHEA 2012 Diekema

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MolecularTes,ngand

Infec,onPreven,on

DanielJ.DiekemaMDD(ABMM)

UniversityofIowaCarverCollegeofMedicine

Disclosures:

ResearchfundingfrombioMerieuxCerexaMerckPfizerPurthread



Objec,ves

• Knowwhichavailablemoleculardiagnos,c

testsholdpromiseforimprovingthediagnosis

andpreven,onofHAIs

• Understandthelimita,onsofmolecular

diagnos,csandhowthoselimita,onsmay

impactdiagnosisandsurveillanceac,vi,es

• Beabletodiscusswithyourlabdirectortheprosandconsofintroducingcertainmolecular

assays.



Caveats:Intheinterestof,me…

• Willemphasizeapplica,ontohealthcareepi

– mostcommonlyencounteredissues

– MDROsC.difficilemoleculartyping• Willnotdiscussspecificproductsindetail

– Subjecttorapidchangeoenmorethanone

usingaspecificapproach

• Emphasizetechnologyavailabletomostlabs



Majorusesofmoleculartes,ngfor

infec,onpreven,on• Detec,onofpathogensandresistance(MDRO)

– Allowearlierinterven,on

– Focusedtherapyisola,ondecoloniza,on

• Typingtoassessgene,crelatedness

– Inves,gatepathogenesistransmissionoutbreaks

– PFGEribotypingrep-PCRmul,locussequencetypingspatyping(S.aureus)



Moleculardetec,onofpathogens

• Directfromsampleorfromculturegrowth

• Advantagesincomparisonwithculture:

– Fasteranaly,cturnaround,me(TAT)

– Increasedsensi,vity(some,mes)

– Bestalterna,vefordifficult-to-growpathogens

PCRornucleicacidamplifica,ontes,ng(NAAT)methods



Moleculardetec,onofMRSAfromnares

Thisshouldbeeasyright?



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



Falseposi,vesaren’ttheonlyissue

• mecALGA251

– UndetectedbycurrentPCR

– WidespreadinUKamongLA-MRSA

– Mul,plespaandMLSTtypes

• I’msurethiswon’teverhappenagain…

Garcia-Alvarezetal.LancetInfectDis2011;11:595-603.

ShoreACetal.An,microbAgentsChemotherJune2011



Themoralofthisstory:

Don’ttossthoseagarplates!• LessvulnerablethanPCRtogene,cvariability

– Emergingclones“emptycassee”variants

• ImprovedPPVhelpsdetectassayproblems• OrganismisavailableforASTtypingetc.

• Greaterflexibilityofuse(sitesample)

• Addingamoleculartesttocultureratherthan

replacingculture



TheimportanceofMSSAdetec,on

• Focusperiopdecoloniza,onofSAcarriers

• Tailoran,microbialprophylaxisforMRSAcarriers

Bodeetal.NEnglJMed2010;362:9-17



• Usesbacteriophagestoamplifysignal

(mustbespecies-specific)

• SimpleimmunoassaydetectsphageAg

• Tes,ng+/-anan,bio,ccanserveasamethodofsuscep,bilitytes,ng

• OnlycurrentFDA-approvedassayisfor

bloodcultures+forGramposi,vecocci

www.microphage.com

Anewtestmethodofinterestnarestestnotyetavailable:



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.



Scenario

Yourlabdirectorcomestoyourmonthlyinfec,oncontrol

mee,ngtoaskforyoursupporttobringinanewreal-,me

PCRtestforMRSAdirectfromnasalswabs.Yourhospital

currentlyscreensalladmissionstoselectedunitshasa4%

admissionprevalenceandhasseena60%declineinMRSA

HAIrateoverthepast5years.Thecurrentscreeningmethod

ischromogenicagarwith~24hrTAT.

Thelabdirectorasks:willadop,ngthenewtestbe

cost-effec,ve?Willitresultinimprovedpreven,onofMRSAtransmissionandinfec,on?



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.



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.



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….



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



Considertwounits

A. 40%adherencetoHHandCPpoor

implementa,onofDAIpreven,onbundles

B. 90%adherencetoHHandCPnearly100%

adherencetoDAIbundlemeasures

• ReduceTATtozeroinunitAyous,llhaveno

impactonMRSAdisease.

• InunitBMRSAdiseasefalls85-90%before

screeningcanbeimplemented.



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.



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.



Quoteexcerptsfromsurveyresponses• MaygobacktoculturefromPCRb/cpromisedcostsavingshaven'tbeen

demonstrated

• DidacomparisonandPCRnotbeerathighercost

• AdoptedPCRbuthadnoimpactonMRSAratesinICU

• Concernsaboutsensi,vityandapplica,ontonon-naressites

• Costplusnotvalidatedfornon-naressamples

• NodiffinperformanceinourhandsTATnotdifferentenough

• ActualTATinourlabnotmuchdifferentforchromvsPCR

• Wouldcostover500KperyearnoproofthatfasterTATcausesfewer

MRSAinfxs

• Wesaveabout450Kperyearbyusingchromogenicagar

• Nostudyshowingreduc,oninMRSAtransmissionformolecularvs24hr

chromogenicculturedetec,on



Lessonslearnedfrommolecular

tes,ngforS.aureus

• Gene,cvariabilityandevolu,onwillbeanongoingchallengeforNAATtests

• Cultureback-upremainsimportant• Analy,cTATmaydifferfromactualTAT

– Knowhowyourlabwillincorporatethetest

• Costsavinges,matesmustbeindividualized:NAATmaynotbetherightchoiceforall



Vancomycin-resistantenterococci

• AnotherMDROwithwell-characterized

resistancegenes(vanA/B/C/D/E/G/L/M)

• vanAandvanBdetec,onmostimportant

– Mobilegene,celementsE.faeciumandE.faecalis

• Problemforrapiddetec,on:reservoir



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.



PerformanceofvanA/vanBscreenvs.broth

enrichedculture

XpertvanA/vanBpackageinsert(www.cepheid.com)

Gazinetal.EurJClinMicrobiolInfectDis2012;31:273-76.

Posi,vepredic,vevalueinmul,centerevalua,on~50%

ForvanBPPVinonestudywas<10%for2diffassaysDon’ttossthoseagarplates!



Lessonslearnedfrommolecular

tes,ngforVRE• Tes,ngsamplesfromcomplexorganism

popula,ons(directfromsamplesofnon-

sterileenvironment)ischallenging• Otherspeciesshareresistancedeterminants

– mecA:foundinS.aureus+coagnega,vestaph

– vanB:foundinVRE+selectedanaerobes



Peleg and Hooper. N Engl J Med 2010;362:1804-13.

Moleculardetec,onofMDR-GNR

Evenmorecomplicated!



Rapidemergenceandspreadofcarbapenemases(NDMKPCVIMIMP)



Obstaclestowidelyavailable

moleculardetec,onofMDR-GNR• Mul,plicityoftargetsemergingnewtargets

– Difficultto“keepup”withFDA-approvedmethods

• Genepresence≠geneexpression – e.g.chromosomalcephalosporinaseinE.coli

• Someimportantphenotypesarecomplex

– CarbapenemRfromstablyde-repressedampC

cephalosporinase+porinmuta,on…

• Therealmofreferralreferenceresearchlabs



Currentapproaches

• PCRtarge,ngmostcommonproblems – e.g.“homebrew”KPCdetec,onassayscommon

– onisolatedcolonieslimitedtargets

• Forscreeningmicroarraymostpromising

Naasetal.JClinMicrobiol2011;Feb16(onlineaheadofprint)

SpecificnucleicacidprobesOnlyhybridizedprobesamplified



MDR-GNRNAATtes,ng:Summary

• Complexity/varietyofresistancedeterminantsisachallengefortestdevelopment

• Fornow“screening”forcarriagebestdone

withselec,veculture(e.g.chromogenicagar) – Laborintensiveandidealscreeningsitesassay

performances,lluncertain

• Performanceofanynewassayinmixedand

complexsamples(e.g.stoolbloodculturebrothrespiratorysecre,ons)willbekey



Moleculardetec,onofC.difficile

• Therighttargetformoleculartes,ng:

– Difficult/laborioustoculture

– Standardtests(EIA)performpoorly(60-70%sens)

– Toxingenepresencecorrelateswithpathogenicity



ToxinEIAs:Poorsensi,vityandPPV

GoldenbergSDetal.JHospInfect2011;79:4-7.



SeveralNAATtestsnowavailable

• MosttargettcdBgene(genenottoxin!)

• Importanttotestonlythosewithdisease

• Sensi,vityandspecificityinmid-90s• Repeattes;ngisnotnecessary

Tableadaptedfrom:CarrollKC.Anaerobe2011;17:170-174.



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.



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



Whatisyourprevalence?

Pooledsensi,vity=90%

Pooledspecificity=96%

Deshpandeetal.ClinInfectDis2011;53:e81-e90.



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.



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



UseofMolecularTyping

• Studypathogenesisofinfec,on – Colonizingandinfec,ngstrains

– Contamina,onversuspathogen

• Assessextentandmodeofpathogentransmission – Effec,venessofinfec,oncontrolefforts

– Outbreakinves,ga,on

• Detectepidemiologicallyimportant

pathogen(e.g.NAP1/BIC.difficile)

Requiresmaintaininganorganismbank



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.



CriteriaforEvalua,ngTypingSystems

• Typeability:Abilitytoobtainan

unambiguousresultforeachisolate

analyzed

• Reproducibility:Abilitytogivethesame

resulteach,meanisolateistested

• Discriminatorypower:Abilityto

differen,ateamongunrelatedstrains

• Cost($$laboreaseofuseetc.)

• Turnaround,me



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)



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.



Typingdatainformsgoodepidemiology

(itdoesn’treplaceit)

• “Indis,nguishable”≠immediatecommonsource

Same:

PFGE

spa

MLST

SCCmecrep-PCR

Zazakovaetal.NEnglJMed2005;352:468.



Typingdatainformsgoodepidemiology

(itdoesn’treplaceit)

• “Different”(typeorspecies)doesnot“rule

out”transmission

– e.g.spreadofplasmid-mediatedESBLs

RegionalKPCspread

MostwereKPNInterspeciestoE.coli

onatleast2occasions

Wonetal.ClinInfectDis2011;53:532.



Moleculartyping:Summary

• Manyacceptablemethodsavailable

• Discussper,nentissueswithyourlabdirector

– TAT:needforinhouseversussendouttes,ng

– Localuseforcluster/outbreaksonly?

– Needforlibrarymethod(e.g.MLSTspa)

• MostwellservedbyPFGErep-PCRMLVA

• Mostimportantpointistointerpretresultsincontext(buggene,csofRepidemiology)



Proteomics

• Matrixassistedlaserdesorp,on/ioniza,on–,me

offlight(MALDI-TOF)

• Producesdetailedprotein

fingerprint

• AccuratespeciesID

– Rapidinexpensive

– Requiresisolatedcolony

• Otherpoten,alapplica,ons?

– Straintyping

– Resistancedetec,on

– Analysisofmixedsamples



Summary

• Moleculartes,ngincreasinglyoffersfaster

moresensi,vedetec,onofHAIpathogens

• Notestisperfect:understandlimita,onsand

implica,ons(diagnosissurveillancecost)

• Closecollabora,onwithlabdirector

– Regularroundsinthemicrolab

– Labpar,cipa,ononinfec,oncontrolcommiee – Knowimplica,onsofICini,a,vesonthelab

shea 2012 diekema

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