The Performance Of Diagnostic Tests

JOURNALOF CLINICALMICROBIOLOGY,Sept.2011,p.S44ŒS48V OL.49,N O.9SUPPL.0095-1137/11/$12.00doi:10.1128/JCM.00698-11 Copyright©2011,AmericanSocietyforMicrobiology.AllRightsReserved. CurrentBestPracticesforRespiratoryVirusTesting ChristineC.Ginocchio 1*andAlexanderJ.McAdam 2DepartmentofPathologyandLaboratoryMedicine,NorthShore-LIJHealthSystemLaboratories,andDepartmentof MolecularMedicine,FeinsteinInstituteforMedicalResearch,HofstraNorthShore-LIJSchoolofMedicine,LakeSuccess, NewYork11042, 1andDepartmentofLaboratoryMedicine,Children™sHospitalBoston,andDepartmentof Pathology,HarvardMedicalSchool,Boston,Massachusetts 2Diagnostictestingforrespiratoryviruseshasbeenrevolutionizedbyrecentadvancesthathavemaderapid andhighlyaccuratetestsaccessibletoclinicallaboratories,anditisimportantthattheseimprovedmethods beutilized.Accuratedetectionofrespiratoryvirusesisimportantinpatientcare,asitguidesboththerapyand infectioncontrolmeasures.Onalargerscale,theCDCanditscollaboratinglaboratoriescollectbothdataand isolatesfromclinicallaboratoriesfornationalsurveillance,andtheuseofhigh-qualitytestsinclinical laboratoriescanimprovethequalityofthesedata. Inthepastdecade,therehasbeenamarkedimprovementin theavailabilityoflaboratoryandpoint-of-caretestsforthe diagnosisofrespiratoryvirusinfections.Commercialmanufac- turershaveintroducednewrapidrespiratoryvirusculture methods,pooledantibodyreagents,rapidantigendirecttests (RADTs),andimprovedspecimencollectiondevices.Most importantamongtheseisthedevelopmentofcommercial, FoodandDrugAdministration(FDA)-approved,andlabora- tory-developednucleicacidampli˚cationtests(NAATs).The introductionofthesenewsystemshascreatednewchallenges forlaboratorydirectors,whomustdecidewhichofthemany teststoofferandwhatspecimentypestoacceptfordiagnostic testing.Thisworkinggroupconsideredseveralcurrentissuesindi- agnostictestingforrespiratoryviruses,includingthebest methodsfordetectionofrespiratoryviruses,samplehandling, turnaroundtime,thescopeandseasonaluseoftests,testing forantiviralresistance,andmonitoringtheperformanceof diagnostictests.Ourgoalsindiscussingtheseissueswere, whenpossible,tocometoaconsensusonthebestpractices andtoraisequestionsforfurtherconsiderationandinvestiga- tion.METHODSFORTHEDETECTIONOF RESPIRATORYVIRUSES Therewasconsensusamongthegroupmembersthattheuse ofNAATsintheroutineclinicalsettinghasdramatically changedourapproachtothediagnosisofviralrespiratorytract infections.Traditionalvirusdetectionmethods,including RADTs,direct˜uorescentantibodytesting(DFA),andvirus culture,canbeeffectivediagnostictoolsbutareofteninferior inassaysensitivity,speci˚city,timetovirusidenti˚cation,and breadthofpathogendetectioncomparedtoNAATs(30). Thetimestoresultsthatarepossiblewiththesetestsvary widely,asdothevirusesthattheycandetect.RADTsare simpletoperformandprovideresultswithin15to30minbut arelimitedtothedetectionofin˜uenzaAvirus,in˜uenzaB virus,andrespiratorysyncytialvirus(RSV)(30,46,51).DFA canbeperformedinaslittleas30to60minandcandetect8 ofthecommonrespiratoryviruses(adenovirus;in˜uenzaA virus;in˜uenzaBvirus;humanmetapneumovirus[hMPV]; parain˜uenzavirustypes1,2,and3[PIV-1,PIV-2,andPIV-3]; andRSV)(26).Rapidcellculture(shellvialorclustertrays) candetectadenovirus,in˜uenzaAvirus,in˜uenzaBvirus, PIV-1,PIV-2,PIV-3,hMPV,andRSV(35).Traditionaltube cellculturecanhaveabroaderscopeofpathogendetection dependingonthecelllinesused.However,itusuallytakes3to 7daystodetectthesevirusesbytraditionaltubeculture, whereasrapidcellculturecangenerallydetect 90%ofthe viruseswithin48h(7).DFAandrapidcellculturemethods canthereforeprovideresultswithinatimeframethatcould affectpatientmanagementiftestingisperformedlocally.How- ever,outsidelargerhospitalsandreferencelaboratories,DFA andrapidcellculturearegenerallynotavailable,andthe resultsvaryandcanbedelayeddependingonvirusviability andthetransittimetoareferencelaboratory. Theaccuracyofthesedifferenttestsalsovarieswidely.The utilityofRADTsisgreatlylimitedbytheirmodestsensitivities (6,11,15,16,24,30,45).Thesensitivityofthesetestsfor in˜uenzavirusesandRSVare10to85%and50to98%, respectively(reference30andreferencestherein).Thespeci- ˚citiesofRADTsaregenerallyreportedtobehigh(6,11,15, 16,24,30,45),butarecentreportsuggeststhatthismightbe incorrect,andthiswarrantsfurtherinvestigation(44).Al- thoughthespeci˚citiesofDFAandrapidcellculturearehigh, thesensitivitiesofthetestsvarybyvirus(andsometimesby viralstrain)fromalowof50%(adenovirusDFAandRSV culture)toahighof 80%(RSVDFAandin˜uenzaAvirus culture)comparedtoNAATs(26,30,31,35,52).Inaddition, althoughthe8virusescommonlydetectedareresponsiblefor alargeportionofviralrespiratorytractinfections,selectcoro- naviruses(229E,OC43,NL63,andHKU-1),parain˜uenzavi- rustype4,rhinovirus,andpotentiallybocavirusarealsosig- ni˚cantcausesofrespiratorydiseaseandaregenerallyonly detectedusingNAATs(4).Forexample,studiesperformed duringtheheightoftheNewYorkCityoutbreakofthe2009 in˜uenzaAvirusH1N1pandemicdemonstratedthattheover- allratesofpositivityforanyrespiratoryvirusintheclinical *Correspondingauthor.Mailingaddress:NorthShore-LIJHealth SystemLaboratories,10NevadaDrive,LakeSuccess,NY11042. Phone:(516)719-1079.Fax:(516)719-1254.E-mail:[email protected] .edu.S44sampleswerehighlydependentonthetestmethodologyand thenumberofviraltargetsdetectedbytheassay(16).Asingle virusormultipleviruses(4%ofthesamples)weredetected withprevalenceratesofapproximately20%forRADTs(3 viruses),20%forDFA(8viruses),35%forrapidcellculture(7 viruses),and63%foracomprehensiveNAAT(15viruses). THEROLESOFRADTS,DFA,ANDVIRALCULTUREIN THEAGEOFNAATS Knowingthecaveatsoftraditionaltestingcomparedto NAATs,thequestionastowhetherthereisstillarolefor RADTs,DFA,andculturearisesTheworkinggroupconsid- eredthisquestioncarefully,anditistherecommendationof thepanelmembersthatRADTsshouldbereplacedbymore sensitiveNAATswheneverpractical.UntilFDA-cleared,easy- to-use,cartridge-basedNAATs(fimolecularpoint-of-care testsfl)becomeavailable,theparticipantsacknowledgethatfor mostcommunityhospitalsandphysician™sof˚ces,RADTsmay betheonlydiagnosticmethodavailable.Theopinionsofthe participantsoftheworkinggroupdifferedabouthowforceful laboratorydirectorsshouldbeintheireffortstoeliminatethe useofRADTs,buttherewasconsensusthatitisnotpossible todiscontinuetheiruseatallinstitutions.Laboratorydirectors, inconjunctionwithinfectious-diseasephysiciansandinfection controlpractitioners,needtoplayakeyroleineducatingtheir medicalstaffsonthelimitationsofRADTs(lowsensitivityand marginalspeci˚city)andtocautionthemontheclinicaland infectioncontrolconsequencesofamisseddiagnosisduetoa negativeRADT.ThoughthecostpertestforNAATsmaybe signi˚cantlyhigher,theeaseofuseandthehighqualityand importanceoftheresult,particularlyfortheinpatientsetting, needtobeconsideredandweighedagainstthetotalcostof patientcare.Thesecostsincludeinappropriateantibioticuse (costandincreasingdrugresistance),increasedlengthofstay withdecreasedreimbursement,increasedancillarytestingdue tothelackofadiagnosis,andpotentialfornosocomialout- breakswithsigni˚cantmorbidityandmortality(25,28).Atthis time,thecombinationofRADTorDFAwithrecourseto rapidviralcultureforRADT-orDFA-negativesamplesmay besuf˚cientforrespiratoryinfectionsthatarenotsevere,such asthoseinpatientswhoarecaredforintheoutpatientsetting. DFAwouldbeapplicableasarapidscreeningtestforhospi- talizedpatients,withadditionalcomprehensiveNAATfor specimensthatarenegativebyDFA.Theutilityofrapidviral cultureislimitedbytherangeofvirusesdetected,variable viabilityofthevirusesduringtransportandstorage,andin- abilitytoeasilydetectmixedinfections.Inaddition,although rapidviruscultureisfasterthanconventionaltubeculture,itis stillgenerallyslowerthanRADT,DFA,andNAAT. Studieshavedemonstratedthatmixedviralinfectionsare detectedin3.3to30.0%ofsampleswhenawidevarietyofviral respiratorypathogensareincludedinthetesting(4,16,39,41). Althoughthesigni˚canceofmixedviralinfectionsstillneedsto beclearlyde˚ned,theclinicalimpactincriticallyillpatients andpatientswithcomorbiditiesorimmunesuppressionmust beconsideredpotentiallysevere.Duringtheheightofin˜u- enzaseason,whenpatientcohortingisoftennecessarydueto largenumbersofpatientswithrespiratoryillness,theconse- quencesofasecondviralinfectioninanalreadyseriouslyill hospitalizedpatientcouldbesubstantial.Inaddition,withthe adventofnewantiviralagents,suchasthosetargetedtothe treatmentofsevererhinovirusinfections,comprehensivetar- geteddiagnosticswillbecomeincreasinglyimportant(42,54). Theworkinggroupagreedthattraditionaltubeculture,al- thoughgenerallytooslowtoimpactpatientmanagement,is stillapplicableincertainsituations,includingthecareofim- munocompromisedpatients,whereadditionalviruses,suchas herpessimplexvirustypes1and2andcytomegalovirus,can playakeyrole.Thesevirusesarenotyetincludedinstandard NAATpanelsforrespiratoryvirusdetection.Othersituations whereculturestillplaysanimportantroleareinproviding clinicalisolatesforepidemiologystudies,fortheestablishment ofvaccinecandidates,toevaluatemechanismsofantiviralre- sistancefornewantiviraldrugs,forclinicaltrialstudies,andto identifypotentialnovelviralagents.Certainviruses,suchas adenovirus,thatdemonstratesigni˚cantdivergence,canbea challengefordetectionbyNAATs,andculturemaybeneces- sarytosupplementNAATs(40). APPROPRIATESAMPLECOLLECTION,TRANSPORT, ANDSTORAGE Respiratoryvirusdetectionishighlydependentonthetype ofsamplecollected,thetimeofcollectionaftertheonsetof clinicalsymptoms,theageofthepatient,andthetransportand storageofthesamplepriortotesting(48,49).Severaldifferent upperrespiratorytractspecimensareapplicablefortesting, includingnasopharyngeal(NP)washes,NPaspirates,andNP swabsplacedinvirustransportmedia(48,49).Thereare limiteddatathatsupporttheuseofcombinednose-throat swabsforin˜uenzaAvirustestingbyNAAT(14).Detectionof 12respiratoryvirusesusingaNAATpanelwassigni˚cantly lesssensitivewithoropharyngealswabspecimens(54.2%)than witheithernasopharyngealswabs(73.3%)ornasopharyngeal washspecimens(84.9%)(33).Thismaybeduetothesubstan- tiallylowerviralloadsintheoropharynxthaninthenasophar- ynx(23). Therewasconsensusamongthemembersoftheworking groupthatuseofNPswabsforspecimencollectionisan importantadvanceintestingforrespiratoryviruses.NP ˜ockedswabs(Copan,Brescia,Italy)havegenerallybeen foundtoyieldspecimensasgoodasnasopharyngealwash specimensfordetectionofrespiratoryvirusesbyNAATor DFA(1,50).Thisispresumablybecause˜ockedswabseffec- tivelycollectandreleaserespiratoryepithelialcellsfromNP specimens(12).NP˜ockedswabsaregenerallyeasiertocol- lectfromadultpatientsandolderchildrenthanNPaspirates orwashes.Forsafetyreasons,thecollectionofanNPaspirate maybeindicatedinyoungchildren.Theuseofnasalororo- pharyngealswabsamplesisanareaofactiveinvestigation,but routineuseofthesespecimensisnotrecommendedatpresent becauseofconcernsaboutsensitivityforvirusdetectionin somestudies(32,38).Lowerrespiratorytractsamples,suchas inducedsputum,protectedbrushsamples,andbronchial alveolarlavage(BAL)samples,maybenecessary,asseveral studieshavedemonstratedthatinsomeseverecasesofin˜u- enza,upperrespiratorytractsamplesarenegativewhilelower respiratorytractsamplesarepositive(29,37,53).Foroptimal results,samplesshouldbecollectedwithin3to5daysof VOL.49,N O.9SUPPL.,2011CONVENTIONALVERSUSMOLECULARMETHODSS45 symptomonset,transportedtothelaboratoryonwetice,and refrigerated(2to8°C)iftestingistobeperformedwithin48h (10,48,49).Iftestingisdelayed,thesamplesshouldbestored frozenat 80°C(48,49).Multiplefreeze-thawsarenotrec- ommended,asthisprocessdecreasestheviraltiter,particu- larlyforculture-basedmethods.Ingeneral,methodstendto performbetterwithpediatricsamples,sincechildrenshedhigher titersofvirusandforlongerperiodsthanadults(2,8,20,27). TESTTURNAROUNDTIMES Testingneedstobecompletedwithinatimeframethatcan affectpatientmanagement(i.e.,initiationordiscontinuationof antiviralsandantibioticsandsupportivecare);provideforef- ˚cientbedutilizationandappropriateinfectioncontrolmea- sures,suchascohorting,toreducenosocomialtransmission; andidentifyoutbreaksituations.Therefore,batchtestingper- formed2to3timesaweekisnotoptimal.Itisrecommended bytheworkinggroupthatresultsofNAATs,RADTs,and DFAtestsbeavailablewithin24hofsamplecollection,al- thoughthecommitteeacknowledgesthatthismaynotbepos- sibleforalllaboratories,especiallyduringtimeswhenstaf˚ng islimited,suchasweekendsandholidays. SCOPEOFRESPIRATORYVIRUSTESTING Shouldlaboratoriesscreenforallrespiratoryviruses,atall timesoftheyear,andinallpatientpopulations?Manyrespi- ratoryvirusesdemonstrateseasonalvariationsinprevalence, particularlyintemperateareas.Forexample,RSVisgenerally detectedfromNovemberthroughMarch,in˜uenzavirusbe- tweenDecemberandApril,andhMPVbetweenDecember andMayintheUnitedStates.Often,laboratoriesrestricttest- ingforspeci˚crespiratoryvirusesduringcertainseasons.This typeofrestrictionisespeciallyindicatedfortestswithlower speci˚city(perhapstheRADTs)outsidethein˜uenzaorRSV seasons,asthepositivepredictivevalueisgreatlydiminished whentheprevalenceoftheseviralinfectionsislow.However, withglobaltravel,manyfiseasonalflvirusesarenowdetected throughouttheyear.Therefore,limitingdetectiontoseasonal periodscanresultinmissinganimportantoutbreak,suchas the2009in˜uenzaAvirusH1N1pandemicintheUnited States,whichbeganattheendofthefitraditional˜useasonfl (April2009)andcontinuedthroughoutthesummermonths. SinceNAATsandviralculturearehighlyspeci˚c,thepositive predictivevalueofthesetestsremainshighduringtimesoflow viralprevalence.However,laboratorydirectorsshouldcon- sidercon˚rmatorytestingofpositiveresultswhenavirusis detectedduringanunusualperiod.Laboratoriesshouldmon- itorthelocalseasonalprevalenceofthevirusesroutinely screenedforandmaketestingdecisionsbaseduponthesedata andadditionalresourcesandepidemiologyinformationpro- videdontheCDCwebsite(http://www.cdc.gov/˜u/).Labora- toriesmustalwayskeepinmindthattestingalgorithmsmustbe adaptabletounexpectedlocal,national,andinternational events,suchasthe2009in˜uenzaAvirusH1N1pandemic. Theworkinggroupagreedthatlimitingtestingforroutine respiratoryvirusestocertainpatientpopulations,suchaschil- dren,isnotthebestclinicalpractice.Forexample,although RSVandhMPVareprimarilydetectedinpediatricsamples (19,22,34),severeRSVandhMPVinfectionshavebeen describedinadultsofallageswithandwithoutunderlying disease,suchaschronicobstructivepulmonarydisease (COPD)orasthma(19,21,22).Inaddition,infectionswith thesepathogenscanhaveatypicalpresentations,suchashMPV pericarditisinanotherwisehealthyadult(13).Therefore,age mayhelptriageinitialtestingbutshouldnotgovernthe˚nal rangeofvirusesincludedindiagnostictestingalgorithms. TESTINGFORANTIVIRALRESISTANCE Currently,FDA-approvedantiviraltherapeuticagentsfor respiratoryviralinfectionsarelimitedtothetreatmentofin- ˜uenza.Theclassesofin˜uenzaantiviralsincludetheadaman- tanes(amatadineandrimantadine)andtheneuraminidase inhibitors(oseltamivirandzanamivir)(17).Theef˚cacyof aerosolizedribavirinfortreatmentofin˜uenzaisnotwell understood,andthisisnotanFDA-approveduseforthe antiviral(43).Thesusceptibilityofin˜uenzavirussubtypesto FDA-approvedantiviralsvariesbyantiviral,withseasonalin- ˜uenzaAvirusH1N1beingsusceptibletotheadamantanes andzanamivirand 99%resistanttooseltamivirandseasonal in˜uenzaAvirusH3N2,2009in˜uenzaAvirusH1N1,and in˜uenzaBvirusbeingsusceptibletooseltamivirandzanami- virand100%resistanttotheadamantanes(17,18,47).During thecourseofthe2009in˜uenzaAvirusH1N1pandemic,cases werereportedinwhichpatientswithunderlyingdiseasedevel- opedoseltamivirresistanceduringprolongedtreatment(3). Limitednosocomialtransmissionofoseltamivir-resistant2009 in˜uenzaAvirusH1N1hasoccurredinimmunocompromised patients(9,36).Theneedforroutinein˜uenzavirusantiviral resistancetestingthereforedependsonthecirculatingstrains andknownresistancepatterns(17,18,47).Duringthe2010Œ 2011in˜uenzaseason,routineresistancetestingwasnotindi- cated,astheantiviralsusceptibilitypatternswerethesamefor thetwocommonsubtypesofcirculatingin˜uenzaAvirus. Duringthe2009Œ2010in˜uenzaseason,varyingsusceptibilities toantiviralsamongcirculatingsubtypesofin˜uenzaAvirus meantthatantiviralsusceptibilitytestingwasneededandthat subtypingcouldbeusedasaguideforselectingappropriate antiviraltherapy.Intheeventofashiftincirculatingstrainsor increasingresistancenotedforastrain,theuseofantiviral resistancetestingshouldbereevaluatedandmaybeindicated forpatientsathighriskforseveredisease. MONITORINGTHEPERFORMANCEOF DIAGNOSTICTESTS Earlyinthecourseofthe2009in˜uenzaAvirusH1N1 pandemic,severalstudiesshowedthattheperformancesofthe RADTs,DFA,andviralcultureweresuboptimal(5,16,24, 45).Theworkinggroupdiscussedthepotentialreasonsforthis declineintestperformance.Thereasonsforthedeclineintest performance,comparedtobothpreviouslypublishedpeer- reviewedarticlesandmanufacturers™claims,asdeterminedin tightlycontrolledclinicaltrials,include(i)comparisonof RADTs,DFA,andcultureperformancetoNAAT,thenew, moresensitivefigoldstandardfl;(ii)testingofpatientslatein thecourseofaninfection,whentheyaresheddingvirusat levelsbelowthedetectionlimitofRADTs,DFA,orculture S46CAMPCLINMICRO J.C LIN.MICROBIOL.butstilldetectablebyNAAT;(iii)testingthefiworriedwellfl; (iv)impropercollection,storage,andtransportofsamples;and (v)performanceoftestingbyuntrainedorpoorlysupervised personnel,especiallyinthecaseofClinicalLaboratoryIm- provementAct(CLIA)waivedtests.Inaddition,antigenic divergenceofnewcirculatingstrainscanleadtoadecreasein detectionbyteststhatrelyonantibodyinteractionswithspe- ci˚cviralepitopesforprimarydetection(RADTsandDFA)or forculturecon˚rmation.Similarly,newRNAorDNAse- quencevariantscanaffecttheperformanceofNAATsdueto primerand/orprobemismatches.OncetestsareFDAcleared/ approved,manufacturersarenotunderanobligationto changetestcomponentsormonitortestperformance.There- fore,assayperformancecansigni˚cantlydeclineovertime withouttheknowledgeoftheuser.Itistheresponsibilityof laboratoriestocontinuallyassesstheperformanceoftheirtests and,whenadeclineinperformanceisnoted,investigatepos- sibleon-sitecausesofpoorperformance,comparealternative tests,andnotifythemanufacturerand,˚nally,theFDAifthe changesaresigni˚cantenoughtopotentiallycauseclinical harm.TheparticipantsstronglyfeelthatanFDAreevaluation processshouldbeinplacewherebymanufacturerscanchange primersand/orprobestoaccommodategeneticvariantsina simpleyetsafewaywithoutthenecessitytoperformfullclin- icaltrialsthatcantakeayeartocomplete. CONCLUSIONSANDFUTURECONSIDERATIONS ItisincreasinglyevidentthatNAATsaresuperiortotradi- tionalvirusdetectionmethodsduetoenhancedsensitivityand speci˚city,abroadrangeofvirusdetection,andrapidturn- aroundtime.However,untilsuchtimeaseasy-to-usepoint-of- careNAATsareavailable,manylaboratorieswillneedto continueusingRADTsorothermethods.Severalquestions remainunansweredabouttheuseofNAATs,andthepartic- ipantsintheworkinggroupagreedthattheyareimportant areasforfutureinvestigation.Whataretheappropriatetest panels?ShouldNAATsdetectonlythecommonrespiratory virusesorthoseforwhichtherearetherapeuticoptions?Al- thoughtherapeuticoptionsmaynotbeavailableformany viruses,theriskofnosocomialspreadinhealthcareinstitu- tionscannotbedismissed.Isitbettertousemix-and-match panels,orisasinglecomprehensivepanelbest?Howdowe interpretmixedinfections,andwhatistherelevanceofdetect- ingavirusinanasymptomaticpatient?Finally,howdowe workwiththeFDAtoestablishasafeyetfastwaytomodify FDA-cleared/approvedtestssothatoptimalreactivityandde- tectioncanbemaintainedasviralstrainsshift?Asclinical microbiologists,ourtaskistoprovidethemostclinicallyrele- vantdiagnosticinformation,andrecentimprovementsintest- ingforrespiratoryvirusessupportusinthistask. Sessiondiscussants: MatthewJ.Binnicker,PaulBourbeau,Lynn Boyer,PaulCompangone,ChristianCoogan,RichardHodinka,Mark LaRocco,DuaneW.Newton,RibhiShawar,GongyiShi,B.Jane Smith,RichardB.Thomson,andHemantC.Vaidya. ACKNOWLEDGMENTSC.C.G.,A.J.M.,andtheothermembersofthegroupsincerelythank GaryDoern,MikeDunne,EllenJoBaron,andthecorporatesponsors fororganizingandsupportingthisimportantmeeting. C.C.G.isonthescienti˚cadvisoryboardsofGen-ProbeandLu- minexMolecularDiagnosticsandhasreceivedresearchfundingfrom Gen-Probe,LuminexMolecularDiagnostics,3M,andDiagnosticHy- brids(Quidel)andhonorariafromGen-Probe,LuminexMolecular Diagnostics,Nanosphere,andMeridian.A.J.M.hasreceivedresearch fundingfrom3MandhonorariafromSaunders-Elsevier. REFERENCES1.Abu-Diab,A.,etal. 2008.Comparisonbetweenpernasal˜ockedswabsand nasopharyngealaspiratesfordetectionofcommonrespiratoryvirusesin samplesfromchildren.J.Clin.Microbiol. 46:2414Œ2417.2.Andresen,D.N.,andA.M.Kesson. 2010.Highsensitivityofarapidimmu- nochromatographictestfordetectionofin˜uenzaAvirus2009H1N1in nasopharyngealaspiratesfromyoungchildren.J.Clin.Microbiol. 48:2658Œ2659.3.Anonymous.2009.Update:in˜uenzaactivity-UnitedStates,September28, 2008ŒJanuary31,2009.MMWRMorb.Mortal.Wkly.Rep. 58:115Œ119.4.Balada-Llasat,J.M.,H.LaRue,C.Kelly,L.Rigali,andP.Pancholi. 2011.EvaluationofcommercialResPlexIIv2.0,MultiCode-PLx,andxTAGre- spiratoryviralpanelsforthediagnosisofrespiratoryviralinfectionsin adults.J.Clin.Virol. 50:42Œ45.5.Blyth,C.C.,J.R.Iredell,andD.E.Dwyer. 2009.Rapid-testsensitivityfor novelswine-originin˜uenzaA(H1N1)virusinhumans.N.Engl.J.Med. 361:2493.6.Borek,A.P.,S.H.Clemens,V.K.Gaskins,D.Z.Aird,andA.Valsamakis. 2006.RespiratorysyncytialvirusdetectionbyRemelXpect,BinaxNow RSV,directimmuno˜uorescentstaining,andtissueculture.J.Clin.Micro- biol.44:1105Œ1107.7.Bourgeois,F.T.,C.Valim,A.J.McAdam,andK.D.Mandl. 2009.Relative impactofin˜uenzaandrespiratorysyncytialvirusinyoungchildren.Pedi- atrics124:e1072Œe1080.8.Casiano-Colo´n,A.E.,B.B.Hulbert,T.K.Mayer,E.E.Walsh,andA.R. Falsey.2003.Lackofsensitivityofrapidantigentestsforthediagnosisof respiratorysyncytialvirusinfectioninadults.J.Clin.Virol. 28:169Œ174.9.Chen,L.F.,etal. 2011.Clusterofoseltamivir-resistant2009pandemic in˜uenzaA(H1N1)virusinfectionsonahospitalwardamongimmunocom- promisedpatientsŒNorthCarolina,2009.J.Infect.Dis. 203:838Œ846.10.Cheng,P.K.,etal. 2010.Performanceoflaboratorydiagnosticsforthe detectionofin˜uenzaA(H1N1)vvirusascorrelatedwiththetimeafter symptomonsetandviralload.J.Clin.Virol. 47:182Œ185.11.Cruz,A.T.,A.C.Cazacu,J.M.Greer,andG.J.Demmler. 2007.Perfor- manceofarapidassay(BinaxNOW)fordetectionofrespiratorysyncytial virusatachildren™shospitalovera3-yearperiod.J.Clin.Microbiol. 45:1993Œ1995.12.Daley,P.,S.Castriciano,M.Chernesky,andM.Smieja. 2006.Comparison of˜ockedandrayonswabsforcollectionofrespiratoryepithelialcellsfrom uninfectedvolunteersandsymptomaticpatients.J.Clin.Microbiol. 44:2265Œ2267.13.David,P.H.,S.Goldberg,andC.Ginocchio. 2009.Acutepericarditiscaused byhumanmetapneumovirus.Infect.Dis.Clin.Practice 17:283Œ285.14.delaTabla,V.O.,etal. 2010.Comparisonofcombinednose-throatswabs withnasopharyngealaspiratesfordetectionofpandemicin˜uenzaA/H1N1 2009virusbyreal-timereversetranscriptasePCR.J.Clin.Microbiol. 48:3492Œ3495.15.Gimeno,C.,etal. 2010.Sensitivityofamarketedimmunochromatographic assayspeci˚callytargetingthepandemicin˜uenzaA/H1N12009virus.Di- agn.Microbiol.Infect.Dis. 68:80Œ82.16.Ginocchio,C.C.,etal. 2009.Evaluationofmultipletestmethodsforthe detectionofthenovel2009in˜uenzaA(H1N1)duringtheNewYorkCity outbreak.J.Clin.Virol. 45:191Œ195.17.Glezen,W.P. 2008.Clinicalpractice.Preventionandtreatmentofseasonal in˜uenza.N.Engl.J.Med. 359:2579Œ2585.18.Gubareva,L.V.,etal. 2010.Comprehensiveassessmentof2009pandemic in˜uenzaA(H1N1)virusdrugsusceptibilityinvitro.Antivir.Ther. 15:1151Œ1159.19.Hall,C.B. 2001.Respiratorysyncytialvirusandparain˜uenzavirus.N.Engl. J.Med. 344:1917Œ1928.20.Hall,C.B.,R.G.Douglas,Jr.,andJ.M.Geiman. 1975.Quantitative sheddingpatternsofrespiratorysyncytialvirusininfants.J.Infect.Dis. 132:151Œ156.21.Hall,C.B.,C.E.Long,andK.C.Schnabel. 2001.Respiratorysyncytialvirus infectionsinpreviouslyhealthyworkingadults.Clin.Infect.Dis. 33:792Œ796.22.Hermos,C.R.,S.O.Vargas,andA.J.McAdam. 2010.Humanmetapneu- movirus.Clin.Lab.Med. 30:131Œ148.23.Hernes,S.S.,etal. 2011.Swabbingforrespiratoryviralinfectionsinolder patients:acomparisonofrayonandnylon˜ockedswabs.Eur.J.Clin. Microbiol.Infect.Dis. 30:159Œ165.24.Kawachi,S.,etal. 2011.Multicenterprospectiveevaluationofanovelrapid immunochromatographicdiagnostickitspeci˚callydetectingin˜uenzaA H1N12009virus.J.Clin.Virol. 51:68Œ72.25.Lambert,S.B.,K.M.Allen,R.C.Carter,andT.M.Nolan. 2008.Thecost VOL.49,N O.9SUPPL.,2011CONVENTIONALVERSUSMOLECULARMETHODSS47 ofcommunity-managedviralrespiratoryillnessesinacohortofhealthy preschool-agedchildren.Respir.Res. 9:11.26.Landry,M.L. 2009.Developmentsinimmunologicassaysforrespiratory viruses.Clin.Lab.Med. 29:635Œ647.27.Landry,M.L.,S.Cohen,andD.Ferguson. 2000.Impactofsampletypeon rapiddetectionofin˜uenzavirusAbycytospin-enhancedimmuno˜uores- cenceandmembraneenzyme-linkedimmunosorbentassay.J.Clin.Micro- biol.38:429Œ430.28.Lee,B.Y.,etal. 2010.Totestortotreat?Ananalysisofin˜uenzatestingand antiviraltreatmentstrategiesusingeconomiccomputermodeling.PLoSOne 5:e11284.29.Lee,N.,etal. 2011.Viralclearanceandin˜ammatoryresponsepatternsin adultshospitalizedforpandemic2009in˜uenzaA(H1N1)viruspneumonia. Antivir.Ther. 16:237Œ247.30.Leland,D.S.,andC.C.Ginocchio. 2007.Roleofcellcultureforvirus detectionintheageoftechnology.Clin.Microbiol.Rev. 20:49Œ78.31.Liao,R.S.,L.L.Tomalty,A.Majury,andD.E.Zoutman. 2009.Comparison ofviralisolationandmultiplexreal-timereversetranscription-PCRforcon- ˚rmationofrespiratorysyncytialvirusandin˜uenzavirusdetectionbyan- tigenimmunoassays.J.Clin.Microbiol. 47:527Œ532.32.Lieberman,D.,A.Shimoni,A.Keren-Naus,R.Steinberg,andY.Shemer- Avni.2009.Identi˚cationofrespiratoryvirusesinadults:nasopharyngeal versusoropharyngealsampling.J.Clin.Microbiol. 47:3439Œ3443.33.Lieberman,D.,A.Shimoni,Y.Shemer-Avni,A.Keren-Naos,andR.Shtain- berg.2010.Respiratoryvirusesinadultswithcommunity-acquiredpneumo- nia.Chest 138:811Œ816.34.McAdam,A.J.,etal. 2004.Humanmetapneumovirusinchildrentestedata tertiary-carehospital.J.Infect.Dis. 190:20Œ26.35.McAdam,A.J.,andA.M.Riley. 2009.Developmentsintissueculture detectionofrespiratoryviruses.Clin.Lab.Med. 29:623Œ634.36.Moore,C.,etal. 2011.Evidenceofperson-to-persontransmissionofoselta- mivir-resistantpandemicin˜uenzaA(H1N1)2009virusinahematology unit.J.Infect.Dis. 203:18Œ24.37.Mulrennan,S.,etal. 2010.Pandemicin˜uenza(H1N1)2009pneumonia: CURB-65scoreforpredictingseverityandnasopharyngealsamplingfor diagnosisareunreliable.PLoSOne 5:e12849.38.Ohrmalm,L.,etal. 2010.Flockednasalswabversusnasopharyngealaspirate fordetectionofrespiratorytractvirusesinimmunocompromisedadults:a matchedcomparativestudy.BMCInfect.Dis. 10:340.39.Pabbaraju,K.,K.L.Tokaryk,S.Wong,andJ.D.Fox. 2008.Comparisonof theLuminexxTAGrespiratoryviralpanelwithin-housenucleicacidampli- ˚cationtestsfordiagnosisofrespiratoryvirusinfections.J.Clin.Microbiol. 46:3056Œ3062.40.Pabbaraju,K.,etal. 2011.Comparisonofasingleplexreal-timeRT-PCR assayandmultiplexrespiratoryviralpanelassayfordetectionofin˜uenza fiAflinrespiratoryspecimens.In˜uenzaOtherRespi.Viruses 5:99Œ103.41.Parrott,R.H.,etal. 1961.Respiratorysyncytialvirus.II.Serologicstudies overa34-monthperiodofchildrenwithbronchiolitis,pneumonia,andminor respiratorydiseases.JAMA 176:653Œ657.42.Patick,A.K. 2006.Rhinoviruschemotherapy.AntiviralRes. 71:391Œ396.43.Preziosi,P. 2011.In˜uenzapharmacotherapy:presentsituation,strategies andhopes.ExpertOpin.Pharmacother. 12:1523Œ1549.44.Sambol,A.R.,etal. 2010.Useofrapidin˜uenzadiagnostictestsunder˚eld conditionsasascreeningtoolduringanoutbreakofthe2009novelin˜uenza virus:practicalconsiderations.J.Clin.Virol. 47:229Œ233.45.Sandora,T.J.,etal. 2010.Testcharacteristicsofcommercialin˜uenza assaysfordetectingpandemicin˜uenzaA(H1N1)inchildren.Pediatr. Infect.Dis.J. 29:261Œ262.46.Selvarangan,R.,D.Abel,andM.Hamilton. 2008.ComparisonofBDDi- rectigenEZRSVandBinaxNOWRSVtestsforrapiddetectionofrespi- ratorysyncytialvirusfromnasopharyngealaspiratesinapediatricpopula- tion.Diagn.Microbiol.Infect.Dis. 62:157Œ161.47.Sheu,T.G.,etal. 2011.Dualresistancetoadamantanesandoseltamivir amongseasonalin˜uenzaA(H1N1)viruses:2008Œ2010.J.Infect.Dis. 203:13Œ17.48.Specter,S. 2009.Clinicalvirologymanual,4thed.ASMPress,Washing- ton,DC. 49.Storch,G.A. 2000.Essentialsofdiagnosticvirology,1sted.ChurchillLiv- ingstone,NewYork,NY. 50.Walsh,P.,etal. 2008.Comparisonofrespiratoryvirusdetectionratesfor infantsandtoddlersbyuseof˜ockedswabs,salineaspirates,andsaline aspiratesmixedinuniversaltransportmediumforroomtemperaturestorage andshipping.J.Clin.Microbiol. 46:2374Œ2376.51.Welch,D.F.,andC.C.Ginocchio. 2010.Roleofrapidimmunochromato- graphicantigentestingindiagnosisofin˜uenzaAvirus2009H1N1infec- tion.J.Clin.Microbiol. 48:22Œ25.52.Wong,S.,K.Pabbaraju,B.E.Lee,andJ.D.Fox. 2009.Enhancedviral etiologicaldiagnosisofrespiratorysysteminfectionoutbreaksbyuseofa multitargetnucleicacidampli˚cationassay.J.Clin.Microbiol. 47:3839Œ3845.53.Yeh,E.,etal. 2010.Preferentiallowerrespiratorytractinfectioninswine- origin2009A(H1N1)in˜uenza.Clin.Infect.Dis. 50:391Œ394.54.Zhang,X.N.,etal. 2010.Rupintrivirisapromisingcandidatefortreating severecasesofEnterovirus-71infection.WorldJ.Gastroenterol. 16:201Œ209.S48CAMPCLINMICRO J.C LIN.MICROBIOL.