Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses

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Universidade de São Paulo

2012

Comparison of fast-track diagnostics

respiratory pathogens multiplex real-time

RT-PCR assay with in-house singleplex assays for

comprehensive detection of human respiratory

viruses

JOURNAL OF VIROLOGICAL METHODS, AMSTERDAM, v. 185, n. 2, pp. 259-266, NOV, 2012

http://www.producao.usp.br/handle/BDPI/42252

Downloaded from: Biblioteca Digital da Produção Intelectual - BDPI, Universidade de São Paulo

Biblioteca Digital da Produção Intelectual - BDPI

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ContentslistsavailableatSciVerseScienceDirect

Journal

of

Virological

Methods

j o ur na l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j v i r o m e t

Comparison

of

fast-track

diagnostics

respiratory

pathogens

multiplex

real-time

RT-PCR

assay

with

in-house

singleplex

assays

for

comprehensive

detection

of

human

respiratory

viruses

夽

Senthilkumar

K. Sakthivel

a,b

_,

_Brett

_Whitaker

a,c

_,

_Xiaoyan

_Lu

a

_,

_Danielle

_B.L.

_Oliveira

a,d

_,

Lauren

J. Stockman

a

_{, Shifaq}

_Kamili

a,c

_,

_M.

_Steven

_Oberste

a

_,

_Dean

_D.

_Erdman

a,∗

a_Division_of_Viral_Diseases,_Centers_for_Disease_Control_and_Prevention,_Atlanta,_GA,_United_States

b_Logistics_Health_{Incorporated,}_La_Crosse,_WI,_United_States

c_Atlanta_Research_and_Education_Foundation,_Atlanta,_GA,_United_States

d_Institute_of_Biomedical_Science,_University_of_São_Paulo,_São_Paulo,_Brazil

Articlehistory:

Received16February2012

Receivedinrevisedform26June2012

Accepted4July2012

Available online 11 July 2012 Keywords:

Respiratoryvirus

Real-timePCR

Multiplex

a

b

s

t

r

a

c

t

Fast-trackDiagnosticsrespiratorypathogens(FTDRP)multiplexreal-timeRT-PCRassaywascompared within-housesingleplexreal-timeRT-PCRassaysfordetectionof16commonrespiratoryviruses.The FTDRPassaycorrectlyidentiﬁed26diverserespiratoryvirusstrains,35of41(85%)externalquality assessmentsamplesspikedwithculturedvirusand232of263(88%)archivedrespiratoryspecimensthat testedpositiveforrespiratoryvirusesbyin-houseassays.Of308prospectivelytestedrespiratory speci-mensselectedfromchildrenhospitalizedwithacuterespiratoryillness,270(87.7%)and265(86%)were positivebyFTDRPandin-houseassaysforoneormoreviruses,respectively,withcombinedtestresults showinggoodconcordance(K=0.812,95%CI=0.786–0.838).IndividualFTDRPassaysforadenovirus, res-piratorysyncytialvirusandrhinovirusshowedthelowestcomparativesensitivitieswithin-houseassays, withmostdiscrepanciesoccurringwithspecimenscontaininglowvirusloadsandfailedtodetectsome rhinovirusstrains,evenwhenabundant.TheFTDRPenterovirusandhumanbocavirusassaysappeared tobemoresensitivethanthein-houseassayswithsomespecimens.Withtheexceptionsnotedabove, mostFTDRPassaysperformedcomparablywithin-houseassaysformostviruseswhileofferingenhanced throughputandeasyintegrationbylaboratoriesusingconventionalreal-timePCRinstrumentation.

Published by Elsevier B.V.

1. Introduction

Respiratoryvirusesareamongthemostimportantcausesof humanmorbidityandmortalityworldwide(Nairetal.,2010;Pavia, 2011). Clinically indistinguishable, respiratory virus infections requireaccurate laboratorydiagnosisto guidetreatment effec-tivelyandpreventiondecisions.Polymerasechainreaction(PCR) andothermolecularassaysarenowroutinelyusedfordiagnosis

夽 Thecontentsofthismanuscriptaresolelytheresponsibilityoftheauthorsand

donotnecessarilyrepresenttheofﬁcialviewsoftheUSCentersforDisease

Con-trolandPrevention(CDC)orDepartmentofHealthandHumanServices(DHHS).

Namesofspeciﬁcvendors,manufacturers,orproductsareincludedforpublichealth

andinformationalpurposes;inclusiondoesnotimplyendorsementofthevendors,

manufacturers,orproductsbytheCDCorDHHS.

∗ Correspondingauthorat:DivisionofViralDiseases,NationalCenterfor

Immu-nizationandRespiratoryDiseases,CentersforDiseaseControlandPrevention,1600

CliftonRoad,N.E.,Atlanta,GA30333,MailstopG-04,UnitedStates.

Tel.:+14046393727;fax:+14046394416.

E-mailaddress:[email protected](D.D.Erdman).

ofrespiratoryvirusinfections (BeckandHenrickson,2010;Kehl andKumar,2009),butthelargeandincreasingnumberofviruses makeslaboratorytestingwithindividual(singleplex)virusassays challenging.Conversely,multiplexPCRassaysthatcombine multi-pleindividualassaysinasinglereactionfacilitatemorerapid,high throughputandcost-effectivetestingandaregenerallypreferred intheclinicalsetting(Elnifroetal.,2000;Jansenetal.,2011).

Numerous laboratory-developed and commercial multiplex PCR assays using different ampliﬁcation platforms have been describedforrespiratoryvirusesandhavebeengenerallyshownto besuperiortotraditionaldiagnosticmethods,suchasvirusculture andantigendetectionforsensitiveandspeciﬁcdetectionof respi-ratoryviruses(Arensetal.,2010;Bibbyetal.,2011;Brittain-Long etal.,2010;Caliendo,2011;Gadsbyetal.,2010;Kimetal.,2009; Lamsonetal.,2006;Mahonyetal.,2007;Raymondetal.,2009). TheUSFoodandDrugAdministration(FDA)hasclearedrecently twocommercialassays,thexTAG®_RVP_Fast_(Luminex_Molecular

Diagnostics,Austin,TX)andFilmArray®_Respiratory_Panels_(Idaho

TechnologyInc.,SaltLakeCity,Utah)(Randetal.,2011),for in-vitrodiagnosticuseformultiplexdetectionofrespiratoryviruses.

0166-0934/$–seefrontmatter.Published by Elsevier B.V.

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260 S.K.Sakthiveletal./JournalofVirologicalMethods185 (2012) 259–266

However,manyoftheseassaysarecostly,requirespecialized lab-oratoryequipmentandusehighlymultiplexedreactionsthatmay bedeﬁcientinindividualassayperformanceandcanbedifﬁcult tomodifywithoutextensiveassayreoptimization(Gunsonetal., 2008).

The FTD respiratory pathogens (FTDRP) multiplex assay kit (Fast-trackDiagnostics,Luxembourg)uses standardcommercial one-stepreversetranscription (RT)-PCRhydrolysisprobe chem-istryandcommonreal-timePCRinstrumentation.TheFTDRPassay consistsof5discreteprimer/probemixesthattogethercover16 commonhumanrespiratoryviruses.Thisstudyreportstheresults ofacomparisonoftheFTDRPmultiplexassaywithapanelof val-idatedin-housesingleplexreal-timeRT-PCRassaysdevelopedat theCentersforDiseaseControlandPrevention(CDC).

2. Materialsandmethods

2.1. Virusesandspecimens

Virusisolatesandarchived clinicalspecimens wereobtained fromCDCcollectionsacquiredduringroutinesurveillanceand out-breakinvestigations.Theseincluded26laboratoryreferencevirus strainsandﬁeldisolatesand265geographically(U.S.,Centraland SouthAmericaandAfrica)andcompositionallydiversespecimens [nasopharyngealandoropharyngealswabs(223),nasalwashesand aspirates(21),sputum(1),lungautopsytissue(1)and unidenti-ﬁed(19)]collectedfromchildrenandadultswithacuterespiratory illnesses(ARIs)acquiredbetween2008and2011andpreviously testingpositiveforrespiratoryvirusesbythein-housesingleplex assays.Allresidualsamplesand extractswerestored at−70◦_C.

Wheneverpossible,archivedspecimenswereselectedtoachievea proportionalrepresentationofviralloads.Forty-sixmockhuman specimensspikedwithmoderate-to-lowconcentrationsofvirus wereavailablefromthe2010QualityControlforMolecular Diag-nostics(QCMD, Glasgow, Scotland) external quality assessment (EQA) programs for rhinovirus/coronavirus, adenovirus, parain-ﬂuenza viruses, human metapneumovirus/respiratory syncytial virus,andinﬂuenzaA&Bviruses(Wallace,2003).Poolednasal washspecimensfrom20consentinghealthynewmilitaryrecruits waskindlyprovidedbyDr.LisaLott,EagleAppliedSciences,L.L.C., SanAntonio,TX.Finally,asubsetof308nasopharyngealaspirates (NPAs)fromanetiologicstudyof1162children<2yearsofage hospitalizedwithARIata tertiaryhospital inSãoPaulo,Brazil, betweenMarch2008andSeptember2010,wereselectedfromthe seasonalpeaksofrespiratoryviruscirculationforeachofthestudy yearsbasedonlocalsurveillancedata.TheNPAswerecollected directlyintoliquidnitrogen,aliquotedandtransferredto−70◦_C

andretaineduntilretrievedforthisstudy.Thisstudywasapproved byinstitutionalreviewboardsattheUniversityofSãoPauloand SantaCasadeMisericórdiadeSãoPauloHospital,Brazil,andCDC.

2.2. Totalnucleicacidextraction

Totalnucleicacid(TNA)extractswerepreparedfromsamples usingtheNucliSENS® _easyMAG® _{(bioMérieux).}_Because_of_their

multiplestudyoriginsandtestinghistories,residualarchived spec-imenextractionvolumesvariedfrom100to300␮LandTNAelution volumesfrom55to100␮L.RNase-freewaterwasaddedtoafew archivedextracts(≤2-folddilution)toobtainsufﬁcientvolumefor comparisontesting.Forprospectivelytestednasalaspirate speci-mens,300␮LofeachsamplewasextractedandtheTNArecovered in210_␮Lofelutionbufferwhichwasthensplitinto3aliquotsand frozenat−70◦_C_until_testing._All_extracts_were_subjected_to

iden-ticalfreeze-thawcyclesforcomparisontesting.Allextractswere

Table1

ComparisonofFTDRPandin-houseassayswith26virusisolates.

Virus(strain) In-house(Ct) FTDRP(Ct)a

AdVC1(Ad.71) Pos(13.7) Pos(16.7)

AdVC5(Ad.75) Pos(18.4) Pos(20.1)

AdVB7(SA-104) Pos(13.8) Pos(18.8)

AdVB14(deWit) Pos(20.8) Pos(23.3)

AdVE4(RI-67) Pos(15.2) Pos(18.2)

CoV229E Pos(10.3) Pos(13.2)

CoVOC43 Pos(13.0) Pos(14.9)

CoVSARS(Urbani) Pos(19.2) n/ac

EV,echovirus6b _Pos_(21.3) _Pos_(15.8)

EV,echovirus11b _Pos_(16.3) _Pos_(15.5)

EV,enterovirus68b _Pos_(21.3) _Pos_(24.6)

HMPVA(CAN97-83) Pos(15.0) Pos(17.0)

HMPVB(CAN98-75) Pos(18.3) Pos(21.2)

InfAH1N1(A/California/09) Pos(14.7) Pos(14.8)

InfAH2N1(A/Japan/57) Pos(27.3) Pos(24.4)

InfB(B/Shanghai/99) Pos(14.6) Pos(15.1)

PIV1(C35) Pos(16.6) Pos(19.1)

PIV2(Greer) Pos(16.9) Pos(15.8)

PIV3(C-43) Pos(15.2) Pos(16.3)

PIV4a(M-25) Pos(16.7) Pos(19.5)

PIV4b(CH19503) Pos(21.5) Pos(21.1)

PeV1b _Pos_(16.0) _Pos_(16.4)

RSVA(Long) Pos(15.0) Pos(15.7)

RSVB(CH93-18B) Pos(15.1) Pos(16.7)

RVA1a Pos(13.4) Pos(15.3)

RVB14 Pos(15.7) Pos(32.0)

a_Unless_otherwise_indicated,_all_other_FTDRP_assays_were_negative.

b_FTDRP_EV/PeV_assay_does_not_distinguish_between_EV_and_PeV.

c _FTDRP_SARS_CoV_assay_not_available_(n/a).

conﬁrmedpositiveforhumanRNasePgenebyreal-timeRT-PCR beforeinclusioninthestudy.

2.3. FTDRPmultiplexassay

The FTDRPmultiplex real-time RT-PCR assay(ver.5, cat. no. FTD2-96/12)consistsof5separateprimer/probemixescovering 16humanrespiratoryvirusesandbromemosaicvirus(BMV),an RNAplantvirusthatservesasaninternalextractioncontrolwhen spikedintothesample(virusprovided);mix#1:influenzaAvirus (InfA),influenzaBvirus(InfB),BMV;mix#2:coronavirus(CoV) NL63,229EandOC43andenterovirus/parechovirus(EV/PeV);mix #3:parainfluenzavirus(PIV)2,3and 4;mix#4: PIV1,human metapneumovirus(HMPV)andhumanbocavirus(HBoV);mix#5: rhinovirus(RV),respiratorysyncytialvirus(RSV)andadenovirus (AdV).Individualassayswithineachpoolaredistinguishedbyuse ofdifferentprobefluorophores,withtheexceptionoftheEVand PeVassays,wherebothprobesareROX-labeledandtherefore can-not be distinguished. Each kit alsocontains a positive plasmid controlpool anddetailed instructionsontest performance.The FTDRPassaywasperformedfollowingthemanufacturer’s recom-mendations.Briefly,194␮Lof2×RT-PCRbufferwascombinedwith 23.3␮Lofeachprimer/probepooland15.5␮Lof25×enzymemix (AgPath-IDTM_One-Step_RT-PCR_Kit,_Applied_Biosystems),_and₁₅_␮L

ofeach mixturewasthen addedto14wellsof aPCRplate(12 samplereactionsplusonepositiveandonenegativeviruscontrol). Ten␮LofsampleTNAextractorcontrolswerethenaddedtothe respectivewellsofeachprimer/probepool.Thefollowingcycling conditionswereperformedona7500FastDxReal-TimePCR Instru-ment(AppliedBiosystems):15minat50◦C,10minat95◦Cand40 cyclesof8sat95◦Cand34sat60◦C.Thresholdcycle(Ct)values weredeterminedbymanuallyadjustingthefluorescencebaseline tofallwithintheexponentialphaseoftheamplificationcurvesand aboveanybackgroundsignal.Apositive testresultwas consid-eredawell-definedcurvethatcrossedthethresholdcyclewithin 40cycles.Positiveandnegativevirusplasmidcontrolsprovidedin thekitwereincludedinallrunstomonitorassayperformance.The

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Table2

ComparisonofFTDRPandin-houseassayswith46samplesfrom5QCMDEQAprograms.

QCMDEQAa _Virus _QCMD_Keyb _In-house_(Ct) _FTDRP_(Ct)

Adenovirus(AdV)

ADV10-02 AdVF41 Pos(113) Neg/Pos(39.6)c _Pos_(39.4)

ADV10-03 AdVC1 Pos(64121) Pos(30.9) Pos(29.2)

ADV10-04 AdVE4 Pos(767) Pos(36.7) Pos(35.1)

ADV10-06 AdVC1 Pos(4055) Pos(34.0) Pos(32.0)

ADV10-08 AdVB34 Pos(1225) Pos(34.0) Neg/Negc

ADV10-07 Novirus Neg Neg Neg

Inﬂuenzavirus(Inf)

INFRNA09-01 InfAsubtypeH1 Pos(29.4) Pos(29.4) Pos(28.9)

INFRNA09-03 InfB Pos(39.2) Pos(38.3) Pos(38.2)

INFRNA09-04 InfAsubtypeH1vd _Pos_(28.7) _Pos_(28.7) _Pos_(25.7)

INFRNA09-07 InfB Pos(32.1) Pos(30.3) Pos(27.5)

INFRNA09-09 InfAsubtypeH1vd _Pos_(32.1) _Pos_(28.8) _Pos_(28.7)

INFRNA09-05 Novirus Neg Neg Neg

Parainﬂuezavirus(PIV)

PINF10-01 PIV1 Pos(33.1) Pos(32.7) Pos(38.1)

PINF10-08 Novirus Neg Neg Neg

Respiratorysyncytialvirus(RSV)&Humanmetapneumovirus(HMPV)

MPV.RSV10-01 RSVA Pos(38.4) Pos(36.7) Neg/Pos(36.8)c

MPV.RSV10-02 RSVB Pos(37.1) Pos(31.7) Neg/Pos(33.1)c

MPV.RSV10-04 RSVA Pos(33.4) Pos(31.1) Pos(30.7)

MPV.RSV10-09 RSVB Pos(39.9) Pos(34.8) Neg/Pos(37.3)c

MPV.RSV10-10 RSVB Pos(32.4) Pos(24.6) Pos(25.4)

MPV.RSV10-11 RSVA Pos(37.3) Pos(33.7) Pos(36.5)

MPV.RSV10-03 HMPVB2 Pos(35.5) Pos(29.7) Pos(32.5)

MPV.RSV10-07 HMPVA1 Pos(39.3) Pos(34.9) Neg/Pos(39)c

MPV.RSV10-08 HMPVA1 Pos(33.2) Pos(29.1) Pos(33.2)

MPV.RSV10-06 Novirus Neg Neg Neg

Rhinovirus(RV)&Coronavirus(CoV)

RV.CV10-01 RVB42 Pos(29.6) Pos(26.9) Pos(36.7)

RV.CV10-02 RVA8 Pos(25.8) Pos(22.5) Pos(24.0)

RV.CV10-03 RVB72 Pos(22.9) Pos(21.5) Neg/Negc

RV.CV10-04 CoV229E Pos(28.5) Pos(27.9) Pos(26.6)

RV.CV10-08 CoV229E Pos(35.0) Pos(34.0) Pos(32.5)

RV.CV10-06 CoVOC43 Pos(31.1) Pos(32.6) Pos(32.2)

RV.CV10-10 CoVNL63 Pos(26.9) Pos(25.7) Pos(24.1)

RV.CV10-11 EVe _Neg _Neg _Neg

a_QCMD_EQA,₂₀₁₀_Quality_Control_for_Molecular_Diagnostics_External_Quality_Assessment_program_samples.

b_QCMD_test_results;_Ct_values_(RV/CoV,_PIV,_RSV/HMPV)_and_genome_copies/mL_(AdV)._QCMD_Ct_values_should_not_be_used_for_method_comparison_or_as_a_target_for

individuallaboratoryassessment.

c _Original_and_repeat_result.

d _Inf_A_subtype_H1v₌_new_variant_pandemic_H1N1_strain.

e_QCMD_EQA_negative_RV_control_sample_contained_{coxsackievirus}_A1.

BMVinternalcontrolwasspikedintoclinicalspecimenstomonitor sampleextractionandreversetranscription.Previouslyextracted TNAsampleswereevaluatedforRNasePonly.

2.4. In-housesingleplexassays

In-housesingleplex real-timeRT-PCR assays forRSV, HMPV, PIV1-4,RV,AdV,HBoVandCoVs(229E,OC43,NL63,HKU1, SARS-CoV)aspreviouslydescribed(Dareetal.,2007;Emeryetal.,2004; Fryetal.,2010;Heimetal.,2003;Kodanietal.,2011;Luetal.,2006,

2008;Morganetal.,2012)wereperformedonaMX3000PQPCR System(AgilentTechnologies)usingAgPath-IDTM_One-Step_RT-PCR

reagents(AppliedBiosystems)withthefollowingcycling condi-tions:45◦Cfor10min,95◦Cfor10minand45cyclesof95◦Cfor15s and55◦Cfor1min.Primer/probesequencesareavailablefromD.E. onrequest.Thein-houseEVandPeVassaysasmodiﬁedfrom previ-ousreports(Kilpatricketal.,2009;Nixetal.,2008)wereperformed onaMX3000PQPCRSystemusingtheSuperScriptIIIPlatinum®

One-StepQuantitativeRT-PCRSystemreagents(Invitrogen)with thefollowingcyclingconditions:50◦Cfor30min,95◦Cfor5min

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and45cyclesof95◦Cfor15s,55◦C(EV)or58◦C(PeV)for45sand 72◦Cfor10s.UniversalInfAandInfBassayswereperformedona 7500FastDxReal-TimePCRInstrumentwithSDSsoftwarever.1.4 (AppliedBiosystems)usingtheSuperScriptIIIPlatinum®_One-Step

QuantitativeRT-PCRSystemwiththefollowingcyclingconditions: 50◦Cfor30min,95◦Cfor2minand45cyclesof95◦Cfor15sand 55◦Cfor30s(StephenLindstrom,CDC,personalcommunication). Following standard operating procedures, all in-house assays wereperformedin25_␮Lﬁnalreactionvolumescontaining5_␮Lof sampleTNAextract.Apositivetestresultwasconsidereda well-deﬁnedcurvethatcrossedthethresholdcyclewithin40 cycles. PositiveandnegativevirusRNAtranscriptorwholevirusextract controlswereincludedinallrunstomonitorassayperformance. 2.5. Statistics

Percent sensitivity and speciﬁcity of the FTDRP assay for prospectivelycollectedspecimens werecalculatedusingthe in-houseassaysasthereferencestandard.Agreementbetweenassays wasmeasuredusing theKappastatistic(Cohen, 1960)where 0 indicatesnoagreementand1indicatesperfectagreement.

3. Results

3.1. Virusisolates

TheFTDRPassaywasfirstevaluatedwithundilutedTNAfrom cultures of 26 respiratory virus strains corresponding to most assaysinthemultiplextoassessassayspecificityandvirusstrain inclusivity(Table1).AlthoughnoFTDRPassayforSARS-CoVwas available,thisviruswastestedtoassessthespecificityoftheother FTDRPCoVassays.HBoVandCoVNL63andHKU1isolateswere notavailablefortesting.Positiveresultswereobtainedwithboth assaysforallviruseswithnocross-reactionsdetected.FTDRPand in-houseassayresultswerewithin3Ctvaluesfor22(88%)ofthe virusestested.Notably,theFTDRPRVassaygavea substantially higherCtvalue(16.3Ct)withoneRVisolate(RV-B14).Serial dilutionsofRV-B14TNAshowedtheFTDRPassaytobe>1000-fold lesssensitivethanthecorrespondingin-houseassaywiththisvirus strain(datanotshown).

3.2. Pooledhumanrespiratoryspecimens

The specificity of the FTDRP assay was further evaluated with pooled nasal wash samples from 20 consenting normal healthyadultstorepresentdiversemicrobialflorainthehuman respiratorytract.Positiveresultswereobtainedwiththein-house assaysforRV(Ct25.0),CoV229E(Ct35.1)andAdV(Ct39.3)which wereconfirmedbyalternateRT-PCRassaysandsequencing.The FTDRPassaywaspositiveforRV(Ct28.3)andCoV229E(Ct34.8), butdidnotdetecttheAdVoninitialorrepeattesting.Allother in-houseandFTDRPassayswerenegative.

3.3. QCMDEQAprogramsamples

Forty-onemockrespiratorysamplesspikedwithlowto mod-eratelevelsof differentvirusesand 5negative controlsamples selectedfrom2010QCMDEQAprogramsforHRV/CoV,AdV,PIV, RSV/HMPVandInfA/B,weretestedtoassessassayperformance againstthereference QCMDassays (Table2).Overall, expected resultswereobtainedwith40(98%)and35(85%)ofpositiveEQA programsampleswiththein-houseandFTDRPassays,respectively. Allprogramnegativecontrolsampleswerenegativebybothassays. Onesample(AdV10-02),withlowconcentrationAdV-F41,was ini-tiallynegativebythein-houseassay,butpositiveonrepeattesting.

TheFTDRPassaygaveexpectedresultswithallPIV(5),CoV(4)Inf Table

3 Comparison of FTDRP and in-house assays with 263 archived respiratory specimens previously positive for respiratory viruses. Virus a In-house + FTDRP + FTDRP % + Ct <30 b Ct ≥ 30 to ≤ 37 b Ct >37 to <40 b Total + FTDRP + FTDRP − FTDRP % + Total + FTDRP + FTDRP − FTDRP % + Total + FTDRP + FTDRP − FTDRP % + AdV 25 17 68% 8 8 0 100% 11 8 3 73% 6 1 5 17% CoV 229E 5 5 100% 3 3 0 100% 2 2 0 100% 0 CoV OC43 7 7 100% 5 5 0 100% 2 2 0 100% 0 CoV NL63 8 8 100% 7 7 0 100% 1 1 0 100% 0 EV/PeV c 8 8 100% 4 4 0 100% 4 4 0 100% 0 HBoV 2 2 100% 2 2 0 100% 0 0 HMPV 26 26 100% 20 20 0 100% 6 6 0 100% 0 Inf A 17 17 100% 12 12 0 100% 5 5 0 100% 0 Inf B 1 1 1 1 100% 10 10 0 100% 1 1 0 100% 0 PIV 1 2 0 1 7 85% 9 9 0 100% 11 8 3 73% 0 PIV 2 13 12 92% 6 6 0 100% 6 5 1 83% 1 1 0 100% PIV 3 3 1 3 0 97% 15 15 0 100% 16 15 1 94% 0 PIV 4 12 12 100% 9 9 0 100% 3 3 0 100% 0 RSV 31 23 74% 12 12 0 100% 11 10 1 91% 8 1 7 14% RV 47 37 79% 38 33 5 87% 9 4 5 44% 0 Total 263 232 88% 160 155 5 97% 88 74 14 84% 15 3 12 20% a Virus co-detections not included in the analysis. b In-house assay results classiﬁed as strong (Ct <30), moderate (Ct ≥ 30 to ≤ 37) or weak (Ct >37) positive. c FTDRP EV/PeV assay does not distinguish between EV and PeV. Eight specimens separately tested positive for EV (3Ct <30; 2Ct ≥ 30 to ≤ 37) and PeV (1Ct <30; 2Ct ≥ 30 to ≤ 37) by in-house assays.

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Table4

ComparisonofFTDRPandin-houseassayswith32archivedrespiratoryspecimenswithsequenceconﬁrmedrhinovirus(RV)orenterovirus(EV).

Virusa _RV _EV _PeV _EV/PeV

In-house(Ct) FTDRP(Ct) In-house(Ct) In-house(Ct) FTDRP(Ct)b

EV,enterovirus68 Neg Neg Pos(31.1) Neg Pos(30.7)

EV,enterovirus68 Neg Neg Pos(28.9) Neg Pos(24.4)

EV,echovirus9 Neg Neg Pos(23.5) Neg Pos(23.4)

EV,coxsackievirusB4 Neg Neg Pos(22.6) Neg Pos(21.6)

EV,coxsackievirusB5 Neg Neg Pos(31.0) Neg Pos(27.1)

RVA18 Pos(17.7) Pos(23.1) Neg Neg Neg

RVA38 Pos(21.1) Pos(24.6) Neg Neg Pos(36.1)

RVA68 Pos(22.8) Pos(22.6) Neg Neg Pos(31.4)

RVB6 Pos(12.9) Pos(24.1) Neg Neg Neg

RVB6 Pos(27.8) Neg Neg Neg Neg

RVB97+C Pos(28.3) Pos(29.5) Neg Neg Neg

RVC Pos(23.2) Pos(22.9) Neg Neg Neg

RVC Pos(26.8) Neg Neg Neg Neg

RVC Pos(17.3) Pos(19.9) Negc _Neg _Pos_(28.3)

RVC Pos(18.6) Pos(25.5) Pos(31.6) Neg Pos(34.6)

a_RV_species_A,_B,_C;_no_{serotype-speciﬁc}_{determination}_for_RV_species_C.

b_FTDRP_EV/PeV_assay_does_not_distinguish_between_EV_and_PeV.

c _Ct_(41.4)_above_assay_cutoff.

A(8)andInfB(2)positivesamples,and3of6(50%)RSV,4of5 (80%)HMPV,5of6(83%)RVand4of5(80%)AdVpositivesamples. RSV(MPV.RSV10-01,MPV.RSV10-02,MPV.RSV10-09)andHMPV (MPV.RSV10-07) positivesamples thatwere negativebyFTDRP assayhadgenerallylowervirusloadsandwerepositiveonrepeat testing.Incontrast,EQAsamplesspikedwithRV-B72(RV.CV10-03) andAdV-B34(ADV10-08)wereconsistentlynegativeandRV-B42 (RV.CV10-01)showedsubstantiallyhigherCtvalues(9.8Ct)by theFTDRPRVassay.

3.4. Archivedclinicalspecimens

Twohundredsixty-fivediverserespiratoryspecimensthat pre-viouslytestedpositiveforrespiratoryvirusesbyin-houseassays wereselectedforcomparisonwiththeFTDRPassay.Ofthese,263 werepositive for at leastone ofthe16 assays available inthe FTDRPmultiplex;twospecimenspositiveforCoVHKU1forwhich therewasnocorrespondingFTDRPassaywerealsotestedtoassess thespecificityoftheotherFTDRPCoVassays (Table3).Because oflimitedavailablesamplevolume,onlyFTDRPmultiplexmixes containingthevirus-specificassaywereperformedandvirus co-detectionsbytheother assaysin each multiplexmix werenot includedintheanalysis.Allspecimenswereconfirmedpositive byin-housesingleplexassaysonretesting.TheFTDRPassay iden-tifiedallspecimensthatwerepositiveforHBoV(2),CoVNL63(8), InfA(17),InfB(11),HMPV(26),PIV4(12)andEV/PeV(5EVand3 PeV);>90%forPIV2(12/13)andPIV3(30/31);85%forPIV1(17/20); 79%forRV(37/47);74%forRSV(23/31);and68%forAdV(17/25). Overall,theFTDRPassayidentifiedcorrectly88%ofthearchived specimenspositiveforrespiratoryvirusesbythein-houseassays

and97%ofspecimenswithlowerCtvalues(<30).Twospecimens positiveforCoVHKU1byin-housesingleplexassaywerenegative bytheFTDRPCoV229E,OC43andNL63assays.

TheFTDRPAdV,RSV andRV assaysgave thelowestrelative sensitivities withthearchived specimens at68%, 74%and 79%, respectively.WiththeexceptionofRV,mostdiscrepanciesoccurred withsamplescontaininglowlevelsofviraltarget.Forexample, mostFTDRPAdVfalse-negativesoccurredwithmoderatetohigh Ctvaluespecimens(meanCt37.3;range33.0–39.5),butthisdid notappeartobeassociatedwithanyparticularAdVtype.Awide rangeofsequence-conﬁrmedAdVtypeswererepresentedamong thearchivedspecimens,includingspeciesB(types3,7and50),C (types2,5,6anduntyped)andF(types40and41),suggestingthat theFTDRPAdVassayisinclusivefor allrecognizedhumanAdV types.Incontrast,FTDRPRVassayfailedtodetect5RVpositive sampleswithlowCtvaluesbythecorrespondingin-houseassay.

Tofurtherassess theFTDRPRV andEV/PeV assays for virus type/straininclusivityandgroupexclusivity,32archivedsamples withhighRV(27)orEV(5)loadsandtypedbypartialVP1and/or VP4/2 RT-PCRand sequencing(protocolsavailable fromX.L. on request)wereretested(Table4).TheFTDRPRVassaygavenegative resultswith4samplesandwas≥10Ctvalueshigherthanthe in-houseassaywith2others,allspeciesBorCRVs.TheFTDRPEV/PeV assaywasalso positivewith4 sequence-confirmed RVpositive specimensofwhich1wasalsopositivebythein-houseEVassay; a secondsamplealsogavean exponentialfluorescence amplifi-cationcurvewiththein-houseEVassay,butwitha>40Ctvalue andwasthereforeclassifiedasEV-negativebasedontestcutoff criteria.AlthoughEVwasnotdetectedin2ofthesesamplesby VP4/2RT-PCR,andPeVwasnotdetectedbythein-houseassay,the

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presenceofthesevirusescouldnotberuledoutdeﬁnitively. Never-theless,themostprobableexplanationfortheseresultsisthatthe FTDRPandin-houseEVassayscross-reactwithsomeRVstrains. Fivesequence-conﬁrmedEV-positivesampleswerepositivebythe FTDRPEV/PeVassaywithnoevidenceofcross-reactionswiththe RVandPeVassays.

3.5. Prospectivelytestedclinicalspecimens

Threehundred-eightnasopharyngealaspiratesselectedfroma studyofinfantsandyoungchildrenhospitalizedwithacute res-piratoryinfectionweretestedprospectivelybybothin-houseand FTDRPassays.Ofthese,277(89.9%)werepositiveforoneormore ofthe16virusesbyeitherthein-housesingleplexorFTDRP mul-tiplexassays,with270(87.7%)positivebythein-houseassayand 265(86%)positivebyFTDRPassayalone(Table5).Overall,the in-houseandFTDRPassaysshowedgoodconcordance(K=0.812,95% CI=0.786–0.838)(Table6).Asseenwiththearchivedspecimens, however,the FTDRPAdV,RSV and RV assays gave consistently lowerdetectionratesthanthecorrespondingin-houseassays,at 43.7%,72.5%and75.5%,respectively,andmissedsomespecimens withhighvirusloads.Coincidently,thesethreeassaysare com-bined in the same reaction mix (mix#5) and had the highest co-detectionrateforthesevirusesbyin-houseassaysat41.9%; fol-lowedbymix#4(PIV1,HBoV,HMPV)at23.8%;mix#2(CoV229E, CoVOC43,CoVNL63,EV/PeV)at16.3%;mix#3(PIV2,PIV3,PIV4) at7.3%;andmix#1(InfA,InfB)at2.2%.Simultaneouspresence ofmultipletargetsinthesamespecimenmayhaveledto compet-itiveinhibitionofampliﬁcationoflessabundanttargetsandmay explainsomelossofassaysensitivity.

TheFTDRP HBoV assay appeared to bemore sensitive than thecorrespondingin-houseassay(Luetal.,2006)withspecimens containinglowlevelsofHBoV.Tofurtherinvestigatethisfinding, limitedsequencingstudieswereperformedusinganewly devel-opedsemi-nestedPCRassayspecificfortheHBoVNS1genethat amplifiesall4recognizedHBoVtypes(protocolavailablefromX.L. uponrequest).Ofthe49specimenspositiveforHBoVbyboth in-houseandFTDRPassays,36of37(meanin-houseCt26.6;range 13.3–37.3)weresuccessfullysequenced(allHBoVtype1).In con-trast,only 2 of4 in-houseassaypositive (mean Ct37.7; range Ct37.3–38.4)/FTDRPnegativeandnoneofthe20FTDRPpositive (meanCt38.2;rangeCt36.3–39.9)/in-housenegativespecimens couldbeconfirmedbyNS1PCRandsequencing.Failuretoresolve thesediscrepanciesmaybedueto(i)a highersensitivityofthe FTDRPassaywithspecimenscontaininglowlevelsofHBoVDNA, possiblyattributabletothelargervolumeofTNAextractusedin theFTDRPassay(10␮Lvs.5␮L),(ii)failureofbothassaystodetect somevariantHBoVstrainsand/or(iii)non-specificamplificationor ampliconcontaminationinthesesamples.

TheFTDRPEV/PeVassayalsoappearedtobemoresensitiveand specificthanthecorrespondingin-houseEVassaywithsome spec-imens.Of18specimenspositivebytheFTDRPEV/PeVassay(mean Ct34.6;range29.9–38.4),and negativebyin-houseEVandPeV assays,9hadrecoverableVP1and/orVP4/2sequences represent-ing8differentEVs(echovirus6,24,30;enterovirus68;poliovirus 1;coxsackievirusA4,B1,B4);3,thatwerealsopositivebyin-house andFTDRPRVassays(Ct<30),hadsequence-confirmedspeciesA RVofwhich2alsohadtype-indeterminateEVsequencespresent; 1gaveafluorescenceamplificationcurvewiththein-housePeV assay,butwithaCtvalue>40andthereforewasclassifiedasPeV negative;and5couldnotbesequenced.Of9samplespositiveby thein-houseEVassayandnegativebytheFTDRPEV/PeVassay,all werestronglypositiveforRV(Ct<30)bybothin-houseandFTDRP RVassaysandwereconfirmedpositiveforRVspeciesAorCbyVP1 and/orVP4/2sequences.All12specimenspositivebythein-house Table

5 Comparison of FTDRP and in-house assays with 308 prospectively tested respiratory specimens. Virus a In-house + FTDRP + In-house + FTDRP − In-house − FTDRP + c In-house − FTDRP − Ct <30 b Ct ≥ 30 to ≤ 37 b Ct >37 to <40 b Total + FTDRP + FTDRP − FTDRP % + Total + FTDRP + FTDRP − FTDRP % + Total + FTDRP + FTDRP − FTDRP % + AdV 38 49 0 221 32 29 3 91% 47 9 38 19% 8 0 8 0% CoV 229E 6 0 1 301 4 4 0 100% 2 2 0 100% 0 CoV OC43 19 4 0 285 14 14 0 100% 6 5 1 83% 3 0 3 0% CoV NL63 19 0 0 289 9 9 0 100% 10 9 1 90% 0 EV/PeV d 36 9 18 245 19 16 3 84% 23 17 6 74% 3 3 0 100% HBoV 49 4 20 235 25 25 0 100% 17 17 0 100% 11 7 4 64% HMPV 50 17 0 241 39 39 0 100% 19 10 9 53% 9 1 8 11% Inf A 29 1 0 278 17 17 0 100% 10 10 0 100% 3 2 1 67% Inf B 14 1 0 293 9 9 0 100% 5 5 0 100% 1 0 1 0% PIV 1 9 4 0 295 7 7 0 100% 5 2 3 40% 1 0 1 0% PIV 2 1 0 0 307 1 1 0 100% 0 0 PIV 3 51 2 0 255 35 35 0 100% 13 12 1 92% 3 2 1 67% PIV 4 8 0 2 298 2 2 0 100% 3 3 0 100% 3 3 0 100% RSV 74 28 0 206 80 68 12 85% 15 6 9 40% 7 0 7 0% RV 80 26 3 199 90 73 17 81% 15 7 8 47% 1 0 1 0% Total 483 145 44 3948 383 348 35 91% 190 114 76 60% 53 18 35 34% a Virus co-detections included in the analysis. b In-house assay results classiﬁed as strong (Ct <30), moderate (Ct ≥ 30 to ≤ 37) or weak (Ct >37 to <40) positive. c FTDRP assay Ct values, median (range): HBoV, 38.3 (36.3–39.9); CoV 229E, 38.3; PIV 4, 39.4 (38.9, 39.9); RV, 34.8 (34.8–37.0); EV/PeV 34.7 (30.0–38.4). d FTDRP EV/PeV assay does not distinguish between EV and PeV. Twelve samples positive by in-house PeV assay were also positive by FTDRP EV/PeV assay.

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Table6

FTDRPandin-houseassaysensitivity,speciﬁcityandKappavalueswith308prospectivelytestedrespiratoryspecimens.

Virusa _FTDRPb _In-houseb _Kappa_statisticc_(95%_CI)

Sensitivity Speciﬁcity Sensitivity Speciﬁcity

AdV 43.7 100.0 100.0 82.0 0.527(0.405–0.648) CoV229E 100.0 99.7 85.7 100.0 0.921(0.767–1) CoVOC43 82.6 100.0 100.0 98.6 0.898(0.798–0.997) CoVNL63 100.0 100.0 100.0 100.0 1.(1–1) EV/PeVd _80.0 _93.2 _66.7 _96.5 _0.676_{(0.559–0.793)} HBoV 92.5 92.2 71.0 98.3 0.756(0.662–0.85) HMPV 74.6 100.0 100.0 93.4 0.822(0.739–0.904) InfA 96.7 100.0 100.0 99.6 0.981(0.946–1) InfB 93.3 100.0 100.0 100.0 0.964(0.893–1) PIV1 69.2 100.0 100.0 98.7 0.812(0.628–0.995) PIV2 100.0 100.0 100.0 100.0 1.(1–1) PIV3 96.2 100.0 100.0 99.2 0.977(0.945–1) PIV4 100.0 99.3 80.0 100.0 0.886(0.728–1) RSV 72.5 100.0 100.0 88.0 0.780(0.702–0.857) RV 75.5 98.5 96.4 88.4 0.780(0.704–0.856) Allassays 77.0 98.8 91.3 96.5 0.812(0.786–0.838)

a_Virus_{co-detections}_included_in_the_analysis.

b_Referenced_to_FTDRP_or_in-house_assay.

c _Kappa_statistic:_<0–0.2₌_poor;_0.21–0.4₌_fair;_0.41–0.6₌_moderate;_0.61–0.8₌_good;_and_0.81–1₌_very_good._CI,_conﬁdence_interval.

d _FTDRP_EV/PeV_assay_does_not_distinguish_between_EV_and_PeV.

PeVassay(meanCt33.8;range27.7–38.4)werealsopositiveby theFTDRPEV/PeVassaywithsimilarCtvalues.

4. Discussion

DiagnosisofARIinbothclinicalcareandpublichealthsettings hasgreatlyadvancedinrecentyearswiththeincreasedavailability ofrapid,sensitiveandspecificmoleculartestsforthe simultane-ousdetectionofmultiplerespiratorypathogens.Somecommercial assaysinparticularthathavereceivedFDA510(k)clearancehave madesubstantialinroadsintothediagnosticlaboratory(Randetal., 2011).However,theseassaysareoftencostly,requirededicated laboratory equipment, use highly multiplexed reactions where individualassayperformancemaybecompromised,and canbe difficulttomodifyquicklyinresponsetotheemergenceofnew medicallyimportant virusstrains, as occurredduring the2009 H1N1influenzapandemic.

The commercial multiplex FTDRP real-time RT-PCR assay addressessomeoftheselimitationsbyofferingacompletekitwith moderatethroughputfordetectionof16respiratoryvirusesthat couldbeeasilyintegratedintotheworkflowoflaboratoriesusing conventionalreal-timePCRplatforms.TheFTDRPassaysetupand runtimerequiresapproximately2.5hfor12samplesandcontrols (assayreagentsarealiquotedin12sampletestunits),excluding sampleextraction,andwithakitlistpriceof$27.34/sample(PCR enzymekitcostsnotincluded).Bycombiningassaysinto5 multi-plexreactionmixes,individualmixescouldmoreeasilymodified ifneededwithoutimpactingtheothermixesandcouldallowfor moreefficienttargetedtestingbasedonepidemiologicfindings.

Inthisstudy,theFTDRPmultiplexassaywascomparedwith in-housesingleplexassayscorrespondingtoeachofthetestviruses. Overall,theFTDRPandin-houseassaysperformedcomparablyfor mostvirusestested,particularlywhentheviruswasabundantin thesample(lowCtvalues).Withexceptionsnotedbelow,most discordantresultswereseenwithsamplescontaininglower con-centrationsofvirus(highCtvalues),suggestingthatdifferences inassaysensitivityneartheirdetectionlimitswasresponsiblefor thesediscrepanciesratherthanfailureofprimer/probe hybridiza-tionduetocriticaltargetsequencemismatches.

FTDRPassaysforRSV,RVandAdVinparticularshowedlower relativesensitivitiesthanthecorrespondingin-houseassayswith

someclinicalspecimens.TheFTDRPRVassayshowedclear evi-denceofdropoutswithsomeRVstrains(seefurtherdiscussion below),andsomeprospectivelytestedspecimenswerenegativefor RSVandAdV,evenwhentheviruseswereabundant.Itisnotable that thesethree FTDRP assaysare combined inthe same reac-tionmixandthesethreevirusesshowedthehighestco-detection ratesbysingleplexin-houseinthesespecimens.Itispossiblethat competingampliﬁcationreactionsinsomespecimenscontaining multiplevirustargetsmayhavereducedthesensitivityofsome assaysforlowabundanttargets.Thismayhavehadamore notice-ableimpactondetectionofAdV,whereadisproportionatenumber ofAdVpositive specimenshadlowervirusloads.Thiswouldbe expectedinapopulationcomprisedofinfantsandyoungchildren wherepersistentlowlevelAdVsheddingiscommon.

Developmentofreal-timeRT-PCRassaysthatcandetectallRV andEVstrainsanddistinguishbetweenbothgroupsis challeng-ingduetotheextensivesequencediversitywithineachgroupand sequencesimilaritybetweensomeEVandRVstrains.Thesedata confirmedpreviousexperiencewiththein-houseEVandRVassays: bothassayscross-reactwithsomeRVandEVstrains,particularlyif presentinhighcopynumber(Luetal.,2008;Obersteetal.,2010). AlthoughthiscomplicatedeffortstoevaluatetheFTDRPEV/PeVand RVassays,severalconclusionscanbedrawnfromthesefindings. TheFTDRPEV/PeVassayappearedtobemoresensitiveandspecific thanthein-houseassayfordetectionofsomeEVstrains,including therecentlyemergentEV68(CDC,2011),althoughcross-reactions withsomeRVstrainsidentifiedinthearchivedsamplecollection couldnotberuledout.Ofparticularconcern,theFTDRPRVassay wasinsensitivewithsomesequenceconfirmedRVspeciesBandC strains.Thisfindingsuggeststhatcriticalprimer/probemismatches withthesevirusessubstantiallydiminishedtargetamplification and/orprobehybridization.Moreextensivetestingofculture puri-fiedRVandEVstrainswillbenecessarytoassessthefullextentof thisdeficiency.

Limiteddiscrepancytestingwasconductedandfocused primar-ily onsampleswithlargedifferences in Ct values betweenthe in-houseandFTDRPassays.Toresolvealldiscrepant resultsfor allassayswouldrequireextensiveconﬁrmatorytestingwith addi-tionalmoleculartestspossessingequalorgreatersensitivitythan thoseevaluatedhere,whichwasbeyondthescopeofthisstudy. Limitedspecimenvolume prevented additionaltesting in some

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cases.Theapparentfalse-positiveresultsbyeitherassayweremost likelytruepositivesoccurringastheresultoftheconditionsnoted above.

Followingcompletionofthiswork,Fast-trackDiagnostics intro-ducedanewversionoftheFTDRPassay(FTDRespiratoryPathogens 21)thatexpandstestingtoincludenewrespiratorypathogensand modiﬁesofsomeoftheexistingassaystoenhanceperformance (MiriamSteimer,Fast-trackDiagnostics,personalcommunication). Giventhesechanges,andthepromisingpotentialoftheFTDRP mul-tiplexassayfordiagnosisofrespiratoryvirusinfections,further studieswithexpandedsamplecollectionsarewarranted.

Acknowledgement

FundingforthisprojectwasawardedbytheHighPriority Pan-demicandSeasonalInﬂuenzaScientiﬁcproposalrequestinitiative.

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