Journal of Medical Virology 84:979985 (2012)Broad Respiratory Virus Detection in InfantsHospitalized for Bronchiolitis by Use of aMultiplex RT-PCR DNA Microarray SystemAntoine Huguenin,1Lauryane Moutte,1Fanny Renois,1Nicolas Leveque,1Deborah Talmud,1Michel Abely,2Yohan Nguyen,1Fabrice Carrat,3and Laurent Andreoletti1*1Medical and Molecular Virology Unit and EA-484, University Hospital Center and Medical School of Reims,Reims, France2Pediatric Department, American Memorial Hospital, University Hospital Center of Reims, Reims, France3UMR-S 707 Medical School of St. Antoine, Paris, FranceNewlyavailablemoleculartoolsallowasensi-tivedetectionofabroadpanelofvirusesinre-spiratorytractspecimens. Inthepresentstudy,the application of a multiplex RT-PCR DNAmicroarray in diagnosis and epidemiologicalsurveyofviralinfectionsininfantshospitalizedfor bronchiolitis was assessed. One hundredandthirty-eight nasopharyngeal aspirates col-lectedfromOctober 2007 toSeptember 2008weretestedbydirectimmunouorescenceandviral culture, acombinationof referencedRT-PCRs andthe DNAmicroarray. One or morevirusesweredetectedin96,126and126ofthespecimens bydirect immunouorescenceandviral culture, RT-PCRs and DNA microarray,respectively(70vs. 91vs. 91%, P0.1). Inconclusion, theuseof this DNAmicroarray in clinical virology prac-tice allows rapid and accurate identicationof common and uncommon viral respiratorypathogensininfantshospitalizedforbronchiol-itis. It should improve theclinical management,theepidemiologicalsurvey,andthepreventionof the nosocomial transmission of respira-toryvirusesinpediatricwards. J.Med.Virol.84:979985, 2012. 2012 Wiley Periodicals, Inc.KEYWORDS: bronchiolitis; infants; RT-PCRDNA microarray; respiratoryviruses; clinical severityparametersINTRODUCTIONBronchiolitis is a common distressing, potentiallylife-threatening respiratory condition that affectsinfants. Hospital admission rates in the USA andEuropeforbronchiolitisarereportedtobearound30per 1,000for childrenyounger than12months andthis rate has increased signicantly over the past10years[SmythandOpenshaw, 2006]. Hospital ad-missionisrequiredforinfantswithmoderatediseasebecause mucus obstruction interferes with feedingand may induce apnoea. In the most severe cases,there is hypoxia and respiratory distress that mayrequire mechanical ventilation [Wainwright, 2010].Bronchiolitis is animportant manifestationof viralGrant sponsor: Reims University Medical Centre; Grant spon-sor: French Army Department (Bourse DGA: DelegationGenerale de lArmement, Ministe`re de la Defense, Topic:Microbiology, infectious diseases; to F.R.).Noneof theauthorsof thepresent manuscript haveacom-mercial or other association that might pose a conict of interest(e.g., pharmaceutical stockownership, consultancy). Thisworkwas supportedinpart byagrant for Clinical andVirologicalResearch(IFR53/EA)fromtheMedicalUniversityandSchoolofMedicine of Reims, France.*Correspondence to: Prof. Laurent Andreoletti, Laboratoire deVirologieMedicaleetMoleculaire,etEA, HopitalRobertDebre,Avenue du General Koenig, 51092 REIMS Cedex, France.E-mail: landreoletti@chu-reims.frAccepted 15 February 2012DOI 10.1002/jmv.23272Published online in Wiley Online Library(wileyonlinelibrary.com). 2012 WILEY PERIODICALS, INC.respiratory tract infections and a large variety of viralpathogensarerecognizedtobeimplicatedinmajorityof thehospitalizedcases[Jacqueset al., 2006; Mah-ony, 2008; Marguet et al., 2009; Wainwright, 2010].Previousstudiesshowedthathumanrespiratorysyn-cytial virus (hRSV) and picornaviruses [enterovirus(EVs)andrhinovirus(HRVs)]aretheleadingetiologi-cal causes of bronchiolitis incohorts of Frenchchil-dren presenting with acute respiratory pathologiesincluding acute wheezing illnesses or bronchiolitisand asthma [Jacques et al., 2006; Jacques et al.,2008a; Frobert et al., 2011]. Humanmetapneumovi-rus (hMPV), parainuenza virus (PIV), coronavirus(HCoV), humanbocavirus(HBoV)havealsobeenfre-quentlydetectedinFrance inacute bronchiolitis inhospitalized children as well as in the community[Jacques et al., 2008b; Marguet et al., 2009; Freymuthet al., 2010].Regardingthediagnosis of respiratorytract infec-tions,virologylaboratorieshavetraditionallyuseddi-rectimmunouorescenceassayandvirusisolationoncellculture[Jarttietal.,2004;SmythandOpenshaw,2006;Mahony,2008].However,becauseofthelimitedscopeandthroughput of theseconventional viral de-tectionmethods, manybronchiolitis cases are nega-tive in routine virological diagnosis [Mahony, 2008].Thedevelopmentofnewmolecularassaysbasedondifferent technologies has allowedasensitive detec-tionofabroaderpanel ofvirusesinrespiratorytractspecimens [Li et al., 2007; Mahony, 2008; Renoiset al., 2010; Frobert et al., 2011; Ginocchio, 2011].Amongthesenewmoleculartools,newsystemsbasedonmultiplexRT-PCRassaysfollowedbylowdensitymicroarrayanalysishave beendevelopedandrecentlyevaluatedinalimitednumberofclinicalsamplestak-en fromhospitalized infants [Frobert et al., 2011;Ginocchio,2011].Thesenewassayscouldallowarap-id detectionand type or subtype identicationof abroad panel of common and newly identied respirato-ry viruses [Frobert et al., 2011; Ginocchio, 2011].Theycouldalsoimprovetheclinical management ofinfants andthe preventionof the nosocomial trans-missioninpediatricwardsandallowthedevelopmentofnewepidemiologicalsurveysystemsforrespiratoryviral infections [Davidet al., 2010]. Theaimof thisstudywastoevaluatetheanalytical andclinical per-formances of a European Community (CE)- and invitrodiagnosis (IVD)-markedcommerciallyavailablemultiplex RT-PCR DNA microarray, the CLART1PneumoVir kit (Genomica SAU, Madrid, Spain),allowing a rapid and simultaneous detection of 17DNAandRNAhumanrespiratoryvirusesinclinicalspecimens.PATIENTS AND METHODSPatients and SpecimensOne hundred and thirty-eight children (sex ratioM/F 1.42) hospitalized in the pediatric department(Reims University Medical Centre, France) fromOctober 2007to September 2008were prospectivelyselected. Theydemonstratedclinicalsignsofbronchi-olitis accordingto the Frenchconsensus conference.Theywere 12months oldat the most (meanage 4months, SD 1.36)andwereadmittedinthepedi-atric unit within3 days of symptoms onset [Davidet al., 2010]. Nasopharyngeal aspiratesampleswerecollectedattheadmissionandroutinelyaddressedtothevirologylaboratoryforrespiratoryvirusesdetec-tionbydirectimmunouorescenceandvirusisolationoncellculture, dividedinaliquotsandthenstoredat808Cuntil processing withthe multiplex RT-PCRDNA microarray.The severityof the bronchiolitis wasretrospectivelyanalyzedusingaset of ve classicalcriteria including intensive care unit admission, O2supply, O2saturationpercentage(pulseoxymetry) athospital admission, O2lengthandlengthof stayatthehospital [Marguetetal., 2009]. Informedconsentwas obtained fromthe infants family. The presentstudy was conducted by the University Medical Hospi-tal of Reims(ChampagneArdenne, France) andwasapproved by the hospital ethics committee.Conventional Respiratory VirusDetection AssaysDirect immunouorescenceassayfor thedetectionofhRSVAandB,inuenzaAandB,PIVs1to3,andadenovirus(AdVs)antigenswascarriedoutasprevi-ously described [Bouscambert et al., 2005; Jacquesetal.,2006].Twohundredmicrolitersofthespecimenwereinoculatedinduplicateonto24-well platescov-ered with monolayers of human diploid broblasts(MRC-5), Rhesus monkey kidney (MA-104) andMadin-Darby canine kidney (MDCK) cells as previ-ouslydescribed[Bouscambertetal., 2005]. Virusiso-lates were typed by direct immunouorescence oninfectedcellmonolayersforhRSVAandB, inuenzaviruses, PIVs, and AdVs and by conventional neutrali-zationassayforEVs[Melnicketal., 1977; Bouscam-bert et al., 2005].Detection of 17 Respiratory Viruses byMultiplex RT-PCR DNA MicroarrayCLART1(CLinical ARrayTechnology) PneumoVirkit V16.3 (Genomica) is based on viral genome-specicfragments amplicationlocatedbetween106328bpby multiplex PCR and its subsequent detection via hy-bridizationwithmicroorganism-specicbindingprobeon low-density microarrays, allowing simultaneousdetectionandidenticationof 17typesandsubtypesof humanrespiratoryviruses (inuenzaAincludingseasonal A/H1N1 and A/H3N2 strains, inuenza B,inuenza C, parainuenza 1, 2, 3, 4A and 4B, hRSV AandB,humanrhinoviruses,adenoviruses,EVsspecieB, humanbocavirus, coronavirus E-229andthehu-manmetapneumovirus AandB) inclinical samples[Renois et al., 2010; Frobert et al., 2011]. The analyseswere performed from 10mL of DNA/RNA extract(NucliSenseasyMAG1, bioMerieux, Lyon, France) in980 Huguenin et al.J. Med. Virol. DOI 10.1002/jmveight-well strips according to the manufacturersinstructions.To conrmthe results obtained with the DNAmicroarray,acombinationofmultiplexandmonoplex(RT)-PCRswasperformedusingthesameDNA/RNAextracts [Freymuth et al., 1997; Eugene-Ruellanet al., 1998; Coiras et al., 2003; Coiras et al., 2004;Lopez-Huertaset al., 2005; Pozoet al., 2007; Gauntet al., 2010].Statistical AnalysesChi-squaretest,McNemarChi-squaretest,Fishersexact test, or Wilcoxonrank-sums test were carriedout as appropriate using the SASsoftware, version9.1.3(SASInstitute, Cary, NC). Resultswereconsid-eredasstatisticallysignicantfortwo-sidedP-values