samnajie

ArchivesofVirology(2018)163:725–730https://doi.
org/10.
1007/s00705-017-3645-1BRIEFREPORTAcomparisonoftheMeltProHPVTestwiththeCobasHPVTestfordetectingandgenotyping14highriskhumanpapillomavirustypesZhitengTang1·YeXu2·NajieSong3·DongqingZou3·YiqunLiao4·QinggeLi2·ChaoPan1Received:11May2017/Accepted:3October2017/Publishedonline:5December2017Springer-VerlagGmbHAustria,partofSpringerNature2017AbstractTheclinicalperformanceofthenewlydevelopedMeltProHPVTest,basedonmulticolormeltingcurveanalysis,wasevaluatedandcomparedwiththecommerciallyavailableCobasHPVTestfordetectionofHPVandgenotypingofHPV-16andHPV-18.
Atotalof1647cervicalsampleswereanalyzedwithbothtests.
Theagreementvalueswere96.
2%forHPVdetection,99.
6%forHPV-16identification,and99.
7%forHPV-18identification.
AllgenotypingresultsfromMeltProHPVTestshowedthatHPV-52,HPV-58,andHPV-16werethemostcommontypesinthisstudy.
Intra-laboratoryreproducibilitystudiesshowed97.
8%agreementwhileinter-laboratoryreproducibilitystudiesshowed96.
9%agreementfortheMeltProHPVTest.
TheMeltProHPVTestandCobasHPVTestarehighlycorrelativeandareusefulformonitoringHPVinfection.
High-riskhumanpapillomavirus(HPV)infectionisahighriskfactorforthedevelopmentofcervicalcancer,whichisthesecondmostcommonmalignanttumorinwomenworld-wide[1].
High-riskHPVdiagnosescombinedwithliquid-basedcytologyanalysisisconsideredtobethemosteffectivemethodforearlycervicalcancerscreening[2].
Thefirstedi-tionofthe"Humanpapillomaviruslaboratorymanual"waspublishedin2009bytheWorldHealthOrganization(WHO)toprovideguidanceforhigh-riskHPVdiagnoses[3].
HPVvaccinationiscurrentlythesafestapproachforpreventingthedevelopmentofcervicalcancer[4].
ThedistributionofHPVtypeandtheindividualriskofeachHPVtypearetwoimportantfactorsthatneededtobeconsideredfordevelopingtheHPVvaccineagainstparticularHPVgenotypeswithinacountry[5].
Convenientandaccuratetechniquesforhigh-riskHPVdetectionandgenotypingareurgentlyneededforHPVclinicaldiagnosesandepidemiologicalstudies.
ThehybridizationcapturedetectionforHPVgeneticDNAoritsPCRampliconisatraditionalapproachforHPVgeno-typing[6–9].
However,itrequiresmanyoperationalsteps,andPCRcontaminationisacommonproblem.
Avarietyofmethodsbasedonreal-timePCRhavebeendevelopedintheHandlingEditor:ZhongjieShi.
ZhitengTangandYeXucontributedequallytothiswork.
ElectronicsupplementarymaterialTheonlineversionofthisarticle(https://doi.
org/10.
1007/s00705-017-3645-1)containssupplementarymaterial,whichisavailabletoauthorizedusers.
*YiqunLiaoyqliao@xmu.
edu.
cn*QinggeLiqgli@xmu.
edu.
cn*ChaoPanpanchao123a@126.
com1ZhongshanHospital,XiamenUniversity,Xiamen,Fujian,China2TheStateKeyLaboratoryofCellularStressBiology,StateKeyLaboratoryofMolecularVaccinologyandMolecularDiagnostics,EngineeringResearchCenterofMolecularDiagnosticsoftheMinistryofEducation,SchoolofLifeSciences,XiamenUniversity,Xiamen,Fujian,China3ZeesanBiotechnologyCompany,Xiamen,Fujian,China4TheStateKeyLaboratoryofMolecularVaccinologyandMolecularDiagnostics,StateKeyLaboratoryofCellularStressBiology,EngineeringResearchCenterofMolecularDiagnosticsoftheMinistryofEducation,SchoolofPublicHealth,XiamenUniversity,Xiamen,Fujian,China726Z.
Tangetal.
pastdecade[10–13].
Comparedwithtraditionalhybridizationcapturedetection,thereal-timePCRplatformpossessestheadvantagesofconvenience,highthroughput,lowtimeandcost,andlowriskoffalse-positiveresultsduetocross-con-taminationofPCR.
Commercialreal-timePCRkitsarenowwidelyadoptedinclinicalHPVdiagnosticandresearchstud-ies.
TheCobasHPVTestisatypicalsystemthathasbeenapprovedbytheU.
S.
FoodandDrugAdministration(FDA).
InapreviousstudywereportedanovelHPVgenotyp-ingmethodbasedonreal-timePCRandmeltcurveanalysis[14].
TheMeltProHPVTestwasdevelopedwiththesamemethod,andcandetectandgenotypethe14mostcommonhigh-riskHPVtypesinasingle-tubereaction.
Inthisstudy,weperformedacomparisonoftheMeltProHPVTestandtheCobasHPVTestforthedetectionandgenotypingof14high-riskHPVtypesinsoutheastChina.
TheprocessesinvolvedinthiscomparisonstudyareshowninFig.
S1.
More-over,reproducibilityisimportantforanynewlydevelopedmethod.
AprotocolforclinicalvalidationofHPVassays,called"VALGENT",wasdevelopedandsuccessfullyappliedforthecomparisonofmanycommercialHPVtests[15–18].
Followingthisguideline,intra-laboratoryandinter-laboratoryreproducibilityexperimentswereperformedinthisstudy.
ForcomparisonoftheMeltProHPVTestandtheCobasHPVTest,atotalof1647residualcervixcellsam-pleswerecollectedfromindividualwomenlivinginthesoutheastofChina.
Pregnantwomenwereexcludedfromthisstudy.
AllsampleswerecollectedatZhongshanHos-pital,XiamenUniversity(Xiamen,Fujian,China)withaThinPrepliquid-basedcytologysystem,in2015,andstoredat-20°Cfor2weeksbeforeanalysis.
Theageofthepatientsisfrom19to65yearsoldwithameanageof32years-oldandamedianof31years-old.
AlloftheexperimentsinthiscomparisonstudywereperformedatZhongshanHospital.
Fortheintra-laboratoryandinter-laboratoryreproducibilitystudyoftheMeltProHPVTest,atotalof540residualcervixcellsampleswerecollectedatZhongshanhospitalbythesameprotocolin2017.
Theintra-laboratoryreproduc-ibilitystudywasperformedatZhongshanHospital,whiletheinter-laboratoryreproducibilitiesstudywasperformedatZhongshanHospitalandtheEngineeringResearchCenterofMolecularDiagnosticsoftheMinistryofEducation,Xia-menUniversity.
ThestudyprotocolwasapprovedbyTheResearchEthicsCommitteeofXiamenUniversity.
Forthecomparisonstudy,1647sampleswereassayedinadouble-blindedfashionusingtheMeltProHPVTest(ZeesanBiotechnologyCo.
,China)andtheCobasHPVTest(RocheDiagnosticsCo.
,Switzerland)onthesameday.
DNAextrac-tionwasautomaticallyperformed,separately,accordingtotheprotocolsofthesetwosystems.
TheMeltProHPVTestdetected14high-riskHPVtypes(HPV-16,18,31,33,35,39,45,51,52,56,58,59,66,and68)andprovidedspecificgeno-typingforall14HPVtypesbasedonmeltcurveanalysis.
Theglyceraldehyde-3-phosphatedehydrogenase(GAPDH)genewasusedasaninternalcontrolintheMeltProHPVTest.
ThePCRreactionanddataprocessingstepsoftheMeltProHPVTestwereperformedintheSLAN96real-timePCRsystem.
TheCobasHPVTestdetectedthesame14high-riskHPVtypesbutonlyprovidedspecificgenotypingforHPV-16andHPV-18,basedonreal-timePCR.
Theβ-globulingenewasusedasinternalcontrolintheCobasHPVTest.
ThePCRreactionanddataprocessingstepsoftheCobasHPVTestwereperformedinaCobas4800real-timePCRsystem.
TheexperimentalconditionsforTheMeltProHPVTestandCobasHPVTestfollowedtheguidelinesoftheirassociatedprotocols.
Duringeachrun,forbothassays,apositiveandnegativecontrolwasincludedtoensureproperPCRreactionsandthattherewasnocarry-overcontamination.
TheHPVgenotypeforeachsamplewasassayedusingindependent,automated-readoutsoftwaresuppliedwiththeMeltProHPVsystemorCobasHPVsystems.
Thediag-nosticresultsfromtheMeltProHPVTestandtheCobasHPVTestwerecomparedtoevaluateagreement.
Foralldis-crepantsamples,theoriginaldatawereprocessedmanually.
ForcaseswithdiscrepantresultsofHPV-16andHPV-18,confirmatorytestingwasperformedusinganHPVtype-specificreal-timePCRassaydiagnostickitdesignedonlyforHPV-16andHPV-18(KehuaLtd.
,Shanghai,China).
Fortheintra-laboratoryreproducibilitystudy,540sam-pleswereassayedbytheMeltProHPVTesttwiceonthesamedayatZhongshanhospital,andtheresultsofthefirstandsecondassaywerecompared.
Fortheinter-laboratoryreproducibilitystudy,thesame540sampleswereassayedoneweeklaterattheEngineeringResearchCenterofMolecularDiagnosticsoftheMinistryofEducation,andtheresultswerecomparedwiththefirstassay'sresultsfromtheintra-laboratoryreproducibilitystudy.
StatisticalanalysisforthecomparisondatawascarriedoutusingtheSPSSstatisticalsoftware(version13.
0,SPSSInc.
,Chicago,IL).
Epidemiologicalprevalence,forthe14high-riskHPVtypes,wasonlycalculatedfromresultsobtainedwiththeMeltProHPVTest,whichwascapableoffullgenotyping.
Wecomparedtheresultsobtainedfrom1647samplesusingtheMeltProHPVTestandtheCobasHPVTestforthedetectionof14high-riskHPVtypeswithoutgenotyping(Table1).
Theoverallagreementbetweenthesetestswas96.
2%(1584/1647),andthekappavaluewas0.
881(95%CI,0.
851-0.
911).
SincetheMeltProHPVTestandtheCobasHPVTestbothprovideHPV-16andHPV-18geno-typing,wealsoevaluatedthegenotypingresultsforHPV-16andHPV-18,withinthecomparisonshowninTable1.
TheagreementforthegenotypingresultsforHPV-16was99.
6%(1641/1647),andthekappavaluewas0.
946(95%CI,0.
902-0.
990).
TheagreementforthegenotypingresultsforHPV-18was99.
7%(1642/1647),andthekappavaluewas7270.
904(95%CI,0.
818-0.
990).
TheresultsfortheHPVdetec-tion,HPV-16genotypingandHPV-18genotypingusingtheMeltProHPVTestandtheCobasHPVTestwerenotsignificantlydifferent(McNemar'sTest,Pvalue=0.
23forHPVdetection,Pvalue=0.
69forHPV-16genotyping,Pvalue=1.
00forHPV-18genotyping).
Forthe63discrepantsamples,weprocessedtheorigi-naldatamanually.
ThequantitativePCRcycle(Cq)valuesforthe22samplesdiagnosedaspositivebytheCobasHPVTestandnegativebytheMeltProHPVTestwereveryclosetothecut-offCqvaluefortheCobasautomatedreadoutsoftware.
Moreover,16casesamongthese22sam-plesshowedweakmeltingcurvesignalsintheMeltProHPVTest,buttheirmeltingcurvereadout(Rm)valueswerelowerthanthecut-offRmvaluefortheMeltProautomatedreadoutsoftware,whiletheother6casesshowednomelt-ingcurvesignals.
Ontheotherhand,theRmvaluesforthe41samplesdiagnosedaspositivebytheMeltProHPVTestandnegativebytheCobasHPVTestwerealsoveryclosetothecut-offRmvaluefortheMeltProautomatedreadoutsoftware.
Moreover,23casesamongthese41sam-plesshowedlateamplificationsignalsintheCobasHPVTest,buttheirCqvaluesoccurredlaterthanthecut-offCqvaluefortheCobasautomatedreadoutsoftware,whiletheother18casesshowednoamplificationsignals.
Sixdiscrep-antsamplesinfectedbyHPV-16and5discrepantsamplesinfectedbyHPV-18werediagnosedagainusingathirdpartycomparisonmethodbasedontype-specificreal-timePCR,andallofthemwereconfirmedtobeHPV-16positiveorHPV-18positive(TableS1).
Avisualcomparisonofthedetectionresultsobtainedfromthe1647samplesusingtheMeltProHPVTestandtheCobasHPVTestisshowninFig.
1.
Atotalof339sampleswerediagnosedasHPVpositivebytheMeltProHPVTest,including83samplesthatwerediagnosedasinfectedwithHPV-16orHPV-18.
Amongthese339sam-ples,78.
5%(266/339)sampleswereidentifiedashavingasingleHPVtypeinfection,whereas21.
5%(73/339)sam-pleswereinfectedbymultipleHPVtypes.
Bycomparison,atotalof321sampleswerediagnosedasHPVpositivebytheCobasHPVTest,including81samplesthatwerediagnosedwithHPV-16orHPV-18infection.
However,becausetheCobasHPVTestcannotgenotypetheother12high-riskHPVtypes,asidefromHPV-16andHPV-18,wecouldnotcalculatethenumberofsamplesinfectedbymultipleHPVtypes.
Thedistributionof14high-riskHPVtypesamongthese1647samplesisshowninFig.
2.
HPV-52(70cases),HPV-58(64cases),andHPV-16(59cases)werethethreemostprevalentHPVtypesinthisstudy.
HPV-31(8cases)andHPV-45(5cases)werethetworarestHPVtypes.
Thedistri-butionsoftheothertypeswereHPV-39(31cases),HPV-68(29cases),HPV-18(27cases),HPV-51(26cases),HPV-56(23cases),HPV-59(20cases),HPV-33(18cases),HPV-66(17cases),andHPV-35(15cases).
Since21.
5%ofthepositivesampleswereinfectedwithmultipleHPVtypes,thetotalnumberofHPVtypecases(412cases)waslargerthanthetotalnumberofHPVpositivesamples(339samples).
Tworepeatsusingparalleldiagnosisofthe540samplesinanintra-laboratorystudyshowed97.
8%agreement,withakappavalueof0.
947(0.
917-0.
977,95%CI).
Theintra-laboratorydataforeachHPVtypeisshowninTableS2.
Theinter-laboratorystudyof540samplesshowed96.
9%agreement,withakappavalueof0.
925(0.
889-0.
961,95%Table1ComparisonofHPVdetection,HPV-16genotyping,andHPV-18genotypingusingtheMeltProHPVTestandtheCobasHPVTestMeltProHPVCobasHPVTotalKappavalue(95%CI)HPV(+)HPV()HPV(+)300413410.
881(0.
851-0.
911)HPV()2212841306Total32213251647MeltProHPVCobasHPVTotalKappavalue(95%CI)HPV-16(+)HPV-16()HPV-16(+)554590.
946(0.
902-0.
990)HPV-16()215861588Total5715901647MeltProHPVCobasHPVTotalKappavalue(95%CI)HPV-18(+)HPV-18()HPV-18(+)243270.
904(0.
818-0.
990)HPV-18()216181620Total2616211647728Z.
Tangetal.
CI).
Theinter-laboratorydataforeachHPVtypeisshowninTableS3.
Regularscreeningforhigh-riskHPVisrecommendedforadultwomanbytheWHOformonitoringthedevelop-mentofcervicalcancer[3].
Inthisstudy,weevaluatedtheclinicalperformanceoftheMeltProHPVTestusing1647samplescollectedfromwomenduringroutinemedi-calexaminations.
TheHPVdetectionresultsandHPV-16andHPV18genotypingresultsusingtheMeltProHPVTestwereinstrongagreementwithdatafromtheCobasHPVTest(Table1).
AllinconsistentcasesbetweentheMeltProHPVTestandtheCobasHPVTestwerefoundtobesamplesinfectedwithalowviralloadofHPV.
61.
9%(16+23cases/63cases)ofthenegativeresultsforthe63discrepantcasesshowedweakdetectionsignalsbelowthecut-offvaluesfortheMeltProandCobasautomatedreadoutsoftware.
BoththeMeltProHPVTestandtheCobasHPVTestweredesignedforthesimultaneousdetectionof14high-riskHPVtypesinasinglereaction,whichmeansthattheampli-ficationprimersusedcannotcompletelymatchthegenesequenceforeachHPVtype.
Differingamplificationprim-ersmightbeanimportantreasonforthediscrepantresultsobtainedforthese63samplesusingthetwomethods.
HPVtype-specificreal-timePCRwasusedasathirddetectionmethodforthesediscrepantsamplesinfectedbyHPV-16orHPV-18(TableS1).
AthirddetectionmethodwasdesignedtocontaintwopairsofprimersthatperfectlymatchedHPV-16orHPV-18andshouldonlydetectthesetwoHPVtypes.
AsshownintableS1,thedetectionresultsforHPVtype-specificreal-timePCRshowedthat11'suspicious'samplesinfectedbyHPV-16orHPV-18werepositive.
HPVdetectionHPVgenotypingHPVnegative(N=1326)HPVnegative(N=1308)HPVpostive(N=321)HPVpostive(N=339)HPV-16(N=32)HPV-18(N=17)HPV-16or18&othertype(N=32)HPV-16(N=36)HPV-18(N=20)HPV-16or18&othertype(N=27)Othertype(N=242)HPV-52(N=48)HPV-58(N=45)HPV-39(N=20)HPV-51(N=17)HPV-68(N=17)HPV-66(N=15)HPV-56(N=14)HPV-59(N=12)HPV-33(N=10)HPV-31(N=5)HPV-35(N=5)HPV-45(N=2)Co-infection(N=46)CobasHPVtestMeltProHPVtestFig.
1Detectionresultsfor1647samplesusingtheMeltProHPVTestandtheCobasHPVTest.
TheleftpanelshowsthestatisticaldatafortheHPVdetectionresults,prasinouspie:HPVnegativesam-ples,pinkpie:HPVpositivesamples.
Therightpanelshowsthesta-tisticaldatafortheHPVgenotypingresults,redpie:HPV-16,yellowpie:HPV-18,greenpie:co-infectionsamplesincludingHPV-16orHPV-18,bluepie:HPV-othertype(fortheMetltProHPVTest,fromdarktolight:HPV-52,58,39,51,68,66,56,59,33,31,35,and45),greypie:co-infectionsamplesexcludingHPV-16andHPV-181618525839685156593366353145204060HPVNumberofHPVtypeFig.
2ThedistributionoftheHPVgenotypingresultsusingtheMeltProHPVTest.
Redbars:HPV-16andHPV-18,bluebars:otherhigh-riskHPVtypes729ComparedtotheCobasHPVsystem,theMeltProHPVTestprovidesfullgenotypinginformationfor14high-riskHPVtypes.
ThismeanstheMeltProHPVTestcanidentifythespecificHPVtypewithinasampleduringthedetectionstep(Fig.
1),whichwillbenefitusersconductingHPVepi-demiologicalstudies.
Inthisstudy,HPV-16,HPV-52,andHPV-58werethethreemostprevalenthigh-riskHPVtypesinthesoutheastofChina(Fig.
2).
Thisconclusioniscon-sistentwithpreviousreportsbyotherresearchers[19,20].
Consequently,HPV-52andHPV-58shouldbeconsideredforcoverageduringHPVvaccinedevelopmentbyChinesescientists.
Thefullgenotypingofhigh-riskHPVcanalsohelpdoctorsdeterminewhetherapatienthasbeenpersis-tentlyinfectedbythesameHPVtypeorinfectedmultipletimesbydifferentHPVtypes.
Insummary,theMeltProHPVTestandCobasHPVTestarecomparable,with96.
2%agreementandakappacoefficientof0.
881forHPVdetection.
AlthoughtheMeltProHPVTestcanidentifymoreHPVtypesthantheCobasHPVTest,theirclinicalperformancesweresimi-larregardingHPVdetection.
Differentcut-offvaluesandthedifferentamplificationprimersmightbethecauseofdiscrepantresultsbetweenthetwomethods.
Thereproduc-ibilityoftheMeltProHPVTestprovedtobestableinbothintra-laboratoryandinter-laboratorystudies.
TheMeltProHPVTestcanprovidefulltypinginformationandisanaccu-rate,high-throughput,andlow-costmethodthatcanbeusedinthefutureforclinicalHPVscreeningandgenotyping.
AcknowledgementsWethankNationalNaturalScienceFoundation(81401724toY.
Liao),KeyProjectofCooperationProgramforUni-versityandIndustryofFujianProvince(2013Y4008toY.
Xu)andCooperationProgramforUniversityandIndustryofXiamenCity(3502Z20173013toY.
Liao)forfinancialsupport.
CompliancewithethicalstandardsConflictofinterestQ.
LiholdsequityinterestinZeesanBiotech.
Allofotherauthorsdeclarethattheyhavenoconflictofinterest.
TheMeltProHPVtestreagentswerekindlyprovidedbyZeesanBiotech.
Humam/animalrightsstatementThisarticledoesnotcontainanystudieswithhumanparticipantsoranimalsperformedbyanyoftheauthors.
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