Introduction HepatitisC isa liverdiseasecaused bythehepatitis C virus HCV.Globally,150millionpeoplehavechronicHCVinfection.An estimated700,000peopledieeachyearfromhepatitisC-related live
Trang 1jou rn al h om ep ag 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
genotype 2.0 assay (LiPA) for the hepatitis C virus genotyping
Wasun Chantratitaa,∗∗, Keum-Soo Songb, Choi GunHoc, Viroj Pongthanapisitha,
Nipa Thongbaipheta, Garanyuta Wongtabtima, Ekawat Pasomsuba,
Kanokwan Angkanavina, Satish Balasaheb Nimseb, Mukesh Digambar Sonawaneb,
Shrikant Dasharath Warkadb, Taisun Kimb,∗
Keywords:
Hcv
a b s t r a c t
Inthisarticle,wedescribethe6HCVGenotyping9Gtestanditsevaluationbyusingclinicalsamplesand plasmidDNAstandards.Intestswith981plasmidDNAstandards,the6HCVGenotyping9Gtestshowed higherthan92.5%sensitivityand99.4%specificity.The6HCVGenotyping9Gtestwascomparedwith theVERSANTHCVGenotype2.0assay(LiPA2.0)fordetectionanddiscriminationofHCVgenotypesin clinicalsamples.Theresultsofbothtestswereverifiedbygenomicsequencing.The6HCVGenotyping9G testdemonstrateda100%agreementwiththesequencingresults,whichwashigherthanLiPA2.0.These resultsindicatethatthe6HCVGenotyping9Gtestcanbeareliable,sensitive,andaccuratediagnostic toolforthecorrectidentificationofHCVgenotypesinclinicalspecimens.6HCVGenotyping9Gtestcan genotypesixHCVtypesin1PCRin30minafterPCRamplification.The6HCVGenotyping9Gtest,thus providecriticalinformationtophysiciansandassistthemtoapplyaccuratedrugregimenfortheeffective hepatitisCtreatment
©2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND
license(http://creativecommons.org/licenses/by-nc-nd/4.0/)
1 Introduction
HepatitisC isa liverdiseasecaused bythehepatitis C virus
(HCV).Globally,150millionpeoplehavechronicHCVinfection.An
estimated700,000peopledieeachyearfromhepatitisC-related
liverdiseasesincludingcirrhosis,hepatocellularcarcinoma(HCC)
andliverfailure(WHOguidelines,2016).Themorbidityand
mor-talityattributabletoHCVinfectioncontinuetoincrease(Lavanchy,
2011).ThetreatmentofHCVinfectionhasbecomemore
compli-catedduetovariousgenotypesandsubtypesofHCV
HCVisclassifiedintosevengenotypes,67confirmedand20
pro-visionalsubtypeswithdistinctpatternsofgeographicdistribution
hanlimsk@empas.com (K.-S Song), ghchoi@bmtchip.com
maymayenator@gmail.com (N Thongbaiphet),
garanyuta@gmail.com (G Wongtabtim), ekawat.pas@mahidol.ac.th (E Pasomsub),
annvirus@yahoo.co.th (K Angkanavin), satish nimse@hallym.ac.kr (S.B Nimse),
mukeshsonawane87@hallym.ac.kr (M.D Sonawane), shrikant.warkad@gmail.com
(Smithetal.,2014).Globally,HCV1isthemostcommon(46.2%), followedbyHCV3(30.1%),HCV2(9.1%),HCV4(8.3%),HCV6(5.4%), andHCV5(0.8%)(Messinaetal.,2015).However,amongAsian countries,thedistributionandsub-typicalcompositionofHCV6are significantlydifferentfromoverallglobalratio.Forexample,HCV6a (24%)andHCV6n(39%)arepredominantinVietnamandMyanmar, respectively(Phametal.,2011;Lwinetal.,2007).InThaipatients HCV6fisthemostcommonHCV6subtype(56%),followedby sub-types6n(22%),and6i(11%)(Akkarathamrongsinetal.,2010).A properantiviraltreatmentcancuretheHCVinfection(LiandLo,
2015).However,theknowledgeaboutHCVgenotypeand subgeno-typeiscrucialfortheappropriatetreatment(Wasitthankasemetal.,
2015).Itisessentialtonotethataccordingtotherecent recom-mendationsby EuropeanAssociation for theStudyof theLiver (EASL),thetreatmentofHCVinfectionsignificantlydepends on particularHCVgenotypeorsub-genotype(EASL,2015).The2016 WHOguidelinesforthescreening,care,andtreatmentofpatients withchronicHCVinfectionrecommendtheidentificationofHCV genotypesforthechoiceoftheprecisetreatmentregimen(WHO guidelines,2016)
Currently,treatmentofHCVhasmadesignificantadvanceswith thehelpofdrugssuchaspegylatedinterferon(PegIFN)-␣,ribavirin, http://dx.doi.org/10.1016/j.jviromet.2016.10.009
Trang 22 W Chantratita et al / Journal of Virological Methods 239 (2017) 1–8
andthedirect-actingantiviralssuchassofosbuvir,simeprevir,
ledi-pasvir,ombitasvir,dasabuvir(Mishraetal.,2015).However,the
choiceofmedicineorthecombinationofdrugsandtheduration
oftreatmentaredifferentforHCVgenotypes1a,1b,2,3,4,5and
6(EASL2015).Therefore,foraccuratetreatmentofhepatitisC,it
isimperativetodetectanddiscriminatethecrucialHCVgenotypes
suchas1a,1b,2,3,4,and6byusingsimpleandaccuratedetection
method
SequenceanalysisofspecificregionssuchasNS5,core,E1,and
5 untranslatedregion(5UTR)isa goldstandardforHCV
geno-typing (Firdaus et al., 2015) However, it is a time-consuming
method,expensive,andrequireshighlytrainedprofessionalto
pro-cessthesamples.Alternativenucleicacidtestssuchasreal-time
PCR(Hawkinsetal.,1997;Beldetal.,2002),restrictionfragment
lengthpolymorphism(Filippoetal.,2012),heteroduplexmobility
analysis(Whiteetal.,2000),andline-probeassay(Caietal.,2013)
arerapidHCVtypingmethods.Theneedsforspecializedand
expen-siveinstrumentshavelimitedtheapplicationsofthesemethodsin
developingcountries.TheVersantHCV2.0assay(lineprobeassay
[LiPA]2.0),AbbottRealtimeHCVGenotypeIIassay,andTrugene
assayarewidelyusedcommercialassaysforHCVgenotyping.A
recentreportonthecomparisonofTrugeneassay,LiPA2.0,and
sequencingindicatethatthesetestscanfailtodifferentiateHCV
subtypes1aand1b,whichcanleadtocriticalerrorsinclinical
prac-ticeforcorrectuseofdirectlyactingantiviralagents(Chuecaetal.,
2016).Furthermore,theLiPA2.0assayandtheAbbottRealtimeHCV
GenotypeIIassayhavelimitationsinidentifyingHCVgenotype6
(Yangetal.,2014)andareexpensive.Thus,forthe
implementa-tionofastricttreatmentregimeinthemanagementofhepatitisC,
thereisaneedtofindaninexpensivegenotypingtestthatisrapid,
simple,andprecise
Inthisstudy,981standardplasmidsampleswereusedto
evalu-atetheperformanceof6HCVGenotyping9Gtest.Allsampleswere
testedunderblindedcodes.Theperformanceof6HCVGenotyping
9Gtestwasalsoevaluatedbycomparingitwiththecommercial
VERSANTHCVGenotype2.0assay(LiPA2.0)(SiemensHealthcare
GmbH,Erlangen,Germany)andHCVsequencingfordetectionof
sixHCVgenotypes1a,1b,2,3,4,and6intheserumsamples.The
principleof6HCVGenotyping9GtestincludesviralRNA
isola-tion,complementaryDNA(cDNA)synthesis,PCRamplification,and
detectionofPCRamplicons.ThecDNAsynthesisandPCR
amplifi-cationaredonesimultaneouslyinsinglePCRtubebyusingRT-PCR
The6 HCVGenotyping9Gtestisa lateralflowstrip membrane
assay,wherein,therequired9Gmembranesareobtainedby
fol-lowingthereported9Gtechnology(Songetal.,2013).ThessDNA
oligonucleotideprobescorrespondingtotherespectiveHCV
geno-typesareimmobilizedonthe9Gmembranesatspecificpositions
TheimmobilizedprobeshybridizewiththecomplementaryCy5
labeledssDNAsinthePCRproductandallowthediscriminationof
sixHCVgenotypes.Itisthefirstreportofitskindtoevaluatethe
performanceof6HCVGenotyping9Gtestanditscomparisonwith
thecommercialHCVgenotypingtest.The6HCVGenotyping9Gtest
isdesignedtoprovidethenecessaryinformationtophysiciansby accuratedetectionofsixHCVgenotypesin30minafterPCR.Thus, the6HCVGenotyping9Gtestcanenabledoctorstoselectthemost effectivetreatmentregimenaspertheEASLrecommendationsfor hepatitisCmanagement
2 Materials and methods
2.1 Materials AllchemicalswerepurchasedfromSigma-AldrichChemicals, Korea.TheoligonucleotideswerepurchasedfromBioneer,Korea Glassfibermembrane(2.5×7.5cm)wasobtainedfromWhatman, Springfield,UK.AllwashingsolventsforthesubstratesareofHPLC gradefromSKChemicals,Korea.Ultrapurewater(18M/cm)was obtainedfromaMilli-Qpurificationsystem(Millipore) Oligonu-cleotideswere linedusingdispenser (BioDotTechnologies,Inc.,
2852AltonPkwy,Irvine,CA92606,USA).Hybridizationwasdone
at25◦C.ThefluorescenceintensitieswererecordedontheBMT ReaderTM,BiometrixTechnologyInc.,SouthKorea.VERSANTHCV Genotype2.0assay(LiPA2.0)(SiemensHealthcareGmbH,Erlangen, Germany)kitwasusedasastandardcommercialproduct 2.2 Compositionofdifferentsolutionsusedfor6HCVgenotyping 9Gtest
a)Hybridizationbuffers(pH=7.4):20%Formamide,0.25%Triton X-100, 6x SSC;b) Washing buffer solution(pH=7.4): 20% For-mamide,0.25%TritonX-100,6xSSC
2.3 StandardDNAsamples ThecDNA standard samples(plasmid DNAs) containing cor-responding 300-bp sequences of the 5UTR of HCV RNA were obtainedfrom Bioneer Inc., Daejeon,Korea The plasmid cDNA sequenceswereuniquetotheHCVgenotypes1a,1b,2,3,4,6a,6f, 6i,and6n.Plasmidstandardsamples(n=981)wereusedforthe testundertheblindedcodes.Allstandardsampleswerediluted with Tris-elution buffer utilized in the regular extraction kits used for clinicalspecimens Thus, theprocess of evaluation by usingclinical sampleswasmimicked in the6 HCVGenotyping 9GtestwithplasmidcDNAstandard samples.The981samples wereseparatedineightdifferentcategoriesas1×106copies/test (7.5×106copies/mL), 1000copies/test (7.5×103copies/mL),
50copies/test (375copies/mL), 10copies/test (75copies/mL),
5copies/test (38copies/mL), 2.5 copies/test (19copies/mL), 1 copy/test (7.5copies/mL), and 0 copy/test (negative samples) Table1depicts thedistributionof981standardsamples corre-spondingtotheHCVgenotypes1a,1b,2,3,4,6a,6f,6i,and6n
ineightconcentrationcategories.Allsamplesweretestedunder blindedcodes.PCRwasperformedonplasmidcDNAsamplesby
Table 1
Trang 3Fig 1.Position of the respective probes on 9G membrane and the experimental protocol of 6 HCV Genotyping 9G Test.
PCR
(probespecificforthedetectionofHCV)probe,andsixotherprobes
specifictotheHCVgenotypes1a,1b,2,3,4,and6,were
immobi-lizedontheglassfibermembranestoproducethe9Gmembranes
accordingtotherecentlyreportedmethod(Songetal.,2013).All
probeswereselectedaccordingtothegeneralizedprobeselection
method(Nimseetal.,2011).Fig.1alsodepictsthesimple
experi-mentalprotocolof6HCVGenotyping9Gtesttoobtainfinalresults
TheHCprobeservesasaninternalstandardforhybridization
control.TheHCVprobeisusedtodetectthepresenceorabsence
ofHCVinthesample.AsshowninFig.2,when6HCVGenotyping 9Gtestshowsthreesignalscorrespondingtoprobesincludingthe
HC,HCV,and1aor2or3or4,asampleisdesignatedasanHCV 1aor2or3or4,respectively.ThesampleisidentifiedasHCV1b
ifthetestshowsfoursignalsforprobesincludingHC,HCV,1a,and 1b,respectively
Whenthetestshowsthreesignalscorrespondingtotheprobes
HC,HCV,and6,thesampleisgenotypedasHCV6aor6f.Whereas,
ifthetestshowsfoursignalscorrespondingtotheprobesHC,HCV, 1a,and6,thesampleisgenotypedasHCV6ior6n.When,6HCV Genotyping9GtestshowstwosignalscorrespondingtotheHCand HCVprobes,thesampleisidentifiedtocontaintheHCVgenotype otherthantheHCV1a,1b,2,3,4,6a,6f,6i,and6n.However,foran HCV-negativesample,thetestshowsonlyonesignalcorresponding
totheHCprobe
Theprocessof6HCVGenotyping9Gtestaftercompletionof cDNAsynthesisfollowedbyPCRamplificationisbrieflydiscussed here.110LofhybridizationsolutionwasaddedtoeachPCRtube containing20LPCRproduct.Then,110Lofthismixturewas loadedintothesampleportofthe6HCVGenotyping9Gtestand allowedtostandfor20minat25◦C.After20min,200Lofwashing solutionwasloadedintothewashingportofthe6HCV Genotyp-ing9Gtestandallowedtostandfor8minat25◦C.After8min, each6HCVGenotyping9GteststripswasscannedontheBMT ReaderTM,(BiometrixTechnologyInc.Chuncheon,SouthKorea)to obtainthefinalresultsasshowninFig.2.Table2andTable3 sum-marizetheresultsofthe6HCVGenotyping9Gtestonstandard samples.Table4andTable5summarizethecomparativeresults
onclinicalsamplesobtainedby6HCVGenotyping9Gtest,LiPA 2.0,andsequencing
2.6 LiPA2.0 LiPA2.0isareversehybridizationlineprobeassay.ViralRNA waspurifiedusingaQIAamp viralRNAminikit(Qiagen,Hilden, Germany)andwassubjectedtoreversetranscription-PCR(RT-PCR) withaVersantHCVamplification2.0kit(manufacturedby Inno-genetics,Ghent,Belgium,forSiemens,Tarrytown,NY,USA).The 240-bp 5UTRand 270-bp corefragmentswere co-amplifiedto
Trang 44 W Chantratita et al / Journal of Virological Methods 239 (2017) 1–8
Table 2
TP: true positive, TN: true negative, FP: false positive, FN: false negative.
Trang 5Fig 2.The final result for HCV genotypes A) 1a, B) 1b, C) 2, D) 3, E) 4, F) 6a or 6f, G) 6i or 6n, and H) HCV negative samples in 6 HCV Genotyping 9G Test.
Overall sensitivity and specificity of the 6 HCV Genotyping 9G Test on 531 standard samples with concentrations of 5 copies/test ∼ 1 × 10 6 copies/test.
Results of 6 HCV Genotyping 9G Test and LiPA 2.0 assay for 79 samples.
Results of LiPA 2.0 Results of 6 HCV Genotyping 9G Test
Trang 66 W Chantratita et al / Journal of Virological Methods 239 (2017) 1–8
Comparison of 6 HCV Genotyping 9G Test and LiPA 2.0 with the sequencing for 19 discordant samples (HCV genotype is given in parenthesis).
3 Results
3.1 Standardsamples
AsshowninTables1and2,981-blindedsamplesweretestedto
evaluatethe6HCVGenotyping9Gtestfortheaccuratedetection
andgenotypingofHCVstrains.ThePCRamplificationtoobtainthe
HCVDNAampliconswasperformedoneachofthe981samples.Out
of981samples,54sampleseachwasintheconcentrationcategory
of1×106copies/testand1000copies/test.Outofthese54samples,
ninesampleswerenegativeandremaining45sampleswerefurther
dividedintofivesampleseachofHCVgenotypes1a,1b,2,3,4,6a,
6f,6i,and6n,respectively.Similarly,therewere99sampleseachin
theconcentrationcategoryof50copies/testand10copies/test.Out
ofthese99samples,ninesampleswerenegative,andremaining90
sampleswerefurtherdividedintotensampleseachofHCV
geno-types1a,1b,2,3,4,6a,6f,6i,and6n,respectively.Therewere225
sampleseachintheconcentrationcategoryof5copies/testand2.5
copies/testand1copy/test.Outofthese225samples,ninesamples
werenegative,andremaining116sampleswerefurtherdivided
into24sampleseachofHCVgenotypes1a,1b,2,3,4,6a,6f,6i,and
6n,respectively.Hence,outof981samples,63samplesweretrue
HCVnegativeand918samplesweretrueHCVpositive
ThedatapresentedinTable2showsthesensitivityand
speci-ficityofeach probeatdifferentconcentrations ofHCVcDNA in
thesamples.The6HCVGenotyping9Gtestcorrectlydetected918
samplesastruepositive(withHCVcDNA)and63samplesastrue
negative(withoutHCVcDNA)ontheHCVlineirrespectiveofthe
concentrationofa cDNAinthesample.TheHCVprobeshowed
almost100%sensitivityandspecificity,respectively.However,as
showninTable2,thesensitivityoftheprobescorrespondingto
theHCV genotypes1a,1b, 2, 3, 4,6a, weresignificantly lower
forthesamplescontainingHCVcDNAwithconcentrationsof2.5
copies/test(54.1–82.4%)and1 copy/test(16.6–76.9%).Forthese
samples,thesensitivity of thesix probes werein the range of
20–75%.However,thesamples containing5 copies/testof HCV
cDNA allprobesdemonstrated thesensitivity andspecificity of 83–100%and99–100%,respectively.Therefore,thelimitof detec-tion(LOD)ofthe6HCVGenotyping9Gtestforthedetectionand discriminationofHCVgenotypes1a,1b,2,3,4,6a,6f,6i,and6n weredecided tobe5copies/test (38copies/mL).Thus, theLOD
of the6 HCVGenotyping 9Gtest is much lower than theLOD (4810IU/mL)reportedforthecommercialassays(Nadarajahetal.,
2007)
TheLODbeingsetto5copies/test,thedataonthe531standard plasmidsampleswasusedforthedeterminationoftheoverall sen-sitivityandspecificityofthe6HCVGenotyping9Gtestasshown
inTable3.Outof531samples,486samples(54samplesperHCV genotypes1a,1b,2,3,4,6a,6f,6i,and6n)weretrueHCVpositive and45samplesweretrueHCVnegative.Allsampleswerecorrectly detectedbyeachprobecorrespondingtotheHCVgenotypes1a,1b,
2,3,4,6a,6f,6i,and6n,respectively.Theprobecorrespondingto theHCVgenotype1asuccessfullyidentifiedtheHCV1ainall54 HCVtruepositivesamples.However,3/477samplesthatdidnot haveanyHCV1acDNAinthemwerefalselydetectedasHCV1a, falsepositiveresults.Outor54truepositivesamplesand477true negativesamplesforHCV1b,theprobeforHCV1bshowedonefalse positiveresultandthreefalsenegativeresult,respectively.Outof
54truepositivesamplesforeachoftheHCVtype2,3,4,and6a,6f, 6i,and6n,theprobescorrespondingtorespectiveHCVgenotypes demonstrated0,2,4,3,2,4,and2falsenegativeresults, respec-tively.However,thefalsenegativerateforprobescorresponding
totheHCVtype2,3,4,and6a,6f,6i,and6nwaslow
ThedatapresentedinTable3showsthesensitivityand speci-ficityofeachprobeusedinthe6HCVGenotyping9Gtestforthe detectionofHCVgenotypesinthestandardplasmidDNAsamples ThesensitivityofallsixprobesfordetectionofHCVgenotypesin thestandardsampleswereintherange92.6-100%.Thespecificity
ofallsixprobeswashigherthan99%.Thesensitivityandspecificity
oftheprobeswereintherange83.3-100%and77.8%-100%, respec-tively,forsamplescontainingmorethan5copies/testofHCVcDNA Theseresultsindicatethat6HCVGenotyping9Gtestcanbeused effectivelyforthedetectionandgenotypingoftheHCVinclinical samples
3.2 Clinicalsamples
AsshowninTable4,6HCVGenotyping9GtestandLiPA2.0 assayshowedconcordanceinthedetectionofHCVgenotypesin 79/98clinicalsamplesanddiscordanceinthedetectionof19/98 samples.Bothtestsshowed80.6%agreementand 19.4% discor-danceintheresultsforthedetectionofHCVgenotypes.Asshown
inTable4,bothtestsclearlyidentified35HCVnegativesamplesas HCVnegative
Outof44HCVpositivesampleswiththeconcordanceofresults
inbothtests,five(11.4%),15(34.1%),13(29.5%),and11(25.0%) samples,wereHCVgenotypes1a,1b,3,and6,respectively.LiPA 2.0assayidentifiedelevenHCV6genotypesamplesasHCV6(c-l) Whereas,6HCVGenotyping9Gtestidentified4(9.1%)and7(15.9%)
Trang 7respectively.The6HCVGenotyping9GtestcansubtypetheHCV6
genotypeintotwocategoriesviz.HCV(6aor6f)andHCV(6ior6n)
The 19/98 (19.4%) samples that showed discordance in the
resultsof6HCVGenotyping9GtestandLiPA2.0assaywere
geno-typedbysequencing.Table4summarizesthecomparisonofthe
resultsofsequencing,6HCVGenotyping9GtestandLiPA2.0assay
Sequencingidentified11(57.9%),1(5.3%),and7(36.8%)samples
outof19samplesasHCVgenotypes1b,3,and6i,respectively.As
showninTable5,6HCVGenotyping9Gtestshowed100%
agree-mentwiththeresultsofsequencingforthedetectionofHCV1b,
HCV3,andHCV6isamples.However,LiPA2.0assayshowed
com-pletedisagreementwithsequencinganalysis.TheelevenHCV1b
sampleswerecorrectlyidentifiedasHCV1bin6HCVGenotyping9G
test.However,oftheelevenHCV1bsamplesLiPA2.0assayfalsely
detectedtwoandninesamplesasHCV1aandHCV6(c-l),
respec-tively.AnHCV3samplewasdesignatedasHCV6(c-l)intheLiPA
2.0assay.AllofthesevenHCV6isamplesweredetectedasHCV(6i
or6n)inthe6HCVGenotyping9Gtest.However,outsevenHCV6i
samples,LiPA2.0assaydetected5and2samplesasHCV1band
HCV1a,respectively
Theseresultsclearlyindicatethat,6HCVGenotyping9Gtestis
superioroverLiPA2.0assayforthegenotypingofHCV.The
over-allsensitivityandspecificityof6HCVGenotyping9Gtestwere
100.0%(94.3–100.0%at95%CI)and100.0%(90.0–100.0%at95%CI),
respectively.Attheindividualsubtypelevel,boththesensitivity
andspecificityofthe6HCVGenotyping9Gtestwere100%.Itwas
observedinthistestthattheLiPA2.0assayhaslimitationsinthe
correctdetectionanddiscriminationoftheHCVgenotypes1a,1b,
and6.TheoverallsensitivityandspecificityofLiPA2.0assaywere
69.8%(56.9–80.7%at95%CI)and100.0%(90.0–100.07%at95%CI),
respectively
4 Discussion
Theevaluationof6HCVGenotyping9GtestforHCVgenotyping
andsubtypingwasconductedon981blindedstandard samples,
withresultsindicatingmorethan92%sensitivityand99%
speci-ficityforthedetectionofHCVgenotypes.Theresultsofthe6HCV
Genotyping9Gtestonthestandardsamplesclearlyindicatethat
thistestcandiscriminatetheHCV1a,1b,2,3,4,andHCV6(6aor
6f,and6ior6n).Essentially,theapplicationofanynewtestcan
beadjudicatedbasedonitsperformanceforthedetectionofHCV
genotypesintheclinicalsamples.Thus,98clinicalsampleswere
usedinthepreliminaryclinicalevaluationofthe6HCVGenotyping
9Gtest
Inthis study,thecommercialLiPA2.0 assayandsequencing
analysiswereusedasthestandardsforthedetectionoftheHCV
genotypesinthe98patientsserumsamplesunderblindedcodes
The6HCVGenotyping9Gtestshowed100.0%concordancewith
sequenceanalysisinthe19samples.However,theLiPA2.0assay
showedcomplete discordancewithsequenceresultsfor the19
samplesthatwerediscordantwith6HCVGenotyping9Gtest
ThelimitationoftheLiPA2.0assayforthecorrectidentification
ofHCV1a,1b,andHCV6isreported(Chuecaetal.,2016).Itwas
observedinthisstudythattheHCVgenotypes1a,1b,2,3,4,and6
alongwiththeirsubtypeshavemorethan95%sequencehomology
Thus,thehighpercentageofsequencehomologyisalikelyreason
forthediscrepanciesintheresultsofLiPA2.0assay
Sequence analysis is considered the gold standard and it is
observedinthisstudythattheresultsof 6HCVGenotyping9G
testwerein100%agreementwithit.Itisimportanttonotethat
accordingtoanearlierreport,the9Gmembranetechnologyused
inthe6HCVGenotyping9GtestenablesdetectionofgenomicDNAs
withaconcentrationof38copies/mL.Moreover,thesensitivityand
specificityof6HCVGenotyping9GtestforthedetectionoftheHCV genotypeviz.1a,1b,2,3,4,and6(6aor6fand6aor6i)inthe standardplasmidsampleswiththeconcentrationhigherthan38 copies/mLwasover94%and99%,respectively.Athigher concen-trationsthesensitivityandspecificityofthe6HCVGenotyping9G testis100.0%
Thecorrect identificationoftheHCV genotypesinthe HCV-infected patients is critical information for physicians to apply theproperdrugregimen.Therefore,itisvitalfortheclinical lab-oratories tousethemostaccuratemethodtoprovidein-depth informationonHCVinfectiontocliniciansforbetterpatientcare
Itisimportanttonotethat6HCVGenotyping9Gtestusessingle RT-PCRfortheidentificationofHCVgenotypesanddeliversfinal resultsinlessthan30minat25◦CafterPCR
5 Conclusion
Theevaluationof6HCVGenotyping9Gtestbyusing981 stan-dardsamplesshowedtheoverallsensitivityandspecificitytobe higherthan92.5%and99.4%,respectively.Theresultsof evalua-tionofthe6HCVGenotyping9Gtestindicatesthatitisareliable, sensitive,andaccuratediagnostictoolforthedetectionand geno-typingHCVgenotypesinclinicalsamples.However,only15clinical sampleswereusedinthisstudy.Therefore,theevaluationofthe
6HCVGenotyping9Gtestwithabroaderrangeofclinical sam-pleswouldbenecessarytosubstantiatethefindingsinthisreport The6HCVGenotyping9Gtestprovidesvaluableinformationto physiciansbydetectinganddiscriminationthesixHCVgenotypes
in30minafterPCR,whichisvital informationforthechoiceof definitivedrugtherapy
Conflicts of interest
Theauthorsdeclarenoconflictofinterest
Acknowledgments
ThisresearchwassupportedbytheMinistryofTrade,Industry
&Energy(MOTIE), KoreaInstituteforAdvancementof Technol-ogy(KIAT)throughtheEncouragementProgramforTheIndustries
of Economic Cooperation Region (Project No.: R0004191) This workwasalsosupported,inpart,byCenterformedicalgenomics, RamathibodiHospital,MahidolUniversity,Bangkok,Thailand
References
distribution of hepatitis C virus genotype 6 subtypes in Thailand J Med Virol.
82, 257–262.
Performance of the New Bayer VERSANT HCV RNA 3.0 assay for quantitation of hepatitis C virus RNA in plasma and serum: conversion to international units and comparison with the Roche COBAS Amplicor HCV Monitor Version 2.0, assay J Clin Microbiol 40, 788–793.
genotyping methods: core, NS5B sequence analysis and line probe assay Int J Mol Med 31 (2), 347–352.
virus genotyping reveals discordances with commercial platforms PLoS One
11 (4), e0153754.
treatment of hepatitis C J Hepatol 63, 199–236.
hepatitis c virus in multi-transfused Colombian patients Virol J 9 (242), 1–8.
for the detection of hepatitis C virus in high risk group population: a
Trang 88 W Chantratita et al / Journal of Virological Methods 239 (2017) 1–8
among individuals infected with hepatitis C virus (HCV) genotypes 1, 2, and 3
by quantiplex HCV RNA assay versions 1 and 2, Roche Monitor assay, and an
in-house limiting dilution method J Clin Microbiol 35, 187–192.
Infect 17, 107–115.
Hepatol 7 (10), 1377–1389.
Hepatitis C virus genotype distribution in Myanmar: predominance of
genotype 6 and existence of new genotype 6 subtype Hepatol Res 37,
337–345.
Hepatology 61, 77–87.
treatment of chronic hepatitis C virus infection: scientific and regulatory
approaches to clinical trial designs Hepatology 62 (4), 1298–1303.
new-generation line-probe assay that detects 5’ untranslated and core regions
to genotype and subtype hepatitis C virus Am J Clin Pathol 128 (2), 300–304.
probe selection method for DNA Chips Chem Commun 47, 12444–12446.
Vietnam: large-scale survey based on sequence determination Jpn J Infect Dis 64, 537–539.
subtypes: updated criteria and genotype assignment web resource.
Hepatology 59, 318–327.
convenient genotyping system Chem Commun 49, 2661–2663.
http://www.who.int/hepatitis/publications/hepatitis-c-guidelines-2016/en/
and southeast Asia PLoS One 10 (5), e0126764.
C virus genotyping by heteroduplex mobility analysis J Clin Microbiol 38 (2), 477–482.
the versant HCV genotype 2: 0 assay (LiPA) and the Abbott RealTime HCV genotype II assay for detecting hepatitis C virus genotype 6 J Clin Microbiol.
52, 3685–3692.