NS5A resistance - associated substitutions in chronic hepatitis C patients with direct acting antiviral treatment failure in Turkey

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NS5A

resistance

–

associated

substitutions

in

chronic

hepatitis

C

patients

with

direct

acting

antiviral

treatment

failure

in

Turkey

Murat

Sayan

a,b,

*

,

Figen

Sar

_ıgül

Y

_ıldırım

c

,

S

_ıla

Akhan

d

,

Arzu

Altunçekiç

Y

_ıldırım

e

,

Göktug

Şirin

f

_,

_Mehmet

_Cabalak

g

_,

_Mehmet

_Demir

h

_,

_Selver

_Can

i

_,

_Gülden

_Ersöz

j

_,

Engin

Alt

_ıntaş

k

,

Fatih

Ensaroglu

l

,

Ayhan

Akbulut

m

,

Alper

_Şener

n

,

Ayd

_ın

Deveci

o a

FacultyofMedicine,ClinicalLaboratory,PCRUnit,UniversityofKocaeli,Kocaeli,Turkey

b

ResearchCenterofExperimentalHealthSciences,UniversityofNearEast,Nicosia,Cyprus

c_Antalya_Education_and_Research_Hospital,_Department_of_Infectious_Disease,_University_of_Health_Sciences,_Antalya,_Turkey d_Faculty_of_Medicine,_Department_of_Infectious_Diseases,_University_of_Kocaeli,_Kocaeli,_Turkey

e

FacultyofMedicine,DepartmentofInfectiousDiseases,UniversityofOrdu,Ordu,Turkey

f

FacultyofMedicine,DepartmentofGastroenterology,UniversityofKocaeli,Kocaeli,Turkey

g

FacultyofMedicine,DepartmentofInfectiousDiseases,UniversityofHatayMustafaKemal,Hatay,Turkey

h

FacultyofMedicine,DepartmentofGastroenterology,UniversityofHatayMustafaKemal,Hatay,Turkey

i

KonyaEducationandResearchHospital,DepartmentofInfectiousDisease,UniversityofHealthSciences,Konya,Turkey

j_Faculty_of_Medicine,_Department_of_Infectious_Diseases,_University_of_Mersin,_Mersin,_Turkey k_Faculty_of_Medicine,_Department_of_{Gastroenterology,}_University_of_Mersin,_Mersin,_Turkey l

FacultyofMedicine,DepartmentofGastroenterology,Universityof _Istinye, _Istanbul,Turkey

m

FacultyofMedicineDepartmentofInfectiousDisease,UniversityofFırat,Elazıg,Turkey

n

FacultyofMedicineDepartmentofInfectiousDisease,UniversityofOnsekizMart,Canakkale,Turkey

o

FacultyofMedicine,DepartmentofInfectiousDiseases,Universityof19Mayıs,Samsun,Turkey

ARTICLE INFO

Articlehistory:

Received26October2019

Receivedinrevisedform19March2020 Accepted24March2020 Keywords: HepatitisC HepatitisCvirus NS-5protein Treatmentfailure Sequenceanalysis ABSTRACT

Objectives:ChronichepatitisC(CHC)isnowamorecurablediseasewithnewdirectactingantivirals (DAA). Althoughhighsustained virologic responserates,failures still occur inDAA regimens. Our objectivein thisstudywastocharacterizethereal-life presenceofclinically relevantresistance– associatedsubstitutions(RASs)intheHCVNS5AgeneinCHCpatientswhoseDAAregimenhasfailed. Methods: The studyenrolled 53 CHC patientswho experienced failure with DAA regimen as the prospectivelongitudinalcohortbetween2017–2019.Genotypicresistancetestingwasperformedviathe viralpopulationsequencingmethodandTheGeno2phenoHCVtoolwasusedforRASanalysis. Results:Themostfrequentfailurecategorywasrelapse(88%)followedbynon-responder(12%).Foratotal of36%ofpatients,RASswasdetectedinNS5A,Y93HwasthemostdetectedRASinGT1binfectedpatients (89%).

Conclusions:ThisstudyestablishesanHCVfailureregistryforTurkeyinwhichsampleswerecombined withclinical,virologicandmoleculardataofadultpatientswhoseDAAtherapyfailed.RASscanoccurin CHCpatientswithDAAtreatmentfailures.EvaluationofRASafterDAAfailureisveryimportantbefore re-treatmentisinitiatedtopreventvirologicfailure.

Introduction

HepatitisCvirus(HCV)istheetiologicagentofchronichepatitis C(CHC)andamajorcauseofcirrhosisandlivercancer(Leeetal., 2012).Since2013,CHChasbecomea morecurablediseaseasa resultofnewdirectactingantivirals(DAA)(Rodriguez-Torresetal.,

2013).In June 2016,sofosbuvir(SOF) [nonstructuralprotein5B (NS5B)polymeraseinhibitor],thecombinationSOFandledipasvir (LDV)[nonstructuralprotein5A(NS5A)inhibitor],the combina-tionofNS5Ainhibitorombitasvir(OBV),dasabuvir(DSV)[NS5B polymerase inhibitor]and paritepravir(PTV) [the nonstructural protein3/4A(NS3/4A)inhibitorboostedwithritonavir(r)(3D) ribavirin (RBV)] started to be used on CHC patients in Turkey (HealthMinistry,2017).However,in2017,thecombinationofan NS3/4Ainhibitorglecaprevir,anNS5Ainhibitorpibrentasvir(GLE/ PIB), and sofosbuvir/velpatasvir/voxilaprevir (SOF/VEL/VOX) started tobeusedaround theworld(EMA, 2019;USD and HS,

*Correspondingauthorat:UniversityHospitalofKocaeli,ClinicalLaboratory, PCRUnit,EskiIstanbulYolu,UmuttepeCampus,41380Izmit,Kocaeli,Turkey.

E-mailaddress:murat.sayan@kocaeli.edu.tr(M.Sayan).

https://doi.org/10.1016/j.ijid.2020.03.061

ContentslistsavailableatScienceDirect

International

Journal

of

Infectious

Diseases

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2019). Today, in Turkey, GLE/PIB has been approved for HCV-infectedpatients(HealthMinistry,2017).

Withextremelyhighcureratesreportedinclinicaltrialsandin real-world data, modern DAAs can effectively cure the vast majority of patients infected with HCV. Sustained virologic response (SVR) rates of 94–100% have been reported with contemporary DAA regimens (Pecoraro et al., 2019; Kumada etal.,2018;Sariguletal.,2019).Althoughalowrateoffailurehas beenobservedwithcurrentDAAregimens,failuresstilloccur(Di Maioetal.,2017a).ThereasonsforfailureoftheDAAregimensare mostlypatientfactorssuchasnonadherencetotreatment, drug interactions withHCV drugs, inappropriate treatment regimen, and inappropriate initialtreatmentduration (Aziz etal., 2018). Besidesthese,virologic factors- resistance-associated substitu-tions(RASs)canaffecttheresponsetothetherapyinCHCpatients withDAAsregimes.Viralfailurecanoccurin 5–10%ofpatients (WylesandLuetkemeyer,2017;Bertollietal.,2018).Inchronically infectedhumans,HCVhashighratesofreplicationdynamicsand viralproductionwithanerror-proneRNApolymerase,providinga favorableenvironmentfortheemergenceandenrichmentofviral nucleotidesubstitutionsthat giveresistancetocertaindrugsor classesofdrugs,particularlyunderthepressureofdrugselection (WylesandLuetkemeyer,2017).

Ingenotype1a(GT1a)infection,baselineNS5ARASshavea13% prevalence,whereasthereisa18%prevalenceinGT1binfection anda 12–17%prevalencein GT3infection(Zeuzemet al.,2017; Hezodeetal.,2018).Patientcharacteristics,includingthepresence of cirrhosis in the patient and any previous HCV treatment regimens(based onnon-NS5A inhibitor),canaffectthe clinical effect of NS5A RAS. After NS5A-based treatmentfailure, many individualshaveHCVNS5A(75–90%)(Wylesetal.,2017).NS5A RASspersistinmostindividualsformorethan2years(Lahseretal., 2018). When NS5A RAS is detected, either the duration of treatmentisprolongedorribavirinmaybeaddedtothetreatment (Sarrazinetal.,2016).Consequently,theresistancetestisthemost importantfactorinthechoiceofthere-treatmentmethod(Hayes etal.,2019).

HCV seroprevalence is about 0.9% in Turkey, whose total populationisaround80million(Data,2019).HCVGT1bisthemost prevalentgenotype,accounting for84%and GT1aisthesecond mostprevalentgenotype,withratioof6.6%ofallinfectionsamong Turkishpatients(Alagoz et al.,2014).In ourcountry, approved regimensforGT1-CHCpatientsare3D+RBVinGT1a,and3Din G1b non-cirrhotic naive patients for 12 weeks. For treatment experiencednon-cirrhoticpatients,LDV/SOFisgivenfor24weeks or LDV/SOF+RBV for 12weeks and a 3D regimenis approved similartothatofnaiveGT1patients. In cirrhoticpatients, both regimens are approvedexcept for child-Pugh Band C patients (Health Ministry, 2017). Unfortunately, the accessibility to the treatmentrate of HCV infection is very poor,but the probable treatmentrateis0.8%forCHCinTurkey(Doreetal.,2014).Onthe otherhand,patientswithHCVinfectioninourcountrycanonlybe treatedwithDAAregimensonceintheirlives.Ifthepatientdoes notrespondtotreatment,orexperiencesrelapseaftertreatmentor breakthrough,thereisnopossibilityofbeingtreatedagain.

Inthisstudy,weaimedtoprovideusefulinformationforthe clinicians themanaging that thereal-life presence of clinically relevant RASs in the NS5A gene in CHC patients whose DAA regimenhasfailed.

Materialandmethods

Patients

Thiswork wasdesigned asaprospectivelongitudinalcohort study,including53CHCpatientswhoseDAAregimenfailed.Cases

has beenfollowed after thecure of HCV RNAwho under DAA regimen.PlasmasampleshavebeenanalyzedonlyforRASsinthe HCVNS5AgeneregionintheHCVRNAreboundedpatientsatthe laboratoryofthePCRUnitoftheUniversityofKocaelifromJuly 2017toSeptember2019.ThecasewhoHCVRNAcuredhasbeen excluded inthestudy. Thetherapy indicationandDAAregimen choiceweremadebyphysiciansaccordingtotheCHCguidelines and Local Health Implementation Guidelines (Health Ministry, 2017;Terraultetal.,2018;Lamperticoetal.,2017).Accordingtothe LocalHealthImplementationGuidelineofTurkeyeachpatientcan be treated once with DAA regime and biopsy application is mandatoryas arule beforethetreatment regimedecision. The treatmentdurationandthedoseofDAAswerechosenaccordingto thesameguidelines.Samplesweretakenfromtwenty-fourclinics locatedin18citiesinallregionsofTurkeyforthestudy:Marmara, Mediterranean, Central Anatolia, Aegean, Black Sea, Eastern AnatoliaandSoutheasternAnatoliaregions.

Afterstoppingthetreatmentat month3,HCVRNAnegative patientswereacceptedasSVR.Patientswereidentifiedashaving relapsedwhentheyshowedavirologicreactivationaftertesting HCV-RNAnegativeattheendoftreatment.PatientswhoseHCV RNAdidnotbecomenegativeduringtreatmentweredefinedas non-responders. Patients whose HCV RNAwas negative during DAAtreatmentandwhoseHCVRNAwaspositivebeforetheendof treatment were defined as viral breakthrough (Terrault et al., 2018).Fibroscan,duetothelimitednumberofpresenceinTurkey andthelackofsocialsecuritycoverage,histologicalscoringwas madeintheliverbiopsybythemodifiedIshakmethodaccordingto thehistologicalactivityindex(grade)andfibrosis(stage)(Ishak etal.,1995).

Plasmasampleswerecollectedfromeachpatientintheprocess ofrecordingthevirologicfailureandstoredat 80CuntiltheRAS analysis.Thepresenceofanti-HCVantibodywastestedthrough commercial ELISAs (Elecsys, Roche Diagnostics, Mannheim, GermanyandAxsym,AbbottLaboratories,AbbottPark,IL,USA). Toobtainthepatients’samplesandtoensuretheanonymoususe of their data in accordance with the Helsinki Declaration and internationalguidelines,writteninformedconsentwasobtained fromallstudypatients.Ethicalapprovalwas obtainedfromthe KocaeliUniversityEthicsCommittee(KOUKAEK12/2016). HCVRNAisolationandquantiﬁcation

HCV RNA was extracted by magnetic particle technologies [QIAsypmhony(QiagenGmBH,Hilden,Germany),AbbottM2000 SP (Abbott Molecular Inc., Des Plaines, IL, USA), and COBAS Ampliprep (RocheMolecularSystems,Inc.Pleasanton, CA,USA) platforms]andquantiﬁcatedbyreal-timePCRassays[artusHCV QS–RGQkit(QiagenGmBH,Hilden,Germany),AbbottRealTime HCVAmpliﬁcationKit(AbbottMolecularInc.,DesPlaines,IL,USA) and COBAS TaqMan HCV kit (Roche Molecular Systems, Inc. Pleasanton,CA,USA)],respectively.

HCVNS5Agenesequencing

For theamplification of theHCV NS5Agene region,specific primer pairs were designed against the HCV reference strain AF483269.1:HCV1a1b_NS5A_F1;3’-TCCCCCACGCACTAYGTGCC-5’ and HCV1a1b_NS5A_R2; 3’-GTGATRTTICCGCCCAT-5’. Protocols applied for the RT-PCR and cycle sequencing were as follows: cDNAsynthesisat48Cfor30minanddenaturationat95Cfor 15minfollowedby50cyclesconsistingofthedenaturationstepat 95Cfor1min,annealingstepat55Cfor1min,extensionat72C for 1minute,and a finalstep at72C for10min.Theextracted ampliconwasapproximately938bpandincludedallknownRASs for theNS5Aregion. Inthesequencingprotocol, HotStartDNA

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polymerase (Finnzymes Oy, Vantaa,Finland) was used.For the purificationofthePCRproducts,aHighPurePCRpurificationkit was used (Roche Diagnostics, Mannheim, Germany). Genotypic resistance testing was performed by the viral population sequencingmethod(NS5Aaminoacidpositionbetween1935.– 2237.).Thecyclesequencingprotocolwasasfollows:35cyclesat 95Cfor20s,50Cfor25s,andfinally,60Cfor2min.Thefinal concentrationsofthesequencingprimerswere0.5mM.BigDye TerminatorCycle SequencingKit(AmershamPharmaciaBiotech Inc.,Piscataway,NewJersey,UnitedStates),POP–7polymerand capillary(36cm)wereusedinthesequencingprocessonanABI PRISM 3130 platform (Applied Biosystems Inc., Foster City, California,UnitedStates).

Resistance-associatedsubstitutiondetection

The Geno2pheno HCV tool (http://coreceptor.bioinf.mpi-inf. mpg.de/)wasusedfortheRASanalysis.Gheno2phenoidentiﬁes the DAA susceptibility for HCV. It is a database speciﬁcally designed for rapid computerassisted virtual phenotyping, and acceptsgenomesequences(inFASTAformat)asinputforsearching known mutations for both licensed and upcoming drugs. The referencestrainforNS5AregionintheGeno2phenotoolwasHCV D90208.

Genotypingresultsof theHCVstrains wereprovidedbythe submitterandmostlybasedoncommercialmethods.However,the HCVgenotypewasreassessedbytheGeno2pheno-HCVtool.Also, weevaluatedtheRASsthatwereassociatedwithDAAfailureinthe lightofthe2018EASLHCVguidelines.Theywerescoredforthe aminoacidposition28.,30.,31.,32.,58.,92.and93.ofNS5Aregion

for daclatasvir, elbasvir, ledipasvir,ombitasvir, pibrentasvirand velpatasvir.

Statisticalanalysis

Sociodemographic, virological and clinical variables were reportedasabsolutenumbers,percentagesandmedians. Quanti-tativedataprocessedwithdescriptivestatisticaltechniques.

Forthedescriptiveanalysis,thefrequenciesandpercentagesof the qualitative variables and the means containing standard deviation and median with interquartile range of quantitative variables were calculated. All analyses were performed on MicrosoftExcel2017.

Results

Thedemographic,clinicalandvirologicdataof53registered patientsaresummarizedinTable1.Thirty-three(75%)weremales, andthemedianagewas31years(range24–77).Allpatientswere domestic;thosewithcirrhosis(6patients,12%,83%inChildAclass and17%inChildBclass).Aliverbiopsywascarriedoutfor32(62%) patientsandthemedianscoreofﬁbrosiswas3(Leeetal.,2012; Rodriguez-Torresetal.,2013;HealthMinistry,2017;EMA,2019; USDandHS,2019).Mostofthepatientswerenaive(69%)and10% had pegylated-interferon experienced treatment. Only 2 (4%) patients had liver transplantation. The therapeutic regimen distributionforHCVgenotypesisshowninTable1.

AfterfailedDAAtherapy,theHCVgenotypewasre-evaluatedin allpatientsand discordance inthegenotypingresults thatwas probablycausingDAAfailurewasidentiﬁedin4(8%)patients.The

Table1

Baselinecharacteristicsofthestudypopulation

Characteristic Studygroup

Numberofpatients,n 53

Age,medianyear(range) 31(24–77)

Gender,male,n(%) 39(75)

Sampling,region/cityofTurkey Marmara; _Istanbul,Kocaeli,Bursa Aegean;Çanakkale

Mediterranean;Antalya,Hatay,Mersin,Adana,Isparta SoutheasternAnatolia;Gaziantep

EasternAnatolia;Elazıg,Van

BlackSea;Samsun,Ordu,Bartın,Amasya CentralAnatolia;Ankara,Konya HCVRNAload,baselinemedianIU/ml(range) 1.7+E6(2.7+E4–4.0+E6) ALTlevel,baselinemedianIU/ml(range) 54(7–283)

Patientwithcirrhosis,n(%) 6(12)

ChildA 5(83)

ChildB 1(17)

Patientwithlivertransplantation,n(%) 2(4) Biopsystatus,medianscore(range),n(%)

Carriedliverbiopsy 32(62)

Fibrosis 3(1–5)

Treatmentstatus,n(%)

Naivepatient 36(69)

Pegylated-interferonexperienced 16(31)

TreatmentchoiceonHCVgenotype,n(%)

GT1a 6(10)

Ombitasvir+paritaprevir/r+dasabuvir+ribavirin 5(83)

Ledipasvir+sofosbuvir 1(17)

GT1b 42(81)

Ledipasvir+sofosbuvir 7(17)

Ledipasvir+sofosbuvir+ribavirin 9(21)

Ombitasvir+paritaprevir/r+dasabuvir 26(62)

GT2 2(4)

Sofosbuvir+ribavirin 2(100)

GT3 3(5)

Sofosbuvir+ribavirin 1(33)

Ledipasvir+sofosbuvir+ribavirin 2(67)

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characteristicsofthesepatientsareshowninTable2.Noneofthem hadRASsintheNS5Aregion.

Theremaining49patientshadbeentreatedwiththeoptimal DAAregimenaccordingtotheguidelines.According totheDAA regime,thedescriptionoffailureis showninTable3.Themost frequentfailurecategorywithDAAwasrelapse(88%).Failureinthe 3Dregimenwashighestwith24(50%)patients,and20(83%)ofthe GT1aandGT1bpatientsrelapsed.Thenumberofpatientsinwhom RASswasdetectedinNS5Awas19(36%)andY93Hwasthemost detected RAS in GT1b patients (89%). The distribution of RASs withinNS5Aseeningenotypes1aand1bHCVpatientsisshownin

Table4.

Table4shows themostfrequenttreatment-selectedRASsin NS5A according to the HCV treatment regimen. In treatment regimewith3D,L28M,Q30R,P32R,P58T,A92S,Y93HRASswere seeninGT1bHCVpatients.L31M,P58S,Y93HRASsweredetected inGT1bHCVpatientstreatedwithLDV+SOF+RBV.L28M,L31V, P58S,Y93HwereseeninpatientsinfectedwithGT1bandtreated with LDV+SOF. However, Q30H/R, H58C, Y93H were seen in patientsinfectedwithGT1aandtreatedwith3D+RBV.

Discussion

Inspiteofthehighef_ﬁcacyofDAAregimens,afailurerateof 5-15% is observed in HCV infected patients (Martínez et al., 2019;Wangetal.,2018).Thefailureisgenerallyassociatedwith theRASs. Thusfar,a signi_ﬁcant amountof real-lifedatahave beenstudiedonthissubject.Atotalof53patientswhoseDAA regimen failed were analyzed in this study. The number of patients inwhich RASs were detected in NS5A was 36%and Y93H was the most detected RAS in GT1b infected patients

(89%).WhentheDAAtreatmentofHCVpatientsisobservedto fail,thepresenceofRASsshouldbeconsidered(74.4%)(Starace etal.,2018).InthestudyofGT1patientsbyMartinezetal.,NS5A RASs were detected in 25.8% of the analyzed sequences (Martínez et al., 2019). In our study, 48 GT1 HCV infected patients failedafter DAAtreatment and19 of themhadRASs (39.5%).Inaddition,relapsewasdetectedin88%of48patients with failure,whiletheother12% werenon-responders.There were no breakthrough cases. Other studies have produced different results in the deﬁnition of failure during DAA treatment(Pawlotsky,2016;DiMaioet al.,2017b).

Firstof all,treatment failurewasthemisclassificationofthe HCVgenotype;fourpatients(8%ofcases)weremisclassifiedand allweretreatedwithinappropriateDAAregimens.Instudiesof failureofDAAregimensinHCVpatients,misclassificationswere seenin 8_–14.9%(Staraceetal.,2018;Ceccherinietal.,2016).In caseswherethecostofDAAregimensishigh,misclassificationof theHCVgenotypeandsubtype,itisveryimportanttoavoidwrong treatmentwiththewrongregimens.Inaddition,thedynamicsof RASshouldbeunderstoodwithinthecirculatorydynamicsofHCV, whichnecessitatesthecorrecttypingoftheHCV.Sequencingof HCVgenomeregionswithphylogeneticanalysisisconsideredthe gold standard of HCV genotyping (Robertson et al., 1998). In countrieswithahighprevalenceofGT1infection,suchasTurkey,it isimportanttotypetheHCVforclinicaldecisions(Alagozetal., 2014).Therefore,thesequencing-basedmethodsforHCVpatients shouldbeconsidered.Perhapsthesefindingsofoursandsimilar studieswouldcauseastrongfeedbacktothetitleofthetypingof HCV diagnostic and treatment guidelines. In addition, correct sequencingmethodsandgenotypeandsubtypedeterminationsin treatment failuresshouldbestudiedbefore resistance testsare performed.

TheprevalenceofRASwasfoundtobehigherinourGT1bthan inGT1apatients.Similarly,theprevalenceofatleastonemajorRAS intwoormoreHCVsiteswasmorefrequentinpatientswithGT1b patientsandinpatientswhodidnotreceiveRBV.ThehighRAS prevalenceinGT1binfectedpatientscanbeexplainedbythefact that most of the patients (n=26, 62%) were treated with 3D according tothe Turkish Ministryof Health rules of treatment regimen. (HealthMinistry,2017).Inaddition,ourGT1apatients weretreatedwith3D+RBVaccordingtothenationalguidelines. TheeffectofNS5ARASisrelativeandcanoftenbeovercomeby increasingthelengthoftreatmentand/orbyaddingRBV(Sarrazin etal.,2016).TheclinicalimpactofbaselineNS5ARASsvariesby

Table2

Demographic,clinicalandvirologicalcharacteristicsofthefourpatientswithmisclassiﬁcationoftheHCVgenotype,causingdirectactingantiviralfailure.

Patient Age Gender Reportedgenotype Identiﬁedgenotype DAAregimen Durationofregimen,months RASinNS5A

1 26 M 1b 3 3D+RBV 12 ND

2 29 M 1b 3a 3D 12 ND

3 34 M 1b 1a 3D 12 ND

4 67 M 2 1b SOF+RBV 24 ND

Abbreviations:M;male,DAA;directactingantiviral,3D;ombitasvir+paritaprevir/r+dasabuvir,RBV;ribavirin,SOF;sofosbuvir,RAS;resistance-associatedsubstitution,ND; notdetected.

Table3

DescriptionoffailureduringthedirectactingantiviraltreatmentinHCVgenotype1a/binfectedpatients.

DAAregimen Patient,n(%) Treatmentmediantime,week(range) Breakthrough,n(%) Non-response,n(%) Relapse,n(%)

LDV+SOF 9(18) 21(8–24) ND 1(12) 8(88)

LDV+SOF+RBV 10(20) 15(12–24) ND 1(10) 9(90)

3D 25(50) 12(12–12) ND 4(17) 19(83)

3D+RBV 4(8) 12(12–12) ND ND 4(100)

Total 48(100) 12(12–12) ND 6(12) 42(88)

Abbreviations:DAA;directactingantiviral,LDV+SOF;ledipasvir+sofosbuvir,RBV;ribavirin,3D;ombitasvir+paritaprevir/r+dasabuvir,ND;notdetermined.

Table4

Themostfrequenttreatment-selectedresistance–associatedsubstitutionsinNS5A accordingtothetreatmentregimen.

DAAregimen GT1a GT1b

LDV+SOF – L28M,L31V,P58S,Y93H LDV+SOF+RBV – L31M,P58S,Y93H

3D – L28M,Q30R,P32R,P58T,

A92S,Y93H 3D+RBV Q30H/R,H58C,Y93H –

Abbreviations:LDV+SOF;ledipasvir+sofosbuvir,RBV;ribavirin,3D;ombitasvir+ paritaprevir/r+dasabuvir,GT;genotype.

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HCVgenotypeandsubtypeandthelargestimpactswereshownin genotype 1a and 3 infections. In GT1a M28A/T/V, Q30E/H/K/R, L31M/V,Y93C/H/NandinGT1bL31I/M/V,Y93HarethekeyNS5A RASs (Wyles and Luetkemeyer, 2017). On the other hand, we publishedpreviousHCVdrugresistanceanalysisresultsinTurkey (Şanlıdag et al., 2017;Sarigulet al.,2017; Altunoket al.,2016; Ertemetal.,2015).ThesestudiesweremostlyrelatedtotheHCV NS3generegion.However,wedonothavesufﬁcientknowledge aboutNS5Ageneregionmutations.Ourﬁndingsinthisstudymay clarifythisissue.

RegardingHCVtreatmentregimens,theprevalenceofRASin SOF-basedregimensislessthanthatof3Dregimens.Thisresult isconsistentwiththehighresistancebarrierofNS5Binhibitors andSOFfailurereportedinthepresence ofRAS(Coppolaetal., 2016;Sarrazin, 2016).In contrast,thehighprevalenceof NS5A RASisassociatedwithalowbarriertotheresistanceofexisting NS5A inhibitors (Sorbo et al., 2018). RASs tend toremain 1-2 years after treatmentfailure for the NS5A region. Inthis real-worldstudy,themostcommonRAS(Y93H)wasdetected(88%) intheNS5Aregionincasesoffailure,especiallyinGT1btreated withtheoptimalDAAregimen.(Sorboetal.,2018).Inparticular, in GT1b failures, complex RAS patterns were observed more frequently (with A92S, L28M, L31M/V, Q30R, P32R, P58S/T, Y93H), while all the GT1a NS5A failures were only seen with H58C,Q30H/R,Y93H.ThesedataappearimportantbecauseNS5A inhibitorsarethemostpopularcomponentsofDAAregimens.In previousstudieswithNS5Ainhibitorcontaining DAAregimens, the amino acid substitutions that produce resistance to NS5A inhibitorshavebeenshowntoaffecttheSVRrate(DiMaioetal., 2017b;Tavaresetal.,2018).Ontheotherhand,inGT1a,asingle RAS can provide high levels of resistance to most NS5A inhibitors,whileinGT1b,thereareonlyhighlevelsofresistance toLDV(Lontoketal.,2015).BecauseNS5AregionRASstendtobe persistent,retreatmentstrategiesshouldinvolveacombination of triple or quadruple DAA regimens with high resistance barriers such as NS5B inhibitors as well as a combination of NS3+NS5Ainhibitors(Terraultet al.,2018).

In conclusion, thisstudy establishedan HCVfailure registry for Turkey in which samples were combined with clinical, virologic and molecular data of adult patients whose DAA therapy failed. RASs can occur in CHC patients with DAA treatment failures. Evaluation of RAS after DAA failure is importantbefore re-treatment is initiated toprevent virologic failure.

Contributionofauthors

Studydesign;MuratSayan,FigenSarıgülYıldırım,SılaAkhan. Writing;MuratSayan,FigenSarıgülYıldırım.

Datacollection;Allofauthors.

Dataanalysis;MuratSayan,FigenSar_ıgülY_ıldırım. Patientsproviding;Allofauthors.

Conﬂictofinterest

Nocompetinginterestsexist.

Fundingsource

Nofundingsourceused.

Ethicalapproval

Ethical approval was obtained from the Kocaeli University EthicsCommittee(KOUKAEK12/2016).

Acknowledgments

We thank Mr. SimonThompson (from Near East University, Nicosia,NorthernCyprus)forEnglishlanguageediting.

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