Clinical Laboratory Testing in the Era of Directly Acting Antiviral

20 Pages·2016·0.84 MB·English

REVIEW
crossm
Clinical Laboratory Testing in the Era of
Directly Acting Antiviral Therapies for
Hepatitis C
D
o
w
n
EleanorM.Wilson,a,bElanaS.Rosenthal,bSarahKattakuzhy,a,bLydiaTang,a lo
ShyamKottilila,c a
d
e
InstituteofHumanVirology,UniversityofMarylandSchoolofMedicine,UniversityofMaryland,Baltimore, d
Maryland,USAa;CriticalCareMedicineDepartmentoftheNIHClinicalCenter,NationalInstitutesofHealth, f
r
Bethesda,Maryland,USAb;NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth, o
m
Bethesda,Maryland,USAc

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SUMMARY.......................................................................................23 Published19October2016 /c
INTRODUCTION.................................................................................23 m
CitationWilsonEM,RosenthalES,KattakuzhyS,
VIRALFACTORS.................................................................................24 TangL,KottililS.2017.Clinicallaboratory r.a
HCVGenotype.................................................................................25 testingintheeraofdirectlyactingantiviral s
ViralLoad......................................................................................27 m
therapiesforhepatitisC.ClinMicrobiolRev
BaselineHCVRNAload....................................................................27 30:23–42. .o
On-treatmentmonitoringofHCVRNAload..............................................28 https://doi.org/10.1128/CMR.00037-16. rg
ResistanceTesting.............................................................................28 Copyright©2016AmericanSocietyfor o/
HOSTFACTORS.................................................................................30 Microbiology.AllRightsReserved. n
IL28BGenotype...............................................................................30
O
IFNL4Genotype...............................................................................31 AddresscorrespondencetoShyamKottilil, c
ViralCoinfection...............................................................................31 [email protected]. to
FibrosisStaging...............................................................................32 b
e
Liverbiopsy.................................................................................32 r
Laboratorystaging.........................................................................34 1
(i)APRI....................................................................................34 9
,
(ii)FIB-4...................................................................................34 2
(iii)FibroTest/FibroSureandHepaScore................................................34 0
1
Transientelastography.....................................................................34 6
DecompensatedCirrhosis....................................................................35 b
Recommendations............................................................................35 y

SafetyMonitoring.............................................................................36 C
O
CONCLUSION....................................................................................36
R
REFERENCES.....................................................................................36
N
AUTHORBIOS...................................................................................41
E
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L

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SUMMARY Directlyactingantiviral(DAA)combinationtherapiesforchronichepatitis N
I
C virus (HCV) infection are highly effective, but treatment decisions remain complex. V
E
Laboratory testing is important to evaluate a range of viral, host, and pharmacologi- R
cal factors when considering HCV treatment, and patients must be monitored during S
I
T
and after therapy for safety and to assess the viral response. In this review, we dis- Y
cuss the laboratory tests relevant for the treatment of HCV infection in the era of
DAAtherapy,groupedaccordingtoviralandhostfactors.
KEYWORDS directlyactingantiviraltherapy,hepatitisCvirus,viralresistance
INTRODUCTION
ThetreatmentofchronichepatitisCvirus(HCV)infectionhaschangeddramatically
overthepast5years.Inthepast,thedurationandcontinuationoftreatmentwith
interferon(IFN)-andribavirin(RBV)-basedtherapieswereguidedbylaboratorytesting
and demographic factors. Those individuals with so-called “unfavorable treatment
characteristics,” including infection by particular HCV genotypes, male sex, distinct
geneticpolymorphisms,advancedhepaticfibrosis(includingcirrhosis),andcoinfection
January2017 Volume30 Issue1 ClinicalMicrobiologyReviews cmr.asm.org 23
Wilsonetal. ClinicalMicrobiologyReviews
with human immunodeficiency virus (HIV), were less likely to achieve a sustained
virologic response (SVR), defined as the absence of detectable hepatitis C virus 12
weeks after the completion of therapy, now synonymous with cure (1–3). The first
approved directly acting antiviral (DAA) agents, which specifically inhibit HCV serine
protease, were introduced in 2011. However, the use of these initial agents was
complicatedbycoadministrationwithpegylatedIFN(pegIFN)andRBV,whichresulted
in severe side effects and required frequent laboratory monitoring throughout pro-
tractedtreatmentcourses.WiththedevelopmentofnewercombinationDAAregimens,
first approved by the U.S. Food and Drug Administration (FDA) in late 2014, patients
havesafeandhighlyeffectiveall-oral,interferon-freeHCVtreatmentoptions,withcure D
o
ratesexceeding90%formostHCVgenotypesandstagesoffibrosis(4–8).Theadvent w
of DAAs has changed the paradigms not only for treatment of HCV but also for the n
lo
relevantassociatedlaboratorytestingaswell. a
d
The DAA agents currently approved for IFN-free combination therapy of HCV e
d
infectionareshowninTable1.TheseagentstargetthreedifferentHCVproteinsviafour
f
r
different pathways. The nucleotide HCV nonstructural protein 5B (NS5B) polymerase o
m
inhibitor sofosbuvir (SOF) (Gilead Sciences, Inc.) (generics are also available in certain
h
countries)istheonlyapprovedmemberofitsclass;auridineanalogchainterminator, tt
p
itisapprovedforcombinationtherapywithotherDAAsorwithpegIFN-RBV(9).There :
/
/
is also an approved nonnucleoside NS5B inhibitor, dasabuvir (DSV; AbbVie, Inc.), that cm
inhibitsthepolymeraseseparatelyfromtheactivesite(10).Theseconddrugtarget,the r
.
a
HCV NS5A protein, is thought to help stabilize infectious HCV replication complex s
m
formation on the surface of the endoplasmic reticulum within hepatocytes (11). Ap-
.
o
provedNS5Ainhibitorsincludeledipasvir(LDV)andvelpatasvir(VEL)(bothfromGilead r
g
Sciences, Inc.) (12, 13), daclatasvir (DCV; Bristol-Myers Squibb) (14), ombitasvir (OMV; /
o
AbbVie,Inc.)(15),andelbasvir(EBR;Merck&Co.,Inc.)(16).Thefinalclassofapproved n

O
second-generation DAAs is the NS3/4A protease inhibitors, which include simeprevir
c
(SMV; Janssen Pharmaceuticals) (17), grazoprevir (GZR; Merck) (18), paritaprevir (PTV; to
b
AbbVie)(19),andasunaprevir(ASV;Bristol-MyersSquibb)(currentlyapprovedinJapan e
r
andRussia)(20).Therearealsoseveralcoformulationsofthesemedicationsthatgreatly 1
9
simplify their administration: LDV and SOF are coformulated as Harvoni (Gilead), GZR ,

2
andEBRarecoformulatedasZepetier(Merck),andOMBandPTVarecoformulatedwith 0
1
ritonavir (inactive against HCV but included to potentiate PTV to facilitate once-daily 6

dosing)asTechnivieandcopackagedwithDSVunderthenameViekiraPak(bothfrom b
y
AbbVie). The American Association for the Study of Liver Diseases (AASLD) and the C
InfectiousDiseasesSocietyofAmerica(IDSA)havetogethercreateddynamicguidelines O
R
(available online at http://www.hcvguidelines.org/) that are regularly updated in re- N
sponse to new data and drug approvals with recommendations and expert opinion E
L
regarding the combination of these medications for the treatment of chronic HCV L

infection(21). U
N
Despite the high efficacy and tolerability of these regimens, treatment decisions I
V
remaincomplex.Afterapatientisfoundtobeseropositive,havingdetectableanti-HCV E
R
antibody,apositivemoleculartestforHCVRNAisrequiredforadiagnosisofchronic
S
HCV infection (22). Depending on the immunological status, between 20 and 45% of IT
Y
personsexposedtoHCVmaynaturallycleartheinfection,usuallyinthefirst6months
afterexposure(23).Cliniciansmustthenuselaboratorytestingtoevaluatearangeof
viral,host,andpharmacologicalfactorswhenconsideringinitiatingtreatmenttoensure
thatpatientsrealizethefullbenefitofnewHCVtherapies.Patientsmustbemonitored
duringandaftertherapyforsafetyandtoassesstheviralresponse(thetestingtimeline
is shown in Fig. 1). In this review, we discuss the laboratory tests relevant for the
treatmentofHCVinfectionintheeraofDAAtherapy,groupedaccordingtoviraland
hostfactors.
VIRALFACTORS
PriortothestartofDAAtherapyforHCV,viraltestingisrequiredfortworeasons:
first,toconfirmchronicHCVinfection(whileitisrare,evenpatientswithdocumented
January2017 Volume30 Issue1 cmr.asm.org 24
ClinicalLaboratoryTestingandDAATherapyforHCV ClinicalMicrobiologyReviews
TABLE1FDA-approvedformulationsforthetreatmentofchronichepatitisCa
Genericname(s) U.S.brand
(concn[mg]) Abbreviation name Dosing Indication(s)
Daclatasvir(60) DCV Daklinza Onetablettakenoncedaily ApprovedfortreatmentofGT1or
GT3infectionwhenusedwith
sofosbuvir,(cid:3)/(cid:4)ribavirin,for12wk
Elbasvir(50)-grazoprevir(100) EBR-GZR Zepatier Onetablettakenoncedaily ApprovedfortreatmentofGT1or
GT4infection,(cid:3)/(cid:4)ribavirin,for
12-wkdurationorfor16-wk
durationforGT1ainfection,
treatment-experiencedpatients D
withNS5Aresistance-associated o
w
variants,andGT4-infection
n
treatment-experiencedpatients lo
Ledipasvir(90)-sofosbuvir(400) LDV-SOF Harvoni Onetablettakenoncedaily ApprovedfortreatmentofGT1,GT4, a
d
GT5,andGT6infection,(cid:3)/(cid:4) e
ribavirin,for12wk,orfor24wkfor d

patientswithGT1ainfectionwith fr
o
compensatedcirrhosis m
Ombitasvir(12.5)-paritaprevir(75) PrO Technivie Twoombitasvir-paritaprevir- ApprovedfortreatmentofGT4,(cid:3)/(cid:4) h
ritonavirtabletstakenonce ribavirin,for12wk,including tt
p
daily(morning) patientswithcompensated :
/
cirrhosis /c
m
Ombitasvir(12.5)-paritaprevir PrOD ViekiraPak Twoombitasvir-paritaprevir- ApprovedfortreatmentofGT1b
r
(75)-ri- ritonavirtabletstakenonce infection,andGT1ainfectionwhen .a
tonavir(50)(cid:3)dasabuvir(250) daily(morning),one usedwithribavirin,for12wk;for s
m
dasabuvirtablettaken patientswithGT1ainfectionand .
o
twicedaily compensatedcirrhosis,treatment r
g
shouldbeextendedto24wk /

Ombitasvir(8.33)-paritaprevir(50)- PrOD ViekiraXR Threefixed-dosecombination ApprovedfortreatmentofGT1b o
n
dasabuvir(200)-ritonavir(33.33) tabletsoncedaily infection,andGT1ainfectionwhen
O
usedwithribavirin,for12wk;for c
patientswithGT1ainfectionand to
b
compensatedcirrhosis,treatment
e
shouldbeextendedto24wk r
1
Simeprevir(150) SMV Olysio Onecapsuletakenoncedaily ApprovedfortreatmentofGT1 9
infectionwhenusedwith ,
2
sofosbuvirfor12-wkduration 0
Sofosbuvir(400) SOF Sovaldi Onetablettakenoncedaily ApprovedfortreatmentofGT1,GT2, 1
6
GT3,orGT4infectionwhen b
combinedwithotherantiviral y

medications. C
O
Sofosbuvir(400)-velpatasvir(100) SOF-VEL Epclusa Onetablettakenoncedaily ApprovedfortreatmentofGT1,GT2,
R
GT3,GT4,GT5,orGT6infection, N
withorwithoutcirrhosis E
(compensated),for12wk;forthose L
L
withadvancedcirrhosis
U
(decompensated),approvedforuse N
withribavirinfor12wk IV
E
aShownareIFN-freetreatmentregimensforchronichepatitisCcurrentlyapprovedbytheFDAasofJuly2016.GT,genotype.
R
S
I
T
Y
infectionformorethanadecadecanoccasionallyclearHCVontheirown);second,to
selectthetreatmentregimenanddeterminetheoptimaldurationoftreatment.Ther-
apeuticregimenselectiondependsupontheHCVgenotypeandsubgenotype,andfor
someregimens,resistancetoDAAsandthebaselineHCVloadmustalsobeconsidered.
Inthissection,wediscussthetestsusedtoassessthesefactorsandthedatasupporting
theserecommendations.
HCVGenotype
There are seven genotypes of HCV, numbered in the order in which they were
discovered, and these distinct genotypes may differ in their genetic sequences by
(cid:2)30%(24,25).Eachgenotypehasmanysubtypes,identifiedbyaletter,alsointhe
order of discovery. People can be infected with more than one HCV genotype,
January2017 Volume30 Issue1 cmr.asm.org 25
Wilsonetal. ClinicalMicrobiologyReviews
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FIG1TimingoflaboratorytestingfortreatmentofhepatitisC.Theschematicshowsthetimingofhost,virus,andsafety /c
laboratory testing prior to, during, and following combination DAA therapy for the treatment of chronic hepatitis C. m
Regimen-specifictestingiscolor-codedaccordingtoregimen.Abbreviations:EOT,endoftherapy;HIV,humanimmuno- r.
a
deficiencyvirus;RAV,resistance-associatedvariant;CBC,completebloodcount;GFR,glomerularfiltrationrate;LFT,liver s
functiontesting;Hgb,hemoglobin.*,indicatedifpatientiscirrhotic;(cid:3),repeatasclinicallyindicated. m
.
o
r
g
/

o
knownasmixedinfection,whichoccursinupto25%ofthosepersonsinfectedvia n

O
bloodtransfusionsorintravenousdruguse,exposureswhichcarrythehighestrisk
c
(24,26).Patientsmayalsobeinfectedwithrecombinantinfections,mostcommonly to
b
subgenotype 2k/1b infections in Georgia and subgenotype 2b/1a infections in the e
r
United States (25). 1
9
HCV genotype may predict disease progression, with genotype 3 infection being ,

2
associated with accelerated fibrosis (27, 28) and, in the era of IFN-based therapy, 0
1
treatment response as well. Dual therapy with pegIFN-RBV cured only 20 to 50% of 6

patientswithgenotype1or4infection,comparedto75to90%ofthoseinfectedwith b
y
genotypes2and3(29–31).DAA-basedregimenshavebecomethestandardofcarefor C
the vast majority of patients; even so, knowledge of genotype, and in some cases O
R
subgenotype,remainsanintegralpartofselectingthemostappropriateDAAregimen N
and duration of treatment. Whether this will change with the approval of the first E
L
pangenotypiccombinationDAAregimen,SOF-VEL,remainstobeseen. L

HCVgenotypeandsubtypetestingisavailablecommercially,althoughthesetests U
N
differ in their approach. The Versant HCV Genotype INNO-LiPA 2.0 assay (Siemens I
V
Healthcare Diagnostics) relies upon a reverse hybridization line probe. The Trugene E
HCV 5=NC (Visible Genetics, Inc.) and M2000 RealTime HCV Genotype 2.0 (Abbott R
S
Laboratories) (FDA approved for HCV genotyping) assays use direct sequencing to IT
Y
differentiategenotypes.Allthreeteststendtobereproducibleandhavehighdegrees
of concordance (32), but in some cases, including cases of mixed infections, non-
genotype 1 subtypes, and recombinant infections, further discrimination with addi-
tional tests can be required (33–36). Most clinical trials, if they report the testing
method,haveusedtheSiemensINNO-LiPA2.0assayforgenotypedeterminations,but
either method is reliable for distinguishing genotypes and subgenotypes in the ma-
jorityofpatients.
Throughout the world, the distribution of HCV genotypes and risk factors for
exposurevary.HCVgenotype1isthepredominantgenotypeintheAmericas,Asia,and
Europe(37),withmostpeoplebeinginfectedwithoneoftwosubgenotypes:subgeno-
types1aand1b.AllFDA-approvedDAAtherapiesareactiveagainstHCVgenotype1.
In initial studies of LDV-SOF (5) and in some studies of EBR-GZR-based (38) and
January2017 Volume30 Issue1 cmr.asm.org 26
ClinicalLaboratoryTestingandDAATherapyforHCV ClinicalMicrobiologyReviews
DCV-based(39)regimens,theauthorsreportedhighresponserates((cid:2)95%)regardless
of the subgenotype. Studies of SOF (40), DCV (41), ombitasvir-paritaprevir-dasabuvir-
ritonavir(PrOD)(6,42),andEBR-GZR(43),however,havereporteddifferencesinactivity
againstsubgenotypes1aand1b,withthemajorityofstudiesreportinglowerresponse
ratesinpatientsinfectedwithgenotype1a(6,41–43).Itisimportanttoidentifypatients
with HCV subgenotype 1a infection, as treatment outcomes may be improved by
increasing the duration of therapy and/or adding RBV (PrOD [6] and EBR-GZR [44]),
neitherofwhichisrequiredfortreatmentofpatientswithgenotype1binfection(42,
45).Patientswithgenotype1binfection,incontrast,tendtodolesswellthangenotype
1a-infectedpatientswhentreatedwithonlySOF-RBV(40,46).Thiscombinationisno D
o
longer recommended for the treatment of any genotype 1-infected patients where w
other, more effective DAA regimens are available. In some developing countries, n
lo
however, dual therapy with SOF-RBV remains the first-line regimen for pegIFN- a
d
ineligiblepatientsforinfectionwithallgenotypes,especiallywheregenericsofosbuvir e
d
isavailable(47).
f
r
Genotypes 2 and 3, as discussed above, were historically easier to treat with o
m
pegIFN-RBV.ManyDAAagents,however,lackinvitroactivityagainstthesegenotypes,
h
with reduced clinical efficacy even when combined with a pangenotypic backbone, tt
p
suchasSOF.Forinfectionwithgenotype2,thecombinationofSOFandRBV(46,48, :/
/
c
49) and/or DCV (50, 51) is highly effective, with cure rates of (cid:5)90 to 100% (48). For m
patientsinfectedwithHCVgenotype3,recommendedtreatmentregimensaresimilar r.
a
to those for genotype 2 infection, with evidence to support treatment with SOF s
m
combinedwithRBV(49),DCV(52),andevenpegIFN-RBV(53).Limiteddatahavealso .
o
suggestedthatwhilethe50%effectiveconcentration(EC50)ofLDVisgreatlyincreased rg
/
ingenotype3infection,LDV-SOFwithRBVmayalsotreatHCVinthosewithgenotype o
n
3infection(54),withtheadvantageofdecreaseddurationandadverseeffects,butthe
O
findings of this one small study have yet to be replicated, and this regimen is not c
t
recommendedbyguidelinesofanymajorprofessionalsociety,althoughitisoccasion- o
b
allyrecommendedbasedonformularyoravailabilityinselectedinstitutions.ManyDAA e
r

regimenshavedemonstratedefficacyingenotype4infection,includingLDV-SOF(55), 1
9
ombitasvir-paritaprevir(PrO)withorwithoutRBV(56),andthecombinationofSOF-RBV ,
2
(57, 58). For genotype 5 and 6 infections, LDV-SOF has shown high efficacy in small 0
1
clinical trials (54, 59), but these data are limited. Table 1 summarizes the currently 6

approved regimens in the United States and Europe and their spectrum of genotype b
y

coverage. C
O
R
ViralLoad
N
Baseline HCV RNA load. HCV RNA testing is required prior to the initiation of E
L
treatmenttoconfirmchronicHCVinfectionand,overthecourseoftreatment,toassess L

treatmentresponse.ThereareseveralapprovedtestsforHCVRNAloadquantification. U
N
Inclinicaltrials,thepreferredtesthasbeeneithertheCobasTaqManHCV,version2.0, I
V
test(CTM2;RocheMolecularSystems),withalowerlimitofquantification(LLOQ)of25 E
R
IU/ml,ortheAbbottRealTimeHCVassay(ART),withaLLOQof12IU/ml,bothofwhich
S
areFDAapproved.Somecomparativeanalyseshaveshownthatthesetestswerehighly IT
correlativeandhavecomparablelinearityforHCVRNAquantificationacrossallgeno- Y
types(60,61).However,recenttestinghasraisedquestionsaboutthecomparabilityof
theresultsofthevarioustestsusedinclinicalpractice,includingCTM2,ART,andthe
newAptimaHCVQuantDxassay(Hologic,Inc.),availableinEuropebutnotcurrently
FDA approved for confirmation of HCV infection, with measurements between tests
varyingwidely,from1.3-to1.8-foldforgenotype1samples(62).Nucleicacidtestsmay
use different methodologies (i.e., PCR-based assays, like ART and CTM2, versus signal
amplification-based branched-DNA-based assays, like the FDA-approved Versant HCV
3.0 assay [Siemens Healthcare Diagnostics]), and therefore, patients should be moni-
tored by using the same test over the course of therapy. Even when patients are
monitored by using the same HCV RNA assay, the HCV set point remains relatively
stablealthoughlesssothantheHIVloadsetpoint.Oneanalysisshowedthat15%of
January2017 Volume30 Issue1 cmr.asm.org 27
Wilsonetal. ClinicalMicrobiologyReviews
those with chronic HCV infection not receiving antiviral therapy had HCV RNA levels
thatvariedbyalogormoreinconsecutivemeasurementsovertime(comparedwith
only4%ofthosewithuntreatedHIVinfection),and44%ofHCV-infectedpatientshad
anHCVRNAloadthatvariedbyatleast0.5logs(63).
Many studies have looked at treatment responses to DAAs stratified by pretreat-
mentHCVRNAmeasurements,asthishadbeenshowntopredicttreatmentresponses
toIFN-basedtherapies(64),buttheexactHCVRNAcutoffvaries.Inaposthocanalysis
of the ION-3 trial restricted to patients with an HCV RNA load of (cid:6)6,000,000 IU/ml,
treatmentresponseratesafter8or12weekswithLDV-SOFweresimilar(65),andthe
LDV-SOFprescribinginformationrecommendsthat8weeksoftherapycanbeconsid- D
o
ered for treatment-naive patients without cirrhosis and with an HCV RNA load of w
(cid:6)6,000,000IU/ml(66).Aseparateanalysisofpublicallyavailabledata(coauthoredby n
lo
oneoftheauthorsofthisreview)foundnoevidencetosupportacutoffof6,000,000 a
d
IU/ml(67).Whilethisspecificrecommendationremainsindispute,otherstudieshave e
d
also suggested that the baseline viral load impacts DAA therapy for HCV infection. A
f
r
lowerproposedHCVRNAloadcutoffof(cid:2)800,000IU/mlhasbeenshowntopredictSVR o
m
rates following 24 weeks of SOF-RBV therapy (40) and 12 weeks of EBR-GZR therapy
h
(43), and an HCV RNA level of (cid:2)2,000,000 IU/ml was shown to predict a favorable tt
p
responseinonestudyofpatientscoinfectedwithHCVandHIVwhoweretreatedwith :/
/
c
DCV-SOFfor8weeks(50). m
On-treatment monitoring of HCV RNA load. Current AASLD-IDSA guidelines rec- r.
a
ommendarepeatHCVRNAmeasurementafter4weeksofcombinationDAAtherapy s
m
(68),asameasureofadherenceonly.Ofpatientswithoutadvancedcirrhosis,mosthave .
o
undetectable HCV RNA by week 4 of therapy, while patients with cirrhosis may r
g
experienceaslowerviraldecline.Theguidelinesgoontostateexplicitlythatthereare /
o
nodatatosupportcessationoftherapyifapatienthasdetectableHCVRNAatweek n

O
4,unlessitrepresentsa(cid:2)10-foldincreasefromthebaselinemeasurement(basedon
c
t
expert opinion) (68). In a large study of patients treated through the Veterans Affairs o
b
Healthcare System, Backus et al. reported that across SOF-based regimens (including e
r
SOF-RBV,SOF-SMV,andSOF-SMV-RBV),patientswhoachievedanHCVRNAloadlower 1
9
than the LLOQ by week 4 of therapy were more likely to go on to achieve SVR, but ,

2
importantly, this analysis included patients who had discontinued therapy prior to 0
1
week4foravarietyofreasons(69).SomeadvocateforHCVRNAtestingattheendof 6

therapy,inordertodifferentiateviralbreakthroughfromrelapseorreinfection,butan b
y
intensive analysis of patients receiving a variety of combination DAA therapies of C
O
variousdurationsdemonstratedthatpatientswhowentontodevelopSVRoccasionally
R
hadresidualviremiadetectedupuntiltheendoftherapy(70),furtherreinforcingthe N
limitationsofHCVRNAmonitoringinpredictingtherapeuticresponseandinguiding E
L
treatmentdecisions. L

U
Measurement of the HCV RNA load at least 12 weeks after the completion of
N
therapy,orSVR12,isusedasasurrogateendpointforcureofHCVinfection.Previously, IV
during the era of IFN-based therapies, SVR was assessed 24 weeks following the E
R
completion of therapy (71), but relapse after 12 weeks following the completion of S
I
combination DAA-based therapy is rare, and the majority of clinical trials now use T
Y
SVR12astheprimaryendpoint(72).MonitoringofHCVRNAlevelsmorethan12weeks
afterthecompletionoftherapyisindicatedonlyifthereisconcernthatapatientmay
have been reinfected; current guidelines do not recommend routine monitoring for
relapse after SVR12 is achieved. In contrast, the HCV antibody test often remains
reactive after successful treatment but these antibodies are not protective against
reinfection.
ResistanceTesting
With high levels of viral replication and an error-prone polymerase, HCV exhibits
broad genetic diversity in chronic infection (73), and some amino acid substitutions
exhibit reduced susceptibility to DAAs in vitro. The presence of resistance-associated
variants (RAVs), also known as resistance-associated polymorphisms or substitutions,
January2017 Volume30 Issue1 cmr.asm.org 28
ClinicalLaboratoryTestingandDAATherapyforHCV ClinicalMicrobiologyReviews
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FIG 2 NS5A inhibitors and NS5A resistance-associated variants (77–80, 87). Numbers denote fold changes in reduced r
g
susceptibilitytotheNS5Ainhibitorfortheindicatedaminoacidsubstitution,roundedtothenearestinteger. /

o
n

O
c
has been shown to predict treatment failure of some DAA-containing regimens (74); to
b
AASLD-IDSAguidelinesandtheFDArecommendthatRAVsshouldbeassessedpriorto e
r
therapyinsometreatmentsituationswhenusingselectedagents(particularlyEBRand, 1
9
ifdesired,DCV)orinthosewhohavepreviouslyfailedDAA-basedtherapies.Whilethe ,

2
potencyofDAAsmaydependuponthegeneticbarriertoresistanceandviralsuscep- 0
1
tibility,theroleofresistancetestinginpredictingtreatmentoutcomesisfarfromclear. 6

There are different methods of testing for RAVs. Some studies and the majority of b
y
clinicallaboratorieshaveusedpopulation-basedsequencing,whichdetectsonlypoly- C
morphisms comprising more than 15 to 25% of the patient’s viral population. In O
R
contrast,clonalsequencinganddeepsequencingcandetectvariantsthatarepresent N
inaslittleas5%and(cid:6)1%ofthepopulation,respectively,dependingonthevolume E
L
ofthesample(73).ItremainsunclearwhethertheproportionofRAVsintheoverallviral L

population is important for treatment outcomes. Qualitative tests for the presence of U
N
RAVs, rather than a quantitative measure of the proportion of the patient’s viral I
V
population comprised of individual RAVs, are available from a variety of clinical E
R
laboratories,includingMonogramBiosciencesandQuestDiagnostics.
S
OneRAVassociatedwithanadverseeffectonthetreatmentresponseistheQ80K IT
Y
polymorphism in NS3, present in up to half of those individuals with genotype 1a
infection at baseline (75), which predicts higher rates of virologic failure in those
receivingSMVincombinationwithIFN-RBV.However,itappearsthisreducedsuscep-
tibilitymaybeovercomebyusingcombinationDAAtherapyforasufficientduration:
in patients receiving standard 12-week, rather than 8-week, courses of combination
therapy with SMV-SOF, the presence of the Q80K polymorphism at baseline did not
alteroutcomes(76).
Polymorphisms in the NS5A region of HCV also confer reduced susceptibility to
NS5A inhibitors in vitro (77–80). Selected NS5A RAVs and the corresponding fold
changesforNS5AinhibitorsareshowninFig.2.ApooledanalysisofdatafromLDV-SOF
phase3clinicaltrialsfoundbaselineNS5ARAVsin(cid:5)16%ofallpatients,butreduced
ratesofSVR(by(cid:5)30%)wereobservedonlyinthosepatientswithpriorHCVtreatment
January2017 Volume30 Issue1 cmr.asm.org 29
Wilsonetal. ClinicalMicrobiologyReviews
experience and NS5A RAVs conferring a (cid:2)100-fold reduction in susceptibility (81). A
subanalysisofdatafromEBR-GZRphase2and3clinicaltrialsshowedreducedefficacy
(by(cid:5)10%)inpatientswithpreexistingNS5ARAVsconferring5-fold-decreasedsuscep-
tibilitytoEBRwhendetectedbypopulation-basedsequencing(38,82).Asubsequent
analysis, this time in patients undergoing retreatment after failing initial combination
DAAtherapywithLDV-SOF,foundthatthoseindividualswithNS5ARAVsidentifiedby
deepsequencing,amoresensitivetechniquethanpopulation-basedsequencing,were
less likely to respond to longer courses of LDV-SOF plus RBV (83), with only 60%
achievingSVR,asopposedto100%ofthosewithoutNS5ARAVsdetected.Ourgroup
has shown that patients with baseline high-level NS5A RAVs ((cid:2)25-fold-reduced sus- D
o
ceptibility to LDV), detected by deep sequencing, were less likely to respond to w
short-durationcombinationDAA-basedtherapies(4weeksoftherapywiththreeorfour n
lo
DAAs) (84). In contrast, we found that similar patients treated with combination DAA a
d
therapycontainingLDV-SOFforatleast6weeks(85)orretreatedafterfailingprevious e
d
short-duration therapy with LDV-SOF for at least 12 weeks (86) achieved SVR at the
f
r
same frequency as patients without NS5A resistance. Another study of SOF-based o
m
therapy, this time SOF-VEL, showed that reduced efficacy was genotype dependent,
h
with marginally lower rates of viral decline in patients with genotype 1a and 3 tt
p
infectionsandRAVs,whilenosuchreductionwasnoteddespitethepresenceofRAVs :
/
/
inpatientswithgenotype1b,2,and4infections(87).Otherstudiesalsosuggestthat cm
increasingthedurationoftherapy,and/oraddingRBV,canovercomethepresenceof r
.
a
baselineNS5ARAVs(81,88). s
m
WhilesomeaminoacidsubstitutionswithintheNS5Bgeneassociatedwithreduced
.
o
susceptibility to SOF and DSV have been reported, their clinical significance remains r
g
unknown. Patients who have failed SOF-based regimens have been reported to have /
o
S282TandV321Isubstitutions(86,89),butinvitrodatasuggestthatthesesubstitutions n

O
confer only slightly reduced ((cid:6)5-fold) susceptibility to SOF (77) and that the S282T
c
substitution may be present only transiently, possibly because this variant exhibits to
b
reducedviralfitnessandisrapidlyreplacedwiththewild-typevirus(89). e
r
Interestingly, NS5A RAVs have been shown to be remarkably stable, persisting for 1
9
monthstoyearsintheabsenceofselectivepressure(90),suggestingthatthesubsti- ,

2
tutionsthatconferreducedsusceptibilitytoNS5Ainhibitorsreplicateandpersistwith 0
1
afitnesssimilartothoseofwild-typeviruses.Incontrast,theprevalenceofNS3RAVs 6

followingtherapywithanNS3inhibitordeclinedovertime(86,90),astheviralvariants b
y
carrying resistance-associated substitutions are outcompeted by variants with wild- C
type NS3 sequences. Current guidelines recommend pretreatment evaluation for the O
R
presenceofNS5ARAVsinapatientwithgenotype1aHCVinfectioniftreatmentwith N
EBR or DCV (if the patient is cirrhotic) is being considered. If NS5A RAVs conferring E
L
(cid:2)5-fold-reducedsusceptibilitytoEBRareidentified(inparticularatpositionM28,Q30, L

L31, or Y93), current recommendations are that the duration of EBR-GZR treatment U
N
should be extended to 16 weeks and that RBV should be added (91). Any other RAV I
V
testing, including testing for substitutions within NS5B, has yet to be supported by E
R
clinicalstudies.
S
I
T
Y
HOSTFACTORS
Aswithvirustesting,itisimportanttoevaluateseveralhostfactorspriortothestart
ofDAAtherapyforHCVinfection.Somecomorbidconditions,likerenaldysfunctionor
anemia,orinfectionsmaycomplicateHCVtreatmentandinfluencetheselectionofa
therapeutic regimen. Other factors, including host genotype and fibrosis staging, can
affect treatment outcomes. In this section, we discuss the tests used to assess these
factorsandthedatasupportingtheserecommendations.
IL28BGenotype
Polymorphisms within or near the IL28B gene have been strongly associated
with prediction of treatment responses to IFN-based regimens (92–94), likely me-
diated by the levels of intrahepatic expression of IFN-stimulated genes (ISGs) (95).
January2017 Volume30 Issue1 cmr.asm.org 30
ClinicalLaboratoryTestingandDAATherapyforHCV ClinicalMicrobiologyReviews
TheextenttowhichIL28BpolymorphismsremainrelevantintheeraofDAA-based
therapiesisunclear.AreportbyBackusetal.onalargecohortofveteranstreated
for HCV infection found that patients with the favorable IL28B (rs12979860) geno-
type CC were more likely to respond to SOF plus pegIFN-RBV; no such difference
was shown for combination DAA treatment with SMV-SOF (69). In a small study of
IFN-free therapy with investigational agents, a favorable CC IL28B genotype was
associatedwithmorerapidhepatitisCvirusdecline(96),butthisearlyresponsedid
not translate into higher SVR rates. The IL28B genotype of study participants has
beenreportedinmultipletrialsofcombinationDAAtherapy,includingLDV-SOF(4,
5, 8, 65), PrOD (7, 42, 45), DCV (50, 51), and EBR-GZR (38, 43, 97), without a D
o
significant impact on SVR. Commercial sequence-based testing is available, and w
n
becauseoftheeffectoftheIL28BgenotypeontheresponsetoIFN-basedtherapies, lo
the IL28B genotype should be evaluated for all patients receiving IFN-based a
d
therapies. This evaluation is not currently recommended for those receiving com- e
d
bination DAA-based IFN-free therapies; IL28B genotypes may play a role in the f
r
o
response to short-course (6 weeks or less) combination DAA-based therapies, but m
this would require further investigation. h
t
t
p
IFNL4Genotype :/
/
c
TheIFNlambda-4gene(IFNL4-∆G)isanexonicdeletionthatiscloselyassociated m
r
and in close linkage disequilibrium with the IL28B genotype. Our group has .a
s
identified a possible association with reduced spontaneous clearance of HCV (98), m
a reduced response to IFN-based regimens (99), and a slower early HCV decay in .o
r
responsetoSOF-RBVtherapy(100).Thispolymorphismhasbeenrarelyreportedin g
/

clinicaltrialsofcombinationDAA-based,IFN-freetherapy,andwhenreported,ithas o
n
not been associated with different rates of viral clearance (85). As such, testing for O
the presence of IFNL4-∆G is not routinely recommended in clinical practice and is c
t
o
not commercially available at this time. Allele-specific probes are available com- b
e
mercially from Applied Biosystems and can be used with PCR-based sequencing r

1
systems, but these probes are not covered under U.S. Clinical Laboratory Improve- 9
,
ment Amendments (CLIA) regulations. 2
0
1
ViralCoinfection 6

b
Because of shared routes of transmission, chronic HCV infection is common in y

C
patientswithHIVinfection,andpatientswithHCVinfectionareroutinelyscreened
O
for HIV (101). Individuals coinfected with HIV and HCV have been shown to have R
N
worseoutcomesthanpersonswithHCVinfectionalone,withmorerapidandmore
E
frequentdevelopmentofcirrhosisandhepatocellularcarcinoma(HCC)(102).While L
L
patients coinfected with HIV and HCV are less likely to respond to immune-based U
HCV therapies, DAA-based therapies appear to maintain high SVR rates similar to N
I
V
thoseobservedforHIV-negativeHCV-infectedpatients(103).Whiletheselectionof
E
a treatment regimen for HCV infection requires thoughtful consideration of the R
S
potential interactions with antiretroviral therapy, HCV treatment outcomes do not I
T
appear to depend upon the patient’s HIV status, regardless of the selected DAA Y
regimen (50, 97, 104).
Similarly,hepatitisBvirus(HBV)andHCValsosharecommonroutesoftransmission,
and patients chronically coinfected with HBV and HCV have accelerated liver fibrosis
and are at increased risk for hepatic decompensation and HCC (105). IFN-based HCV
therapies also have activity against HBV, and patients with inactive or resolved HBV
infectionwereatriskforHBVreactivationwithIFN-basedregimens;thishasalsobeen
reported, albeit infrequently, in patients treated with DAA-only regimens (106). Cur-
rently, AASLD-IDSA guidelines and other professional organizations recommend
screening of patients for HBV prior to DAA-based therapy with a hepatitis B virus
surfaceantigentest(22),butthereisnoconsensusonthebestwaytomonitorpatients
forHBVreactivationduringoraftertreatment.
January2017 Volume30 Issue1 cmr.asm.org 31
Wilsonetal. ClinicalMicrobiologyReviews
FibrosisStaging
Although DAAs have demonstrated nearly universal efficacy regardless of most
baseline demographic characteristics, fibrosis staging remains an important part of
pretreatment evaluation, as treatment outcomes continue to be impacted by the
degreeofliverfibrosis.ThismaybedueinparttotheimpactofhepaticfibrosisonHCV
clearanceanddrugdelivery.Individualswithadvancedhepaticfibrosismayexperience
reduced drug delivery due to venous shunting, limited drug uptake secondary to
fibroticchanges,decreaseddrugmetabolismfromreducedliverfunction,andimpaired
immunesignalingpathways(107).However,giventhedirectantiviralactivityofDAAs,
thesignificanceofthehostimmuneresponseinachievingSVRremainsunclear.Most D
o
clinical trials have divided patients into two categories: no cirrhosis (stage 0 to 3 w
n
fibrosis)andcompensatedcirrhosis(4,5,108).DuetodisparitiesinSVRsbetweenthese lo
groups,thepresenceofcirrhosismaychangetherecommendeddurationoftreatment a
d
or warrant the addition of RBV (109). In addition, from a clinical perspective, it is e
d
important to be aware if a patient has cirrhosis or advanced fibrosis (stage 3 fibrosis) f
r
o
because surveillance for hepatocellular carcinoma and esophageal varices are recom- m
mended,regardlessoftreatmentoutcome.Itisalsoimportanttodistinguishbetween h
t
patients with compensated and those with decompensated cirrhosis, as this may tp
:
change the recommended treatment. While patients with advanced fibrosis have a //
c
clearbenefitfromachievingSVR,itremainsunknownwhetheroutcomesvaryforthose m
r
treated at early, as opposed to moderate, stages of fibrosis. At this point, the main .a
s
reason for distinguishing between early and moderate stages of fibrosis is to identify m
whichpatientsmeetinsurancestandardsfortreatment,asmanyinsurancecompanies .o
r
intheUnitedStatesrestrictaccesstoDAAs,reservingthemforpatientswithmoderate g
/

oradvancedfibrosis(110).Theevaluationsmostcommonlyusedfortheevaluationof o
n
hepaticfibrosisareshowninTable2. O
Liverbiopsy.The“goldstandard”forstagingofliverfibrosisisliverbiopsy.Patients c
t
o
undergo a percutaneous, transvenous, or surgical/laparoscopic procedure to obtain a b
e
needlecorebiopsyspecimen.Optimaloutcomeshavebeenidentifiedwhentheneedle r

1
gaugeis16,thecorelengthis3cmafterfixation,andthreeseparatecoresaretaken 9
,
toreducesamplingerrors(111).Thespecimensarefixedandparaffinembeddedand 2
0
undergo staining with hematoxylin and eosin (H&E) to determine the degree and 1
6
extent of hepatic inflammation and the presence of disease-specific abnormal cells.
b
SpecimensarealsostainedwithMasson’strichromeinordertodeterminetheextent y

C
and nature of fibrosis. Scoring systems have been developed to help standardize the O
classificationofthedegreeofhepaticinflammationandfibrosis. R
N
TheIshak(modifiedKnodell)scoringsystemwasdevelopedin1995inanattempt E
togradetheintensityofhepaticnecroinflammatoryactivityandstagehepaticfibrosis L
L
andarchitecturalalteration.Forgrading,scoresaregivenforpathologicalassessment U
N
of the degree of periportal or periseptal interface hepatitis (piecemeal necrosis),
I
V
confluent necrosis, focal (spotty) lytic necrosis, apoptosis and focal inflammation, as E
wellasportalinflammation.Forstaging,architecturalchanges,fibrosis,andcirrhosisare R
S
takenintoaccountbyassessingfibrousexpansionwithinportalareas,thepresenceand I
T
extentofseptation,andbridging.Thestageoffibrosisrangesfrom0to6,withstage Y
5or6indicatingcirrhosis(112).
TheMetavirscoringsystemwasspecificallydesignedandvalidatedforindividuals
with chronic HCV infection. By using this system, pathologists assess the degree of
histologicalactivitythroughassessmentofthenumberofnecroinflammatoryfociper
lobule (focal lobular necrosis) and alteration of the periportal plate in portal tracts
(piecemealnecrosis).HistologicalactivityisscoredasA0toA3(whereA0isnoactivity
andA3issevereactivity).Afibrosisscoreisdeterminedbasedontheextentofportal
fibrosisandthedegreeofseptationandisreportedasascoreofF0toF4(whereF0is
nofibrosisandF4iscirrhosis)(113).
Althoughliverbiopsyisconsideredthegoldstandard,thisstatusisbeingcalled
into question, as this methodology has many limitations and challenges. Because
January2017 Volume30 Issue1 cmr.asm.org 32