Hepatitis C virus genotypes and associated risk factors in the state of Pará, Northern Brazil

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Hepatitis

C

virus

genotypes

and

associated

risk

factors

in

the

state

of

Pará,

Northern

Brazil

Geison

Luiz

Costa

de

Castro

_,

_Ednelza

_da

_Grac¸a

_Silva

_Amoras

_,

Mauro

Sérgio

Moura

de

Araújo

_,

_Simone

_Regina

_Souza

_da

_Silva

_Conde

_,

Antonio

Carlos

R.

Vallinoto

a_{FederalUniversityofPará(UFPA),BiologicalSciencesInstitute,VirologyLaboratory,Belem,PA,Brazil} b_{FederalUniversityofPará(UFPA),InstituteofHealthSciences,SchoolofMedicine,Belem,PA,Brazil} c_{JoãodeBarrosBarretoUniversityHospital,Belem,PA,Brazil}

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Articlehistory:

Received1March2020 Accepted22June2020 Availableonline28July2020

Keywords: HCV Genotypes Subgenotypes Pará Brazil

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Background:Despitetheemergenceofmoreeffectivetherapies,hepatitisCvirus(HCV) infec-tionremainsaseriouspublichealth problematthegloballevel.Currently,thisvirusis classifiedintosevengenotypesand67subgenotypes,whichinturnaredistributed hetero-geneouslyinBrazilandworldwide.Studieshaveshownthatthisgeneticdivergenceresults indifferencesintheprogressionofchronicdiseaseassociatedwithHCVinfectionandits treatment.

Objective:TheaimofthisstudywastoreportthefrequencyofHCVgenotypesinthestateof Pará,NorthernBrazil,andtoassesstheassociationbetweengenotypeanddifferentclinical andlaboratorycharacteristics,aswellasriskfactorsforinfection.

Method:Datafrom85medicalrecordsofuntreatedpatientswhohadchronichepatitisC infectionwereanalyzed;thepatientswereevaluatedattwohospitalsinBelem,Pará,Brazil.

Results:Circulationofgenotypes1and3wasdetected,withahigherprevalenceofgenotype 1(75.3%)thangenotype3(24.7%).Inaddition,therewasapredominanceofsubgenotype 1b(60.34%)comparedto1a(20.69%)and3a(18.97%).Reuseofneedlesand/orglasssyringes wassignificantlyassociatedwithinfectionbyHCVgenotype1thangenotype3;however, thesmallnumberofpatientsinfectedwithgenotype3mayhavebiasedtheresults.No associationsbetweengenotypeandtheevaluatedclinicalandlaboratorycharacteristics wereobserved.

Conclusion:ThisstudyreinforcesthedifferencesinthedistributionofHCVgenotypesin BrazilandshowednoassociationbetweenHCVgenotypeandprogressionofchronic hep-atitisCinthestudiedgroup.

©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddress:vallinoto@ufpa.br(A.C.Vallinoto).

https://doi.org/10.1016/j.bjid.2020.06.010

1413-8670/©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Hepatitis C virus (HCV) infection has a significant global

impactonpublichealth.Itaffectsapproximately71million

peoplearoundtheworldandhascausedthedeathofmore

than 400,000 carriers per year due to complications from

theinfection,includinghepaticcirrhosisandhepatocellular carcinoma.1,2

HCVbelongs tothefamily Flaviviridae andhas a

single-strandedRNA genome withapproximately 9.6kbin length

that encodes a polyprotein of around 3000 amino acid

residues,which,inturn,issubsequentlycleavedtoform struc-turalandnonstructuralviralproteins.3

HCV viral RNA polymerase has a high error propensity

duringsuccessivereplications, resulting in high nucleotide substitution rates in the RNA chainsof the viral particles produced and, consequently, in wide genotypic variability.

DuetogenomicsequencedifferencesHCVisclassifiedinto

sevengenotypesand67subgenotypeswithdifferentdegrees ofdivergenceatthegenomelevel.4–6

DifferentHCVgenotypesshowcharacteristicdistributions indifferentregionsofthe world.Genotype1hasthe high-estglobalprevalence,followedbygenotypes3,4,and2.The remainingcasesofhepatitisC,approximately5%,arecaused bygenotypes5and6.Inaddition,genotype7wasisolatedin CanadafromasinglepatientfromCongo.7–9

SeveralstudieshaveshownthatHCVgenotypeshave

con-siderablerelevance inthe clinical managementofpatients withchronicviralinfectionandclinicalevolutionofthe dis-ease,suchasriskofprogressiontodecompensatedcirrhosis10

andoccurrenceofcomorbidities,suchashepaticsteatosis,11

aswellasdistictefficacyofdifferenttreatmentregimens.12,13

ThesedivergencesmakeHCVgenotypeanalysisanimportant

toolthatshouldbeconsideredinthecontextofchronicHCV infection.Therefore,theaimofthisstudywastoreportthe frequencyofHCVgenotypesinthestateofPará,NorthRegion ofBrazil,andtoassesstheassociationbetweenHCVgenotype withdifferentclinicalandlaboratorycharacteristicsandrisk factorsforchronicinfection.

Materials

and

methods

Studypopulationanddataanalysis

A total of 85 chronic HCV carriers treated at the Santa

Casa de Misericórdia do Pará Foundation and at João de

Barros Barreto University Hospital of the Federal

Univer-sityofParábetween2013and 2016were evaluated.Patient inclusion criteriawere asfollows: HCVchronic carrier

sta-tus, age over 18 years old, detectable HCV RNA, absence

of treatment during the collection period, and availability

of clinical and biochemical parameters related to

hep-atic function. Exclusion criteria were as follows: previous

diagnosis of autoimmune hepatitis, and coinfection with

hepatitis Bvirus (HBV) or humanimmunodeficiency virus

(HIV).

Ethicalaspects

This study was approved by the Research Ethics

Commit-tee ofJoãode BarrosBarretoUniversity Hospital(protocols 962.537/2015and2.165.948/2017)andbytheResearchEthics

CommitteeoftheSantaCasadeMisericórdiadoPará

Foun-dation (protocol 772.782/2014). All patients who agreed to participateinthestudysignedaninformedconsentform.

Biologicalsamples

Forthegenotypeanalysisandbiochemicaltests,venousblood fromeachpatientwascollectedintubescontainingseparator geltoobtainserum,intubescontaining ethylenediaminete-traaceticacidanticoagulant,andintubescontainingsodium citrate.

Liverbiopsysampleswereobtainedfromthepatientsafter medicalindicationforevaluationofpossiblechangesinthe liverparenchyma.Toobtainthespecimens,aTrucutneedle wasusedwithultrasoundguidance.Hepaticbiopsyspecimens wereexaminedattheDepartmentofPathology,Federal Uni-versityofPará(UFPA),usingthecriteriaforhistopathological diagnosisaccordingtotheMETAVIRscaleclassification.14

Clinicalandlaboratoryassessment

The eligible patientsunderwent clinical evaluationsat the

respective health services of each hospital and answered

questionnairesaboutriskfactorsforinfection,riskof

wors-ening, and history of liver disease. Abdominal ultrasound

procedures, upper gastrointestinal endoscopy, liver biopsy, andevaluationofbiochemicalmarkersofliverfunctionand liverdamagewerealsoperformed.Allobtainedclinicaland laboratorydatawereenteredintomedicalrecordsand subse-quentlystoredinadatabaseforanalysis.

Viralloadquantificationandgenotypedetermination

ThequantitativeanalysisofHCVviralload(VL)and

determi-nationofviralgenotypesandsubgenotypeswereperformed

attheCentralLaboratoryofthestateofPará(LACEN,forits

acronym in Portuguese). VL was determined using RT-PCR

(AMPLICORMONITOR®,RocheMolecularSystems).Theviral

genotypesandsubgenotypesweredeterminedbysequencing

the5’untranslatedregionofHCVusingtheLinearArray Hep-atitisCVirusGenotypingTest(LiPA–LineProbeAssay–Roche Diagnostics).

Statisticalanalysis

BioEstatversion5.4andSPSSversion22(SPSSInc.,Chicago, IL,USA)wereusedforstatisticalprocessingofthedata. Qual-itativeparameterswerecomparedusingthechi-squaretest, Fisher’sexacttest,andtheGtest.Initially,the

Kolmogorov-Smirnovtestwasusedtoevaluatenormalityofcontinuous

variables, which were compared using Student’s t-test or

Table1–Generaldataoftheanalyzedpatients.

Female(n=36) Male(n=49) Total(n=85)

Genotype 1 27(42.19%) 37(57.81%) 64(75.29%) 3 9(42.86%) 12(57.14%) 21(24.31%) Subgenotype(N=58) 1a 4(33.33%) 8(66.67%) 12(20.69%) 1b 15(42.85%) 20(57.15%) 35(60.34%) 3a 6(54.54%) 5(45.45%) 11(18.97%) Meanage 56.28±9.82 51.9±7.58 55.22±9.47

Table2–DataonriskfactorsforHCVacquisition.

Riskfactor N(Total) Total N(G1) G1 N(G3) G3 p-Value

Injectabledrugsb _{82 7(8.86%) 64 5(7.81%) 18 2(11.11%) 0.6402} Inhalantdrugsb _{82 8(10.13%) 64 6(9.38%) 18 2(11.11%) 0.9889} Reuseofequipmentb _{82 20(25.32%) 63 21(33.33%) 19 1(5.26%) 0.0173}c Tattoob _{83 8(10.00%) 64 5(7.94%) 19 3(15.79%) 0.3790} Bloodtransfusion Before1993a _{84 24(29.63%) 64 18(28.13%) 20 7(35%) 0.7590} After1993b _{84 9(11.11%) 64 7(10.94%) 20 3(15%) 0.6941} Surgeries Before1993a _{79 35(44.87%) 60 27(45.00%) 19 8(42.11%) 0.9652} After1993a _{82 20(25.32%) 62 15(24.19%) 20 7(35.00%) 0.5104} Hemodialysisb _{83 4(4.94%) 63 4(6.35%) 20 0 0.5678} a _{Chi-squaretest.} b _{Fisher’sexacttest.}

tothelackofanassociationbetweenthefactorsevaluated, wasestablishedwithatwo-tailedp-valuelessthan0.05.

Results

Themeanageoftheevaluatedpatientswas55.22±9.47years.

Themolecularanalysisdetectedgenotypes 1and 3butno

genotypes 2,4, 5, 6or 7. In addition, no mixedgenotypes weredetected.Genotype1wasdetectedin64(75.3%)cases, whereasgenotype3wasdetectedin21(24.7%)cases.Among

the58samplesinwhichtheHCVsubgenotypewasanalyzed,

12(20.69%)containedsubgenotype1a,35(60.34%)1b,and11 (18.97%)subgenotype3a(Table1).

Themainidentifiedrisk factor forHCV acquisitionwas

surgerybefore1993,followedbytransfusions,othersurgeries afterthatyear,and reuseofneedlesand/or glasssyringes; the latterrisk factor had a statisticallysignificant associa-tionwithviralgenotype1infection.Viralgenotype3infection wassignificantlyassociatedwithsharingneedlesand/orglass syringes (p=0.0173). Theother factors were not associated withanyofthegenotypes(Table2).

All85patientsunderwentclinicalevaluations,61(81.33%)

endoscopy, and 77 (90.59%) abdominal ultrasound. In the

context of the clinical assessment, systemic hypertension

(SAH)wasthemostfrequentclinicalfeatureintheevaluated patients,occurringin28(34.15%)of82patientswhowere

eval-uatedforcomorbidities.However,whencomparingthedata

ofpatientswithdifferentgenotypes,nosignificantdifferences wereobservedinthefrequenciesofanyofthevariables eval-uated.Liverbiopsieswereperformedin66(77.65%)patients.

Nodifferenceswereobservedbetweengenotypesneitherin

thedistributionofnecroinflammationscores(p=0.8652)nor inthedegreeofliverfibrosis(p=0.288)(Table3).

Additionally,theanalysisofbiochemicalmarkersandVL showed no statistically significant differences between the observedvaluesofpatientsinfectedwithneithergenotype1 andnorgenotype3(Table4).

Discussion

The genotypicdistributionofHCV follows patterns related to both geographic and cultural factors.8,9 _{In the present}

study,onlygenotypes1and3weredetected,andtherewas

a higherprevalenceofgenotype 1(74.7%)than genotype3

(25.3%).Infact,thestudiesperformedbyPetruzzielloetal.8

andBlachetal.9_{showedapredominanceofthesegenotypes}

inBrazil,withgenotype1presentingaprevalenceof64.8%and genotype3aprevalenceof30.2%.Additionally,thesestudies showedtheoccurrence,evenatlowfrequency,ofgenotypes2 (4.6%),4(0.2%),and5(0.1%)inthecountry,unlikewhatwas foundinthisstudy.

Although the distributions of HCV genotypes are well

establishedinBrazil,theyvarybetweenregionsandstates.

Even so, the predominance of genotype 1 over genotype

3 was also observed in several states, including Ceará,15

Pernambuco,16 _{Rio Grande do Sul,}17,18 _{Santa Catarina,}19

Amapá,Paraná,RiodeJaneiro,RondôniaandSãoPaulo,17_in

additiontostatesintheMidwestregion.17,20

InthestateofPará,prevalenceratesof65.7%and23.3% hadbeenpreviouslyobservedinpatientsforgenotypes1and

Table3–Clinicalandhistopathologicaldataofthepatients.

Clinicalcharacteristic N(Total) Total N(G1) G1 N(G3) G3 p-Value

Systemicarterialhypertensiona _{82 28(34.15%) 61 23(37.70%) 21 5(23.81%) 0.3727}

Diabetesmellitustype2b _{82 13(15.85%) 61 9(11.11%) 21 4(19.05%) 0.4606}

Hepatomegalyb _{82 10(12.20%) 62 8(12.90%) 20 2(10.00%) 1.0000} Splenomegalya _{82 14(17.07%) 62 8(12.90%) 20 14(30.00%) 0.1541} Esophagealvaricesb _{61 12(19.67%) 46 8(17.39%) 15 4(26.67%) 0.4665} Hepaticsteatosisa _{77 16(17.20%) 59 10(16.95%) 18 6(33.33%) 0.2429} Cirrhosisb _{85 16(17.98%) 64 12(18.75%) 21 4(19.05%) 0.9973} Necroinflammatoryactivityc A0 66 5(7.58%) 51 3(5.88%) 15 2(13.33%) 0.8652 A1 33(50.00%) 26(50.98%) 7(46.67%) A2 23(34.84%) 18(35.29%) 5(33.33%) A3 5(7.58%) 4(7.85%) 1(6.67%) Degreeoffibrosisc F0 66 4(6.06%) 51 2(3.92%) 15 2(13.33%) 0.2880 F1 21(31.82%) 17(33.33%) 4(26.67%) F2 19(28.79%) 18(35.29%) 2(13.33%) F3 17(25.75%) 10(19.61%) 6(40.00%) F4 5(7.58%) 4(7.85%) 1(6.67%) a _{Chi-squaretest.} b _{Fisher’sexacttest.} c _GTest.

Table4–LaboratorymarkersaccordingtoHCVgenotype.

Marker N(G1) G1 N(G3) G3 p-Value AST(U/L)a _{60 69.58±40.80 20 76.78±70.70 0.9690} ALT(U/L)a _{60 80.27±54.60 20 81.05±68.84 0.7684} GGT(U/L)a _{59 104.22±94.00 17 78.73±77.17 0.1315} FA(U/L)b _{54 118.57±55.00 19 117.31±64.96 0.9352} TB(mg/dL)a _{57 0.79±0.50 19 1.22±1.35 0.1133} DB(mg/dL)a _{54 0.25±0.18 19 0.32±0.29 0.3926} IB(mg/dL)a _{53 0.58±0.37 19 0.90±1.15 0.1716} TP(g/dL)b _{54 7.59±0.93 17 7.20±1.13 0.1616} Albumin(g/dl)b _{56 4.22±0.65 17 4.13±0.52 0.5954} Globulin(g/dL)a _{54 3.43±0.86 16 3.26±0.90 0.5291} PTA(%)b _{47 89.71±16.95 15 82.58±17.35 0.1633} Platelets(×103₎b _{60 202.20±67.73 21 205.15±98.52 0.8795} VL(log10)b _{57 5.59±0.69 19 5.31±0.92 0.1740}

ALT,alanineaminotransferase;AST,aspartateaminotransferase;GGT,gammaglutamyltransferase;FA,alkalinephosphatase;TB,totalbilirubin; DB,directbilirubin;IB,indirectbilirubin(IB);totalproteins(TP);PTA,prothrombintimeofactivity.

a _{Mann–Whitneytest.} b _Ttest.

3,respectively21_{;theseprevalenceratesaresimilartothose}

foundin the present study.In addition, other studies also

foundapredominanceofgenotype1comparedtogenotype

3inblooddonors,and thefrequenciesofgenotype1(from 89.1%22_to93.1%23_{)werehigherthanthosefoundinpatientsin}

thisstudy.Unlikethepresentstudy,genotype2wasdetected instudiesconductedbySawadaetal.23andGuimarãesetal.21 atfrequenciesof0.6and4.4,respectively,indicatingthe pres-enceofthesegenotypesinthestate.

In the present study, a predominance of subtype 1b

(61.1%) over 1a (20.4%) and 3a (18.5%) was demonstrated.

Thesefindingsarenotinagreementwiththedistributionof subgenotypesobservedinBrazil,whicharecharacterizedby anequalprevalencebetweenthe3subtypes:31%,33.4%,and 30.2%,forsubgenotype1a,1band3a,respectively.9_The

pre-dominanceofsubgenotype1bhasalsobeenobservedinthe

statesofAmapá,Pará,PernambucoandRoraima.16,17,21

TherearealsodiscrepanciesbetweentheHCVsubgenotype distributionsobservedinthisstudyandthosefoundinother Brazilianstates.InthestatesofGoiás,Paraná,RioGrandedo

Sul,and SãoPaulo,therewas apredominanceof

subgeno-type1aandalessremarkablerepresentationofsubgenotype 1b.17,20_{Inaddition,inthestateofRiodeJaneiroandthe}

Fed-eralDistrict,genotype3apredominatedoversubgenotypes1a and1b.17,20_{Intheanalysisoftheevaluatedriskfactors,there}

wasasignificantassociationbetweenHCVgenotype1chronic infectionandsharingofglasssyringesand/orneedles,butthe smallnumberofpatientswithgenotype3couldhave compro-misedpowertoshowanassociation.Consideringotherrisk factors,althoughPetruzzielloetal.13_{showedthatillicitdrug}

usewasassociatedwithgenotype3,thisassociationwasnot foundinthepresentstudy,inaccordancewithdatareported byGarcía-MontalvoandGalguera-Colorado.24

In terms of clinical characteristics, some studies have

shown an association between genotype 3 and hepatic

steatosis,25–27_{whichwasnotobservedinthepresentstudy.}

Onepossible cause forthis difference is the small sample size,especiallythosewithviralgenotype3.Associationsof HCVgenotypeswithotherclinicalcharacteristicsare contra-dictory,accordingtodatafromtheliterature.Otherauthors

showed thatHCV genotype 3infection,compared toother

genotypeinfections,maybeassociatedwithmorerapid pro-gression ofliver disease and greater chance of developing cirrhosisandhepatocellularcarcinoma.25,28,29_Inturn,itwas

shownthatpatientswiththisgenotypepresentedamilder

degreeoffibrosisthan didpatientswithothergenotypes.30

Moreover,althoughahigherriskofhepatocellularcarcinoma

has been reported to be associatedwith genotype 1

com-paredtogenotype3,31_{otherstudiesdidnotfinddifferences}

betweendegrees offibrosis inpatients with differentHCV

genotypes,32,33_{asinthepresentstudy.}

Ingeneral,dataontheassociationbetweenHCVgenotypes andbiochemicalmarkersarealsocontroversial.Whilesome studies,suchasthepresentstudy,havenotfound associa-tions betweenany ofthemarkersand viral genotypes,24,33

othersobservedhigherlevelsofaspartateaminotransferase

(AST), alanine aminotransferase (ALT) and alkaline

phos-phatase(AP)34inpatientswithviralgenotype1comparedto thosewithgenotype3.35

Inaddition,somestudieshaveshownthatHCVgenotype1 wasassociatedwithahigherVL,13,35_{whileothershaveshown}

thatthisparameterwasindependentofviralgenotype,33_as

found in the present study.Finally, the small sample size couldbealimitationofthepresentstudyandhighlightsthe needforcontinuousstudiesusinglargersamplesizestodraw

conclusionsontheassociationbetweenHCVgenotypesand

progressionofchronichepatitisC.

Conclusion

This study demonstrates the predominance of HCV

geno-type 1 over genotype 3 in the state of Pará, especially in patientswhoreportedsharingglasssyringesand/orneedles. Thisstudyalsoreinforcestheexistenceofdifferencesinthe prevalenceofviralgenotypesandsubgenotypesindifferent regions ofBrazil, which has relevance, especiallyin terms oftreatment.Another importantaspectofthisstudy isthe comparisonperformedintermsofepidemiological,clinical andlaboratorycharacteristicsamongpatientswithdifferent

genotypes,which showedthatinthe studiedregion,

geno-typehadnosignificantinterferenceintheclinicalcourseof thedisease,whichwarrantsfurtherinvestigationswithlarger samplesizestoconfirmthispreliminaryevidence.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Funding

ThestudywassupportedbyfundingfromtheNationalCouncil forScientificandTechnologicalDevelopmentofBrazil(CNPQ #480128/2013-8;#301869/2017-0)andtheFederalUniversityof

Pará(PROPESP/PAPQ/2019).

Acknowledgments

Wethankallthepatientsaskedandwillingtoparticipatein thestudy.

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