Human T cell lymphotropic virus type 1 infection among men who have sex with men in Central Brazil

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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

Human

T

cell

lymphotropic

virus

type

1 infection

among

men

who

have

sex

with

men

in

Central

Brazil

Lisie

Souza

Castro

a,∗

_,

_Grazielli

_Rocha

_de

_Rezende

a

_,

_Fernanda

_Rodas

_Pires

_Fernandes

a,1

_,

Larissa

Melo

Bandeira

a

_,

_Marco

_Antonio

_Moreira

_Puga

a

_,

_Tayana

_Serpa

_Ortiz

_Tanaka

a

_,

Sabrina

Moreira

dos

Santos

Weis-Torres

a

_,

_Ana

_Carolina

_Paulo

_Vicente

b

_,

_Koko

_Otsuki

b

_,

Ana

Rita

Coimbra

Motta-Castro

a,c

a_Universidade_Federal_de_Mato_Grosso_do_Sul,_Faculdade_de_Ciências_{Farmacêuticas,}_Alimentos_e_Nutric¸ão,_Laboratório_de_Imunologia Clínica,MatoGrossodoSul,MS,Brazil

b_Fundac¸ão_Oswaldo_Cruz,_FIOCRUZ,_Laboratório_de_Genética_Molecular_de_{Microrganismos,}_Rio_de_Janeiro,_RJ,_Brazil c_Fundac¸ão_Oswaldo_Cruz,_FIOCRUZ,_Mato_Grosso_do_Sul,_MS,_Brazil

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Articlehistory:

Received1August2018 Accepted12November2018 Availableonline6December2018

Keywords:

HTLV-1

Menwhohavesexwithmen Sexualriskbehavior Molecularepidemiology Brazil

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HumanTcelllymphotropic virustype 1(HTLV-1)wastheﬁrstretrovirusdiscoveredin humansandisendemicinseveralpartsoftheworld.Becauseofriskbehaviors,mainly sexual,menwhohavesexwithmen(MSM)areathighriskofacquiringHTLV-1infection. Across-sectionalstudywasperformedtoestimatetheprevalenceofHTLV-1infection,to characterizegeneticallyHTLV-1sequencesandtoidentifyriskbehaviorsassociatedwith thisinfectionamongMSMinCentralBrazil.Atotalof430MSMwereenrolledinthisstudy andthreewereshowntobeHTLV-1infected,prevalenceof0.7%(95%conﬁdenceinterval: 0.4–0.9).PhylogeneticanalysisshowedthatallHTLV-1positivesamplesbelongedto Cos-mopolitansubtypeTranscontinentalsubgroupA.AlthoughtheprevalencerateofHTLV-1 infectionfoundinthisstudywassimilartothatobservedamongBrazilianblooddonors, additionalHTLV-1preventiveinterventionsneedtobefurtherimplementedbecausethis populationisengagedinhigh-risksexualbehavior.

Introduction

Thehuman Tcell lymphotropic virus type1 (HTLV-1) is a retrovirus belonging to Retroviridae family, Orthoretrovirinae

subfamilyand Deltaretrovirusgenus. Two maindiseases are

∗ _{Corresponding}_author.

E-mailaddress:[email protected](L.S.Castro).

1 _Present_address:_Ministério_da_Agricultura,_Pecuária_e_{Abastecimento/Laboratório}_Nacional_{Agropecuário,}_{MAPA/LANAGRO,}_Minas

Gerais,Brazil.

associatedwiththevirus,adultT-cellleukemiaandthe neuro-logicaldiseaseHTLV-1-associatedmyelopathy/tropicalspastic paraparesis (HAM/TSP), besides inﬂammatory syndromes.1

ThemostimportantroutesofHTLV-1transmissionwerefound tobeverticallyfrommothertochild(breastfeeding),sexual intercourse,andbloodcontact.1,2

https://doi.org/10.1016/j.bjid.2018.11.003

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Worldwide,thenumberofcarriersofHTLV-1infectionis estimatedtobeatleastﬁvetotenmillioninfected individ-uals.SouthwesternJapan,Caribbeanislands,theMiddleEast, Australo-Melanesia, Sub-Saharan Africa, and South Amer-ica are considered endemic areas of this infection. Based onanalyses oflong terminalrepeat(LTR) regionofseveral HTLV-1 isolates worldwide, seven different viral subtypes weredeﬁned(a–g).ThemostwidelycirculatingHTLV-1strain aroundtheworldbelongstotheCosmopolitan(a)subtype.3

Besidesgeographicallocation,theprevalenceofHTLV-1/2 also varies depending on the population groups in Brazil. HTLV-Iprevalencerateamongblooddonorswas0.48%ina studyconductedindifferentbloodcentersacrossthecountry.4

Furthermore,high-riskgroupsincludesexworkers,injection drugusers,Japaneseimmigrants,andmenwhohavesexwith men(MSM).2,5,6

Duetoriskbehaviors,suchasgreaternumberoflifetime malepartnersandunprotectedsexualintercourse,MSMare consideredhighlyvulnerableforacquiringsexually transmit-tedinfections(STIs).7,8 _There_is_a_paucity_of_information_on

theprevalenceofHTLV-1/2amongMSM.Studiesinthis pop-ulationaremorecommonlyrelatedtoHIVinfection.9,10

ConsideringtherelevanceofHTLV-1infectionandlackof datarelatedtoinfectionamongMSM,theaimofthepresent studywastoassessHTLV-1infectionprevalenceandto iden-tifythecirculatingHTLV-1typesandsubtypesinMSMfrom CentralBrazil.

Materials

and

methods

Studypopulation

Thiscross-sectionalstudywasperformedinCampoGrande city(786,797inhabitants),theStatecapitalofMatoGrossodo Sul,CentralBrazil,fromNovember2011toSeptember2013. AconveniencesamplingofMSMwasselectedprimarilyfrom entitiesrelatedtothisgroupsuchastheMatoGrossodoSul StateAssociationofTravestitesandTranssexuals(ATMS-MS) andReferenceCenterofHumanRightsinthePreventionand CombatofHomophobia(CentrHo).Inaddition,public(square, parks,streets,gay prideparades,etc.)andprivate (saunas, nightclubs, brothels, etc.) locations were also included as recruitment sites of participants. Males who self-reported havinghadsexualintercoursewithmenwithin12 months priortothestudyandolderthan18yearsofagewere eligi-bletoparticipate.Theminimumsamplesizecalculatedwas 402participants,basedonthemeanprevalenceof2%for anti-HTLV-1/2amongMSM,6,7_a_{signiﬁcance}_level_of_95%_(˛_<_0.05),

1.5%ofaccuracyand20%non-respondent.

Afterreadingandsigningtheinformedconsentform,all consentingparticipantswere interviewedbytrained health professionalusingaquestionnairetoobtaindataaboutstudy subjectsandanalyzeitsassociationwithHTLVinfection.The variables tested included socio-demographic data (such as age,maritalstatus,educationallevel,familyincome);sexual behaviors(suchasweeklynumberofsexualpartners, engage-mentindiversesexualpractices,condomuseinlastsexual intercourse,historyofsexualcoercion);beingsexworker (his-toryofhavingeverexchangedmoneyor goodsforsex,age

ofsexwork initiation,condomuse duringsexwithclients and/ornon-paying partners,and placeofsexwork);useof drugs(injectionandnon-injectiondrugs);alcohol consump-tion; having tattoo and/or piercing, and blood transfusion history.

Aftertheinterview,bloodwascollectedfromallsubjects and serum samples were testedfor HTLV-1/2-speciﬁc anti-bodiesbyenzyme-linkedimmunosorbentassay(ELISA)using commercialkit(GoldELISAHTLV-I/II,REMInd.Com.,Brazil). PositivesamplesintheELISAscreeningtestwereconﬁrmedby Westernblot(HTLVBLOT2.4,MPDiagnostics,Singapore).All HTLV-1positivesampleswerealsotestedbyELISAforHIV-1/2 infection(Murex,Kyalami,SouthAfrica).

In exchange for participation, the subjects received condoms, lubricantsand risk reductioncounselingon sex-ually transmitted infections. Those participants found to be infected with HTLV-1 were subsequently referred to an infectious disease clinic forfurther clinical and laboratory assessment.

Theprotocolusedinthepresentstudywasapprovedby theEthicsCommitteeonHumanResearchoftheFederal Uni-versityofMatoGrossodoSul,underprotocolnumber180.909 CAAE06886712.3.0000.0021.

Statisticalanalysis

Data wereenteredand analyzedusingEPI-INFO3.5.1 (Cen-ters for DiseaseControl and Prevention, Atlanta, GA,USA) statistical software package and SPSS Statistics Data Edi-tor(StatisticalPackageforSocialScience,Chicago,IL,USA), respectively.Prevalencewascalculatedwith95%confidence interval(CI).Chi-squaretestandFisher’sexacttestwereused tocomparecategoricalvariables.Ap-value<0.05wasdefined asstatisticallysignificant.

HTLV-1molecularcharacterization

ProviralDNAwasobtainedfromwhole-bloodsamplesofthe HTLV-1infected individualsusing theDNA Genomic Puriﬁ-cationKit(Wizard® Genomic,Promega,Madison,WI,USA), according tomanufacturer’s instructions.Theampliﬁcation oftaxandlongterminalrepeat(5LTR)regionsofthe HTLV-1proviralDNA bynested-PCR wascarriedout asdescribed previously.11,12_PCR_products_of_tax₍₄₆₈_bp)_and₅_LTR₍₄₈₅_bp)

region were puriﬁedusing IllustraTM _GFXTM _PCR_DNA _and

GelBandPuriﬁcationKit(GEHealthcare),accordingto manu-facturer’sinstructions.Theampliconsweresequencedusing BigDyeTerminatorCycleSequencingReadyReactionKitand ABI1373(AppliedBiosystems,FosterCity,CA,USA).

Phylogeneticanalysis

The 5 LTR sequences were aligned using BioEdit v7.2.3 (DepartmentofMicrobiology,NorthCarolinaStateUniversity, NC,USA)andeditedbyuseofClustalWprogram.MEGA soft-ware(version6)andTamura-Neimodelwereusedtoconstruct aMaximumLikelihood(ML)tree,whichwasevaluatedby ana-lyzing1000bootstrapreplicates.GenBankaccessionnumbers

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oftheHTLV-1sequencesnewlyreportedinthisstudyareas follows:HSH13(KM023762)andHSH76(KM023763).

Results

Ofthe530MSMinvitedtoparticipateofthestudy,430 con-sentedtoanswerthequestionnaireandtohaveabloodsample collected.Medianageofparticipantswas 23years,ranging from18to70years.Havinglessthan10yearsofformal educa-tionwasreportedby24.9%andlowfamilyincomeby21.6%of subjects.Majorityweresingle(87.4%)andfromCampoGrande city (77.7%).Alcoholconsumptionwas reportedbymostof thesubjects(81.6%).Useofoneormoretypesofillicit non-injectiondrugswasreportedby48.8%ofparticipants;among thosereported,marijuanawasthemostcommonlyuseddrug (77.1%).Onlyﬁvesubjectsofthestudy(1.2%)reportedillicit injectiondruguse.Presenceoftattoosand/orbodypiercings wasreportedby52.3%ofsubjects.Historyofbloodtransfusion wasinformedby30MSM(7.0%),ofwhom18receivedablood transfusionbefore1993.

Out ofthe totalMSM surveyed,37.0% weresexworkers andpublicplaceswerethemostfrequentlocationofsexwork (66.7%).Meanageofsexualinitiationandsexworkdebutwere 14.8years (standarddeviation[SD]=±3.044)and 16.7years (SD=±3.289), respectively. Majority of individuals reported havinghad morethansevenclientsintheweekbeforethe interview,andthefrequencyofirregularcondomusewiththe lastclientwas32.1%.Ontheotherhand,abouthalfofthe par-ticipantsusedcondomregularlywiththeirsteadypartner.Part ofMSM(35.8%)reportedhavingengagedinavarietyof sex-ualpractices,includingmasochism(35.9%),rimming(53.6%), groupsex(10.5%),watersports(28.1%),sharedsextoys,ﬁsting andothers(20.3%).SixteenpercentoftheMSMgroupsuffered sexualcoercion.Signiﬁcantdifferencesinrelationto commer-cialsexworkerandmultiplesexualpartners(morethan10) wasfoundbetweenHTLV-1-infectedandnon-infectedMSM.

TheprevalenceofHTLV-1infectionamongthestudy popu-lationwas0.7%(3/430;95%CI:0.4–0.9).Allsamplesconﬁrmed anti-HTLV-1-reactivitywithcompleteproﬁlesinWesternBlot test.Nosamplehadpositivereactionforanti-HTLV-2 antibod-ies.Table1showsriskbehaviorcharacteristicsofallHTLV-1

positiveMSM.NoneofHTLV-1-infectedsubjectswere HIV-1/2-infected.

Phylogeneticanalysisof5LTRregionofHTLV-1sequences was possible in samples of two infected MSM (HSH13 and HSH76). Their sequences, clustered in Transcontinen-tal subgroup (A) of Cosmopolitan HTLV-1a subtype, were closely related to other Latin American strains, mainly from Brazil, and two more strains from Portugal and India (Fig. 1). In addition, only one of the three samples (HSH28) could not be ampliﬁed in 5 LTR region of HTLV genome, only in tax region. After sequencing, this isolate was subjected to BLAST (Basic Local Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/Blast.cgi)tocharacterize HTLV-1viral subtype.Thisprocedure detectedthe same subtype identiﬁedpreviouslyinanothersequencesofthestudy,the CosmopolitanHTLV-1asubtypeTranscontinentalsubgroupA (datanotshown).

Discussion

Thisistheﬁrstcross-sectionalsurveyofaMSMconvenient sampletoinvestigateHTLV-1infectioninCampoGrande, Cen-tralBrazil.The0.7%(95%CI:0.4–0.9)prevalencerateofHTLV-1 infection foundin this study was similar tothat observed amongBrazilianblooddonors(0.48%),aswell asinan iso-latedAfro-Brazilianslave-descendantcommunityinCentral Brazil (0.5%) and inMSM from Campinas, Southeast Brazil (1.5%;95%CI:0.5–3.0).4,6,13_{Nevertheless,}_the_prevalence_rate

foundinpregnantwomeninMatoGrossodoSulState(0.11%) waslowerthanthatreportedinthisstudy.14

Accordingtoseveralriskbehaviorsassessedinthisstudy, mainly through sexual exposures, there is a high vulner-ability forSTIs acquisition inthese individuals. Signiﬁcant differencesinrelationtocommercialsexworkerwerefound betweenHTLV-1-infectedandnon-infectedMSM.Greater vul-nerability and social marginalization was observed in this group ofpopulation.8,10 _Furthermore,_MSM _who _exchanged

moneyforsexpresentagreaterriskforacquiringSTIsthan femalesexworkersandothernon-sexworkersMSMdueto combinationofhighriskbehaviors,suchashavingmultiple

Table1–Sociodemographicandriskbehaviorcharacteristicsofthethreeanti-HTLV-1infectedMSM,CampoGrande– MS,2012–2013.

Characteristics ID-13 ID-28 ID-76

Age(years) 61 19 24

Education(years) 5 12 9

Maritalstatus Single Single Single

Bloodtransfusionbefore1993 Yes No No

Frequencyofdrinkingalcoholinthelast4weeks Drinksatleastonceamonth Drinksatleastonceaweek Drinksatleastonceaweek

Everusedanillicitdrug Yes No No

Sexworker Yes Yes Yes

Placeofsexwork Residence Street Street

Ageatﬁrstreceivedmoneyforsex(years) 16 15 18

Numberofsexualpartnersinthelastweek 2 20 15

Condomusewithclients Always Almostalways Always

Condomusewithsteadypartner Always Almostalways Always

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J APON ÊS Subg rupo B Subg rupo A SUBTIPO COSMOPOLIT A − HTL V 1a TRANSCONTINENT AL

Fig.1–Phylogenetictreeconstructedusingisolatesbasedonlongterminalrepeat(5LTR)regionof485bpofHTLV-1, including48sequencesavailableinGenBankandtwosequencesdescribedinthisstudy(HSH-13andHSH-76)byMaximum Likelihoodmethod.Thenumbersonthetreedenotebootstrapvalue(1000replicates).Onlybootstrapvaluesof50%ormore areshown.

sexualpartners,unprotectedsexualintercourseandreceptive analintercourse.8,15

RegardingMSMpopulation,greaternumberoflifetimesex partnersisahigh-risk behaviorfactor forHTLV-1infection accordingtosomestudies.7,16 _A_{cross-sectional}_surveillance

studyforHIVamongMSMfromtendifferentBraziliancities reported multiple sexual partners and inconsistent use of condomwithcasualandcommercialpartners.9_{Additionally,}

othersstudiesconductedindifferentcountrieshavepointed rectalmucosalinjuryduringintercourseasariskfactorfor HTLV-1andotherSTIsinthispopulation.7,16,17

In the past, blood transfusion was an important route ofHTLV-1transmission,withaseroconversionrateranging from 40 to 60%.1 _In _1993, _serological _screening _for

HTLV-1/2becamemandatoryinBrazilianbloodbanks,interrupting thistransmissionroute.Historyofbloodtransfusion before 1993was found inone infectedMSM (ID-13), as shown in

Table1.AlthoughresultsaresuggestiveofHTLVtransmission viatransfusion,thethreeinfectedMSMmayhaveacquired theinfectionthroughsexualintercourse,giventheyweresex workers,agroupknowntobeatriskforacquiringSTIs.4,8,15

The positive samples were classiﬁed as belonging to the Transcontinental (A) subgroup ofthe Cosmopolitan (a)

subtype. This subgroup of HTLV-1 is considered the most prevalent in Brazil.3,18 _Phylogenetic _analysis _showed _that

HTLV-1strains isolated fromthe present studywere found withintheBrazilianandSouthAmericanclusters.Inaddition, twoHTLV-1sequencesoutofLatinAmericancountries,one sequencefrom anIndiancommercialsexworkerandother fromaPortugueseinjectingdruguser(IDU)clusteredwiththe studystrains.

Oneofthepositivesampleswasampliﬁedonlyinthetax

region ofHTLV-1 (HSH28), probablydue tothe low HTLV-1 proviralload,sincethattaxregionismorereadilyPCR ampli-ﬁedthanotherHTLV-1regions.Accordingtopreviousstudies, the highsensitivityand widedynamicrange ofHTLV-1tax

sequencedetectionallowfordeterminationofabroadrange ofHTLV-1proviralloads.19,20

Somelimitationsfoundinthisstudyshouldbeconsidered. First,relatedtotheveracityofthestudysubjects’responses about risk behaviors. Second, MSM from this study were recruited from gay-related public and private places, who may not be representative of the whole MSM community. Furthermore, a convenience sample was selected because themethodrespondent-drivensampling(RDS)couldnotbe implementedforrecruitmentofthisstudypopulation.Third,

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thesmallnumberofHTLV-1casesmayhavecausedalimited power to evaluate some statistical associations of risk. In addition,verticaltransmissionofHTLV-1infectionamongthe studiedMSMcouldnotbeanalyzedforlackofinformation aboutthisroute.Despitetheselimitations,thestudyprovides importantinformationabout HTLV-1infection amongMSM groupinCentralBrazil.

Insummary,the prevalenceofHTLV-1infectioninMSM wassimilartotheprevalenceamongBrazilianblooddonors. However,thehigh sexualriskbehaviorfoundinthis study populationmakesthemmoresusceptibletoacquiringHTLV-1 infectionthanBrazilianblooddonorsingeneral.TheHTLV-1 subtypeidentiﬁedinthestudiedindividualsisconsistentwith thosepreviouslyfoundinBrazil.Therefore,educational meas-uresforcontrollingthespreadofHTLV-1andother STIsin thispopulationareneededandcouldbeachievedthroughthe implementationofspeciﬁcpreventiveactivitiesamongMSM inCentralBrazil.

Ethics

approval

Theauthors assert that all procedurescontributing tothis work comply with the ethical standards of the relevant nationalandinstitutionalcommitteesonhuman experimen-tationandwiththeHelsinkiDeclaration.

Funding

ThisworkwassupportedbyConselhoNacionalde Desenvolvi-mentoCientíﬁcoeTecnológico(CNPq),Grant/AwardNumber: 134191/2012-9;andFundac¸ãodeApoioaoDesenvolvimento doEnsino,CiênciaeTecnologiadoEstadodeMatoGrossodo Sul(FUNDECT/MS),Grant/AwardNumber:23/200.283/2009.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

Theauthors acknowledgeall the MSMwho participated in thisstudyandtheimportantcontributionofentities’ mem-bersthatcollaboratedwiththisproject:ReferenceCenterfor HumanRightsinthePreventionand Combatof Homopho-bia(CentrHo) and MatoGrossodo SulStateAssociation of TravestitesandTranssexuals(ATMS-MS).

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