Chlamydia trachomatis in human immunodeficiency virus-infected men treated at a referral hospital for sexually transmitted diseases in the Amazonas, Brazil☆

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

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

Original

article

Chlamydia

trachomatis

in

human

immunodeficiency

virus-infected

men

treated

at

a

referral

hospital

for

sexually

transmitted

diseases

in

the

Amazonas,

Brazil

夽

Alex

Panizza

Jalkh

_,

_Angelica

_Espinosa

_Miranda

_,

_Jose

_Camilo

_{Hurtado-Guerreiro}

_,

Lorena

Angelica

Castano

Ramos

_,

_Guiseppe

_Figliuolo

_,

_Jussimara

_Maia

_,

Cintia

Mara

Costa

_,

_Rajendranath

_Ramasawmy

_,

_Luiz

_Carlos

_de

_Lima

_Ferreira

a_{Fundac¸ãoMedicinaTropicalDoutorHeitorVieiraDourado,Manaus,AM,Brazil}

b_{InfectiousDiseasesUnit,FederalUniversityofEspíritoSanto,Vitória,ES,Brazil}

c_{UniversidadeNiltonLins,Manaus,AM,Brazil}

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Articlehistory:

Received28February2013 Accepted19June2013

Availableonline9November2013

Keywords:

Chlamydiatrachomatis

Real-timepolymerasechain reaction(RT-PCR)

HIV-infectedmen Amazonas

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b

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Objectives:TheaimofthepresentstudywastodeterminetheChlamydiatrachomatis

preva-lenceandtoidentifythedemographic,behaviouralandclinicalfactorsassociatedwithC.

trachomatisinhumanimmunodeficiencyvirusinfectedmen.

Study:This was a cross-sectional study of C. trachomatis prevalence among human

immunodeficiencyvirus-infectedmenenrolledattheOutpatientclinicofacquired immu-nodeficiencysyndromeoftheFundac¸ãodeMedicinaTropicalDr.HeitorVieiraDouradoin Manaus,Amazonas,Brazil.C.trachomatisdeoxyribonucleicacidfromurethralsampleswas purifiedandsubmittedtorealtimepolymerasechainreactiontoidentifythepresenceofC. trachomatis.

Results:A total of 276 human immunodeficiencyvirus-infected men were included in

thestudy.The prevalenceofC. trachomatisinfection was12%(95% confidenceinterval 8.1%–15.7%).Themeanageoftheparticipantswas34.63(standarddeviation10.80)years.Of the276humanimmunodeficiencyvirus-infectedmen,93(56.2%)hadmorethanonesexual partnerinthepastyearand105(38.0%)reportedhavingtheirfirstsexualintercourseunder theageof15years.Menhavingsexwithmenandbisexualsamountedto61.2%ofthe stud-iedpopulation.Atotalof71.7%hadreceivedhumanimmunodeficiencyvirusdiagnosisin thelastthreeyearsand55.1%wereusingantiretroviraltherapy.FactorsassociatedwithC.

trachomatisinfectioninthelogisticmodelwerebeingsingle(p<0.034),menhavingsexwith

men(p<0.021),andhavingprevioussexuallytransmitteddiseases(p<0.001).

Conclusion:ThehighprevalenceofC.trachomatisinfectionamonghumanimmunodeficiency

virus-infectedmenhighlightsthatscreeninghumanimmunodeficiencyvirus-infectedmen

forC.trachomatis,especiallyamongmenhavingsexwithmen,isparamounttocontrolthe

spreadofC.trachomatisinfection.

©2013 ElsevierEditoraLtda.Allrightsreserved.

夽_{Sourcesofsupport:Fundac¸ãodeMedicinaTropicalDr.HeitorVieiraDourado.}

∗ _{Correspondingauthorat:AlamedaAlaska1051,ap.601,Ed.PortoSeguro,PontaNegra,Manaus,AM,Brazil.}

E-mailaddresses:ajalkh@gmail.com,alpajal@hotmail.com(A.P.Jalkh). 1413-8670/$–seefrontmatter©2013 ElsevierEditoraLtda.Allrightsreserved.

Introduction

GenitalChlamydiatrachomatis(CT)infection,themost preva-lent sexually transmitteddisease (STDs) inthe world, is a majorpublichealthproblem.1,2_{Asymptomaticinfectionsare} commonandareassociatedwithhighprobabilityof acquir-ing human immunodeficiency virus (HIV) infection.3–5 _The screeningofHIV-infectedindividualsforCTisakeystrategy forreducingthetransmissionofHIV.2,3_{Fewstudieshavebeen} conductedonCTinfectionamongmeninBrazil.6_Astudyin HIV-negativemenattendingSTDsclinicshaveestimatedthe incidenceofurethralCTinfectiontobe13.1%.7_Studies con-ductedinHIV-infectedmenoutside ofBrazilhavereported prevalenceratesof9%8_{and2–10%inHIV-infectedmenandin} menwhohavesexwithmen(MSM),2,9_{respectively.}

There is no mandatory reporting system for CT infec-tions in Brazil,8 _{and the lack of population-based studies} in HIV-infected patients hinders efforts to document the problem,implementpriorityinterventionsandevaluatetheir efficacy.ScreeningforCT infectionin HIV-infected individ-uals is importantfor monitoringSTDs and forelaborating preventivemeasuresaswellastreatmentassistancetothis population.

Thepresentstudyaimedtodeterminetheprevalenceof CTinfectionandassociatedriskfactorsinHIV-infectedmen followedinareferralhospitalspecializingininfectiousand tropicaldiseasesinManaus,Amazonas,Brazil.

Methods

Apopulationbasedcross-sectionalstudyamongHIV-infected menwasconductedatthereferralhospitaloftheFundac¸ão de Medicina Tropical Dr. HeitorVieira Dourado (FMT-HVD) inManaus,capitalcityofthestateoftheAmazonas,Brazil from November 1,2009 to November 1, 2010. HIV-infected menwereinvitedtoparticipateinthe study bysigningan informedconsentformasapprovedbytheethicalcommittee oftheFMT-HVD.Eachparticipantansweredaquestionnaire concerning socio-demographic factors, age of first sexual intercourse, number of sexual partners over the past 12 months, regular use ofcondoms, sexual behaviours, prior symptomsofSTDs.SignsandsymptomsofSTDonphysical examinationwerealsorecordedandadequatetreatmentwas provided.

Theinclusioncriteriawerethefollowing:maleindividuals withpositive serologyfor HIV1–2, sexually active,over 18 yearsofageandagreedtoaurethralsamplecollection. Vul-nerablemalepatients(mentaldiseasesandyoungerthan18 yearsold)wereexcludedfromthestudy.

The sample size was calculated for estimating the prevalence of CT infection in Human immunodeficiency virus/acquiredimmunodeficiencysyndrome(HIV/AIDS) peo-plewitha95%confidenceinterval(CI)andabilateralsizeof 0.5%.Thesamplesizewascalculatedbasedonanaveragerate of6.0%withavariationof±3.0%whichgeneratedanumber of270patients.Assumingalossof10%,thefinalsamplesize was297patients.Ofnote,theFMT-HVDisthereferral cen-treforAIDSandallthepatientswithHIV/AIDSfromthestate

ofAmazonasarereferredtothatcentre. Anaverageof310 HIV-infectedmenistreatedyearlyfrom2001to2010atthe FMT-HVDandintheyear2010,atotalof530HIV-infectedmen wereenrolled.

Urethral samples were collected by inserting a small brush (Cavi-brush) in the distal urethra, between 1.5 and 2cm,androtatingitfivetimesclockwise.Thesampleswere then placed in tubes containing 400␮L of buffer solution (10mMTris–HCLand1mMEDTA)andkeptoniceuntilthey were transportedtothelaboratory,wheretheywerestored at −20◦_{C until processing fordeoxyribonucleic acid (DNA)}

purification.DNApurificationwasperformedusingthe com-mercialDNA extractionkit Nucleo-SpinTissue –Machery– NagelTM_{followingthecompany’sinstructions.Forthe}

detec-tion of CT DNA by RT-PCR, the following pair of primers amplifying a fragment of 241 base pairs CT plasmid DNA was used: KL15-TCCGGAGCGAGTTACGAAGA-3 and KL2 -AATCAATGCCCGGGATTGGT-3. Briefly, 5␮L of DNA sample and3␮Lofeachprimer(10pmol/␮L)wereaddedtoafinal vol-umeof25␮Lcontaining12.5␮LofMaxima®_{SYBRGreen/ROX}

qPCRMasterMix(2×).Fortheinitialconfirmationofthe ampli-consizethePCRproductwaselectrophoresedin1.5%agarose gelandrevealedwithethidiumbromideunderUV. Further-more,samplewasconsideredpositivewhenRT-PCRanalysis showeduniquepeakdissociationcurveat78◦C.

The data were stored and analyzed using the Epi Info software,version3.5.2,andtheStatisticalPackageforSocial Science(SPSS)version16.0forWindows.Chi-squaretestwith Yatescorrectionwasusedtoassessforpossibleassociations between clinical,demographic or risk behavioursvariables andCTinfection.Fisherexacttestwasappliedwhen appro-priate.Theoddsratioalongwiththe95%confidenceinterval (95%CI)wascalculatedforvariablestoestimatethedegreeof associationbetweenCTinfectionandpotentialriskfactors. Themultivariatelogisticregressionanalysiswas appliedto examinetheindependenteffectofeachdemographic, clin-icalor riskbehaviourvariableson CTinfection,controlling forallothervariablessimultaneously.Allvariablesthatwere moderatelyassociatedwithasignificanceofp≤0.15 inthe univariateanalysiswereconsideredforinclusioninthe multi-variatemodel.Inthefinalmodelanalysis,onlythosevariables thatremainedsignificantwithp<0.05wereconsidered.

Results

Ofatotalof276HIV-infectedmenincludedinthestudy,33 wereinfectedwithCTinfectionshowingaprevalenceof12% (95%CI8.1%–15.7%).Nourethralsymptomswereobservedin anyoftheHIV-infectedmen.Baselinecharacteristicsofthe participantsareshowninTable1.Themeanageof partici-pantswas34.6years(SD=10.8)and36.6%were29yearsoldor less.Morethan80%ofthe276HIV-infectedmenhadupto11 yearsofschooling.Atotalof63.8%reportedalcoholuseand 23.9%illicitdrugsabuse.

Concerning risk behaviours, as shown in Table 2, 93 (56.2%) participantsreported havingmorethan onesexual partnerinthepastyear,and105(38.0%)hadtheirfirstsexual intercourseundertheageof15years.Priortothediagnosis ofHIVinfection,230(83.3%)patientsadmittedirregularuse

Table1–PrevalenceofCTinfection(RT-PCR)anditsassociationwithsocio-demographicfactorsamong276HIV-infected mentreatedattheFMT-HVD,2009–2010.

DemographicIndices n(%) CTpositive CTnegative p-Value

Age 18–29years 101(36.6) 2 22 0.3785a 30–49years 151(54.7) 17 134 ≥50years 24(8.7) 2 22 Race/colour Mulatto 210(76.1) 21 189 0.066a White 48(17.4) 11 37 Black 18(6.5) 1 17 Maritalstatus Single 158(57.2) 25 133 0.034a Married/Livingtogether 109(39.5) 8 101 Divorced 7(2.5) 0 7 Widower 2(0.8) 0 2 Education Primary 97(35.1) 11 86 0.111a Secondary 125(45.3) 11 114 College 52(18.8) 11 41 Illiterate 2(0.8) 0 2 Alcoholconsumption Yes 176(63.8) 24 152 0.335b No 100(36.2) 9 91

Useofillicitdrugs

Yes 66(23.9) 12 54 0.0837b

No 210(76.1) 21 189

a _{Pearson’schi-squaredtest(consideredsignificantwhenp≤0.05).} b _{ExtendedFisher’sexacttest(consideredsignificantwhenp≤0.05).}

ofcondoms.Afterthediagnosis,206(74.6%)reportedalways usingcondoms.However,179(64.9%)subjectsreportedhaving previous STDs. A history of STDs was strongly associated (p<0.001)withthedetectionofCT.Regardingsexual orienta-tion,homosexualsandbisexualstogethermadeupto61.2% of the studied population. Multivariate logistic regression showedthatMSM[OR=2.51(95%CI1.12–6.64)andprevious STD [OR=3.46 (95% CI 1.96–5.72) were independent risk factorsforCTinfectionamongHIV-infectedmen.

Table3showsvariablesrelatedtoHIVstatus.Noneofthe variablesstudiedshowedanyassociationwithCTinfection.A totalof71.7%hadreceivedtheHIVdiagnosisinthelastthree yearsand55.1%wereusingantiretroviraltherapy.

Discussion

OurstudyprospectivelyinvestigatedCTinfectionamong HIV-infectedmenintheAmazonasthroughtheRT-PCRtechnology toprovide arapiddiagnosis forimprovingmanagement of patientssoastolimitthespreadofCT.InBrazil,limiteddata are availabledemonstratingthe precisepattern ofthe epi-demiologicalbehaviourofCT infection inmen,6,10 _{and the} statusofthisinfectionwithinthepopulationofHIV-infected menisunknown.

Theprevalence ofCT urethral infection in HIV-infected menobservedinthisstudywas12%.Theobservedprevalence issimilartothatobservedinnon-HIV-infectedmenreceiving

careatsixSTDclinicsinBrazil,6_{andinHIV-infectedmenin} UnitedKingdomandUSA.8,11

STDsareofprimaryimportancebecauseoftheemergence ofAIDS. Theseinfections enhancethe sexualtransmission ofHIV3,12,13_{andareassociatedwithearlierandmoresevere} symptomsinHIV-infectedindividuals.14–16_{Furthermore,they} cancauseseriouscomplications,resultinginchronicdiseases and evendeath.Infectionsinmenoftencause problemsin thegenitourinarytractthatcanresultinmaleinfertility.In women,theconsequencescanbeextremelysevere,including chronicpelvicpain,infertility,andcervicalcancer.16,17

None of the HIV-infected men with CT infection in this study showed any urethral symptoms, which is con-sistent with most previous studies. A high proportion of asymptomaticpatientshasbeenobservedinCTinfections18 indicatinganeedforscreeninginthispopulation.11,19–21_The routinetestingofpatientsremainsacontroversialissue,given thatsomestudiesshowahigherprevalenceofCTinfection amongindividualswithurethralsymptoms.8,19,22–24

Several studies have reported higher prevalence of CT infection in young individuals (<25 years of age),23 _{but it} was notobserved inthis study.Similar towork previously published by Iwuji et al. in 2008 inthe United Kingdom,8 other studies havereported common associationsbetween CTinfectionsandreportsofmultiplepartners,irregularuse ofcondomsandahistoryofSTDs.25,26_{Here,onlyahistoryof} STDswasfoundtobeassociatedwithCTinfection.Notably, Fioravanteet al.25 _{observedaprevalence of5%of} chlamy-dialinfectionamongmaleasymptomaticconscripts.Thismay

Table2–Sexualbehavioursof276patientswithHIV+/AIDSsortedbyCTurethralinfectionstatusasdetectedbyPCR.

Variables n(%) CTpositive CTnegative p-Value

Onsetofsexualactivity

≤15years 105(38.0) 7 98 0.078a

15–19years 151(54.7) 22 129

≥20years 20(7.3) 4 16

No.ofsexualpartnersinthepastyear

None 24(8.7) 3 21 0.068a

1 97(35.1) 5 92

2–9 93(33.7) 14 79

≥10 62(22.5) 11 51

UseofcondomsbeforeHIV+diagnosis

Sometimes 230(83.3) 32 198 0.091a

Always 7(2.5) 0 7

Never 39(14.2) 1 38

UseofcondomsafterHIV+diagnosis

Nosex 34(12.4) 5 29 0.766a Sometimes 29(10.5) 4 25 Always 206(74.6) 24 182 Never 7(2.5) 0 7 Sexualorientation MSMb _{101(36.6) 14 87 0.021}a Bisexual 68(24.6) 13 55 Heterosexual 107(38.8) 6 101 PreviousSTDs Yes 179(64.9) 30 149 0.001a No 97(35.1) 3 94

a _{Pearson’schi-squaredtest(consideredsignificantwhenp≤0.05).} b _{Menwhohavesexwithmen.}

Table3–VariablesassociatedwiththepresenceofHIVamong276patientssortedbyCTurethralinfectionstatusas detectedbyPCR.

Variables n(%) CTpositive CTnegative p-Value

TimesinceHIVdiagnosisa

≤3years 198(71.7) 23 179 0.330c 4–6years 38(13.8) 6 28 7–9years 15(5.4) 0 15 ≥10years 25(9.1) 4 21 UseofARTb Yes 152(55.1) 25 127 0.5778d No 124(44.9) 8 116

PeriodreceivingARTb

<1year 79(52.0) 25 54 0.509c

1–2years 16(10.5) 0 16

2–3years 12(7.9) 0 12

>3years 45(29.6) 8 37

TCD4Lymphocytecount(cells/mm3₎

<200 103(37.3) 13 90 0.310d

200–349 68(24.6) 10 58

350–500 41(14.9) 4 37

>500 40(14.5) 6 34

Noresultsavailable 24(8.7) 0 24

a _{Relatedtoall276participantsinthestudy.}

b _{Relatedto152patientswhoreceivedantiretroviraltherapy(ART).} c _{Pearson’schi-squaredtest(consideredsignificantwhenp≤0.05).} d _{ExtendedFisher’sexacttest(consideredsignificantwhenp≤0.05).}

reflecttheprevalenceinthegeneralpopulationofmale Brazil-ians,whichislowerthantheprevalenceinHIV-infectedmen. A significant association between CT urethral infection andsexualorientation(MSM)wasnotedinthis study. Sev-eral studies have demonstrated that a high prevalence of STDs,9,27_{bacterialSTDs,}14_anorectalCT,22–24,28_{andCTurethral} infection14,28 _{are directlyrelatedtothesexualbehaviourof} MSM.

Thepossibility ofresponse bias may be a limitation in thisstudyduetothetendencytoprovidesociallyacceptable answers.However,suchbiaseswouldresultin underestima-tion ofriskattitudes and behaviours.Inaccuracies ofrecall ofcondomuse, ageoffirstintercourse,andnumberof sex-ual partners may also have occurred. Another limitation isprobably fewHIV-infected individuals comingfrom high socioeconomicclasseswhomayseekprivateclinicsfor treat-mentandthuspotentiallyintroducingpatientselectionbias. However,astreatmentdeliveredforfreeinpublichospitals inBrazilcomparedtotheexpensivecostinprivatehospitals, selectionbiasmayhavebeennegligible.

Thefindingsfromthisstudyhaveimportantimplications foreducationandpreventioneffortsdirectedtowardsyoung HIV-infectedmen,mainlyMSM.Acomprehensiveapproach wouldpromotesexualresponsibilityand,atthesametime, improveyoungmen’sunderstandingofsexualhealthrisksto curbthespreadofCTinfection.

ThelackofclinicalsymptomsinCTinfectionimpairsthe identificationofinfectedindividuals.Thisstudyunderscores theneedforscreeningthroughlaboratorialteststoidentify theseindividualsforrapidtreatment,tolimitthespreadand alsotoavoidchronicsymptomsandsevereconsequencesfor theindividual.Thistopicisofextremeimportanceforhealth policies,asithasasignificantimpactonfamilypolicyand hasconsequencesforthesexualandreproductivehealthof boththepopulationingeneraland,morespecifically,the HIV-infectedpopulation.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WeacknowledgethecontributionsofDra.HelineSilvaLira, Dra.CarolinaMarinhodaCosta,Dra.MarlyMarquezdeMelo andTechnicianElizabethMonteiroforperforminglaboratory procedures.

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