Canine visceral leishmaniasis : incidence and risk factors for infection in a cohort study in Brazil.

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ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j ourn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

Canine

visceral

leishmaniasis:

Incidence

and

risk

factors

for

infection

in

a

cohort

study

in

Brazil

Wendel

Coura-Vital

a,b,c,f,∗

_,

_Alexandre

_Barbosa

_Reis

c,d,f

_,

Levi

Eduardo

Soares

Reis

c,d

_,

_Samuel

_Leôncio

_Braga

c,d

_,

_Bruno

_Mendes

_Roatt

c,d,f

_,

Rodrigo

Dian

de

Oliveira

Aguiar-Soares

c,d,f

_,

_Marcos

_José

_Marques

e

_,

Vanja

Maria

Veloso

c

_,

_Mariângela

_Carneiro

a,b

a_{Pós-Graduac¸}_ão_em_Ciências_da_Saúde:_Infectologia_e_Medicina_Tropical,_Faculdade_de_Medicina,_Universidade_Federal_de_Minas_Gerais,

AvenidaAlfredoBalena190,CEP30130-100BeloHorizonte,MinasGerais,Brazil

b_Laboratório_de_{Epidemiologia}_de_Doenc¸_as_Infecciosas_e_{Parasitárias,}_Departamento_de_{Parasitologia,}_Instituto_de_Ciências_Biológicas,

UniversidadeFederaldeMinasGerais,CampusPampulha,CEP31270-901BeloHorizonte,MinasGerais,Brazil

c_Laboratório_de_Pesquisas_Clínicas,_Ciências_{Farmacêuticas,}_Escola_de_Farmácia,_Universidade_Federal_de_Ouro_Preto,_Campus

Universitário,CEP35400-000OuroPreto,MinasGerais,Brazil

d_Laboratório_de_{Imunopatologia,}_Núcleo_de_Pesquisas_em_Ciências_Biológicas,_Instituto_de_Ciências_Exatas_e_Biológicas,_Universidade

FederaldeOuroPreto,CampusUniversitário,CEP35400-000OuroPreto,MinasGerais,Brazil

e_Laboratório_de_{Parasitologia}_Básica,_Departamento_de_Ciências_Biológicas,_Universidade_Federal_de_Alfenas,_37130-000_Alfenas,_Minas

Gerais,Brazil

f_Instituto_Nacional_de_Ciência_e_Tecnologia_em_Doenc¸_as_Tropicais,_Salvador,_Bahia,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received5March2013

Receivedinrevisedform9July2013 Accepted19July2013

Keywords: Riskfactors Incidence

Caninevisceralleishmaniasis Leishmaniainfantum Cohortstudy PCR-RFLP

a

b

s

t

r

a

c

t

ZoonoticvisceralleishmaniasisinBraziliscausedbyLeishmaniainfantumparasitesand istransmittedbysandfliesofthePhlebotominaefamily.Dogsarethemainurban reser-voirsandrepresentthemajorsourceofcontagionforthevectors.Studieshaveshownthat mostinfecteddogsarepolymerasechainreaction-positivemonthsbeforeseroconversion. Herein,wedescribeacohortstudydesignedtoidentifytheincidenceofandriskfactors forL.infantuminfectionasdetectedbypolymerasechainreaction-restrictionfragment lengthpolymorphism.Todeterminetheriskfactorsforinfection,weconducteda base-linecaninesurvey(n=1443)fromwhichdogswereselectedforthecohortstudy(n=282) involvingthreeevaluationsoverthecourseofa26-monthfollow-upperiod.Serology, moleculartests,andastructuredquestionnairewereused.Theriskfactorsforinfection wereidentifiedbymeansoftheCoxregressionmodel.Theoverallinfectionincidence was5.8per100dog-months(95%confidenceinterval5.1–6.5).Increasedriskof infec-tionwasassociatedwiththepresenceofpreviouscasesofcaninevisceralleishmaniasisin thedomiciles(hazardratio[HR]1.4;95%confidenceinterval[CI]1.1–1.8)andunplastered housewalls(HR3.6;95%CI1.6–8.1).Theseriskfactorssuggestthatinsecticidesprayingin cracksandcrevicesinunplasteredwallscanreducebitingrateswithinandaroundhomes. Furthermore,ourresultsdemonstratethattheVisceralLeishmaniasisControland Surveil-lanceProgramshouldadoptenvironmentalmanagementmeasuresinhomeswithprevious casesofcaninevisceralleishmaniasis,becausethesehomesaremorelikelytomaintainthe transmissioncycle.

∗ Correspondingauthorat:LaboratóriodeEpidemiologiadasDoenc¸asInfecciosaseParasitárias,Parasitologia,InstitutodeCiênciasBiológicas,Campus Pampulha,UniversidadeFederaldeMinasGerais,AvenidaAntônioCarlos,6627,CEP31270-901BeloHorizonte,MinasGerais,Brazil.

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1. Introduction

VisceralleishmaniasisiscausedbyLeishmaniadonovani orL.infantum,protozoanparasites that are transmitted tohumanandanimalhostsbythebiteofphlebotomine sandﬂies(Chappuisetal.,2007).Dependingonwhether or not a reservoir host is present, there are two basic typesofepidemiologicalcycles:zoonotic,generallycaused by L. infantum,and anthroponotic, generally caused by L. donovani (Palatnik-de-Sousa et al., 2001). Dogs are themain urban reservoirsof L. infantumand represent themajorsourceof contagionfor thevectorsbyvirtue of the high prevalence of infection and intense cuta-neousparasitism (Molinaet al., 1994; Giunchettietal., 2006).

Caninevisceralleishmaniasis(CVL)ispresentin approx-imately 50 countries, mainlyin South America and the Mediterraneanregion(Solano-Gallegoetal.,2009; Dantas-Torreset al., 2012).Epidemiologicalstudiesin endemic areashavedemonstratedthattheprevalenceofinfection asdeterminedbymoleculartechniques(50–80%)ishigher thantheseroprevalence(10–30%)(Solano-Gallegoetal., 2009;Wang etal., 2011).A cohortstudy conductedby Olivaetal.(2006)showedthatmostofthestudyanimals were polymerase chain reaction (PCR)-positive months before seroconversion. The CVL infection rate depends onseveralfactorsincludingthelengthof the transmis-sionseason,thevectordensity, thesusceptibilityof the dog population, dog behavior, the degree of exposure tovectors,anddogowners’attitudestowardprevention (Banethetal.,2008;Dantas-Torresetal.,2012). Prevent-ingtheexpansionand urbanizationof zoonotic visceral leishmaniasis (ZVL) requires that the risk factors asso-ciated with human and canine infection be identified. Somecross-sectional serologicalsurveyshavesuggested thatcaninesusceptibilitytoinfectionisassociated with dogsize,furlength,age,andhousingconditions( Franca-Silva et al., 2003; Almeida et al., 2009; Galvez et al., 2010;Cortes etal., 2012).During a previously reported cross-sectional study carried out in an urban area of Brazil (Belo Horizonte, southeastern Brazil), we evalu-ated theprevalenceof and risk factors associated with L.infantuminfection in dogs,asidentified by meansof a molecular test (polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP) (Coura-Vital et al., 2011b). Some studies have evaluated the risk factors associated with L. infantum infection in dogs, however few have used a cohort studies (Belo et al., 2013),whichisthemostappropriateobservationaldesign to establish causal inference. Recently in cohort study we demonstrated that dogs with PCR positive for L. infantumshowed approximatelytwicethe risk of sero-conversionasthosethatwerePCRnegative(Coura-Vital et al., 2013).Herein, we report theresults of a cohort studydesigned toidentifytheincidenceofandrisk fac-torsfor L.infantuminfectionas identifiedbyPCR-RFLP; we evaluated thedomiciliary and peridomiciliary envi-ronments, the socioeconomicstatus of theowners, the typeofcareprovidedfortheanimals,andspecificanimal behaviors.

2. Methods

2.1. Baselinesurvey(cross-sectionalstudy)

The rationale for and organization of the study and the data collection methods have been described else-where(Coura-Vitaletal.,2011b).Brieﬂy,across-sectional studywascarried outinthenorthwest sanitarydistrict (36,874km2₎_of_Belo_Horizonte_in_2008._According_to_a cen-susconductedbytheBrazilianInstituteofGeographyand Statistics,thehumanpopulationofthisareawas331,362 in2000.Thecaninepopulationcomprised20,883animals in2008,accordingtotheZoonosisControlManagement. Atthetimeofthisstudy,theseroprevalenceinBelo Hori-zonte(7.6%)wassimilartothatinthenorthwestsanitary district(7.8%)(PBH,2007).WithanexpectedCVLpositivity inthestudyareabetween5%and10%,aconﬁdenceinterval (CI)of95%,andanestimatedprecisionof1.5%,therequired samplesizeforthestudywasapproximately1500animals. Atotalof918householdswerevisited,and1443dogswere includedinthebaselinesurvey(Coura-Vitaletal.,2011b). 2.2. Follow-upcohortstudy

We then conducted a follow-up cohort study con-sisting ofthree evaluations.EvaluationIwasconducted 10 monthsafterthebaseline survey(April2009),and a totalof282seronegative/PCR-negativedogswereenrolled. Thisnumberofdogswassimilartothenumberof PCR-positivedogsinthecross-sectionalstudy.Householdswith seronegative/PCR-negativedogswereselectedby proxim-itytotheseronegative/PCR-positivedogs.Thedogswere selectedfrom222owners,allofwhomwereinterviewed; alldogswereclinicallyexamined,andbloodwascollected byvenipuncture.EvaluationIIwasconducted16months after thebaseline survey (October2009), and 225dogs weretested.EvaluationIIIwascarriedout26monthsafter the baseline survey (August 2010), and 178dogs were tested.AllthedogsincludedinevaluationsIIandIIIwere subjectedtothesameproceduresusedinevaluationI.

Toevaluatelosstrendsduringthecohortstudy,we com-paredthesex,size, andfurlengthoftheincludeddogs withthesamevariablesfortheunincludeddogsineach evaluation.

2.3. Dataandsamplecollection

Atrainedresearchteaminterviewedtheownersofthe studyanimalsusingapreviouslytestedstructured ques-tionnairethatsoughtinformationregarding(i)theowner’s knowledgeaboutthehumandisease(i.e.,formof transmis-sionandclinicalsignsofhumanvisceralleishmaniasis); (ii)theowner’sknowledgeaboutthevector (characteris-ticsandpresenceinthedomicileandperidomicile);(iii)the owner’sknowledgeaboutthereservoirhost (epidemiolog-icalimportanceofthehost,clinicalsignsofCVL,anddog care);(iv)theowner’ssocioeconomiccharacteristics(per capita/familyincomeandschooling);(v)thecharacteristics of the domicile, annexes, and surroundings (i.e., struc-tureofroof,ﬂoor,andwalls;numberofrooms,including

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bedrooms;numberofresidents;andpresenceoftrees [par-ticularlybananatrees],rubble,manure,exposedgarbage, dry leaves, anda vegetable garden);(vi)themethodof garbagedisposal(collected,burned,orburied);and (vii) thepresenceofotherdomesticanimals(birds,cats,and cat-tle).Thefollowinginformationwascollectedforeachdog: age,sex,size,furlength,breed,behavior(habitsrelatedto theplacewherethedogsleptandspentmostofitstime, e.g.,street,residence,orbackyard),dogcare,clinical exam-inations,pasthistoryofvaccination,andserologicalexams previoustoleishmaniasis.

A sample of peripheral blood (5mL) was collected bypuncture ofthebrachiocephalic vein, andan aliquot wastransferredtoaglassvialcontainingsufficient anti-coagulant(ethylenediaminetetraaceticacid)toachievea final concentration of 1mg/mL. The blood sample was centrifuged(1500–1800×g;20min), and thebuffycoat fraction containingthe leukocytes wasremoved, resus-pended in an equal volume of 10mM Tris–HCl buffer (supplemented with 1mM ethylenediaminetetraacetic acid),andstoredat−70◦_C_until_PCR-RFLP._The_remaining portionofthebloodsamplewastransferredtotwoseparate filterpapers forsubsequentenzyme-linked immunosor-bentassay(ELISA)analysis.

2.4. ELISA

Each of theeluates fromthe blood dried onthe ﬁl-ter paperswastestedbymeansoftwo ELISAprotocols. The ﬁrst protocol used a Canine Leishmaniasis EIE Kit (Bio-Manguinhos/Fiocruz, Rio de Janeiro, Brazil), which employs a soluble antigen from promastigote forms of L. major-like parasites (ELISA-L. major-like). This sero-logy was performed in the Laboratory of Zoonosis of theBeloHorizonte HealthDepartment accordingtothe manufacturer’s instructions. The second ELISA proto-col was performed in parallel with soluble L. infantum (MHOM/BR/1070/BH46)antigenandwascarriedoutinthe LaboratoryofImmunopathologyoftheFederalUniversity ofOuroPretoasdescribedbyCoura-Vitaletal.(2011b). 2.5. PCR-RFLP

DNA was extracted fromthe buffycoat fractions by meansofaWizardGenomicDNAPuriﬁcationKit(Promega Corporation,Madison,WI,USA)accordingtothe manu-facturer’s instructions. Theprimers usedto amplifythe conservedregionoftheLeishmaniakDNAminicirclewere asfollows:forward,5-GGG(G/T)AGGGGCGTTCT(G/C)CG AA-3;reverse,5-(G/C)(G/C)(G/C)(A/T)CTAT(A/T)TTACAC CAACCCC-3 (Passosetal.,1999).AsinglePCRproduct of120bpwasgenerated (Degraveetal.,1994).PCRwas performedasdescribedbyCoura-Vitaletal.(2011b).

PCRamplicons(5␮L)weredigestedfor3hat37◦Cin 1UofHaeIII(Invitrogen,Carlsbad,CA,USA)in1×buffer (10mM Tris–HCl, 10mM MgCl2 [pH 7.5]) and enough Milli-Qwater tobring theﬁnal volumeto 15.0_␮L/well (MicroAmp FastOptical96-WellReactionPlate,Applied Biosystems, Foster City,CA, USA) (Volpini et al., 2004). Restrictionfragments,togetherwitha25-bpDNAladder (Invitrogen,SãoPaulo,Brazil),underwentelectrophoresis

in10%polyacrylamidegelsat40mAin89mMTrisbase (pH8.0),89mMboricacid,and2mM ethylenediaminetet-raaceticacid.Bandsweredetectedbysilverstaining,and thepatternswerecomparedwiththoseobtainedwithDNA fromL.(L.)amazonensis(strainMHOM/BR/1973/M2269),L. (Viannia)braziliensis(strainMHOM/BR/1975/M2903),and L. (L.) infantum(strain MHOM/BR/1972/BH46) from the DNAreferencelibraryattheLaboratoryof Immunopathol-ogyoftheFederalUniversityofOuroPreto.

2.6. Statisticalanalysis

DatabasesweregeneratedwithEpiDatasoftware (ver-sion3.2,EpiDataAssociation,Odense,Denmark)bydouble entryoftheresultsandweresubsequentlycorrected, com-pared, and analyzed with Stata software (version 11.0, StataCorp,CollegeStation, TX,USA). Theincidenceof L. infantum infection as indicated by PCR-RFLP was esti-matedper100dog-months.TheCoxproportionalhazard modelwasusedtoevaluatetheriskfactorsassociatedwith infectionbyL.infantum.Variablesthatwerestatistically significantbutexhibitedcolinearitywereexcludedfrom themultivariateanalysis,andcategoricalvariables were transformedintodummyvariables.Theunivariateanalysis wasperformedbymeansofKaplan–Meirsurvivalanalysis followingbyalog-ranktesttoexaminetheassociations betweeneachvariableandtimetoinfection.A multivari-ableadjustedmodelwasfittedwiththevariablesthatwere statisticallysignificantatp<0.25inunivariateanalyses.A step-by-stepbackwardselection procedurewasusedto selectthevariablesandtoproducethefinalmultivariate regressionmodel.Onlyadjustedvariablesshowinga signif-icantassociation(p<0.05)withtheoccurrenceofinfection byL.infantumremainedinthefinalmodel.Thestrengthof associationwasdeterminedbyahazardratioanda95%CI. TheSchoenfeldtestwasperformedtotesttheproportional hazardsassumption.

2.7. Ethics

Thestudywasapprovedby theEthicsCommitteein AnimalExperimentationoftheFederalUniversityofOuro Preto(protocolno.083/2007),oftheFederalUniversityof MinasGerais(protocolno.020/2007),andoftheBelo Hori-zonteCityCouncil(protocolno.001/2008).Allprocedures inthisstudywereconductedaccordingtotheguidelinesset outbytheBrazilianAnimalExperimentalCollege(federal lawnumber11794).Ownersofdogsparticipatinginthe projectwereinformedoftheresearchobjectivesandwere requiredtosignaninformedconsentformbeforesample anddatacollection.

3. Results

3.1. Characteristicsofthedogsandhousingconditions Thegender,size,andfurlengthofthedogsinthecohort study(50.0%female,50.0%mediumsized,and57.1%short fur)weresimilartothoseinthebaselinesurvey.Themean agewas54.3months(standarddeviation[SD]41.7),the median(interquartilerange[IQR])was48months(IQR24;

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Table1

Characteristicsofthedogsinthecohortstudy(n=282),Brazil2010.

Variable No. % Gender Female 141 50.0 Male 141 50.0 Size Small 81 28.7 Medium 141 50.0 Big 60 21.3 Furlength Long 161 57.1 Short 121 42.9 Age ≤24months 97 34.4 >24and≤84months 123 43.6 >84months 62 22.0

Originoftheanimal

Districtofresidence 158 56.0

Otherdistrict 124 44.0

Dogstayedpredominantlyinthebackyard

No 35 12.4

Yes 247 87.6

Sleepingplace

Insidethehouse 46 16.3

Inthebackyard 236 83.7

Hadaccesstothestreet

No 253 89.7

Yes 29 10.3

Veterinarycheckups

Yes 141 50.0

No 141 50.0

72).Onehundredtwenty-threedogs(43.6%)werebetween 24and84months.Mostoftheanimalslivedandsleptinthe backyard(87.6and83.7%,respectively)ratherthaninside theresidence.Only29(10.3%)ofthedogshadeasyaccess tothestreet.Halfofthedogswereregularlycheckedbya veterinarian(Table1).

Atotal of222householdswereselected.Theyhad a meanof1.3(SD0.8)dogsperhousehold(1–7dogs/house) andmedianof1(IQR1;1).Mostofthedwellings(142; 63.9%)weredetachedhouses,216(97.3%)hadplastered walls,183(82.4%)hadagarden,and100%wereservedby asewagemain.Garbagewascollectedatleast3timesper weekfrom202(91.8%)residences.Themeannumbersof roomsandbedroomsperhousewere7.2(SD2.8)and2.6 (SD0.9),respectively.Eachdwellinghadanaverageof4.1 (SD1.8)residents(datanotshown).

3.2. Lossestofollow-up

PriortoevaluationsIIandIII,57and47dogswerelost tofollow-uprespectively.Thereasonsforlossesto follow-up were euthanasia (seroconversion), death, change of address,household closed,refusal, and dogescape. The closedhouseswerevisited3timesbeforethedogswere consideredlosttofollow-up.Comparisonsofthe charac-teristicsoftheevaluateddogsandthoselosttofollow-up ineachevaluationphasedidnotrevealanydifferences. 3.3. IncidenceandriskfactorsforL.infantuminfection

Among282seronegativeandPCR-RFLP-negativedogs, 234showedfailureeventsasindicatedbyPCR-RFLPduring

Table2

Failureevents(PCR-RFLP),dog-monthsoffollow-up,andincidencerates with95%CIs,Brazil2010.

Follow-up PCR-RFLP

No.offailure events

Dog-months Incidencerate (95%CI)a EvaluationIb ₁₀₉ ₃₅₅₇ _3.1_(2.5–3.7) EvaluationIIc ₁₁₂ ₄₃₆ _25.7_{(21.3–30.9)} EvaluationIIId ₁₃ ₆₇ _19.4_{(11.1–33.1)} Total 234 4061 5.8(5.1–6.5) a_Per₁₀₀_dog-months.

b₁₀_months_after_the_baseline_survey. c₁₆_months_after_the_baseline_survey. d₂₆_months_after_the_baseline_survey.

follow-up(26months),andtheoverallincidencefor4061 dog-monthswas5.8per100dog-months(95%CI5.1–6.5). Theﬁrstevaluationshowed109events(PCR-RFLP),andan incidenceof3.1per100dog-months(95%CI2.5–3.7). Eval-uationsIIandIIshowed112and13events,withincidences of25.7(95%CI21.3–30.9)and19.4per100dog-months (95%CI11.1–33.1),respectively(Table2).Theresultsofthe preliminaryselectionofthevariablesfromtheunivariate analysis(p<0.25)aswellasthehazardratios(HR), num-beroffailureevents,andincidencesper100dog-months areshowninTable3.Thevariablesselectedtobuildthe ﬁnalmodel(p<0.15)weredogfurlength(long/short), pre-viouscaseofCVLinthehousehold(yes/no),unplastered housewalls(yes/no),regulargarbagecollection(<3×per week/≥3×perweek),accesstothestreet(yes/no),and pre-viousserologicalexaminationforCVL(yes/no).Infection withL.infantum(PCR-RFLP)wasassociatedwithaprevious caseofCVLinthehousehold(HR1.4;95%CI1.1–1.8)and unplasteredhousewalls(HR3.6;95%CI1.6–8.1)(Table4). 4. Discussion

Thepresentinvestigationshowedthattheconditionof thedomicileandthepresenceofa previouscaseofCVL inthehouseholdwereassociatedwithearlyL.infantum infectionindogs.Thus,itwaspossibletodeterminethe roleofhouseholdstructuresasapredictorofcanine infec-tionoccurrenceinurbanareas.Theseresultsarerelevant becausetheyallowabetterunderstandingofhow house-holdstructuremayaffecttheurbanizationofZVLandthus whatcontrolmeasuresmightbeadoptedtomore effec-tivelycontrolthespreadofthedisease.

Studieshaveshownthatmolecularmethodsnotonly lead to more accurate Leishmania detection ( Solano-Gallego et al., 2009) but also reveal the presence of protozoanDNAveryearly,beforeseroconversionand clin-icalmanifestations(Quinnelletal.,2001;Olivaetal.,2006; Coura-Vitaletal.,2011a).Inexperimentallyinfecteddogs, seroconversioncantakefrom1to6months(Morenoand Alvar, 2002),whereas in naturallyinfected animals,the mediantimetoseroconversionis10.5months(range,4–22 months) (Olivaet al., 2006).These factsemphasize the importance ofearlydetection of infectionbymolecular methods.ForCVLdiagnosis,PCRcanbeperformedon sam-ples froma broadrange oftissueswithvarious degrees ofsensitivity(Mannaetal.,2004;DiMuccioetal.,2012).

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Table3

UnivariateanalysisusingKaplan–MeiersurvivalanalysisandtheCoxregressionmodel,todetermineriskfactorsforLeishmaniainfantuminfection,Brazil 2010.

Variable No.offailureevents No.ofmeasured Timeatrisk Incid./100dog-month HR 95%CI p

Vector/reservoir Seenthevector

No 220 267 3859 5.7 1

Yes 14 15 210 6.7 1.47 0.85–2.53 0.17

PreviouscaseofCVLinthehousehold

No 162 198 2918 5.5 1

Yes 72 84 1151 6.3 1.35 1.02–1.78 0.04

Housingconditions Unplasteredhousewalls

No 228 276 4000 5.7 1

Yes 6 6 70 8.6 3.37 1.49–7.63 0.00

Regulargarbagecollection

≥3×/week 216 257 3682 5.9 1 <3×/week 15 22 343 4.4 0.63 0.37–1.05 0.08 Treesinbackyard No 122 152 2236 5.4 1 Yes 112 130 1832 6.1 1.17 0.91–1.52 0.22 Characteristicsofdogs Furlength Long 95 121 1747 5.4 1 Short 139 161 2322 6.0 1.21 0.93–1.57 0.16

Dogstayedpredominantlyinthebackyard

No 27 35 543 5.0 1

Yes 207 247 3526 5.9 1.40 0.93–2.08 0.10

Hadaccesstothestreet

No 207 253 3681 5.6 1

Yes 27 29 387 7.0 1.38 0.92–2.05 0.12

PreviousserologicalexaminationforCVL

Yes 179 217 3161 5.7 1

No 48 55 755 6.4 1.38 1.00–1.91 0.05

HR,hazardratio;CI,conﬁdenceinterval;CVL,caninevisceralleishmaniasis.

Althoughtheperipheralblood isnot themostsensitive sample, it is themost useful for mass surveysbecause samplecollectionislessinvasiveandtheuseof periph-eralbloodallowsserologytobeperformedconcurrently (Lachaudetal.,2002a,b),allowingthedetectionofmostof theinfecteddogs.

Theincidenceof L.infantuminfectionasdetectedby PCR-RFLPwashigh;attheendof26monthsoffollow-up, almostallthedogshadbeeninfected.Theseresultsindicate that Belo Horizonte is an area of active CVL transmis-sion.TheincidenceofCVLisanimportantepidemiologic parametertoconsiderinprioritizingtargetcontrolareas. DetectionofDNAbyPCReffectivelyassistsinCVL diagno-sis,mostlyfordogswithuncertainserologystatusorfor dogsthathavenotseroconverted(Martinezetal.,2011).

CVL is currently spreading to non-endemic areas in Europe (Dereure et al., 2009)and recently emerged in NorthAmerica(Petersen,2009).Furthermore,anincrease in the prevalence of ZVL has been observed in urban areas,whichmaybeattributedtohighpopulationdensity,

increasedmigration,environmentalchanges, inadequate livingconditions,andthepresenceofvectorandreservoir inthedomesticenvironment(Desjeux,2004;Oliveiraetal., 2008).BecausedogsarethemainreservoirsforL.infantum inhumans,weinvestigatedtheriskfactorsassociatedwith L.infantuminfectionindogs.Identiﬁcationoffactors associ-atedwithCVLmightbeusefulforidentifyingareasofhigher ZVLrisk,becauseinfectionindogsgenerallyprecedesthe occurrenceofhumancases(Nunesetal.,2010).

WeobservedthatcanineinfectionbyL.infantumwas associatedwiththepresenceofunplasteredhousewalls. Sandfliesarecrepuscularandnocturnal,andduringthe day,theyrestincomparativelycool,humidareas,including houses, latrines, basements, and wall fissures ( Killick-Kendrick,1999).Sandfliescanhideincracksinunplastered walls, which facilitatescontact betweenthevector and dogs.TheoccurrenceofLutzomyialongipalpisincracksand crevicesofhousewallshasbeenobservedinendemicareas ofnortheasternBrazil(DeaneandDeane,1962).Ina case-controlstudyconductedinNepal,cracksinhousewalls

Table4

RiskfactorsforLeishmaniainfantuminfectionindogs,Brazil2010.

Variable Crudehazardratio(95%CI) Adjustedhazardratio(95%CI)

PreviouscaseofCVLinthehousehold 1.3(1.0–1.8) 1.4(1.1–1.8)

Yesversusno

Unplasteredhousewalls 3.4(1.5–7.6) 3.6(1.6–8.1)

Noversusyes CI,conﬁdenceinterval.

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werefound to beassociated withelevated human vis-ceralleishmaniasisrisk(Bernetal.,2000).Acase-control studybyCostaetal.(2005)inTeresina,Brazil,revealedno associationbetweenriskof infectionbyL.infantumand unplasteredwalls,ceiling,floor,andwatersupply; how-ever,thelackofsewerageorregularcollectionofgarbage wasassociatedwithincreasedZVLincidence.These find-ingsdemonstratethatbasicpublicservicesareimportant incontrollingthespreadofthedisease(Costaetal.,2005). Inthecurrentstudy,almostallhouseholdsevaluatedhad sewerageandgarbagecollection,makingitimpossibleto detecttheinfluenceofthesefactorsoncanineinfection.

Housingconditionsgenerallyreﬂectsocioeconomic sta-tus.Thecross-sectionalstudyonwhichthiscohortstudy wasbaseddemonstratedthatonefactorassociatedwith L.infantuminfection(asindicatedbyPCR-RFLP)wasthe socioeconomic status of the owner (Coura-Vital et al., 2011b).Indeed, Oliveira et al. (2006) demonstrated an associationbetweenZVL and familyincome in theBelo Horizontemetropolitanarea.Ourdataarealsoconsistent withliterature conﬁrmingthat ZVL is more incident in areasofprecarioussocioeconomicstatus(Wernecketal., 2007).

InBrazil,themainstrategiesusedbytheVisceral Leish-maniasis Control and Surveillance Program have been vectorcontrolandcullingofinfecteddogs.However,these measureshave beenfoundtobeinsufﬁcientto contain diseasedissemination(Harhayetal.,2011).Inthisstudy, weobservedthatdogsresidingin householdswith pre-viouscaseofCVLshowedahigherriskofinfectionthan dogsresidinginhouseholdswithnopreviouscases. Simi-larresultswereobservedinastudyconductedinTeresina, Brazil,whereinhouseholdswithahistoryofdogremoval bytheprogram,theoddsofhavingatleastoneinfecteddog were5timestheoddsindwellingswithnohistoryofdog removal(daSilvaetal.,2012).Thisresultmayhavebeen duetoanenvironmentfavorabletothedevelopmentof thevectorinthedomiciles,causingtheoccurrenceofnew cases.AccordingtodaSilvaetal.(2012),simplyremoving aseropositivedogfromahousehold,withouttaking envi-ronmentalmanagementmeasures,doesnotpreventfuture infectionsinotheranimalsinthathousehold.

Onelimitationofcohortstudiesis losstofollow-up; however,inthecurrentstudy,thedogslosttofollow-updid notdiffersubstantiallyfromthosethatremained,as indi-catedbycomparisonofseveralcharacteristics.Therefore, theeffectoflosstofollow-upwasminimized.Thepresent studywasdesignednottoevaluatearepresentative sam-pleofBeloHorizontebuttoassesstheincidenceofand identifyriskfactorsforL.infantuminfection.However,the northwestsanitarydistrictisrepresentativeofthecityin termsofbuildings,commerce,residences,andgreenareas. 5. Conclusion

Weidentiﬁeddomicilecharacteristicsthatwere asso-ciated with an increased risk of canine infection by L. infantum.Theidentiﬁcationoftheseriskfactorsindicates thenecessityforinsecticidesprayingincracksandcrevices inthewallsof housestoreducebitingrateswithinand aroundhouses.Furthermore,ourresultsdemonstratethat

theVisceralLeishmaniasisControlandSurveillance Pro-gramshouldadoptenvironmentalmanagementmeasures inhomeswithpreviouscasesofCVLbecausethese house-holdsaremorelikelytomaintainthetransmissioncycle. Acknowledgements

This study was supported by the following grants: PNPD/Institutional/2011, DECIT/MS/CNPq/BR/grant: 57 6062/2008-1;FAPEMIG/BR/grant:CBB-APQ-3073-4.01/07, CNPq/BR/grant: 472554/2007-7; PPSUS/MS/ CNPq/ FAPEMIG/ SES-MG/grant CBB-APQ-00356-10; FAPEMIG/ PPM and FederalUniversity of Ouro Preto. Thefunders hadnoroleinstudydesign,datacollectionoranalysis,the decisiontopublish,orthepreparationofthemanuscript. ABRandMCaregratefulforCNPqfellowships,andWCV isgratefultothePNPD/CAPESfellowships.Wealsothank thestaffoftheSecretariaMunicipaldeSaúdedeBelo Hori-zonte, Minas Gerais, for cooperation, logistical support, andspecialdedicationtothiswork.

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