w w w . e l s e v i e r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Original
article
Are
opossums
a
relevant
factor
associated
with
asymptomatic
Leishmania
infection
in
the
outskirts
of
the
largest
Brazilian
cities?
César
Omar
Carranza-Tamayo
a,∗,1,
Guilherme
Loureiro
Werneck
b,1,
Gustavo
Adolfo
Sierra
Romero
a,1aNúcleodeMedicinaTropical,UniversidadedeBrasília(UnB),CampusUniversitárioDarcyRibeiro,Brasília,DF,Brazil
bDepartamentodeEpidemiologia,InstitutodeMedicinaSocial,UniversidadeEstadualdoRiodeJaneiro(UERJ),RiodeJaneiro,RJ,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received27July2015 Accepted17November2015 Availableonline8February2016
Keywords: Visceralleishmaniasis Brazil Didelphisalbiventris Leishmanin
a
b
s
t
r
a
c
t
Apopulationsurveywasconductedtoexploretheprevalenceandfactorsassociatedwith
LeishmaniainfectionintheFercalregionoftheFederalDistrict.TheFercalregionisagroupof
neighborhoodsinBrasíliainwhichthefirstcasesofvisceralleishmaniasisweredescribed.
Leishmaniainfectionwasestablishedbyapositiveleishmanintest.Althoughothertests
wereperformedinthestudy(animmunochromatographicassay(Kalazardetect®)anda
molecularassay),onlytheleishmaninskintestprovidedsufficientresultsforthe measure-mentofthediseaseprevalence.Dataontheepidemiological,clinicalandenvironmental characteristicsofindividualswerecollectedalongwiththediagnostictests.Aftersampling andenrollment,sevenhundredpeoplefrom2to14yearsofagewereincludedinthestudy. TheprevalenceofLeishmaniainfectionwas33.28%(95%CI29.87–36.84).Thefactors
associ-atedwithLeishmaniainfectionaccordingtothemultivariateanalysiswereageofmorethan
sevenyearsandthepresenceofopossumsnearthehome.Ageisaknownfactor
associ-atedwithLeishmaniainfection;however,thepresenceofwildanimals,asdescribed,isan
understudiedfactor.Thepresenceofopossums,whichareknownreservoirsofLeishmania,
inperi-urbanareascouldbethelinkbetweentheruralandurbanoccurrenceofvisceral leishmaniasisintheoutskirtsoflargestBraziliancities,assuggestedbypreviousstudies.
©2016ElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mailaddress:rhasec@unb.br(C.O.Carranza-Tamayo).
1 Allauthorscontributedequallytothiswork. http://dx.doi.org/10.1016/j.bjid.2015.11.013
1413-8670/©2016 Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/).
Introduction
Visceralleishmaniasis(VL)isanexpandingzoonosisinBrazil andLatinAmerica.Theinfectionhasprogressedfromrural areastothesuburbsoflargecities;thereisapossibilityof humans becoming infected in the urban areas ofsome of the largest cities in Brazil. The urbanization of VL is well described.1ThefirstBraziliancitiestoexperiencethis
phe-nomenonhaveexperienced40yearsofVLendemicity,2and
thefirstautochthonouscasesofVLinBrasília,occurringin theFederalDistrict,weredescribedin2005byourgroup.3
InBrazil,severalstudieshavefailedtodetectthefactors associatedwith positive testing for Leishmania.4–8 Arecent
meta-analysisreviewedLatin-Americanpublications,mostof whichwereBrazilian,lookingforfactorsassociatedwithVL anddescribedanassociationofolderage,presenceofdogsin thehouse,highercanineseropositivityinnearbyareas,lower socioeconomicstatus,andhighlyvegetatedareaswith
Leish-mania(L.) infantum infection.The association ofLeishmania
infectionandgendervariedaccordingtothetestperformed. Thefactorsassociatedwithseropositivitywerenotidentical tothose associatedwithdelayedskintestreactivity.9
Leish-maniainfectionwasfoundtobesignificantlyassociatedwith
variablessuchaslivinginthesame houseofaVLpatient, havingrelativeswithLV,orbeingover23monthsofage.10–13
Mostofthesestudieswereconductedinareaswithalengthy endemicity of VL. As noted by Belo et al., many studies assessingtheriskfactorsforVLarequestionedonthebasis ofanumberoffactors,suchastherelianceonsmallorpoorly representative samples, the strategiesto avoid refusals or notdescribingthemagnitudeoflosses,theuseofcombined diagnostictestsfordiagnosinginfections,andthecontrolof confounding variables and issues related to the statistical analysis.
Thisstudy aimsto describe the magnitude and factors associated with Leishmania infection in the area ofFercal, aconglomerationofneighborhoodsinwhichwepreviously described thefirst humancasesofVL inBrasíliaand from whichmostofthecasesofthediseasewerereportedbetween 2005and2010.
Patients,
material
and
methods
Thisresearchwasanobservational,cross-sectionalanalytical studyaimingtoestimatetheprevalenceofLeishmania infec-tioninthestudiedarea.
Studyareaandpopulation
Thearea identified asFercal, is locatedinthe administra-tiveregionofSobradinhoIIintheoutskirtsofBrasília,Brazil. Between 2005and2010, this regioncontributed morethan half ofthe casesof autochthonous VL in the Federal Dis-trict. According to census, the population of Brasília was 2455.903inhabitants in2010, and Fercalestimated popula-tionwas32,000people.Fercalcomprisesseveralcommunities separatedbymountainousareasofresidualforestandfarms locatedinaarearichinvegetationand watersources.The
occupationofthisregionhasgraduallyoccurredforabout60 years,originatingnearalimestonefactory.Thestudywas con-ductedbetween2007and2008andcoveredthecommunities ofAltoBelaVista,Bananal,BoaVista,Catingueiro,Córregodo Ouro,Curvas,EngenhoVelho,FercalIandII,QueimaLenc¸ol, Ribeirão-Pedreira, andRuadoMato,all ofwhichare inthe Fercalregion.
Samplesize
Assuming that there were approximately 5000 residents withinthe chosenagerange, anestimated15%prevalence rateofinfection,aprecisionof5%,asamplesizeof134 peo-plewouldbenecessarytobeincludedinthestudy.Because thisstudy wouldbethebeginningofafollow-upstudy,the samplesizewasinflatedbyafactorof5androundedto700 participants.Thenumberofsubjectsfromeachcommunity wascalculatedtakingintoaccountthenumberofhouseholds inthatcommunity,sothat73individualsfromAltoBelaVista, 132fromBananal,80fromBoaVista,20fromCórregodoOuro, 65 from Curvas,117from Engenho Velho,66 from FercalI, 27fromFercalII,57fromQueimaLenc¸ol,16from Ribeirão-Pedreiraand47fromRuadoMatowereincluded.
Randomization
Ineachparticipatingcommunitythehousestobecontacted wererandomlyselected.Onlyoneparticipantfromeachhouse was included.Houseswithout individualsinthetarget age rangeorincaseparticipationinthestudywasnotaccepted anotherhousewasrandomizedinthesamecommunity. Inclusioncriteria
The study included Fercalresidents agedtwo to 14 years, who fulfilled the followinginclusion criteria and agreedto participateinthestudyafterapprovalfromtheirparentsor guardians.Thefollowinginclusioncriteriawereused: resid-ingcontinuouslyformorethanoneyearinthestudyarea,no historyofsymptomaticleishmaniasis,andno contraindica-tionstobloodextractionortoadministrationofaleishmanin skintest(LST).
Laboratorytests
Theleishmanintest:Montenegro’santigencontainsalysate of promastigotes of L. (L.) amazonensis, strain MHOM/BR/ 73/M2269, batch 01/07 manufactured by the Centro de Produc¸ãoePesquisadeImunobiológicos(CPPI)fromthe Insti-tutodeSaúdedoParaná.Forthetest,0.1mLofthesuspension was intradermally inoculated in the forearm. The reading was performed 48–72h afterthe inoculation bymeasuring thediameter(inmillimeters)ofdermalindurationwiththe ballpoint-pen technique.Anindurationequalorsuperiorto 5mmwasconsideredpositive.Theimmunochromatographic assayKalazardetect®(InBiosInternational,Seattle,WA)was
performedonserumsamplesoftheincludedparticipants.The assayisbasedonthecolorshiftduetothepresenceof anti-bodiesagainsttherecombinantK39antigen.Theprocedures wereperformedaccordingtothemanufacturer’sindications.
Theserumandbuffersolutionweretestedatroom temper-atureusinganELISA plate asabase.Ribbonsand 20Lof serum were placed in the wells of the plate. Three drops ofbuffersolutionwereaddedtoeachwell,andthereading wasconductedafter10minofreaction. Positivitywas con-sideredwhentwolineswereevident(controlandpositive). Polymerasechain reactionswere performed on samplesof 300Lofwholeblood.Oligonucleotideswereusedtoamplifya targetof120bpoftheconservedregionoftheDNAminicircle. Thespecificsequencesoftheprimerswere:hm1–CCGCTA CCCCCCTTTTACACCAAC,MW=7504.8g/mol;hm2–GGG GAGGGGCGTTCTGCGAA,MW=6602.0g/mol;andhm3– GGCCCACTATATTACACCAACCCC,MW=7577.8g/mol.14
DNAextractionwasperformedusingthecommercialWizard GenomicPurificationKit® (Promega,USA),accordingtothe
manufacturer’sinstructions.Theelutionvolumewas100L. Eachreactionincludedtwonegativecontrolsandonepositive controlextractedfromculturedpromastigotesofL.infantum.
Thepreparationoftheamplificationmixturewasperformed inanisolatedenvironment,irradiatedwithUVlightpriorto mixingandfreeofDNA-amplifiedproductsorbiological sam-ples.Themixingwas performedinafinal volumeof10L containing1LofthesampleDNA,0.2mMofdNTPs,10mM ofTris–HCl(pH8.6),50mMofKCl,1.5mMofMgCl2,12pmolof
eachprimerand0.25UTaqpolymerase(Promega,USA).The followingamplificationconditionswereused:initial denatur-ationat95◦Cfor5min,39cyclesofdenaturationat95◦Cfor
30s,annealingat66◦Cfor30s,extensionat72◦Cfor30sand
finalextensionat72◦Cfor30s.PCRwasperformedusinga
PerkinElmerGeneAmpPCRSystem2400(PerkinElmerCorp., Norwalk,CT,USA).Afteramplification,3Lofthesamplewas subjectedtoelectrophoresisona6%polyacrylamidegeland stainedwithsilvertovisualizetheamplifiedproducts. Casedefinition
Apositiveresultbyanyofthediagnostictestsadministered definedaninfectedcase.
Statisticalanalysis
A database was constructed using SPSS 16.0 (SPSS Inc., Chicago,IL,USA) containingdataon subject identification, clinicalexamination,animalhusbandry(petsandlivestock), houseand neighborhoodcharacteristics, andresultsofthe tests.ThestatisticalanalysiswasperformedusingPearson’s chi-squaretest tocomparethe categoricalvariables.When indicatedtwo-tailedFisher’sexacttestwasused.Tocompare meansofvariableswithanormaldistribution,Student’sttest wasused,andifthesevariablesshowedanon-Gaussian distri-butiontheMann–WhitneyUtestwasused.Thesignificance levelwassetatp<0.05.Therelationshipbetweeninfection
withLeishmania(positivitytotheLST)andtherecordeddata
wasexploredwithunivariatelogisticregression.Apredictive modelwasselectedtoidentifythevariablesassociatedwith
theriskofLeishmaniainfectiondetectedbytheMontenegro
skintestpositivity.Thevariablesusedwerethosethatwere biologically or epidemiologically plausibly associated with theoutcome.Initially,weusedaone-stepunivariatelogistic regression,whichenabledtheselectionofthevariablesthat
wouldbefurtherevaluatedinthemultivariateanalysisofthe sametype,incompliancewiththecriterionvalueofp<0.20. Priortotheentryofthevariablesinthemultivariate analy-sis,acollinearitytestwasperformed,and,subsequently,the interactionsbetweenthevariablesthatshowedstatistical sig-nificancewereassessed.Allthetestswereconductedwitha 95%levelofconfidence.
Ethicalissues
Theparentsorlegalguardiansofindividualsincludedinthe studywereinformedofthepossiblerisksofthestudy proce-dures,andthosewhoagreedtoparticipatesignedaninformed consentform.TheprojectwasapprovedbytheEthics Com-mitteeoftheFacultyofMedicineoftheUniversityofBrasília (CEP-FM)–CEPFM073/2006process–on28/03/2007.Thestudy compliedwiththerecommendationsoftheHelsinki Declara-tionandresolutions196/1996and251/1997fromtheNational BoardofHealthonresearchinvolvinghumans.
Results
Sevenhundredsubjectswereincluded.Thedistributionofthis samplebythespecificcommunityofresidenceisshownin Table1.Theparticipants’agerangedfromtwoto14years, with a mean of 8.63±3.17 years and a median of9 years (Q1/4=6.0 and Q3/4=11.0). Most of the evaluated subjects (590/700,84.3%)werebornintheFederalDistrict.The aver-agelengthofresidenceintheareawas7.38±3.5years,with amedianof7years(Q1/4=5.0and Q3/4=10.0).Ofthe chil-drenand adolescentsevaluated, 104(14.9%)hadpreviously livedindistrictswithtransmissionofVL,asdeterminedby thedatanotificationsoftheMinistryofHealthatthetimeof thestudy.Mostoftheparticipants(56/104,53.8%)hadlivedin endemicareasofthenortheastregion,followedby37(35.6%) whoreportedhavinglivedinthestateofGoiás.In85(12.1%) cases,atleastonedoghadbeencollectedbystaffof environ-mentalsurveillanceagencyforsuspectedVL.Twelvesubjects (1.7%)reportedatleastonecaseofVLinthefamily.No chil-dren or teenagers with fever,hepatomegaly, splenomegaly, orpallorwerefound.Duringthestudy(2007–2008),no indi-viduals wereclassified asasuspectedcaseofVL,andthey wereallclassifiedasasymptomatic(n=700).Oneparticipant hadaskinlesion,whichwasnotcompatiblewithcutaneous leishmaniasis.
Regardingthecharacteristicsofthedwellings,10yearswas themedianageofthehouses(Q1/4=5.0andQ3/4=13.0).Most of the houses were constructedof masonry(92.9%), and a fewhouses werewooden(6.7%).Thisconditionoccurredin allthecommunities,withtheexceptionofQueimaLenc¸ol,in which26.3%ofthedwellingswere builtofwood.Allofthe assessedhouseholdshad accesstoservices providing elec-tricityandwatersupply.Publicsewerserviceswerefoundin onlyoneareaoftheFercalIcommunityandintwohousesof QueimaLenc¸ol(whichdrainedtothesewersystemofthelocal school).Thenumberofresidentsperhomerangedfromtwo to13people,withamediannumberoffourpeople(Q1/4=4.0 andQ3/4=6.0).Inallofthehouses,thepresenceof annoy-ing insects inside the home was reported. In response to
Table1–Studysampledistributionaccordingtogenderandcommunitiesofresidence.
Community Gender Total
Male(%) Female(%)
AltoBelaVista 34(47) 39(53) 73
BananalI 37(56) 29(44) 66 BananalII 36(55) 30(45) 66 BoaVista 39(49) 41(51) 80 CórregodoOuro 9(45) 11(55) 20 Curvas 33(51) 32(49) 65 EngenhoVelho 66(56) 51(44) 117 FercalI 35(53) 31(47) 66 FercalII 14(52) 13(48) 27 QueimaLenc¸ol 32(68) 25(32) 57 Ribeirão–Pedreira 7(44) 9(56) 16 RuadoMato 27(57) 20(43) 47 Total 369(53) 331(47) 700
questioning,59(8.4%)oftheparentsandcaregiversreported thattheyprotectedtheirchildrenusingmosquitonetsinthe bedroom,andknowledgeregardingtheuseofmosquitonets impregnatedwithinsecticideswasreportedbythepopulation. Inthepreviousyear,insecticidehadbeensprayedbyhealth workersin37(5.3%)homesoftheevaluatedchildrenand ado-lescents.Somesprayinghadbeenperformedinhouseswith seropositivedogsafteracanineserologicalsurvey.Aminority ofthesprayingwasperformedduetoconvenience.Atleast onedoglived in305(43.6%)ofthe dwellings.Thisnumber rangedfromzerotosevendogsperhousehold.Theoverall averagewas 0.7±1dog/house.When onlythe houses with the presenceof dogs were evaluated, that value increased to 1.61±0.92dogs/house, witha median of onedog/house (Q1/4=1and Q3/4=2).Oftheevaluated dogs,the predomi-nantbreedwasmongrel,whichwasfoundin387(90.4%)of thecases.Thedogshadamedianageoftwoyears.Maledogs weremorenumerousthanfemales(248vs.180).Thedogswere foundpredominantly inthe FederalDistrict, withatotalof 414(96.7%)dogs,andmorethanahalfofthemwerebornin thecommunityofresidence.Theparticipantsin316(45.1%) householdsreportedkeepinganimalsother thandogs.The mostfrequentlyreportedanimalswerechickens(27.9%),other birds(14.6%),cats(13.6%),pigs(5.6%),andcattle(3.3%).In225 dwellings(32.1%),theparentsorguardiansoftheindividuals assessedreportedthepresenceofmammalsinthevicinity. Asthecommunitiesweremoredistantfromtheurbanarea ofSobradinhoII,thepresenceofwildanimalsbecamemore frequent.Thepresenceofopossums(28.9%),monkeys(5.7%), foxes(1.7%),andarmadillos(1.4%)wasreported.Thisstudy didnotdefinethespecieofopossum.IntheFercalareathe prevalentspeciesisDidelphisalbiventris.
ThediagnostictestsresultsareshowninTable2.The preva-lenceofinfection,asdetectedbyapositiveleishmaninskin test,wasnothomogeneousinallofthecommunities, ran-gingfrom19.2%(AltoBelaVista)to53.8%(BoaVista),withthe highestvaluesbeingfoundinthecommunitiesmostremote fromthe urbanarea ofSobradinho. Thepositivityratewas higheramongparticipantsagedeighttofourteenyears(39.7%) comparedtothegroupofchildrenuptosevenyearsofage (22.5%)(X2=21.86,p<0.001).Amongthefourchildrenwitha
positivePCRtest, twowerepositiveand twowerenegative forLST.
Thestatisticalanalysisofthefactorsassociatedwiththe outcomewasperformedusingtheresultsoftheLSTbecause ofthelowpositivityoftheappliedserologicalandmolecular tests.Theunivariateanalysisshowedthatthepredominant associatedfactorwasthepresenceofdomesticorwild ani-malsinoraroundthehouses,asshowninTable3.
Fourteenvariablesweresubjectedtomultivariateanalysis afterevaluationaccordingtotheMenardandMyerscriteria ofcollinearity.Thefinalresultshowedthathumaninfection definedbypositiveLSTwasassociatedwithagegreaterthan sevenyears(OR=3.04,95%CI1.86–4.99)andwithpresenceof opossumsaroundthehouses(OR=1.46,95%CI1.02–2.10).The other variableswerenotindependentlyassociatedwiththe outcome,asseeninTable4.
Discussion
Thestudydemonstratedtheexistenceofasymptomatic
infec-tionbyLeishmaniaspp.intheFercalarea.Themagnitudeof
asymptomatic infectioninthe studyarea wasgreater than expected(33.28%).Someofthestudiedcommunitieshada prevalencesimilartoendemicregionsinwhichtransmission hasbeendemonstratedfordecades.Fewstudieshave investi-gatedtheextentofinfectioninareaswithrecentVLcases.The prevalenceofinfectionwassimilartothatreportedbyCunha etal.,whoevaluatedasymptomaticchildrenandadultsinan areaofrecentintroductionofVLinthestateofBahia.15Inthe
city ofNatal,Northeast Brazil,fiveyearsafterthereportof thefirstcaseofhumanVL,Jeromeetal.reporteda38% pos-itivityoftheneighborsandcohabitantsofpeoplewithVL.16
InthecityofTeresina,alsoinNortheastBrazil,asurveyusing identicalmethodologytothatusedinthisstudy,Gouveaetal. describedaprevalenceofinfectionof36.7%.8
Fercal isarecentlysettled territory;therefore, the deci-siontoselectchildrenandadolescentsforthestudyavoided biasrelatedtoaparticipanthavingpreviouslylivedinareasof leishmaniasistransmission.Becausealeishmanintestcould remain positive aftermany years ofcontact withendemic
Table2–PrevalenceofLeishmaniainfectionaccordingtothetypeoftestused(n=700).
Test Positive Prevalence CI95%a
Leishmaninskintest 233 33.28% 29.87–36.84
PCR 4 0.57% 0.18–1.37
Leishmaninskintest+PCRb 235 33.57% 30.17–37.15
Kalazardetect® Zero Zero 0–0.46
a 95%confidenceintervaloftheprevalence. b Positivitytoanyappliedtest.
Table3–UnivariateanalysisoffactorsassociatedwithLeishmaniainfectioninFercal,Brasília–DF.
Variables OR IC95% p
Malegender 1.33 0.97–1.83 0.07
BorninamunicipalitywithreportingofautochthonousVL 1.12 0.71–1.77 0.63
Age(inyears) 1.12 1.06–1.18 <0.01
Morethansevenyearsold 2.27 1.60–3.21 <0.01
ResidencetimeintheFercalregion(inyears) 1.06 1.02–1.11 0.01
TimeofresidenceinFercalregionmorethansevenyears 1.47 1.07–2.01 0.02
LivedinacitywithreportedautochthonousVL 1.31 0.84–2.01 0.23
FamilyhistoryofVL 2.03 0.65–6.37 0.22
LivingathomewithdogswithsuspectedVL 0.92 0.57–1.50 0.75
Withoutbednet 1.15 0.64–2.05 0.64
Ageofthehouse(inyears) 1.03 1.01–1.05 0.01
Ageofthehouse(>9years) 1.44 1.05–1.98 0.02
Numberofresidentsinthehouse 1.04 0.94–1.14 0.47
Elevationofhouse(inmeters) 1.00 0.99–1.01 0.66
Housenotsprayedwithinsecticidesduringthelastyear 0.92 0.46–1.84 0.81
Numberofdogsinhouse 1.27 1.09–1.48 <0.01
AtleastonedogborninamunicipalitywithautochthonoustransmissionofVL 1.59 0.45–5.61 0.47 PresenceofdogsdisplacedfrommunicipalitieswithindigenoustransmissionofVL 1.28 0.58–2.84 0.55
Dogswhostayinthebackyard 1.50 1.09–2.08 0.01
PresenceofdogswithsignsconsistentwithcanineVL 0.89 0.25–3.11 0.85
Horsebreeding 2.61 1.20–5.67 0.02 Rabbitbreeding 0.60 0.16–2.19 0.44 Cattlebreeding 2.70 1.17–6.25 0.02 Henbreeding 1.29 0.91–1.82 0.15 Catbreeding 1.26 0.81–1.98 0.31 Nogallinaceouspoultry 1.35 0.88–2.09 0.17 Raisingducks 2.86 0.90–9.12 0.08 Raisingturkeys <0.01 0 0.99 Pigbreeding 2.21 1.16–4.24 0.02 Presenceofopossums 1.52 1.08–2.14 0.02 Presenceofmonkeys 0.96 0.49–1.90 0.91 Presenceoffoxes 0.66 0.18–2.48 0.54 Presenceofarmadillos 3.06 0.85–10.95 0.09
Table4–MultivariatelogisticregressionanalysisofvariablesassociatedwithLeishmaniainfectioninFercal,Brasília–DF.
Variables OR IC95% pvalue
Malegender 1.35 0.97–1.88 0.08
Age>7yearsold 3.04 1.86–4.99 <0.01
TimeofresidenceinFercalregion(>7years) 0.63 0.40–1.00 0.05
Ageofthehouse(>9years) 1.26 0.90–1.77 0.18
Numberofdogsinhouse 1.09 0.87–1.38 0.45
Dogswhostayinthebackyard 1.19 0.76–1.87 0.45
Horsebreeding 1.27 0.43–3.73 0.66 Cattlebreeding 1.06 0.35–3.23 0.92 Henbreeding 0.87 0.57–1.32 0.50 Nogallinaceouspoultry 1.22 0.77–1.93 0.41 Raisingducks 2.07 0.57–7.45 0.27 Pigbreeding 1.32 0.55–3.20 0.54 Presenceofopossums 1.46 1.02–2.10 0.04 Presenceofarmadillos 1.76 0.45–6.85 0.42
areas,theselectionofyoungpeoplehelpstoreducethatbias, duetodecreasedexposuretime.Theleishmanintestwhich wasselectedtodefinethestateofLeishmaniainfectionhasthe followingadvantages:itisinexpensive;itissimpletoapply andread;noequipmentorlaboratoryfacilitiesarerequired fortheapplication,readingandinterpretation;anditenables themeasurementofinfectionbyLeishmaniaspeciescausing VLinareaswithoutthepresenceofcutaneousleishmaniasis (CL).Somestudieshavesuggestedthatpeoplewitha nega-tiveleishmanintesthadeithernocontactwiththeparasite orhavehadcontactwithoutdevelopingreactivitytothetest. ThelatterareindividualsmorelikelytobecomeillwithVL.17
TheFercalarea hassporadiccasesofCL,and thereareno reportsofthemucosalformofleishmaniasis.From2000to 2008,307casesofCLwere reportedinBrasília,and among them,twocaseswereconsideredautochthonousfromthe Fer-calregion.18Inpopulationsthathavehadrecentorsporadic
casesofCL,theprevalenceofasymptomaticinfection identi-fiedbyapositiveLSThasbeenshowntorangebetweenzero and3.8%19;inareas ofhighendemicity includingCortede
PedraintheStateofBahia,theprevalenceincreasesto47%.20
Thesefindingssuggestthatthepositiveresultsofthisstudy reflectthemagnitudeofL.infantuminfection.
Thefindingofzeroprevalencewhenan immunochromato-graphicassayisusedisindisagreementwithpreviousstudies ofasymptomaticinfectionbyLeishmania.Thistesthasbeen usedinBrazilforthispurpose,andvariableprevalencerates havebeenobservedinstudiesperformed.Astudyconducted in MinasGerais showed that the rapid test had low posi-tivityratesinpopulationsurveys,respectively5.6%and4.3% inSabará4and4,3%inPorteirinha.6InMontesClaros–MG,
nopositivecaseswerefoundamongblooddonorswith posi-tiveserologicaltestsforLeishmania.21Themoleculartestused
inthis study had lower performancethan those described previously. In Brazil, the positivity of this test has varied between7.2%inTeresina(Piauí)22and29.7%inBeloHorizonte
–MG.23Thesestudieswereperformedinrelativesofpeople
withVL,whichcouldexplainthehigherpositivityratewhen comparedwithsurveysinrandomlyselectedasymptomatic individuals.Thesmallparasiticloadinasymptomaticpatients mayexplainthelowperformanceofourmoleculartests.4The
findingthatnotallpatientswithapositiveserologictesthave positiveDNAtestsshouldnotnecessarilybeinterpretedasa lackofspecificity,butasademonstrationoftherapid circula-tionoftheparasiteintothebloodstream,whichcouldexplain the association ofa positive leishmanintest with asymp-tomaticinfection.Positivemoleculartestsmightbepresentin asymptomaticindividualswithnegativeserologyaswell.22,24
Theestimatedprevalence(33.3%,95%CI29.9–36.8%)inthe Fercalregionwasbasedontheresultsoftheleishmanintest. Thisprevalenceisinlinewithprevioussurveysinareasof recentintroductionofthediseaseinBahiaandRioGrandedo Nortewherepositivityratesof32%and38%,respectively,were found.15,16Inareasoflong-lastingVLtransmissioninBrazil,
theprevalencehasvariedfrom2.9to71%.22,23 We
hypoth-esizethattheprevalenceidentifiedinthisstudyisaresult recentintroductionofbyLeishmaniaspeciesinfectioninthe area,wherethereiswidespreadsusceptibilityofdwellers.This explainstheasymptomaticinfectionofalargenumberof sus-ceptibleindividuals,particularlythosewithnopriorcontact
withendemicareas,suchaschildrenandadolescentsbornin theareaoftherecentintroductionoftheparasite.
Fourteencharacteristicswerepotentiallyassociatedwith theprevalenceofLeishmaniainfectionmeasuredbyLST.After themultivariatelogisticregressionanalysis,thetwovariables thatremainedassociatedwiththeoutcomewereolderageand presenceofopossumsintheneighborhood.Previousstudies havedescribedtheassociationvariable magnitudebetween ageandasymptomaticVL.However,mostofthemagreethat riskbeginstoincreasebefore10yearsofage.InItaly,Biglino etal.studiedLeishmaniainfectionusingWesternblotanalysis andreportedthattheageassociatedwiththeconditionwas over65years.25OurstudycorroboratesthefindingsofBadaró
andcolleagues,whodescribed,inJacobinacityofBahia,Brazil, thatpositivityfortheleishmanintestbegintoincreaseatthe ageofsevenyears.26InthemunicipalityofRaposa,in
Maran-hão, Brazil,Caldas etal.indicated 23monthsasthecutoff ageassociatedwiththeriseinprevalence,showingthat chil-dreninendemicareasareinfectedatanearlyage.Inanother municipalityinMaranhão,thecutoffagewasfiveyears,and 56.7%ofchildrenandadolescentswerepositiveforthe leish-manintest,comparedto35% ofthe childrenyounger than five(p<0.001).11InTeresina,inPiaui,Gouveaetal.reported
anassociationofagewithapositiveleishmanintest; how-ever, this association disappeared when the outcome was seropositivity.8 Inanarea ofrecentintroductionofthe
dis-easeinthestateofBahia,Cunhaetal.foundnoassociationof infectionwithageortheassessedcharacteristics.15
Addition-ally,astudyconductedinalow-endemicityarea,Arac¸atuba, SãoPaulobyBarãoetal.showednoassociationofVLwith age.12Regarding thepresenceofopossumsintheassessed
dwellings,thisanimalwasreportedmostfrequently.Anopen questionwasaskedtoassessthisvariable,andthequestion wasintendedtopertainonlytomammalsandtothepresence ofwildanimalsaroundthehomes.Thepresenceofopossums wasreportedinanevenhigherproportionthanthatoffarm animals, and opossumswere seen morefrequentlyaround housesthanotheranimals,exceptfordogs.Theroleofthis marsupialasareservoirofLVhasbeenshown,27,28andsome
authors consider the speciesto bea potential reservoirin urbanareas.28 Althoughthefirstreferences toitspotential
asareservoirwereintheearly1980s,inBrazil,itsrolehas rarelybeenstudied,andtherearemorefrequentreportsin ColombiaandVenezuela.29–31Theopossumhasbeenlinkedto
thetransmissionofL.infantumchagasiandthespecies
associ-atedwithCL.32,33InColombia,theopossumandawildrodent
wereshowntobethebestdietarysourcesofLutzomyiaevansi
(theVLvectorinnorthernColombia).31InRiodeJaneiro,this
marsupialhasbeenassociatedwithcanineVL.34Asreported
inthestudyconductedinBeloHorizonte,theopossumcould representanimportantreservoirofVL,tobeconsideredin thecontrolmeasuresforthisinfection.28Onestudyofcases
ofVLinBeloHorizontefrom1999to2000showed thatthe presenceofhouseholdanimalswasrelatedtotheoccurrence ofthediseaseandconsideredthepossibilityofmarsupial– inadditiontodogs,horses,cattleandpigs–involvementin VLtransmission.35Arecentstudyperformedinconservation
unitsaroundBrasíliafoundDidelphisalviventriswithpositive moleculartestsforLeishmania,confirmingthepresenceofthis speciesasapotentialreservoirandsuggestingthatasylvatic
cycle could bebeginning to settle inthis area.36 Complete
understandingoftheroleofopossumsintheepidemiology ofleishmaniasisshouldprovideappropriateinformationfor controlactivities.
Thisstudyhighlightstheimportanceanddistributionof asymptomatic infection by Leishmania inan area ofrecent introductionofVL.Newstudiesthatovercomethelimitations facedinthisresearcharewarrantedinordertocheckthe con-sistencyofourfindings.Thisstudysuggeststhatopossums couldbethelinkinthetransmissionofVLbetweenruraland urbanareas.Theroleplayedbyopossumsinattractingvectors fromwildtourbanareasshouldbefurtherexploredinfuture studies.Wesuggest thatspecificstudiesshouldbedirected atunderstandingtheroleofsynanthropicanddomestic ani-malsotherthandogsinareasofrecenttransmissionofVL. Theinvestmentinreducing theasymptomatic infection by
L.infantumismeaningfulconsideringthatco-infectionwith
Leishmania/HIVisincreasinginBrazil,withahigherfatality
rate;atleastsomeofthoseco-infectedindividualsacquired
Leishmaniainfectionatanearlierphaseoflife.37
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
ToRenataRiberode Sousa,technicianatthe Laboratoryof LeishmaniasisoftheTropicalMedicineCenterofthe Univer-sityofBrasília,forhervaluablecontributiontothelaboratory work;toJoséBarbosaBezerra,forhiscollaborationinthefield work;andtotheofficersoftheEpidemiologicalSurveillance UnitoftheStateSecretaryofHealthofFederalDistrict(SES-DF) fortheirsupportduringthefieldwork.
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1. WerneckGL.Forum:geographicspreadandurbanizationof visceralleishmaniasisinBrazil.CadSaudePublica. 2008;24:2937–40.
2. DrumondKO,CostaFAL.Fortyyearsofvisceralleishmaniasis intheStateofPiaui:areview.RevInstMedTropSPaulo. 2011;53:3–11.
3. Carranza-TamayoCO,CarvalhoMS,BredtA,etal. AutochthonousvisceralleishmaniasisinBrasília,Federal District,Brazil.RevSocBrasMedTrop.2010;43:396–9. 4. MorenoEC,MeloMN,LambertucciJR,etal.Diagnosing
humanasymptomaticvisceralleishmaniasisinanurbanarea oftheStateofMinasGerais,usingserologicalandmolecular biologytechniques.RevSocBrasMedTrop.2006;39:421–7. 5. SilveiraFT,LainsonR,DeSouzaAA,etal.Furtherevidences
onanewdiagnosticapproachformonitoringhuman Leishmania(L.)infantumchagasiinfectioninAmazonianBrazil. ParasitolRes.2010;106:377–86.
6. RomeroHD,SilvaLdeA,Silva-VergaraML,etal.Comparative studyofserologictestsforthediagnosisofasymptomatic visceralleishmaniasisinanendemicarea.AmJTropMed Hyg.2009;81:27–33.
7. OliveiraAL,PaniagoAM,SanchesMA,etal.Asymptomatic infectioninfamilycontactsofpatientswithhumanvisceral
leishmaniasisinTrêsLagoas,MatoGrossodoSulState,Brazil. CadSaudePublica.2008;24:2827–33.
8.GouveaMV,WerneckGL,CostaCH,deAmorimCarvalhoFA. FactorsassociatedtoMontenegroskintestpositivityin Teresina,Brazil.ActaTrop.2007;104:99–107.
9.BeloVS,WerneckGL,BarbosaDS,etal.Factorsassociated withvisceralleishmaniasisintheAmericas:asystematic reviewandmeta-analysis.PLoSNeglTropDis.2013;7:e2182, http://dx.doi.org/10.1371/journal.pntd.0002182.
10.JeronimoSM,TeixeiraMJ,SousaA,ThielkingP,PearsonRD, EvansTG.NaturalhistoryofLeishmania(Leishmania)chagasi infectioninNortheasternBrazil:long-termfollow-up.Clin InfectDis.2000;30:608–9.
11.CaldasAJ,CostaJM,SilvaAA,VinhasV,BarralA.Riskfactors associatedwithasymptomaticinfectionbyLeishmaniachagasi innorth-eastBrazil.TransRSocTropMedHyg.2002;96: 21–8.
12.BarãoSC,deFonsecaCamargo-NevesVL,ResendeMR,da SilvaLJ.Humanasymptomaticinfectioninvisceral
leishmaniasis:aseroprevalencestudyinanurbanareaoflow endemicity.Preliminaryresults.AmJTropMedHyg.
2007;77:1051–3.
13.NascimentoMDSB,SouzaEC,daSilvaLM,etal.Prevalência deinfecc¸ãoporLeishmaniachagasiutilizandoosmétodosde ELISA(rK39eCRUDE)eintradermorreac¸ãodeMontenegroem áreaendêmicadoMaranhão,Brasil.CadSaudePublica. 2005;21:1801–7.
14.DegraveW,FernandesO,CampbellD,BozzaM,LopesUG.Use ofmolecularprobesandPCRfordetectionandtypingof Leishmania—amini-review.MemInstOswaldoCruz. 1994;89:463–9.
15.CunhaS,FreireM,EulalioC,etal.Visceralleishmaniasisina newecologicalnichenearamajormetropolitanareaof Brazil.TransRSocTropMedHyg.1995;89:155–8.
16.JeronimoSM,OliveiraRM,MackayS,etal.Anurbanoutbreak ofvisceralleishmaniasisinNatal,Brazil.TransRSocTrop MedHyg.1994;88:386–8.
17.IbrahimME,LambsonB,YousifAO,etal.Kala-azarinahigh transmissionfocus:anethnicandgeographicdimension.Am JTropMedHyg.1999;61:941–4.
18.CarvalhoMSL,BredtA,MeneghinERS,Flebotomíneosde OliveiraC.(Diptera:Psychodidae)emáreasdeocorrênciade leishmaniosetegumentaramericananoDistritoFederal, Brasil,2006a2008.EpidemiolServSaude.2010;19:227–37. 19.SalomónOD,SosaEstaniS,MonzaniAS,StuderC.Brote
epidémicodeleishmaniasiscutáneaenPuertoEsperanza, Misiones,1998.Medicina(BAires).2001;61:385–90. 20.NoronhaEF[dissertation]Avaliac¸ãodoefeitodousode
mosquiteirosimpregnadoscominseticidasobrea
transmissãodeleishmaniosetegumentaramericanaemárea decolonizac¸ãoantiga.Brasília.Brasília:Universidadede Brasília;2003,140pp.
21.UriasEVR,CarvalhoSFG,OliveiraCL,etal.Prevalênciade adultosinfectadosporLeishmanialeishmaniachagasientre doadoresdesanguedoHemocentroRegionaldeMontes Claros,MinasGerais,Brasil.RevBrasHematolHemoter. 2009;31:348–54.
22.CostaCH,StewartJM,GomesRB,etal.Asymptomatichuman carriersofLeishmaniachagasi.AmJTropMedHyg.
2002;66:334–7.
23.VianaLG,deAssisTS,OrsiniM,etal.Combineddiagnostic methodsidentifyaremarkableproportionofasymptomatic Leishmania(Leishmania)chagasicarrierswhopresent modulatedcytokineprofiles.TransRSocTropMedHyg. 2008;102:548–55.
24.RieraC,FisaR,UdinaM,GallegoM,PortusM.Detectionof Leishmaniainfantumcrypticinfectioninasymptomaticblood donorslivinginanendemicarea(Eivissa,BalearicIslands,
Spain)bydifferentdiagnosticmethods.TransRSocTropMed Hyg.2004;98:102–10.
25.BiglinoA,BollaC,ConcialdiE,TrisciuoglioA,RomanoA, FerroglioE.AsymptomaticLeishmaniainfantuminfectioninan areaofnorthwesternItaly(Piedmontregion)wheresuch infectionsaretraditionallynonendemic.JClinMicrobiol. 2009;48:131–6.
26.BadaróR,JonesTC,Lorenc¸oR,etal.Aprospectivestudyof visceralleishmaniasisinanendemicareaofBrazil.JInfect Dis.1986;154:639–49.
27.HansonWL,ChapmanWLJr,HendricksLD.Leishmania donovaniintheopossum(Didelphismarsupialis).JParasitol. 1980;66:700–1.
28.SchalligHD,daSilvaES,vanderMeideWF,SchooneGJ, GontijoCM.Didelphismarsupialis(commonopossum):a potentialreservoirhostforzoonoticleishmaniasisinthe metropolitanregionofBeloHorizonte(MinasGerais,Brazil). VectorBorneZoonoticDis.2007;7:387–93.
29.CorredorA,GallegoJF,TeshRB,etal.Didelphismarsupialis,an apparentwildreservoirofLeishmaniadonovanichagasiin Colombia,SouthAmerica.TransRSocTropMedHyg. 1989;83:195.
30.ScorzaJV,RezzanoS,CesarMarquezJ.Didelphismarsupialis: reservorioprimáriodeLeishmaniaspp.enlaciudadde Trujillo,Venezuela.RevCubanaMedTrop.1984;36:194–200. 31.AdlerGH,BecerraMT,TraviBL.FeedingsuccessofLutzomyia
evansi(Diptera:Psychodidae)experimentallyexposedto
smallmammalhostsinanendemicfocusofLeishmania chagasiinnorthernColombia.Biomedica.2003;23:396–400. 32.YoshidaEL,CorreaFM,PachecoRS,MomenH,GrimaldiJunior
G.LeishmaniamexicanainDidelphismarsupialisauritainSão PauloState,Brazil.RevInstMedTropSaoPaulo.1985;27:172. 33.LainsonR,ShawJJ,ReadyPD,MilesMA,PovoaM.
LeishmaniasisinBrazil:XVI.Isolationandidentificationof Leishmaniaspeciesfromsandflies,wildmammalsandmanin northParaState,withparticularreferencetoL.braziliensis guyanensiscausativeagentofpian-bois.TransRSocTropMed Hyg.1981;75:530–6.
34.CabreraMA,PaulaAA,CamachoLA,etal.Caninevisceral leishmaniasisinBarradeGuaratiba,RiodeJaneiro,Brazil: assessmentofriskfactors.RevInstMedTropSaoPaulo. 2003;45:79–83.
35.OliveiraCD,Diez-RouxA,CesarCC,ProiettiFA.Acase–control studyofmicroenvironmentalriskfactorsforurbanvisceral leishmaniasisinalargecityinBrazil,1999–2000.RevPanam SaludPublica.2006;20:369–76.
36.CardosoRM,deAraújoNNSL,RomeroGAS,etal.Expanding theknowledgeaboutLeishmaniaspeciesinwildmammals anddogsintheBraziliansavannah.ParasitVectors. 2015;8:171,http://dx.doi.org/10.1186/s13071-015-0780-y. 37.LindosoJA,CotaGF,daCruzAM,etal.Visceralleishmaniasis
andHIVcoinfectioninLatinAmerica.PLoSNeglTropDis. 2014;8(9):e3136,http://dx.doi.org/10.1371/journal. pntd.0003136.
