Trypanosoma
cruzi
III
causing
the
indeterminate
form
of
Chagas
disease
in
a
semi-arid
region
of
Brazil
Kiev
Martins
a,
Cle´ber
de
Mesquita
Andrade
b,
Andressa
Noronha
Barbosa-Silva
a,
Gerson
Barbosa
do
Nascimento
c,
Egler
Chiari
d,
Lu´cia
Maria
da
Cunha
Galva˜o
a,d,
Antonia
Cla´udia
Ja´come
da
Caˆmara
a,e,*
a
GraduatePrograminPharmaceuticalSciences,CenterforHealthSciences,FederalUniversityofRioGrandedoNorte,Natal,Brazil
b
GraduatePrograminHealthSciences/DINTER/UERN,FederalUniversityofRioGrandedoNorte,Natal,Brazil
c
StateDepartmentofPublicHealthofRioGrandedoNorte,Caico´,Brazil
d
DepartmentofParasitology,InstituteofBiologicalSciences,FederalUniversityofMinasGerais,BeloHorizonte,Brazil
e
DepartmentofClinicalandToxicologicalAnalyses,CenterforHealthSciences,FederalUniversityofRioGrandedoNorte,RuaGal.GustavoCordeirodeFarias s/n28AndarPetro´polis,59012-570Natal,RN,Brazil
1. Introduction
Chagasdiseaseisasylvaticenzooticdiseasedistributedwidely inLatinAmerica.Itistransmittedinnaturebetweentriatomines and mammals and has become an anthropozoonosis due to anthropic changes in the natural environment.1,2 Increasing
humanoccupationof thesemi-aridenvironmenthasunleashed triatominedispersion, expanding thetransmission cycle of the parasitetohumandwellings.3ThenortheastofBrazilencompasses
extensiveareasofterritorywithasemi-aridclimate,constituted bytheCaatingabiome,andhasa large quantityoflow quality housinginruralareas.Thesefactorspromotethecolonizationby insectsoriginatingfromthewildenvironment.3,4Thisregionisthe
center of dispersion and has high concentrations of Triatoma brasiliensisandTriatomapseudomaculata.3,5Similarly,highratesof naturalinfectionbyTrypanosomacruziinPanstrongyluslutzimay playanimportantrole,6togetherwithwildmammalsfoundinthis
region,whichhavebeenindicatedashostsindifferentareas.7–9
T. cruzi populations show a high degree of intraspecific variability,asdetectedbybiological,biochemical,immunological, andgeneticmarkers.10Recently,consensuswasreachedamong
researcherstorenameisolatesofT.cruzibyassigningthemoneof six discrete typing units (DTUs; TcI–TcVI) based on different geneticmarkers.11Ofnote,DTUsIIandIIIwereformerlyknownas
TcIIbandTcIIc.12
Geographically, TcI is dispersed widely in the Americas, maintainsmoreaffinitywithmarsupials thanothermammals,13
andisfoundthroughouttherangeoftriatominedistribution,such thatthisDTUisassociatedwithsylvaticanddomesticcycles.Recent studies have confirmed the presence of the TcI genotypes in
InternationalJournalofInfectiousDiseases39(2015)68–75
ARTICLE INFO
Articlehistory:
Received13March2015
Receivedinrevisedform8August2015 Accepted13August2015
CorrespondingEditor:EskildPetersen, Aarhus,Denmark.
Keywords: Trypanosomacruzi Discretetypingunits Chronicchagasicpatients Clinicalforms
Geneticdiversity
SUMMARY
Objective:Trypanosomacruziissubdividedintosixdiscretetypingunits(DTUs),TcI–TcVI.Theprecise
identificationofeachcancontributetotrackingwildDTUsthatinvadethedomiciliaryenvironment.
Methods:TwentyT.cruzistocksisolatedfrom16chagasicpatients,twoPanstrongyluslutzi,oneGalea
spixii,andoneEuphractussexcinctus,fromdifferentlocalitiesintheStateofRioGrandedoNorte,Brazil,
werecharacterizedbygenotypingthe30regionofthe24SarRNAgene,themitochondrialcytochrome
oxidasesubunit2gene,andthesplicedleaderintergenicregion.
Results:TcIIIwasidentifiedin18.7%(3/16)ofpatientsfromdifferentmunicipalities,aswellasinP.lutzi,
G.spixii,andE.sexcinctus,indicatingtheconnectionbetweenthesylvaticanddomesticcyclesinthis
Braziliansemi-aridregion.TcIandTcIIwerealsodetected,in37.5%(6/16)and43.8%(7/16)ofpatients,
respectively.TheseDTUswereassociatedwithcardiac,digestive,andindeterminateclinicalforms,while
TcIIIwasidentifiedonlyinpatientswiththeindeterminateform.
Conclusions: TheoccurrenceoftheseDTUsrevealsimportantphylogeneticdiversityinT.cruziisolates
fromhumans.TcIIIisreportedforthefirsttimeinnortheasternBrazil.Thesefindingsappeartoindicate
anoverlapbetweenthesylvaticanddomestictransmissioncyclesoftheparasiteinthisregion.
ß2015TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.
ThisisanopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
* Correspondingauthor.Tel.:+55843342-9827;fax:+55843342-9833. E-mailaddress:[email protected](A.C.J.d.Caˆmara).
ContentslistsavailableatScienceDirect
International
Journal
of
Infectious
Diseases
j o urn a l hom e pa ge : ww w. e l s e v i e r. c om/ l o ca t e / i j i d
http://dx.doi.org/10.1016/j.ijid.2015.08.012
1201-9712/ß2015TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Argentina,Brazil,Bolivia,Chile, Colombia,Mexico,Panama, Para-guay,FrenchGuiana,Venezuela,andtheUSA.14,15Humaninfection withTcIinBraziliscommonlyconcentratedintheAmazonbasin,in thenorthernpartofSouthAmericaandCentralAmerica.16Insome
countries,likeColombia,TcIisassociatedwithchagasic cardiomy-opathy,17whileinothersitshowslowpathogenicity.18ThisDTUhas
beenfoundfromArgentinatotheUSA,whileTcII–VIaredistributed from the Amazon basin to southern Argentina. However, it is possibletofindregionswhereallsixDTUsarepresent,suchasin Colombia,whereTcIisreportedpredominantly,togetherwithlow proportionsofDTUsII,III,IV,andVI.19–23Thewidegeneticdiversity
ofTcIintheAmericashasbeenestablished,ashasthegeographical distributionofTcIgenotypesthroughouttheAmericas,confirming thatitismostprevalentinColombia.24
TcIIispredominantinthesouthernandcentralregionsofSouth America,butitstrueextentremainsunclear.Ithasmostlybeen isolatedindomestictransmissioncyclesfromvectors,primates, andsporadicallyfromothermammals.25,26ThisDTUisfoundin different clinical forms and can be associated with distinct symptoms and varying degrees ofpathological processesin all theclinicalformsofthedisease.27
TcIIIismostlyassociatedwiththesylvaticcycleinBrazil,with theterrestrialnicheandDasypusnovemcinctusfoundoveravast range from western Venezuela to the Argentine Chaco.28,29
Althoughrareindomestic transmissioncycles, TcIIIoccurs ata relatively high frequency in the sylvatic environment and is associatedalmostexclusivelywithterrestrialtransmissioncycles andfossorialmammaliangenera,includingtheCingulata (arma-dillos)and terrestrialmarsupials.30–33So far,fewvectorspecies
have been incriminated in the sylvatic transmission of TcIII. Panstrongylus geniculatus and Triatoma rubrovaria, both mainly sylvatic vectors, are often,although not exclusively,associated withterrestrialecotopes.34Althoughinfrequent,theoccurrenceof
TcIIIindomestictransmissioncyclesimpliesitsroleasanagentof humandisease.28Recentdatahaveshownsporadicacutecasesin
humanswithintheAmazonbasin.29Inthestudyarea,TcIIIwas
identified in T. brasiliensis from the peri-domicile and P. lutzi captured in the wildenvironment. TcIII wildpopulations have alwaysshownahighlevelofhomozygosity,whichisinconsistent withclonal propagation, although it is unclear whether this is explainedby intra-lineagerecombination orgene conversion.34 TcIIIisalsooccasionallyisolatedfromdomesticdogs30,31andother
uncommonreservoirsand vectors.6,7 ThisDTU therefore
repre-sentsanimportantfocusforstudy28,ashumanpopulationsare expandingintopreviouslyundisturbedcyclesofnatural transmis-sion and secondary vector species are re-emerging from the sylvaticenvironmentfollowingtheeradicationofmajordomestic species.35,36
ThediversityofnaturalT.cruzipopulationsinvolvingTcI,TcII, and TcIIIcirculatingamong sylvaticand peri-domiciliary trans-missioncycleshasbeendemonstratedinthewestmesoregionof theStateofRioGrandedoNorte.6HumaninfectionwithTcIand
TcIIwasreportedatthesametimethatTcI,TcII,and TcIIIwere isolatedfromT.brasiliensisandP.lutzi.6,7,32ThepresenceofTcIIIin
triatominevectorssuchasT.brasiliensishaspromptedthesearch for these DTUs in humans to be extended, with the aim of understandinghowtheperi-domesticanddomesticenvironments areconnectedtothewildinthesemi-aridregion.
2. Methods 2.1. Studyarea
TheStateofRioGrandedoNorte(RN)islocatedinnortheastern Brazil between 484905300S and 685805700S, and 3585800300W and
3883601200W.Ithasapopulationofoverthreemillioninhabitants.
ThepredominantbiomeisBraziliansavanna(Caatinga),described asa‘whiteforest’duetothelackofwater,whichmeanstheplants are leafless over the dry months. This biome presents a large number of mammals, birds, reptiles, and shrubby plants, and undergoes longperiodsof droughtover theyears. A variety of triatomineendemicspecieshavebeenreportedoverthelastfew decades,includingT.brasiliensis,T.pseudomaculata,Panstrongylus megistus,Rhodniusnasutus,andP.lutzi,allfoundinthesemi-arid regionofthestate.4,5,32
2.2. OriginofT.cruzi
Atotalof266chagasicpatientswereidentified.Thesepatients had positive ELISA (Chagatest recombinant ELISA v. 3.0 kit), positivehemagglutinationinhibition(HAI)(Chagatest hemagglu-tination inhibition, screening A-V kit; Wiener Lab, Rosa´rio, Argentina),and/orpositiveindirectimmunofluorescence(IIF)test results.TheHAIhasasensitivityof100%andspecificityof98.7% according to the manufacturer37. The IIF, with a titer of 1:40
consideredthecut-offpointforthismethod,wasperformedusing epimastigotesofT.cruziY(DTUII)11strainasantigen,maintained
in acellularcultureand fixedwith20%formaldehyde. Afterthe exclusionofheartdiseases(ischemic,valvular,andhypertensive), allpatientsaged23to88 yearswereenrolledinthestudy.The same clinical–epidemiological protocol wasapplied throughout thephysicalexamination,takingintoaccountthepatient’shabits and concomitant diseases, including thepresence of signs and symptomsgeneralorspecifictothecardiovascularand gastroin-testinal systems. These patients were examined clinically and submitted to complementary tests, including an electrocardio-gram,chestX-ray,andcontrastradiographyoftheesophagusand colon.Theywereclassifiedaccordingtotheclinicalformofthe diseaseascardiac,digestive,orindeterminate,asrecommendedby the Brazilian Consensus on Chagas Disease.38 The algorithm
developedforpatientclassificationissummarizedinFigure1. ThisstudywasapprovedbytheResearchEthicsCommitteeof the State University of Rio Grande do Norte (COEP/UERN No. 027.2011)inMossoro´,RioGrandedoNorte,Brazil,andinformed consentwasobtainedfromtheparticipants.TheoriginofT.cruzi andthepatient’sepidemiologicalandclinicalcharacteristicsare presentedinTable1.
2.3. IsolationandcultureofT.cruzi
Parasite isolation fromhumans wasperformed by hemocul-ture39 and from triatomine bugs and sylvatic mammals by
xenoculture methods.40 Hemoculture was performed for all chagasicpatientsandT.cruziwasisolatedfrom20ofthem(20/ 266,7.5%).In ordertominimizeparasite selection,T.cruziwas maintained in culture for a short period with three or four successivepassagesinLIT(LiverInfusionTryptose)medium.Only 16patientsreturnedfortheclinicalexaminations.Fouradditional isolateswereobtainedfromthesylvaticenvironment,onefrom GaleaspixiiWagler1831,onefromEuphractussexcinctusLinnaeus 1758,andtwofromP.lutzi,andthesewereincludedinthestudy. Cultures ofT. cruziepimastigotesat 106/mlwerewashed three
times in Krebs–Ringer–Tris pH7.3, centrifugedat 2000g for 15minat48C,andwerethenstoredat208CuntilDNAextraction and genetic characterization. Genomic DNA from the cultured parasite was obtained by phenol–chloroform method and was usedasthetemplateforPCRassays.41
2.4. GeneticcharacterizationofT.cruziDTUs
The protocol used for genotyping was that proposed by D’A´vilaetal.,42usingathree-stepassay(Figure2).Polymorphism
analysis of the mitochondrial cytochrome oxidase subunit 2 (COII)genewasperformedwithTcmit-10(50
-CCATATATTGTTG-CATTATT-30) and Tcmit-21 (50-TTGTAATAGGAGTCATGTTT-30)
primersdesignedtoamplifyafragmentof375basepairs(bp) fromT.cruzimaxicircleDNA.42,43Onthebasisoftherestriction
mapof COIIsequences, the AluIrestriction endonucleasewas chosen for use in restriction fragment length polymorphism
(RFLP)analysis ofthe mitochondrialCOIIgene.This markeris abletodistinguishhaplotypeA(TcI)andhaplotypeC(TcII)from otherDTUswithhaplotypeB(TcIII–VI).AmplificationoftheD7 domainofthe24S
a
rRNAgenewasachievedbyPCRwithprimers D71 (50-AAGGTGCGTCGACAGTGTGG-30) and D72 (50-TTTTCA-GAATGGCCGAACAGT-30),followingpreviouslydescribed
proto-cols.44
Figure1.Flowchartforpatientclassification,asrecommendedbytheBrazilianConsensusonChagasDisease.38
Table1
Geographicalorigin,hosts,andgenetictypingbythreemarkersofTrypanosomacruzistocksfrompatientswithdifferentclinicalforms,vectors,andreservoirs T.cruzistocks Host Clinicalform Age,years Sex 24SarDNAa(bp) COIIb
(haplotype/bp)
SL-IRc (bp)
DTU Municipalityd
1150 Human Indeterminate 44 Male 110 A/30,81,264 150 TcI Caico´ 1816 Human Indeterminate 45 Female 110 A/30,81,264 150 TcI Caico´ 2137 Human Cardiac 32 Female 110 A/30,81,264 150 TcI S.Melo 2549 Human Indeterminate 27 Female 110 A/30,81,264 150 TcI Apodi 3188 Human Digestive 62 Male 110 A/30,81,264 150 TcI Assu CBS202 Human Cardiac 88 Male 110 A/30,81,264 150 TcI Carau´bas
240 Human Cardiac 29 Male 125 C/81,212 150 TcII Caico´
1317 Human Indeterminate 58 Male 125 C/81,212 150 TcII DSR 2934 Human Indeterminate 44 Male 125 C/81,212 150 TcII Caico´
3973a Human Cardiac 72 Male 125 C/81,212 150 TcII Acari
RN26 Human Digestive 56 Male 125 C/81,212 150 TcII SNN
RN79 Human Cardiac 43 Female 125 C/81,212 150 TcII SNN
SM73 Human Cardiac 64 Female 125 C/81,212 150 TcII S.Melo 105 Human Indeterminate 39 Female 110 B/81,294 200 TcIII Caico´ CBS195 Human Indeterminate 40 Female 110 B/81,294 200 TcIII Carau´bas SM76 Human Indeterminate 67 Female 110 B/81,294 200 TcIII S.Melo Gs3 G.spixii - Adult Male 110 B/81,294 200 TcIII Carau´bas Es18 E.sexcinctus - Adult Male 110 B/81,294 200 TcIII Carau´bas
Pl0213 P.lutzi - Adult Male 110 B/81,294 200 TcIII SNN
Pl0812 P.lutzi - Adult Female 110 B/81,294 200 TcIII SNN Col1.7G2*46
Human - - - 110 A/30,81,264 150 TcI
JG*47 Human - - - 125 C/81,212 150 TcII RN19*6 Human - - - 110 B/81,294 200 TcIII AM64*29 Human - - - 117/119 B/81,294 200 TcIV 3253*e Human - - - 110+125 B/81,294 150 TcV CL-Brener*48 T.infestans - - - 125 B/81,294 150 TcVI
bp,basepairs;Gs3,Galeaspixii;Es18,Euphractussexcinctus;Pl082andPl0213,Panstrongyluslutzi.
aSoutoandZingales.44
b Freitasetal.43;D’A`vilaetal.42
c
Burgosetal.45
d
DSR,DixSeptRosado;S.Melo,SeverianoMelo;SNN,SerraNegradoNorte.
e
3253,Lages-Silvaetal.unpublisheddata.
*
T.cruzistrainsandclonesusedascontrols.
K.Martinsetal./InternationalJournalofInfectiousDiseases39(2015)68–75 70
The amplification of the spliced leader intergenic region (SL-IRac) genes was achieved with primers TcIII forward (50-CTCCCCAGTGTGGCCTGGG-30) and UTCC reverse (50
-CGTAC-CAATATAGTACAGAAACTG-30).ThePCRstrategywiththeSL-IRac
genewasdevisedtodistinguishpopulationsbelongingtoT.cruziIII (ampliconsof200bp)fromT.cruziI,T.cruziII,andhybridstrains, whichpresentfragmentsofapproximately150to157bp.45The PCRproductswereanalyzedbyelectrophoresison6.0% polyacryl-amide gels and the DNA fragments were visualized by silver staining.
3. Results
GeneticprofilesoftheT.cruzihumanstocksshowedgenetic diversity.Threehumanstocksamplified fragment110bprDNA, COIIhaplotypeB,andSL-IRac200bp,correspondingtoTcIII.TcI was identified in six patients presenting 110bp rDNA, COII haplotypeA,andSL-IRac150bp.SevenTcIIstockswereobtained fromhumansshowing125bprDNA,COIIhaplotypeC,andSL-IRac 150bp(Figure3).AllstocksobtainedfromP.lutzi,G.spixii,andE. sexcinctusalsoshowedprofilescorrespondingtoTcIII(Table1).
Themeanageofthe16patientswas50.6years.Patient2549
triatominebugs.Nevertheless,themostincisivefactorappearstobe thelackofanyborderbetweentheruralandsylvaticzones,which allowsthevectorsandsynanthropicmammalstoremaininclose contactwithhumans.Itislikelythattheabsenceoflimitshinders epidemiologicalsurveillanceactionsthatshouldbepermanent.
PatientsinthepresentstudyinfectedwithTcIII(105,CBS195, andSM76)onlypresentedtheindeterminateclinicalform,which wasalsodiagnosedina patientfromtheStateofMinasGerais, Brazil.42ThesedatasuggestthatTcIIIhaslowpathogenicpower,
eveninpatientsoverthemeanage(SM76,67yearsold),andsince thethreeTcIII-infectedpatientslivedindifferentmunicipalities, withnoparentageorevidenceoforalinfection,itisprobablethat theinfectionwastransmittedbythevector.AstudyinColombiaof chronicpatientsinfectedbyTcIII,inwhommixedinfectionwith TcI and TcII was detected,50 demonstrated that it is hard to
distinguishtheroleofeachDTUin theheartdiseasesanalyzed. PreviousfindingshaveshownthatTcIII(formerlyknownasTcIIc) maybeunderreportedfrombothdomesticandsylvatic transmis-sion cycles because certain genotyping methodologies fail to distinguish between TcIV (TcIIa) and TcIII.51 TcIII has been associated with terrestrial ecotopes from different reservoirs andvectors.7,33IntheBrazilianAmazon,Venezuela,andColombia,
TcIisthepredominantDTUandtheprincipalcauseofbothacute and cardiac Chagas disease, but not of the ‘mega’ syndromes, whilstTcIIIcausessporadicacutecasesofChagasdiseaseinthe Brazilian Amazon basin.49 Outbreaks of acute Chagas disease
causedby bothTcIandZ3(TcIII orTcIV)have beenreported.52 However,datafromVenezuelanlocalitieshaveshownthatTcIis responsible for human acute Chagas infection and for causing severeheartfailure,53in contrasttoanoutbreakofacute cases caused uniquely by the genotype TcIII/Z3.29 Three stocks from
chronicchagasicpatients(onewithanasymptomaticform,two withacardiac–digestiveform)werefoundtobecloselyrelatedto Z3/TcIII,54 highlighting the need for the characterization of
epidemiological and clinical traits associated with different genotypesofChagasdiseaseintheregionunderstudy.
Inthisstudy,TcIwasassociatedwithchronicchagasicpatients presenting cardiac, digestive, and indeterminate clinical forms. Humaninfection withTcIhas been reportedinthe State ofRio GrandedoNorte,althoughwithnoassociationwithclinicalforms.6
TherecentdescriptionoftheimportanceofTcIinthedevelopmentof cardiomyopathies in Argentina21 and cardiac alterations in
Colombia50,55hasdrawnattentiontothepossibilitythatthisDTU contributestothepathogenicityofChagasdisease.InColombiaand Venezuela, TcIisolates have been associated not onlywith low parasitemiaandverymildclinicalsymptoms,butalsowithsevere acutesymptomaticcasesanddeath.50,53TcIhaplotypes TcIaand
TcId have been identified in different tissues from a heart-transplanted Chagas cardiomyopathy patient with reactivation, indicatinghistotropism.14Ahighpercentageofisolatesbelongingto
TcIhaplotypeTcIdwereidentifiedinVenezuelanpatientsinfected withoraloutbreaksandtherewasevidenceofmulticlonalinfections
Figure3.ARFLPanalysisofthemitochondrialCOIIgeneintheTrypanosomacruziisolatesbelongingtodifferenthaplotypes,obtainedbypolyacrylamidegelelectrophoresis. DigestionoftheDNAwithAluIgeneratesthreeRFLPpatternsfortheT.cruzistrains:restrictionfragmentsof264,81,and30bpareclassifiedasTcI(mitochondrialhaplotype A;lane1,Col1.7G2clone),restrictionfragmentsof212and81bpareclassifiedasTcII(mitochondrialhaplotypeC;lane2,strainJG),andrestrictionfragmentsof294and 81bpareclassifiedasTcIII–VI(mitochondrialhaplotypeB;lane3,strainRN19;lane4,strainAM64;lane5,strain3253;lane6,CLBrenerclone).Lanes7–13showT.cruzi samplesofpatients2137,3188,RN26,RN79,105,CBS195,andSM76.(B)Analysisof24SarRNAofT.cruziisolatesfromchronicchagasicpatientsandcontrols.Lane1, Col1.7G2clone(110bp);lane2,JG(125bp);lane3,RN19(110bp);lane4,AM64(117/119bp);lane5,3253(110and125bp);lane6,CLBrenerclone(125bp); lanes7–13,T.cruzisampleofpatients2137,3188,RN26,RN79,105,CBS195,andSM76.(C)TheSL-IRacgenewasusedtoseparateisolatesofTcIIIandTcIVfromotherDTUs. Lanes1,2,5and6:controlsCol1.7G2clone,JG,3253,andCLBrenerclone(fragmentsof150–157bp);lanes4and5:controlsRN19andAM64(fragmentsof200bp);lanes7– 13:T.cruzisampleofpatients2137,3188,RN26,RN79,105,CBS195,andSM76;laneM:molecularsizemarker;laneNC:negativePCRcontrol.
K.Martinsetal./InternationalJournalofInfectiousDiseases39(2015)68–75 72
inpatients,triatominevectors,andreservoirsintheregionsofthese outbreaks,suggestingmultipleinfectionsinreservoirsfromurban andruralareas.56Furtherstudiesareneededtodeterminewhether
the differentclinical formsfound here arerelevanttosome TcI haplotypes,immuneresponsesofpatients,ormultipleinfectionsby differentDTUsnotdetectedbythemethodologyused.
Inthis study, the distributionof TcIinpatients fromseveral municipalities, who did not share food sources capable of transmittingT.cruzi,suggeststhattransmissioninthesemi-arid area is vectorial and is not associated with oral outbreaks, as reportedpreviouslyforthisDTU.57SinceTcIhasbeendetectedin
humans,T.pseudomaculata,andT.brasiliensis7,58,itisbelievedthat theTcIpresentherecouldbeassociatedwiththeTriatomagenus actingasvectors.
Chagasic patients with TcII presented cardiac, digestive, or indeterminate clinical forms, corroborating previous findings in whichDTUIIpopulationshavebeenshowntoplayamajorrolein Brazilianchagasicpatients.18,27,42ThisDTUisconsideredcommon
inhumans,withhighhomologyamongdifferenthostsandlocations inthesemi-aridregion.32T.brasiliensis,avectorcapableofactingasa
linkbetweensylvaticanddomesticcycles,hasregularlybeenfound tobeinfected.6TcIIhasalsobeenobservedinCingulata,Rodentia,and
Primata,whicharedistributedinmanyAmericanregions,andthese alsooccasionallycarryTcIII.26Theseordersarecommoninthe
semi-aridregionandareprobablyinvolvedasreservoirsofthisDTU. Thedatapresentedheredemonstratethattransmissioncycles of T. cruzi in the semi-arid region can sustain epidemiological patternsoftheparasitethataredifferenttothoseofotherregions ofthecountry.Moreover,TcIIIwasidentifiedforthefirsttimein northeasternBrazilasacausativeagentofchronichumandisease; no similar reports from other states of the region have been verified.Thesefindingsalertustothecomplexityofcontrollingthe transmission of the parasite in such areas, where overlapping sylvaticandperi-domiciliarycyclesoccur.
Acknowledgements
ThisworkwassupportedbytheNationalCouncilforScientific and Technological Development(MCT/CNPq Grant No. 472251/ 2010-4andMCTI/CNPq/MS-SCTIE-DecitGrantNo. 404056/2012-1)andtheCoordinationfortheImprovementofHigherEducation Personnel (CAPES) – National Incentive Program for Basic Parasitology (Programa Nacional de Incentivo a` Parasitologia Ba´sica;Grant No.23038.005288/2011-48).LMCG wasgranteda visiting researcher fellowship by CNPq and KMwas granted a graduationfellowshipbyCAPES.Theauthorsaregratefultothe Serido´ EcologicalStation,theChicoMendesInstitutefor Biodiver-sity Conservation (Instituto Chico Mendes de Conservac¸a˜o da Biodiversidade,ICMBio),theStateSecretaryforPublicHealthofRio Grandedo Norte,Brazil,municipalauthorities, andlocal health teamsfortheirunconditionalsupportinthedevelopmentofthis study. The authors also wish to thank Prof. Adalberto Antonio Varela-Freire(inmemoriam)andPhilipS.P.Badizforhiscritical readingandrevisionofthemanuscript.
Conflict of interest: The authors declare that there are no conflictsofinterest.
Contributions: Concept and design (ACJC, EC, LMCG), data collection(KM,ANBS,CMA,GBN),dataanalysisandinterpretation (KM, ACJC, EC, LMCG), drafting of the manuscript (KM, ACJC, LMCG),criticalreviewofthemanuscript(ACJC,EC,LMCG),final approvalofmanuscriptforpublication(KM,ANBS,CMA,GBN,EC, LMCG,ACJC).
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