Chagas cardiomyopathy : the potential effect of benznidazole treatment on diastolic dysfunction and cardiac damage in dogs chronically infected with Trypanosoma cruzi.

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ContentslistsavailableatScienceDirect

Acta

Tropica

j ourna 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 / a c t a t r o p i c a

Chagas

cardiomyopathy:

The

potential

effect

of

benznidazole

treatment

on

diastolic

dysfunction

and

cardiac

damage

in

dogs

chronically

infected

with

Trypanosoma

cruzi

Fabiane

M. Santos

a,b

_,

_Ana

_L.

_Mazzeti

a

_,

_Sérgio

_Caldas

d

_,

_Karolina

_R.

_Gonc¸

_alves

a

_,

Wanderson

G. Lima

c

_,

_Rosália

_M.

_Torres

e

_,

_Maria

_Terezinha

_Bahia

a,c,∗

a_Laboratório_de_Doenc¸_as_{Parasitárias,}_Escola_de_Medicina,_Universidade_Federal_de_Ouro_Preto,_Morro_do_Cruzeiro,_CEP_35400-000,_Ouro_Preto,_Minas

Gerais,Brazil

b_Departamento_de_Farmácia_e_Nutric¸_ão,_Universidade_Federal_do_Espírito_Santo,_Alto_{Universitário,}_CEP_29500-000,_Alegre,_Espírito_Santo,_Brazil c_Departamento_de_Ciências_Biológicas,_Instituto_de_Ciências_Exatas_e_Biológicas,_Universidade_Federal_de_Ouro_Preto,_Morro_do_Cruzeiro,_CEP_35400-000,

OuroPreto,MinasGerais,Brazil

d_Fundac¸_ão_Ezequiel_Dias,_Rua_Conde_Pereira_Carneiro,_80,_Gameleira,_Belo_Horizonte,_Minas_Gerais,_Brazil

e_Faculdade_de_Medicina,_Universidade_Federal_de_Minas_Gerais,_Avenida_Alfredo_Balena,_190,_Santa_Eﬁgênia,_CEP_30130-100,_Belo_Horizonte,_Minas_Gerais,

Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25November2015 Receivedinrevisedform24April2016 Accepted15May2016

Availableonline20May2016

Keywords: Trypanosomacruzi Benznidazole

KinetoplastDNAfollow-up Diastolicdysfunction

a

b

s

t

r

a

c

t

CardiacinvolvementrepresentsthemaincauseofmortalityamongpatientswithChagasdisease,andthe relevanceoftrypanocidaltreatmenttoimprovingdiastolicdysfunctionisstilldoubtful.Inthepresent study,weusedacaninemodelinfectedwiththebenznidazole-sensitiveBerenice-78Trypanosomacruzi straintoverifytheefficacyofanetiologictreatmentinreducingtheparasiteloadandamelioratingcardiac muscletissuedamageandleftventriculardiastolicdysfunctioninthechronicphaseoftheinfection.The effectofthetreatmentonreducingtheparasiteloadwasmonitoredbybloodPCRandbloodculture assays,andtheeffectofthetreatmentontheoutcomeofhearttissuedamageandondiastolicfunctionwas evaluatedbyhistopathologyandechoDopplercardiogram.Thebenefitofthebenznidazole-treatment inreducingtheparasiteburdenwasdemonstratedbyamarkeddecreaseinpositivebloodcultureand PCRassayresultsuntil30dayspost-treatment.Atthistime,thePCRandbloodcultureassaysyielded negativeresultsfor82%ofthetreatedanimals,comparedwithonly36%oftheuntreateddogs.However, aprogressiveincreaseintheparasiteloadcouldbedetectedintheperipheralbloodforoneyear post-treatment,asevidencedbyaprogressiveincreaseinpositiveresultsforboththePCRandtheblood cultureassaysatfollow-up.Theparasiteloadreductioninducedbytreatmentwascompatiblewiththe lowerdegreeoftissuedamageamonganimalseuthanizedinthefirstmonthaftertreatmentandwith theincreasedcardiacdamageafterthisperiod,reachinglevelssimilartothoseinuntreatedanimalsat theone-yearfollow-up.Thetwoinfectedgroupsalsopresentedsimilar,significantlysmallervaluesfor earlytissueseptalvelocity(E’SIV)thanthenon-infecteddogsdidatthislatertime.Moreover,inthe treatedanimals,anincreaseintheE/E’septaltissuefillingpressureratiowasobservedwhencompared withbasalvaluesaswellaswithvaluesinnon-infecteddogs.Thesefindingsstronglysuggestthatthe temporaryreductionintheparasiteloadthatwasinducedbybenznidazoletreatmentwasnotableto preventmyocardiallesionsanddiastolicdysfunctionforlongaftertreatment.

1. Introduction

Chagasdiseaseisavector-borneparasiticinfectioncausedby

thekinetoplastidprotozoanTrypanosomacruziandisanimportant

∗ Correspondingauthorat:LaboratóriodeDoenc¸asParasitárias,sala203,Escola deMedicina,UniversidadeFederaldeOuroPreto,OuroPreto,MG,35400-000,Brazil. E-mailaddresses:mtbahia@nupeb.ufop.br,fabiane.santos@ufes.br(M.T.Bahia).

causeofend-stagecardiomyopathyandlossofdisability-adjusted

life years (DALYs)among young,economically active adults in

endemiccountries(Coura,2007;Martins-Meloetal.,2012).An

esti-mated14,000peoplediefromChagasdiseaseeachyear,mainly

duetoa chroniccardiacconditionthatmaybeassociated with

otherchronicdiseases,furtherincreasingmortality(Schmunisand

Yadon,2010;Guarientoetal.,2011).

Therole ofparasite persistence in thepathogenesis of

Cha-gasheart diseasehasbeendemonstrated bythe presenceof T.

http://dx.doi.org/10.1016/j.actatropica.2016.05.007

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cruziintheheart,alongwithalow-gradebutrelentless

inﬂamma-toryprocessandmyocardialautoimmuneinjury(Gutierrezetal.,

2009;Marin-Netoetal.,2009;Caldasetal.,2013).However,the

immunopathologicalmechanismsinvolvedinthepathogenesisof

chagasiccardiomyopathyandtheeffectoftrypanocidaltherapyon

theclinicalcourseofpatientswithchronicChagasheartdisease

havenotbeencompletelyelucidated.

Themyocardialabnormalities observedin thechronicphase

oftheT.cruziinfectionareextremelyvariable,rangingfrommild

forms,suchasdigitiformapicalaneurysmsandabnormalitiesofthe

leftventriculardiastolicfunctiononly,tosigniﬁcantcardiac

cham-berdilatationcoupledwithseveresystolicdysfunction(Iannietal.,

2001;Miglioreetal.,2004).Echocardiographyisawell-established

testinclinicalpracticethatprovidesparametersbywhich

chaga-sicpatientscanbeanalyzedandstratiﬁed(Rassietal.,2014).Early

diagnosisofdiastolicdysfunctionisagoodtoolforthe

manage-mentofpatientswithChagascardiomyopathyandcanimprove

prognosis(Cianciullietal.,2006;Garcia-Alvarezetal.,2010).

Anumber of clinicaltrialshave demonstrated thebeneﬁcial

effectofetiologictreatmentwithbenznidazoleintheacuteand

recent chronic phases of Chagas disease(Andradeet al., 1996;

Andradeetal.,2004;SosaEstanietal.,1998).However,no

deﬁni-tiveconsensushasbeenreachedconcerningwhetherbenznidazole

treatment signiﬁcantly reduces the parasite burden or

symp-tomsintheestablishedchronicphaseofthedisease(Marin-Neto

etal.,2009).TherecentlypublishedresultsoftheBENEFIT

clin-ical trialshowed that benznidazole treatment of patients with

establishedChagascardiomyopathywasabletoreduce(although

only transiently) blood parasite levelsbut didnot signiﬁcantly

reducecardiacclinicaldeteriorationthrough5yearsoffollow-up

(Morilloetal.,2015).Pre-clinicalstudieshavedemonstratedthe

beneﬁcial effectof etiologictreatmentonreducing tissue

dam-age(Garcia etal., 2005; Bahiaet al.,2012; Caldas etal., 2014)

andonelectrocardiographicalterations(Caldasetal.,2013).

Oth-ershavedemonstratedthatbenznidazoletreatmentwasableto

induceamildimprovementinsystolicdysfunctionindogs

chron-ically infected with T. cruzi (Santos et al., 2012). However,the

authorsshowedthatthisimprovementinsystolicheartfunction

inbenznidazole-treateddogswasnotaccompaniedbyprevention

ofgrowthofthecardiacchambers,includingtheleftatrialvolume,

aparameterthat mayalsobeevaluatedtoassessleft

ventricu-lardiastoliccardiacfunction.Consideringtheseantecedents,the

presentinvestigationwasundertakentobetterelucidatewhether

trypanocidaltherapy withbenznidazolein thechronicphase of

Chagasdiseasewouldbeeffectiveinpreventingorreducingthe

leftventricular diastolicdysfunctionand tissue damageas well

asinreducingtheparasiteburdenimmediatelyandforoneyear

post-treatment.

2. Methods

2.1. Parasite

TheBerenice-78T.cruzistrain(T.cruziII),isolatedby

xenodiag-nosisin1978(LanaandChiari,1986)fromtheﬁrstreportedhuman

caseofChagasdisease,wasusedinthisstudy

2.2. Experimentalanimals

Thirty4-month-oldmongreldogsfromtheKenneloftheOuro

PretoFederalUniversity(UFOP),MinasGeraisState,Brazil,were

usedinthisstudy.Theanimalswerefedwithcommercialchow

andwateradlibitum.Beforethestudy,theanimalsweredewormed

andvaccinatedagainstseveralinfectiousdiseases.Allprocedures

andexperimentalprotocolswereconductedinaccordancewiththe

guidelinesoftheCOBEA(BrazilianCollegeofAnimal

Experimenta-tion)andwiththeapprovaloftheEthicsCommitteeforAnimal

ExperimentationofUFOP(Protocolnumber2008/08).Theinfected

animalswereinoculatedintraperitoneallywith4000blood

trypo-mastigotesperkgofbodyweightandthenweredividedintotwo

experimentalgroups:(i)11dogsthatweretreatedwith

benznida-zoleat7.0mg/kgbid(Q12)for60daysand(ii)11dogsthatwere

maintainedasuntreated controls.Anadditional8animalswere

maintainedasanon-infected/healthycontrolgroup.

2.3. Drugandtreatmentscheme

The drug benznidazole (Bz;

N-benzyl-2-nitro-1-imidazolacetamide) was synthesized at LAFEPE, Pernambuco,

Brazil.ThetreatmentschemewaspreviouslydescribedbyGuedes

et al. (2002). In all chronic-stage therapeutic schemes, oral

treatmentwasinitiated120dayspost-infection.

2.4. Assessmentofparasiteclearance

TheparasiteloadwasmonitoredbybloodPCRandbloodculture

assaysperformedbeforetreatmentandinthe1st,6thand12th

monthspost-treatment.Additionally,quantitativereal-timePCR

(qPCR)wasperformedonhearttissuesamplesfromtheanimals

euthanizedinthe1stand12thmonthspost-treatment.

2.4.1. BloodPCR

For the PCR assays of the blood, 10mL of blood was

col-lectedfromeachanimalduringfollow-upevaluationsinthe4th

month of infection (before treatment) and at the 1st, 6th and

12thmonthspost-treatment.Thebloodsamplesweremixedwith

an equal volume ofa 6Mguanidine hydrochloride-0.2MEDTA

solution and were stored for two weeks at room temperature,

followed by boilingfor 15min beforeDNA wasextracted from

200_␮Laliquotstakenfromeachsample(Guedesetal.,2002).PCR

ampliﬁcationwasperformedinatotalvolumeof20␮L

contain-ing0.1%TritonX-100;10mMTris-HCl(pH9.0);75mMKCl;5mM

MgCl2;0.2mM(each)dATP,dTTP,dGTPanddCTP(Sigma

Chem-icalCo.);1␮LofTaqDNApolymerase(Invitrogen,USA);20pmol

ofS35(5-AAATAATGTACGGG(T/A)GAGATGCATGA-3)andS36(5

-GGGTTCGATTGGGGTTGGTGT-3)primers;and2␮LofDNAforeach

sample (Ávilaet al.,1991).Thereactionmixturewassubjected

to35cyclesofampliﬁcationinanautomaticthermocycler

(Bio-cycler).Thetemperatureproﬁlewasasfollows:denaturationat

95◦Cfor1min(withalongerinitialtimeof5minat95◦C),65◦C

for1minforprimerannealingand72◦Cfor1minforextension,

withaﬁnalincubationat72◦Cfor10mintoextendtheannealed

primers.ThePCRproductswerevisualizedby6%polyacrylamide

gelelectrophoresis,followedbysilverstaining.AllDNAextraction

stepsandreactionmixturesusedforPCRweremonitoredand

com-paredwithpositiveandnegativecontrols.ThePCRanalysiswas

considerednegativeafterthreefailedDNAextractionsforagiven

sample.

2.4.2. Bloodculture

Parasite detection was performed by culturing 10mL blood

samples collectedin parallel with theblood usedfor theDNA

extractionandPCRampliﬁcation.Bloodcultureassayswere

per-formedfor treated and control/untreateddogs,as describedby

Guedesetal.(2002).Theculturesweremaintainedat28◦C,

homog-enized weekly, and examined monthly for up to 120days for

parasitedetection.

2.4.3. Quantitativereal-timePCR

DNAextractionfromtheleftatriumwasperformedat1and12

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Kit(Promega)withcertainmodiﬁcations(Caldasetal.,2012).Using

thepGEM-TEasyVectorSystem(Promega,Madison,WI,USA),a

plasmidwasconstructedbycloningthe182bpampliconofT.cruzi

satelliteDNA.Theampliconwasgeneratedusingtheprimers

TCZ-F5-GCTCTTGCCCACAMGGGTGC-3,whereM=AorC,andTCZ-R

5-CCAAGCAGCGGATAGTTCAGG-3,asdescribedbyCummingsand

Tarleton(2003).Therecombinantplasmidwaslinearizedby

diges-tionwithNdeI,andthetargetsequencewaspuriﬁedandquantiﬁed

usingaQubit®2.0Fluorometer(LifeTechnologies).TheDNAcopy

numberwascalculated,and100␮Lof1×1012_cloned_copies_of_T.

cruziDNAwasmixedwith100␮LofDNA(5-foldconcentrated)

fromanon-infecteddog.Thecaninehousekeeping␤-actingene

wasusedastheendogenouscontrolforqPCRnormalization.

Stan-dardcurvesweregeneratedfromﬁveserialdilutionsofthemixed

DNAinwater(1:10),rangingfrom5×109_to₅_×₁₀5_copies/_␮L_for

T.cruziand1×107_to₁_×₁₀3_arbitrary_units/_␮L_for_␤-actin.

AssaysofqPCRwereperformedusingSYBRGreenMasterMix

(AppliedBiosystems)accordingtothemanufacturer’sinstructions

and thespeciﬁcprimers TCZsat-F5

-YCTCTGACTCCCACCATTCA-3, where Y=C+T, and TCZsat-R 5-GCACTCGGCTGATCGTTT-3

(InvitrogenTM_),_designed_by_us_for_T._cruzi_DNA₍₈₉_bp)

ampliﬁca-tion.Thecanine_␤-actinendogenouscontrol(54bp)wasampliﬁed

usingthesenseprimer5-CCACTTTCCTGTCTTACCCAA-3 andthe

antisenseprimer 5-AATTAACCACCCACGGTGTT-3 (Guedesetal.,

2010)(InvitrogenTM_)._DNA_samples_were_10-fold_diluted_in_water

beforeuse.Cyclesofampliﬁcationwerecarriedoutina7500Fast

Real-TimePCRSystem(AppliedBiosystems).Thecyclesconsisted

ofaninitialdenaturationfor10minat95◦C,followedby40cycles

of95◦Cfor15sand62◦Cfor1min,withﬂuorescenceacquisition.

Ampliﬁcationwasimmediatelyfollowedbyameltingcurve

pro-gramwithaninitialdenaturationat95◦Cfor15s,coolingto60◦C

for1minandthenastepwisetemperatureincreaseof0.3◦C/sfrom

60to95◦C.Each96-wellreactionplatecontainedastandardfor

thecurvesandtwonegativecontrols,consistingofreactionswith

T.cruziand _{␤-actin-speciﬁc}primerswithoutDNAandreactions

withDNAfromanon-infecteddog.EachDNAsamplewas

quan-tiﬁedinduplicate fortheT.cruziand␤-actintargets.Themean

valuesforT.cruziDNAwerenormalizedbythedataobtainedfor

␤-actin,asfollows:normalizedvalue=(meanT.cruziDNA/mean

␤-actinDNA)×4×106_(expected_mean_value_for_␤-actin).

2.5. Histopathologyandmorphometricanalysis

Afragmentofapproximately1.0cm×1.0cm×0.2cmfromthe

middleoftherightatrialwallwascollectedforhistopathological

analysisat1and12 monthspost-treatmentaftereuthanasia of

thedogs.Thetissuefragmentswereﬁxedina10%buffered

for-malinsolution,dehydrated,clearedandembeddedinparafﬁn.The

blockswerecutinto4␮m-thicksectionsandstainedwith

hema-toxylinandeosin(H&E)forassessmentoftheinﬂammationorwith

Masson’strichromeforquantitativeevaluationofﬁbrosis.Twenty

ﬁeldsfromeachH&EorMasson’strichrome-stainedsectionwere

randomlychosenat a 40× magniﬁcation,giving a total areaof

1.49×106_␮m2 _of _analyzed_myocardium._Images_were_obtained

usingaLeicaDM5000microcameraand theLeicaApplications

SuitesoftwarebuiltintotheLeicaQ-WinplusVSimageanalyzer.

Theinﬂammatoryprocesswasevaluatedbasedonthenumberof

cellnucleiquantiﬁedinthemyocardialmusclefromnon-infected

andinfectedanimals.Theareaofﬁbrosiswasquantiﬁedusingthe

imagesegmentationfunction.Allpixelswithbluehuesin

Mas-son’strichrome-stainedsectionswereselectedtobuildabinary

image,and thetotal areaoccupiedbyconnectivetissue in

non-infectedandT. cruzi-infecteddogswassubsequently calculated.

Theinfectedanimalswereconsideredtohavearelevant

inﬂam-matoryprocessoranareaofﬁbrosiswhenthecellnucleusnumber

ortheﬁbrosisareaindicatedbybluepixelswasgreaterthanthe

Fig.1.Inﬂuenceofbenznidazoletreatmentontheparasiteload.Percentageof pos-itivebloodcultureandbloodPCRassaysobtainedbeforetreatmentaswellasat1,6 and12monthspost-treatmentwith7mg/kgbenznidazoletwicedailyfor60daysin dogsinfectedwiththeBerenice-78Trypanosomacruzistrain.IF:untreatedinfected animals.IF-T:benznidazole-treatedinfectedanimals.

medianvalueplusonestandarddeviation(SD)quantiﬁedinthe

non-infectedgroup.

2.6. Echocardiography

All animals were submitted to Doppler echocardiographic

exams before infection and in the 1st and 12th months

post-treatment(i.e.,6and18monthspost-infection).Theanimalswere

injectedintraperitoneallywiththeanestheticthiopentalsodium

(0.03g/mL0.8%salinesolution)andplacedonaninsulated

elec-tric surface,where theanesthetizeddogswere positionedwith

theirlimbsperpendicularlyorientatedtothebody(Santosetal.,

2012).Theleftventriculardiastolicfunctionwasmeasuredbased

onthemitralinﬂowpattern,pulmonaryvenousﬂowandpulsed

Doppler tissue imaging. The parameters were monitoredusing

a moving Cypress echocardiographic instrument and are well

described inearlydiastolic dysfunctioninChagas heartdisease

(Garcia-Alvarezetal.,2010).M-mode,two-dimensionalandpulsed

Dopplerechocardiographicstudieswereperformedoneach

ani-malaccordingtothemethoddescribedbytheAmericanSocietyof

Echocardiography(Schilleretal.,1989;Kerenetal.,1985).

2.7. Statisticalanalysis

Themeansofthehistologicalinﬂammatoryprocess

quantiﬁca-tionandechocardiographicparameterswereanalyzedviaanalysis

ofvariance,followedbyTukey’smultiplecomparisontests.Inall

cases, differences wereconsidered as signiﬁcantwhen p<0.05.

ThecorrelationsoftissueDopplerdiastolicdysfunction

parame-terswiththeintensityoftheﬁbrosisandinﬂammationintheright

atriumwereveriﬁedbythePearsonlinearcorrelationtest.

Statisti-calcalculationswereperformedusingGraph-PadPrismsoftware.

3. Results

3.1. Bloodparasiteclearance

Toevaluatetheefﬁcacyofbenznidazoleinclearingthe

para-sitism,detectionofT.cruziinthebloodsampleswasperformedby

PCRandbloodculturebeforetreatmentandinthe1st,6thand12th

monthspost-treatment.Thedogsweretreatedfor60consecutive

daysintheearlychronicphase(4thto6thmonthsafterinfection).

Beforethe4thmonthofinfection,whenthetreatmentbegan,the

summationsofthepositiveresultsveriﬁedbyPCRandblood

cul-tureweresimilarandgreaterthan70%intheinfectedgroups(8of

the11treatedanimalsand9ofthe11untreatedanimals)(Fig.1).

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Fig.2.EffectoftreatmentonthedevelopmentofcardiacdamageinexperimentalChagasdisease.Quantitativeanalysisoftheparasiteload(A),rightatrialinﬂammation (B)andﬁbroticarea(C)observedinsamplesoftheleftatriafrommongreldogsinfectedwith4×103_blood_{trypomastigotes}_of_the_Berenice-78_Trypanosoma_cruzi_strain_per

kgofbodyweight.Thedogswereeuthanizedatthe1stand12thmonthspost-treatment(i.e.,6thand18thmonthspost-infection).Thegraphrepresentsthemean±SDof resultsobtainedfromanimalsincludedinthecontrolnon-infected(NI),untreatedinfected(IF)andtreatedinfected(IF-T)groups(magnification40x,andis10␮m).(D,F,H, J,LandN)Hematoxylinandeosinstainingforinflammationassessment;(E,G,I,K,MandO)Masson’strichromestainingforfibrosisassessment;(D,E,JandK)myocardial sectionsfromanon-infecteddog;(F,G,LandM)sectionsfromanuntreatedinfecteddog;(H,I,N,O)sectionsfromatreatedinfecteddog.

thisevaluationhadbeenconﬁrmedashavingT.cruziinfectionat

theacutestageofthediseasebyaperipheralbloodexam.

Immediatelyafterthetrypanocidalchemotherapy,82%(9of11)

oftheanimalsoriginallyinfectedwithT.cruzihadnegativeresults

intheblood PCRassay.Thenegativeresultswereveriﬁedinall

bloodculturetests,conﬁrmingtheimmediatereductionofthe

par-asiteloadinducedbybenznidazoletreatment.Incontrast,atthis

time,theparasiteoritskinetoplastDNA(kDNA)couldbedetected

in82%(9of11)oftheanimalsthatwereinfectedanduntreated

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Fig.3.Influenceofbenznidazoletreatmentonventriculardiastolicfunction:mitralinflow.Evaluationofmitralinflowinmongreldogs,performedbeforeinfectionwith Trypanosomacruzi(whitecircles)aswellas1and12monthspost-treatmentwithbenznidazole(blackcircles).Theresults(mean±SD)representthoseobtainedfromanimals fromthecontrolnon-infected,untreatedinfectedandbenznidazole-treatedinfectedgroups.*Statisticalsignificance(p<0.05,asassessedusingTukey’stest).

cardiactissuethaninperipheralblood,butatsigniﬁcantlylower

levelsin thetissue samplesobtainedfrom thetreated animals

relativetothelevelsobservedintheuntreatedinfectedanimals.

Consideringtheresultsofthereal-timePCR,themeanparasitism

detected30daysaftertreatmentwas45.83×103_copies_of_T._cruzi

DNA/30mgofcardiactissueintreateddogsand58,032.51×103

copiesofT.cruziDNAinthosewithouttreatment(Fig.2A).

Thefollow-upevaluationatoneyearpost-treatmentrevealed

anincreaseinbloodparasitismbecausetheparasiteoritskDNA

couldbedetectedin40%(2of5)ofthebloodsamplescollectedin

the6thmonthaftertreatmentandin60%(3of5)ofthosecollected

inthe12thmonthpost-treatment.Thesameresultwasdetectedin

80%(4of5)andin100%(5of5)ofthebloodsamplescollectedfrom

theuntreatedanimalsinthe6thand12thmonthspost-treatment,

respectively(Fig.1).T.cruziDNAcouldbedetectedatthesame

levelsinallsamplesofcardiacmuscletissuefromtheanimalsin

bothinfectedgroups,namely,treatedornottreated.Atthistime,

couldbedetected202.52×103_copies_of_T._cruzi_DNA/30_mg_of

car-diactissueintreateddogsand88.86×103_copies_of_T._cruzi_DNA_in

thosewithouttreatment(Fig.2A).

3.2. Myocardialinﬂammatoryprocess

Toassesstheeffectivenessofbenznidazoletreatmentin

ame-lioratingheart muscletissuedamage, a quantitativeanalysisof

inﬂammation and ﬁbrosis was performed in the 1st and 12th

monthspost-treatment.Nostatisticallysigniﬁcantvariation was

observed in the rightatrial inﬂammatory process betweenthe

infected groups at 1 month post-treatment. However, in the

benznidazole-treatedanimals,approximately20%fewer

mononu-clear inﬂammatory cells and 64% less intrafascicular collagen

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Table1

EvaluationofleftventriclediastolicfunctionbymitralinﬂowanalysiswithDopplerechocardiographyperformedonmongreldogsat6and18monthspost-infectionwith Trypanosomacruzi,respectivelyat1and12monthspost-treatmentwithbenznidazole,aswellasonnoninfectedanimals.

Variable Experimentalgroups

1monthpost-treatment 12monthspost-treatment

NI IF-NT IF-T NI IF-NT IF-T

E(cm/s) 0.70±0.1 0.93±0.1a,c _0.71_±_0.05b _0.78_±_0.25 _0.66_±_0.1 _0.84_±_0.1

A(cm/s) 0.43±0.1 0.51±0.06 0.46±0.11 0.54±0.14 0.48±0.10 0.53±0.08 E/Aratio 1.64±0.14 1.85±0.30 1.63±0.39 1.45±0.15 1.44±0.47 1.62±0.30 DT(ms) 126.0±35.33 106.0±39.56 104.0±28.06 135.0±15.87 139.2±34.83 110.4±16.82 NI:noninfectedcontrolgroup;IF-NT:infectednontreatedgroup;IF-T:infectedbenznidazole-treatedgroup.

a_Indicates_signiﬁcant_difference_relative_to_the_noninfected_control_group_in_the_{corresponding}_time. b_Indicates_signiﬁcant_difference_relative_to_the_infected_nontreated_group.

c _Indicates_signiﬁcant_difference_relative_to_the_infected_{benznidazole-treated}_group.

Table2

EvaluationofleftventriclediastolicfunctionbypulmonaryvenousﬂowanalysiswithDopplerechocardiographyperformedonmongreldogsat6and18monthspost-infection withTrypanosomacruzi,respectivelyat1and12monthspost-treatmentwithbenznidazole,aswellasonnoninfectedanimals.

S(cm/s) 0.49±0.1 0.71±0.22 0.64±0.16 0.53±0.07 0.47±0.06 0.46±0.13

D(cm/s) 0.4±0.14 0.43±0.11 0.51±0.21 0.42±0.08 0.3±0.03 0.35±0.09

S/Dratio 1.26±0.2 1.65±0.20 1.33±0.27 1.3±0.27 1.59±0.21 1.37±0.46

Arvelocity(cm/s) 0.26±0.01 0.29±0.06 0.31±0.04 0.31±0.00 0.29±0.07 0.31±0.05

Arduration(ms) 66.0±0.00 68.0±3.46 62.67±15.32 67.5±9.0 74.0±8.25 77.6±6.54

NI:noninfectedcontrolgroup;IF-NT:infectednontreatedgroup;IF-T:infectedbenznidazole-treatedgroup.

Table3

EvaluationofleftventriclediastolicfunctionbytissueDoppleratbasalsegmentsperformedwithDopplerechocardiographyonmongreldogsat6and18monthspost-infection withTrypanosomacruzi,respectivelyat1and12monthspost-treatmentwithbenznidazole,aswellasonnoninfectedanimals.

E’SIV(cm/s) 0.20±0.06 0.19±0.06 0.18±0.04 0.23±0.11 0.08±0.03a _0.09_±_0.04a

A’SIV(cm/s) 0.10±0.02 0.13±0.06 0.10±0.03 0.16±0.14 0.07±0.02 0.10±0.03 E’/A’SIVratio 2.09±0.53 1.64±0.67 2.07±0.74 1.7±0.51 1.43±1.06 0.94±0.61 E/E’SIVratio 3.85±1.61 5.73±3.01 4.24±1.26 4.15±1.94 8.76±2.70 11.26±4.91a

E’LAT(cm/s) 0.25±0.05 0.33±0.06c _0.22_±_0.06b _0.25_±_0.05 _0.16_±_0.03* _0.18_±_0.06

A’LAT(cm/s) 0.14±0.03 0.16±0.03c _0.10_±_0.03b _0.14_±_0.05 _0.07_±_0.01 _0.11_±_0.04

E’/A’LATratio 1.90±0.36 2.08±0.57 2.31±0.52 1.95±0.53 2.33±0.75 1.71±0.51 E/E’LATratio 2.84±0.76 2.92±0.56 3.54±1.0 3.19±0.73 4.22±1.12 4.86±1.03 NI:noninfectedcontrolgroup;IF-NT:infectednontreatedgroup;IF-T:infectedbenznidazole-treatedgroup.

a_Indicates_signiﬁcant_difference_in_relation_to_the_noninfected_control_group_in_the_{corresponding}_time. b_Indicates_a_signiﬁcant_difference_relative_to_the_infected_nontreated_group.

c _Indicates_a_signiﬁcant_difference_relative_to_the_infected_{benznidazole-treated}_group.

animals had signiﬁcantly higher ﬁbrosis levels than the

non-infecteddogsdid(Fig.2BandC).Inalaterevaluation,at12months

post-treatment,similarlevelsofrightatriallesionsweredetected

intreatedanduntreatedinfectedanimals,andthelevelsinboth

groupsweresigniﬁcantlyhigherthanthosepresentinnon-infected

dogs(Fig.2BandC).

3.3. Leftventriculardiastolicdysfunction

Theevaluationoftheleftventriculardiastolicfunctionwas

car-riedout by assessing the mitral inﬂowand pulmonary venous

ﬂowandbytissueDopplerimaging,whichwereaccomplishedby

performingDopplerechocardiographicexamsbeforetheinfection

andatthe1stand12thmonthspost-treatment.Beforeinfection,

allparameters evaluated weresimilaramong the non-infected,

untreatedinfectedandtreatedinfectedanimals(Figs.3–5).

Themitralinﬂowanalysis,whichinvolvementmeasurementof

theearlydiastolicmitralﬂowvelocity(E),latediastolicmitralﬂow

velocity(A),E/AratioanddecelerationtimeoftheEwave(DT),

showedthattheonlydifferencesamongthegroupsoccurredatthe

1stmonthpost-treatment,withgreatermedianvaluesofEinthe

untreatedinfectedanimals(0.93cm/s)comparedwiththetreated

infected(0.71cm/s)andnon-infected(0.70cm/s)animals(p<0.05).

Therewasnovariationintheparametersofthemitralinﬂow

anal-ysisamongthegroupsatthe12thmonthpost-treatment,andthe

valuesdidnotvarywithrespecttothebaselinedataateither1or

12monthspost-treatment(Table1andFig.3).

Theevaluationofpulmonaryvenousﬂowatthe1stand12th

monthspost-treatmentrevealedsimilarvaluesamongthegroups

forallofthefollowingparameters:peakSwaveinﬂowpulmonary

velocityduringventricularsystole(S),peakDwave inﬂow

pul-monaryvelocityduringtheearlyphaseofventriculardiastole(D),

thecorrespondingS/Dratio,peakretrogradepulmonaryvein

veloc-ityoratrialreversevelocity(Arvelocity)anddurationofretrograde

pulmonaryveinoratrialreverseduration(Arduration)(Table2).

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Fig.4.Inﬂuenceofbenznidazoletreatmentonleftventriculardiastolicfunction:tissueDopplerattheinterventricularseptum.Analysisofleftventriculardiastolicfunction bytissueDopplerattheinterventricularseptum(SIV),performedonmongreldogsbeforeinfectionwithTrypanosomacruzi(whitecircles)aswellas1and12months post-treatmentwithbenznidazole(blackcircles).Theresults(mean±SD)representthoseobtainedfromanimalsfromthecontrolnon-infected,untreatedinfectedand benznidazole-treatedinfectedgroups.*Statisticalsigniﬁcance(p<0.05,asassessedusingTukey’stest).

monthspost-treatmentwasnotperformedduetotheabsenceof

differencesamongthegroups.

The study of tissue Doppler at the basal segments showed

averagevaluesforthepeakearly(E’)andlate(A’)diastolic

myocar-dialvelocitiesobtainedattheseptal(SIV)andlateral(LAT)wall

positions,theratiosof E’/A’ SIVandE’/A’ LAT, and theE/E’SIV

and LAT ratiosto estimate theleft ventricular ﬁlling pressure.

Earlyand latetissuelateralvelocities(E’LATandA’LAT)atthe

1stmonthpost-infectionweresimilarbetweennon-infectedand

infected animals, although the treated infected and untreated

infectedgroups showedsimilardifferences asfortheirbaseline

data(p>0.05).Afterward,theevaluationatthe12thmonth

post-treatmentrevealedaworsereductionintheE’SIV(p<0.05)andE’

LAT(p<0.01)inboththetreatedandtheuntreatedinfected

ani-malscomparedwiththeirbaselinedata.Thenon-infectedanimals

showedsimilarvaluesfortheseparameterscomparedwiththeir

baselinedataandstillexhibitedahigherE’SIVthaneitherofthe

infectedgroupsdid(p<0.05)andahigherE’LATthantheuntreated

infectedgroupdid(p<0.05).Theestimateoftheleftventricular

ﬁllingpressurebasedontheE/E’septalratio(E/E’SIV)showedan

increaseinthetreatedinfectedanimalscomparedwiththeir

base-linedata(p<0.05),highervaluesthanobservedinthenon-infected

dogs(p<0.05),anddatathatwereintermediaterelativetothedata

fortheuntreatedinfectedgroupatthe12thmonthpost-treatment

(Table3,Figs.4and5).

Tostudytheinﬂuenceofthetissueinﬂammatoryprocessonthe

developmentofleftventriculardiastolicdysfunction,

intrafascicu-larcollagendepositionandthelevelsofmononuclearinﬂammatory

cellswereassessedbasedonthetissueDopplerparametersusing

the Pearson linear correlation test. Fibrosis and mononuclear

inﬂammatorycellswerecorrelatedwiththeE’SIVvelocity,E’/A’

SIVratioandE/E’SIVratio(p<0.01).TheE/E’LATratiowasstill

cor-relatedwiththeinﬂammatorycells(p<0.01).ThevaluesoftheE/E’

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Fig.5.Inﬂuenceofbenznidazole-treatmentonleftventriculardiastolicfunction:tissueDoppleratthelateralwallposition.Analysisofleftventriculardiastolicfunction bytissueDoppleratthelateralwallposition(LAT),performedonmongreldogsbeforeinfectionwithTrypanosomacruzi(whitecircles)aswellasat1and12months post-treatmentwithbenznidazole(blackcircles).Theresults(mean±SD)representthoseobtainedfromanimalsfromthecontrolnon-infected,untreatedinfectedand benznidazole-treatedinfectedgroups.*Statisticalsigniﬁcance(p<0.05,asassessedusingTukey’stest).

thoseoftheE’SIVandE’/A’SIVwereproportionallylowerasthe

levelsoftissueinﬂammationincreased(Fig.6).

4. Discussion

Benznidazoletreatmenthasrecentlybeenspeciﬁcally

recom-mended for all patients with Chagas disease, even though the

experimentalandclinicalevidence thatis currentlyavailableis

insufﬁcienttosupporttheroutine useof etiologictreatmentin

chronicpatients(Viottietal.,2006;Marin-Netoetal.,2009).

We used a canine model infected with the

benznidazole-sensitiveBerenice-78straintoverifythedrug’sefﬁcacyinreducing

the parasiteload and ameliorating cardiac muscletissue

dam-ageand left ventricular diastolic dysfunction.Here, the beneﬁt

ofbenznidazole treatmentinreducing theparasiteburdenwas

demonstratedbythemarkeddecreaseinpositiveresultsforblood

cultureandPCRassaysuntil30dayspost-treatment.Atthattime,

thePCRandbloodcultureassayshadnegativeresultsfor82%of

thetreatedanimals,comparedwithonly36%oftheuntreateddogs.

Nevertheless,aprogressiveincreaseinpositiveresultsforboththe

PCRandthebloodcultureassayswasdetectedthroughoutthe

eval-uationperiod.Overall,ourresultsshowthat thenegativeblood

PCRorbloodcultureresultsobtainedimmediatelyafterthe

eti-ologic treatmentmaybepredictivenotof a cure,but ratherof

only a transitoryreduction in theparasite burden;this ﬁnding

suggeststhattheparasitesremain,butat levelsbelow the

lim-itsofdetectionofthemethodsused.TheqPCRdataforthetissue

samplesfrombenznidazole-treateddogsatonemonthafter

treat-mentandat12monthspost-treatmentsupportthishypothesisby

showingthepersistenceoftheparasiteinthetissuesoftreated

animals,andeventhoseinwhichtheparasitewasnotdetected

intheperipheralbloodbyPCRorbybloodculture.Theresultsof

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Fig.6. Influenceofmusclecardiactissuedamageonleftventriculardiastolicdysfunction.Pearsoncorrelationamongthefibroticareas(collagen)andinflammation(cells) intherightatriumaccordingtoE’SIV,E’/A’SIV,andE/E’SIVandbetweeninflammatorycellsandE/E’LAT.

monthsaftertreatmentconﬁrmthepersistenceofparasitisminthe

benznidazole-treateddogs.

Our previous studies in dogs infected withthe Berenice-78

strain showedthat benznidazole treatmentin theacute phase

(20–30daysafterinfection)wasabletocure75–100%ofanimals,

reducingtissuedamageandpreventingelectrocardiographic

alter-ations(Guedesetal.,2002;Caldasetal.,2013).Theseobservations

suggestthatthetreatmentfailuredetectedinthepresentstudy

wasnotduetothegeneticresistanceoftheBerenice-78strainto

thedrug,butrathertoaphenotypiccharacteristic.This

hypothe-sisisinlinewiththeobservationsofothers,showingthatinvivo

resistancetomultiplecompoundscanvarywiththehostspecies

andbecomesmoreevidentwithanincreasinglengthofinfection

(DosSantosetal.,2008;Caldasetal.,2008;Bustamanteetal.,2013).

AccordingtoBustamanteetal.(2013),failureinetiologictreatment

couldalsoberelatedtothetissuedistributionofparasitesandthe

inaccessibilityofcertaincellsortissuestothedrug.

However,theresultsofexperimentalstudiesshouldbeanalyzed

withcaution,asitisunclearwhichexperimentalmodelisthemost

appropriatefortranslationintohumans.Anumberof

experimen-talstudieshavealreadydemonstratedtheeffectsoftherouteof

infection;thesizeoftheinocula;andtheanimal’sweight,sex,and

ageontheoutcomesofanimalinfection.AccordingtoChatelain

andKonar(2015),achronicChagasdiseasemodelwouldideally

offerIgGnegativity,negativeparasitemia,positiveserology,

sur-vival,andmeasuresofinﬂammationmarkersintheheartandother

organs.Alongtheselines,thedogmodelshowsprogressiontoa

chronicphasethatcloselyreﬂectshumandisease,soitmaybe

con-cludedthatthisisoneofthebestavailableinvivomodelsforChagas

disease(Andrade,1984;Lanaetal.,1992;Bahiaetal.,2002;Guedes

etal.,2010;Santosetal.,2012;Caldasetal.,2013).

In the present study, the parasite load reduction induced

by treatment in the early chronic phase was compatible with

thelessertissuedamageamong animalseuthanizedintheﬁrst

month after treatment. At this time, a higher level of ﬁbrosis

wasdetectedin thecardiacmuscletissue amongtheuntreated

infectedanimals(2750.442±2369.27collagen/74,931.3␮m2₎

rel-ativetotheﬁbrosisdetectedinthetreateddogs(1017.57±368.95

collagen/74,931.3␮m2_)._However,_the_large_SDs_in_cardiac

dam-agedetectedamongtheuntreatedinfectedanimalspreventedthe

detection of signiﬁcant differences between untreated infected

and treated infected animals, but only the untreated infected

animalshadﬁbrosislevelssigniﬁcantlyhigherthanthosein

non-infectedanimals.Consistentwiththeparasiteloadsverysimilar

levelsof ﬁbrosiscouldbe detectedbetweenuntreated infected

(3369.48±1098.12 collagen/74,931.3␮m2₎_and _treated _infected

(3966.65±995.70collagen/74,931.3␮m2₎_animals_at₁₂_months

aftertreatment.Theseﬁndingsallowtoformulatethehypothesis

thatintheabsenceofcure,thetreatmentresponsemaybe

ben-eﬁcialintheearlystagesaftertreatment;however,thelaterlack

ofcontroloftheparasitewouldallowtissuedamagetooccur.Our

ﬁndingsareinagreementwiththeresultsoftherecentlyconcluded

BENEFITclinical trial,in which it was foundthat benznidazole

therapyinpatientswithestablishedChagascardiomyopathy

sig-niﬁcantly(buttransiently)reducedserumparasitelevelsthrough5

yearsoffollow-up,althoughcardiacclinicaldeteriorationwasnot

signiﬁcantlyreduced(Morilloetal.,2015).Basedontheresultsof

theBENEFITstudyandthoseofthepresentwork,thehighlevels

(94%)ofbloodparasiteDNAclearanceobservedintheMolinaetal.

(2014)studymaysimplyreﬂectatransientreductionofthe

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loadorparasitologicalcure,andassuchdonotguaranteeapositive

clinicaloutcomeinthemediumtolongterm.

Toassesswhetherthetransientreductionintheparasite

bur-denbybenznidazoletreatmentwouldaffecttheoutcomeofcardiac

diastolicfunction,theleftventriculardiastolicfunctionwas

veri-fiedbasedonthemitralflow,pulmonaryvenousflowandtissue

Doppler.Intheclinicalsetting,evaluationofdiastolicventricular

functioniscomplicatedbythecoexistenceofmorethanoneofthe

factorsthataffectdiastolicﬁlling,andforthisreason,anintegrated

approach involvingvarious Dopplerechocardiographic

parame-tersforevaluationandquantiﬁcationoftheﬁllingpressuresisthe

bestwaytoassessdiastolicfunction.Achangeindiastolic

func-tionmaycontributesigniﬁcantlytotheproductionofsymptoms

invariouscardiacdiseases,eveninthepresenceofnormalsystolic

function(Doughertyetal.,1984;Sonnenblick,1988;Aguirreetal.,

1990).Ithasalsobeenshownthatdiastolicdysfunctioncanhave

anearlyonset, precedingtheoccurrenceofsystolicdysfunction

(Hirota, 1980;Brutsaert, 1983;Nishimura etal., 1989). Accord-ingtoCianciullietal.(2006),alowerE/Aratioandalengthening

ofthedecelerationtimeofearlydiastolicﬁlling(DT)revealedan

earlydisorderoftheleftventriculardiastolicfunctioninpatients

withChagasdisease,andtheseearlydiastolicdisorderscanalso

bedetectedina greatnumberofpatientswithotherheart

dis-eases.However, the assessment of diastolic function by mitral

Dopplersufferssigniﬁcantlimitations,asthecurvechanges

accord-ingtoage,heartrateandhemodynamicconditions,whereastissue

Dopplerislesssensitivetoloadchanges(Nishimuraetal.,1990;

AyuelaandVilchez,2004).

ThepresentstudyshowedthattissueDopplerwasthebesttool

todemonstratetheworseningintheleftventriculardiastolic

func-tionintheinfectedanimals.Theabsenceofsigniﬁcantdifferences

inthetissueDopplerparametersintheinfectedgroupsbetween

theirbaselineandsixmonthspost-infectionrevealsthatthehigher

E’LATandA’LATvelocitiespresentedbytheuntreatedinfected

animalscomparedwiththetreatedinfecteddogsat30days

post-treatment may have occurred due to an elevation of preload

(end-diastolic stress), and not tothe infection by T. cruzi.This

conceptwasalsodemonstrated,buttoagreater degree,bythe

highest Emitral velocity inthese sameanimals becausemitral

inﬂowparametersaremorepreloaddependentthantissueDoppler

variablesare(Oliveiraetal.,2009).Theseearlyalterationsdidnot

remainat thetime of thelater evaluation, at 12 months

post-treatment, whereas an interesting reduction of the E’ SIV and

E’ LAT velocities occurred in both infected groups, beyondthe

E/E’ratioelevationthatoccurredinthetreatedinfectedanimals.

Theselaterﬁndingsareinagreementwiththedeﬁnitionof

dias-tolicdysfunction,whichistheinabilitytoacceptbloodﬂowatall

oraninabilitytodosowithoutacompensatoryincreaseinleft

atrialpressure (Staufferand Gaasch,1990).The reduction ofE’

(SIVandLAT)reﬂectsimpairedmyocardialrelaxation,whereasthe

growthoftheE/E’ratioreﬂectstheelevationoftheleft

ventric-ularﬁllingpressure(Dokainish,2007).ThelowerE’velocityand

thelengtheningoftheE/E’ratioaccordingtotheworseningof

car-diomyopathydysfunctionwereinagreementwiththeincreasein

ﬁbrosisandtheprogressionofheartinﬂammationinthe

experi-mentaldogsevaluatedinthiswork,aswellaswiththestratiﬁcation

ofheartdysfunctioninchagasicpatientsdemonstratedinother

work(Nascimentoetal.,2013;Garcia-Alvarezetal.,2010).

Interestingly,ourpreviousstudy(Santosetal.,2012)showed

that benznidazole treatment in early chronic Chagas disease

slightlyameliorated,butwasunabletoefﬁcientlyprevent,

long-term left ventricular systolic cardiac dysfunction. In fact, only

untreatedinfectedanimalsexhibitedsigniﬁcantlyworsevaluesfor

classicalparametersforassessingsystolicdysfunctionrelativeto

non-infecteddogs.However,bothT.cruzi-infectedgroups(treated

ornot)experiencedasigniﬁcantdecreaseinthesecardiacsystolic

functionparametersat18monthspost-infection(12monthsafter

treatment)comparedwithbaselinevalues.Inaddition,thesame

previousworkstillrevealedthatbenznidazoletreatmentofchronic

T.cruziinfectionwasunabletopreventthelong-term

develop-mentofcardiomegaly,includinganincreaseintheleftatrialvolume

(Santosetal.,2012).Inthepresentstudy,theabsenceofsigniﬁcant

beneﬁtsofbenznidazoletreatmentata chronicstageofT. cruzi

infectionintermsofamelioratingcardiomyopathyevolutionwas

furtherclariﬁed,andtheresultswereinagreementwithour

previ-ousﬁndingsfortheleftatrialvolume,aparameterwhoseincrease

mayberelated toleftventriculardiastolic dysfunction.Despite

thelimitednumberofanimalsusedineachexperimentalgroup

(11benznidazole-treatedinfectedanimals,11untreatedinfected

animalsand 8non-infectedcontrols),theseﬁndings permitthe

conclusionthatthecardiacstructuralandfunctionalalterationsin

theinfectedanimalswereassociatedwiththeparasiteloadinthe

heart.

An overall analysis of ourresults indicatesthat the

tempo-rarysuppressiveactivityof treatmentwithbenznidazoleduring

thechronicstageofChagasdiseaseinBerenice-78T.cruzi

strain-infectedmongreldogswasnotabletoaffecttheoutcomeofthe

myocardial pathophysiological degenerative process. The

post-treatmentparasitereboundinthelongtermandthedevelopment

ofdiastolicdysfunctioncorrelatedwithheartlesionsshowedthat

benznidazoletreatmentintheearlychronicphasewasnot

efﬁ-cientatpreventingtheprogressionofcardiomyopathydiseasein

dogsinfectedwiththeBerenice-78T.cruzistrain,eventhougha

slightimprovement insystolicdysfunctionhadpreviously been

shown.Basedourpresentﬁndingssuchtherapeuticinterventions

mustaimataprofoundandsustained,notshortterm,suppression

oftheparasiteloadofinfectedpatients.

Conﬂictofinterest

Theauthorsdeclarethattheyhavenoconﬂictofinterest.

Acknowledgments

This work receivedﬁnancial support from the Fundac¸ãode

AmparoaPesquisadoEstadodeMinasGerais(Fapemig);Conselho

NacionaldeDesenvolvimentoCientíﬁcoeTecnológico(CNPq);

Uni-versidade Federalde OuroPreto andresearch fellowshipsfrom

Conselho NacionaldeDesenvolvimento CientíﬁcoeTecnológico

(M.T.B.).

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