Real-time PCR strategy for parasite quantification in blood and tissue samples of experimental Trypanosoma cruzi infection.

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ContentslistsavailableatSciVerseScienceDirect

Acta

Tropica

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

Real-time

PCR

strategy

for

parasite

quantiﬁcation

in

blood

and

tissue

samples

of

experimental

Trypanosoma

cruzi

infection

Sérgio

Caldas

c,d

_,

_Ivo

_Santana

_Caldas

c

_,

_Lívia

_de

_Figueiredo

_Diniz

c

_,

_Wanderson

_Geraldo

_de

_Lima

a,c

_,

Riva

de

Paula

Oliveira

b,c

_,

_Alzira

_Batista

_Cecílio

d

_,

_Isabela

_Ribeiro

e

_,

_André

_Talvani

a,c

_,

Maria

Terezinha

Bahia

a,c,∗

a_Departamento_de_Ciências_Biológicas,_Universidade_Federal_de_Ouro_Preto,_Campus_{universitário,}_Morro_do_Cruzeiro,_Ouro_Preto,_Minas_Gerais,_Brazil

b_Departamento_de_Evoluc¸_ão,_{Biodiversidade}_e_Meio_Ambiente,_Universidade_Federal_de_Ouro_Preto,_Campus_{universitário,}_Morro_do_Cruzeiro,_Ouro_Preto,_Minas_Gerais,_Brazil c_Núcleo_de_Pesquisas_em_Ciências_Biológicas,_Universidade_Federal_de_Ouro_Preto,_Campus_{universitário,}_Morro_do_Cruzeiro,_Ouro_Preto,_Minas_Gerais,_Brazil

d_Fundac¸_ão_Ezequiel_Dias,_Rua_Conde_Pereira_Carneiro,_80,_Gameleira,_Belo_Horizonte,_Minas_Gerais,_Brazil e_Drugs_for_Neglected_Disease_Initiative,₁₂₀₂_Geneve,_Switzerland

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28July2011

Receivedinrevisedform30April2012 Accepted8May2012

Available online 18 May 2012 Keywords: Trypanosomacruzi Real-timePCR Experimentalmodel Inﬂammation Chemotherapy

a

b

s

t

r

a

c

t

Thelackofanaccuratediagnosishasbeenaseriousobstacletotheadvancementoftheanti-Trypanosoma cruzichemotherapyandlong-terminfectioncanresultindifferenthealthriskstohuman.PCRsare alter-nativemethods,moresensitivethanconventionalparasitologicaltechniques,whichduetotheirlow sensitivitiesareconsideredunsuitableforthesepurposes.Theaimofthisstudywastoinvestigatea sensitivediagnosticstrategytoquantifybloodandcardiactissuesparasitesbasedonreal-timePCRtools duringacuteandchronicphasesofmurineChagasdisease,aswellastomonitortheevolutionof infec-tioninthosemiceunderspecifictreatment.Inparallel,freshbloodexamination,immunologicalanalysis andquantificationofcardiacinflammationwerealsoperformedtoconfrontandimprovereal-timePCR data.Similarprofilesofparasitemiacurveswereobservedinbothquantificationtechniquesduringthe acutephaseoftheinfection.Incontrast,parasitescouldbequantifiedonlybyreal-timePCRat60and 120daysofinfection.Incardiactissue,real-timePCRdetectedT.cruziDNAin100%ofinfectedmice, andusingthistoolasignificantPearsoncorrelationbetweenparasiteloadinperipheralbloodandin cardiactissueduringacuteandchronicphaseswasobserved.LevelsofserumCCL2,CCL5andnitricoxide werecoincidentwithparasiteloadbutfocalanddiffusemononuclearinfiltrateswasobserved,evenwith significant(p<0.05)reductionofparasitismafter60daysofinfection.Later,thismethodologywasused tomonitortheevolutionofinfectioninanimalstreatedwithitraconazole(Itz).Itz-treatmentinduceda reductionofparasiteloadinbothbloodandcardiacmuscleatthetreatmentperiod,butaftertheendof chemotherapyanincreaseofparasitismwasdetected.Interestingly,inflammatorymediatorslevelsand heartinflammationintensityhadsimilarevolutiontotheparasiteload,inthegroupofanimalstreated. Takentogether,ourdatashowthatreal-timePCRstrategyusedwassuitableforstudiesofmurineT.cruzi infectionandmayproveusefulininvestigationsinvolvingexperimentalchemotherapyofthedisease andthebenefitsoftreatmentinrelationtoparasitismandinflammatoryresponse.

1. Introduction

TheAmericantrypanosomiasisorChagasdisease,discoveredin

1909byCarlosJustinianoRibeirodasChagas,isazoonosiscaused

Abbreviations: TNF-␣,tumornecrosisfactor-alpha;CCL5,regulatedupon acti-vation,normalTcellexpressedandsecreted;CCL2,monocytechemoattractant protein-1;NO,nitricoxide;FBE,freshbloodexamination;d.i.,daysofinfection; Itz,itraconazole.

∗ Correspondingauthorat:Laboratóriodedoenc¸adeChagas,DECBI/NUPEB, Uni-versidadeFederaldeOuroPreto,ICEB,CampusMorrodoCruzeiro,35400-000Ouro Preto,MG,Brazil.Tel.:+553135591690;fax:+553135591680.

E-mailaddress:mtbahia@nupeb.ufop.br(M.T.Bahia).

bytheprotozoanTrypanosomacruzi(Chagas,1909).Thiszoonosis

istypicalofLatinAmericaextendingtothesoutheasternofthe

UnitedStatesofAmerica.Signiﬁcantadvanceshavetakenplacein

thecontrolofvectorialandtransfusionaltransmissionofthe

dis-easeinsomepartsoftheendemicarea,particularlybytheSouthern

Coneinitiative that ledtointerruption ofvector-to-human and

human-to-human propagationof the disease in Uruguay,Chile

andBrazilinrecentyears(Diasetal.,2002;Schoﬁeldetal.,2006).

However,duetomigrationalmovementsofpeoplefromendemic

countries, Chagas disease has been reported in non-endemic

areaswhere thecongenitaltransmission,blood transfusionand

organ transplantplays an important role (Gascon et al., 2007).

Therefore,therearestillmanychallengestoachievefullcontrol

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oreliminationofthedisease,mainlyduetotheunevenprogress

ofthevectorial/transfusional controlprogramsinotherpartsof

thecontinent,limitations ofdiagnosticmethods andsigniﬁcant

limitationsofcurrentchemotherapyavailable(Urbina,2010).

Chagasdiseasebegins withan acutephase characterizedby

thepresenceofparasitesinthebloodstreamanddifferenttissues

oftheinfectedindividual.Nonspeciﬁcsymptomsandmyocarditis

arecommonfeaturesduringtheearlystageoftheinfection(Dias,

1992).InﬁltrationofTcellsandmacrophagesintothehearttissue

duringtheacutephaseoftheinfectionisessentialforcontrolling

theparasitereplication(Hardisonetal.,2006).Additionally,cardiac

parasitismwasapparentlyrelatedtotheincreasedexpressionof

cytokinesandchemokines(Villaltaetal.,1998;Talvanietal.,2000),

inwhichthechemokinescorrelatedintheuptakeandkillingofthe

intracellularparasitesbyinducingNOsynthaseactivation

enhanc-ingNOproductionbymacrophagesandcardiomyocytes(Villalta

etal.,1998;Teixeiraetal.,2002;TalvaniandTeixeira,2011).

Molecularassayshavebeenwidelyusedforthediagnosisand

monitoringofdiseaseprogressionandtherapyoutcomein

Cha-gasdisease(Moseretal.,1989;Junqueiraetal.,1996;Kirchhoff

etal.,1996;Marconetal.,2002).PCRsarealternativemethods,

moresensitivethanconventionalparasitologicaltechniques,such

ashemoculture, whichduetotheirlowsensitivitiesare

consid-eredunsuitable for these purposes (WHO, 2002). For instance,

nested-PCR(N-PCR)hasdemonstratedgreatersensitivitythan

con-ventionalPCRandithasbeendescribedasanextremelysensitive

methodforthediagnosisofChagasdisease(Marconetal.,2002).

However,thedisadvantageofthistechniqueisthetimerequired

fortheirachievementandthegreatriskoffalsepositivesresults

causedbycontaminatingamplicons(Pironetal.,2007).

In contrast, real-time PCR uses ﬂuorescent dyes or probes

allowingthe continuousmonitoring of the reactionduring the

ampliﬁcationprocess, insteadof theamountoftarget

accumu-latedafteraﬁxednumberofcycles.Thiscompletelyrevolutionizes

thewaytoapproach the quantiﬁcationof DNA and RNA.Once

thenucleicacidampliﬁcationanddetectionstepsareperformed

inone tube,theriskof releasingampliﬁednucleicacidstothe

environmentandcontaminationofsubsequentassaysisverylow

compared to therisk conferred by conventional PCR or N-PCR

(Cockerill,2003;BankowskiandAnderson,2004).Thus,the

com-binationofexcellentsensitivityandspeciﬁcity,lowcontamination

riskandgreaterspeedofworkhasmadethetechnologyof

real-timePCRahighlyapplicabletool,usefulformanypurposes,such

aslaboratorydiagnostics,geneexpressionanalysis,quantiﬁcation

ofparasitesandmanyotherapplicationsintheﬁeldofresearch

(Duffyetal.,2009;deFreitasetal.,2011).Theaimofthisstudy

wastostandardizeanaccuratereal-timePCRstrategyfor

detec-tionandquantiﬁcationofT.cruziDNAinthebloodandheartof

infectedmiceduringtheacutephaseandchronicdisease

transi-tion,assessingthecorrelationbetweenbloodandtissueparasitism,

inﬂammatorymediatorsCCL2,CCL5,nitricoxideandthe

applica-bilityofthisapproachtomonitorthecourseofinfectionduringthe

treatmentofmice.

2. Materialsandmethods

2.1. Parasiteandinfection

TheVL-10strainofT.cruzi,DTUII(Morenoetal.,2010)whichis

resistanttobenznidazoletreatment(FilardiandBrener,1987)has

beenmaintainedcryopreservedinliquidnitrogenatLaboratoryof

Chagasdisease,UniversidadeFederaldeOuroPreto(UFOP),Brazil.

ThirtyfemaleSwissmice(age,3–4weeks;weight,18–22g)were

inoculatedingroupswith5× 103_bloodstream_forms_of_T._cruzi_by

intraperitonealrouteinatotaloftwoindependentexperiments.

All procedures and experimental protocols were conducted in

accordance withtheCOBEA (Brazilian Schoolof Animal

Exper-imentation) guidelines for the use of animals in research and

approvedbytheEthics CommitteeinAnimalResearch atUFOP

(number2009/17).

2.2. Bloodandtissueparasitequantiﬁcation

Blood parasitequantiﬁcation wereperformed at 10, 12, 14,

16,20, 30, 40,50, 60,and 120daysafterinfection(d.i.) (n=6).

Twomethodswere usedfor measurementof blood parasitism:

(i)microscopicfreshbloodexamination(FBE):5␮Lofbloodwere

collectedfromthemouse’stailandthenumberofparasiteswas

estimatedasdescribedbyBrener(1962).(ii)Real-timePCR:200␮L

ofbloodwascollectedfromorbitalvenoussinusofanimalsand

mixedto35␮Lof129mMsodiumcitratesolution(DOLES,BR).The

collectedmaterialwassubjectedtoDNAextraction.Theextracted

DNAwasfrozenat−20◦_C_until_required._In_both_methods_(i)_and

(ii),curveswereplottedusingparasitemiaaverageofsixmice.

Thecurvesofcardiacparasitismweregeneratedbyreal-time

PCR.Wecollected30mgofhearttissueofanimalsat10,16,30,60

and120d.i(n=6).Thismaterialwasstoredat₋₇₀◦CuntilDNA

extraction. Theextracted DNAwasfrozenat −20◦_C_for _later _T.

cruziquantiﬁcationbyreal-timePCR.Curveswereplottedusing

parasitismaverageofsixmice.

2.3. Treatmentschedule

Groups of 24 miceinoculated intraperitoneally with5×103

trypomastigotes were treated with Itraconazole (Itz):

4-[4-[4- [4-[[2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-

1,3-dioxolan-4-yl]methoxy]phenyl]-1-piperazinyl]phenyl]-2,4-dihydro-2-(1-methylpropyl)-3H-1,2,4-Triazol-3-one. Treatments

werestartedonthe10thdayafterinfectionandwereadministered

bygavagefor20consecutivedays.Itzweresuspendedinwater

with4%methylcellulose(Sigma)andwereadministeredindosesof

100mg/kgbodyweight.Thistreatmentprotocolwasstandardized

previouslybyToledoetal.(2003).

2.4. DNApreparationandreal-timePCR

TheextractionoftotalgenomicDNAfrombloodandtissueof

animalsinfectedwithT.cruziwasperformedusingthe

commer-cialkit (Wizard® _Genomic_DNA _{Puriﬁcation} _Kit,_Promega) _with

modiﬁcationsforDNAextractionfromcardiactissueofmouse.The

modiﬁcationconsistedintheincubationof30mgofsampleat55◦C

for2hwith120␮Lof0.5MEDTAsolution(pH8.0)(Sigma®_),₅₀₀_␮L

ofnucleiclysissolution(Promega)and9␮LofproteinaseK(fungal)

(InvitrogenTM₎_at₂₀_mg/mL_followed_by_maceration_and_treatment

according tothemanufacturer’s speciﬁcations for extractionof

tissueDNA.DNAwasquantiﬁedbyspectrophotometer

(Pharma-ciaBiotechGenequant)andtheconcentrationswereadjustedto

25ng/␮L.

ThePCRreactionwereperformedin10␮Lcontaining50ngof

genomicDNA,5␮LofSYBR®_Green_PCR_Mastermix_(Applied

Biosys-tems)andeither0.35␮MforT.cruzi195basepairs(bp)repeat

DNA-speciﬁcprimersor0.50␮Mofmurine-speciﬁctumor

necro-sisfactor-␣ (TNF-␣)primers. Theprimersfor T.cruzi repetitive

DNA (TCZ-F5-GCTCTTGCCCACAMGGGTGC-3,where M=Aor C

andTCZR5-CCAAGCAGCGGATAGTTCAGG-3)amplifya182-bpas

describedbyCummingsandTarleton(2003).Primersformurine

TNF-␣ (TNF-52415-TCCCTCTCATCAGTTCTATGGCCCA-3and

TNF-54115-CAGCAAGCATCTATGCACTTAGACCCC-3)amplifya170-bp

product(CummingsandTarleton,2003).Thecyclingprogram

con-sistedofaninitialdenaturationat95◦Cfor10min,followedby

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acquisitionat64.3◦C.Ampliﬁcationwasimmediatelyfollowedby

ameltprogramwithaninitialdenaturationof15sat95◦C,

cool-ingto60◦Cfor1minandthenastepwisetemperatureincreaseof

0.3◦C/sfrom60to95◦C.

Each96-wellreactionplatecontainedstandardcurveandtwo

negativecontrols.Negativecontrolsconsistedofareactionwith

T.cruzi-speciﬁcormurine-speciﬁcprimerswithoutDNAandalso

withbloodortissueDNAfromnon-infectedmice.EachDNAsample

wasquantiﬁedinduplicate.ThemeanquantiﬁcationvaluesforT.

cruziDNAwerenormalizedbythedataobtainedwiththe

murine-speciﬁc(TNF-␣) primersasfollows:normalized value=(meanT.

cruziDNA/meanTNF-␣DNA)×1000,where“1000”correspondsto

theexpectedvalueforTNF-␣from200␮Lofbloodor30mgofheart

tissue.Theefﬁcienciesofampliﬁcationweredetermined

automat-icallybytheStepOneTM _Software_v2.0_by_calculating:_efﬁciency

(E)=10(−1/slope)₍_Stordeur_et_al.,₂₀₀₂_).

2.5. T.cruzistandardcalibrationcurve

Standard curvesweregenerated fromﬁveserial dilutionsin

water(1:10)ofDNAextractedfrombloodandtissuestandards

con-taining5_{× 10}6_{parasites/0.1}_mL_of_blood_and₁₀6_parasites/30_mg

ofhearttissue, respectively.Thelimitsofdetectionof parasites

wereveriﬁedby6serialdilutions(1:10)of100parasiteequiv.(from

bloodandcardiactissuestandards)inDNA(25ng/␮L)fromblood

andhearttissueofhealthymice.Theterms“bloodstandard”and

“tissuestandard”refertoDNAextractedfromoptimizedvolumes

ofbloodsamplesandmassofhearttissuespecimensfromhealthy

micespikedwithaknownquantityofepimastigotes(VL-10strain).

Thestandardsweregeneratedinsufﬁcientquantitiesforall

real-timePCRassays.

2.6. Histopathologyandmorphometricanalysis

Forhistopathologicalanalysis,experimentalmicewere

sacri-ﬁcedondays10,30and120(6animals/group/day).Hearttissues

wereﬁxedin10%formalinandembeddedinparafﬁn.Blockswere

cut into 4␮m sections and stained by Hematoxylin and Eosin

(H&E)forinﬂammationassessment.Twentyﬁeldsfromeachslide

wererandomlychosenat40×magniﬁcationperformingatotalof

1.49×106_␮m2_analyzed_myocardium_area._Images_were_obtained

throughaLeicaDM5000Bmicrocamera(LeicaApplicationSuite,

model2.4.0R1)andprocessedbythesoftwareLeicaQwinV3image

analyzer.Theinﬂammatoryprocesswasevaluatedbythe

correla-tionindexbetweenthenumberofcellsobservedinmyocardium

musclefromnon-infectedandinfectedanimals(Caldasetal.,2008).

2.7. Immunoassays

Immunoassayswereperformedusingplasmafromall

exper-imental animalsto detectmonocyte chemoattractant protein-1

(MCP-1/CCL2) and Regulated upon Activation, normal T-cell

expressedand secreted (RANTES/CCL5). Brieﬂy, ﬂat-bottom

96-well microtiter plates (Nunc) were coated with 100␮L/well of

the CCL2 and CCL5 speciﬁc chemokine monoclonal antibodies

(0.2␮g/mLand2.0␮g/mL,respectively)for18hat4◦Candthen

washedwithPBSbuffer(pH7.4)containing0.05%Tween20(wash

buffer).Nonspeciﬁcbindingsiteswereblockedwith300␮L/wellof

1%BSAinPBS.Plateswererinsedwithwashbuffer,and50␮L/well

ofsamples andstandards addedfollowed byincubation for2h

atroomtemperature. Sevenpoint standardcurves using 2-fold

serial dilutions with 1% BSA in PBS, and high CCL2 and CCL5

standardsof250pg/mLand2000pg/mLwereused,respectively.

Plateswerethenwashedand100_␮L/welloftheappropriateCCL2

(50ng/mL)andCCL5(400ng/mL)biotinylateddetection

antibod-iesdiluted in blocking buffercontaining0.05% Tween 20 were

added for 1hat room temperature. Plateswere, then, washed

andstreptavidin–horseradishperoxidase(0.1␮g/mL)wasadded

for30minofincubationatroomtemperature.Finally,plateswere

washedand100␮L/wellofthesubstratesolution–1:1mixture

ofcolorreagentA(H2O2)andcolorreagentB

(Tetramethylbenzi-dine)–wasaddedandafter30minofadarkincubationatroom

temperature,thereactionwasstoppedby50␮L/wellof1MH2SO4

solution.Plateswerereadat450nmwithwavelengthcorrection

at570nminaspectrophotometer(MicroplateReader,model680,

BioRad).AllsampleswereassayedinduplicateusingDuoSet®_ELISA

DevelopmentSystem,RandDSystems®_,_Minneapolis,_MN_systems.

2.8. Nitricoxidedetection

Nitritelevelsintheplasmaofmiceweredeterminedusingthe

GriessreactionasanindexofNOproduction(Vespaetal.,1994).

Brieﬂy,50␮Lofplasmafromeachanimalwasdilutedwith50␮L

ofwaterandtreatedwith15␮LofamixofNADPH,FADandnitrate

reductase(atconcentrationsof1U/mL–Sigma®_)._About₁₈_h_after

incubationat37◦C,samplesweredeproteinatedwith10␮Lofzinc

sulfate(300g/L–Sigma®₎_and_centrifuged_at₂₀₀₀_×_g_for₅_min.

50_␮Lofsupernatantsampleswascombinedinaﬂat-bottom

96-wellmicrotiterplates(Nunc)witha1:1mixtureof1%sulfanilamide

in 2.5%H3PO4 and 0.1%naphtylethylenediamidein 2.5%H3PO4

(Sigma®_)._Plates_were_incubated_for₁₀_min_at_room_temperature,

andtheabsorbancewasmeasuredat550nmusingtheautomated

MicroplateReader,model680,BioRad.Nitriteconcentrationswere

determinedbyusingastandardcurveofsodiumnitritefrom125

to1␮M.

2.9. Statisticalanalysis

Datawereexpressedasmean±standarddeviation.Statistical

differenceforparasitologicaldataamongvariousgroupsofmice

atdifferentdaysaswellascytokinelevelsandintensityof

inﬂam-mationweredeterminedbythenonparametricTukey’sMultiple

ComparisonTest.Mann–Whitneytestwasusedwhentwogroups

ofanimalswereanalyzed.Pearson’scorrelation(r)wasalsoused

toevaluatetheassociationofparasitisminbloodandhearttissues.

Valuesofp<0.05wereconsideredsigniﬁcant.

3. Results

3.1. Standardcurvesandsensitivityofreal-timePCRassay

Theprimarygoalofthis studywastostandardizeastrategy

basedonreal-timePCRforasensitiveandreproducible

quantiﬁ-cationofparasiteloadinperipheralbloodandhearttissueduring

acuteandchronicexperimentalChagasdiseaseinmice,aswell

asitsapplicationinthemonitoringofexperimental

chemother-apy.Thetissueandbloodstandardscurvesweregeneratedfrom

ﬁveserialdilutionsofDNAfrom106_parasites/30_mg_of_heart

tis-sueand5×106_{parasites/0.1}_mL_of_blood_for_the_{quantiﬁcation}_of

T.cruzi(basedonourpreviousexperiencewithFBEshowing

maxi-mumparasitemiasaround3–4millionparasitesper0.1mLblood).

TheTNF-␣DNAwasassignedasanarbitraryvalueof103_in_both

bloodandtissuestandards.Fig.1AshowsampliﬁcationcurvesofT.

cruziandTNF-␣DNAinfourlogdilutions.Fig.1Bshowsthe

stan-dardcurvesgeneratedfromthelinearregionofeachampliﬁcation

curve.Theefﬁciency(E)andrvaluesforthehearttissuesamples

weresimilar.Fig.1C illustratestypicalmelting curvesobserved

afterampliﬁcationofT.cruziandinternalcontrol.Thetemperature

ofmelting(Tm)observedfortheparasitewas_∼81◦Candforthe

internalcontrolwas∼79.3◦_C_in_either_DNA_extracted_from_blood

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Fig.1.StandardcurvegeneratedwithDNAextractedfrombloodofhealthymicespikedwithTrypanosomacruziepimastigotes(VL-10strain).TheblacklinesrefertoT.cruzi DNAandthegraylinestoTNF-␣DNA(referencegene).(A)CurvesweregeneratedwithT.cruziandTNF-␣primersfromfiveserialdilutionsinwater(1:10)ofDNAextracted frombloodstandardscontaining5× 106_{parasites/0.1}_mL_of_blood._(B)_Standard_curves_were_generated_from_the_linear_region_of_each_{amplification}_curve._Efficiency_of

ampliﬁcationforeachprimersetwasdeterminedusingtheequation:efﬁciency(E)=10(−1/slope)_,_being_T._cruzi_E₌_93.989%_and_R2₌_0.997_and_TNF-␣_E₌_93.07%_and_R2₌_0.993.

(C)TypicalmeltingcurvesweregeneratedafterampliﬁcationofT.cruziandTNF-˛DNAshowingpeaksaround81◦Cand79.3◦C,respectively.

Fig.2. LimitsofdetectionforbothTrypanosomacruziDNAfromblood(graysquares) andhearttissues(blacksquares)ofmice.Thelimitofdetectionwas0.1parasite equiv./50ngofDNAinbothtissues(bloodandheart).

Fig.2showsthesensitivityofreal-timePCRtodetectT.cruziDNA

frombloodandhearttissueofmice.Thelimitsofdetectionwere

0.1parasiteequiv./50ngDNA.Table1representsthe

reproducibil-ityoftwoindependenttestsofreal-timePCRwiththebloodand

hearttissuesfromsixmice.TheresultsofthePCRreproducibility

wereexpressedinparasiteequiv./50ngDNAfrom0.1mLofblood

and30mgofhearttissue.

Table1

Reproducibilityofquantiﬁedproductfromtwoindependentreal-timePCRtests performedwiththebloodandhearttissuesfromsixmice.

Parasiteequiv./50ngDNA

Bloodsample Run1 Run2 Mean (S.D.) 1 0.128 0.120 0.124 (0.01) 2 0.520 0.530 0.525 (0.01) 3 0.818 0.825 0.821 (0.01) 4 10.012 9.879 9.946 (0.09) 5 5.082 5.567 5.324 (0.34) 6 3.965 4.081 4.023 (0.08) Cardiacmusclesample

1 2.673 2.975 2.824 (0.21) 2 2.272 2.388 2.330 (0.08) 3 1.616 1.651 1.634 (0.02) 4 3.962 4.050 4.006 (0.06) 5 0.927 0.775 0.851 (0.11) 6 3.130 3.057 3.093 (0.05)

3.2. QuantitativeanalysisofT.cruziloadinbloodandcardiac

tissues

Next,weassessedtheparasiteloadduringtheacuteandchronic

disease transition by real-time PCR and FBE. The parasitemia

proﬁles curves weresimilar for both quantiﬁcation techniques

during the acute phase of the infection. In contrast, parasites

couldbe quantiﬁedonly by real-time PCR at a ratioof 159.38

and 174.63parasites/0.1mLofblood at60 and 120d.i.,

respec-tively(Fig.3).Inaddition,real-timePCRdetectedparasiteDNAin

bloodandcardiactissuesin100%ofinfectedmiceinevery

analy-sisperformed,whileFBEshowedlowandintermittentparasitemia

quantiﬁcationat40(16.6%)and50(33%)d.i.andnegativeresults

at60and120d.i.asshowedinFig.4.

Inthenext,correlationsbetweenbloodandcardiactissues

par-asiteburdenwasevaluatedbyreal-timePCRondays10,16,30,60

and120afterinfection.Duringtheacutephaseofinfection

para-siteswereeasilydetectedinboth,bloodandcardiactissues,being

theirburdensigniﬁcantlylarger(p<0.05)inbloodsamplethanin

cardiactissue (Fig.5).Interestingly, after60 d.i.parasites were

moreeasilydetectedincardiac tissuethan inperipheralblood.

Fig.3.Averagenumberofparasitesdetectedbyreal-timePCR(blackcolumns)and freshbloodexamination-FBE(whitecolumns)onpredetermineddaysafter infec-tioningroupsofsixmiceinfectedwiththeVL-10strainofT.cruzi.Datashowthe averagesoftwoindependentexperiments.Asteriskdenotesasigniﬁcantdifference, asdeterminedbytheMann–Whitneytest(p<0.05).

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Fig.4. Percentageofpositiveresultsbyfreshbloodexamination(FBE)andreal-time PCRinblood(PCR–blood)andhearttissues(PCR–heart)ofmiceinfectedwith 5×103_{trypomastigotes}_of_T._cruzi_(VL-10_strain)_at_different_days_after_infection.

Thedatarepresenttheaverageoftwoindependentexperiments.

Consideringtheresultsofreal-timePCR,theaveragenumberof

par-asitesdetectedinthehearttissueat60and120d.i.were2608.83

and925.31parasites/30mgofcardiactissueandintheperipheral

bloodwere159.38and174.63parasites/0.1mLofblood,

respec-tively.TherewasasigniﬁcantPearsoncorrelationbetweenparasite

loadpresentintheperipheralbloodandinthecardiactissue

dur-ingacuteandchronicphasesofdisease(p<0.001andrvalueof

0.8068).

Later, in the group of treated animals (Fig. 6), at 30 d.i.

(end of chemotherapeutic schedule), the PCR performed with

DNA extracted from heart (PCR-H) detected an average of

416.69parasites/30mgofhearttissueandthePCRperformedwith

DNAfromblood(PCR-B)foundanaverageof1.16parasites/0.1mL

ofblood.Afterthisperiod,thelevelsofparasitism(inbloodand

heart)increasedagain,beingequivalentat60and120d.i.by

real-timePCRdetection.The“+”signat120d.i.indicatesapositiveresult

byFBE,detectedafterexhaustiveanalysissincetheparasitecount

Fig.5. Bloodandhearttissueparasitismdetectedbyreal-timePCRinblood(PCR– blood)andhearttissues(PCR–heart)ofmiceinfectedwith5×103_{trypomastigotes}

ofTrypanosomacruzi(strainVL-10)atdays10,16,30,60and120afterinfection.The datarepresenttheaverageoftwoindependentexperiments.Asteriskdenotesa sig-niﬁcantdifference,asdeterminedbytheMann–Whitneytest(p<0.05).Theinsert representthecorrelationanalysisoftissueandbloodparasitism,beingPearson (r)=0.8068andp<0.001.

Fig.6.ParasitesdetectedbyFreshBloodExamination(FBE)andbyreal-timePCR inblood(PCR–blood)andincardiactissues(PCR–heart)ofmiceinoculatedwith 5×103_{trypomastigote}_forms_of_Trypanosoma_cruzi_(VL10_strain)_and_treated_with

Itraconazole.Thetreatmentwasperformedondays10–30post-infection.Different symbolsindicatesigniﬁcantdifference(p<0.05).

bythemethodofBrener (1962)showednegativeresults.These

datashowedthattheVL-10strainofT.cruziwasfullyresistantto

Itz,sincealltreatedanimalsshowedparasitismupto90daysafter

treatment.

3.3. Immunologicalanalysisandquantiﬁcationofcardiac

inﬂammation

Correlatingwiththeincreasinginparasiteburden,serumlevels

ofCCL2(Fig.7A)andCCL5(Fig.7B)wereproportionallyelevated

duringtheearlyacutephase(10d.i.)oftheinfectionpeakingat30

d.i.withserumlevels∼6-fold(CCL2)and∼3-fold(CCL5)higher

compared tonon-infectedmice,except forthetreated animals,

whichshowedsimilarlevelstothosenon-infectedanimals.

How-ever,atthe90daysaftertreatment,thelevelsofthesechemokines

increasedandbecamesimilartothosenon-treatedinfected

ani-mals,whichwere3and2-foldlowers,respectively,thanthelevels

observedat30d.i.,butyethighercomparedtonon-infectedmice.

Levelsofnitricoxide(NO)werealsomeasuredinbothphasesof

infection,asshowedin Fig.7C. At30d.i. serumconcentrations

ofNO reached∼3.3-fold higherthanthose observedtohealthy

mice.Interestingly,whentheparasitismwascontrolled,therewas

adecreaseinNOlevelsreachingsimilarlevelstohealthyanimals

at120d.i.,aswellasthetreatedones,whichalwayshadNOlevels

similartothosenon-infectedmice.

Besides,attheendoftreatment(30d.i.),treatedinfected

ani-mals(Fig.8C)showedlessinﬂammatoryinﬁltratethannon-treated

infectedcontrol(Fig.8D)butsigniﬁcantlyhigher(p<0.001)than

the healthy mice (Fig. 8B). Furthermore, the partial protection

observedafterthetreatmentwasnotmaintainedthroughoutthe

courseofinfection(Fig.8A)and,at120d.i.,weobservedhigh

cel-lularityininfectedanimals,regardlessoftheyhavebeentreatedor

nottreated.

4. Discussion

In research laboratories, PCR has been proposed to be an

alternativetoolforT.cruziquantiﬁcationsinceitismoresensitive

thanthetraditionalparasitologicaltechniques,suchasfreshblood

examination,xenodiagnosisandhemoculture(Moseretal.,1989;

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Fig.7. CCL2(A),CCL5(B)andnitricoxide(C)detectedintheplasmaofmiceinfectedwithT.cruzi(I),non-infected(NI)andtreatedinfected(TI)at10,30and120daysof infection(d.i.).Differenceswereconsideredsigniﬁcantatp<0.05.

ThedisadvantageofconventionalPCR,however,istimeconsuming

andhighriskoffalsepositiveresultsduetocarry-over

contamina-tion(sincetheampliﬁedproductisdetectedbygelelectrophoresis)

alongwiththeimpossibility ofperformingquantitativeanalysis

(Piron etal.,2007),oncethecalculationofampliﬁedproductis

limitedtotheplateauphaseoftheampliﬁcationreaction.

In this context, the quantitative real-time PCR is emerging

asanappropriatemoleculartoolformonitoringparasiteloadin

experimentalT.cruzi infections.Real-time PCRacquires dataat

eachcycleofthePCRreaction,allowingthecalculationof

prod-uctamountfromthelog-linearregionoftheampliﬁcationcurve

(CummingsandTarleton,2003).Asatargetforampliﬁcation,

satel-liteT.cruziDNA(representedin104_to₁₀5_copies_in_the_parasite

genome)highlyconserved(Gonzalezetal.,1984;Moseretal.,1989;

Eliasetal.,2005)wasusedtoprovideaccurateandefﬁcientPCR

based-measurements.PrimersetusedforT.cruziDNA(Cummings

andTarleton,2003)iscapableofamplifyingatandemlyrepeated

genomicsequenceof195basepairs(Gonzalezetal.,1984;Moser

etal.,1989).Theinternalcontrol(asinglecopyofthemouseTNF-␣

gene)wasusedtocorrectvariationsininitialsamplesamount,DNA

recovery,and samples loading(Cummings andTarleton, 2003).

Theoptimizationoftheabovementionedreal-timePCRstrategy

allowedarapid,reproducibleandsensitivequantiﬁcationofT.cruzi

directlyinbloodandhearttissuesofmice,aswellasmonitoringthe

courseofinfectioninanimalsunderspeciﬁctreatment.The

proto-colwasfollowedbyconfirmationoftheamplificationspecificityby

analysisofmeltingcurves.

Inthiswork,PCRinhibitorswhichcaninterferewiththe

ampli-ﬁcationcyclesandalter thereactionefﬁciencywereminimized

withtheuseofanoptimizedextractionprotocolfollowedby

dilu-tionoftheextractedDNAinordertoobtaintheconcentrationof

25ng/␮LofDNA.Theendpointofthecurve(100parasiteequiv.)

wasdilutedwith25ng/␮LofDNAtomakesurethatthehigh

sen-sitivityobservedin thetest wasnot favored bydilution ofthe

standardsinwater.Thus,itwasfoundthatdilutionofthestandards

inwaterorDNAdoesnotinterferewiththetestsensitivity.The

Fig.8.AnalysisofhistologicalsectionsofheartsfrommiceinfectedwithVL-10strainofTrypanosomacruzi.(A)CellularityofthemyocardialsectionsofmiceinfectedwithT. cruzi(I),non-infected(NI)andtreatedinfected(TI)at10,30and120daysofinfection(d.i.).SectionsofmyocardiumofNI(B),TI(C)andI(D)at30d.i.(H&E,40×magniﬁcation). Differenceswereconsideredsigniﬁcantforp<0.05.

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standardcurvesgeneratedforquantiﬁcationofbloodandtissue

parasitesshowedampliﬁcationefﬁcienciesabove90%.

SincetheDNArecoveryfrombloodandtissuewassigniﬁcantly

variable,itwasimportanttohaveaninternalcontroltonormalize

theamountofsamplebeinganalyzedineachreal-timePCRassay

(CummingsandTarleton,2003).TheDNAofTNF-␣presentinthe

standardsample(generatedwith200␮Lofbloodor30mgofheart

tissue)wasassignedasanarbitraryvalueof103_,_since_we_do_not

needtoknowtheabsolutevalueofTNF-_␣,buttheexpectedvalue

in200␮Lofbloodor30mgofcardiactissuetotherebynormalize

thesampleloadingerrors.Theuseofareferencegeneallowedus

toobtainreproduciblequantiﬁcationofT.cruziinbloodandheart

samples.Wedemonstratedthereproducibilityoftheassayby

per-formingthequantiﬁcationofthesamegroupofbloodandheart

tissuessamplesintwoPCRtestsconductedatdifferenttimes.

ToverifywhetherthequantiﬁcationofT.cruziDNAwouldreﬂect

thenumberofliveparasitespresentinbloodsamples,their

quan-tiﬁcationwasalwaysperformedinparallelbyFBEandreal-time

PCR.Similarproﬁlesofparasitemiawereobservedinbothmethods

ofquantiﬁcation;however,thereal-timePCRwasabletoquantify

parasitesat60and120d.i.whenparasitemiawaslow.

Further-more,theFBEdoneinparallelwithPCRdemonstratesnoinhibition

andnodrugsinterferenceinthePCRquantiﬁcationofparasitemia.

Thestandardizationofreal-timePCRusing200␮Lofblood

con-tributedtothehighersensitivityofthistechniquecomparedwith

theFBE,whichrequires5␮Lofblood(mainlybecauselarger

vol-umesofbloodaffecttheperceptionandparasitesquantiﬁcationby

theobserver).However,wecannotdisregardthehighefﬁciencyof

FBEformonitoringtheparasitemiaduringtheacutephaseof

infec-tionand/orwhentheparasitemiaishigh.Thegreatadvantageof

PCR,however,isitsapplicationinmonitoringofparasitesinboth

bloodandcardiactissueofmicewithhighsensitivity.Attheendof

treatment,onlythereal-timePCRwasabletodetectandquantify

parasitism,aswellasduringthechronicphaseofinfection.

Besides,therewasa positive correlationbetweenblood and

tissueparasitism(r=0.8068andp<0.001)detectedbyreal-time

PCR.Previously,ithasalsobeendescribedacorrelationamongthe

parasiteburden,theintensityofinﬂammatoryprocessesandthe

severityofthediseaseinbothhumansandexperimentalanimals

(Zhangand Tarleton,1999;Pérez-Fuentesetal.,2003;Schijman etal.,2004;Benvenutietal.,2008).Althoughparasiteisthemain

triggerofcardiaclesions,theimmune systemof thevertebrate

host exerts a decisive role in the development of lesions. The

effectiveimmunedefenseofthevertebratehostrequiresaﬂow

directedandprecisepositioningofeffectorscellstowardthe

infec-tionsite(Luster,2002).ChemokinessuchasCCL2andCCL5,have

beenobservedinhumanandmurinemacrophagesand

cardiomy-ocytesinfectedbyT.cruzi(Villaltaetal.,1998;Alibertietal.,1999;

Machadoetal.,2000).Severalauthorshavedescribedtheroleof

thesechemokinestoleukocyteattractionanditslikelyinvolvement

incontrollingthegrowthofT.cruziandNOproduction(Sallusto

et al.,1998; Villaltaetal., 1998; Alibertiet al.,1999; Machado etal.,2000;Coelhoetal.,2002;Talvanietal.,2009;Talvaniand Teixeira,2011).Probablythehighparasitismintheheartandblood

(mainlyobservedwithin30d.i.intheinfectednon-treated

ani-mals),exertedinﬂuenceintheincreasedexpressionofCCL2and

CCL5bymacrophagesandactivatedCD4+_and_CD8+ _T_cells,

con-tributing to thedirected migration of leukocytes to the injury

site.AccordingtoPaivaetal.(2009),thechemokineCCL2is

pro-ducedin large quantitiesin the heart of miceinfected withT.

cruziandplaysanimportantroleinthedestructionofparasites

bymacrophages.Infectedcardiomyocytescoupledtotheinﬂuxof

monocytes/macrophagestotheinfectedhearttissue,probably

con-tributetotheincreasedproductionofchemokinesand NO.This

viewalsoexplainsthelackofstimulationoftheseinﬂammatory

moleculesintreatedanimalsat30d.i.sincetheparasitismwas

controlledbythedrugatthistime. Ontheotherhands,NO,as

describedbyseveralauthors(Saefteletal.,2001;Machadoetal.,

2008)exertstrypanocidalactivitybutalsocausedamagetoinfected

tissue,whichcanbeaggravatedbytheinﬂuxofCD8+_T_cells_rich_in

granulysinandperforin(Stegelmannetal.,2005),directedtothe

siteofinfectioninaCCL5-dependentmanner.However,inparallel

withthedevelopmentofthespeciﬁcimmuneresponse,parasitism

levelswerecontrolledandtherespectivestimulusforthe

expres-sionofchemokines,cytokinesandNO,partially,reduced.Probably

thisisonereasonbywhichanimalshavelowlevelsofCCL2,CCL5

and NOat 120d.i.,regardlessof whethertheywere treatedor

non-treated.Buttheprevalenceofcardiacinﬂammationat120d.i.

(inbothtreatedandnon-treatedmice)suggestsanimbalancein

theimmuneresponseoftheseanimals.Therefore,infectionwith

T.cruzishouldnotbelinkedinvariablytoadistinctparasiteload,

butaﬁnebalancebetweenresponsesthat,althoughessentialfor

hostresistance,mayalsocontributetoimmunopathology(Roggero

etal.,2002).

Finally,theexperimentalmodelofT.cruziinfectionusedinthis

studyinassociationwithrealtimePCR,asaprimarytool,was

suit-ableshowingtobeusefulininvestigationsinvolvingexperimental

chemotherapyandinﬂammatoryresponse.Besides,the

combina-tionofhighsensitivityandspeciﬁcity,lowcontaminationriskand

greaterspeedofworkconﬁrmthereal-timePCRtechnologyasa

moderntool toassist instudiesinvolvingthecourseofChagas’

infectioninexperimentalchemotherapy.

Acknowledgments

This work received ﬁnancial support from the Drugs for

NeglectedDiseaseInitiative(DNDi;Geneva,Switzerland),theUBS

OptimusFoundationofSwitzerland,RedeMineiradeBioterismo

(FAPEMIG),UniversidadeFederaldeOuroPreto,andresearch

fel-lowshipsfromConselhoNacionaldeDesenvolvimentoCientíﬁcoe

Tecnológico(Bahia,M.T.andTalvani,A.),fromFundac¸ãodeAmparo

àPesquisadoEstadodeMinasGerais(Caldas,S.)andCoordenac¸ão

deAperfeic¸oamentodePessoaldeNívelSuperior(CAPES)(Caldas,

I.S.).

References

Aliberti,J.C., Machado, F.S.,Souto,J.T.,Campanelli, A.P., Teixeira,M.M., Gazz-inelli, R.T.,Silva, J.S.,1999. Beta-Chemokinesenhance parasiteuptakeand promotenitricoxide-dependentmicrobiostaticactivityinmurine inﬂamma-torymacrophagesinfectedwithTrypanosomacruzi.InfectionandImmunity67, 4819–4826.

Bankowski,M.J.,Anderson,S.M.,2004.Real-timenucleicacidampliﬁcationin clin-icalmicrobiology.ClinicalMicrobiologyNews26,9–15.

Benvenuti,L.A.,Roggerio,A.,Freitas,H.F.,Mansur,A.J.,Fiorelli,A.,Higuchi,M.L.,2008. ChronicAmericantrypanosomiasis:parasitepersistenceinendomyocardial biopsiesisassociatedwithhigh-grademyocarditis.AnnalsofTropicalMedicine andParasitology102,481–487.

Brener,Z.,1962.Therapeuticactivityandcriterionofcureonmiceexperimentally infectedwithTrypanosomacruzi.RevistadoInstitutodeMedicinaTropicalde SaoPaulo4,389–396.

Caldas,I.S.,Talvani,A.,Caldas,S.,Carneiro,C.M.,Lana,M.,Guedes,P.M.M.,Bahia, M.T.,2008.BenznidazoletherapyduringacutephaseofChagasdiseasereduces parasiteloadbutdoesnotpreventchroniccardiaclesions.ParasitologyResearch 103,413–421.

Chagas,C.,1909.Novatripanosomíasehumana:estudossobreamorfologiaeo cicloevolutivodoSchizotrypanumcruzin.gen.n.sp.,agenteetiológicodenova entidademórbidadohomem.MemoriasdoInstitutoOswaldoCruz1,159–218. Cockerill,F.R.,2003.Applicationofrapid-cyclereal-timepolymerasechain reac-tionfordiagnostictestingintheclinicalmicrobiologylaboratory.Archivesof PathologyandLaboratoryMedicine127,1112–1120.

Coelho,P.S.,Klein,A.,Talvani,A.,Coutinho,S.F.,Takeuchi,O.,Akira,S.,Silva,J.S., Can-izzaro,H.,Gazzinelli,R.T.,Teixeira,M.M.,2002. Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi try-pomastigotes induce invivo leukocyterecruitment dependent on MCP-1 productionbyIFN-gamma-primed-macrophages.JournalofLeukocyteBiology 71,837–844.

Cummings,K.L.,Tarleton,R.L.,2003.RapidquantitationofTrypanosomacruziinhost tissuebyreal-timePCR.MolecularandBiochemicalParasitology129,53–59.

(8)

deFreitas,V.L.,daSilva,S.C.,Sartori,A.M.,Bezerra,R.C.,Westphalen,E.V.,Molina, T.D.,Teixeira,A.R.,Ibrahim,K.Y.,Shikanai-Yasuda,M.A.,2011.Real-timePCR inHIV/TrypanosomacruzicoinfectionwithandwithoutChagasdisease reacti-vation:associationwithHIVviralloadandCD4level.PLoSNeglectedTropical Diseases5,e1277.

Dias,J.C.,1992.EpidemiologyofChagasdisease.In:Wendel,S.,Brener,Z.,Camargo, M.S.,Rassi,A.(Eds.),Chagasdisease(AmericanTrypanosomiasis):itsimpacton transfusionandclinicalmedicine.ISBTBrasil,SãoPaulo,pp.49–80.

Dias,J.C.,Silveira,A.C.,Schoﬁeld,C.J.,2002.TheimpactofChagasdisease con-trolin LatinAmerica: areview. MemoriasdoInstituto OswaldoCruz97, 603–612.

Duffy,T.,Bisio,M.,Altcheh,J.,Burgos,J.M.,Diez,M.,Levin,M.J.,Favaloro,R.R., Freilij,H.,Schijman,A.G.,2009.Accuratereal-timePCRstrategyformonitoring bloodstreamparasiticloadsinchagasdiseasepatients.PLoSNeglectedTropical Diseases3,e419.

Elias,M.C.,Vargas,N.,Tomazi,L.,Pedroso,A.,Zingales,B.,Schenkman,S.,Briones, M.R.,2005.Comparativeanalysisofgenomic sequencessuggeststhat Try-panosomacruziCLBrenercontainstwosetsofnon-intercalatedrepeatsof satelliteDNAthatcorrespondtoT.cruziIandT.cruziIItypes.Molecularand BiochemicalParasitology140,221–227.

Filardi,L.S.,Brener,Z.,1987.SusceptibilityandnaturalresistanceofTrypanosoma cruzistrainstodrugsusedclinicallyinChagasdisease.TransactionsoftheRoyal SocietyofTropicalMedicineandHygiene81,755–759.

Gascon,J.,Albajar,P.,Canas,E.,Flores,M.,Prat,J.,Herrera,R.N.,Lafuente,C.A., Lucia-rdi,H.L.,Moncayo,A.,Molina,L.,Munoz,J.,Puente,S.,Sanz,G.,Trevino,B., Sergio-Salles,X.,2007.Diagnosis,managementandtreatmentofchronic Cha-gas’heartdiseaseinareaswhereTrypanosomacruziinfectionisnotendemic. RevistaEspanoladeCardiologia60,285–293.

Gonzalez,A.,Prediger,E.,Huecas,M.E.,Nogueira,N.,Lizardi,P.M.,1984. Minichromo-somalrepetitiveDNAinTrypanosomacruzi:itsuseinahigh-sensitivityparasite detectionassay.ProceedingsoftheNationalAcademyofSciencesoftheUnited StatesofAmerica81,3356–3360.

Hardison,J.L.,Wrightsman,R.A.,Carpenter,P.M.,Kuziel,W.A.,Lane,T.E.,Manning, J.E.,2006.TheCCchemokinereceptor5isimportantincontrolofparasite repli-cationandacutecardiacinﬂammationfollowinginfectionwithTrypanosoma cruzi.InfectionandImmunity74,135–143.

Junqueira,A.C.,Chiari,E.,Wincker,P.,1996.Comparisonofthepolymerasechain reactionwithtwoclassicalparasitologicalmethodsforthediagnosisofChagas diseaseinanendemicregionofnorth-easternBrazil.TransactionsoftheRoyal SocietyofTropicalMedicineandHygiene90,129–132.

Kirchhoff,L.V.,Votava,J.R.,Ochs,D.E.,Moser,D.R.,1996.ComparisonofPCRand microscopicmethodsfordetectingTrypanosomacruzi.JournalofClinical Micro-biology34,1171–1175.

Luster,A.D.,2002.Theroleofchemokinesinlinkinginnateandadaptiveimmunity. CurrentOpinioninImmunology14,129–135.

Machado,F.S.,Martins,G.A.,Aliberti,J.C.,Mestriner,F.L.,Cunha,F.Q.,Silva,J.S., 2000.Trypanosomacruzi-infectedcardiomyocytesproducechemokinesand cytokinesthattriggerpotentnitricoxide-dependenttrypanocidalactivity. Cir-culation102,3003–3008.

Machado,F.S.,Souto,J.T.,Rossi,M.A.,Esper,L.,Tanowitz,H.B.,Aliberti,J.,Silva,J.S., 2008.Nitricoxidesynthase-2modulateschemokineproductionbyTrypanosoma cruzi-infectedcardiacmyocytes.MicrobesandInfection10,1558–1566. Marcon,G.E.,Andrade,P.D.,deAlbuquerque,D.M.,Wanderley,J.S.,deAlmeida,E.A.,

Guariento,M.E.,Costa,S.C.,2002.Useofanestedpolymerasechainreaction (N-PCR)todetectTrypanosomacruziinbloodsamplesfromchronicchagasic patientsandpatientswithdoubtfulserologies.DiagnosticMicrobiologyand InfectiousDisease43,39–43.

Moreno,M.,D’avila,D.A.,Silva,M.N.,Galvão,L.M.,Macedo,A.M.,Chiari,E.,Gontijo, E.D.,Zingales,B.,2010.Trypanosomacruzibenznidazolesusceptibilityinvitro doesnotpredictthetherapeuticoutcomeofhumanChagasdisease.Memorias doInstitutoOswaldoCruz105,918–924.

Moser,D.R.,Kirchhoff,L.V.,Donelson,J.E.,1989.DetectionofTrypanosomacruzi byDNAampliﬁcationusingthepolymerasechainreaction.JournalofClinical Microbiology27,1477–1482.

Paiva,C.N.,Figueiredo,R.T.,Kroll-Palhares,K.,Silva,A.A.,Silverio,J.C.,Gibaldi,D., Pyrrho,A.S.,Benjamim,C.F.,Lannes-Vieira,J.,Bozza,M.T.,2009.CCL2/MCP-1

controlsparasiteburden,cellinﬁltration,andmononuclearactivationduring acuteTrypanosomacruziinfection.JournalofLeukocyteBiology86,1239–1246. Pérez-Fuentes,R.,Guégan,J.F.,Barnabé,C.,López-Colombo,A.,Salgado-Rosas,H., Torres-Rasgado,E.,Briones,B.,Romero-Díaz,M.,Ramos-Jiménez,J., Sánchez-Guillén, M.C.,2003. Severity ofchronic Chagasdisease is associated with cytokine/antioxidantimbalanceinchronicallyinfectedindividuals. Interna-tionalJournalforParasitology33,293–299.

Piron,M.,Fisa,R.,Casamitjana,N.,Lopez-Chejade,P.,Puig,L.,Verges,M.,Gascon,J., Prat,J.,Portus,M.,Sauleda,S.,2007.Developmentofareal-timePCRassayfor Trypanosomacruzidetectioninbloodsamples.ActaTropica103,195–200. Roggero,E.,Perez,A.,Tamae-Kakazu,M.,Piazzon,I.,Nepomnaschy,I.,Wietzerbin,

J.,Serra,E.,Revelli,S.,Bottasso,O.,2002.Differentialsusceptibilitytoacute Try-panosomacruziinfectioninBALB/candC57BL/6miceisnotassociatedwith adistinctparasiteloadbutcytokineabnormalities.ClinicalandExperimental Immunology128,421–428.

Saeftel, M., Fleischer, B., Hoerauf, A., 2001. Stage-dependent role of nitric oxideincontrolofTrypanosomacruziinfection.InfectionandImmunity69, 2252–2259.

Sallusto,F.,Lanzavecchia,A.,Mackay,C.R.,1998.Chemokinesandchemokine recep-torsinT-cellprimingandTh1/Th2-mediatedresponses.ImmunologyToday19, 568–574.

Schijman,A.G.,Vigliano,C.A.,Viotti,R.J.,Burgos,J.M.,Brandariz,S.,Lococo,B.E.,Leze, M.I.,Armenti,H.A.,Levin,M.J.,2004.TrypanosomacruziDNAincardiaclesions ofArgentineanpatientswithend-stagechronicchagasheartdisease.American JournalofTropicalMedicineandHygiene70,210–220.

Schoﬁeld,C.J.,Jannin,J.,Salvatella,R.,2006.ThefutureofChagasdiseasecontrol. TrendsinParasitology22,583–588.

Stegelmann,F.,Bastian,M., Swoboda,K., Bhat, R.,Kiessler, V.,Krensky,A.M., Roellinghoff,M.,Modlin,R.L.,Stenger,S.,2005.CoordinateexpressionofCC chemokineligand5,granulysin,andperforininCD8+Tcellsprovidesahost defensemechanismagainstMycobacteriumtuberculosis.JournalofImmunology 175,7474–7483.

Stordeur,P.,Poulin,L.F.,Craciun,L.,Zhou,L.,Schandené,L.,deLavareille,A.,Goriely, S.,Goldman,M.,2002.CytokinemRNAquantiﬁcationbyreal-timePCR.Journal ofImmunologicalMethods259,55–64.

Talvani,A.,Coutinho,S.F.,Barcelos,L.S.,Teixeira,M.M.,2009.CyclicAMPdecreases theproductionofNOandCCL2bymacrophagesstimulatedwithTrypanosoma cruziGPI-mucins.ParasitologyResearch104,1141–1148.

Talvani,A.,Ribeiro,C.S.,Aliberti,J.C.,Michailowsky,V.,Santos,P.V.,Murta,S.M., Romanha,A.J.,Almeida,I.C.,Farber,J.,Lannes-Vieira,J.,Silva,J.S.,Gazzinelli, R.T.,2000.Kineticsofcytokinegeneexpressioninexperimentalchagasic car-diomyopathy:tissueparasitismand endogenousIFN-gammaasimportant determinants of chemokinemRNA expressionduring infectionwith Try-panosomacruzi.MicrobesandInfection2,851–866.

Talvani,A.,Teixeira,M.M.,2011.InﬂammationandChagasdisease:some mecha-nismsandrelevance.AdvancesinParasitology76,171–194.

Teixeira,M.M.,Gazzinelli,R.T.,Silva,J.S.,2002.Chemokines,inﬂammationand Try-panosomacruziinfection.TrendsinParasitology18,262–265.

Toledo,M.J.,Bahia,M.T.,Carneiro,C.M.,martins-ﬁlho,O.A.,Tibayrenc,M.,Barnabé,C., Tafuri,W.L.,Lana,M.,2003.Chemotherapywithbenznidazoleanditraconazole formiceinfectedwithdifferentTrypanosomacruziclonalgenotypes. Antimicro-bialAgentsandChemotherapy47,223–230.

Urbina,J.A.,2010.SpeciﬁcchemotherapyofChagasdisease:relevance,current lim-itationsandnewapproaches.ActaTropica115,55–68.

Vespa,G.N.,Cunha, F.Q.,Silva,J.S., 1994.Nitricoxideis involvedincontrolof Trypanosomacruzi-inducedparasitemiaanddirectlykillstheparasiteinvitro. InfectionandImmunity62,5177–5182.

Villalta,F.,Zhang,Y.,Bibb,K.E.,Kappes,J.C.,Lima,M.F.,1998.Thecysteine-cysteine familyofchemokinesRANTES,MIP-1alpha,andMIP-1betainducetrypanocidal activityinhumanmacrophagesvianitricoxide.InfectionandImmunity66, 4690–4695.

WorldHealthOrganization(WHO),2002.ControlofChagasdisease.SecondReport oftheWHOExpertCommittee.WHOTechnicalReportSeries.905,Geneva,109p. Zhang,L.,Tarleton,R.L.,1999.Parasitepersistencecorrelateswithdiseaseseverity andlocalizationinchronicChagas’disease.JournalofInfectiousDiseases180, 480–486.