ISSN 0036-4665 ISSN 1678-9946 on lines3.amazonaws.com/zanran_storage/ · IV ENDEREÇO INSTITUTO DE...

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ISSN 0036-4665ISSN 1678-9946online

Established: 1959.The year 2010 is the 51st anniversaryof continuous publication

UNIVERSIDADE DE SÃO PAULO - BRAZILFACULDADE DE MEDICINA

InstitutodeMedicinaTropicaldeSãoPauloDirector: Prof. Dr. Claudio Sergio Pannuti

EDITOR‑IN‑CHIEF EMERITUSEDITORS Prof.Dr.ThalesF.deBrito Prof.Dr.LuisRey(FoundingEditor)AssociateEditors:Prof.Dr.MarcelloFabianodeFranco Prof.Dr.CarlosdaSilvaLacaz Prof.Dr.PedroPauloChieffi

EDITORIALBOARDAlanL.deMelo(BeloHorizonte,MG)AlbertoDuarte(S.Paulo,SP)AngelaRestrepoM.(Medellin,Colombia)AnnaSaraS.Levin(S.Paulo,SP)AntonioA.Barone(S.Paulo,SP)AntonioCarlosNicodemo(S.Paulo,SP)AntonioSesso(S.Paulo,SP)AntonioW.Ferreira(S.Paulo,SP)BarnettL.Cline(NewOrleans,USA)CarlosF.S.Amaral(BeloHorizonte,MG)CelsoGranato(S.Paulo,SP)CesarA.CubaCuba(Brasília,DF)CésarNaquiraV.(Lima,Peru)ClarisseM.Machado(S.Paulo,SP)ClaudioS.Pannuti(S.Paulo,SP)CláudioSantosFerreira(S.Paulo,SP)DaltonL.F.Alves(BeloHorizonte,MG)EridanCoutinho(Recife,PE)ErnestoHofer(RiodeJaneiro,RJ)EuclidesA.Castilho(S.Paulo,SP)EufrosinaS.Umezawa(S.Paulo,SP)FanHuiWen(S.Paulo,SP)FernandoA.Corrêa(S.Paulo,SP)FernandoMontero‑Gei(SanJosé,CostaRica)

FlairJ.Carrilho(S.Paulo,SP)GilBenard(S.Paulo,SP)GiocondaSan‑Blas(Caracas,Venezuela)GovindaVisvesvara(Atlanta,USA)HeitorF.AndradeJr.(S.Paulo,SP)HenriqueL.Lenzi(RiodeJaneiro,RJ)HiroGoto(S.Paulo,SP)IsesA.Abrahamsohn(S.Paulo,SP)JoãoCarlosPintoDias(BeloHorizonte,MG)JoãoRenatoRebelloPinho(SaoPaulo,SP)JoséEduardoLevi(S.Paulo,SP)JoséM.R.Zeitune(Campinas,SP)JuliaMariaCosta‑Cruz(Uberlândia,MG)JulioLitvoc(S.Paulo,SP)LuizCaetanodaSilva(S.Paulo,SP)LuizCarlosSevero(P.Alegre,RS)LuizJacinthodaSilva(Campinas,SP)LuizT.M.Figueiredo(Rib.Preto,SP)LygiaB.Iversson(S.Paulo,SP)MarcosA.Rossi(RibeirãoPreto,SP)MarcosBoulos(S.Paulo,SP)M.A.Shikanai‑Yasuda(S.Paulo,SP)MariaI.S.Duarte(S.Paulo,SP)MariaL.Higuchi(S.Paulo,SP)

MarioMariano(S.Paulo,SP)MirianN.Sotto(S.Paulo,SP)MoisésGoldbaum(S.Paulo,SP)MoysésMincis(S.Paulo,SP)MoysésSadigursky(Salvador,BA)MyrthesT.Barros(S.Paulo,SP)NilmaCintraLeal(Recife,PE)PauloC.Cotrim(SãoPaulo,SP)PauloM.Z.Coelho(BeloHorizonte,MG)PedroMorera(SanJosé,CostaRica)ReginaAbdulkader(S.Paulo,SP)RicardoNegroni(B.Aires,Argentina)RobertH.Gilman(Baltimore,USA)RobertoMartinez(Rib.Preto,SP)SemíramisGuimarãesF.Viana(Botucatu,SP)SilvinoA.Carvalho(S.Paulo,SP)SilvioAlencarMarques(Botucatu,SP)SumieHoshino‑Shimizu(S.Paulo,SP)ThelmaS.Okay(S.Paulo,SP)TsutomuTakeuchi(Tokyo,Japan)VenâncioA.F.Alves(S.Paulo,SP)VicenteAmatoNeto(S.Paulo,SP)ZiltonA.Andrade(Salvador,BA)

ExecutiveBoard‑Librarians: MariadoCarmoBertheRosa;SoniaPedrozoGomes;MariaÂngeladeCastroFígaroPinca;CarlosJoséQuinteiro

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Thepurposeofthe“RevistadoInstitutodeMedicinaTropicaldeSãoPaulo”(JournaloftheSãoPauloInstituteofTropicalMedicine)istopublishtheresultsofresearchwhichcontributesignificantlytoknowledgeofalltransmissiblediseases.

REVISTADOINSTITUTODEMEDICINATROPICALDESÃOPAULO(JOURNALOFTHES.PAULOINSTITUTEOFTROPICALMEDICINE).

SãoPaulo,SP‑Brasil,1959‑v.ilust.28cm

1959‑2010,1‑521973‑2002(supl.1‑12)2003(supl.13‑on‑lineonly)2005‑2010(supl.14‑16)

ISSN0036‑4665ISSN1678‑9946online

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Rev. Inst. Med. trop. S. Paulo Vol. 52 No. 6 P. 289-346 November-December, 2010


ADDRESSINSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO

Av. Dr. Enéas de Carvalho Aguiar, 47005403-000 São Paulo, SP - Brazil

Phone/Fax: 55.11.3062.2174; 55.11.3061-7005e-mail: [email protected]

SUBSCRIPTIONSFOREIGN COUNTRIESOne year (six issues) ........ U$ 200.00Single issue ...................... U$ 50.00

CONTENTS

EDITORIALArticlespublishedintheNovember/December2010issueofthe“RevistadoInstitutodeMedicinaTropicaldeSãoPaulo”‑T.deBRITO,P.P.CHIEFFI&M.F.FRANCO.....................................................................................................................................................................................289

TOxOPLASMOSISPreliminaryreportofHIVandToxoplasma gondiioccurrenceinpregnantwomenfromMozambique‑S.P.B.L.SITOE,B.RAFAEL,L.R.MEIRELES,H.F.ANDRADEJÚNIOR&R.THOMPSON.................................................................................................................................291

MICROBIOLOGYProfileofpatientswithBaggio‑Yoshinarisyndromeadmittedat“InstitutodeInfectologiaEmilioRibas”‑E.A.GOUVEIA,M.F.ALVES,E.MANTOVANI,L.K.OYAFUSO,V.L.N.BONOLDI&N.H.YOSHINARI.............................................................................................................297

AIDSNeurologiccytomegaloviruscomplicationsinpatientswithAIDS:retrospectivereviewof13casesandreviewoftheliterature‑C.ALMEIDASILVA,A.C.PENALVADEOLIVEIRA,L.VILAS‑BOAS,M.C.D.S.FINK,C.S.PANNUTI&J.E.VIDAL...................................305

LEPROSYLeprosycontrol:perspectives&epidemiologicalandoperationalaspects‑A.GONÇALVES,G.G.MANTELLINI&C.R.PADOVANI................311

VIROLOGYAdenovirusrespiratoryinfection:significantincreaseindiagnosisusingPCRcomparingwithantigendetectionandculturemethods‑E.STROPARO,C.R.CRUZ,M.C.DEBUR,L.R.VIDAL,M.B.NOGUEIRA,S.M.ALMEIDA,L.A.PEREIRA,I.ROTTA&S.M.RABONI.....317

PARASITOLOGYMelanoides tuberculataasintermediatehostofPhilophthalmus gralliinBrazil‑H.A.PINTO&A.L.deMELO.....................................................323

CASEREPORTTreatmentofseverechromoblastomycosiswithitraconazoleand5‑flucytosineassociation‑V.S.ANTONELLO,M.C.APPELDASILVA,E.CAMBRUZZI,D.A.KLIEMANN,B.R.SANTOS&F.QUEIROZ‑TELLES.........................................................................................................329

EnvenomationbyMicrurus coralsnakesinBrazilianAmazonregion:reportoftwocases‑P.P.O.PARDAL,J.S.O.PARDAL,M.A.C.GADELHA,L.S.RODRIGUES,D.T.FEITOSA,A.L.C.PRUDENTE&H.W.FAN.....................................................................................333

BRIEFCOMMUNICATIONEvaluationofEnterovirus71immunestatusinSãoPauloState,Brazil‑A.LUCHS,A.CILLI,D.H.RUSSO,F.F.COSTA,R.C.C.CARMONA&M.C.S.T.TIMENETSKY............................................................................................................................................................................................339

AUTHORINDEx.....................................................................................................................................................................................................343

SUBJECTINDEx.....................................................................................................................................................................................................345

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ENDEREÇOINSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO

Av. Dr. Enéas de Carvalho Aguiar, 47005403-000 São Paulo, SP - Brasil

Fone/Fax: 55.11.3062.2174; 55.11.3061-7005e-mail: [email protected]

Rev. Inst. Med. trop. S. Paulo Vol. 52 No. 6 P. 289-346 Novembro-Dezembro, 2010

CONTEÚDO


EDITORIALArtigospublicadosnofascículodenovembro/dezembrode2010da“RevistadoInstitutodeMedicinaTropicaldeSãoPaulo‑T.deBRITO,P.P.CHIEFFI&M.F.FRANCO.....................................................................................................................................................................................289

TOxOPLASMOSEDescriçãopreliminardaocorrênciadeinfecçãopeloHIVetoxoplasmoseemmulheresgrávidasemMoçambique‑S.P.B.L.SITOE,B.RAFAEL,L.R.MEIRELES,H.F.ANDRADEJÚNIOR&R.THOMPSON...........................................................................................................291

MICROBIOLOGIAPerfildospacientescomSíndromeBaggio‑Yoshinariadmitidosno“InstitutodeInfectologiaEmílioRibas”‑E.A.GOUVEIA,M.F.ALVES,E.MANTOVANI,L.K.OYAFUSO,V.L.N.BONOLDI&N.H.YOSHINARI.............................................................................................................297

AIDSComplicaçõesneurológicascausadaspelocitomegalovirusempacientescomaids:estudoretrospectivode13casoserevisãodaliteratura‑C.ALMEIDASILVA,A.C.PENALVADEOLIVEIRA,L.VILAS‑BOAS,M.C.D.S.FINK,C.S.PANNUTI&J.E.VIDAL...................................305

LEPRAControledahanseníase:perspectivaseaspectosepidemiológicoseoperacionais‑A.GONÇALVES,G.G.MANTELLINI&C.R.PADOVANI.....311

VIROLOGIAInfecçãorespiratóriaagudaporadenovírus‑comparaçãodosmétodosdePCReimunofluorescênciaindiretaparaoseudiagnósticoedadosclínicosdospacientesinfectados‑E.STROPARO,C.R.CRUZ,M.C.DEBUR,L.R.VIDAL,M.B.NOGUEIRA,S.M.ALMEIDA,L.A.PEREIRA,I.ROTTA&S.M.RABONI.................................................................................................................................................................317

PARASITOLOGIAMelanoides tuberculata comohospedeirointermediáriodePhilophthalmus gralli noBrasil‑H.A.PINTO&A.L.deMELO..................................323

RELATOSDECASOSTratamentodecromoblastomicoseseveracomaassociaçãoitraconazolee5‑flucitosina‑V.S.ANTONELLO,M.C.APPELDASILVA,E.CAMBRUZZI,D.A.KLIEMANN,B.R.SANTOS&F.QUEIROZ‑TELLES.........................................................................................................329

EnvenenamentoporcoraldogêneroMicrurus naAmazôniabrasileira:relatodedoiscasos‑P.P.O.PARDAL,J.S.O.PARDAL,M.A.C.GADELHA,L.S.RODRIGUES,D.T.FEITOSA,A.L.C.PRUDENTE&H.W.FAN.....................................................................................333

COMUNICAÇÃOBREVEAvaliaçãodasituaçãoimunitáriadoEnterovírus71noEstadodeSãoPaulo,Brasil‑A.LUCHS,A.CILLI,D.H.RUSSO,F.F.COSTA,R.C.C.CARMONA&M.C.S.T.TIMENETSKY..........................................................................................................................................................339

ÍNDICEDEAUTORES..........................................................................................................................................................................................343

ÍNDICEDEASSUNTOS.........................................................................................................................................................................................345

Rev. Inst. Med. Trop. Sao Paulo52(6):289, November-December, 2010doi: 10.1590/S0036-46652010000600001

EDITORIAL

ARTICLES PUBLISHED IN THE NOVEMBER/DECEMBER 2010 ISSUE OF THE “REVISTA DO INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO”

IntheNovember/December2010issueoftheRevistadoInstitutodeMedicinaTropicaldeSãoPaulo(JournaloftheSãoPauloInstituteofTropicalMedicine)GOUVEIAet al.studiedtheprofileofpatientswith Baggio‑Yoshinari syndrome (BYS) who underwent hospitalinternmentfromJuly1990toJuly2006.BYSisanewBraziliantick‑bornediseasecausedbyBorrrelia burgdorferisensulatomicroorganismthat resembles Lyme disease except for its epidemiological, clinicalandlaboratorialparticularities.From60patients’recordswithpositiveserology to B. burgdorferi, 19 were diagnosed as having BYS. ThebeginningofsymptomsinBYSgroupvariedfromonedaytosixyearsfromtheonsetofthediseaseandfourpatientswereincludedinacutediseaseand15inlatentstage.Unspecificsymptomsasfever(78.9%)andlymphadenomegaly(36.8%)wereidentifiedinalmostallcases.Sixpatientshadskinlesions(31.5%),sixarthralgiaorarthritis(31.5%)andeightneurological symptoms (42%).Twopatients showedantibodiesdirectedtoB. burgdorferiexclusivelyincerebralfluid.

LUCHSet al. evaluatedantibodiestoEnterovirus71(EV71)inSãoPauloStateduringthe1995‑2005period.NeutralizingantibodiestoEV71wereobservedin12.4%(55/442)ofserasamplessuggestingthatEV71isuncommoninthisregionbutthatthereisarelativelyhighsusceptibilitytoEV71relateddiseases.NeutralizingantibodiestoEV71wereobservedin8.7%(21/241)ofSãoPaulometropolitanareaserasamplesand16.9%(34/201)oftheserasamplesfromothermunicipalities.DisseminationofEV71isworryingincountryandcoastalareaswithoutadequateaccesstopipedwaterorsanitation.

SITOEet al. studiedHIVandToxoplasma gondii occurrencein150pregnantwomen fromMozambiquebyELISAandsixcerebrospinalfluid(CSF)samplesfromAIDSpatientswithencephalitis.HIVstatuswasconfirmedandCD4bloodcountswereobtainedfromHIV‑positiveindividualsandcomparedtothosewhowereHIV‑negative(31.3%vs10.9%).The data may indicate an interaction of HIV and T. gondii. Prevalenceofbothdiseasesincreaseswithage,butthisismoreclearlyseenfortoxoplasmosis.InHIVpatientssufferingfromencephalitisCSFserologyshowedthat33%ofspecificIgGCSFhadahighavidity.Lowerprevalenceratesofbothinfectionsinoldergroupscouldbeexplainedbymoredeathsintheinfectedgroups.UsingCD4countsasamarkeroftimeofHIVinfectionandcorrectingforage,patientswithcontactwithT. gondiihadfewerCD4cells,suggestingprolongedHIVdiseaseorothercauses.ToxoplasmaIgGprevalenceishigherinHIV+positivegroupswhichcouldbeascribedtoHIV‑andT. gondii-associatedriskfactors.The low incidenceofToxoplasma IgG inyoungeragegroups showsthat transmission couldbe related tobetter access to cyst‑containingmeatinadulthood.

PINTO&MELOreportedforthefirsttimeinBrazilMelanoides tuberculata as intermediatehostofPhilophtalmus gralli.Melanoides tuberculata that naturally harbored trematode larvae were collectedat the Pampulha dam, Belo Horizonte, during malacological surveysconducted from 2006 to 2010. From 7164 specimens of Melanoides tuberculata collected 25 (0.35%) were infected by cercariae of theMegalurous group, genus Philophthalmus. Successful experimentalinfectionsofGallus gallus domesticus wereobtainedandadultparasitesrecoveredfromthenictitatingmembranesofchickenswereidentifiedasPhilophthalmus gralli.

ANTONELLO et al. reported a case of treatment of severechromoblastomycosis with an association of itraconazole and5‑flucytosine.

STROPAROet al. aimingtoanalyzethesensitivityandspecificityoflaboratorialmethodsavailablefortheadenovirus(ADV)respiratoryinfectioncomparedantigendetectionbyindirectimmunofluorescenceassay (IF) and a specific nested polymerase chain reaction (PCR) inrespiratorysamplescollectedfromhospitalpatients.Molecularmethodswerefoundtobeusefulforrapiddiagnosisofadenovirusinfectionswithhighersensitivitythanantigendetectionspermittingasignificantincreaseindiagnosesofadenovirusinfection.

GONÇALVES et al. studied perspectives, epidemiological andoperationalaspectsofleprosycontrolandconcludedthatifaprojectionismade towards thefact that themaintenanceof thedisease’presentincidencelevelsconstituteeconomicandsanitarychallengesthatconfrontissuesrangingfromtheneoliberalmodelofglobalsocietalorganizationtospecificcompetencesofactionstakenbyhealthteamsinthefield.

ALVESDASILVAet al. reportedaretrospectivereviewof13casesofneurologiccytomegaloviruscomplicationsinAIDSpatientswhicharerarelydescribedintheantiretroviraltherapy(HAART)period.Althoughinfrequent,distinctneurologicalsyndromescausedbyCMVcontinuetocausehighmorbidityinAIDSpatients.Survivaldependsontheuseofeffectiveantiviral therapyagainstCMVandtheearly introductionofHAART.

PARDALet al. reportedtwocasesofprovencoralsnakesbitesinBelém,ParáwhicharescarcelydescribedintheAmazonregionwithabroadspectrumofclinicalmanifestations.Thefirst,moreseverecasewascausedbyMicrurus surinamensis andthesecond,withmildsignsofenvenomation,byMicrurus filiformis.

ThalesdeBRITO,EditorPedroPauloCHIEFFI,AssociateEditorMarcelloF.FRANCO,AssociateEditor

RevistadoInstitutodeMedicinaTropicaldeSãoPauloon line.

Publications from 1990 to the present data are now available on:http://www.scielo.br/rimtsp

PASTISSUES1959-1989(PDF)www.imt.usp.br/portal/

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Rev. Inst. Med. Trop. Sao Paulo52(6):291-295, November-December, 2010doi: 10.1590/S0036-46652010000600002

(1)InstitutoNacionaldeSaúde,MinistériodaSaúdedeMoçambique.(2)Lab.Protozoologia,InstitutodeMedicinaTropicaldeSãoPaulo,SãoPaulo,SP,Brasil.Correspondenceto:HeitorFrancodeAndradeJr.,Lab.Protozoologia IMTSP,Av.Dr.EneasdeCarvalhoAguiar470,05403‑000SãoPaulo,SP,Brasil.Phone+55.11.3061‑7010.Fax

+55.11.3088‑5237.E‑mail:[email protected]

PRELIMINARy REPORT OF HIV AND Toxoplasma gondii OCCURRENCE IN PREgNANT wOMEN FROM MOzAMBIqUE

SoniaPaulaBeneditoLuisSITOE(1),BernardeteRAFAEL(1),LucianaReginaMEIRELES(2),HeitorFrancodeANDRADEJR.(2)&RicardoTHOMPSON(1)

SUMMARY

Toxoplasmosis,aprotozoandisease,causesseverediseaseinfetusesduringpregnancyanddeadlyencephalitisinHIVpatients.Thereareseveralstudiesonitsseroprevalencearoundtheworld,butstudiesfocusingonAfricancountriesarelimitedinnumberandmostlyanecdotal.WestudiedtwogroupsofsamplesfromMozambiquebyELISA,usingserumsamplesfrom150pregnantwomenandsixCerebrospinalfluid(CSF)samplesfromAIDSpatientswithencephalitis.HIVstatuswasconfirmed,andCD4bloodcountswereobtainedfromHIV‑positivepregnantwomen.IgGseroprevalenceofthegroupasawholewas18.7%(28/150),withahigherprevalence inHIV‑positive individualscompared to thosewhowereHIV‑negative (31.3%, [18/58]vs.10.9%, [10/92])patients.Thesedatamaybebiasedduetocumulativeeffectsofexpositionaffectingdiseaseprevalence.Ifcorrected,thisdatamayindicateaninteractionofHIVandT. gondii.Prevalenceofbothdiseasesincreaseswithage,butthisismoreclearlyseenfortoxoplasmosis(p<0.005)thanHIVinfection,possiblyexplainedbyhighertransmissionofHIVafterchildhood.InHIVpatientssufferingfromencephalitis,CSFserologyshowedthat33%ofspecificIgGCSFhadahighavidity,whichwasinaccordancewiththedatafromthegroupofpregnantwomen.Lowerprevalenceratesofbothinfectionsinoldergroupscouldbeexplainedbymoredeathsintheinfectedgroups,resultinginanartificiallylowerprevalence.UsingCD4countsasamarkeroftimeofHIVinfection,andcorrectingforage,patientswithcontactwithT. gondiihadfewerCD4cells,suggestingprolongedHIVdiseaseorothercauses.ToxoplasmaIgGprevalenceishigherinHIV+groups,whichcouldbeascribedtoHIV‑andT. gondii‑associatedriskfactors,suchasexposuretohigherandmorediversesocialcontacts.ThelowincidenceofToxoplasmaIgGinyoungeragegroupsshowsthattransmissioncouldberelatedtobetteraccesstocyst‑containingmeatinadulthood,asenvironmentaltransmissionduetooocystsisusuallyblamedforhigherincidenceinchildren.Takentogether,thesedatasupporttheurgentneedofresearchintoxoplasmosisinAfrica,especiallyinthepresenceofHIVepidemics.

KEYwORDS:AustralAfrica;Toxoplasmosis;HIV;Serology;Pregnantwomen.

INTRODUCTION

Toxoplasma gondiiinfectionoccursworldwide,anditisoneofthemostcommoninfectionsinhumans.Theinfectionismainlyacquiredbyingestionofundercookedorrawmeatcontainingviabletissuecysts,orbyingestionoffoodandwaterthatiscontaminatedwithoocystsshedbyfelids(DUBEY&JONES,2008).Althoughthecourseoftheprimaryinfectionisusuallysubclinicalandthevastmajorityoftheinfectedhumanpopulation remainsasymptomatic, the infectioncancausesignificantmorbidityandmortalityincertaingroups(WEISS&DUBEY,2009).Toxoplasmosiscancauseseverediseaseinfetusesofacutelyinfectedpregnantwomen(MONTOYA&REMINGTON,2008).Additionally,thereactivationoflatentinfectionoccursinimmunecompromisedpatients,causinglife‑threateningdisease,especiallyencephalitis(PASSOSet al.,2000).EncephalitisduetoreactivatedtoxoplasmosisisoneofthemostcommonopportunisticneurologicalinfectionsinAIDSpatients,typically

observedin the laterstagesofhumanimmunodeficiencyvirus(HIV)infection(MASCHKEet al.,2000).Itsincidenceisdirectlyrelatedtotheprevalenceofanti‑T. gondiiantibodiesinthegeneralpopulation(PASSOSet al.,2000).Theprevalenceoftoxoplasmosis(between30%and60%inmostcountries)varieswidelyindifferentregionsoftheglobe(TENTERet al.,2000).Fewstudiesonthisprevalencehavebeenconductedinsub‑SaharanAfrica(SHIMELISet al.,2009,LINDSTRON et al.,2006)orinAustralAfrica.Instead,studieshavemostlybeenconductedinSouthAfrica(HARIet al.,2007),wherethediseasewasfirstdescribedinthe1950s(BECKER,1954;FASSER,1955).

In Mozambique, physicians anecdotally report congenitaltoxoplasmosis orAIDS encephalitis, but the true occurrence oftoxoplasmosis,whichiscurrentlyestimatedbyanti‑T. gondii antibodiesinpregnantwomenandpatientswithHIV/AIDSisunknown.Webelievethat defining the occurrence of toxoplasmosis, using anti‑T.gondii

SITOE,S.P.B.L.;RAFAEL,B.;MEIRELES,L.R.;ANDRADEJr.,H.F.&THOMPSON,R.‑PreliminaryreportofHIVandToxoplasma gondiioccurrenceinpregnantwomenfromMozambique.Rev.Inst.Med.Trop.SaoPaulo,52(6):291‑5,2010.

292

antibodiesduringantenatalcareinpregnantwomenorinAIDSpatientsinourcommunity,wouldreducetheriskoftreatabledamagetotheCNSandthehighmorbidityofthistypeofpatient(MASCHKEet al.,2000).

The objective of this study was to determine the occurrence oftoxoplasmosisinsamplesfrompublichealthsystemhospitalsandclinicsfromtheMaputoarea,bydetectingT. gondii‑specificIgGantibodiesfromwomeninthefirsttrimesteroftheirpregnancy,andalsobytiteringtheseantibodiesandtheiravidityinCerebrospinalfluid(CSF)samplesfromAIDSpatientswithsuspectedencephalitis.

MATERIALSANDMETHODS

Samples:We studied 150 serum samples from pregnant womenseekingfirsttrimesterprenatalcare.SerumsampleswerealsotestedforHIVinfection,andCD4countsweredeterminedinpositivesamples.CSFfromHIV‑positivepatientswithsuspectedencephalitisfromHospitalCentraldeMaputo,thelargepublichealthfacility,wereavailableforconfirmingToxoplasmaencephalitisusingspecificantibodydetectionandavidityassays.ThisstudywasperformedwithapprovaloftheEthicsCommissionofHCMandINSandinformedconsentwasobtainedfrompatientsortheirrelativesbeforecollection.Allsampleswereimmediatelyprocessedandstoredat‑20°Cuntiluse.

Enzyme Linked ImmunosorbentAssay (ELISA): Serumsamples were examined by immunoenzymatic assay (ELISA) usingthecommercialkits Toxoplasma gondii IgGELISA(TOXG‑013)andToxoplasma gondii IgM µ‑capture ELISA (T0XM‑016) (ELISA‑IBLImuno‑Biological Laboratories®, Hamburg, Germany) to identifythe presence of immunoglobulin IgG and IgM, according to themanufacturer’sinstructions.

CerebrospinalFluid(CSF)IgGAvidity:TheIgGavidityassaywasperformedaselsewheredescribed(MEIRELESet al.,2004)withminormodifications.ELISAmicroplates(Multiwellplate/polystyrene,Sigma)werecoatedwith100mL/wellofasolutioncontaining10mg/mLoftheT. gondiiantigenextractdilutedin6MureapH7.0,incubatedovernightat4°Candblockedforonehourat37°Cwith2%fatfreedrymilk.UndilutedCSFwasappliedtotwowellsandincubatedforonehourat37°C.Afterthis,onewellwaswashedwith100mLof6MureapH7.0,andincubatedat37°Cfor10min.After theincubation,boundIgGwasrevealedbyspecificperoxidaseconjugateando‑phenylenediamine.TheA

492nmwas

determinedin thecontrolandurea treatedwellsbyspectrophotometry(MultiskanMS).TheavidityofeachsamplewascalculatedasthepercentofA

492nmresistanttourea.Sampleswithmorethan75%A

492nmresistanceto

ureatreatmentwereconsideredtohavehighavidity.SampleswithcontrolwellvaluesgreaterthanthemeanplusthreeSDofA

492nmofnegativewells

wereconsideredpositiveforT. gondiiIgGandtoxoplasmosis.

Statistics:Comparisonsof frequenciesofeventsbetweengroupswere performed using two‑tailed Chi‑square tests. Comparisons ofquantitativedata,suchasCD4+counts,wereperformedusingStudent’st‑tests.Ageevolutionwastestedusinglinearregressionandgoodnessto fit or r2, with confidence interval comparison between slopes andintercepts.Differenceswereconsideredsignificantwhentheprobabilityofequalitywaslessthan0.05(p<0.05).WeusedforallcalculationsthestatisticalsoftwareGraphPadPrism5.0(GraphPadSoftware,SanDiegoCaliforniaUSA,www.graphpad.com).

RESULTS

We studied two groups of samples using ELISA methods.Thepopulationsstudiedincludedserumsamplesfrom150pregnantwomenfrompublicprenatalcareclinicsand6CSFsamplesfromAIDSpatientswithencephalitisfromthe“HospitalCentraldeMaputo”.HIVstatuswasconfirmedbyserology,andCD4bloodcountswereobtainedforHIV‑positivepregnantwomen.Theageofthesubjectsrangedfrom15to50yearsold,withanaverageof32years.

Theoveralloccurrenceofanti‑ToxoplasmaIgGantibodies,asshownin Fig. 1, was 18.7% (28/150) with only one case (3.6%, 95% C.I.0.09‑16)associatedwithanti‑T. gondiiIgMantibodies.ToxoplasmosisoccurrencewasmoreprevalentinHIV‑positivepatients(31.3%,18/58),whencomparedtoHIV‑negativepatients(10.9%,10/92)(p<0.005),witha3.6OddsRatioforToxoplasmainfectioninHIV‑infectedindividuals.In samples from HIV patients with encephalitis, cerebrospinal fluidserologyshoweda33%(95%CI4‑77%)occurrenceofspecificanti‑T. gondii IgGofhighavidityinCSF,whichwasexpectedaccordingtotheoccurrenceinthisgroup,andbasedonourdatafromthepregnantwomen.

TheoccurrenceofbothHIVorToxoplasmainfectionsincreaseswithage,asshowninFig.2,butthisismoreclearlyseenintoxoplasmosis(p<0.005)thaninHIVinfection(p<0.05),withsimilarslopes,butdifferentintercepts.

Looking for interaction between both infections, we sorted thepatientsaccordingtoHIVstatustolookforoccurrenceofToxoplasma-specific antibody, or according tooccurrenceofToxoplasma specificantibodyand frequencyofHIV infection inagegroups,as shown inFig.3.TherewasaclearincreaseofToxoplasmaoccurrenceaccordingtoagegroupsinbothHIV‑negative(Fig.3A)andHIV‑positivegroups(Fig.3B),butthecurveswerequitediverse,duetoadifferenceinageoffirstcontactwiththeagent.Thedifferenceininterceptswassignificant(p<0.004),withoutadifferenceintheirslopes.Thus,HIV‑infectedpregnantwomenappearedtohavehadcontactwithT. gondiiatloweragesthannon‑infectedwomen,withadifferenceofalmost13years,butafterthis,theincreaseofoccurrencewithagewassimilarforbothgroups.AftersortingbyoccurrenceofToxoplasma-specificantibody,theoccurrence

Fig.1‑ProportionofToxoplasma-specificantibodydetectioninserafrompregnantwomen

inMozambique,intotalandsortedaccordingtoHIVstatus,andCSFfromencephalitisAIDS

patients.Barsrepresent95%confidenceinterval.InHIVinfectedwomen,theoddsratiofor

toxoplasmainfectionis3.6timeshigher.


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ofHIVinfectionclearlyincreasedwithageinpregnantwomenwithoutToxoplasma antibodies(Fig.3C),butthiswasnotobservedinpregnantwomenwithT. gondiiantibodies,whopresentedwithahigherfrequencyofHIVinfectiondespiteitsagegroup(Fig.3D).

HIVevolution,asmeasuredbyCD4+cellcounts,wasevidentinthedatashowninFig.4.TheprogressionofHIVdisease,asindicated

bylowCD4meancounts,appearstobemoreintenseinToxoplasmainfectedpatients.ThisfactisnotassociatedwiththeageeffectorwithdelayedHIVdiseaseinToxoplasma‑freepersons,asitwasclearlyseeninyoungeragegroups(<30yearsold),whereasolderpeopledidnotshowthesameT. gondii infection effectintheirCD4+cellcounts.

DISCUSSION

Ourdataconcerningthefrequencyofanti‑T. gondiiantibodieswassimilartootherfindingsinAfrica,andsufferedfromthesameproblemsof sampling populations as similar reports (ZUMLA et al., 1991,LINDSTRONet al.,2006,NABIASet al.,1998).Theplanningofanadequate and reliable search for toxoplasmosis isurgentlyneeded inAfrica,duetotheHIVepidemicandtheriskofseverelethalordisablingopportunisticToxoplasmaencephalitis(MAKUWAet al.,1999).

Westudiedarelativelyuniformpopulation,withoutagedifferencesandof thesameethnicity.Theoccurrenceofbothdiseases increasedwithsubjectage,whichisexpectedwithconstantexposure.ThisfactcouldbeexplainedbyahighertransmissionofHIVduringadulthoodinoursamplepopulation.Stableorslightlyloweroccurrencesofbothinfections in older groups could be explained by biased selection ofnon‑infected survivors (resulting from death of infected patients), adefectofcross‑sectionalstudiesandtheprevalenceofHIVinfection.HIVoccurrencewashigherthantoxoplasmosisinourpopulation,but

Fig.2‑ProportionofHIV‑andToxoplasma‑specificantibodydetectioninserafrompregnant

womenfromMozambique,intotalandsortedaccordingtoagegroup.

Fig.3‑OccurrenceofHIVortoxoplasmosisinagegroups,sortedfortheotherdiseaseserologyinpregnantwomenfromMozambique.AandB:ToxoplasmosisoccurrenceaccordingtoHIV

statusA:HIVnegativesamples,B:HIVpositivesamples.CandD:HIVoccurrenceaccordingtotoxoplasmosisstatusC:Toxoplasmosisnegativesamples,D:Toxoplasmosispositivesamples.

Regressionwasestimatedascontinuouslinesandnumbersareexpressedaspositive/totalineachpoint.


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toxoplasmosisoccurrencewashigher intheHIV‑infectedpopulation,suggestinganinteractionbetweendiseases.ThesedatacouldbebiasedaccordingtothecumulativeeffectofdeathsduetoHIVthatsimilarlycould affect occurrence measure.This association of toxoplasmosisandHIVwasseenelsewhereinAfrica(ZUMLAet al.,1991),buttherearefewstudiesofthenon‑HIV‑infectedpopulation(SHIMELISet al.,2009, LINDSTRON et al., 2006).We found a higher toxoplasmosisoccurrence in the HIV‑positive groups as compared to HIV‑negativepregnantwomenthatcouldbeascribedtocommonorassociatedriskfactorsforbothinfections,suchasexposuretobothsexualcontactsandmeatconsumption.Thisfactisalsoshowninanti‑T.gondiiIgGfrequencybyagegroup,withadelayoftoxoplasmosisriskinoldergroupsoftheHIV‑negative population, which suggests that the transmission couldbe related to reduced exposure to cyst‑containing meat in adult life,andnotanenvironmentaltransmissionduetooocysts(whichisusuallyassociated with a higher incidence in children) (BAHIA‑OLIVEIRAet al., 2003). Several explanations for the interaction between bothinfectionsresultinginhighToxoplasmainfectioninHIVpatientshavebeenpreviouslyproposed(LIN&BOWMAN,1992).OnepossibilityisanincreaseinriskybehaviorinToxoplasmainfectedindividualsthatleadstoincreasedexposuretoHIVinfection.Suchachangeinbehaviorcould be due to parasite‑driven personality changes, as described inToxoplasma-infectedindividualsintheCzechRepublic(FLEGRet al.,1996).AnotherplausibleexplanationisthatToxoplasma infectionisamarkerofexposuretoriskysocialcontactsorhabits,whichcorrelateswithearlyHIVinfection.Theaccesstoinfectedmeatwouldbeonlyamarkerofmigrationfromsmallisolatedvillagestobiggercitiesortheimprovementofqualityoflifebybetterincome,resultinginmoresocialcontacts,whichcouldalsobeassociatedwithHIVinfection.

ThelowerCD4countsfoundinHIVpatientswhoalsopresentedwith anti‑T. gondii antibodies, as compared to HIV patients withoutthose antibodies, suggests intense HIV disease progression in thisgroup.ProgressioncouldbecausedbyHIVinfectionofmoreCD4cellswithhigherviralloadsandprogressivediseaseinT. gondiichronicallyinfectedpatients.ToxoplasmainfectioncanpromoteanincreaseinCD4cellsbyastrongspecificimmuneresponse(DENKERS&BUTCHER,

2005),ornonspecificCD4cellproliferationinducedbytoxoplasmosis(PURNERet al.,1998).MalariahassimilareffectsinHIVpatientswithmoresevereinfections,especiallyininfantsandpregnantwomen.ThereisevidencethatHIVprogressioncouldbemorerapidinmalariapatients,duetoincreasesintransientviralloadinmalarialbouts(REITHINGERet al.,2009).

ManagementofHIVepidemicshasbeenestablishedinindustrializedcountrieswithoutfrequenttransmissiblediseases,butmustbereevaluatedinAfrica,wherehyperendemicdiseases suchasmalaria increase thecomplexityofhostdiseaseinteractionsthataltertheimmunesystem’scontrolstrategiesforalldiseases(STILLWAGGON,2009).Dataobtainedelsewhere may not apply to areas with other hyperendemic diseasesthatenhanceHIVtransmissionordiseaseprogression.Ourpreliminarydataincludestoxoplasmosisinthosediseases.ThisperversecircuitofmeatthatcantransmitToxoplasma,therebyincreasingtheseverityofHIV,andwithmalaria,resultinginlongerlifewithhigherviralloadsandmoretransmission,increasestheburdenofHIVinAfrica.Ourdatasuggests that Toxoplasma infection could also affect HIV evolution,eitherbyincreasingtheriskofrapidevolutionofHIVinfectionorasanopportunisticinfection,withdisablingorlethalencephalitis.

ThisdatasupportstheurgentneedofresearchintoxoplasmosisinAfrica,especially in thepresenceofHIVepidemics.Theoccurrenceand peculiarities of toxoplasmosis in Mozambique revealed by thisstudyemphasizetheimportanceofincreasingpublicawarenessabouttheinfectioninMozambique.Furtherstudiesoftoxoplasmosis,ineitherMaputo or other areas of Mozambique, such as Chokwé and CapeDelgado,areneededtoimproveourknowledgeontheriskofcongenitaltoxoplasmosisandToxoplasmaencephalitis inHIV/AIDSpatients.AprogramforserodiagnosisandpreventionoftoxoplasmosisinpregnantwomenorHIV‑positivepatientscouldreducetheburdenanddisabilitiesinToxoplasma infectionsinMozambique.

RESUMO

DescriçãopreliminardaocorrênciadeinfecçãopeloHIVetoxoplasmoseemmulheresgrávidasemMoçambique

Toxoplasmose,umaprotozoonose,causadoençagraveemfetosdemulheresgrávidascominfecçãoagudaeencefaliteletalemportadoresde HIV.Apesar de muitos estudos sobre sua prevalência no Mundo,existem apenas alguns relatos da toxoplasmose naÁfricaAustral,geralmenteanedóticos.EstudamosporELISAdoisgruposdeamostrasdeMoçambique,usando150amostrasdesorosdemulheresgrávidaseseisamostrasdeLiquidoCefalorraquidiano(LCR)depacientescomAIDSeencefalite.OestadodainfecçãopeloHIVfoiconfirmadoeacontagemdecélulasCD4+nosanguefoiobtidadaspacientesgrávidasinfectadaspeloHIV.Nogrupodasgestantes,IgGantiT.gondii foiencontradaem18.7% (28/150), mais freqüente em pacientes HIV positivas (31.3%,18/58)doqueemHIVnegativas(10.9%,10/92).Aocorrênciadeambasasdoençasaumentacomaidade,maisclaramentevistanatoxoplasmose(p<0.005)doquenainfecçãopeloHIV,devidomaiortransmissãodoHIVapósainfância.NaencefaliteempacientesHIV+,asorologiadoLCRmostrouumaocorrênciade33%deIgGespecificadealtaavidez,queestádeacordocomaocorrêncianestegrupoetário,baseadonosdadosdenossasgestantes.Amenorocorrênciadeambasasinfecçõesemgruposetáriosmaisidosospodeserexplicadapelamortalidadecumulativapor

Fig. 4 ‑ Blood CD4+ cells counts in HIV‑pregnant women from Mozambique Maputo,

accordingtotoxoplasmosisserologyandagegroups.Opensymbols=Toxoplasma negative

Closedsymbols=Toxoplasma positive.Significantdifferencesaremarkedas (*)and its

absenceas(NS).BarsrepresentmeanvaluesandSEM.


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qualquercausanosgruposmaisidosos,resultandoemmenorocorrênciarelativa.Usandoas contagensde célulasCD4+comomarcadoresdaprogressãodainfecçãopeloHIVecorrigindoparagruposetários,asgestantesHIV+comcontatocomT. gondiitemmenoresníveisdecélulasCD4+doqueasgestantesHIV+semcontatocomT.gondii.AocorrênciamaiordatoxoplasmoseemgestantesHIV+podeseratribuídaafatoresderiscosemelhantes,comoexposiçãoamaiorcontatosocial.Abaixaocorrênciadatoxoplasmoseemgruposmaisjovenspodeserelacionarcommenoracessoacarnecontendocistos,jáqueatransmissãoambientalporoocistosestáassociadaàmaiorincidênciaemcrianças.TodosestesdadosreforçamanecessidadedepesquisadatoxoplasmosenaÁfricaAustral,especialmentenapresençadaepidemiapeloHIV.

ACKNOwLEDGEMENTS

ThisworkwassupportedbyInstitutoNacionaldeSaúde‑MinistériodaSaúde‑Mozambique,LIMHCFMUSPandCNPqPróAfrica(Proc.No490374/2008‑5).ThisworkissubmittedonbehalfofS.Sitoe,B.RafaelandR.Thompson,ascontactwithMaputoauthorsbecomeverydifficultafterAugust2009.Thecorrespondingauthorwasresponsiblefortheanalysisandthesubmissionofthearticle,inconsiderationthatthisworkwascommunicatedasanabstractatthe13thInternationalCongressofProtistology,2009,Búzios,anditwasnecessarytocommunicatethedatainapublishedpaperinatimelyfashion.Allcontactedauthorswereinagreementwiththisarrangement.

REFERENCES

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7.HariKR,ModiMR,MochanAH,ModiG.Reducedriskof toxoplasmaencephalitisinHIV‑infectedpatients‑aprospectivestudyfromGauteng,SouthAfrica.IntJSTDAIDS.2007;18:555‑8.

8.Lin DS, Bowman DD. Toxoplasma gondii: anAIDS enhancing cofactor. MedHypotheses.1992;39:140‑2.

9.Lindström I, Kaddu‑Mulindwa DH, Kironde F, Lindh J. Prevalence of latent andreactivatedToxoplasma gondiiparasitesinHIV‑patientsfromUganda.ActaTrop.2006;100:218‑22.

10.Makuwa M, Loemba H, BeuzitY, Livrozet JM, Belec L. Synthèse intrathécaled’anticorpsanti‑Toxoplasma gondiiaucoursdelatoxoplasmosecérébraleassociéeausidaafricain.BullSocPatholExot.1999;92:95‑8.

11.Maschke M, Kastrup O, Esser S, Ross B, Hengge U, HufnagelA. Incidence andprevalence of neurological disorders associated with HIV since the introductionofhighlyactiveantiretroviral therapy (HAART). JNeurolNeurosurgPsychiatry.2000;69:376‑80.

12.MeirelesLR,GalisteoAJJr,PompeuE,AndradeHFJr.Toxoplasma gondiispreadinginanurbanareaevaluatedbyseroprevalenceinfree‑livingcatsanddogs.TropMedIntHealth.2004;9:876‑81.

13.Montoya JG, Remington JS. Management of Toxoplasma gondii infection duringpregnancy.ClinInfectDis.2008;47:554‑66.

14.NabiasR,NgouamizokouA,Migot‑NabiasF,Mbou‑MoutsimbiRA,Lansoud‑SoukateJ.EnquêtesérologiquesurlatoxoplasmosechezlesconsultantsducentredeP.M.I.deFranceville(Gabon).BullSocPatholExot.1998;91:318‑20.

15.Passos LN,Araújo Filho OF,Andrade Junior HF.Toxoplasma encephalitis inAIDSpatientsinSãoPauloduring1988and1991.Acomparativeretrospectiveanalysis.RevInstMedTropSaoPaulo.2000;42:141‑5.

16.PurnerMB,BerensRL,TomavoS,LecordierL,Cesbron‑DelauwMF,KotzinBL,et al.StimulationofhumanTlymphocytesobtainedfromToxoplasma gondii-seronegativepersonsbyproteinsderivedfromT. gondii.JInfectDis.1998;177:746‑53.

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Received:15June2010Accepted:17September2010

LIBRARYOFTHESÃOPAULOINSTITUTEOFTROPICALMEDICINE

website:www.imt.usp.br/portalAddress:BibliotecadoInstitutodeMedicinaTropicaldeSãoPaulodaUniversidadedeSãoPaulo

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Telephone:55113061‑7003‑Fax:55113062‑2174

TheLibraryoftheSãoPauloInstituteofTropicalMedicine(IMTSPLibrary)wascreatedonJanuary15,1959inordertoserveallthosewhoareinterestedintropicaldiseases.Toreachthisobjective,weselectandacquirebydonationand/orexchangeappropriatematerialtobeusedbyresearchersandwemaintaininterchangebetweenInstitutionsthoroughtheJournaloftheSãoPauloInstituteofTropicalMedicine,sincetheLibraryhasnofundstobuilditsownpatrimony.

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Financialsupport:FundaçãodeAmparoàPesquisadoEstadodeSãoPaulo(FAPESP).(1)HospitalEstadualMárioCovas.(2)LaboratóriodeInvestigaçãoMédicaemReumatologiadoHospitaldasClínicasdaFaculdadedeMedicinadaUniversidadedeSãoPaulo(LIM‑17HCFMUSP),SãoPaulo,SP,Brasil.(3)InstitutodeInfectologiaEmílioRibas,SãoPaulo,SP,Brasil.Correspondenceto:NatalinoHajimeYoshinari,FaculdadedeMedicinadaUniversidadedeSãoPaulo,Av.Dr.Arnaldo455,3ºandar,sala3184.E‑mail:[email protected]

PROFILE OF PATIENTS wITH BAggIO-yOSHINARI SyNDROME ADMITTED AT “INSTITUTO DE INFECTOLOgIA EMILIO RIBAS”

EmyAkiyamaGOUVEIA(1),MayraFernandaALVES(2),EleniceMANTOVANI(2),LuizaKeikoOYAFUSO(3),VirgíniaLuciaNazarioBONOLDI(2)&NatalinoHajimeYOSHINARI(2)

SUMMARY

Theaimofthisstudywastoevaluatetheepidemiological,clinicalandlaboratorialprofileofpatientswithBaggio‑YoshinariSyndrome(BYS),whounderwentinternmentattheInstitutodeInfectologiaEmilioRibasinSãoPaulo,Brazil,duringtheperiodfromJuly1990toJuly2006.BYSisanewBraziliantick‑bornediseasecausedbyBorrelia burgdorferisensu latomicroorganismsthatresemblesfeaturesofLymedisease(LD),exceptforitsepidemiological,clinicalandlaboratorialparticularities.From60patients’recordswithpositiveserologytoB. burgdorferidonebyELISAandWestern‑blottingmethods,19caseswerediagnosedashavingBYS,accordingtocriteriaadoptedatLIM‑17HCFMUSP,theBrazilianReferenceLaboratoryfortheresearchofBYS.Theother41remainingpatientsdisplayedmiscellaneousinfectionsorauto‑immuneprocesses.ThebeginningofsymptomsinBYSgroupvariedfromonedaytosixyears,fromtheonsetofthedisease.Fourof19patientswereincludedinacutediseasestage,and15inlatent.Generalunspecificsymptomswereidentifiedinalmostallcases,withhighfrequenciesoffever(78.9%)andlymphadenomegaly(36.8%).Sixpatientshadskinlesions(31.5%);sixarthralgiaorarthritis(31.5%)andeightneurologicalsymptoms(42%).Interestingly,twopatientsshowedantibodiesdirectedtoB. burgdorferiexclusivelyincerebrospinalfluid.SinceBYSisanewemergentBrazilianzoonosisanditsdiagnosisissometimescomplex,allthenewknowledgeaboutBYSmustbescatteredtoBrazilianMedicalspecialists,aimingtoteachthemhowtodiagnosethisamazingtick‑bornediseaseandtoavoiditsprogressiontochronicirreversiblesequels.

KEYwORDS:LymeDisease;Lymedisease‑likeillness;Baggio‑YoshinariSyndrome;Tick‑bornediseases;Borrelia burgdorferi;Brazil.

INTRODUCTION

LymeDisease (LD) is a frequent zoonosis transmittedby Ixodes ricinus complex ticks, caused by Borrelia burgdorferi sensu latocomplexmicroorganisms,constitutedbyatleast11spirochetespecies.B. burgdorferisensu strictoisidentifiedintheUnitedStatesofAmerica(USA) and Eurasia, while B. afzelii and B. garinii in Europe andAsia2,8,36,37.SkinandjointsymptomsarepredominantintheUSA,whileneurologicaldisordersaremorefrequentinEurope2,13,21.

A similar zoonosis called Lyme disease‑like Illness or Masters’Disease,alsoknownasSouthernTick‑AssociatedRashIllness(STARI)hasbeenreportedinthesouthofUSA27,causedbyB. lonestari,whichisaspirocheteuncultivableinBSKmediumandgeneticallysimilartoB. theileri, found incattle.STARI is transmittedby theAmblyomma americanum tick, and the clinical picture is characterized by theappearance of erythema migrans (EM), without jeopardy of internalorgans.Furthermore,patientswithSTARIdonotdevelopantibodiestoB. burgdorferi.

TheproposalofLDresearchinBrazilemergedin1989,withthegatheringofamultidisciplinaryteam39.ThegroupintendedtoidentifypatientsinBrazilandtoperformmicrobiologicalanddiagnostictests.In1990,theLaboratóriodeInvestigaçãoemReumatologiadoHospitaldasClínicasdaFaculdadedeMedinadaUniversidadedeSãoPaulo(LIM‑17HCFMUSP)wasconsideredbytheBrazilianHealthDepartmentasReferenceLaboratorytoresearchandtoassistsuspectedpatients.

In1992,thefirstcasesofBrazilianLDweredescribed40,admittedtotheInstitutodeInfectologia EmilioRibas¨(IIER).Theseyoungpatientspresentedfever,erythemamigrans(EM)andarthritis,andthediagnosiswasconfirmedattheLIM‑17HCFMUSPLabbyELISAandWestern‑blotting(WB)assays,usingB. burgdorferisensu strictoantigensofNorthAmerican origin, originally isolated from the tick Ixodes scapularis. Sincethen,manyothercaseswerereported,allofthemexhibitingsimilarclinicalfeaturesofLD,includingpresenceofEMandsystemicarticular,neurologicalandcardiacsymptoms5,11,34,41,42,43,45,47.

However, along the years, many distinguishing features were

GOUVEIA,E.A.;ALVES,M.F.;MANTOVANI,E.;OYAFUSO,L.K.;BONOLDI,V.L.N.&YOSHINARI,N.H.‑ProfileofpatientswithBaggio‑Yoshinarisyndromeadmittedat“InstitutodeInfectologiaEmilioRibas”.Rev.Inst.Med.Trop.SaoPaulo,52(6):297‑303,2010.

298

identifiedinBrazil.B. burgdorferi hadneverbeenisolatedorculturedin BSK, even when many enriched media were employed to culturebiological materials from patients12,38, potential reservoir animals1 ortickscollectedatriskareas6.

Ticks from Ixodes ricinus complex, the main invertebrate hostsinvolvedinBorreliatransmissioninUSAandEurasia,hadneverbeenfound biting people at risk areas. On the contrary, it was reportedemergenceofBrazilianLDinalaboratoryresearcher,accidentallybittenbyAmblyomma cajennensetick(YoshinariNH‑personalcommunication).TicksofgenusRhipicephaluswerealsosuspectedtotransmitspirochetes,duetocoexistenceofantibodiestoB.burgdorferi andBabesia bovisinBrazilianLDpatients44.

Molecular Biology studies (PCR) performed by COSTA10 andBARROS4 using human and ticks samples revealed negative results,whenprimerstoamplifyconservativespecificgenesofB. burgdorferiwereused.TheprimersemployedweretargetedtoidentifyoutersurfaceproteinA(OspA)22andgenesformajorflagellinFlaBandminorflagellinFlaA20,aswellasforribosomal16SrRNA3.

Clinically,BrazilianLD‑likepatientspresent increasedfrequencyof relapsing episodes, especially if thedisease is unrecognized at anearlystageorwronglytreated.Furthermore,inmostofcases,antibiotictreatmentisnotefficienttoceaserecurrentdiseasesymptoms.Allergicmanifestationsincludingacquiredangioedema,havebeenalsodescribedalongthediseaseprogression17,42,45,47.

Laboratorial assays todiagnoseBrazilianLD‑like illness is averycomplex question, since antibodies against B. burgdorferi sensu lato identifiedbyELISAorWestern‑blottingproceduresofferlowsensitivityandspecificity42,47.Inthisrespect,whenbothassaysareemployedtogetherinBrazilianpatients,thepositivityreachesonly65%ofcases,whileinanormalcontrolgroupthefrequencyisaround16%25.However,serologicaldiagnosticprocedureforLDdiagnosisinUSAbasedontwo‑tieralgorithmusingELISAorIFA(immunofluorescenceassay)asscreeningtestsandWBasconfirmatoryassayisalsoconsideredinadequateforclinicalpurposes19.AnotherimportantlaboratorialaspectinBrazil isthedemonstrationofautoimmunedisorders,includingtheemergenceofantinuclearantibodies(ANA),anticardiolipin,anti‑neutrophilcytoplasmicantibodies(ANCA),andantineuronalantibodiesinthebloodofpatients17,42.

MANTOVANI et al.24 had described structures resemblingMycoplasmaspp,Chlamydia sppandlongbacterium‑likemicroorganismsintheperipheralbloodofpatientsbyelectronmicroscopyanalysis.Sinceserologic and PCR assays did not confirm presence of these latentbacteria,researchersfromLIM‑17HCFMUSPproposedthatetiologicalagentfoundinBrazilcouldbeexpressedatatypicalmorphologies46.

MANTOVANI26 in her PhD thesis, presented to Faculdade deMedicinadaUniversidadedeSãoPaulo,identifiedtheetiologicalagentasspirochetebelongingtoB. burgdorferisensu latocomplex,executingaPCRassay,employingaconservedgenethatsynthesizestheflagellarhook(flgE)30(unpublisheddata).OtherpreliminaryunpublishedresultsalsoshowedthatticksfromgenusRhipicephalusanddomesticanimalslike horses and bovines were infected by microorganisms and canparticipate in the epidemiological process of this emergent Braziliantickbornedisease.

Duetothesemanyparticularities,thistick‑bornezoonosisfoundinBrazil,wasassumedtobeanexoticinfectiousdisease,andthus,inanattempttodistinguishitfromclassicalLDwasnamedBaggio‑YoshinariSyndrome(BYS)16.Currently,BYSisdefinedasanewBraziliantick‑borne disease that imitates clinical and laboratorial features of LD,causedbyB. burgdorferisensu latospirochete,possiblyatitsatypicalmorphologiesandbeingtransmittedbyticksofgenusRhipicephalusandAmblyomma, thusnotbelongingtoIxodes ricinuscomplex.

We assume that Brazilian biodiversity and distinct geographicconditions,especiallytheoccurrenceofexotictickspeciesandanimalreservoirs,originatedmutantB. burgdorferiadaptedtosurvivein thecountry.MutantBorreliaswithgenetic changesat itsdifferentgeneshasbeenpreviouslydescribedbyMALAWISTAet al.23,SADZIENEet al.31,32,33,FINGERLEet al.14,MOTALEBet al.28,CHARONet al.9.Theexistenceofmutantspirocheteexpressedatatypicalcysticmorphologies,possiblywithoutperiplasmicflagellaanddecreasedexpressionofoutersurfaceproteins(Osp),justifyalltheparticularitiesfoundinBYS.

DuetoexistenceofmanydistinguishingfeaturesinBYS,mostofthemneverdescribedbefore,itiseasytounderstandthedifficultiesfoundbyBrazilianphysicianstodiagnosethisnewzoonosis.Infact,itisnotaneasytaskduetotheoccurrenceofmanypitfallsduringBYSpatients’examination.Additionally, we have to understand that this emergentzoonosisisaspecificBrazilianhealthproblemandwecannotimportanswersfromcountrieswhereLDexists.

Our researchers point out that B. burgdorferi can originate twodistinctdiseases:LDandBYS,possiblyaccordingtogeographicalandbiodiversity conditions. Interestingly, B. burgdorferi had never beenidentifiedinSouthAmericabefore,andevenintheSouthernhemisphere,suggestingtheexistenceofmanyunansweredquestionsconcerningtherelationshipbetweenspirochetes,ticks,reservoiranimalsandecologicalconditions.Inthisrespect,wepostulatethatthiscomplexrelationshipisassociatedtoemergenceofdifferentB. burgdorferimutants,whichinturn,causesdistinguishingclinicalandlaboratorialmanifestations.

Theaimofthisstudywastocharacterizetheepidemiological,clinicalandlaboratorialprofileofBYSpatients,admittedtotheIIER,whichisareferencehospitalfor infectiousdiseases.ThefrequencyofBYSisgrowingfast,andaswesaidbefore,wehavetoproducenewknowledgetoanswermanyunsolvedquestionsconcerningthisemergentzoonosis.Inthisrespect,weintendtodiscusspracticalmedicalquestionsemergedafteranalyzingthecasuisticofBYSadmittedtoIIER,aimingtohelpphysicianstodealwiththisoriginalandamazinginfectiousdisease.

MATERIALSANDMETHODS

FromJuly1990toJuly2006,theIIERsent171serumand/orCSFsampleswithsuspicionofBYStoLIM‑17HCFMUSPLab,toperformserologicaltestsforB. burgdorferi,throughtheIndirectimmunosorbentassay(ELISA)29andamodifiedWesternBlotting(WB)18,40.

The diagnosis of BYS was based on the previously describedcriteria24,withfinalexclusionofinfectiousdiseasesandautoimmunedisordersthatcouldleadtofalse‑positiveserology.

Fromthe171seraorCSFsamplessubmittedtoantibodyresearch,it


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waspossibletorecoverandtoanalyze60patientrecords.Afterexclusionof specific infectious or autoimmune diseases, 19 patients (31.6%)fulfilleddiagnosticcriteriaandremainedwithfinaldiagnosisofBYS.Inthissubgroupofpatients,thefollowingaspectswereexamined:gender,age,origin,tickbiteorpositiveepidemiology,cityandstatewheretheinfectionprobablywasacquired,andclinicalprofile.

RESULTS

Characterization of 60 patients with positive serology to B. burgdorferi: Description of the group of 60 patients with positiveserology to B. burgdorferi is detailed inTable 1. Nineteen patientsremainedasBYS(31.6%)and41patients(68.4%)werediagnosedashavingotherdiseases.

ThefollowingdiseaseshadfalsepositiveserologyforB. burgdorferi:rheumaticorautoimmunediseases(n=11;18.3%);leptospirosis(n=4;6.6%);denguefever(n=2;3.3%);tuberculousmeningoencephalitis(n=2;3.3%)andmiscellaneousdiseases (onepatient/eachdisease),includingBrazilianSpottedFever,HIV,salmonellosis,meningococcemia,pneumococcal meningitis, Toxocara canis meningitis, herpes virusmeningitis,meningitisassociatedtomastoiditisandtropicalpyomyositis.Twelvepatients(20%)remainedundiagnosed.

Clinicalandepidemiologicalprofileofthe19patientswithfinaldiagnosisofBYS: Fromthe19individualswithfinaldiagnosisofBYS,53%werewomenand47%men,andthemeanagewas26years(4to68years).Fourpatientswereidentifiedatacutediseasestage(untilthreemonthsfromdiseaseonset)andfifteenatlatentphase.

Regardingthepatientsorigin,thirteenwerelivinginSãoPaulocity,twocamefromSãoBernardodoCampo,andtheotherfourwerefromCotia,Guarulhos,PiraporadoBomJesusandJuquitiba,allmunicipalitiesbelongingtotheSãoPauloState.

Inonlyfiveoutof19patients(25%),itwaspossibletodeterminetheexactregionwheretheinfectionwasacquired:theurbanareaofSãoPaulocity(twopatients),Juquitiba,CotiaandtheregionaroundBillingsdam.

Signs and symptoms of 19 patients with BYS: The meantimebetweenthetickbiteepisodeandtheappearanceoffirstsymptomsvariedfromonedaytosixyears.General“flu‑like”symptomswereobservedin15patients(78.9%).Feverwasthemostfrequentsymptom,viewedin11cases(57.8%),isolateormultiplelymphadenomegalywasfoundinsevenpatients(36.8%)andheadachewasthethirdmostcommonsymptom,followedbythepresenceofchills,vomit,odynophagia,diarrhea,cough,hyperhidrosis,andconjuctivalhyperaemia(Fig.1).

Sixpatients(31.5%)hadskinlesions.Itchingacuteerythemamigranswasobservedinonecase(5.2%),annularsecondaryerythemainthreecases(15.7%)andpetechiae‑likelesionsintwocases(10.5%).

Regarding the disorders of the locomotor apparatus, six patients(31.5%)presentedarthralgiaorarthritis,andsixpatients(31.5%)hadmyalgia.

Eightpatients(42%)presentedneurologicaldisorders.Meningitiswasthemostcommonfinding,observedinfivecases(26.3%).Itwasanisolatedsymptominonlyonepatient,threecaseshadmeningoencephalitisandthefifthpatientwasassociatedtocranialandperipheralneuritis.Fourpatientsunrolledisolateormultiplecranialneuritiswithsymptomsofdysarthria,diplopia,facialnervepalsy,eyelidptosis,etc.Fourpatientshadperipheral sensitiveand/ormotorneuropathy.Sevenoutofeightpatientspresentedmultipleneurologicalmanifestations(Table2),whichisaprominentfeatureofneurologicalinvolvementrelatedtoBYS.

Laboratorialfindingsof19patientswithBYS: AllstudiedpatientspresentedantibodiesagainstB. burgdorferi. TwocaseshadantibodiesdetectedbyELISAonlyinthecerebrospinalfluid(CSF).Amonganother

Table1Finaldiagnosisofthe60patientswithpositiveserologytoB. burgdorferi

admittedatIIER

FinalDiagnosis N(%)

BYS 19(31.6%)

Rheumaticand/orautoimmunedisease 11(18.3%)

Leptospirosis 4(6.6%)

Dengue 2(3.3%)

Tuberculosismeningoencephalitis 2(3.3%)

Brazilianspottedfever 1(1.6%)

HIV 1(1.6%)

Salmonellosis 1(1.6%)

Meningococcemia 1(1.6%)

Pneumococcalmeningitis 1(1.6%)

Toxocara canis meningitis 1(1.6%)

Herpesvirusmeningoencephalitis 1(1.6%)

Meningitissecondarytomastoiditis 1(1.6%)

Epiduralempyema 1(1.6%)

Tropicalpyomyositiswithosteomyelitis 1(1.6%)

Othercauses 12(20.0%)

Total 60(100%)

Fig. 1 ‑ List of general symptoms presented by the 19 patients with Baggio‑Yoshinari

syndromeadmittedtoIIER.


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17cases,nine(52.9%)hadantibodyidentifiedbyWBtoB. burgdorferi,five(29.4%)byELISA,andonlythreepatients(17.5%)demonstratedantibodiesbyWBandELISAassaysatthesametime.

Eleven out of 19 patients had the cerebrospinal fluid (CSF)analysis performed. Six samples exhibited a discreet increaseof lymphomononuclear cells (less than 200 cells/mL) and sevenshowed higher CSF protein concentration. Isolated increased proteinconcentrationwasdemonstratedinonecase.

Other laboratorial exams were conducted with all samples toexcludeinfectiousorautoimmunediseases.Haemogramwasnormalinallcases.SixpatientsperformedVDRLtest(CSFand/orblood),threeweresubmittedtoserologicanalysisforrickettsiosis,fourpatientsforbrucellosis, threepatientsfor typhoidfeverandfive toanti‑HIVtest.Theseandotherspecificexamswerenegative.

OnepatientwithBYShadpositiveANAwithhomogenouspattern(>1/200)andanothercasepresentedpositiveanti‑smoothmuscleandanti‑peroxidaseantibodies.ThesedatadidnotnullifydiagnosisofBYS,sincethissyndromecanpresentautoimmunefeatures.

Treatmentofthe19patientswithBYS:Fifteenof19patientsweretreatedwithantibioticsandthemeanperiodoftreatmenttimewas22days,rangingfromsevento46days.Infourcasestherewasnoinformationconcerningatherapeuticschemeonthepatients’records.Theantibioticsofchoicewere:ceftriaxone(42%),amoxicillin(16%),ceftriaxoneassociatedwithdoxycycline(11%),erythromycinandtetracycline(5%).

DISCUSSION

Aftercarefulepidemiological,clinicalandlaboratorialanalysis,19out60(32%)patientswithpositiveserologytoB. burgdorferi,admittedatIIERremainedwithfinaldiagnosisofBYS.Theremaining41patients(68%)wereidentifiedashavingautoimmunedisease(N=11;18.3%),miscellaneousspecificinfectiousdiseases(N=18;30%)andtheother12patients(20%)stayedundiagnosed.Inotherwords,thisoriginalresearchisveryimportantforBrazilianphysicians,sinceforthefirsttimeitwaspossibletoestimatethat32%ofBYSsuspectedpatientswithpositiveserologyforB. burgdorferi displayedfinaldiagnosisofBYS.

Asreportedbefore,serologyforB. burgdorferimustbeinterpretedcarefully, since it cross reacts with autoimmune and other infectiousdiseases42.Among these diseases we have rheumatoid arthritis,scleroderma, systemic lupus erythematosus, syphilis, leptospirosis,leishmaniasis, tuberculosis, acute virosis, etc.This manuscript alsocontributed to add a new list of infectious diseases that cross‑reactwith BYS as dengue, Brazilian spotted fever, HIV, salmonellosis,meningococcemia,Toxocara canismeningitis,tropicalpyomyositis,etc.

The main learning of this article is to understand that BYS isan infectious disease of increasing interest for Brazilian physicians.However,sinceitsdiagnosisisverycomplexanddifficult,theBrazilianphysicianmustbewellpreparedtorecognizeit.Certainly,itisnotaneasytask,mainlyatcurrentdays,whenpatientanamnesisisdonequicklyandoften,replacedbyexpensivecomplementarysubsidiaryexams.

Itisimportanttonotethatonlyfouroutof19caseswereatacutediseasestage,whenthepatientsstillrememberedrecentepidemiologicalandclinicaldataasthosesymptomsrelatedtotickbitehistoryorvisitstoatriskareas.Onthecontrary,itiseasytounderstandhowdifficultwastodiagnosetheremaining15BYSpatientsbelongingtolatentdiseasestage,whounderwentBYSsymptomsaftermonthsoryearsfromacutediseaseonset.

Clinically,BYS,differenttoLD,isrecognizedatacuteandlatentrelapsingstage.ThediagnosisandtreatmentofBYSatearlystageisofcriticalrelevancetoavoidzoonosisprogressiontolatentstage,whenthediseasecanrevealirreversiblesequels.Nowadays,itisshownthatmisdiagnosedorwronglytreatedpatientsatacutediseasestageinthepast,canprogresstochronicidiopathicrheumaticorneurologicaldiseases.Unfortunately,theBrazilianGovernmentdoesnotconsideracutestageofBYSofcompulsorynotification,sinceitdoesnotkillpeopleatthisphase.Conversely,theeconomiccoststotreatsequelsofrelapsingsymptomsfromlatentstageareenormous.

BrazilianpatientspresentthetypicalEMattickbitesiteinnearly50%ofcases.Fewcases from thisarticlehadEM,possiblybecauseclinicaldatawasgatheredretrospectively,andaswesaidbefore,patientsfromlatentstagedonotinformspontaneouslyearliersymptoms,andphysiciansduring theanamnesisarenot trained to inquireaboutEMorannularsecondarylesionshappenedataremotedate.Articularand

Table2Descriptionofthenervoussystemdisordersfoundin19patientswithBYSadmittedtotheIIER

Case CNS PeripheralNervousSystem

1 Patellarreflexasymmetryandwalkdifficulty

2 Meningoencephalitis,ataxia Dysarthria

3 Meningoencephalitis Diplopia

4 Shouldersandlegsweakness,walkdifficulty,hypoesthesiatrembling

5 Meningoencephalitis Rightcentralfacialnerveparalysis,muscularweaknesslevelIVofleftlegandlevelIIIofrightarm

6 Rightperipheralfacialnerveparalysis,walkdifficulty,muscularweaknessofthelegs,paresthesiainbootsandgloves

7 Meningitis Lefteyelidptosis,fixedmydriasis,dysarthria,weaknessofrightlegstrengthlevelIV

8 Meningitis


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neurologicalsymptomsoccurinnearly35%ofBYSpatientsandsimilarfrequencieswereobservedinthepresentstudy.

Importantly,patientsfromthepresentresearchexhibitedthesamepatternofneurologicalinvolvementreportedinpreviousmanuscripts,asthepredominanceofmeningitis,isolatedorassociatedtoperipheralorfacialneuritis.OneofthehallmarkfeaturesofBYSistheoccurrenceof simultaneous multiple neurological involvement or its associationwith arthritis, identified in nearly 50% of patients. Furthermore, thefrequenciesoftypicalrecurrentannularsecondaryskinlesionsand flu‑likesymptoms¨decreaseasthediseaseprogresses,bringingadditionaldifficultiestoidentifylatentBYS34.

Currently,manyotherminorormajorsymptomsareconsideredusefultodiagnoseBYS.PatientswithBYSdisplaymanysubjectivesymptomsasthoserelatedtochronicfatigue,fibromyalgia,cognitivedisturbanceoremotional,laborandsocialdisabilities.Additionally,thesepatientscanmentionpresentorpastrelevantpsychiatriccomplaints(depression,panicsyndrome,schizophrenia‑like,suicidalattempt),cardiac(arrhythmias,cardiomegaly)orocularsymptoms(uveitis,opticneuritis,retinalarteritis,papilledema)47.Also,skinlesionsarenotrestrictedtothepresenceofEM or annular secondary lesion. Localized scleroderma‑like lesion,benignBlymphocytoma,acrodermatitischronicaatrophicans(ACA)andpanniculitishavebeendescribedinBrazil15,47.AllthesesymptomsalreadyidentifiedinBYS,mustberememberedbyBrazilianphysicianswhentheyareperformingapatient’sanamnesisandphysicalexamination.

Otherimportantlearningofthismanuscriptistoacceptthatisolateddetection of antibodies to B. burgdorferi is not diagnostic for BYS.Indeed,wesawthattwothirdsofpatientswithpositiveserologyhadautoimmuneorother infectiousdiseases.Also, negative serology foridentificationofB. burgdorferiinfectiondoesnotruleoutthehypothesisof this tick borne zoonosis, since sensibility of both specific assaystogether(ELISAplusWB)islow,approximately65%.Despiteallthedifficulties,theserologyforsearchofB.burdorferi infectionisstillanimportantdiagnosticparameterifinterpretedcarefully,alwaysconjugatedtocarefulanamnesisandphysicalexaminations.

Sometimes,mainlyatacutediseasestage,theserologicalresultsareunnecessary,especiallyifthepatientbecameillaftertickbitehistoryandpresentedEM.Similarly,theemergenceofarthritisorneurologicalsymptomsafterweeksormonthsfollowingthediseaseonset,mustberelatedtoBYSanddeservespecifictreatment,evenwhenantibodiestoB. burgdorferiareabsent.ItisimportanttonotethatantibodytitersintheseraofBYSpatientsarelowandoscillating,possiblybecausewehaveusedantigensofNorthAmericanorigin.Also,ascommentedbefore,B. burgdorferiwasneverculturedor isolated inBrazil.Additionally,Brazilianetiologicalagentseemstobeexpressedatcysticformwithsuppression/inhibitionofmanygenesandproteins,explainingthelowrepertoireofantibodies toB. burgdorfericonfirmedbyWBanalysis.AllthesefeaturesindicatethatBrazilianB. burgdorferiisaspirochetewhichisverywelladaptedtosurviveinvertebrateandinvertebratehosts,justifyingoccurrenceofrelapsingsymptoms.

SearchingforantibodiestoB. burgdorferiinCSFisagooddiagnosticoption,mainlyifwearedealingwithaneurologicpatientwithpreviousnegative serology for B. burgdorferi done in the serum.Two of 16patientsofthisstudyhadantibodiesexclusivelyinCSF.Interestingly,

accordingtoourunpublishedexperience,cerebrospinalfluidfromnormalindividuals,mostlywithheadache,doesnotrevealantibodiesagainstB. burgdorferi. Unfortunately,thefindingofantibodiestoB. burgdorferiinCSFisequallyunspecific,sincecross‑reactivityhappenswithmanyneurological infections like syphilis, tuberculosis, herpes virus, HIV,HTLV,etc (unpublisheddata).

The question concerning autoimmunity in BYS deserves furthercomments. Often, patients with BYS exhibit symptoms present inrheumatic diseases like fever, arthritis, myalgia, muscle weakness,Raynaud´s phenomenon, sicca syndrome complaints, vasculitis,photosensibility, neuritis, meningitis, ocular manifestations, skinrashes, etc.Also, laboratorial findings can mimicry autoimmunedisorders by appearance ofANA, anti cardiolipin antibodies,antineutrophil cytoplasmic antibodies (ANCA), antitireoperoxidaseantibodies, hypergammaglobulinemia, anti neuronal antibodies, etc.Forthesereasons,physiciansmustbeawaretodistinguishBYSfromautoimmnunediseases.Additionally,wehavefoundanemia,leucopenia,thrombocytopenia,transaminasesincreaseandbilirrubinemia,mainlyinBYSpatientssuspectedofco‑infectionswithothertick‑bornediseaseslikebabesiosis,ehrlichiosis,bartonellosisandrickettsiosis7,35.

Weassume that the frequencyofBYSat the IIER in thestudiedperiodishigherthandemonstratedinthismanuscript.Firstofall,weexcludedpatientswithsymptomsofBYSwithoutpositiveserologyforB. burgdorferi,evenwhenautoimnuneorother infectionswereruledout.Also,itisplausibletoadmitthatamongthe12undiagnosedpatients(20%)withpositiveserologyforB. burgdorferi,withoutautoimmuneorotherspecificinfection,anewcarefulanamnesisofthepasthistoryorfollowuplookingforeventualrelapsingsymptomsinthefuture,couldcontributetoanincreaseinthefrequencyofBYSpositivity.

A few number of patients offered safe information about wheretheywereinfected.Itwasnotasurprisethattwopatientsacquiredthedisease inside theurbanareaofSãoPaulo,becausemanyperipheralurbanregionsexhibitrichvegetationwithpresenceofwildanddomesticanimals.PreviousreportshadalreadydemonstratedBYSonset,withinSão Paulo city, showing that this zoonosis is also a cosmopolitandisease42,43.Recently,wehaveperformedPCRforB. burgdorferiusingflgEprimerandpreliminaryunpublishedresultsconfirmedthatbovinesandhorsesparticipateintheBYStransmission(unpublisheddata).AlsoB. burgdorferihasbeenidentifiedinticksfromgenusRhipicephalus,whichinfestthesedomesticanimals(unpublisheddata).ThesereportsconfirmthatepidemiologyofBYSisverydifferentfromthoseobservedinLD,explainingwhythezoonosisbehaviorissodifferentinourcountry.

Ingeneral,peopleareinfectedcasuallyatsmallruralmunicipalitiesaroundSãoPaulocityor in forestedareasclose to thebeaches. It isimportanttoknowthatprematuretickremovalfromtheskinbefore24hoursavoidsspirochetetransmission.Peoplemustwearclearclothes,boots,tickrepellentsandsearchcarefullyforticksaftervisitingatriskareas.

Asfarasthetreatmentisconcerned,15outof19patientswithBYSweresubmittedtoantibiotictherapy,mostofthecaseswithceftriaxone2g/day, given over an average of 22 days. Unfortunately, it was notpossibletoobtainadditionalinformationabouttheotherfourpatients’records,neitherabouttheevolutionofthepatientstreatedwithantibiotics.


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Currently,acutecasesofBYSaretreatedwithdoxycycline100mgb.i.d.oramoxicillin,500mgeachq.i.dfor30days.Relapsingsymptomsaretreatedwithceftriaxone,2ga/dayfor30dayswithadditionaluseofdoxycycline100mgtwiceadayforthreemonthsorcontinuoususeofdoxycyclineforfourmonths.Ingeneral,ceftriaxoneorpenicillin20millions/day/IVareindicatedwhenneurologicalsymptomsarepresent.Hydroxychloroquine400mg/daygivenforaprolongedperiodoftimeseemstobeausefuldrugtoavoidappearanceofrecurrentsymptoms.Unfortunately,wearenot sureabout the real roleand importanceofantibiotictreatmentatlatentstageofBYS,becauseautoimmunedisordersareequallyobservedatthisdiseasestage.Otherwise,spirocheteshavebeenidentifiedbyPCRprocedureinsomepatientswithBYSatlatentstage,afteryearsfromdiseaseonset.Surprisingly,insomecases,patientsreportedprevioustreatmentwithantibiotics(unpublisheddata).

TheprevalenceofBYS inBrazil isunknown,but thenumberofsuspectcasesisgrowingfast,sincephysiciansofdifferentspecialtiesarestartingtorecognize thisnewtick‑bornedisease.However, ithasbeenaverydifficulttasktospreadknowledgeonBYS,becausemedicalstudents are accustomed to learn about LD in International MedicalTextbooks,andweknowthatthisNorthAmericanandEurasianzoonosisiscompletelydifferent.TheachievementofknowledgeonBYShasbeendoneslowlyduetoitscomplexity.WebelievethatthismanuscriptcanbeveryhelpfulforBrazilianphysicians,inhelpingthemavoidpitfallsusuallyfoundinBYSpatients.

Unfortunately, often we are censured by external referees sinceweciteourownarticles.However,wehavetounderstandthatthisisanecessaryproceduresincewearedealingwithanewBrazilianinfectiousdiseaseandweneedtodevelopourownlearning,insteadofbringingknowledgefromforeigncountries.

BYS must receive great attention from Public Health managersduetoitshighmorbidityandpropensitytocauseirreversiblesequels.Researches and results on BYS have been an enormous scientificchallenge, which demand extensive cooperation with experts ofdifferentareas,inanattempttofulldisclosurethisintriguingBraziliantickbornezoonosis.Finally,ithasnotbeenaneasymissiontoassumeresponsibilitiestoresearchandtotransmitknowledgeofthisunbelievabledisease,mainlybecauseitinvolvesdeepconceptualchangesconcerninghostversusmicroorganismrelationship.

RESUMO

PerfildospacientescomSíndromeBaggio‑Yoshinariadmitidosno“InstitutodeInfectologiaEmilioRibas¨

O objetivo do estudo foi avaliar o perfil epidemiológico, clínicoe laboratorial dos pacientes com Síndrome Baggio‑Yoshinari (SBY),internadosnoInstitutodeInfectologiaEmilioRibas,SãoPaulo,Brasil,noperíododejulhode1990ajulhode2006.SBYéumanovadoençatransmitidaporcarrapatos,causadapelaBorrelia burgdorferisensulato,queseassemelhaaDoençadeLyme(DL),excetopelasparticularidadesepidemiológicas,clínicaselaboratoriais.Apartirdosregistrosde60pacientescomsorologiapositivaparaB. burgdorferipelosmétodosdeELISAeWestern-blotting,19casosforamdiagnosticadoscomoSBY,deacordocomcritériosadotadospeloLIM‑17HCFMUSP,laboratóriodereferênciaparaapesquisadeSBYnoBrasil.Osoutros41pacientes

restantesforamclassificadoscomotendoinfecçõesdiversasouprocessosauto‑imunes.OiníciodossintomasnogrupoSBYvarioudeumdiaaseisanos.Quatrodos19pacientesforamincluídosnafaseagudadadoençae15nafaselatente.Ossintomasgeraisinespecíficosforamidentificadosem quase todos os casos, com altas freqüências de febre (78,9%) elinfoadenomegalia (36,8%). Seis pacientes tiveram lesões de pele(31,5%);seisartralgiaouartrite(31,5%)eoitosintomasneurológicos(42%). Curiosamente, dois pacientes apresentaram anticorpos paraB. burgdorferiexclusivamentenolíquidocefalorraquidiano.Umavezque a SBY é uma zoonose emergente brasileira e seu diagnóstico écomplexo, todooconhecimentonovo sobreSBYdeve serdifundidoparaosmédicosbrasileiros,comoobjetivodeensiná‑losadiagnosticarestasurpreendentedoençatransmitidaporcarrapatos,evitando‑seasuaprogressãoparasequelascrônicasirreversíveis.

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21.KristoferitschW.Lymeborreliosis inEurope.Neurologicdisorders.RheumDisClinNorthAm.1989;15:767‑74.

22.LieblingMR,NishioMJ,RodriguezA,SigalLH,JinT,LouieJS.Thepolymerasechainreactionfor thedetectionofBorrelia burgdorferi inhumanbodyfluids.ArthritisRheum.1993;36:665‑75.

23.MalawistaSE,MontgomeryRR,WangXM,FuLL,GilesSS.GeographicclusteringofanoutersurfaceproteinAmutantofBorrelia burgdorferi.PossibleimplicationsofmultiplevariantsforLymediseasepersistence.Rheumatology(Oxford).2000;39:537‑41.

24.Mantovani E, Costa IP, Gauditano G, BonoldiVLN, Higuchi ML,Yoshinari NH.DescriptionofLymedisease‑likesyndromeinBrazil.IsitanewtickbornediseaseorLymediseasevariation?BrazJMedBiolRes.2007;40:443‑56.

25.MantovaniE,GauditanoG,BonoldiVLN,YoshinariNH.Análiseclínicaesorológicade pacientes com síndrome infecto‑reacional Lyme‑símile. Rev Paul Reumatol.2007;6:29.

26.MantovaniE.IdentificaçãodoagenteetiológicodadoençadeLyme‑símilebrasileira(síndrome Baggio‑Yoshinari). [tese]. São Paulo: Faculdade de Medicina daUniversidadedeSãoPaulo;2010.

27.Masters E, Granter S, Duray P, Cordes P. Physician‑diagnosed erythema migransanderythemamigrans‑likerashesfollowingLoneStartickbites.ArchDermatol.1998;134:955‑60.

28.MotalebMA,SalMS,CharonNW.ThedecreaseinFlaAobservedinaflaBmutantofBorrelia burgdorferioccursposttranscriptionally.JBacteriol.2004;186:3703‑11.

29.NakaEN,CostaIP,ArãoCSB,SoaresCO,YoshinariNH.Pesquisadeanticorposanti‑Borreliaeanti‑BabesiaesorodecriançascommanifestaçõesclínicaseepidemiologiacompatíveiscomadoençadeLyme‑similenoEstadodeMatoGrossodoSul.RevBrasReumatol.2008;48(2):74‑85.

30.Ojaimi C, Davidson BE, Girons IS, Old IG. Conservation of gene arrangement andanunusual organizationof rRNAgenes in the linear chromosomesof theLymediseasespirochaetesBorrelia burgdorferi,B. gariniiandB. afzelii.Microbiology,1994;140:2931‑40.

31.SadzieneA,ThomasDD,BundocVG,HoltSC,BarbourAG.Aflagella‑lessmutantofBorrelia burgdorferi.Structural,molecular,andin vitro functionalcharacterization.JClinInvest.1991;88:82‑92.

32.SadzieneA,BarbourAG,RosaPA,ThomasDD.AnOspBmutantofBorrelia burgdorferihas reduced invasiveness in vitro and reduced infectivity in vivo. Infect Immun.1993;61:3590‑6.

33.SadzieneA,Thomas DD, BarbourAG. Borrelia burgdorferi mutant lacking Osp:biologicalandimmunologicalcharacterization.InfectImmun.1995;63:1573‑80.

34.ShinjoSK,GauditanoG,MarchioriPE,BonoldiVLN,Costa IP,ManovaniE,et al.NeurologicalmanifestationsinBaggio‑Yoshinarisyndrome(BrazilianLymedisease‑likesyndrome).BrazJRheumatol.2009;49:492‑505.

35.SpolidorioMG,LabrunaMB,MachadoRZ,Moraes‑FilhoJ,ZagoAM,DonateleDM,et al.Surveyfortick‑bornezoonosesinthestateofEspiritoSanto,southeasternBrazil.AmJTropMedHyg.2010;83:201‑6.

36.SteereAC.Lymedisease.NEnglJMed.1989;321:586‑96.

37.SteereAC.Lymedisease.NEnglJMed.2001;345:115‑25.

38.TalhariS,SantosMNS,TalhariC, FerreiraLCL,SilvaJrRM,ZelgerB,et al.Borrelia burgdorferi¨sensulato¨inBrazil:occurrenceconfirmedbyimmunohistochemistryandfocusfloatingmicroscopy.ActaTropica.2010;115:200‑4.

39.YoshinariNH,SteereAC,CossermelliW.RevisãodaborreliosedeLyme.RevAssocMedBras.1989;35:34‑8.

40.YoshinariNH,BarrosPJ,CruzFCM,OyafusoLK,MendonçaM,BaggioD,et al. ClínicaesorologiadadoençadeLymenoBrasil.RevBrasReumatol.1992;32(Supl.):57.

41.YoshinariNH,OyafusoLK,MonteiroFG,BarrosPJ,CruzFC,FerreiraLGE,et al.DoençadeLyme.RelatodeumcasoobservadonoBrasil.RevHospClinFacMedSaoPaulo.1993;48:170‑4.

42.YoshinariNH,BarrosPJL,FonsecaAH,BonoldiVL,Barros‑BattestiDM,SchumakerTT.BorreliosedeLyme.Zoonoseemergentedeinteressemultidisciplinar.Newslab.1995;12:90‑104.

43.Yoshinari NH, Barros‑Battesti PJ, Gauditano G,FonsecaAH. Report of 57 cases ofLyme‑likedisease(LLD)inBrazil.ArthritisRheum.1999;43(Suppl):S188.

44.YoshinariNH,AbrãoMG,BonoldiVLN,SoaresCO,MadrugaCR,ScofieldA,et al..Coexistenceofantibodiestotick‑borneagentsofBabesiosisandLymeborreliosisinpatientsfromCotiaCounty,StateofSãoPaulo,Brazil.MemInstOswaldoCruz.2003;98:311‑8.

45.Yoshinari NH, Gonçalves R. Doença de Lyme‑símile no Brasil. DiagnTratamento.2003;8:61‑70.

46.YoshinariNH,VasconcelosSA,TiribaAC,GauditanoG,MantovaniE,BonoldiVLN.Reportofunusualpresenceoflatentmicroorganismsinanimals:arisktoresearchandhealthofemployees?BrazJRheumatol.2009;49:517‑28.

47.YoshinariNH,MantovaniE,BonoldiVLN,MarangoniRG,GauditanoG.DoençadeLyme‑símilebrasileiraousíndromeBaggio‑Yoshinari:zoonoseexóticaeemergentetransmitidaporcarrapatos.RevAssocMedBras.2010;56:363‑9.

Received:7April2008Accepted:3September2010







[email protected]


(1)DepartamentodeInfectologia,InstitutodeInfectologiaEmílioRibas,SãoPaulo,SP,Brasil.(2)DepartamentodeNeurologia,InstitutodeInfectologiaEmílioRibas,SãoPaulo,SP,Brasil.(3)UnidadedePesquisaClínicaemRetrovirosesHumanas,UniversidadedeCampinas,SãoPaulo,SP,Brasil.(4)LaboratóriodeVirologia,InstitutodeMedicinaTropicaldeSãoPaulo,UniversidadedeSãoPaulo,SãoPaulo,SP,Brasil.(5)ServiçodeExtensãoaoAtendimentodePacientesHIV/Aids“CasadaAids”,DivisãodeClínicadeMoléstiasInfecciosaseParasitarias,HospitaldasClinicasdaFaculdadedeMedicina

daUniversidadedeSãoPaulo,SãoPaulo,SP,Brasil.Correspondenceto:JoséE.Vidal,ServiçodeNeurologia,InstitutodeInfectologiaEmílioRibas,Av.Dr.Arnaldo165,01246‑900SãoPaulo,SP,Brasil.E‑mail:[email protected]

NEUROLOgIC CyTOMEgALOVIRUS COMPLICATIONS IN PATIENTS wITH AIDS: RETROSPECTIVE REVIEw OF 13 CASES AND REVIEw OF THE LITERATURE

CamilaALMEIDASILVA(1),AugustoC.PENALVADEOLIVEIRA(2,3),LucyVILAS‑BOAS(4),MariaCristinaD.S.FINK(4),CláudioS.PANNUTI(4)&JoséE.VIDAL(2,5).

SUMMARY

NeurologicaldisorderscausedbyCytomegalovirus(CMV)inpatientswithAcquiredImmunodeficiencySyndrome(AIDS)arerarelyreportedintheHighlyActiveAntiretroviralTherapy(HAART)period.TheobjectiveofthisstudywastodescribethemainclinicalandlaboratoryfeaturesofpatientswithCMV‑relatedneurologicalcomplicationsinHIV‑infectedpatientsadmittedtoareferralcenterinSãoPaulo,Brazil.CMVdiseaserequirestheidentificationofthevirusinthecerebrospinalfluid(CSF)usingPolymeraseChainReaction(PCR).ThirteencaseswereidentifiedbetweenJanuary,2004andDecember,2008.Themedianageofpatientswas38yearsandnine(69%)weremen.AtadmissionallpatientswereawareoftheirHIVstatusandonlyfour(31%)patientswereonHAART.PatientswhowerenotonantiretroviraltherapybeforeadmissionreceivedHAARTwhileinpatients.CMVdiseasewasthefirstAIDS‑definingillnessineight(62%)patients.Theneurologicsyndromesidentifiedwerediffuseencephalitis(n=7;62%),polyradiculopathy(n=7;54%),focalencephalitis(rhombencephalitis)(n=1;8%),andventriculo‑encephalitis(n=1;8%).Seven(54%)patientspresentedextra‑neuralCMVdiseaseandfour(31%)hadretinitis.ThemedianofCD4+T‑cellcountwas13cells/µL(range:1‑124cells/µL).Overallin‑hospitalmortalitywas38%.Eightpatientsusedganciclovirorfoscarnet(in‑hospitalmortality:50%) and five patients used ganciclovir and foscarnet (in‑hospital mortality: 20%). None of the patients fulfilled the diagnosiscriteriaofimmunereconstitutioninflammatorysyndrome.Fourpatientswerelosttofollow‑up,andthreepatientspresentedimmunerecoveryanddiscontinuedsecondaryprophylaxis.Althoughinfrequent,distinctneurologicalsyndromescausedbyCMVcontinuetocausehighmortalityamongAIDSpatients.SurvivaldependsupontheuseofeffectiveantiviraltherapyagainstCMVandtheearlyintroductionofHAART.

KEYwORDS:Humanimmunodeficiencyvirus;AIDS;Cytomegalovirus;Encephalitis;Polyradiculopathy.

INTRODUCTION

HighlyActiveAntiretroviralTherapy (HAART)hasdecreased themorbidityandmortalityrate inHumanimmunodeficiencyvirus(HIV)patients with complications caused by Cytomegalovirus (CMV)5,18.ReactivationoftheinfectioninAcquiredImmunodeficiencySyndrome(AIDS) patients usually presents itself as retinitis or gastrointestinalinvolvement, whereas other neurologic syndromes are considerablyless common5. In this area, two Brazilian studies showed that thesecomplicationsare rare in theHAARTperiod28,33.However,neurologiccomplications causedbyCMVcontinue tooccur for several reasons:1)patientsfailtouseHAART,eitherbecausetheyarenotincareorarenon‑adherent;2)antiretroviralresistance;and3)lateHIVdiagnosis(latepresenters).Furthermore,theindividualclinicalimpactofneurologicaldisorderscausedbyCMVcontinuestoberelevant.Inthisretrospectivestudy,wereportonacaseseriesofHIV‑infectedpatientswithencephalitis,rhombencephalitis, ventriculoencephalitis, and/or polyradiculopathysecondarytoCMV,anddiscussdiagnosisandtherapeuticissues.

PATIENTSANDMETHODS

We performed a retrospective study between January, 2004 andDecember,2008.AlladultHIV‑infectedpatientsadmittedtotheInstitutode Infectologia Emílio Ribas, São Paulo, Brazil, with a neurologicaldisorder caused by CMV were included. CMV disease required theidentification of the virus in the cerebrospinal fluid (CSF) usingPolymeraseChainReaction(PCR).Forthispurpose,wereviewedtherecordsoftheLaboratoryofVirologyattheInstitutodeMedicinaTropicaldeSãoPaulo,wheretheCSF‑PCRforthediagnosisofCMVinfectionofthepatientsatourinstitutionisperformed.Wereviewedtheclinicalchartsofallpatients.ThisstudywasapprovedbytheInstitutionalReviewBoardoftheInstitutodeInfectologiaEmilioRibas.

Clinicalsyndromesweredefinedasfollows:1)diffuseencephalitis:progressiveencephalopathyordementiasyndrome;2)focalencephalitis(rhombencephalitis):focalsignsindicativeofbrainstemand/orcerebellainvolvement;3)ventriculo‑encephalitis:lethargyormentalconfusion,

ALMEIDASILVA,C.;PENALVADEOLIVEIRA,A.C.;VILAS‑BOAS,L.;FINK,M.C.D.S.;PANNUTI,C.S.&VIDAL,J.E.‑NeurologicCytomegaloviruscomplicationsinpatientswithAIDS:retrospectivereviewof13casesandreviewoftheliterature.Rev.Inst.Med.Trop.SaoPaulo,52(6):305‑10,2010.

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nystagmus, and unilateral or bilateral cranial nerve palsies; and 4)polyradiculopathy: progressive ascending bilateral weakness, distalsensoryloss,arreflexia,andbladderand/oranalsphincterdysfunction.Thesedefinitionswereadaptedfromstudiespublishedelsewhere17,27,29.

The CMV DNA amplification was performed using a standardPCR technique that amplified the gB gene, according with CHOU& DENNISON6. HIV infection was documented by HIV‑1 antibodyenzyme‑linkedimmunosorbentassay(ELISA)andWesternblotanalysis.

MedianwascomparedusingWilcoxonranksumtestwithcontinuitycorrection.

RESULTS

Atotalof13HIV‑infectedpatientswithneurologicalcomplicationssecondary toCMVwere included.All caseswithpositiveCSFPCRforCMVDNApresentedclinicalmanifestationcompatiblewithCMVdisease(nofalsepositivecaseswereidentified).Table1showsthemainclinicalandlaboratoryfeaturesofourpatients.Nine(69%)weremenandmedianagewas38years(range:28‑58years).AllpatientswereawareoftheirHIVstatusbeforetheadmission.ThemediandiagnosisofHIVinfectionwas36months(range:1‑216months).Ineight(62%)patients,CMVdiseasewasthefirstAIDS‑definingillness.NoneofthepatientspresentedhistoriesofCMVdiseasebut all of themhaddocumented

historyofCMV infectionwith antibodydetection (CMVIgG).Four(31%) patients were on HAART at admission: two had extensivemultidrugresistanceandtworeportedirregulartreatment.Theclinicalsyndromesidentifiedwere:encephalitis(n=7,54%),polyradiculopathy(n=7,54%),focalencephalitis(rhombencephalitis)(n=1,8%),andventriculo‑encephalitis (n = 1, 8%).Three (23%) patients presentedpolyradiculopathyandencephalitissimultaneously.Seven(54%)patientspresentedextra‑neuralCMVdisease,includingfour(31%)withretinitis,although ophthalmoscopic examination was performed in all cases.InonlyonecasethediagnosisofCMVneurologiccomplicationwasconsideredintheemergencyroomofourinstitution.

ThemedianofCD4+T‑cellcountwas13cells/µL(range:1‑124cells/µL).InCSF,thetotalleukocytecountmedianwas3cells/mm3(range:1‑3150cells/mm3),glucosemedianwas50mg/dL(range:10‑89mg/dL),andproteinmedianwas113mg/dL(range:25‑914mg/dL).NormalCSFwasobservedinonlyonecase.Thequantitativepp65antigenemiaassaymedianwas10positivecellsper400,000neutrophils(range:0‑96positivecellsper400,000neuthophils).

Braincomputedtomography(CT)imagingshowedhypodensebi‑frontalareasinthewhitematterofcases7,9,10.Magneticresonanceimaging(MRI)wasperformedinfourpatients:cases9and10showedhyperintense bi‑frontal areas on T2‑weighted and fluid attenuatedinversion recovery images (FLAIR), confirming the CT findings;

Table1Mainclinicalandlaboratorycharacteristicsof13HIV‑infectedpatientswithneurologicCytomegaloviruscomplications

Case Age/Sex AIDS‑definingcondition

Clinicalsyndrome Extra‑neuralCMVdisease

CD4+cell/mL pp65antigen Treatment In‑hospitaloutcome

1 36/M Toxoplasmosis Polyradiculopathy Not 2 56cells Ganciclovir+Foscarnet

Alive

2 41/M Toxoplasmosis Polyradiculopathy Not 46 72cells Ganciclovir Death

3 30/M NeurologicCMV Polyradiculopathy Colitis 54 NA Ganciclovir Death

4 29/M NeurologicCMV Diffuseencephalitis+Polyradiculopathy

Not 124 96cells Ganciclovir+Foscarnet

Alive

5 29/M NeurologicCMV Encephalitis+Polyradiculopathy

Not 2 NA Ganciclovir Death

6 38/F NeurologicCMV Diffuseencephalitis Not 37 NA Ganciclovir Alive

7 58/F NeurologicCMV Diffuseencephalitis Not 14 Negative Ganciclovir Alive

8 44/F NeurologicCMV Ventrículo‑encephalitis Retinitis 13 NA Ganciclovir+Foscarnet

Alive

9 45/M Toxoplasmosis Diffuseencephalitis Retinitis,Esophagitis

1 NA Ganciclovir Death

10 58/M NeurologicCMV Diffuseencephalitis Retinitis 9 Negative Ganciclovir Alive

11 53/F Wastingsyndrome

Diffuseencephalitis+Polyradiculopathy

Pneumonitis 12 11cells Foscarnet Alive

12 28/M NeurologicCMV Myeloradiculopathy Gastriculcer 14 9cells Ganciclovir+Foscarnet

Alive

13 34/M Toxoplasmosis Focalencephalitis(Rhombencephalitis)

Retinitis 12 Negative Ganciclovir+Foscarnet

Death

M=male;F=female;NA=notavailable


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case 8 showed hyperintense periventricular areas on T2‑weightedimages,FLAIRandenhancementinthistopographyaftergadoliniumadministration;andthecase13showedonehyperintenseimageonT2‑weightedandFLAIRinthebrainstem.

Mediantimeofhospitalizationwas60days(range:13‑111days).Mediantimetoanti‑Cytomegalovirustreatmentwas28(range:6‑60)days.Medianuse timeof thesedrugswas28(range:6‑56)daysand30(range:16‑60)daysinpatientswithmonotherapyandcombinationtherapy,respectively.Five(63%)ofeightpatientsthatusedganciclovirorfoscarnetdiedandone(20%)offivepatientsthatusedganciclovirplusfoscarnetdied.However,thisdifferencewasnotsignificant(p =0.56).Thedecisiontousecombinationtherapyconsideredthehematologicalandrenalfunctionofthepatientsandtheneurologicstatus.Thus,thepatientswithmoreseveredisease receivedganciclovirandfoscarnet.Afterreviewingthehistoryoftreatment,allpatientswereonHAARTwithinthefirsttwoweeksofadmission.Thetwopatientswithtriple‑classresistancereceivedanon‑suppressiveregimen.

Overall in‑hospital mortality was 38% (5/13): three as a resultof nosocomial pneumonia and two secondary to CMV encephalitis.Autopsywasnotperformedinanycase.Onepatientdiedduetoacuterenalfailureaftersixmonthsofdischarge,whenhehasbeenreceivingmaintenancetherapywithfoscarnetandtakingtenofovir,lamivudine,and efavirenz (CD4 = 326 cells/µL;VL < 50 copies/mL). None ofthe patients fulfilled the diagnosis criteria of immune reconstitutioninflammatorysyndrome(IRIS)32.Nevertheless,thiscomplicationcouldnot be completely overlooked in all cases. One year after discharge,fourpatientshadatleastoneoutpatientevaluationandthreewerelostto follow‑up.Among the three patients available one year after thediagnosis,tworeceivedganciclovirplusfoscarnetintheinductionphaseandtheotheronereceivedganciclovir.However,allthesethreepatientsreceivedsecondaryprophylaxiswithganciclovir.TheCD4+T‑cellcountmedianofthethreeavailablepatientswas189cells/µL(range:180‑214cells/µL),allpresentedHIV‑1viral<50copies/mL,andadequateuseofHAART.Inallcases,CMVtherapywasdiscontinuedafterCD4+Tcellcountexceeded100cells/µLforsixmonths.

Table2showstimesfromadmissiontodiagnosis,admissiontodeath,anddiagnosistodeathofourpatients.Themediantimefromadmissiontodeathwas68days(range:8‑57days).Themediantimefromcorrectdiagnosistodeathwas44days(range:6‑104days).Themediantimesfromadmission todiagnosiswas9.5days (range:5‑25days)amongsurvivalsand23days(range:8‑57days)amongdeaths(p =0.09).

DISCUSSION

WefoundthatneurologicCMVcomplications,althoughuncommon,continuetocauseimportantmortalityinoursetting.Ofnote,allpatientswereawareof theirHIVinfectionstatusandmostpatientspresentedCMV disease as initialAIDS defining illness.A subset of patientsobtainedclinicalandlaboratoryimprovementandwerealiveoneyearafterthediagnosisofneurologicCMVcomplication.

Retinitis is themostcommonmanifestationofCMVinfection inpatientswithAIDSanditsdiagnosisisusuallyclinical.However,thespectrumofneurologicCMVcomplicationsisbroadandtheirdiagnosisandtreatmentismorechallenging.Inthepresentstudy,similartosome

reportsofthepreHAARTperiod2,26,approximately30%ofpatientswithneurologicCMVcomplicationspresentedconcomitantCMVretinitis.Forthisreason,routineophthalmologicevaluationshouldbeperformedin all these patients. However, most patients will have simultaneousinvolvementofotherorgansatautopsy11.

ClinicalstudiesperformedindevelopedcountriesintheHAARTerareportedaprevalenceofCMVencephalitis<2%21.Similarfigureshavebeenreportedinhospital‑basedstudiesinBrazil28,33.

As in the pre‑HAART period, all patients included in this studypresentedwithverylowCD4+Tcellcounts.Most(~70%)hadnotbeenusingHAART.ThispicturereflectsfailuresinHIVtherapy,particularlyinBrazil,adevelopingcountrywithauniversalandfreeaccessanti‑retroviral program. It is important to reinforce that these seriousneurologicCMVcomplicationsarebestpreventedbyusingHAARTtomaintaintheCD4+Tcellcounts>100cells/µL5.

NeurologicCMVcomplicationwasthefirstAIDS‑definingconditionin~60%ofourpatients.Conversely, thisfigurewasonly~10% inthepreHAARTperiod2,26.ThisfindingsuggeststhatCMVshouldbeincludedinthedifferentialdiagnosisofneurologiccomplaintsinAIDSpatientswithsevereimmunosuppression,andwithouthistoryofpreviousAIDS‑definingconditions.

PresumptiveclinicaldiagnosisofsomeneurologicCMVsyndromescan be made on the basis of a combination of signs and symptoms,imaging, and CSF findings24,25. Distinct clinical syndromes such asventriculo‑encephalitis and polyradiculopathy were well describedin classical studies of the pre HAART period. However, neurologic

Table2Timesfromadmissiontodiagnosis,admissiontodeath,anddiagnosistodeath

in13HIV‑infectedpatientswithneurologiccytomegaloviruscomplications

Case Timefromadmission

todiagnosis(days)

Timefromadmissiontodeath(days)

Timefromdiagnosistodeath(days)

1 5 Alive Alive

2 14 118 104

3 23 68 45

4 11 Alive Alive

5 57 101 44

6 25 Alive Alive

7 15 Alive Alive

8 9 Alive Alive

9 8 14 6

10 10 Alive Alive

11 7 Alive Alive

12 7 Alive Alive

13 28 37 9


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complicationsduetoCMVarenotusuallyconsideredintheemergencyroom,aswasobservedinthepresentstudy.Incontrast,CMVencephalitismaybedifficulttodifferentiatefromthoseofHIVencephalitis.

AneuropathologystudyofHIV‑infectedpatientsreported28(17%)casesofCMVencephalitis,mostofthemwithpre-mortemdiagnosisofHIVdementia,showingthatCMVhasbeenunderestimatedasacauseofneurocognitvedisorders15.Ourresultsreinforcetheimportanceofin vivodiagnosisofCMVencephalitisandtheneedtoincludethisetiologyinthedifferentialdiagnosisofpatientswithclinicalsuspicionofHIVdementiaandsevereimmunosuppression.Ontheotherhand,encephalitishasbeendescribedeveninpatientsalreadyreceivingganciclovir23.

CMV polyradiculopathy causes a Guillain‑Barré‑like syndromecharacterizedbyurinaryretentionandprogressivebilaterallegweakness.The differential diagnosis in HIV‑infected patients includes otheropportunisticinfections(tuberculosis,herpessimplexvirus‑HSV‑type2,varicella‑zostervirus,andtoxoplasmosis),syphilis,andlymphoma26.

Rhombencephalitis is an unusual manifestation of CMV diseaseandwhenisolateditsdiagnosisismoredifficult.TheirmaindifferentialdiagnosisincludesHSV‑1,tuberculosisandlymphoma27.

NeurologicCMVcomplicationsusuallyoccurinpatientswithverylowCD4cellcounts(<50cells/µL)andareoftensimultaneouslypresentat other sites (retina, gastrointestinal tracts, blood, etc)20. However,6/13patientsof thepresent seriesdidnotpresent extra‑neuralCMVdiseases,exceptbythepresenceofpp65antigenemiainthreeofthem.Antigenemia was not performed in five patients; of those, two hadretinitis,onehadcolitisandtheothertwohadnotanyextra‑neurologicdisease.ThepresenceofCMVviremiaisanimportantfactorbutitisnotrequiredin thesettingof“compartmentalizedinfections”suchascolitis, esophagitis or neurologicdisease.Thedeterminationofpp65antigenemiadependsonatechnicianandtheabsenceofantigenemiaisnotequaltotheabsenceofviremia,sinceCMV‑PCRinbloodsamplesisamoresensitivemethod.CMVcomplicationsrestrictedtothenervoussystemshouldbeconsideredintheadequateclinicalandimmunologicalcontext.Inthissetting,minimallyinvasivediagnosisusingCSF‑PCRforCMVDNAisparticularlyuseful.Thistoolshowed62‑100%sensitivityand89‑100%ofspecificityinCMVencephalitisandpolyradiculopathy29,andpresentpositiveandnegativepredictivevaluesrangedbetween86‑95%and95‑98%,respectively7‑9.

AlthoughneurologicCMVcomplicationsareunusual, it is likelythat they are under‑diagnosed or ‑reported in developing countriesbecauseofdifficultaccesstoreferencecenterswithdiagnosticsupport,including molecular diagnosis and neuroimaging.The results of thepresentstudysuggestthatCMVmightbemorefrequentlydiagnosedwhenappropriatediagnosticprocedurescanbeemployed.Furthermore,atimelydiagnosisandprompttreatmentiscriticalforanadequateclinicalresponse.However,theabsenceofspecifictreatmentinmostlow‑andmiddle‑incomecountriesconstitutesanunsolvedproblemthatlimitstheoutcomeforthesepatients.

Examination of CSF is non‑specific for the diagnosis of CMVencephalitis due to their variable profile. CMV encephalitis rarelycausespleocytosis;ifapleocytosisoccursitisusuallylowgradeandconsistspredominantlyof lymphocytesandmonocytes.On theother

hand,CSFabnormalitiesinpatientswithCMVpolyradiculopathycanmimicthepatternofacutebacterialmeningitis,includingapleocytosiswith polymorphonuclear leucocyte preponderance, a raised proteinconcentration and hypoglycorrhachia or a reduced CSF:plasmaglucoseratio24,25.Somestudiesreported50‑76%ofpatientswithCMVpoliradiculopathy and “typical” polymorphonuclear preponderantpleocytosis21.InourstudyweconfirmabroadspectrumofCSFpatternsinCMVpolyradiculopathy.

InthepreHAARTperiod,theoverallprognosisofneurologicCMVcomplicationswaspoor,independentoftreatment,withsurvivalofonetofourmonths2,3,11,26.Forexample,inaretrospectivestudyof103casespublished with CMV polyradiculopathy, the mean survival time foruntreatedandtreatedpatientswithganciclovirwas5.4and14.6weeks,respectively1.Incontrast,inthepresentstudy,theuseofHAARTalongwith thespecific treatmentforCMVresulted inaneffective immunerecoverythatimprovedtheoutcomeofasubsetofpatients.

BothganciclovirandfoscarnetareknowntopenetratewellintotheCSF.Forexample,therearereportsthatCSFganciclovirconcentrationswere24‑67%oftheconcurrentplasmaconcentrationsandfoscarnetwasreportedtodistributetotheCSFinaconcentrationvaryingfrom13to68%oftheconcomitantplasmaconcentration18.ThereisnodataaboutcidofovirCSFconcentrationsasapercentageofplasmaconcentration.Nevertheless,cidofovirCSFpenetrationislow12.

TheefficaciesandtoxicitiesofapprovedtreatmentsforCMVretinitishave been established by randomized prospective trials. In contrast,therehavebeennoprospectiverandomizedtrialsoftreatmentforCMVneurologic disease5.The largest series published was an open non‑comparativemulticentrestudyof31patientswithCMVencephalitisofmyelitis,60%ofthemwithpreviousCMV2.Thisstudywasperformedin the pre HAART period, and all patients received ganciclovir plusfoscarnet by a media of six weeks.Approximately 75% of patientspresented clinical improvement or stabilization and 32% of patientsdiscontinuedoneofthetwodrugsbecauseofside‑effects.Themediansurvivaltimewasonlythreemonths.

Treatment recommendations to neurologic CMV complicationsarediverse,butmostsuggestganciclovir, foscarnetorbothagents incombination9,13,14,17,19,27,29,34andhighlightthatinitiatingtherapypromptlyiscritical foranoptimalclinical response.Someauthorsrecommendcombinationtherapyinallcases11,20orinpatientswhohavereceivedpriorantiviraltherapyforCMVorinpatientswithdiseaseprogressionundermonotherapy22,34.Inthepresentstudyweobservedthatmostpatientswithcombinationtherapyweredischargedalive,butitwasnotsignificantlyhighercomparedwiththosereceivingmonotherapy.Nevertheless,thechoicebetweenoneortwoanti‑CMVdrugswasarbitrary,butusuallycombination treatment was indicated in patients with more severeneurologicdisease.

Althoughthereisfewdatathatsupporttheuseofcidofovirinthetreatment of CMV‑related neurological disorders31, its use could beconsidered in those failing or intolerant of ganciclovir and foscarnettherapy.

Thedurationofinductiontreatmentisvariableintheliterature(3‑8weeks)11,27,29.Wepreferatleastfourweeks,butinsomecasesaresponse


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willbenotedafterthisperiod.AcloseclinicalfollowupandCSF‑PCRforCMVDNAcanguidetheoptimaldurationoftreatmentonanindividualbasis.However,theretrospectivenatureofourstudyandthesmallnumberof cases evaluatedprecludes anydefinitive recommendationabout theoptimaltreatmentofneurologicCMVcomplicationsinAIDSpatients.

Maintenancetherapyoptionsincludeone(valganciclovir,ganciclovirorfoscarnet)ortwodrugs(valganciclovirorganciclovirplusfoscarnet).Maintenancemonotherapyseemstobeparticularlyadvisableinpatientswithoutprevioususeofganciclovirwhere thepossibility toobtain anadequateimmunereconstitutionduetoHAARTisarealisticone.AlthoughmaynotbereadilyavailableinmostdevelopingcountriesandmostdatawasextrapolatedfromCytomegalovirusretinitis,theuseofvalganciclovir(anorallyadministeredprodrugofganciclovir)seemtobethepreferredregimentomaintenance4.Theuseofmaintenancetherapywithfoscarnetis feasible but cumbersome since it requires a permanent line for itsadministrationandfrequentmonitoringofelectrolytesandkidneyfunction.Althoughtheoptimaltimetodiscontinuemaintenancetherapyisunknown,someauthors suggest that canbe safelydiscontinued inpatientswithinactivediseaseandsustainedimmunereconstitution(CD4+Tcellscount>100cells/mLfor3‑6monthsonHAART)17,20,22.ThreeofourpatientsdiscontinuedmaintenancetherapyaftersixmonthswithCD4+Tcellcount>100cells/mL.RecommendedtherapyregimensareshowninTable3.

Incontrasttocytomegalovirusimmunerecoveryuveitis4,IRISseemstobeveryrareinpatientswithneurologiccytomegaloviruscomplications.Inaccordancewiththis,noneoftheincludedpatientsinthisstudyfulfilledthediagnosiscriteriaofIRIS32.

Inconclusion,neurologicCMVcomplicationsareuncommonbutdistinct diseases and continue to cause high mortality in our setting.CSF‑PCR for detection of CMV DNA permits a timely diagnosis.Althoughthereisnoadefinitivechoicetreatment,theuseofspecificantiviralsassociatedtoHAARTimprovedtheoutcomeofasubsetofpatients.

RESUMO

ComplicaçõesneurológicascausadaspeloCitomegalovírusempacientescomaids:estudoretrospectivode13casoserevisãoda

literatura

As complicações neurológicas causadas pelo Citomegalovírus

(CMV)empacientescomaidssãoraramenterelatadasnaeraHAART.OobjetivodesteestudofoidescreverasprincipaiscaracterísticasclínicaselaboratoriaisdepacientescomcomplicaçõesneurológicasassociadasaoCMVempacientescomaidsadmitidosemcentrodereferênciaemSao Paulo, Brasil.A doença citomegálica precisou da identificaçãodovírusnolíquormedianteareaçãoemcadeiadapolimerase(PCR).Trezecasosforamidentificadosentrejaneirode2004edezembrode2008.A mediana da idade foi 38 anos e nove (69%) eram homens.Naadmissão,todosospacientessabiamdoseustatussorológicoparao HIV e apenas quatro (31%) pacientes usavam HAART.A doençacitomegálica foiaprimeiradoençadefinidoradeaidsemoito (62%)pacientes.As síndromes neurológicas identificadas foram: encefalitedifusa(n=7;62%),polirradiculopatia(n=7;54%),encefalitefocal(romboencefalite)(n=1;8%),eventrículo‑encefalite(n=1;8%).Sete(54%) pacientes apresentaram doença citomegálica fora do sistemanervoso e quatro (31%) tiveram retinite.A mediana da contagem decélulasCD4+foi13células/µL.Amortalidadeglobalduranteainternaçãofoi38%.Oitopacientesusaramgancicloviroufoscarnet(mortalidade:50%) e cincopacientesusaramganciclovir e foscarnet (mortalidade:20%).Nenhumpacienteapresentoucritériosdiagnósticosdasíndromeinflamatória de reconstituição imunológica. Quatro pacientes foramperdidosdoacompanhamentoambulatorialetrêspacientesapresentaramreconstituiçãoimunológicaedescontinuaramasprofilaxiassecundárias.Embora raras, as particulares síndromes neurológicas causadas peloCMVcontinuamcausandoelevadamortalidadeempacientescomaids.AsobrevidadependedousodeterapiaantiviralefetivacontraoCMVeaintroduçãooportunadoHAART.

ACKNOwLEDGEMENTS

We would like to thank David Clifford,Washington UniversitySchoolofMedicine,St.Louis,USA,andtwoanonymousrefereesforvaluablecomments.

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Table3TherapeuticregimensforneurologicCytomegaloviruscomplications*

Typeoftherapy Patientswithrenaldysfuncion Patientswithsevereneutropeniaorthrombocytopenia

PatientspreviouslytreatedwithantiviralsforCMVorwithdiseaseprogression

Inductiontherapy Ganciclovir5mg/kgIVevery12hr Foscarnet90mg/kgIVevery12hr Ganciclovir5mg/kgIV+foscarnet90mg/kgIVevery12hr

Maintenancetherapy Valganciclovir900mg/dayPOevery12hrorganciclovir5mg/kg/dayIV

Foscarnet90‑120mg/kg/dayIV Valganciclovir900mg/dayPOevery12hrorganciclovir5mg/kg/dayIV+foscarnet90‑120mg/kg/dayIV

*AdaptedfromReferences2,4,5,17,20,22.


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6.ChouSW,DennisonKM.AnalysisofinterstrainvariationincytomegalovirusglycoproteinBsequencesencodingneutralization‑relatedepitopes.JInfectDis.1991;163:1229‑34.

7. CinqueP,VagoL,TerreniMR,BryttingM,MarenziR,CastagnaA,et al.DiagnosisofCytomegalovirusinfectionofthenervoussysteminAIDSbypolymerasechainreactionanalysisofcerebrospinalfluid.ScandJInfectDis.1995;99(Suppl.1):92‑4.

8.CinqueP,BaldantiF,VagoL,TerreniMR,LilloF,FurioneM,et al. Ganciclovirtherapyfor cytomegalovirus (CMV) infection of the central nervous system inAIDSpatients:monitoringbyCMVDNAdetectionincerebrospinalfluid.JInfectDis.1995;171:1603‑6.

9.CinqueP,CleatorGM,WeberT,MonteyneP,SindicC,GernaG,et al.DiagnosisandclinicalmanagementofneurologicaldisorderscausedbycytomegalovirusinAIDSpatients.JNeurovirol.1998;4:120‑32.

10.CinqueP,BossolascoS,BestettiA,SalaS,PierottiC,LazzarinA.Molecularstudiesofcerebrospinalfluidinhumanimmunodeficiencyvírustype1‑associatedopportunisticcentralnervoussystemdiseases‑anupdate.JNeurovirol.2002;8(Suppl.2):122‑8.

11.CollazosJ.OpportunisticinfectionsoftheCNSinpatientswithAIDS:diagnosisandmanagement.CNSDrugs.2003;17:869‑87.

12. CundyKC.Clinicalpharmacokineticsoftheantiviralnucleotideanaloguescidofovirandadefovir.ClinPharmacokinet.1999;36:127‑43.

13. Enting R, de Gans J, Reiss P, Jansen C, Portegies P. Ganciclovir/foscarnet forcytomegalovirusmeningoencephalitisinAIDS.Lancet.1992;340:559‑60.

14.FletcherCV,BalfourHHJr.Evaluationofganciclovirforcytomegalovirusdisease.DICP.1989;23:5‑12.

15.GoplenAK,LiestolK,DunlopO,BruunJN,MaehlenJ.Dementia inAIDSpatientsinOslo: theroleofHIVencephalitisandCMVencephalitis.ScandJ InfectDis.2001;33:755‑8.

16.GozlanJ,SalordJM,RoulletE,BaudrimontM,CaburetF,PicardO,et al. RapiddetectionofcytomegalovirusDNAincerebrospinalfluidofAIDSpatientswithneurologicdisorders.JInfectDis.1992;166:1416‑21.

17.Griffiths P. Cytomegalovirus infection of the central nervous system. Herpes.2004;11(Suppl.2):95A‑104A.

18.KedharSR,JabsDA.Cytomegalovirusretinitisintheeraofhighlyactiveantiretroviraltherapy.Herpes.2007;14:66‑71.

19.MamidiA,DeSimoneJA,PomerantzRJ.CentralnervoussysteminfectionsinindividualswithHIV‑1infection.JNeurovirol.2002;8:158‑67.

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21.MaschkeM,KastrupO,EsserS,RossB,HenggeU,HufnagelA.IncidenceandprevalenceofneurologicaldisordersassociatedwithHIVsincetheintroductionofhighlyactiveantiretroviraltherapy.JNeurolNeurosurgPsychiatry.2000;69:376‑80.

22.MaschkeM,KastrupO,DienerHC.CNSmanifestationsofcytomegalovirusinfections.CSNDrugs.2002;16:303‑15.

23. Mastroianni CM, Ciardi M, Folgori F, Sebastiani G,VulloV, Delia S, et al.CytomegalovirusencephalitisintwopatientswithAIDSreceivingganciclovirforcytomegalovirusretinitis.JInfect.1994;29:331‑7.

24.McCutchanJA.CytomegalovirusinfectionsofthenervoussysteminpatientswithAIDS.ClinInfectDis.1995;20:747‑54.

25.McCutchanJA.ClinicalimpactofCytomegalovirusinfectionsofthenervoussysteminpatientswithAIDS.ClinInfectDis.1995;21(Suppl.2):S196‑201.

26.MillerRF,FoxJD,ThomasP,WaiteJC,SharvellY,GazzardBG,et al.AcutelumbosacralpolyradiculopathyduetocytomegalovirusinadvancedHIVdisease:CSFfindingsin17patients.JNeurolNeurosurgPsychiatry.1996;61:456‑60.

27.MoulignierA.AtteintesdusystémenerveuxcentraletinfectionparleVIH‑1.RevNeurol(Paris).2006;162:22‑42.

28. Oliveira JF, Greco DB, Oliveira GC, Christo PP, Guimaraes MD, Oliveira RC.NeurologicaldiseaseinHIV‑infectedpatientsintheeraofhighlyactiveantiretroviraltreatment:aBrazilianexperience.RevSocBrasMedTrop.2006;39:146‑51.

29.PortegiesP,SolodL,CinqueP,ChaudhuriA,BegovacJ,EverallI,et al.GuidelinesforthediagnosisandmanagementofneurologicalcomplicationsofHIVinfection.EurJNeurol.2004;11:297‑304.

30. SacktorN.Theepidemiologyofhumanimmunodeficiencyvirus‑associatedneurologicaldiseaseintheeraofhighlyactiveantiretroviraltherapy.JNeurovirol.2002;8(Suppl.2):115‑21.

31. Sadler M, Morris‑Jones S, Nelson, M, Gazzard, BG. Successful treatment ofcytomegalovirus encephalitis in anAIDS patient using cidofovir.AIDS.1997;11:1293‑4.

32. ShelburneSA,MontesM,HamillRJ.Immunereconstitutioninflammatorysyndrome:moreanswers,morequestions.JAntimicrobChemother.2006;57:167‑70.

33.Vidal JE, Penalva de OliveiraAC, Fink MC, Pannuti CS,Trujillo JR.Aids‑relatedprogressivemultifocalleukoencephalopathy:aretrospectivestudyinareferralcenterinSaoPaulo,Brazil.RevInstMedTropSaoPaulo.2008;50:209‑12.

34.WhitleyRJ,JacobsonMA,FriedbergDN,HollandGN,JabsDA,DieterichDT,et al.GuidelinesforthetreatmentofcytomegalovirusdiseasesinpatientswithAIDSintheeraofpotentantirretroiraltherapy.ArchInternMed.1998;158:957‑69.

Received:29January2010Accepted:8October2010


(1)CollectiveHealthArea,MedicineCollege,CampinasCatholicUniversity(Puccamp),Campinas,SP,Brazil.(2)RehabilitationAmbulatory,DepartmentofGeriatrics,UniversityofBern,HospitalNetzBernZiegler,Bern,Switzerland.(3)DepartmentofBiostatistics,UniversidadeEstadualPaulista(Unesp),BotucatuCampus,Botucatu,SP,Brazil.Correspondence to: Aguinaldo Gonçalves, Rua Luverci Pereira de Souza 1151, Cidade Universitária, 13083‑730 Campinas, SP, Brasil. Phone: +55 19 3289 5022. E‑mail:

[email protected]

LEPROSy CONTROL: PERSPECTIVES & EPIDEMIOLOgICAL AND OPERATIONAL ASPECTS

AguinaldoGONÇALVES(1),GlaucaG.MANTELLINI(2)&CarlosRobertoPADOVANI(3)

SUMMARY

Objectives:Asastartingpoint,avastvarietyof200technicalpapersanddocumentspublishedduringthetenyears1999‑2008,fromBrazilianandinternationalorganizationsdedicatedtothecontrolofleprosy,wastaken.Astudywasthenundertakentoinvestigateitsfutureevolutivepossibilitiesbyemployingresourcesobtainedfromscenarioanalyses.Design:Themethodologicalreconstructioninusewasofaqualitativenature,basedonabibliographicreviewandcontentanalysistechniques.Thelatterwereemployedinadocumental,categorical,contingent,frequency‑basedformat,incompliancewithappropriateandpertinentconditions.Results:Nowadays,importantelementsonepidemiologicalandoperationalaspectshavebeenregained,aswellasrespectiveperspectives.Conclusions:Aprojectionismadetowardsthefactthatthemaintenanceofthedisease’spresentincidencelevelsconstituteeconomicandsanitarychallengesthatconfrontissuesrangingfromtheneoliberalmodelofglobalsocietalorganizationtospecificcompetencesofactionstakenbyhealthteamsinthefield.

KEYwORDS:Leprosy;Control;Epidemiology.

INTRODUCTION

Leprosycontinues tobeagreatPublicHealthprobleminBrazil,as it is recognizedevenby sectoral authorities, those responsible forthecontrolofdiseaseinourcountry1.Effortshavebeenhamperedbygapsininformationrelatingtoimportantspecificaspects,exacerbatedby the absenceof appropriate instruments for evaluation and routinesystematicanalysis20.

Theexplorationofpossiblehealthevolutionswithinthenextfewyears has been drawn to attention in several important publishingmaterials,includingtheofficialperiodicalissuedbytheWorldHealthOrganization(WHO)whopublishedareview13onthehealthdimensionof31previousstudiesonglobalscenarios.Thereviewshowedthreepossibleprospectivealternativesexpected: i) infectiousdiseases; ii)medicaltechnology,andiii)sustainedhealth.Focusingonthefirst,itdealswith thereemergenceofformerdiseasesor theappearanceofnewones,andstemsfromsocial,politicalandeconomicfactorswhichleadtohumanmovementsthatfacilitatenewcontactwithmicrobes,whichareassociatedtochangesconcernedwithproductiveactivities,such as deforestation, road construction and changes in irrigationsystems.Microbial resistance,personalbehaviorandenvironmentalpressuresrelatetothesecondperiod,which,ifaccompaniedbysocialbalance,willgothroughatimewhen:a)publicpoliciesshallactuallyprotectthenaturalneedsoffuturegenerations;b)globalsurveillanceand monitoring shall lead to the eradication of disease; c) despite

theagingprocessof theworld’spopulation,healthwillbeeasilyathandand,“moreover,disparitiesamongpoorandrichcountriesshalleventuallydisappear”.

Under this line of scenario analysis, leprosy control has beenintenselyanalyzedbysomeexperts14.Infact,anepidemiologicalmodelwasadoptedbasedonfigures regardingnewdetectedcasesobtainedfrom 1985, using national data published by theWHO’sWeeklyEpidemiologicalRecords,workingwith14countrieswheresuchvalueswereequalorgreaterthan2000in1998.Therespectivetemporalserieswereshowntoberatherunstable,thusreflecting‑althoughwithsomedelay‑changesthatwereintroducedtothepoliciesofcontrol.Threebasicevolutiveincidencestandardswereobtained:i)stableorrising,asforthecasesofBrazilandIndia;ii)asuddenrise,asforMyanmar,Nepal,andMadagascar,andiii)adecline,asforthePhilippines,China,andVietnam;fluctuationsinGuineaweretooharsh.Itwasobservedthat,asseveralpossiblescenariosaresimulated,thetimecorrespondingtothereductionof50%wassevento14years,accordingtodifferentiatedinterventionsoftheBCGvaccination,universaladoptionofmultidrugtherapy(MDT)and socioeconomic changes; “the most important conclusion is theslowrhythmofdisappearanceinallthescenariostakenintoaccount”.GiventhattheeliminationofsuchconditionreceivedWHOattention,whichdiffersfromconceptsconferredamongothers,itisrecognized15that“sickpeoplewithnocurrenturgefor treatment, thoughpossiblyphysicallyimpaired,wereremovedfromformalrecords”and,therefore,willcontinuetocoexistwiththedisease.

GONÇALVES,A.;MANTELLINI,G.G.&PADOVANI,C.R.‑Leprosycontrol:perspectives&epidemiologicalandoperationalaspects.Rev.Inst.Med.Trop.SaoPaulo,52(6):311‑5,2010.

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METHODOLOGY

As a starting point, a vast variety of 200 technical papers anddocumentspublishedduring the tenyears1999‑2008,fromBrazilianandinternationalorganizationsdedicatedtothecontrolofleprosy,wastaken.Astudywasthenundertakentoinvestigateitsfutureevolutivepossibilitiesbyemployingresourcesobtainedfromscenarioanalyses.Themethodologicalreconstructioninusewasofaqualitativenature,basedonabibliographicreviewandcontentanalysistechniques.Thelatterwereemployedinadocumental,categorical,contingent,frequency‑basedformat,incompliancewithappropriateandpertinentconditions.

EPIDEMIOLOGICALASPECTS

The reasons for the inaccuracyof the informationobtained fromregisteredprevalencestudieshavebecomeofcommonknowledge.Inadditiontotheso‑calledepidemiological“iceberg”duetolowcoveragebyhealthservices,thefirstquestionposedreferstopatientsinrelapseordrug‑resistantbacilluscarriers,whoarebothordinarilydischargedfromhospital.Inconsistencieswhendefiningcasesanddiagnosesformulatedunder different criteria make comparisons between successive yearsor different regions a difficult experience, “if not impossible”. Pointprevalence rates, i.e., a day defined in the year, as it is employed inleprosy,arealsoknowntobeaffectedbythedurationofthecondition,astheydonotconsiderpatientswhoundergotreatmentslastinglessthanoneyear(currentlythepaucibacillaryandprobablythemultibacillaryinthefuture).

Indignation isconcentrated inanofficialdocument issuedby theWHO22,whichquotes “non‑existingpatients” regarding a “diagnosisvalidationstudy”carriedoutinseveralregionsofIndia:two“individualswithsignificantclinicalexperience”reviewedpatientsinitiallyevaluatedbyhealthprofessionalsinordertocheckthediagnosticaccuracyandtheydiscoveredthatapproximately30%ofdiseasedpersonsinDelhi’sclinichospitalscouldnotbeconfirmedattheaddressesprovided,which,inafewcases,wereevenfictitious.Theauthorbeginsbyquestioningthequalificationof“validation”ascribedtoresearch,asthiscategorydoesnotsolelyimplyconsiderationwithanindependentevaluator,butalsowithindependentinstruments,which,forleprosy,consistofskinsmear,biopsy,andapplicationofPolymeraseChainReaction(PCR)inordertodetectbacillaryDNA.

The secondcriticism isdirected towards the terminologycreatedfor“non‑existingpatients”,aseveryoneworkingwith leprosyknowsthat many “existing patients” use the mistaken address strategy tominimize the effects brought on by stigma and ostracism either forthemselvesortheirfamilies.Thethirdfactorthatgeneratedconfusionwastherecommendationaccordingtowhich1/3ofthediseasedshouldbedischargedfromtherecordwhen,incompliancewiththepreviouslymentionedargument,thesepatientsaretheonesingreaterneedofrecordandfollow‑up.

The lack of acquiescence would be even greater if informationcirculatedaboutafewcurrentpracticesthatarelegitimatedasMDTisadopted.Forinstance,theimmediatedischargeoftheactiverecordofpatientspresentingincompletetreatmentwhohavebeenabsentfromahealthcareunitforoverayear,asrevealedduringtheconsiderationofthefavorableimpactofsuchatherapeuticschemeintheRegionalHealth

DepartmentofJuizdeFora,aninlandcityinBrazil,withnearlyhalfamillioninhabitants23.

Controlofpatients’contactscontinuestobeanimportantquestiontoberesolved:inBrazil,eveninepidemiologicallywellsurveilledregions,itisusuallynothigherthan1/3ofregisterednotifications26.

Inspecificreferenceto theAfricanreality, ithasbeenfoundout3thatofficialfigurescanoftenprovidean“overoptimistic”imageofthesituation.Asquotedbytheauthors,afairexampleisnoticingthatthenumberofrecordsinmanycountries,attheendoftheyear,issmallerthantheoneofmultibacillarycasesdetectedduringtheperiod.Well,ifleprosytakesonanyimportance,thatisowingtotheincapacitiesitpresents:these“aresupposedtoattractalotmoreattentionthantheypresentlyreceive”.

AsSOUTAR24puts it,“it ispossible tosuggest thatpast failuresrelated to other control initiatives, such as, for example, the ones ofmalaria and tuberculosis, contain lessons thatWHO might as wellperceiveasfarasleprosyeliminationisconcerned”.

OPERATIONALASPECTS

Perseveratingthroughthespecificitiesofleprosy,SAUNDERSON21providesquiteindispensableadditionalelements.Amongstthe“manifoldchallenges”thatareboundtoappear,heenvisagesthatthefirsttoshowupwithrecentconcernistomaintainsufficientlytrainedhealthprofessionalsasmuchastakingactionagainstthediseaseinendemiccountries.

Withinthepresentedcontext,itisclearthattheongoingglobalizedneoliberal model features the shrinking of the State, deletion of theworker’ssocialandoccupationalwelfareconquests,supporttotheactionofgeneraliststothedetrimentofthoseholdingspecificcompetences,thefragilityofhumanbasesineconomicrelations‑includingthethirdsector,devotedtoservicerendering‑,finally,byawiderangeoffactorsthatleadustoeasilyunderstandthat“manyexperiencedclinicians,fromimmediateassistanceleveluptothescopesofsupervisionandbackupwillnolongerbeworkingwithleprosypatientsinthenextfivetotenyears”(inBrazil,wecansaythatthissituationhasalreadybeentrueforatleastthesameperiodoftime).Therefore,thecontroloftheentireindividualhealthcarenetworkisputatstake‑fromdiagnosisuptothehandlingofcomplications‑eitheronaquantitativeorqualitativebasis,bothbeingaspectsofthehighestrelevance.

As a complement, following Saunderson’s thinking, how shouldyoungprofessionalslearntoexamine,treatandguideleprosycarriers?Throughdigital technology resourcesprovidedbydistance learning?Teleconferencesinvadingthecyberspace?

Quitestraightforwardly in thatrespect, ina textpublishedbytheWHOBulletin10whereanyconflictofinterestisdenied,theexistenceofnegativeeffectsinthe“campaign”toeliminatethediseasearerecognized,whichshouldbeconfrontedintheimmediatefuture,focusingonthosethatrefer tothepersonneleducationagenda,researchandinteractionamong services. The key category in such analysis is the mistakecommitted in understanding the proposal in terms of, if ever beingeliminated,thediseasefallingbackintoimportance.Inanapparentlyingenuouspragmatism, the following isput intoquestion:“Whocan


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pursueacareerregardingadiseasethatisconsideredtobeextinct?”

Thementionedscaleofdifficultiesreferstotheconditionstointegratecontrolinlocalhealthcareunits,aguidelineadoptedbymostcountries‑ due to process economic reasons.Also at this level, it is true thatqualifiedprofessionalsarescarceaswellasthefacilitiesrequired(theyquotethecaseofaHinduprovincewherethereisahealthcarecenterfor200thousandpeoplewhenthesefiguresamountto30thousandinotherregions).Surveillanceneedstobethecentralstrategytobedeveloped,mainlyinareaswhereintegrationisfound,evenindifferentmomentsofdeployment:eitherincidencemustbemeaningfullyraisedthroughanincreaseindetectionorabandonmentthroughthedifficultiestogoahead.Withinthiscontext,anadditionalchallengeappears:non‑governmentalorganizationsareparticularlyinvitedtoredesignrespectiveperformancesforthesubstitutionofverticalactions.

As a consequence, they predict hindrances to occur in activitieswhicharetobemaintained,i.e.intheidentificationofcases,treatmentapplication, incapacity prevention, and determination as well as theconductionofneurallesions.Inthefirstaspect,theyadmitthatdiagnosis,althoughsimple,callsforcompetenceintheformulationofdifferentialsaswellasintheperceptionofneuralinvolvement(theyhighlightthefactthatanestheticchangesarenotfoundinoneoutofthreemultibacillarypatients).

As a complement, nosographic peculiarities become priorities aswell,mainlythefollowing:i) increasing demand for recognition in communities and services,

ofcasespresentingahighbacillaryload,whichcallforatleast24monthsofMDT,asitisofcommonknowledge;

ii) tendencyfortheadoptionofoperationalschemesdevoidofintakesupervision,contrarilytowhatisadoptedinrelationtotuberculosis,abiologicallysimilarmycobacteriosisthatpresentsfailedadherenceinunmonitoredregimens;

iii)gradually increasing theurge toassess the relapse ratefiveyearsaftertheintroductionofthedrug;

iv)theeventofreactivephenomenainoneoutofthreemultibacillarypatients, requiringprimaryhealthcareunits to supply immediateaccesstomakecorticoidsavailableandareferencesystemformorecomplexcenters,forwhichspecialconditionsarerequired;

v) preventionofincapacities,devisedtolastforthepatient’swholelife,whichiscriticaltoachievecontrolsuccessasevennowadaysmosttreatmenteventscompriseulcerationsandotherrespectiveoutcomes;

vi)the fight against stigmatization and insertion in public socialinclusionprograms,inadditiontoareductioninpovertyandmisery‑conditionsassociatedwiththehighestratesofdisease.

Asapowerfulmanifestationofreality,WorldHealthOrganizationstoodoutas itpublishedadocumentregardingtheglobalstrategytoreduceleprosyinthemorbidityofcountriesandalsothemaintenanceofcontrolactivities.27Clearly,itssenseis,ononeside,toconfirmtheprevious Organization documents in relation to classic actions takenagainst the endemic disease; dialectically, when pursuing such aconservativeprinciple,itinnovatesandindoingsoimpliesrecognizingthattheconditionfollowsitsshamefulcourseinthemidstofpopulations.Thecentralideasareactuallysurprising:i) Inthreeyears(2001to2003),onlybyconsideringofficialdataand

disregardingtheoldepidemiologicalprevalence“iceberg”18,nearly

twomillioncitizenswereinfectedworldwide!ii) The campaign‑prone approach must be banned! More clearly

to “maintain control activities in the long run, the ‘campaign‑like’ approach must be reinterpreted towards a long‑term servicemaintenanceprocessagainstleprosy,whichisabletodeliverhighquality,integratedservices”.

iii)“Specific competence in leprosy as well as its control must bemaintainedwithinbothnationalandsubnationallevels”.

Aboveall,voicesquestioningtheresolutiveMDTvalidityconcernedwithdiseasecontrolandmainlyphysicalsequelaehavebeenheardonceagain,inanewapproachofwell‑knowncontroversiesthatappearedatatimewhensuchstrategywasintroducedasaroutine,redeemingmeasure9.Someofthempointtotheabsenceofevidenceonhowitreducesleprosyincidence and to thedifficulty in separating its owneffects from theactioncausedbyconfoundingfactors2.Specifically,theystatethatnoconsensushasbeenreachedbyexpertsconcerningitsearlyadministrationandpreventionofincapacities.

Infact,thisissuehadalreadybeencloselyexaminedanddrawnintoincisiveconclusion25.Evenin thesense thatWHOascribes to“cure”asthe“successfulconclusionofagivenevolutionwithMDT”,ifcureisattainedinlevelsupto67%forpaucibacillarypatientsand38%forthemultibacillaryandsuchsituationcannotbeduetodrugresistance,thenthemedicationatissueisnotabletosolvetheendemicsituation.Furthermore,itissaidthatthecurebyMDT,therefore,inadditiontobeingmistaken,leadstoseriousoutcomes,suchasdrawingattentionawayfrommoreseveresituationsassociatedwiththedisease.Forthesakeofclarityanddepth:“leprosyshallremainahealthissueuntilthequalityoflifeofthoseaffectedbythisconditionismeaningfullyenhanced”.

Then,howtorebuildit?Itisemphaticallysustained6thatthefutureofcollectiveissuesposedbyleprosywillonlyfindasolutionthroughtheintegrationtogeneralhealthcareservices.Indeed,reportstheretoarenotscarceinrespectivetechnicalliterature,eitherinBrazil7orinothercountries17andevenintermsofoperationalresearch,withalongdescriptionabouthowanon‑governmentalorganizationtookactioninIndia19.

PERSPECTIVES

It is this effervescence of a leper’s singular situation, which isvisiblebeforeperceptiveeyesthatthediseasetendstofollowthrough.Everlastingforsomanyindividualsinthepast,presentandfuture,suchsituationscannotbeforgottenorsolelydealtwithbymeansofbandaging.Theymakeupelementsfromgreaterunitstobeconsideredinanon‑segmentedmood.Theyareinseparablepartsofsubjectswho,ontheirturn,interactinsociety.Theylivetogetherevenwhenwallsandfencesarebuiltin‑between.

In other terms, a reference is made here to the need to get overpreviously mentioned gaps and stumbling blocks, the search for anddiscoveryofotherprophylacticmeasuresandtherapies,whicharenotonlycenteredinmedicativeactionagainstthebacillusinamechanicalandisolatedway.

As a solution, there is the synthesis that combines research infundamentalscienceandinvestigativedevelopmentdevotedtopractical


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controlaspects28.Thisspeechwouldrefertotheevolutionofmicrobialgenomes, pathogen‑host interactions, susceptible genetics, biologicalinstrumentsagainst transmissionblocks,drugresistance,detectionofsolublecytokines,comparativegenomics,molecularepidemiologyatlastdirectly,bacillaryneurotropismandimmunologyasprioritiesrequiringhighdedicationandfinancing.

Withinthiscontext,therediscoverymadebythecontributionoftheacademiccommunityplayedbysystematicphysicalactivitypracticeforprimary,secondaryandtertiaryleprosypreventionwasaconquestthatisdisclosedinfew,remotepartsoftheworld.Asanelementusedpreviouslytoitsfullextentandsuccessfullyfordecadesinasmuchasitreferstonon‑transmissible diseases, it has only recently been reconsidered inrelationtotransmissibleillnesses.Bydeconstructingmisunderstandingsderivedfrompreviousviews8,leprosyhasdeservedspecialattentioninsucharespectthatifitisnottakenintoaccountasapriorityinpoorcountries,itishardlysupposedtobetakenseriouslyindevelopedones,whereitisnotthepriorityofhealthpolicies.Animportantwaytobetaken,inthatsense,istheonepointedoutinBrazilbyMONTEIRO&GONÇALVES16intheirinvestigationinahospitalcontext.Theyfoundoutthat,althoughphysicalactivitymaybepresentedasariskfactorforthediseaseoutbreakanddevelopment,italsomeans,ontheotherhand,aprotectivefactorconcernedwithneuritisevents.Itisalsoundeniablethattherestillisalottobeknowninthatrespectaswell.

Itisworthquotingthatitisamatterofprovidingpeoplewithpracticalintervention,which,underspecificinteractionfeatures,outdoesisolatedpedagogic,playful,competitiveorbiologicaspects.Whenincorporatingnewperspectives,personalaswellassocialaspectsofinclusioninsocietyshouldbedealtwith.Specifically,therehabilitationprocesswouldloseitsstrictmeaningof“habilitatingagain”butitwouldstarttofavorpersonalautonomybyrespectingthecharacteristicsofwhatisexperiencedbyeachindividual.11

As recalled12, the guidance “philosophy” towards inclusion goes

through an enhanced autonomy of people in disadvantage, withinterventional participation as well as collaborative participation.Objectively,socialenvironmentsdonotneedtobemandatorilyadaptedbecausewhentheseusersaretreatedfortheirdisadvantages,theybecomeactivefromtheirpersonaldiversitiesinneedsandexpectations.Inotherwords,focuspassesbyandgoesbeyondstrictlyenvironmentaladaptations.4

CONCLUSIONS

1. It was possible to specify a way to apply the qualitativemethodology of scenario construction in order to give a sense offuture to the evolution of leprosy control among us in distinguishedepidemiologicalandoperationalaspects.

2.Fromdifferentperspectives, including thatof theWorldHealthOrganization,itisestimatedthat,unlikereiteratingforecasts,leprosyisnotsupposedtobeeliminatedasapublichealthprobleminthenearfuture.

3. Within this unsuccessfulness, in opposition to previousmanifestations made by sectoral leaders, Brazil is one of the mostremarkablecountries.Severalauthorsmention,amongrespectivecauses,thelowprioritizationconferredupontheactionsofdiseasecontroltofollowthroughandputthehealthservicenetworkintopractice.

4. It is recognized that the internationally adopted strategy ofmultidrug therapy presented positive outcomes as to its complete

application (i.e. the cure, according to theWHO) toover10millionpeople.However,importantshortcomingsremain,mainlynon‑reductionofendemicdiseaseandnegativebalancegeneratedbycampaign‑likeactivityadoptedbythestrategy.Inrelationtothat,itisrecognizedasbeingabsolutelynecessarytoovercomesomeofitsmostseriousconsequences.Reductionandnon‑replacementofqualifiedprofessionalsisoneofthese,aswellastheprogressiveabsenceofpublicpermanenthealthservicesincontrolofthedisease.

5.The number of infected people is great in comparison to thereduction of specialized centers, which has been observed regardingtreatment units for the disease and its derivatives. Clearly phrased,thegreatestfearisthatthepopulationgrowssimultaneouslywiththedecreasedresourcesandinterestforthepurposesofhandlingandcontrol.

6.Apossiblealternativethatisinterpretedasasolutionmightbepoliticaldecisionstowardsmakingcontemporaryscientificandsocialdevelopmentalsosuitableforspecificissuesofthedisease.

RESUMO

Controledahanseníase:perspectivaseaspectosepidemiológicoseoperacionais

Objetivos: A partir de acervo de 200 textos acadêmicos e dedocumentos de organismos nacionais e internacionais voltados aocontroledahanseníasepublicadosnoperíodode1999a2008,procurou‑seestudarrespectivaspossibilidadesevolutivasfuturas,empregando‑seossubsídiosdorecursodeanálisedecenários.Métodos:Areconstruçãometodológicaadotadafoidenaturezaqualitativa,fulcradanastécnicasderevisãobibliográficaeanálisedeconteúdo.Estaúltimafoiempregadanatipificaçãodocumentalcategorialfrequencialcontigencial,deacordocomdevidafundamentaçãopertinente.Resultados: Recuperaram‑seelementosatuaisimportantesdenaturezaepidemiológicaeoperacional,bemcomoderespectivasperspectivas.Conclusões: Projeta‑sequeamanutençãodoscoeficientesde incidênciadadoençacolocareptoseconômicosesanitáriosadesafiardesdeomodeloneoliberaldeorganizaçãosocietáriamundialatécompetênciasespecíficasdasaçõesdasequipesdesaúdeemcampo.

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5.Feenstra P. “Elimination” of leprosy and the need to sustain leprosy services,expectations, predictions and reality. Internat J Lepr Other Mycobact Dis.2003;71:248‑56.

6.FeenstraP,VisschedijkJ.Leprosycontrolthroughgeneralhealthservices‑revisitingtheconceptofintegration.LeprRev.2002;73:111‑22.


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10.LockwoodDNJ,SuneethaS.Leprosy:toocomplexadiseaseforasimpleeliminationparadigm.BullWorldHealthOrgan.2005;83:230‑5.

11.Marques CA. Implicações políticas da institucionalização da deficiência. Edu Soc.1998;19:34‑41.

12.MarquesUM,CastroJAM,SilvaMA.Actividade físicaadaptada:umavisãocrítica.RevPortuguesaCiênDesp.2001;1(1):73‑79.

13.MartensP,HuynenM.A futurewithouthealth?healthdimension inglobal scenariostudies.BullWorldHealthOrgan.2003;81:896‑901.

14.MeimaA.The impact of multidrug therapy on trends in transmission. In: ScientificWorking Group, Report on Leprosy. Geneva:World Health Organization; 2003.p.42‑5.

15.MeimaA,SmiothWC,VanOortmarssenGJ,RichardusJH,HabbemaJD.Thefutureincidenceofleprosy:ascenarioanalysis.BullWorldHealthOrgan.2004;82:373‑80.

16.Monteiro HL, GonçalvesA. Saúde coletiva e atividade física no contexto desubdesenvolvimento:evidênciaseperspectivesparasuperaçãodoatraso.RevBrasMedEsporte.2000;6(5):180‑7.

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18.OpromollaDV,NobregaRC,GonçalvesNNS,PadovaniSHP,PadovaniCR,GonçalvesA.Estimativadaprevalênciadahanseníasepelainvestigaçãoemdemandainespecíficadeagênciasdesaúde.RevSaúdePública.1990;24:l78‑85.

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20.Ramos Jr, ANR, Heukelbach J, Gomide M, Hinders DC, Schreuder PAM.Investigaçõesemsistemasde serviçosde saúdecomo ferramentaparaoalcancedeprogramasdecontroledahanseníasemaisefetivosnoBrasil.CadSaúdeCol.2008;16:147‑68.

21.SaundersonP.Learning tomanage leprosyafter2005:preservingcriticalknowledgeandexploitingnewtechnology.LeprRev.2005;76:2‑4.

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27.WorldHealthOrganization.Globalstrategyforfurtherreducingtheleprosyburdenandsustainingleprosycontrolactivities.Planperiod:2006‑2010.Availablefrom:http://www.searo.who.int/LinkFiles/Guidelines_1‑Global_Strategy_Plan_period_06‑10.pdf

28.Young DB. Leprosy ‑ new opportunities in basic science. In: ScientificWorkingGroup,ReportonLeprosy.Geneva:WorldHealthOrganization;2003.p.36‑40.

Received:8February2010Accepted:11August2010




Telephone:55113061‑7003‑Fax:55113062‑2174





(1)ProgramadePós‑GraduaçãoemSaúdedaCriançaedoAdolescente,UniversidadeFederaldoParaná.Curitiba,Paraná,Brasil.(2)DepartamentodePediatria,UniversidadeFederaldoParaná,Curitiba,Paraná,Brasil.(3)LaboratóriodeVirologia,HospitaldeClínicasdaUniversidadeFederaldoParaná.Curitiba,Paraná,Brasil.(4)DisciplinadeDoençasinfecciosas,UniversidadeFederaldoParaná,Curitiba,Paraná,Brasil.Correspondenceto:SoniaMaraRaboni.LaboratóriodeVirologia,HospitaldeClínicas,UniversidadeFederaldoParaná,R.PadreCamargo280,2oandar,sala202,AltodaXV,82060‑240

Curitiba,PR,Brasil.E.mail:[email protected]

ADENOVIRUS RESPIRATORy INFECTION: SIgNIFICANT INCREASE IN DIAgNOSIS USINg PCR COMPARINg wITH ANTIgEN DETECTION AND CULTURE METHODS

EleniceSTROPARO(1),CristinaR.CRUz(2),MariadoCarmoDEBUR(3),LuineR.VIDAL(3),MeriB.NOGUEIRA(3),SergioM.deALMEIDA(3),LucianeA.PEREIRA(3),IndianaraROTTA(3)&SoniaMaraRABONI(3,4)

SUMMARY

Adenovirus(AdV)respiratoryinfectionsareusuallydescribedasbeingassociatedwithhighmortalityrates.Laboratorydiagnosisisessentialfortheestablishmentoftheappropriatetherapy,andforguidingtheimplementationofpreventivemeasuresinordertopreventthespreadoftheinfection.Aimingtoanalyzethesensitivityandspecificityofthelaboratorialdiagnosismethodsavailable,wecomparedantigendetectionbyindirectimmunofluorescenceassay(IF),andaspecificnestedpolymerasechainreaction(PCR),todetectAdVinrespiratorysamplescollectedfrompatientsadmittedtohospitalwithacuterespiratorydisease.Positivesampleswereinoculatedintoacellculturetoconfirmtheresults.Weanalyzed381samplesfromthenasopharyngealaspiratescollectedduringtheyear2008;ofthese,2.6%testedwerepositiveforadenovirusthroughIFand10%throughPCR;positiveisolationwasobtainedin40%and26%ofthesecases,respectively.Mostinfectedpatientswerechildrenundersixmonthsofage,anddespiteofthefactthatasignificantnumberofpatientsrequiredintensivecare,themortalityratewaslow(5%).Inconclusion,molecularmethodswerefoundtobeusefulforrapiddiagnosisofadenovirusinfectionswithhighersensitivitythanantigendetection;theirintroductionpermittedasignificantincreaseindiagnosesofadenovirusinfections.

KEYwORDS:Adenoviruses; Polymerase chain reaction; Diagnosis; Pediatric patients; Immunocompromised patients;Acuterespiratoryinfections.

INTRODUCTION

Humanadenovirusesareresponsibleforvariousdifferentclinicalsyndromes,includinggastroenteritis,respiratorydisease,conjunctivitis,hemorrhagic cystitis and exanthema11,18. There are 51 knownimmunologicallydistinctAdVserotypesthatcaninfecthumans,whicharegroupedintosixspecies(formerlycalledsubgenera)AtoF,basedontherelativenucleicacidhomology,fiberproteincharacteristicsandotherbiochemicalandbiologicalproperties6,whichdifferintissuetropismandpatternsofdisease30.Thissubdivisionhassomeclinicalrelevance,asdistinctAdVspeciesshowapreferenceforspecificorgans:C,EandsomeBspeciestypicallyinfecttherespiratorytract;otherBspeciesinfecttheurinarytract;speciesAandFtargetthegastrointestinaltract;andspeciesD,theeyes19.ThedegreeofseverityofthediseasecausedbyAdValsodependsontheageandimmunestatusoftheinfectedindividual;althoughavarietyofothersocialandphysicalfactorsmayalsocausemoreseveredisease.Inimmunocompetentpatients,AdVisresponsibleforseveralacute,usuallyself‑limiteddiseases,aswellaspersistentinfection3.Thediseasemayvaryfrommildtosevere,disseminated,orfatal;theoverallfatalityrateisbetween18%and26%17.Despiteitsrelativelycommonoccurrenceininfantsandyoungchildren,fatalpneumoniacanoccurin

healthyadults.Inimmunocompromisedpatients,AdV‑associatedcasefatalityrateshavebeenreportedtobeashighas60%inpatientswithpneumoniaand50%inthosewithhepatitis,comparedtofatalityratesof15%forpneumoniaand10%forhepatitisinimmunocompetentpatients15.Infectionisfarmoresevereintheimmunocompromisedpopulation,withacasemortalityrateashighas80%inhematopoieticstemcelltransplant(HSCT)recipients26.

ThediagnosisofAdVdiseasehastraditionallybeenperformedusingantigen detection by indirect immunofluorescence and culture‑basedtechniques.Viralantigendetectionofrespiratoryvirusesisusefulformostrespiratoryviruses,butthismethodologyhasalowersensitivitythancellcultureforthediagnosisofAdV.Whileeffectiveatdetectingend‑organ infection in symptomatic patients, cell culture lacks thesensitivityfordetectinglowlevelsofcirculatingviruses,andmayrequireweekstodeliverdefinitiveresults14.Moleculardiagnosticassaysofferadvantages in terms of speed, sensitivity and the ability to quantifyviruses7andimprovedmethodsofdiagnosisareneededpartucularlyinimmunocompromisedpatients21.TheyhavebeenparticularlyusefulfortheAdVdetectioninperipheralblood‑afindingthathasbeencorrelatedwiththeriskofprogressiontodisseminateddiseaseinHSCTpatients

STROPARO,E.;CRUZ,C.R.;DEBUR,M.C.;VIDAL,L.R.;NOGUEIRA,M.B.;ALMEIDA,S.M.;PEREIRA,L.A.;ROTTA,I.&RABONI,S.M.‑Adenovirusrespiratoryinfection:significantincreaseindiagnosIsusingPCRcomparingwithantigendetectionandculturemethods.Rev.Inst.Med.Trop.SaoPaulo,52(6):317‑21,2010.

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infectedwithAdV8,27.Inaddition,theabilitytomonitorviralloadofferspotential formonitoring responsiveness toantiviral therapy,which isbecomingmorepractical29.However,thepaucityoftreatmentoptionstocontroltheseinfectionsemphasizestheurgentneedforwell‑controlledclinicaltrialsofthecurrentlydevelopedtherapeuticmodalities16.

Thepresentstudyhastwopurposes:tocompareantigendetectionandPCRmethodforthediagnosisofAdVinfectionsinpatientshospitalizeddue to acute respiratory infection, and to report on the clinical andepidemiologicalfindingsinAdVinfectedpediatricpatients.

MATERIALANDMETHODS

Clinicalspecimens:Weconductedaprospective,descriptiveandcross‑sectionalstudy.Nasopharyngealaspirates(NPA)collectedfromchildrenandimmunosuppressedpatientswithacuterespiratoryinfectionshospitalizedattheHospitaldeClínicas/UniversidadeFederaldoParaná(HC‑UFPR) in2008wereassessed.Aftercollectionand theadditionof transportmedium,samplesweresent toavirology laboratoryandimmediatelyprocessed.First,theywerecentrifugedat1,500gfor10to15minutesat4°C.Thesupernatantwasseparatedintotwoportions:onetobeusedforvirusisolationincellcultureandoneformolecularbiologytests(PCR).Additionally,cellsinthesedimentwerewashedandplacedonglassslides,fixedincoldacetonefor10minutes,andprocessedforindirectimmunofluorescenceassay(IF).

Indirectimmunofluorescenceassays(IF)onrespiratorysamples:Detection of viruses (respiratory syncytial virus ‑ RSV, influenzaA‑FLUA, influenzaB ‑FLUB,AdV, andparainfluenzagroupviruses‑ PIV) in cells shed into the respiratory tract was performed by IFas previously reported28, using commercially available monoclonalantibodies(ChemiconInternational Inc.,Temecula,CA),according tothemanufacturer’ssuggestions.

Theslideswereexaminedinafluorescencemicroscopewithepi‑illumination.Allclinicalspecimenswerereadbytwoobservers.Samplesthat seemeddoubtfulordiscrepantwere readat least twice tocheckfor reproducibility. Samples have indeterminate results when it wasimpossibletodefinethepresenceorabsenceoffluorescentcells,duetounspecificcoloration(background).

PCR: DNA extraction was performed following the methodpreviously reportedbyCASASet al. (1995)4, basedonguanidiniumthiocyanateextractionandisopropanolprecipitation.ThePCRmethodused,specifictoAdV,wasthenestedPCR,andprimersweredesignedwithinthehexonproteingeneoftheAdVgenome,capableofdetectingtheDNAof51typesofhumanAdVinawiderangeofclinicalsamples,reportedbyAVELLÓNet al.(2001)1.ThePCRreactionwasperformedfollowingtheguidelinespreviouslyreportedwithsomemodifications.Briefly,theamplificationwascarriedoutwith2.5mLDNAextractedin a 25 µL reaction mixture containing 10xBuffer, 1.5mM MgCl2,0.2mMdNTP's(Boehringer,Mannheim,Germany),10pmolofeachprimers(ADHEX1FandADHEX2R)and1UofTaq DNApolymerase(Invitrogen,Inc,California,USA)forthefirstreaction.Themixtureswereoverlaidand30cyclesofamplificationwereperformedinathermocycler.Eachcycleconsistedofdenaturationfor60sat94°C,annealingfor60sat50°C,andprimerextensionfor60sat72°C.Inthefirstcycle,thedenaturationstepwasfortwominat94°Candelongationwasextended

tosixmin.Forthenestedreaction,1mLofprimaryamplificationproductwasaddedto24mLofanewreactionmixture,similartotheprimaryamplificationbutcontaining theprimersADHEX2FandADHEX1R,usingthesamecycleconditions.Aftertheamplificationwascompleted,5mLofthereactionmixturewasanalyzedon1%agarosegelcontainingethidiumbromideandwasvisualizedunderUVlighttodetectanexpectedproductof168bp.

Cell culture: Cell culture was performed on continuous humanepithelial carcinoma cell lines (HEP‑2).Tubes with 80% confluentmonolayerswereinoculatedwith0.2mLofhomogenizedsamplestreatedwith antibiotics and antifungals.The inoculum was absorbed to themonolayerinthespaceofonehourat37ºC.Cellswerefedwith2mLof2%fetalcalfseruminBasalMediumEagle(SigmaInc),andmonitoredtodetect thecytopathiceffect(CPE)forfourweeks.WhenCPEwasobserved,themonolayerwasscrapedandtestedforAdVantigenbyIFwithaspecificmonoclonalantibody.Tubeswerediscardedasnegativeafter30dayswithoutCPE.

Ethics: Clinical and epidemiological data fromAdV infectedpediatricpatientswererevisedfrommedicalrecords.TheEthicsReviewBoardoftheHC‑UFPRapprovedthestudy.

RESULTS

Atotalof381sampleswereanalyzedduring2008.Twohundredtwenty‑nine (229/381 ‑ 34%) samples tested were positive to one ormorerespiratoryviruses(Fig.1).

ConsideringtheresultsoftheresearchofAdV,PCRwaspositivein 38/381(10%) samples, IF was positive in 10/381 (2.6%) andindeterminatein2/381(0.5%).Atotalof46indeterminateorpositivesamples(foursampleswerepositivebyPCRandIFI)wereinoculatedincellculture,andAdVwasisolatedin11(24%)cases(Fig.2).Nine(9/46‑19.5%)patientswereundergoingbonemarrowtransplantationandother37/46(80.5%)werehospitalizedinpediatricunits.Recordsfrom37pediatricpatientswerereviewed.

The majority of the children were male (59.4%), and 81% wereunder twenty‑fourmonthsold.Fever, coughand tachypneawere themostcommonlyreportedclinicalmanifestations,andthemostfrequent

Fig.1‑Respiratoryviraldetectioninpatientshospitalizedbyacuterespiratoryinfections

during2008year–HC/UFPR(HC/UFPR:HospitaldeClínicas/UniversidadeFederaldo

Paraná).


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medicaldiagnoseswerebronchopneumoniaandtracheobronchitis.Oneormoreriskfactorsorcomorbiditieswerepresentin64.9%ofpatients,mainly represented by prematurity, pulmonary, genetic and cardiacdisorders.Among genetic disorders, Down syndrome was the mostfrequent, with four patients. Epidemiological and clinical data frompediatricpatientsareshowninTable1.

Eleven(11/37‑29.7%)patientsrequiredadmissiontointensivecareunitforamediantimeofninedays(rangefromfourto19days),and19%(7/37)patientsneededmechanicalventilation.Antibioticswereusedin67.6%(25/37),oxygenin86.5%(32/37)andthemedianhospitalstaywassevendays(withatotalrangeoftwoto85days).Bloodcultureswerecollectedfrom36patients.Ofthese,94.4%(34/36)werenegative;onewaspositiveforPseudomonas aeruginosa(justthechildadmittedtosepsis);andanotherwaspositiveforcoagulasenegativestaphylococcus(probablybycontamination).Theoverallmortalityratewas5%.

Thepresenceofviralcoinfectionwas21.6%(8/37):fourcasesofco‑infectionwithAdenovirus/Enterovirus,twowithAdenovirus/Respiratorysyncytialvirus,andtwowithAdenovirus/Parainfluenza3virus.

DISCUSSION

Despiteitsassociationwithsevererespiratoryinfections,theuseofspecificdiagnostic laboratorytestsforviralagents in thecaseofARIsislimitedduetofinancialandtechnicalproblems,orduetothefactthatvirusesareunderestimatedasagentsofrespiratoryinfectionsrequiringhospitalization22.Inthepresentstudy,one‑thirdofthesamplestested were positive for one or more respiratory virus analyzed byantigendetection,andwedemonstratedanincreaseof2%to10%inthedetectionofAdVinclinicalsamples,comparingtheresultsofIFandPCR.The cell culture results demonstrated ahigher sensitivitythanIF,andalowersensitivitythanPCRresults,aswasexpected.Theresultswereconsistentforthemajorityofthetests.Nevertheless,somesampleshavepositiveIFwithnegativeisolationandPCR.ThismayhaveoccurredbecausevirusisolationandPCRwerenotperformedinthesameperiodoftheIF,andconsequentlytherewasalossofproperclinicalmaterialafterfreezing,orelse,couldhaveoccurredduetoafalse‑positiveIF.False‑positiveresultsmustbeconsideredwhenthereaderfoundcellswithcharacteristiccoloration,andwhentheresultwasnotconfirmedbyothermethods.Itisimportanttomentionthat

Table1Pediatricpatientsinfectedwithadenovirus:demographicandclinicaldata

Data N %

Agerange

<6months 9 24.3

>6months‑12months 12 32.4

>12months‑24months 9 24.3

>24months 7 18.9

Gender

Male 22 59.4

Female 15 40.6

Diagnosis

Bronchopneumonia 14 37.8

Bronchitis 13 35.2

Laryngotracheitis 3 8.1

Acutebronchiolitis 2 5.4

Asthma 2 5.4

Metabolicdisorder 1 2.7

Sepsis 1 2.7

Febrilneutropenia 1 2.7

Symptoms

Cough 28 75.7

Tachypnea 28 75.7

Fever 27 73

Wheezing 17 45.9

Vomiting 8 21.6

Cyanosis 6 16.2

Diarrhea 3 8.1

Apnea 1 2.7

Riskfactors/comorbidity

Yes 24 64.9

No 13 35.1

Geneticdisorder* 7/24 18.9

Pulmonarydisorder 7/24 18.9

Cardiacdisorder 6/24 16.2

Prematurity 6/24 16.2

Neurologicdisorder 3/24 8.1

HIV 2/24 5.4

Other** 4/24 10.8

*DownSyndrome:4;**Hematologicdisorder:1;Leukemia:1;Endocrinopathy:1;Nephopathy:1.

Fig.2‑Comparisonofimmunofluorescenceassay,PCRandcellculturetodetectadenovirus

inpatientswithrespiratoryinfections*.(*Adenovirusindeterminateandpositivesamplesby

immunofluorescence(IF)assayand/orPCRarecomparedtocellcultureresults).


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alltheslidesaresubmittedtodouble‑blindreadingbytworesearchers.Evenso,readingerrormaybeconsidered.

DetectionofviralantigenbyIFisfast,butoftenlackssensitivityin detecting some viruses, and its confirmation by viral culture maysometimes be necessary10.Viral culture was recognized as the goldstandardfortestingviralrespiratorypathogens.However,thismethodis generally slow, often requiring more than 14 days for deliveryof results24.Although the combination of these two techniques canincreasethepercentageofpositiveresults,ithasbeenreportedthatasignificantnumberofspecimensremainsnegativedespiteclinicalandepidemiologicalsuspicionofviral infection9,12,13.Over thepastyears,there have been an increasing number of reports on the progress ofmolecularbiologytechniquesforthediagnosisofviralinfections;thesewillprobablybethegoldstandardmethodscurrentlyemployedinroutinelaboratorytests.Moleculartechniqueshavesignificantlycontributedtotherapididentificationoftheviralagentassociatedwithacuterespiratoryinfections, allowing for the quick adoption of therapeutic measuresandpreventivestrategiestoavoidthespreadofthediseasewithhightransmissibility20,23.

RecordanalysisshowedtheclinicalanddemographicprofileofAdVinfectedpatients:theinfectionoccurredmainlyinmalepediatricpatients,mostofthemlessthantwoyearsofage;andin33%ofthecases,patientsshowednoriskfactors,otherthantheiryoungage.Regardlessofthelowmortalityrateobserved(5%),twenty‑sevenpercentofthepatientsneededintensivecare,whichcorroboratestheseriousnessoftheseinfections.Despitethewideuseofantibiotics,bacterialinfectionwasconfirmedinonlyonepatient.However,viralcoinfectionwasdetectedin20%oftheadenoviruspositivesamples,which,accordingly,previousreportsmayhavecontributedtohighhospitaladmissionrates2,5,25.

The introduction of molecular methods in routine diagnosis hasshown thatAdV is more frequently involved with severe respiratoryinfectionsinpediatricpatientsthanpreviousstudiesbasedonantigendetectionandcellculturemethodshaveindicated.RapidandsensitivemolecularmethodsusedtoidentifyAdVinfectionareneededbecauseoftheirrapidspreadandhighhospitalmortalityrate.Inaddition,theuseofmolecularteststodetectrespiratoryvirusesinroutinelaboratoryinvestigationsinhospitalizedpatientswillassistintheintroductionofpreventivemeasurestoavoidviruses’disseminationandtominimizethemisuseofbroadspectrumantibioticsinthisenvironment.

RESUMO

Infecçãorespiratóriaagudaporadenovirus‑comparaçãodosmétodosdePCReimunofluorescênciaindiretaparaoseu

diagnósticoedadosclínicosdospacientesinfectados

Infecções respiratórias porAdenovírus (ADV) são geralmentedescritasassociadascomaltamortalidade.Odiagnóstico laboratorialé essencial para o estabelecimento da terapêutica adequada e paraorientaraimplantaçãodemedidaspreventivasevitandoapropagaçãodainfecção.Comoobjetivodeanalisarasensibilidadeeaespecificidadedosmétodosdeavaliaçãodediagnósticolaboratorial,foicomparadaadetecçãodeantígenoporimunofluorescênciaindireta(IF)comareaçãoemcadeiadapolimeraseespecífica(PCR)paradetectarAdVemamostrasrespiratóriascoletadasdepacientesinternadoscomdoençarespiratória

aguda.As amostras com resultados positivos foram inoculadas emculturacelular.Foramanalisadas381amostrasdasecreçãonasofaríngeacoletadasduranteoanode2008,dasquais2,6%forampositivaspelaIFe10%pelaPCR,isolamentopositivofoiobtidoem40%e26%doscasospositivospelostestesanteriores,respectivamente.Amaioriadospacientesinfectadoseramcriançascommenosdeseismesesdeidade,eapesardofatodequeumnúmerosignificativodepacientesnecessitoudecuidadosintensivos,ataxademortalidadefoibaixa(5%).Emconclusão,osmétodosmolecularessãoúteisparaodiagnósticorápidodeinfecçõesporadenovíruscommaiorsensibilidadedoqueadetecçãodoantígeno,asuaintroduçãonarotinapermitiuumaumentosignificativonodiagnósticodeinfecçõesporadenovírus.

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27.TeramuraT,NayaM,YoshiharaT,KanochG,MorimotoA,ImashukuS.Adenoviralinfectioninhematopoieticstemcelltransplantation:earlydiagnosiswithquantitativedetection of the viral genome in serum and urine. Bone Marrow Transplant.2004;33:87‑92.

28.Vidal LR, Siqueira MM, Nogueira MB, Raboni SM, Pereira LA,Takahashi GR, et al.TheepidemiologyandantigeniccharacterizationofinfluenzavirusesisolatedinCuritiba,SouthBrazil.MemInstOswaldoCruz.2008;103:180‑5.

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Received:14July2010Accepted:8October2010







[email protected]


LaboratóriodeTaxonomiaeBiologiadeInvertebrados,DepartamentodeParasitologia,InstitutodeCiênciasBiológicas,UFMG.BeloHorizonte,MG,Brasil.Correspondenceto:Dr.AlanLanedeMelo,LaboratóriodeTaxonomiaeBiologiadeInvertebrados,DepartamentodeParasitologia,InstitutodeCiênciasBiológicas,UFMG.C.P.486,30123‑

970BeloHorizonte,MinasGerais,Brasil.E‑mail:[email protected]

Melanoides tuberculata AS INTERMEDIATE HOST OF Philophthalmus gralli IN BRAzIL

HudsonAlvesPINTO&AlanLanedeMELO

SUMMARY

Melanoides tuberculata thatnaturallyharboredtrematodelarvaewerecollectedatthePampulhadam,BeloHorizonte(MinasGerais,Brazil),duringmalacologicalsurveysconductedfrom2006to2010.From7,164specimensofM. tuberculatacollected,25(0.35%)wereinfectedbycercariae,whichhavebeenmorphologicallycharacterizedasbelongingtotheMegalurousgroup,genusPhilophthalmus.Excystedmetacercariaewereusedforsuccessfulexperimental infectionofGallus gallus domesticus, andadultparasitesrecoveredfromthenictitatingmembranesofchickenswereidentifiedasPhilophthalmus gralli.ThisisthefirstreportofP. gralliinM. tuberculatainBrazil.

KEYwORDS:Philophthalmus gralli;Melanoides tuberculata;Eyefluke;Brazil;Snailintermediatehost.

INTRODUCTION

Melanoides tuberculata (Müller, 1774), an exotic species ofsnail introduced in Brazil in the late 1960s35, has been found inseveral Brazilian states9. Studies related to the interaction betweenM. tuberculata and some species of Biomphalaria Preston, 1910,which transmit Schistosoma mansoni Sambon, 1907 in the countryhave reported that endemicpopulationsof planorbids coexistswiththese thiaridsdespite theirdisplacement12,13,32.Other studiesdidnotconfirmthecoexistenceofplanorbidswith thiarids,butverified thesignificantdeclineorcomplete disappearanceofBiomphalariaspeciesaftertheintroductionofthiarids14,15.However,thepossibleuseofM. tuberculataasabiologicalcontrolstrategyagainstschistosomiasismustbeconsideredwithcautionbecauseofpossibledamagetothenativefauna(asanalienspecies),andthepotentialofM. tuberculatatoactasanintermediatehostforparasitesofmedicalandveterinaryimportanceinBrazilarenotfullyunderstood.SomestudieshavealreadyreportedthefindingofM. tuberculatathatharborPleurolophocercouscercariaeinBrazil4,5,33.RecentlythiscercariantypefoundinM. tuberculatafromthestateofMinasGeraiswasidentifiedasCentrocestusformosanus (Nishigori,1924)28.

The aim of this study is to report the natural infection of M. tuberculata byanothertypeoftrematodelarvaeinBrazil,cercariaeofPhilophthalmusLooss,1899,whichhavebeenusedforexperimentallifecyclestudiesallowingthemorphologicalidentificationoftheorientaleyeflukeP. gralliMathisandLeger,1910.

MATERIALSANDMETHODS

Molluskswerecollectedduring26randommalacologicalsurveys

(overminimumintervalsofonemonth),conductedfrom2006to2010atPampulhadam,aneutrophicartificialwaterbodywithanareaof260hectaresandatotalwatervolumeof12millionm3locatedinthenorthernregionofthecityofBeloHorizonte,inthestateofMinasGerais,Brazil.The mollusks were obtained with a scoop net and long forceps, andwerepackedandtransportedtothelaboratory,thenplacedindividuallyinplasticreceptaclescontaining5mLoftapwaterandleftovernightatroomtemperature.Thethiaridswereexaminedwithastereomicroscopebefore and after artificial photostimulation. Emerged cercariae werestudiedundera lightmicroscopewithvitalstainswhilealive(0.05%neutralred,0.05%Nileblue,0.05%alizarinred),orafterbeingfixedin10%formalin,stainedwithaceticcarmine,clearedinbeechwoodcreosoteandmountedinCanadabalsam,accordingtoMELO(2008)22.Tostudyintramolluscanparasiticstages,naturallyinfectedsnailswerecrushedbetweentwoglassplatesanddissectedunderastereoscopicmicroscope;thelarvaefoundwerecollectedandstudiedalive.

Young specimens of Gallus gallus domesticus (Linnaeus, 1758)(n=5)wereorally administeredwith twentymechanically excystedmetacercariaeeach.Thechickensweresacrificedandnecropsiedatfourweeks after infection, according to the local animal experimentationethicscommittee(CETEA/UFMG).Ovigerousflukeswererecoveredfromthenictitatingmembranesandconjunctivalsacsofthechickensandwerepressedbetweenglassslides,fixedincold10%formalinandstainedandmountedasdescribedabove.

Measurementsofthedevelopmentalstagesobtainedwereperformedwithamillimeteredeyepiece.Tenlarvaespecimensand13adultparasitesweremeasured.Drawingsweremadeincameralucidaandphotographicdocumentationwasperformedwithadigitalcameraattachedtoalightmicroscope.Thecercariaewereidentifiedandcharacterizedaccordingto

PINTO,H.A.&MELO,A.L.de‑Melanoides tuberculataasintermediatehostofPhilophthalmus gralliinBrazil.Rev.Inst.Med.Trop.SaoPaulo,52(6):323‑7,2010.

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previouslypublisheddescriptions7,10,30,31,34.Adultparasiteswereidentifiedwiththeaidoftaxonomicalkeysandtheearlierdescriptionsofseveralauthors1,6,8,11,19,21,24,25,29.Measurementsaregiveninmicrometers(µm).

The specimens studied were deposited in the collection of theDepartmentofParasitology(DPIC),UFMG,underaccessionnumber5926a‑m;e5927.

RESULTS

In all, 7,164 specimens of M. tuberculata were collected andexamined.Adifferenttypeofcercariaemergedfrom25M. tuberculataspecimens(0.35%)whichwaspreliminarilycharacterizedasbelongingto the Megalurous group (Fig. 1a). Larvae tended to emerge duringthemorning,insmallnumbers,andwereveryactive.Theypresentedelongatedbodymeasurementsof,onaverage,535(420‑580)longby128(110‑140)widewithaconstrictionattheleveloftheventralsucker.Thesubterminaloralsuckeris58(50‑69)longby55(49‑65)wideandfollowedbyalongprepharynx,amuscularpharynxandanesophagusbifurcatingintotwoblindcaecathatreachtheposteriorendofthebody.Theventralsuckerwasequatorial,withacircumferenceaveraging68(65‑78)longby75(60‑80)wide.Thegenitalprimordiumwasformedbytwolongitudinalcellmassesdorsaltotheventralsucker.Numerouscystogenous cells were observed.The tail was simple and slender,averaging434(302‑485)longby54(36‑62)wide,withadhesiveglandsattheterminalregion.Thesecercariaeencystedrapidlyatthebottomofthereceptacleorontheshellofthemollusks.Metacercariaeusuallyhadatypicalpyriformshapeaveraging315(300‑380)longby210(184‑236)wide(Fig.1b).

Duringthedissectionofnaturallyinfectedsnails,matureandyoungrediae were found.Young rediae (Fig. 1c) were characterized by anelongatedbodythataveraged375(334‑457)longby78(68‑96)wideandamuscularpharynxthataveraged53(48‑63)longby45(38‑53)wide,withatail‑likeprocessattheposteriorendofthebody.Maturerediae(Fig.1d)werecharacterizedbyasac‑likeelongatedbodythataveraged946 (653‑1,203) long by 178 (155‑206) wide.A muscular pharynxmeasured73(63‑88)longby74(57‑75)wide.Dark‑coloredintestinalcaecumthatwereonaverage397(273‑512)longby50(27‑68)wide,extendedupthehalfofthebody.Thebirthporewaslocatedontheanteriorbody,belowthelevelofthepharynx.Apairofappendagesandtail‑likestructureswaspresentattheposteriorregion.Germballsandcercariaewereobservedatdifferentdevelopmentalstages.ThemorphologicalandbiologicalcharacteristicsoftheselarvaeandoftheintramolluscanstagesmakeitpossibletoidentifytheseMegalurouscercariaeasbelongingtothetrematodesofthegenusPhilophthalmus.

In the experimental infection, 23 adult parasites were obtainedfromthenictitatingmembranesandconjunctivalsacs(Fig.2a)ofallofthechickens,withameanintensityofinfection7(2‑12)parasites.Therecoveredspecimens(Fig.2d)haveanelongatedbody,thatisanaverageof3,610(3,100‑4,070)longby1,020(860‑1,210)wide.Theoralsuckersubterminalis,onaverage,330(316‑339)longby401(374‑421)wide.Theprepharynxisverysmallorabsent.Themuscularpharynxislocatedposteriortotheoralsucker,andisonaverage299(279‑316)longby339(326‑358)wide.Theratioofthetransversediameteroftheoralsuckertothatofthepharynxis1:0.5‑1.05(1:0.86).Theesophagusissmall,bifurcatingattheborderoftheventralsucker.Theventralsucker(acetabulum)islocatedontheanteriorthirdofbodyandisanaverageof

Fig.1 ‑Developmental stagesofPhilophthalmus gralli obtained fromnaturally infected

Melanoides tuberculata inBrazil.Cercaria(a),metacercaria(b),youngredia(c)andadult

redia(d).Scalebars=100µm.

Fig.2‑DevelopmentalstagesofPhilophthalmus gralliobtainedfromchickensexperimentally

infectedwithlarvaeemergedfromnaturallyinfectedMelanoides tuberculatafromBrazil.

Thesephotosfeatureparasitesintheconjunctivalsacofachicken(a),amatureegg(b),a

miracidium(c),anadultparasite(d).Scalebars=50µm(b,c);500µm(a,d).


325

514(479‑542)longby514(479‑542)wide.Theratioofthetransversediameteroftheoralsuckertothatoftheacetabulumis1:1.22(1:1.5).Thecirruspouchiselongated,averaging758(507‑1,000)by120(86‑464),withaseminalvesicleextendingposteriorlytotheventralsucker.Thetestesareovalandaresituatedintandem;thetestesarelocatedatthepositionpostovarian,intercecal,intheposteriorpartofbody.Theanteriortestismeasures,onaverage,222(179‑263)longby403(279‑453)wide.Posteriortestismeasures,onaverage,216(174‑247)longby351(268‑421)wide.Theshapeoftheovaryvariedfromroundtooval,andissituatedmediallyinapretesticularandpost‑uterineposition.Theovarymeasures237(211‑258)longby266(245‑300)wide.Thegenitalporeismediallylocatedattheesophagealbifurcation.Thelocationofthe uterus is post‑acetabular, pretesticular and intercecal (presentingmatureeggsthatcontainedmiracidiawitheyespots).Thevitellariaare

bilateral,extracecal,tubular,andextended87%(73‑97%)ofthedistancefromtheanteriortestistotheventralsucker.Matureeggs(Fig.2b)arenon‑operculatedandarelocatedatthedistaluterinecoils.Thematureeggswereanaveragesizeof135(120‑145)longby61(53‑65)whenfreshand73(60‑87)by32(27‑36)afterstaining.Themiracidium(Fig.2c)isanaveragesizeof131(92‑171)longby53(38‑62)wide,andhadapreformedrediastage.Themainexcretoryductsextendeduptheanteriorpartofthebody.Theexcretoryporeisterminal.

The morphometric data related to adult parasites obtainedexperimentally in the present study were compared with otherneotropical recordsofPhilophthalmus and the resultsarepresentedin theTable 1, represented as the amplitude followed by the meaninbrackets(inmicrometers)whenavailable.Themorphologicaland

Table1MeasurementsofPhilophthalmus gralliobtainedfromchickensthatwereexperimentallyinfectedwithtrematodeslarvaeemergedfromMelanoides tuberculatainBrazil,andcomparedwithSouthAmericanrecordsofPhilophthalmus.Morphometricdatainmicrometersarepresentedasamplitudefollowedbythemeanin

brackets.

Philophthalmus gralli Philophthalmus lachrymosus

Philophthalmus semipalmatus

Presentstudy Muniz‑PereiraandAmato,1993

Díazet al.,2002 Freitas,1955 Pintoet al.,2005 NasirandDíaz,1972

Locality BeloHorizonte, Maricá, AguasantaandYaguaracal

Manguinhos, FozdoIguaçu, LagunadelPeñon

MG,Brazil RJ,Brazil Venezuela RJ,Brazil PR,Brazil Venezuela

Host Gallus gallus Anas bahamensis Gallus gallus Casmerodius albus Hydrochaeris Catoptrophorus

domesticus Amazoneta brasiliensis

domesticus hydrochaeris semipalmatus

n 13 9 25 6 10 –

Body L 3100‑4070(3610) 2120‑3710(2710) 2564‑3384 4190‑4620 3400‑4250(3730) 2624‑4475

W 860‑1210(1020) 604‑1280(834) 512‑1205 1380‑1640 850‑1530(1080) 960‑1794

Oralsucker L 316‑339(330) 204‑329(260) 237‑297 300‑310 220‑290(260) 216‑363

W 374‑421(401) 277‑421(332) 287‑378 360‑430 260‑330(300) 253‑485

Ventralsucker L 479‑542(514) 343‑549(418) 388‑544 610‑690 610‑720(670) 917

W 479‑542(514) 343‑494(391) 409‑505 – 630‑770(700) 958

OS/VS 1‑1,22 1‑1,1‑1,3 1‑1,3 1‑2 1‑2 1‑2,75

Pharynx L 279‑316(299) 183‑293(226) 227‑323 310‑350 190‑210(200) 225‑333d

W 326‑358(339) 183‑403(277) 227‑333 380‑460 90‑150(110) –

Ovary L 211‑258(237) 88‑219(153) 126‑222 200‑210 150‑280(190) 188‑394d

W 245‑300(266) 88‑256(181) 151‑252 180‑220 150‑290(210) –

Anteriortestis L 179‑263(222) 161‑416(258) 272‑378 360‑480 140‑270(230) 188‑297

W 279‑453(403) 234‑445(329) 424‑530 460‑550 280‑460(370) 206‑563

Posteriortestis L 174‑247(216) 161‑438(248) 272‑464 380‑480 150‑360(280) 188‑454

W 268‑421(351) 241‑504(319) 404‑505 450‑530 280‑470(360) 216‑669

Eggs L 60‑87(73) 64‑90(70) 74‑80 94‑97 90‑110(100) 60‑69

W 27‑36(32) 22‑40(33) 25‑35 38‑42 21‑40(30) 18‑30

Typeofvitellaria tubular tubular tubular follicular follicular follicular

Extensionofvitellaria % 73‑97(87) 79‑89 – – 71‑90(78.6) –

d=diameterL=length;W=width


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biologicalcharacteristicsoftheparasitereportedheremadepossiblethe identification of the eyefluke Philophthalmus gralli Mathis andLeger,1910.

DISCUSSION

Most of trematodes of the family Philophthalmidae Looss, 1899are cosmopolitan eyeflukesofbirds andmammals, andhave alreadybeen reported to infect human beings20,26. In Brazil, two speciesof Philophthalmus have been reported only in vertebrate hosts.Philophthalmus lachrymosus Braun, 1902 was described in Rio deJaneiro (RJ, Brazil) in the brown‑hooded gull, Larus maculipennis(Lichtenstein,1823).Thespecieshasalsobeenfoundinthegreategret,Casmerodius albus egretta(Gmelin,1789) fromthesamestate11,aswellasthecapybara,Hydrochaeris hydrochaeris Linnaeus,1766inFozdoIguaçu(PR,Brazil)29.Anotherspecies,Philophthalmus gralliMathisandLeger,1910wasinitiallydescribedinGallus fromAsia,andhasalsobeenregisteredinthewhite‑cheekedpintail,Anas bahamensis Linnaeus,1758andintheBrazilianteal,Amazonetta brasiliensis Boetticher,1929inMaricá (RJ, Brazil)24.More recently, P. gralli have been found inostriches, Struthio camelus Linnaeus,1758inCaratinga(MG,Brazil)36.Despite these reports,mollusksnaturally infectedbyPhilophthalmus havestillnotbeenreportedinBrazil.

OriginallydescribedinG. gallus domesticusinVietnam,thebiologicalcycleofP. gralli hasprimarilybeenelucidatedinNorthAmerica,wherethedevelopmentalstagesandtheparticipationofthiaridsmollusksinitstransmissionhavebeendescribed1,2,3,6,37.Sincethen,severalstudieshaveconfirmedtheparticipationofM. tuberculataasanintermediatehostofP. gralliindifferentcountries,suchastheUSA27,Jordan17,18,Mexico31,theUnitedArabEmirates16,SaudiArabia19,Venezuela8andZimbabwe23.ThebiologicalandmorphologicalcharacteristicsofP. grallireportedhereareinaccordancewiththosedescribedbytheseauthors,differingfromP. lachrymosusandP. semipalmatus(NasirandDíaz,1972)mainlybytheirsmallerventralsucker,thetype(tubular),andthelongerlengthofvitellaria.

In Brazil, the possible involvement of M. tuberculata in thetransmissionofPhilophthalmus hasbeensuggested24,29,36,however,itsoccurrencehasnotbeenpreviouslyverifiedinnature.Inthepresentstudy,theparticipationofM. tuberculatainthebiologicalcycleofP. gralliinBrazilisconfirmed,butthenaturaldefinitivehostofP. gralliinPampulhadamremainsunknown.GiventhatM. tuberculataiswidespreadinBrazilandparticipatesinthelifecycleofPhilophthalmus inthecountry,theimpactofintroducingandspreadingthesethiaridsaroundthecountrymustbebetterevaluated,withanaimtowardpreventingfuturecasesofphilophthalmiasis.

RESUMO

Melanoides tuberculata comohospedeirointermediáriodePhilophthalmus grallinoBrasil

Melanoides tuberculata naturalmente infectados por larvas detrematódeosforamcoletadosnarepresadaPampulhaBeloHorizonte,MinasGerais,Brasilduranteestudosmalacológicosrealizadosentre2006e2010.De7164exemplaresdeM. tuberculatacoletados,25(0,35%)apresentavam‑se infectados por cercárias que foram caracterizadas

morfologicamente como pertencentes ao grupo Megalura, gêneroPhilophthalmus. Metacercárias desencistadas foram utilizadas comsucesso para a infecção experimental de Gallus gallus domesticus eparasitosadultosrecuperadosdamembrananictitantedasavesforamidentificadoscomoPhilophthalmus gralli.EsteéoprimeirorelatodeP. gralliemM. tuberculatanoBrasil.

ACKNOwLEDGMENTS

ToMrAirtonLobofortechnicalassistanceandtoConselhoNacionalde Desenvolvimento Científico eTecnológico (CNPq) for financialsupportandscholarshiptoHudsonAlvesPinto.

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gralli(Trematoda:Philophthalmidae)inVenezuela.RevBiolTrop.2002;50:629‑41. 9.FernandezMA,ThiengoSC,SimoneLRL.Distributionoftheintroducedfreshwatersnail

Melanoides tuberculatus(Mollusca;Thiaridae)inBrazil.Nautilus.2003;117:78‑82.10.Fisher FM, WestAF. Cercaria megalura Cort, 1914, the larva of a species of

Philophthalmus.JParasitol.1958;44:648.11.Freitas JFT. Sobre dois trematódeos parasitos de aves: Philophthalmus lachrymosus

Braun,1902eRenicola mirandaribeiroi n.sp.ArqMusNac.1955;42:585‑610.12.FreitasJR,SantosMBL.Currentadvancesonthestudyofsnail‑snailinteractions,with

specialemphasisoncompetitionprocess.MemInstOswaldoCruz.1995;90:261‑9.13.GiovanelliA,SilvaCLPAC,LealGBE,BaptistaDF.Habitatpreferenceoffreshwater

snailsinrelationtoenvironmentalfactorsandthepresenceofthecompetitorsnailMelanoides tuberculatus(Müller,1774).MemInstOswaldoCruz.2005;100:169‑76.

14.GiovanelliA,Vieira MV, Silva CLPAC. Interaction between the intermediate host

of schistosomiasis in Brazil, Biomphalaria glabrata (Say, 1818) and a possiblecompetitor,Melanoides tuberculata (Müller,1774): afield study. JMollusStud.2005;71:7‑13.

15.GuimarãesCT,SouzaCP,SoaresDM.Possiblecompetitivedisplacementofplanorbidsby

Melanoides tuberculatus inMinasGerais,Brazil.MemInstOswaldoCruz.2001;96(Suppl):173‑6.


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17. IsmailNS,IssaI.LifecycleofPhilophthalmus gralli (Trematoda:Philophthalmidae)in

AzraqOasis,Jordan.JpnJParasitol.1987;36:53‑62.18. IsmailNS,SalibaEK.StudiesonlarvalstagesofdigenetictrematodesofMelanoides

tuberculata (Muller)snailsfromAzraqoasis,Jordan.RivParassitol.1985;46:263‑71.19.KalantanAMN,ArfinM,Al‑ArefiHA,BobshaitHI,HamadahSA,Al‑ThawabFH,et al.

OccurrenceoflarvalPhilophthalmus gralli(MathisandLeger,1910)infreshwatersnail, Melanoides tuberculatus (Muller) fromAl‑Hafuf, SaudiArabia and itsdevelopmentintoadultinvariousexperimentalhosts.ParasitolInt.1997;46:127‑36.

20.KanevI,NollenPM,VassilevI,DimitrovV.RedescriptionofPhilophthalmus lucipetus

(Rudolphi,1819)(Trematoda:Philophthalmidae)withadiscussionofits identityandcharacteristics.AnnNatMusWien.1993;94/95B:11‑34.

21.KanevI,RadevV,FriedB.FamilyPhilophthalmidaeLooss,1899.In:JonesA,BrayR,

GibsonD,editors.KeystotheTrematoda. Wallingford:CABIInternational;2005.v.2,p.87‑97.

22.MeloAL.Caracterizaçãodelarvasdetrematódeosemergentesdemoluscosdulciaquícolas.

In:Amaral RS,Thiengo SC, Pieri OS, organizadores.Vigilância e controle demoluscosdeimportânciaepidemiológica:diretrizestécnicas:ProgramadeVigilânciaeControledaEsquistossomose(PCE).2.ed.Brasília:MinistériodaSaúde;2008.p.71‑80.

23.MukaratirwaS,HoveT,CindziZM,MaonongaDB,TaruvingaM,MatengaE.First

reportofafieldoutbreakoftheorientaleye‑fluke,Philophthalmus gralli(Mathis&Leger1910),incommerciallyrearedostriches(Struthio camelus)inZimbabwe.OnderstepoortJVetRes.2005;72:203‑6.

24.Muniz‑PereiraLC,AmatoSB.Philophthalmus gralli (Digenea:Philophthalmidae)parasite

ofAnas bahamensis andAmazonetta brasiliensis,fromlagoonsofMaricácounty,RiodeJaneiro,Brazil.MemInstOswaldoCruz.1993;88:567‑9.

25.NasirP,DíazMT.AvianflukesofVenezuela.RivParassitol.1972;33:245‑76.26.Nollen PM, Kanev I.The taxonomy and biology of philophthalmid eyeflukes.Adv

Parasitol.1995;36:205‑69.27.NollenPM,MurrayHD.Philophthalmus gralli:identification,growthcharacteristics,

andtreatmentofanorientaleyeflukeofbirdsintroducedintothecontinentalUnitedStates.JParasitol.1978;64:178‑80.

28.PintoHA,MeloAL.Melanoides tuberculata(Mollusca:Thiaridae)asanintermediatehostofCentrocestus formosanus (Trematoda:Heterophyidae) inBrazil.RevInstMedTropSaoPaulo.2010;52:207‑10.

29.PintoRM,SantosLC,TortellyR,MenezesRC,MoraesW,JuvenalJC,et al.Pathology

andfirstreportofnaturalinfectionsoftheeyetrematodePhilophthalmus lachrymosusBraun,1902(Digenea,Philophthalmidae)inanon‑humanmammalianhost.MemInstOswaldoCruz.2005;100:579‑83.

30.RadevV, Kanev I, Gold D. Life cycle and identification of an eyefluke from Israel

transmittedbyMelanoides tuberculata (Müller,1774).JParasitol.2000;86:773‑6.31.ScholzT,Aguirre‑MacedoML,DiazdeLeonATSF,DitrichO.Larvalstagesoftrematodes

inMexicanfreshwatermollusc:areviewofpresentstateandmethodologyforfutureresearch.In:Salgado‑MaldonadoG,García‑AldreteAN,Vidal‑MartínezVM,editors.Metazoanparasitesintheneotropic:asystematicandecologicalperspective.México:InstitutodeBiología,UniversidadNacionalAutónomadeMéxico;2000.p.77‑100.

32.SilvaRE,MeloAL,PereiraLH,FredericoLF.Levantamentomalacológicodabacia

hidrográficadolagoSoledade,OuroBranco(MinasGerais,Brasil).RevInstMedTropSaoPaulo.1994;36:437‑44.

33.ThiengoSC,FernandezMA,BoaventuraMF,GraultCE,SilvaHFR,MattosAC,et al.

FreshwatersnailsandschistosomiasismansoniinthestateofRiodeJaneiro,Brazil:I‑MetropolitanMesoregion.MemInstOswaldoCruz.2001;96(Suppl):177‑84.

34.Urabe M. Cercariae of a species of Philophthalmus detected in a freshwater snail,

Semisulcospira libertina,inJapan.ParasitolInt.2005;54:55‑7.35.VazJF,TelesHMS,CorreaMA,LeiteSPS.OcorrêncianoBrasildeThiara (Melanoides)

tuberculata(O.F.Müller,1774)(Gastropoda,Prosobranchia),primeirohospedeirointermediáriodeClonorchis sinensis.RevSaúdePública.1986;20:318‑22.

36.VerocaiGG,LopesLN,BurliniL,CorreiaTR,SouzaCP,CoumendourosK.Occurrence

ofPhilophthalmus gralli(Trematoda:Philophthalmidae)infarmedostrichesinBrazil.TropAnimHealthProd.2009;41:1241‑2.

37.WestAF. Studies on the biology of Philophthalmus gralli Mathis and Leger 1910

(Trematoda:Digenea).AmMidlNat.1961;66:363‑83.





Telephone:55113061‑7003‑Fax:55113062‑2174





(1)DepartmentofInfectiousDiseasesofHospitalNossaSenhoradaConceição,PortoAlegre,RS,Brazil.(2)DepartmentofInternalMedicineofHospitalNossaSenhoradaConceição,PortoAlegre,RS,Brazil.(3)DepartmentofPathologyofHospitalNossaSenhoradaConceição,PortoAlegre,RS,Brazil.(4)DepartmentofCommunityHealthandInfectiousDiseasesofHospitaldeClinicas,UniversidadeFederaldoParaná,Curitiba,PR,Brazil.Correspondenceto:VicenteSperbAntonello,MD,HospitalFêmina,ServiçodeControledeInfecçãoHospitalar,RuaMostardeiro17,91430‑001PortoAlegre,RioGrandedoSul,Brasil.

E‑mail:[email protected]

CASE REPORT

TREATMENT OF SEVERE CHROMOBLASTOMyCOSIS wITH ITRACONAzOLE AND 5-FLUCyTOSINE ASSOCIATION

VicenteSperbANTONELLO(1),MarceloCamposAPPELDASILVA(2),EduardoCAMBRUzzI(3),DimasAlexandreKLIEMANN(1),BrenoRiegelSANTOS(1)&FlávioQUEIROz‑TELLES(4)

SUMMARY

Chromoblastomycosisisachronichumanmelanizedfungiinfectionofthesubcutaneoustissuecausedbytraumaticinoculationofaspecificgroupofdematiaceousfungithroughtheskin,oftenfoundinbarefootedagriculturalworkers,intropicalandsubtropicalclimatecountries.Wereport thecaseofamalepatientpresentingaslow‑growingpruriginous lesionon the limbs for20years,mistreatedoverthattime,whichwasdiagnosedandsuccessfullytreatedaschromoblastomycosis.Besidestheprevalenceofthisdisease,treatmentisstillaclinicalchallenge.

KEYwORDS:Chromoblastomycosis;Itraconazole;5‑Flucytosine;Subcutaneousmycoses.

INTRODUCTION

Chromoblastomycosis(CBM)isachronichumanmelanizedfungiinfectionofsubcutaneoustissuecausedbytraumaticinoculationofaspecificgroupofdematiaceous fungi (usuallyFonsecaea pedrosoi orCladophialophora carrionii)throughtheskin1,14.Ithasbeendescribedworldwide, but it is most commonly seen in tropical or subtropicalclimates18. Itmaybeencounteredas anoccupational‑relateddisease,mainlyinmalebarefooted‑agriculturalworkers21.

Differential diagnoses may include infectious diseases, such asblastomycosis, paracoccidioidomycosis, leishmaniasis and verrucosetuberculosis, and non‑infectious disorders, as sarcoidosis andpsoriasis13,18.WereportapatientfromthestateofRioGrandedoSulwith CBM diagnosed by histology, and who responded successfullytooralitraconazole(ITZ)and5‑flucytosine(5‑FC)combinedtherapy.

CASEREPORT

A55‑year‑oldmaleagriculturalworkerpresentedwitha20‑year‑old,slow‑growing,pruriginousanderythematousplaquewithwell‑definedbordersandcoveredby“blackdots”,extendingfromtheleftankletotheleftmidthigh(Fig.1‑A).Hismedicalhistorywasremarkableonlyfor thediagnosisof insulin‑dependentdiabetesmellitussincehewas40yearsold.Severalointmentshadbeenused to treat thecondition,aswellasoralITZ,fluconazole(FCZ)andterbinafine(TBF),withno

major improvement. No fungal growth was obtained after culture ofbiopsyspecimens.Melanizedfungicellswereseeninhematoxylinandeosin tissue sections as shown in Fig. 1‑B. Clinical and histologicalfindings were compatible with the diagnosis of severe CBM, withpresenceofclusteredmuriformcells.Thediseaseseverity,accordingtoQUEIROZ‑TELLESet al. scorewassevere18.TreatmentwithoralITZ400mgdailyand5‑FC2gramsq.i.d.was initiated.Within twomonths,dramaticclinicalimprovementwasobserved,andbytheendof12monthscompleteclinicalhealingofthelesionwasachieved(Fig.2).Follow‑upofthelaboratorialexamswereperformedeverythreemonthsandincludedbloodcellcount,creatinineandliverfunctionpanels,andshowednoalterationsuntilthelastconsultation(Table1).

DISCUSSION

CBMisatherapeuticchallengeforwhichthereisnotreatmentofchoice.Severaldrugoptionsare suggested,basedon reportsofnon‑comparativestudiesandcaseseries,ratherthanrandomizedcontrolledtrials7,11,18.Treatmentmaydependontheetiologicalagent,sizeandextentofthelesions,thepatient’sindividualtolerance,statusoftheimmunesystemandeconomicfeatures11,butisoftenassociatedwithlowcureratesandhigh relapse rates18.Besides that, clinical,mycologicalandhistopathologicaldatacanbeusedtoguideappropriateantifungaldrugchoice.

Smalllesionsintheearlystagescanbetreatedwithsurgicalresection

ANTONELLO,V.S.;APPELDASILVA,M.C.;CAMBRUZZI,E.;KLIEMANN,D.A.;SANTOS,B.R.&QUEIROZ‑TELLES,F.‑Treatmentofseverechromoblastomycosiswithitraconazoleand5‑flucytosineassociation.Rev.Inst.Med.Trop.SaoPaulo,52(6):329‑31,2010.

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withcurettageanddesiccation.Treatmentswithcarbondioxidelaser,cryotherapy and topical heat alsohavebeen reported10,11,18.However,recurrencesarecommon.Moderateandsevereforms,withwidespreadlesions, usually require systemic treatment.While amphotericin B,thiabendazole, 5‑FC and ketoconazole are variably effective in thiscondition,ITZandTBFdemonstratedthebestresultsathighdosesfor6‑12months1,7,14,21.However,acureisdifficulttoachieve,andprolongedremissionisanacceptableoutcome.Giventhehighrelapserateofthedisease,therapeuticcombinationsmayincreasethecurerate18.Inourpatient,systemicantifungal treatmentwithITZassociatedwith5‑FCwasusedfor12monthswithexcellentresponse.

Due to the lack of new antifungal compounds, in the 1970s thecombination of antifungal drugs was considered for treatment ofsubcutaneousmycoses.5‑FC‑apyrimidinederivative,withmainactionininhibitingthesynthesisofnucleicacidsofthefungalcells,activein vitro and in vivo againstyeasts (Candida albicans andCryptococcus neoformans,Aspergillus sp.anddematiaceousfungi)3,17,23‑wastheonlydrugthathadnoactivitydirectlytothefungalmembraneandthatcouldbecombinedwithotherantifungalsubstances5,15,16,20.

Based on the additive effect observed in vitro, BOPP introducedin1976thecombinationofintravenousamphotericinBwith5‑FCinthe treatmentofCBM8.Thiscombinationprovedtobeadvantageous

Fig.1‑A.Erythematousplaquewithwell‑definedbordersandcoveredby“blackdots”;B.Melanizedfungicellsinhematoxilin‑eosintissuesections.

Fig.2‑Completeclinicalhealingofthelesionafter12monthsoftreatmentwithItraconazole

and5‑flucytosine.

Table1Laboratorialexamsofthechromoblastomycosispatient

Exams\Date 08/28/09 10/19/09 12/17/09 04/06/10 06/18/10 10/04/10

RBC* 4,79mil 4,74mil 4,84mil 4,92mil 4,75mil 5,22mil

Hb** 12,5 13,3 13,6 13,7 13,3 13,9

Leucocytes 8550 6350 7180 8070 8170 10330

Creatinine 1,06 1,12 1,2 1,19 1,11 1,07

TGP 12 23 18 17 14 19

TGO 13 17 20 19 20 18*RBC=Redbloodcells;**Hb=Hemoglobin.

ANTONELLO,V.S.;APPELDASILVA,M.C.;CAMBRUZZI,E.;KLIEMANN,D.A.;SANTOS,B.R.&QUEIROZ‑TELLES,F.‑Treatmentofseverechromoblastomycosiswithitraconazoleand5‑flucytosineassociation.Rev.Inst.Med.Trop.SaoPaulo,52(6):329‑31,2010.

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over monotherapy with 5‑FC12. However, the related nephrotoxicityof amphotericin B and prolonged period of hospitalization for drugadministrationhavemotivatedthesearchforlesstoxicregimens.Theassociationoftwooralcompounds,5‑FCandthiabendazole,wastriedwithenthusiasticresults2,22.

The combination of ITZ and 5‑FC, although assessed in a smallnumber of patients, was very effective even in severe forms ofsubcutaneousmycoses6,9,19.Pharmacologicaldataonantifungaldrugsdemonstratedanadditiveeffectagainstfungi‑where5‑FCactsbycausingsuppressionoftheyeast’sDNAsynthesisandITZactingonthefungi´scytoplasmaticmembrane‑byinhibitingthesynthesisofergosterol,animportantsubstanceforfungalgrowth4.Despiteaninsufficientnumberofcasestocomposeadetailedcomparison,thecombinedtherapyofITZand5‑FCmaybeanexcellentoptionforsevereorunresponsivecasesofCBMaftermonotherapywithITZorterbinafine.Duetothedifficultyinacquiring5‑FCinBrazilatpresent,theaccomplishmentofacomparativestudyinvolvingalargernumberofpatientsismissing.

Finally,itisimportanttoconsiderthattreatmentsuccessdependsalsoonanearlydiagnosisandthechoiceoftheappropriateantifungalagent.

RESUMO

Tratamentodecromoblastomicoseseveracomaassociaçãoitraconazolee5‑flucitosina

Cromoblastomicose é uma infecção fúngica crônica do tecidosubcutâneocausadapelainoculaçãotraumáticadeumgrupoespecíficodefungosatravésdapele,encontradoseventualmenteemtrabalhadoresdo campo descalços em países de clima tropical e subtropical.Relatamos aqui o caso de um paciente do sexo masculino com umalesãodermatológicadecrescimentolentoepruriginosanosmembrosinferiores por 20 anos, diagnosticada e tratada com sucesso paracromoblastomicose.Apesardaprevalênciadestadoençaemnossaregião,otratamentoaindaéumdesafio.

CONFLICTSOFINTEREST

Nothingtoreport.

REFERENCES

1.Ameen M. Chromoblastomycosis: clinical presentation and management. Clin ExpDermatol.2009;34:849‑54.

2.Bayles MAH. Chromomycosis. In: Bailliere’s clinical tropical medicine andcommunicablediseases.Tropicalfungalinfections. London: BailliereTindall;1989.p.45‑70.

3.BennetJE.Flucytosine.AnnInternMed.1977;86:319‑21.

4.BennetJE.Antifungalagents.In:BruntonLL,LazoJS,ParkerKL,editors.Goodman&Gilman´s:thepharmacologicalbasisoftherapeutics.11thed.NewYork:McGraw‑Hill;2005.p.1225‑40.

5.BlockER,BennettJE.Thecombinedeffectof5‑fluorocytosineandamphotericinBinthetherapyofmurinecryptococcosis.ProcSocExpBiolMed.1973;142:476‑80.

6.BolzingerT,PradinaudR,Sainte‑MarieD,DupontB,ChwetzoffE.Traitementdequatrecas de chromomycose à Fonsecaea pedrosoi par l’association 5‑fluorocytosine‑itraconazole.NouvDermatol.1991;10:462‑6.

7.BonifazA, Paredes‑SolisV, SaulA.Treating chromoblastomycosis with systemicantifungals.ExpertOpinPharmacother.2004;5:247‑54.

8.BoppC.Therapyofchromoblastomycosiswithanewmethod.MedCutanIberoLatAm.1976;4:285‑92.

9.Borelli D.A clinical trial of itraconazole in the treatment of deep mycoses andleishmaniasis.RevInfectDis.1987;9(Supl.1):S57‑S63.

10.CastroLG,PimentelER,LacazCS.Treatmentofchromomycosisbycryosurgerywithliquidnitrogen:15years’experience.IntJDermatol2003;42:408‑12.

11.Garnica M, Nucci M, Queiroz‑Telles F. Difficult mycoses of the skin: advances inthe epidemiology and management of eumycetoma, phaeohyphomycosis andchromoblastomycosis.CurrOpinInfectDis.2009;22:559‑63.

12.LopesCF,ResendeMA,AlvarengaRJ,MoreiraYK.Associaçãode5‑fluorocitosinaeanfotericinaBnotratamentodacromomicose.MedCutanIberoLatAm.1979:7:1‑7.

13.MartínezRL,TovarLJM.Chromoblastomycosis.ClinDermatol.2007;25:188‑94.

14.MatteSMW,LopesJO,MeloIS,EspadimLER,PintoMS.CromoblastomicosenoRioGrandedoSul:relatode12casos.RevSocBrasMedTrop.1997;30:309‑11.

15.MedoffG,KobayashiGS,KwanCN,SchlessingerD,VenkovP.Potentiationofrifampicinand5‑fluorocytosineasantifungalantibioticsbyamphotericinB(yeast‑membranepermeability‑ribosomalRNA‑eukaryoticcell‑synergism).ProcNatlAcadSci USA.1972;69:196‑9.

16.PolakAM.Determinationdelasynergieentrela5‑fluorocytosineetl’amphotericineBaumycosesdedifferentesmodèlesin vitro etin vivo.BullSocFrançMycol Med..1974;3:175‑8.

17.PolakA,ScholerHJ.Modeofactionof5‑fluorocytosineandmechanismsofresistance.Chemotherapy.1975;21:113‑30.

18.Queiroz‑Telles F, Esterre P, Perez‑Blanco M,Vitale RG, Salgado CG, BonifazA.Chromoblastomycosis: an overview of clinical manifestations, diagnosis andtreatment.MedMycol.2009;47:3‑15.

19.Queiroz‑TellesF;PurimKS,FillusJN,BordignonGF,LameiraRP,VanCutsemJ, et al.ItraconazoleinthetreatmentofchromoblastomycosisduetoFonsecaea pedrosoi.IntJDermatol.1992;31:805‑12.

20.ResendeMA.AçãosinérgicadaanfotericinaBe5‑fluorocitosinaemFonsecaea pedrosoi.RevInstMedTropSaoPaulo. 1979;21:344‑6.

21.SilvaACCM,SaraNetoA,GalvãoCES,MarquesSG,SaldanhaACR,PedrosoeSilvaCMP,et al..CromoblastomicoseproduzidaporFonsecaea pedrosoinoEstadodoMaranhão.I‑Aspectosclínicos,epidemiológicoseevolutivos.RevSocBrasMedTrop.1992;25:37‑44.

22.SolanoAE,HidalgoHH,CastroAC,Montero‑GeiF.Tratamientodelacromoblastomicoseconlaassociaciónthiabendazoley5‑fluorocitosina,seisanosdeseguimento.MedCutIberoLatAm.1983;11:413‑8.

23.VandeveldeAG,MauceriAA,JohnsonIIIJE.5‑fluorocytosineinthetreatmentofmycoticinfections.AnnIntMed.1972;77:43‑51.








[email protected]


(1)HospitalUniversitárioJoãodeBarrosBarreto,UniversidadeFederaldoPará,Belém,Pará,Brasil.(2)LaboratóriodeHerpetologia,CoordenaçãodeZoologia,MuseuParaenseEmílioGoeldi,Belém,Pará,Brasil.(3)InstitutoButantan,SecretariadeEstadodaSaúdedeSãoPaulo,SãoPaulo,SP,Brasil.Correspondenceto:PedroPereiradeOliveiraPardal.HospitalUniversitárioJoãodeBarrosBarreto/CentrodeInformaçõesToxicológicas.RuadosMundurucus,4487.Guamá.66073‑000

Belém,Pará,Brasil.Tel.:55‑913249.6370.E‑mail:[email protected]

ENVENOMATION By Micrurus CORAL SNAKES IN THE BRAzILIAN AMAzON REgION: REPORT OF TwO CASES

PedroPereiradeOliveiraPARDAL(1),JoseanaSilvadeOliveiraPARDAL(1),MariaApolôniadaCostaGADELHA(1),LíliamdaSilvaRODRIGUES(1),DarlanTavaresFEITOSA(2),AnaLúciadaCostaPRUDENTE(2)&HuiwenFAN(3)

SUMMARY

TwocasesofprovencoralsnakebiteswerereportedinBelém,ParáState,Brazil.ThefirstcasewasasevereonecausedbyMicrurus surinamensis.Thepatientrequiredmechanicalventilationduetoacuterespiratoryfailure.ThesecondcaseshowedjustmildsignsofenvenomationcausedbyMicrurus filiformis.BothpatientsreceivedspecificMicrurusantivenomandweredischargedwithoutfurthercomplications.CoralsnakebitesarescarcelyreportedintheAmazonregionandthereisabroadspectrumofclinicalmanifestations,varyingfromextremelymildtothosewhichmayrapidlyleadtodeathifthepatientisnottreatedassoonaspossible.

KEYwORDS:Envenomation;Coralsnakes;Micrurussurinamensis;M. filiformis;BrazilianAmazon.

INTRODUCTION

Coral snakes are the main representatives of the Elapidae in theAmericas, the genus Micrurus being the most important in terms ofpublichealth4,6.Duetotheirlargegeographicaldistribution,coralsnakesmaybefoundindiverseenvironments6,13.InBrazil,severalspeciesofMicrurusarefoundthroughoutthewholecountryandagreatnumberofthemliveinthelowlandrainforestortropicaljungle6.TheAmazonregionharborsthelargestnumberofspeciesofcoralsnakes,suchasMicrurus spixiiandM. lemniscatus,andtheonlyspeciescloselyassociatedwithwaterenvironments,theaquaticcoralsnake,M. surinamensis6,22.ThesespecieslivesympatricallyintheAmazonbasin31.Coralsnakesarelargelyfossorialandseldomseen,eveninverydenseareas.

Incontrasttoviperids,thefangsofcoralsnakesareshort,hollowstructuresthatarepermanentlyfixedinpositionontheanteriormaxillarybones,whichisafeatureofproteroglyphousdentition.Theshortandsmall‑sized fangs usually represent low risk to individuals wearingfootwearandclothing.Mostcoralsnakebitesinhumanbeingsoccuronthehandsandusuallyinvolveasnakethatwasintentionallypickedupandhandled.ThismayexplainthelownumberofMicrurussnakebitesrecorded.In2007,of22,763snakebitesreportedinBrazil,136wereattributedtothegenusMicrurusandwerewithoutfatalities5.MostcaseswerereportedintheSouthernregion,whereM. corallinusispredominant,11occurredinParáState.

StudiesonMicrurusspecifictoxinsarelimitedduetothefactthatthese animals are difficult to capture and keep in captivity, and are

knowntoproduceonlyasmallamountofvenom.ThegenusMicruruspossesses a potent neurotoxic venom, which causes postsynapticblockageof neuromuscular transmissionbybinding competitively tothe acetylcholine receptors38.Thevenomof M. corallinus alsohas aneurotoxinwithpresynapticactivity.Theseneurotoxinsmainlycausecranialnerveparalysisinenvenomedindividuals,leadingtoso‑calledmyasthenicorneurotoxicfacies.

Thefirstsignofperipheralnervoussysteminvolvementisusuallyptosis,often followedbyophthalmoplegia,dysarthriaanddysphagia.Thedescendentprogressionofenvenomingmayleadtodyspnea.Deathcanbeaconsequenceofmuscleparalysisandrespiratoryarrest.SpecificantivenomisproducedbytheimmunizationofhorseswithM. corallinusand M. frontalis venoms. For other species, including the Micrurusspeciesof theAmazonregion,studieson theirmechanismsofactionandtheneutralizingeffectsofantivenomsarerarelyavailable2.ClinicaldescriptionsofpatientsbittenbyMicrurusarestillmuchneededandthereforewedescribetwocasesinwhichtheseverityofenvenomationvariedconsiderably.

CASEREPORTS

Case 1:An 18 year‑old male, Biology student, was reportedlywalkingonauniversitycampusbelongingto theUniversityofRuralAmazon,Belém,ParáState,whenhefoundan80‑cmcoralsnake(Fig.1).Inanattempttocapturetheanimal,hewasbittenontheleftthumb.Immediately,thepatientpulledthesnakeoffsotheanimaldidnotholdontothesiteofthebite.Onlyonefangmarkwasvisible.Hedidnot

PARDAL,P.P.O.;PARDAL,J.S.O.;GADELHA,M.A.C.;RODRIGUES,L.S.;FEITOSA,D.T.;PRUDENTE,A.L.C.&FAN,H.W.‑EnvenomationbyMicruruscoralsnakesintheBrazilianAmazonregion:reportoftwocases.Rev.Inst.Med.Trop.SaoPaulo,52(6):333‑7,2010.

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complainofhavinganypainbuthementionedparesthesiaatthebitesitethatwentupalongthewholelimbafewminutesaftertheaccident.Hehadbeensenttothereferencehospital,JoãodeBarrosBarretoUniversityHospital,wherehearrivedwithin20minutesafterthebitetookplace,bringing with him a M. surinamensis coral snake. Upon arrival, hecomplainedofblurredvisionanddifficulty inspeaking,walkingandopeninghiseyelids.Onexamination,hewasinrespiratorydistress(40mpm),foamingatthenoseandmouth,consciousbutunabletospeak.Hewasnotcyanosedorinshock.Thepulserateandbloodpressurewerenormal(100/80mmHg).HewasintubatedandtransferredtotheIntensiveCareUnit(ICU)wherehewasimmediatelyputonaventilator,followedby IV administration of neostigmine methylsulfate (0.5mL = 5 mg),precededbyatropinesulfate(1mL=0.25mg),and100mLofMicrurus

antivenom,producedbytheButantanInstitute(SãoPaulo)inampoulesof10mLcontaining:F(ab’)2horseantibodies,1mLneutralizingatleast10mgofM. frontalisreference‑venominmice.IntheICUthepatientwasfoundtobeunconscious.Thechestwasclearandheartsoundswerenormal.Initiallaboratorystudieswerenormal(Table1).Approximately12hrsafterthedevelopmentofneurologicalsymptoms,thepatientwasalert and following commands with a normal physical examination.Neostigmineadministrationdidnotapparentlyreverseanyneurologicalmanifestation.Thepatientcouldbeweanedfromartificialventilation48hoursafterantivenominfusion.Nofurthermedicationwasrequiredandthepatientwasdischargedinagoodconditiononday3.

Case2:A19year‑oldmalewashandlingacoloredsnakewhich

Fig.1‑Micrurus surinamensis,specimenresponsibleforsevereenvenoming(Case1).

Table1LaboratorialdatafromapatientbittenbyMicrurus surinamensis(Case1)

TestDate

11/April/2006(beforeAVtherapy*)

12/April/2006(24hrsafterAVtherapy)

13/April/2006(withoutartificialventilation)

Arterialbloodgasanalysis

pH 7.36 7.51 7.41

pCO2(mmHg) 32 26 40

pO2(mmHg) 379 198 283

HCO3(mEq/L) 18 21 25

TotalCO2(mmol/L) 19 ‑‑ 26

BE(mmol/L) ‑6 +1

Std.BE ‑6 ‑1 +1

Std.BC 20 ‑‑ 25

Sat.O2(%) 100 100 100

FiO2(%) 40 50 50

Hemoglobin(g/dL) 13.2 ‑‑ ‑‑

Totalleukocytes(count/mm3) 18,500 ‑‑ ‑‑

Platelets(count/mm3) 230,000 ‑‑ ‑‑*Patientwasalreadyonartificialventilation.

Fig.2‑Micrurus filiformis,specimenresponsibleformildenvenoming(Case2).


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hadbeencapturedthedaybeforeinthevillageofBoaVistaintheAcarámunicipality,ParáState.Hereportedthathehadbeenbittenonaleft‑handfingerandcomplainedofmildpain.Hestatedthatapproximatelyone hour after the accident he noticed a mild swelling on the bittenlimbandcomplainedofepigastricpain.Hementionedtwoepisodesofvomiting.Uponphysicalexaminationeverythingseemednormalapartfromasmallpuncturewoundonthefourthfingerofthelefthandwithverymildedema.Hehadnoblurredvision,ptosisorothersymptomofneurotoxicity.Hewasgiven100mLofMicrurusantivenomandfullyrecoveredwhileinthehospital.Hewasdischarged24hoursaftertheantivenom therapywithonlymildedemaon thehand,and remainedasymptomatic.Thesnakewasidentifiedasa50‑cmspecimenofMicrurus filiformis (Fig.2)bytheHerpetologyLaboratoryoftheEmílioGoeldiMuseum,Belém,ParáState.

DISCUSSION

Two cases of human Micrurus envenoming are here presented,onecausedbyM. surinamensisandtheotherbyM. filiformis,whichshow the potential for severe envenoming and the variability of theclinical manifestations. In Brazil, Micrurus envenoming is relativelyrareandmostcasesaredue toM. corallinus,M. lemniscatusandM. frontalis6,9,20,32,39.

Althoughbroadlydistributedacrossthewholecountry,thenumberofMicrurusaccidentsisnegligible,whencomparedtocasesinvolvingBothropsandCrotalussnakes4.Therarityofhumanaccidentsisattributedtothetypeofdentitionandenvenomingwhichwouldresultfromabiteonasmallpartofthebody,suchasfingers,whichenablesthesnaketoholdonandtoinoculateitsvenomintotissuesbymeansofachewingmotion25.Contrarytothiscommonview,Case1illustratesthecapabilityofthecoralsnakeindeliveringapotentiallyseverebitewithouthavingto“chew”onthevictimforalongperiodoftime.Thus,envenomingshould be diagnosed by the presence of the signs and symptoms ofneurotoxicity.Fangmarksmayormaynotbeevidentandwhentheyarepresent,theyareusuallyslightandmaybeseenasscratchmarks.Eventheabsenceofvisiblefangmarksdoesnotprecludethepossibilityofabitewithvenominoculation25.

Micrurusvenomsareknowntopossessneurotoxicproperties37.Inmostsymptomaticcasesneuromuscularparalysisisthemostprominentandiscausedbypostsynapticmotorend‑plateblockageofacetylcholinereceptors,whichproducessimilareffectstothoseseeninmyastheniagravis18andcurarepoisoning24.Thispreventsnervesfromstimulatingmusclecontractionandleadstoparalysis.SimilaractionsaredescribedforotherElapidaevenoms,suchasNajaandBungarusspp14,19.

Thetwocasesreportedherepresenteddifferentclinicalaspects.Thefirstprogressedrapidlytorespiratorydistresswhiletheothershowedonly mild edema without any neurological manifestations up to 24hrsafterbite.Thisbroadspectrumofclinicalmanifestationsinsnakebites is generally known to occur. Cases of patients bitten by elapidsnakesthatdidnotreceiveantivenomanddidnotpresentneurologicalmanifestationshavebeenreportedandcharacterizedasasymptomaticordry‑bites6.ItisimportanttopointoutthattherearefewstudiesavailabletounderstandtheexactmechanismofactionofMicrurusvenoms,andsome variation in their composition may be present among differentspecies.MostexperimentalstudiesinvolveM. frontalisandM. corallinus

venoms,sincethesetwospeciesarethemostfrequentcauseofMicrurusenvenoming inBrazil33.M. surinamensisvenom isknown tocontainpostsynaptic toxins33.OneexperimentalstudywithMicrurusvenomsfromtheAmazon,includingM. spixii,M. averyi,M. lemniscatusandM. surinamensis,showednocoagulantactivitybutedematogeniceffects,except for M. surinamensis2. Another study evaluated the biologicalandenzymaticactivitiesofM. surinamensisvenombutnotthoseofM. filiformis8.

Antivenomadministrationisrecommendedasthemostefficacioustreatment for envenoming by coral snakes6,17. Specific Micrurusantivenoms,producedinBrazilbytheButantanInstitute(SãoPaulo)andtheEzequielDiasFoundation(MinasGerais),areproducedinhorsesimmunizedwiththevenomsofM. corallinusandM. frontalis29.However,somestudiessuggestthattheneutralizingcapacityofantivenomsmaybe improvedby includingvenomfromotherMicrurus species in thevenompoolusedintheimmunizationprotocol1,10,15,36.

The assumption that commercial antivenomsmaybeused in thetreatment of bites by any Brazilian Micrurus species, even for otherSouthAmerican coral snakes contrasts with the scarcity of clinicalstudies on envenoming by Micrurus coral snakes, which are limitedto case reports3,6,21,27,40. Unusual symptoms of envenoming by M. lemniscatushavebeendescribed21inwhichthepatienthadseverepaininthebittenlimb,despiterepeateddosesofpotentanalgesics,andnoresponse to anti‑cholinesterase or specific antivenoms was observed,either.Incontrast,ourpatientshadnointenselocalpain,buttheCase2patientpresentedmildlocalswellingandpain,whichisinaccordancetoexperimentalreportsthathaveshownthatsomeMicrurusvenomsmayinducemyotoxicityandlocallesions2.

ReportsonElapidae snakebites inAustraliahavesuggested thatpressureimmobilizationbandagingoftheaffectedlimbcouldimpedetheegressoftoxinsfromthesiteofthebiteanddelaytheonsetoflife‑threateningsystemicinvolvement23,35.Ontheotherhand,experimentalstudieshavesuggestedthatattainingtherecommendedpressurelevelsistechnicallyverydifficultandtheeffectiveuseofthismethodmightrequireextensive trainingandexperience26.Becauseof thisand timewastageconcerns,inBrazilemphasisisgiventoimprovingantivenomsupplyanddistribution,andinstructionsarethatallvictimsshouldbetransportedtothenearestmedicaltreatmentfacilityassoonaspossible.

Neurologic signs and symptoms of envenoming can be apparentinminutes,orbedelayedforaslongas12hours.InCase1,therapidonset of clinicalmanifestations indicates the severity of envenomingand the necessity of prompt intervention, not only with antivenom,butalsowithintensivecaresupportivemeasures.Moreover,improvedmethodsofreversingtheeffectsofneurotoxicityareneeded.Evenwhenadministered early, antivenoms did not promptly reverse paralysis.Improvement inneurological signswasnot seenuntil 24hours afterantivenomadministration.Ancillarytreatmenthasbeenrecommendedwithanti‑cholinesterasedrugs;EdrophoniumChloridecouldbenefitthosepatientsbittenbyacoralsnakewithpostsynapticneurotoxicvenom41,andneostigminehaddemonstratedthepotentialtoreverseneuroparalysis9,39.Evenwhenusedinseveraloccasionsinclinicalpractice,nostandardprotocolhasbeenfollowed.Ourpatient,forinstance,hadreceivedahalfdoseofthatusedinotherstudies5.ThefailureofneostigmineinreversingneurotoxicityinCase1maybepartiallyattributedtoaninsufficientdose.


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On the other hand, the very mild symptoms observed in Case 2suggestthatcoralsnakebitesarenotnecessarilyalwayssevere.Thereisnogenerallyacceptedclinicalclassificationforseverityofaccidents,andanempiricaldoseof10ampoulesofantivenomisusuallygiven,regardlessofseverity.Areviewoftheclassificationofseverityisthusneededtoadjusttheamountofantivenomtotherealnecessityincaseof mild, moderate or severe envenoming, as exists for Bothrops andCrotalusenvenoming.

CoralsnakesarewidelydistributedinalmostallmajorecologicalregionsoftheNewWorld,andagreatnumberliveinthelowlandrainforest,particularlyintheAmazonregionofBrazil22.Thisareaharborsthelargestnumberofspeciesofcoralsnakes,suchasMicrurus spixiiandM. lemniscatus,aswellastheonlyspeciescloselyassociatedwithwaterenvironments,theaquaticcoralsnake,M.surinamensis30.FromalltheMicrurusspp.thatarefoundinBrazilianAmazon,M. surinamensisisoneofthelargestingirthandlength,reachingupto1,350mm6.Itinhabitshumidtropicalforestsandsecondaryforests,nearwaterflowsoftheAmazonasandOrinocoRivers,intheNorthernandCentralpartsofSouthAmerica11,12,14,30.Aproteomicanalysisofthevenomofthefish‑eatingM. surinamensisprovidesanoverviewofitsproteincomposition,thespecificpharmacologicalfeaturesofwhichdifferfromotherMicrurusspecies28.Micrurus filiformisisalsobroadlydistributedintheAmazonbasinandmaybefoundinsecondaryforests,lowaltitudemountains,openfields,andhumidforests7,16,30.

Ingeneral,coralsnakesarenotabundantinnature,thusthescarcityofspecimensisonereasonwhytheyarenotwellknown,andtheirelusivehabitsareakeyfortheirsuccessinatleastpartiallyavoidingdecimationbyhumans.But,ascolorfulanddistinctivesnakes,theysometimesappealtohumanstobehandledandobserved.Withtheincreasingspreadofthehabitofkeepingsnakesaspets,itisimportanttoidentifyelapidsanddistinguishthemfromfalsecoralsnakes,andalsotospreadinformationaboutfirstaidandtreatment.

CONFLICTOFINTEREST

Theauthorsofthisstudyarenotpartofanyassociationnordotheyhavecommercialrelationshipsthatmightrepresentconflictsofinterestinthewritingofthisstudy

RESUMO

EnvenenamentoporcoraldogêneroMicrurus naAmazôniabrasileira:relatodedoiscasos

Dois acidentes por coral verdadeira são descritos em Belém,Pará.OprimeirocasofoidecorrentedeenvenenamentoporMicrurus surinamensis, no qual a vítima necessitou ventilação mecânica porinsuficiênciarespiratória.Osegundo,causadoporMicrurus filiformis,apresentouapenasmanifestaçõesleves.Ambosospacientesreceberamsoroantielapídicoespecíficoeevoluíramsemcomplicações.Acidentespor coral verdadeira na regiãoAmazônica são raramente descritos epodemcursarcomumlargoespectrodealterações,quevariamdesdequadrosdeenvenenamentomuitolevesatémanifestaçõescomriscodeóbito.

ACKNOwLEDGMENTS

The authors would like to thank all the health professionals ofHospitalUniversitárioJoãodeBarrosBarreto,whocontributedtothequalityofpatients’medicalassistance.TheauthorsarealsogratefultoDr.ReginaDórea,forkindlyreviewingthearticle.

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20.MachadoJC,RosenfeldG.Achadosanátomo‑patológicosemnecroscopiadepacientefalecidoporenvenenamentoelapídico.MemInstButantan.1970;35:41‑53.

21.ManockSR,SuarezG,GrahamD,Avila‑AgueroML,WarrellDA.NeurotoxicenvenomingbySouthAmericancoral snake (Micrurus lemniscatus helleri): case report fromeasternEcuadorandreview.TransRSocTropMedHyg.2008;102:1127‑32.

22.MelgarejoAR.SerpentespeçonhentasdoBrasil. In:Cardoso JLC,FrançaFOS,FanHW,MálaqueCMS,HaddadJrV.AnimaispeçonhentosnoBrasil.Biologia,clínicaeterapêuticadosacidentes.SãoPaulo:Sarvier;2003.p.33‑61.

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36.TanakaGD,FurtadoMFD,PortaroFCV,Sant’AnnaAO,TambourgiDV.DiversityofMicrurussnakespeciesrelatedtotheirvenomtoxiceffectsandtheprospectiveofantivenomneutralization.PLOSNeglTropDis.2010;4:e622.

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38.VitalBrazilO,FontanaMD.Açõespré‑juncionaisepós‑juncionaisdapeçonhadacobracoral Micrurus corallinus na junção neuromuscular. Mem Inst Butantan. 1983‑4;47/48:13‑26.

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Received:26November2007Accepted:8October2010




Telephone:55113061‑7003‑Fax:55113062‑2174





AdolfoLutzInstitute,VirologyCenter,EntericDiseasesLaboratory,SãoPaulo,SP,Brazil.Correspondenceto:AdrianaLuchs,MariadoCarmoSampaioTavaresTimenetsky,InstitutoAdolfoLutz,Virologia,AvDrArnaldo355,CerqueiraCésar,01246‑902SãoPaulo,SP,Brasil.

Tel.:+551130682909;fax:+551130853505.E‑mails:[email protected];[email protected]

BRIEF COMMUNICATION

EVALUATION OF ENTEROVIRUS 71 IMMUNE STATUS IN SÃO PAULO STATE, BRAzIL

AdrianaLUCHS,AudreyCILLI,DeniseH.RUSSO, FernandaF.COSTA, RitadeCássiaC.CARMONA&MariadoCarmoS.T.TIMENETSKY

SUMMARY

AntibodiestoEnterovirus71(EV71)wereevaluatedinSãoPauloStateduring1999‑2005.ThetiterofneutralizingantibodiesagainstEV71wasdeterminedbymicroneutralizationassay,andatiterof≥1:8wasdefinedasindicativeofprotectedimmunity.NeutralizingantibodiestoEV71wereobservedin12.4%(55/442)ofserasamples,alowprotectiverate,suggestingthatEV71infectionisuncommoninthisregion,butthatthereisarelativelyhighsusceptibilitytoEV71relateddiseases,whichisworryingconsideringtherecentAsianoutbreaks.Also,asignificantlocation‑specificdifferenceinseropositivitywasobserved.NeutralizingantibodiestoEV71wereobservedin8.7%(21/241)ofSãoPaulometropolitanareaserasamples,and16.9%(34/201)oftheserasamplesfromothermunicipalities.AhighnumberofBrazilianresidentsliveincountryandcoastalareaswithoutadequateaccesstopipedwaterorsanitation.ThissituationmaycontributetotheEV71disseminationinthesezones.TheanalysisofenvironmentalsamplescouldpossiblymakeavaluablecontributiontostudiesontheepidemiologyofEV71.

KEYwORDS:Enterovirus71;Neutralizingantibodies;Immunestatus.

Enterovirus 71 (EV71) belongs to the enterovirus genus of thePicornaviridaefamily,anditsinfectionsaremanifestedmostfrequentlyasamildexanthemaknownashand,footandmouthdisease(HFMD)5.Otherwise, among the enteroviruses, (except polioviruses), EV71infections are the most important type because they are frequentlycomplicatedbyneurologicdiseases,includingencephalitis,meningitisandepidemicpoliomyelitis‑likesyndromes,thathavegeneratedinterestandsubsequentpublichealthimplications9.

EV71hasemergedasasignificantpathogenwithpotentialtocauselargeoutbreaks.Thelast10‑13yearshaveseenashiftinthefrequencyofreportedEV71associatedwithencephalitisfatalities,inparticularduringoutbreaksinMalasyain1997andTaiwanin19981.SeveralsmallerscaleoutbreakshavebeenalsorecordedinWesternAustralia,Korea,JapanandSingapore1.During2006,thereoccurredmultiplereportsoflarge‑scaleHFMDoutbreaksinIndia,Thailand,HongKong,Malaysia,Brunei1,and,morerecently,inChina,in200820.However,EV71activityisnotjustconfinedtoSouthEastAsia.During1983‑2005,atotalof270casesofEV71werereportedinUnitedStates,anditappearedamongthe15mostcommonserotypesduringthe2000‑2005period11.InBrazil,prospectivestudiesshowedthatEV71wasthecauseof6%ofacutepoliomyelitis‑likediseaseand5%ofGuillain‑Barrésyndrome4,19.

TheserumneutralizingantibodyresponseisthemajorindicatorofEV71 infection andprotective immunity. InBrazil, few studieshavebeen published on the detection of neutralizing antibodies to EV71;

in particular there is concern about its involvement in acute flaccidparalysis(AFP)afterpolioviruseradication,whichisfoundconcentratedinthecityofBelém,intheNorthernregionofthecountry2,7.AslimitedinformationisavailableaboutthelevelofimmunityagainstEV71inSãoPauloState,inthecountry´sSoutheasternregion‑especiallyaftertheAsianoutbreaks‑serologicaldatafrom1999‑2005arepresentedinthisstudy.PreviousEthicsCommitteeapprovalwasgrantedbytheAdolfoLutzInstitute(Ref.12∕05).Thiswasananonymousunlinkedstudyandinformedconsentwasnotrequired.

SerumsamplesfrompatientswithsuspectedenterovirusinfectionsareroutinelysenttotheEntericDiseasesLaboratoryoftheAdolfoLutzInstitute,thesolefacilityresponsibleforEnterovirussurveillanceinSãoPauloState,inordertoconductviralinvestigation.ThelevelofimmunityagainstEV71wasinvestigatedinatotalof442randomlyselectedserasamples,obtainedfromaconvenientretrospectivesampling.Eachserumrepresentsone individual,andnoconsecutivesampleswereobtainedfromthesameindividualindifferentyears.

Theindividualsweredividedinthefollowingagestrata(inyears):0‑5(n =155;35.1%),preschoolandkindergartenchildren(themainlygroup affected by severe EV71 infections); 6‑15 (n = 103; 23.3%),school‑agedchildrenandthoseofthemaximumagethatcarriesthroughAFPmonitoring;and>15(n =184;41.6%),adultindividuals.Inthiswork, two panels of clinical samples were used.The first panel wasof 241 serum samples from eight different municipalities in the São

LUCHS,A.;CILLI,A.;RUSSO,D.H.;COSTA,F.F.;CARMONA,R.C.C.&TIMENETSKY,M.C.S.T.‑EvaluationofEnterovirus71immunestatusinSãoPauloState,Brazil.Rev.Inst.Med.Trop.SaoPaulo,52(6):339‑41,2010.

340

Paulo metropolitan area (Diadema, Guarulhos, Itapecerica da Serra,MogidasCruzes,Osasco,SantaIsabel,SãoBernardodoCampoandSãoPaulo),andthesecondpanelwascomposedof201serumsamplesfrom35distinctSãoPauloStatemunicipalities,includingcountrysideandcoastalareas.

ThetiterofneutralizingantibodiesagainstEV71wasdeterminedbymicroneutralizationassay.Seraweredilutedtwo‑fold,from1:8to1:1024,intriplicate,andeachdilutionwasincubatedfortwohat36°Cwith a 100 × 50% cell culture infectious dose of EV71/ BrCr strain(PAHO‑WHO).Thevirus‑serummixtureswereaddedtoRDcells(humanrhabdomyosarcoma‑ATCC‑CCL‑136),andaftera5‑dayincubationat36°C,thecytopathiceffectwasassessedbyphasecontrastmicroscopy.Atiterof≥1:8wasdefinedasindicativeofprotectedimmunity,anddatawereanalyzedbyEpiInfoversion3.4.3(CDC,Atlanta,GA,USA).

Overall, 12.4% (55/442; 95% IC 9.0‑15.0%) of tested sampleshad neutralizing antibodies to EV71, and 87.6% (387/442; 95% IC83.9‑90.1%)werenegative.AdifferentscenariohasbeenreportedinGermany in two distinct studies: (i) a non‑age‑specific evaluation ofantibodiesintheyearsof1997‑2007withaseroprevalenceof60.3%5,and(ii)anage‑adjustedevaluationofindividuals≥oneyearduring2006with a seroprevalence of 42.8%15. InTaiwan, a seroepidemiologicalstudyperformedin1997(pre‑epidemic),showedratesofabout60‑70%inadultsandchildrenolderthansixyearsofage3;andaseropositiverateof50‑60%inpregnantwomeninresultsfrom2006to2008(post‑epidemic)13.InChina,EV71antibodiesweredetectedin44.2%ofhealthychildrenagedbetween1‑6in20058.

A significant location‑specific difference in seropositivity wasobserved: 8.7% (21/241; 95% IC 5.1‑12.3%) of the São Paulometropolitanareasamples,and16.9%(34/201;95%IC11.8‑22.2%)ofthesamplesfromothermunicipalitiesshowedneutralizingantibodiestoEV71(p=0.014).Enterovirusarecommonlyacquiredbyfecal‑oralcontamination, therefore numerous variables may be associated withEnterovirusseroprevalenceincludingsourcesofdrinkingwater,sewagesystems(orlackthereof),socioeconomicfactors,andbehaviorrelatingtohygiene.Infact,severalEnterovirusinfectionsandviralgastroenteritisoutbreakshavebeenmicrobiologicallylinkedtocontaminatedmunicipalwatersupplies6,17,includinginBrazil16.

ThemetropolitanregionofSãoPaulohasrelativelyhighcoveragelevelsforwatersupplythroughhouseconnections(98.4%)andadequatesanitation(81.2%),whileahighnumberofBrazilianresidentsliveincountry and coastal areas without adequate access to piped water orsanitation10.Thissituationmaycontribute to theEV71disseminationin these zones. Similar data were displayed in an age‑specific studyof seroprevalence of hepatitisA conducted in centralTunisia, where21.3%ofschoolchildrenlivingintheurbanareasand87.7%ofthoselivinginruralareashadantibodiestohepatitisA12.Itisapublichealthpriority to improvewater quality and sanitation coverage.Moreover,albeitindirectly,theanalysisofenvironmentalsamplescouldpossiblymakeavaluablecontributiontostudiesontheepidemiologyofEV71.

Nocorrelationbetweenagegroupsandseropositivitywereobserved.There was no significant difference in EV71 immunity in preschoolchildrenaged0‑5years(11.0%;17/155;95%CI6.1‑15.9%),schoolingchildrenaged6‑15years(14.6%;15/103;95%CI7.3‑20.8%),oradults

>15years(12.5%;23/184;95%CI7.3‑16.7%).Asimilarpicturehasbeen reported inTaiwanandGermany. InTaiwan,during1996‑1997(thepre‑epidemicperiod),inaserosurveywhere≈12%ofthechildren<fiveyearshadantibodiestoEV71,thisseroprevalencereached≈50%in childrenaged5‑12years14. InGermany,during2006, therewas asignificantincreaseintheEV71seroprevalencefrom12%amongonetofouryears‑oldsto49%amongfivetonineyears‑olds15.InanotherstudyconductedinGermany,theseroprevalenceincreasedwithage,butrangedfrom36.6%inpreschoolchildrenaged0‑6years,to67.2%and75%inthoseaged10‑15yearsandadults,respectively5.

In Brazil, two studies among children ≤ 15 years of age wereconducted in the city of Belém.The first study, conducted in 1998,showedthat59.2%ofthechildrentestedpositivetoEV71antibodies,althoughtheseropositivitydecreasedwithagefrom45.4%inchildren<threeyearsto10.9%inchildrenaged12‑15years7.Thesecondstudy,carriedoutbetween1998and2001,presented57.1%ofseropositivitytoEV71,butonly20.2%forchildren<threeyears2.

Toanalyzetheimmunitylevel,threeneutralizingtiterrangesweredefined:1:8‑1:16 (low level),1:32‑1:128 (medium level), and1:256‑1:1024(highlevel).Theanalysisof theimmunitylevel inrelationtotheageoftheindividualsshowedthatthenumberofserawithmediumneutralizingantibodieslevelstoEV71washighestinthe0‑5yearagegroup(4.5%;7/155),followedbythe>15(1.6%;3/185),andthe6‑15(1.0%;1/103)agegroups.ThelowimmunityleveltoEV71increasedwith age from 6.4% (10/155) in children aged 0‑5 years to 12.6%(13/103)inchildrenaged6‑15years,butdeclinedto10.8%(20/185)inindividualsover15years.NohighneutralizingantibodieslevelstoEV71wereobserved.Similardatawasdescribed inGermany,whereEV71seropositiveindividualsgenerallyshowedlowerneutralizingantibodiestiters when compared to CoxsackieA 1615.This same study showedthat thenumberof individualswithhigh levelsofEV71neutralizingantibodies declined with age, suggesting that the reinfection of theelderlyisrare;otherwise,thenumberofindividualswithhighlevelsofneutralizingantibodiesshouldincreasewithage15.

TheEV71seropositivityshowedadifferentfrequencyforeachyear:14.3% (3/21) in1999;30.0% (24/80) in2000;4.0% (2/50) in2001;4.1%(3/73)in2002;11.0%(11/100)in2003;9.2%(10/109)in2004;and22.2%(2/9)in2005.Unfortunately,thenumberofseraineachyearwasflimsy,impairingthestatisticalanalysis.Inaddition,caserecordsofEV71infectionarenotsufficientlydetailedtolookfortemporaltrendsinoccurrenceofEV71diseasesacrossthecountry,andlaboratoryrecordsofserodiagnosedEV71aretoofewtoaidinterpretation.

The serologicaldataobtained in this study showeda low rateofprotectiveEV71antibodiesinthepopulationofSãoPaulo,suggestingthatEV71infectionisuncommoninthisregion,andarelativelyhighsusceptibilityoftheinhabitantstoEV71relateddiseases.Inaccordancewith the serological data obtained in this study, EV71 isolations inBrazil were very low during the 1999‑2005 period11,18, as well as inprevious times4,19. It is worth mentioning that our results correspondto442randomlyselectedsamplesofsera,obtainedfromaconvenientretrospective sampling from the Enteric Diseases Laboratory, andpresentingdifferentsymptomatologies.Therefore,thenumberofseraineachagegroupand/oryearmaybefairlysmall,andthestudydoesnot represent the prevalence in the general population. It is possible

LUCHS,A.;CILLI,A.;RUSSO,D.H.;COSTA,F.F.;CARMONA,R.C.C.&TIMENETSKY,M.C.S.T.‑EvaluationofEnterovirus71immunestatusinSãoPauloState,Brazil.Rev.Inst.Med.Trop.SaoPaulo,52(6):339‑41,2010.

341

thatabroadersamplesizewouldberequiredtoconfirmthesefindings.

The transmission of strains between countries is made easier byglobalization,andthatwouldfavoranEV71infectionincreaseinmanycountries in thenearfuture,as therearenoeffectivesteps topreventits transmission, nor good therapeuticmeasures to control its deadlycomplications.TheonlymeansofpreventingandcontrollingthespreadofEV71duringoutbreaksisbypublichealthsurveillance.Currently,there isnovaccineavailable for thisviral infection21.SeveralstudieshaveshownthatinfantsofpreschoolageareatthehighestriskofsevereEV71infection13,andthetransmissionofthisvirusislowerinotheragegroups14.ThehighlypathogenicpotentialofEV71‑inadditiontoitseasilytransmittablenature‑arethecausesofaverycommonpediatricdisease21.Thesefactstogethersuggestthatpublichealthmeasuresshouldfocusonthepopulationmostatrisk.

EventhoughtheimportanceofEV71hasalreadybeendefined,morestudiesarerequiredinordertoimprovetheknowledgeofseveralkeyaspectsrelatedtothisvirusinBrazil.

RESUMO

AvaliaçãodasituaçãoimunitáriacontraEnterovírus71noEstadodeSãoPaulo,Brasil

AnticorposparaEnterovírus71(EV71)foramavaliadosnoEstadodeSãoPaulodurante1999‑2005.OtítulodeanticorposneutralizantescontraEV71foideterminadopeloensaiomicroneutralização,eumtítulode≥1:8foidefinidocomoindicadordeimunidadeprotetora.Anticorposneutralizantes para EV71 foram observados em 12,4% (55/442) dasamostrasdesoro,umabaixataxadeproteção,sugerindoqueainfecçãopeloEV71éincomumnestaregiãoequeexistealtasusceptibilidadeadoençasrelacionadasaoEV71,oqueépreocupanteconsiderandoosrecentessurtosasiáticos.Ainda,foiobservadadiferençasignificativanasoropositividadeemrelaçãoàlocalização,onde8,7%(21/241)e16,9%(34/201) das amostras provenientes da região metropolitana de SãoPaulo,edemaismunicípios,respectivamente,apresentaramanticorposneutralizantesparaEV71.Umgrandenúmerodebrasileirosviveemáreas ruraiseàbeira‑mar, semacessoadequadoàáguaencanadaousaneamento. Essa situação pode contribuir para a disseminação deEV71nessasregiões.AanálisedeamostrasambientaispoderiagerarcontribuiçãovaliosaparaestudossobreaepidemiologiadaEV71.

AUTHORS’CONTRIBUTIONS

ALandMCSTTconceivedanddesignedthestudy;AL,AC,DHRandFFCperformedthemicroneutralizationassay;AL,ACandDHRanalyzedthedata;ALwrotethemanuscript;RCCCconductedlaboratorysupervision.Allauthorscontributedtothepreparationandrevisionofthemanuscript,readandapprovedthefinalversion.ALandMCSTTareguarantorsofthepaper.

REFERENCES

1.Bible JM,PantelidisP,ChanPKS,TongCYW.Geneticevolutionof enterovirus71:epidemiologicalandpathologicalimplications.RevMedVirol.2007;17:371‑9.

2.Castro CMO, CruzACR, Silva EE, Gomes MLC. Molecular and seroepidemiologystudiesofenterovirus71infectioninthestateofPará,Brazil.RevInstMedTropSPaulo.2005;47:65‑71.

3.ChangLY.Enterovirus71inTaiwan.PediatrNeonatol.2008;49:103‑12.

4.Da Silva EE,Winkler MT, Pallansch MA. Role of enterovirus 71 in acute flaccidparalysisaftereradicationofpoliovirusinBrazil.EmergInfectDis.1996;2:231‑3.

5.Diedrich S,WeinbrechtA, Schreier E. Seroprevalence and molecular epidemiologyofenteroviris71inGermany.ArchVirol.2009;154:1139‑42.

6.Ehlers MM, GrabowWO, Pavlov DN. Detection of enteroviruses in untreated andtreateddrinkingwatersuppliesinSouthAfrica.WaterRes.2005;39:2253‑8.

7.Gomes MLC, De Castro CMO, Oliveira MJ, Da Silva EE. Neutralizing antibodiestoenterovirus71inBelém,Brazil.MemInstOswaldoCruz.2002;97:47‑9.

8.Guo XB, Zhu SL,Wang DY. Seroepidemiology investigation of HEV71 in healthychildrenof1‑6yearsoldinthreecountiesofChinain2005.ZhongguoJiHuaMianYi.2009;15:141‑4.

9.Hsiung GD,Wang JR. Enterovirus infections with special reference to enterovirus71.JMicrobiolImmunolInfect.2000;33:1‑8.

10.IBGE [website]. Instituto Brasileiro de Geografia e Estatística. http://www.ibge.gov.br/home/estatistica/populacao/trabalhoerendimento/pnad2009/pnad_sintese_2009.pdf.Access:20/09/2010.

11.Khetsuriani N, Lamonte‑FowlkesA, Oberst S, Pallansch MA; Centers for DiseaseControlandPrevention.Enterovirussurveillance‑‑UnitedStates,1970‑2005.MMWRSurveillSumm.2006;55:1‑20.

12.LetaiefA,KaabiaN,GahaR,BousaadiaA,LazragF,TrabelsiH,et al.Age‑specificseroprevalenceofhepatitisAamongschoolchildrenincentralTunisia.AmJTropMedHyg.2005;73:40‑3.

13.LuoST,ChiangPS,ChaoAS,LiouGY,LinR,LinTV,et al.Enterovirus71maternalantibodiesininfants,Taiwan.EmergInfectDis.2009;15:581‑4.

14.OoiEE,PhoonMC,IshakB,ChanSH.Seroepidemiologyofhumanenterovirus71,Singapore.EmergInfectDis.2002;8:995‑7.

15.Rabenau HF, Richter M, Doerr HW. Hand, foot and mouth disease: seroprevalenceof CoxsackieA16 and Enterovirus 71 in Germany. Med Microbiol Immunol.2010;199:45‑51.

16.Rigotto C,Victoria M, MorescoV, Kolesnikovas CK, CorrêaAA, Souza DS, et al.Assessmentofadenovirus,hepatitisAvirusandrotaviruspresenceinenvironmentalsamplesinFlorianopolis,SouthBrazil.JApplMicrobiol.2010;109:1979‑87.

17.ScarcellaC,CarasiS,CadoriaF,MacchiL,PavanA,SalamanaM,et al.Anoutbreakofviralgastroenteritislinkedtomunicipalwatersupply,Lombardy,Italy,June2009.EuroSurveill.2009;14(29):pii:19274.

18.Silva HR,Tanajura GM,Tavares‑Neto J, Gomes MLC, LinharesAC,VasconcelosPFC,et al.SíndromedameningiteassépticaporenterovíruseLeptospira spemcriançasdeSalvador,Bahia.RevSocBrasMedTrop.2002;35:159‑65.

19.TakimotoS,WaldmanEA,MoreiraRC,KokF,PinheiroFP,SaesSG,et al.Enterovirus71infectionandacuteneurologicaldiseaseamongchildreninBrazil(1988‑1990).TransRSocTropMedHyg.1998;92:25‑8.

20.YangF,RenL,XiongZ,LiJ,XiaoY,ZhaoR,et al.Enterovirus71outbreakinthepeople´sRepublicofChinain2008.JClinMicrobiol.2009;47:2351‑2.

21.Xu J, QianY,Wang S, Serrano JMG, LiW, Huang Z, et al. EV71: an emerginginfectiousdiseasevaccinetargetintheFarEast?Vaccine.2010;28:3516‑21.

Received:14August2010Accepted:8October2010







[email protected]

343

AUTHOR INDEX - VOLUME 52

ABANTO,E.P.,31ABRAHAMSOHN,I.A.,83ABRANTES,T.R.,193ACARDI,S.A.,187AKINTONWA,A.,43ALBUQUERQUE,M.C.M.,3ALENCAR,J.,17ALMEIDA,M.F.de,75ALMEIDA,S.M.de,317ALMEIDASILVA,C.,305ÁLVAREZ,C.,269ALVES,M.F.,297ALVES,S.H.,161AMATONETO,V.,171AMORIM,R.M.S.,57ANDRADEFILHO,J.S.,288ANDRADEJr.,H.F.de,291ÂNGELO,A.M.,129ANTONELLO,V.S.,111,329APPELDASILVA,M.C.,329AQUINO,J.L.B.,113AQUINO,R.T.R.,243ARAUJO,M.R.E.,133ARAÚJO,P.R.B.,63ARAÚJO,S.M.,69AWODELE,O.,43BARROS‑MAZON,S.de,225BASTOS,R.P.,83BELONE,A.F.F.,273BERTOLINI,D.A.,25BONOLDI,V.L.N.,297BORRASCA,V.L.,133BURATTINI,M.N.,203CABRERA,R.,157,269CÁCERES,A.G.,269CAMARGO,L.M.A.,203CAMBRUZZI,E.,329CAMPOS,M.B.,259CAPILLA,J.,145CARLI,M.S.,151CARMONA,R.C.C.,339CARNEIRO,L.A.,259CARNEIRO,L.B.,273CARVALHO,M.E.de,151CAVERO,Y.A.,31,37CESARETLI,Y.,215CHAGAS,E.J.,259CHIEFFI,P.P.,243CHUQUIPIONDO,G.,269CILLI,A.,339COELHO,K.I.R.,151COKER,H.A.B.,43COLAÚTO,C.,125COLLI,C.M.,69COLTURATO,V.R.,281CORBETT,C.E.P.,203,259CORDEIRO,Q.,203CORREA,I.A.,51CORTEZ,J.,169CORTI,M.,279

COSTA,C.R.,139COSTA,F.F.,339COSTA,R.A.,129COSTA,S.F.,133COSTA,S.S.,3CRUZ,C.R.,317CUNHA,E.M.S.,231DALLEGRAVE,E.,267DAUAR,R.F.,285DEBORTHOLISANTOS,E.,285DEBUR,M.C.,317DELGRANDE,J.C.,113DIAS,D.F.,225DIAZ,S.Y.,151DISANTI,S.M.,281DORTA,M.L.,83DUARTE,A.M.R.C.,11DUARTE,L.S.,203EDUARDO,J.M.,211EDUARDO,M.B.P.,183ELKHOURI,M.,203ENDO,S.,107ESPINOZA,Y.A.,31,37EVANGELISTA,M.S.N.,237FALAVIGNA,D.L.M.,69FALAVIGNA‑GUILHERME,A.L.,69FAN,H.W.,333FARAH,J.F.M.,113FEITOSA,D.T.,333FERNANDES,O.F.L.,139FERREIRA,A.W.,63FERREIRA,E.C.,69FIGUEIREDO,F.B.,193FINK,M.C.D.S.,305FRANÇA,J.,111FUC,G.,253GADELHA,M.A.C.,333GAITÉN,Y.G.,197GAMA,A.R.da,75GARCIAPARRA,M.,89GATTÁS,V.L.,175GAZZONI,A.F.,145GELLI,D.S.,183GERHARDT,J.,285GESTINARI,L.M.S.,3GIL‑SANTANA,H.,17GONÇALVES,A.,311GONZÁLEZ,M.E.,169,221GORZONI,M.L.,163GOUVEIA,E.A.,297GRAEFF‑TEIXEIRA,C.,267GRIGALIUNAS,R.,183GUARRO,J.,145GUILHERME,E.V.,69GUIMARÃES,A.E.,17GUIMARÃES,M.C.A.,101,211GUTIÉRREZ,C.A.,31,37HADDADJr.,V.,47,51HEINS‑VACCARI,E.M.,161HENRY,M.A.C.A.,113

HERBELLA,F.A.M.,113HERNÁNDEZ,M.,169,221HUAPAYA,P.E.,31INCANI,R.N.,169,221INOUE,J.,281ITO,F.H.,231ITOU,T.,231JAKABI,M.,183JIMÉNEZ,S.,31,37KAWANO,T.,101KLIEMANN,D.A.,329KOBAYASHI,Y.,231LADERA,P.,269LAINSON,R.,259LARA,M.C.C.S.H.,231LAURENTI,M.D.,259LEITE,R.M.,151LEMOS,M.F.,25LEVIN,A.S.,133LEVIN,M.J.,115LIMA,G.F.M.C.,281LIMA,G.M.C.A.,83LIMA,L.V.R.,259LIMA,M.A.,11LIMAVERDE,E.M.,253LIMAVERDE,F.A.,253LIMAVERDE,F.A.A.,253LIZAMA,R.S.,197LOPES,G.I.S.L.,183LOPREATOFILHO,R.,183LUCENA,P.A.,139LUCHS,A.,339LUNA,E.J.A.,175MACHADO,C.M.,281MACHARIA,J.C.,95MADEIRA,M.F.,193MANGUEIRA,C.P.,119MANTELLINI,G.G.,311MANTOVANI,E.,297MARIO,D.A.N.,161MARTÍNEZ,J.M.,197MARTINS,C.R.F.,57MARTINS,F.O.,3MARTINS,H.S.,203MARTINS,R.M.B.,57MASSAIA,I.F.D.S.,163MATTAR,R.,125MATTIA,S.,69MAYAYO,E.,145MEIRELES,L.R.,291MELO,A.L.de,207,323MENDES,G.S.,3MENDIOLAMARTINEZ,J.,89MENTZ,M.B.,267MIGOTTO,A.E.,47MONTALVOALVAREZ,A.M.,89MONTEIRO,M.doS.,125MONZOTEFIDALGO,L.,89MOURÃO,J.A.,129MOUTA‑CONFORT,E.,193

344

MUNIZ,C.,101MURTA,M.,17MUTISO,J.M.,95MUTISYA,R.M.,95NASCIMENTO,F.S.,225NASCIMENTO,L.D.,193NASCIMENTO,M.J.C.,281NASELLO,A.G.,243NASSAR,A.F.C.,231NATAL,D.,101NEGRONI,R.,279NETO,A.S.,253NEVES,F.A.R.,57NOGUEIRA,M.B.,317NUNURA,J.,107OHLWEILER,F.P.,101,211OLIVEIRA,F.M.,145OLIVEIRA,L.C.M.de,247OLIVEIRA,M.A.P.,83OSUNKALU,V.O.,43OYAFUSO,L.K.,297OZKAN,O.,215PADOVANI,C.R.,311PAES,A.,119PALMIERI,O.,279PALUDO,M.L.,69PANNUTI,C.S.,305PANTANALI,C.A.R.,113PARDAL,J.S.O.,333PARDAL,P.P.O.,333PASQUALOTTI,M.A.,243PASSOS,S.R.L.,193PASSOS,X.S.,139PAULA,A.M.R.de,183PAUVOLID‑CORRÊA,A.,17PAYROL,J.A.,197PELLEGRIN,A.O.,17PELLEGRINO,D.,285PENA,G.P.,288PENALVADEOLIVEIRA,A.C.,285,305PEREIRA,A.J.C.S.,83PEREIRA,L.A.,317PEREIRA,L.I.A.,83PEREYRA,S.,107PINEDO,R.,269PINHO,J.R.R.,25,119PINTO,H.A.,207,323PINTO,S.A.,83PIRES,A.S.,83PIRES,S.L.,163PONTES,J.P.J.,247PORFÍRIO,F.M.V.,285

PREVIDELLI,I.T.S.,69PRUDENTE,A.L.C.,333QUEIROZ‑TELLES,F.,329RABONI,S.M.,317RAFAEL,B.,291RAIOL,T.,57RAMALA.,C.,269RIBEIRO,M.C.S.A.,243RIBEIRO,P.C.,25RIBEIRO,R.B.,203RIBEIRO‑DIAS,F.,83RISTORI,C.A.,183RODRIGUES,D.S.,51RODRIGUES,L.S.,333RODRIGUES,V.L.C.C.,151RODRIGUEZ,A.,107RODRÍGUEZ,D.A.,197ROJAS,C.A.,31ROLDÁN,W.H.,31,37ROMANO‑LIEBER,N.S.,11ROMANOS,M.T.V.,3ROSA.P.S.,273ROSSI,C.L.,225ROTTA,I.,317ROWLANDS,R.E.G.,183RUBINSKY‑ELEFANT,G.,69RUSSO,D.H.,339SAKAI,T.,231SAKUMA,H.,183SALAZAR,D.,107SALAZAR,Y.D.,169SALLES,E.F.,145SALOMÓN,O.D.,187SANDOVAL,E.A.,187SANTINI,M.S.,187SANTOS,A.F.dos,125SANTOS,B.R.,111,329SANTOS,C.R.,51SANTOS,N.,3SANTOS,S.L.,94SANTOS,S.O.,151SANTOS,V.A.dos,203SANTURIO,J.M.,161SARACENI,C.P.,25SATO,G.,231SCHEID,L.A.,161SCHUBACH,T.M.P.,193SERRA‑FREIRE,N.M.,17SEVERO,L.C.,145SHIKANAI‑YASUDA,M.A.,151SHOJI,Y.,231SILVA,A.D.da,237

SILVA,C.C.P.,51SILVA,E.E.,17SILVA,I.R.da,151SILVA,J.M.K.da,125SILVA,J.S.,17SILVA,M.R.R.,139SILVA,P.R.da,151SILVEIRA,F.L.,47SILVEIRA,F.T.,259SILVEIRA,J.F.da,115SITNIK,R.,119SITOE,S.P.B.L.,291SOARES,A.R.,3SODRÉ,M.M.75SOLIS,H.,107SOUZA,C.A.de,225SOUZA,L.K.H.,139SOUZA‑DIAS,M.B.,133STEMPLIUK,V.A.,133STROPARO,E.,317TAKAHASHI,F.Y.,101,211TARACHA,E.,95TARTAGLINO,L.,187TATTO,E.,151TAVARES,F.N.,17THOMPSON,R.,291TIMENETSKY,M.C.S.T.,339TORRESV.,D.B.157TREVIZOLI,J.E.,57TÚLIO,S.A.,151VALDÉSFERNÁNDEZ‑CALIENES,A.,197VALDÉSIGLESIAS,O.,89VÁSQUEZ,T.,107VEGA,S.,269VERONESE,F.J.V.,253VIDAL,J.E.,285,305VIDAL,L.R.,317VIEIRA,G.H.F.,129VIEIRA,R.H.S.F.,129VIGORITO,A.C.,225VILAS‑BOAS,L.,305VILLAFAÑE,M.F.,279VILLALOBOS,E.C.M.,231VILLARES,C.A.,125WANDERLEY,D.M.V.,151WOODS,W.J.,273YAMASHIRO,J.,133YONESHIGUE‑VALENTIN,Y.,3YOSHINARI,N.H.,297ZANNIN,M.,51ZENKNER,F.M.,111

345

SUBJECT INDEX - VOLUME 52

Achatina fulica, 211 InS.Paulo,Br.,211

Acutefebrileillness,237 Dengue,237

Adenoviruses,317 Culturemethods,317 PCR,317

Aelurostrongylus abstrusus, 211 Achatina fulica, 211 InS.Paulo,Br.,211

AIDS,279,305 Cytomegalovirus,305 Rupioidhistoplasmosis,279 InArgentina,279

Angiostrongylus costaricensis, 267 Swissmice,267

Annona muricata, 129 Antibacterialeffect,129

AntiHBsAg,25 Resistance,25

Antioxidants,43 Rifampicin,43

Arbovirus,11,17 InPortoPrimavera,Br.,11 InPantanal,Br.,17

Baggio‑Yoshinarisyndrome,297 Tick‑bornedisease,297

Batspecies,75 Rabies,75

Biomphalaria tenagophila, 101 Schistosoma mansoni,101

Borrelia burgdorferi, 297

Botulism,183 InBrazil,183 Diagnosis,183 Epidemiology,183

Candida albicans, 161 Killertoxins,161

Candida dubliniensis, 161 Killertoxins,161

Candida spp.,139 Adherenceability,139 Exoenzymeactivity,139

Centrocestus formosanus, 207 Melanoides tuberculata,207

Chagasdisease,151,269 Acute,151,269 PeruvianAmazon,269 SãoPauloState,Br.,151

Chiroptera,75 InBrazil,75

Chromoblastomycosis,329 Itraconazole,329 5‑Flucytosine,329

Clostridium botulinium, 183 InBrazil,183

Clostridium difficile, 133 Pseudo‑outbreak,133

Cnidarians,47 Skinlesions,47 BrazilianCoast,47

Cryptococcus spp.,145 Unusualmorphology,145

Culicidae,17 InPantanal,Br.,17

Cutaneousleishmaniasis,95 Immuneresponses,95

Cytomegalovirus,305 AIDS,305

Dengue,94,163,237 Control,94 Elderlypatient,163 Syndromicsurveillance,237 InFederalDistrict,Br,237

Diarrhea,133 Clostridium difficile,133

Echinaster (Othilia) echinophorus,89 Leishmanicidalactivity,89

Enterococcus gallinarum,111 Meningitis,111

Enterovirus‑71,339 Immunestatus,339 InSãoPaulo,Br.,339

Envenomation,47,51,215,333 Cnidarians,47 InBrazilianCoast,47 Coralsnake,333 Pufferfish,51 InBahia,Br.,51 InSantaCatarina,Br.,51 Scorpionstings,215

Fungusandliturgy,288

HBVDNAquantification,119 HepatitisB,119

Helicobacter pylori, 125,203 Seroprevalence,203 WesternAmazon,Br.,203 Stoolantigentest,125

Hemodialysis,57 HepatitisCvirus,57 Genotypes,57

HepatitisB,25,119,247 Medicalstudents,247 InParaná,Br.,25 Mutations,25 RealtimePCR,119

HepatitisC,57 Genotypes,57 Hemodialysis,57

Histoplasmosis,279 AIDS,279 InArgentina,279

HIV,285 Cerebraltuberculoma,285

Hyponatremia,253 Visceralleishmaniasis,253

Influenza,175 Vaccination,175 InBrazil,175

JorgeLobo’sdisease,273 InAcre,Br.,273

Leishmania amazonensis, 89 Echinaster (Othilia) echinophorus, 89

Leishmania (L.) infantum chagasi, 259 InAmazonianBrazil,259

Leishmania major, 95 Infectioninmice,95

346

Leishmania spp.,83 Interferongamma,83 Isolation,83

Leishmaniasis,83 Experimental.83

Leprosy,311 Control,311

Lutzomyia longipalpis,187 Leishmaniasis,187 InArgentina,187

Lymedisease‑likeillness,297

Lymphoma,221 Strongyloides stercoralis,221

Malaria,197,281 Cubanmedicinalplants,197 Stemcelltransplantation,281

Marinealga,3 Metapneumovirus,3

Melanoides tuberculata, 207,323 Centrocestus formosanus, 207 InBrazil,207 Philophthalmus gralli,323

Meningitis,111,169 Immunocompetenthost,111 Staphylococcus warneri, 169

Metapneumovirus,3 Ulva fasciata,3

Micrurus,333 Envenomation,333

Moringa oleifera, 129 Antibacterialeffect,129

Nissenfundoplication,113 Gastroesophagealreflux,113

PCR,317 Adenoviruses,317

Philophthalmus gralli, 323 Melanoides tuberculata, 323

Pufferfish,51 Poisoning,51 InBahia,Br.,51 InSantaCatarina,Br.,51

Rabies,75,231 InBrazil,75 Vaccine,231

Rattus norvegicus, 243 Toxocara canis,243 Behavioralchanges,243

Refluxdisease,113 Chagasdiseasepatient,113

Review,175,305 Cytomegalovirus,305 Influenzavaccination,175

Rifampicin,43 Antioxidantsactivity,43

Rupioidhistoplasmosis,279 AIDSpatient,279 InArgentina,279

Schistosoma mansoni,101 Biomphalaria tenagophila, 101 ValeRibeiradeIguape,Br,101

Scorpionstings,215 InTurkey,215

Staphylococcus warneri, 169 Hyperinfection,169 Strongyloides stercoralis,169 Meningitis,169

Stemcelltransplantation,225,281 Malaria,281 Toxoplasmosis,225

Strongyloides stercoralis,221 Hyperinfection,221 Lymphoma,221

Sylvatictriatomines,157 Intra‑domiciliarycapture,157 InLaConvenciónProvince,157

Toxocara, 69 Antibodies,69

Toxocara canis, 243 Rattus norvegicus, 243 Behavioralchanges,243

Toxocariasis,31,37,69 Andeancommunities,Peru,31 Seroprevalence,31 InSouthBrazil,69 Urbanareas,69 InYurimaguas,Peru,37 Epidemiology,37

Toxoplasma gondii, 63,291 MultipleAntigenPeptides,63 Pregnantwomen,291 HIV,291 Mozambique,291

Toxoplasma infection, 225 Serologicaldiagnosis,225 Stemcelltransplantation,225

Toxoplasmosis,63,107 Acuteinfection,63 Immunocompetent,107 Serology,63

Triatomines,157 InPeru,157

Trypanosoma cruzi, 171 Ystrainorigin,171

Tuberculosis,285 HIV,285

Ulva fasciata, 3 Antiviralactivity,3

Vaccines,175,231 Influenza,175 InBrazil,175 Rabies,231

Visceralleishmaniasis,187,193,253,259 Canine,193,259 InAmazonianBrazil,259 Diagnosis,193 Hyponatremia,253 Lutzomyia longipalpis, 187

Yellowfever,11 Monkeys,11

REVISTADO

INSTITUTO DE MEDICINA TROPICALDE SÃO PAULO

VOLUME 522010

Rev. Inst. Med. trop. S. Paulo vol. 52 n. 1-6 p. 1-346 January-December, 2010

III

CONTENTSOFVOLUME52

No.1‑January/February,2010PRESSRELEASE.........................................................................................................................................................................................................1

VIROLOGYAntiviralactivityofthegreenmarinealgaUlva fasciataonthereplicationofhumanmetapneumovirus‑G.S.MENDES,A.R.SOARES,F.O.MARTINS,M.C.M.ALBUQUERQUE,S.S.COSTA,Y.YONESHIGUE‑VALENTIN,L.M.S.GESTINARI,N.SANTOS&M.T.V.ROMANOS.............................................................................................................................................................................................................3

CirculationofantibodiesagainstyellowfevervirusinasimianpopulationintheareaofPortoPrimaveraHydroelectricPlant,SãoPaulo,Brazil‑M.A.LIMA,N.S.ROMANO‑LIEBER&A.M.R.C.DUARTE.........................................................................................................................11

PreliminaryinvestigationofculicidaespeciesinsouthPantanal,Brazilandtheirpotentialimportanceinthearbovirustransmission‑A.PAUVOLID‑CORRÊA,F.N.TAVARES,J.ALENCAR,J.S.SILVA,M.MURTA,N.M.SERRA‑FREIRE,A.O.PELLEGRIN,H.GIL‑SANTANA,A.E.GUIMARÃES&E.E.SILVA..................................................................................................................................................17

HEPATITISCharacterizationofahepatitisBvirusstraininsouthwesternParaná,Brazil,presentingmutationspreviouslyassociatedwithanti‑HBSresistance‑D.A.BERTOLINI,P.C.RIBEIRO,M.F.LEMOS,C.P.SARACENI&J.R.R.PINHO...............................................................................25

TOxOCARIASISSeroprevalenceofhumantoxocariasisinAndeancommunitiesfromthenortheastofLima,Peru‑Y.A.ESPINOZA,P.E.HUAPAYA,W.H.ROLDÁN,S.JIMÉNEZ,E.P.ABANTO,C.A.ROJAS,Y.A.CAVERO&C.A.GUTIERREZ.............................................................................31

Humantoxocariasis:aseroepidemiologicalsurveyintheAmazoniancityofYurimaguas,Peru‑W.H.ROLDÁN,Y.A.CAVERO,Y.A.ESPINOZA,S.JIMÉNEZ&C.A.GUTIÉRREZ....................................................................................................................................................37

RIFAMPICINTOxICITYModulatoryactivityofantioxidantsagainstthetoxicityofrifampicinin vivo‑O.AWODELE,A.AKINTONWA,V.O.OSUNKALU&H.A.B.COKER.............................................................................................................................................................................................................43

ANIMALENVENOMATIONSkinlesionsinenvenomingbycnidarians(Portugueseman‑of‑warandjellyfish):etiologyandseverityofaccidentsontheBraziliancoast‑V.HADDADJUNIOR,F.L.SILVEIRA&A.E.MIGOTTO...........................................................................................................................................47

Clinicalandepidemiologicalstudyof27poisoningscausedbyingestingpufferfish(Tetrodontidae)inthestatesofSantaCatarinaandBahia,Brazil‑C.C.P.SILVA,M.ZANNIN,D.S.RODRIGUES,C.R.SANTOS,I.A.CORREA&V.HADDADJUNIOR...................................................51

BRIEFCOMMUNICATIONHepatitisCvirusgenotypesinhemodialysispatientsintheFederalDistrict,Brazil‑R.M.S.AMORIM,T.RAIOL,J.E.TREVIZOLI,F.A.R.NEVES,C.R.F.MARTINS&R.M.B.MARTINS................................................................................................................................................57

No.2‑March/April,2010PRESSRELEASE.......................................................................................................................................................................................................61

ACKNOwLEDGEMENTTOREVIEwERS.....................................................................................................................................................62

TOxOPLASMOSISHighdiagnosticefficiencyofIgM‑ELISAwiththeuseofmultipleantigenpeptides(MAP1)fromT. gondii ESA(SAG‑1,GRA‑1andGRA‑7)inacutetoxoplasmosis‑P.R.B.ARAÚJO&A.W.FERREIRA.......................................................................................................................................63

TOxOCARIASISSerological,clinicalandepidemiologicalevaluationoftoxocariasisinurbanareasofsouthBrazil‑C.M.COLLI,G.RUBINSKY‑ELEFANT,M.L.PALUDO,D.L.M.FALAVIGNA,E.V.GUILHERME,S.MATTIA,S.M.ARAÚJO,E.C.FERREIRA,I.T.S.PREVIDELLI&A.L.FALAVIGNA‑GUILHERME...................................................................................................................................................................................69

RABIESUpdatedlistofbatspeciespositiveforrabiesinBrazil‑M.M.SODRÉ,A.R.daGAMA&M.F.deALMEIDA..........................................................75

LEISHMANIASISLeishmaniaspp.parasiteisolationthroughinoculationofpatientbiopsymaceratesininterferongammaknockoutmice‑M.A.P.OLIVEIRA,A.S.PIRES,R.P.BASTOS,G.M.C.A.LIMA,S.A.PINTO,L.I.A.PEREIRA,A.J.C.S.PEREIRA,I.A.ABRAHAMSOHN,M.L.DORTA&F.RIBEIRO‑DIAS............................................................................................................................................................................................................83

LeishmanicidalactivityofEchinaster (Othilia) echinophorus crudeextract‑M.GARCIA‑PARRA,L.MONZOTE‑FIDALGO,J.MENDIOLA‑MARTINEZ,A.M.MONTALVO‑ALVAREZ&O.VALDÉS‑IGLESIAS............................................................................................89

SubcutaneousimmunizationagainstLeishmania major-infectioninmice:efficacyofformalin‑killedpromastigotescombinedwithadjuvants‑J.M.MUTISO,J.C.MACHARIA,R.M.MUTISYA&E.TARACHA...........................................................................................................................95

IV

SCHISTOSOMIASISTheeffectsofaexperimentalinfectionfromafocusoftransmissionofSchistosoma mansoniinapopulation of Biomphalaria tenagophila(Orbigny,1835),intheregionof“ValedoRibeiradeIguape”,Brazil‑M.C.A.GUIMARÃES,C.MUNIZ,F.Y.TAKAHASHI,F.P.OHLWEILER,T.KAWANO&D.NATAL.............................................................................................................................................................101

CASEREPORTDisseminatedtoxoplasmosisinanimmunocompetentpatientfromPeruvianAmazon‑J.NUNURA,T.VÁSQUEZ,S.ENDO,D.SALAZAR,A.RODRIGUEZ,S.PEREYRA&H.SOLIS...............................................................................................................................................................107

Enterococcus gallinarummeningitisinanimmunocompetenthost:acasereport‑V.S.ANTONELLO,F.M.ZENKNER,J.FRANÇA&B.R.SANTOS.............................................................................................................................................................................................................111

LETTERTOTHEEDITORNissenfundoplicationforthetreatmentofgastroesophagealrefluxdiseaseinpatientswithChagasdiseasewithoutachalasia‑C.A.R.PANTANALI,F.A.M.HERBELLA,M.A.C.A.HENRY,J.L.B.AQUINO,J.F.M.FARAH&J.C.DELGRANDE.......................................113

TRIBUTEMarianoJorgeLevin‑J.F.SILVEIRA............................................................................................................................................................................115

SUMMARYOFTHESISEcosystemappliedtothedenguecontrolatlocallevel:anapproachbasedinthesocialreproduction‑S.L.SANTOS.................................................94

BOOKREVIEwPatologia.Processosgerais‑M.FRANCO,M.R.MONTENEGRO,T.deBRITO,C.E.BACCHI&P.CARDOSODEALMEIDA........................106

No.3‑May/June,2010PRESSRELEASE‑T.deBRITO&P.P.CHIEFFI..................................................................................................................................................117

HEPATITISAreal‑timequantitativeassayforhepatitisBDNAvirus(HBV)developedtodetectallHBVgenotypes‑R.SITNIK,A.PAES,C.P.MANGUEIRA&J.R.R.PINHO.............................................................................................................................................................................119

BACTERIOLOGYValidationofarapidstoolantigentestfordiagnosisofHelicobacter pyloriinfection‑J.M.K.daSILVA,C.A.VILLARES,M.doS.MONTEIRO,C.COLAÚTO,A.F.dosSANTOS&R.MATTAR..................................................................................................................125

Antibacterialeffect(in vitro)ofMoringa oleiferaandAnnona muricataagainstGrampositiveandGramnegativebacteria‑G.H.F.VIEIRA,J.A.MOURÃO,A.M.ÂNGELO,R.A.COSTA&R.H.S.F.VIEIRA............................................................................................................................129

Pseudo‑outbreakofClostridium difficileassociateddiarrhea(CDAD)inatertiary‑carehospital‑M.B.SOUZADIAS,J.YAMASHIRO,V.L.BORRASCA,V.A.STEMPLIUK,M.R.E.ARAÚJO,S.F.COSTA&A.S.LEVIN..............................................................................................133

MYCOLOGYDifferencesinexoenzymeproductionandadherenceabilityofCandidaspp.isolatesfromcatheter,bloodandoralcavity‑C.R.COSTA,X.S.PASSOS,L.K.H.SOUZA,P.A.LUCENA,O.F.L.FERNANDES&M.R.R.SILVA............................................................................................139

UnusualmorphologiesofCryptococcusspp.intissuespecimens:reportof10cases‑A.F.GAZZONI,F.M.OLIVEIRA,E.F.SALLES,E.MAYAYO,J.GUARRO,J.CAPILLA&L.C.SEVERO...........................................................................................................................................145

TRYPANOSOMIASISOnanacutecaseofChagasdiseaseinaregionundervectorcontrolintheStateofSãoPaulo,Brazil‑D.M.V.WANDERLEY,V.L.C.C.RODRIGUES,R.M.LEITE,S.Y.DIAZ,M.E.CARVALHO,S.O.SANTOS,E.TATTO,M.S.CARLI,K.I.R.COELHO,P.R.SILVA,S.A.TÚLIO,I.R.SILVA&M.A.SHIKANAI‑YASUDA..........................................................................................................................151

Geographicaldistributionandintra‑domiciliarycaptureofsylvatictriatominesinLaConvenciónProvince,Cusco,Peru‑D.B.TORRESV.&R.CABRERA.............................................................................................................................................................................................................157

TECHNICALREPORTDifferentiationofCandida dubliniensisfromCandida albicans withtheuseofkillertoxins‑L.A.SCHEID,D.A.N.MARIO,E.M.HEINS‑VACCARI,J.M.SANTURIO&S.H.ALVES..........................................................................................................................................161

CASEREPORTDengueinanelderlypatient‑M.L.GORZONI,I.F.D.S.MASSAIA&S.L.PIRES....................................................................................................163

Staphylococcus warneri meningitisinapatientwithStrongyloides stercoralishyperinfectionandlymphoma.Firstreportofacase‑R.N.INCANI,M.HERNÁNDEZ,J.CORTEZ,M.E.GONZÁLEZ&Y.D.SALAZAR..............................................................................................169

LETTERTOTHEEDITOROriginofthe“Ystrain”ofTrypanosoma cruzi - V.AMATONETO.............................................................................................................................171

V

No.4‑July/August,2010EDITORIALArticlespublishedintheJuly/August2010issueoftheRevistadoInstitutodeMedicinaTropicaldeSãoPaulo‑T.deBRITO,P.P.CHIEFFI&M.F.FRANCO.....................................................................................................................................................................................173

REVIEwEffectivenessoftheBrazilianinfluenzavaccinationpolicy,asystematicreview‑E.J.A.LUNA&V.L.GATTÁS......................................................175

MICROBIOLOGYBotulisminBrazil,2000‑2008:epidemiology,clinicalfindingsandlaboratorydiagnosis‑R.E.G.ROWLANDS,C.A.RISTORI,G.I.S.L.LOPES,A.M.R.dePAULA,H.SAKUMA,R.GRIGALIUNAS,R.LOPREATOFILHO,D.S.GELLI,M.B.deP.EDUARDO&M.JAKABI.................................................................................................................................................................................................................183

LEISHMANIASISLutzomyia longipalpisbehaviorandcontrolatanurbanvisceralleishmaniasisfocusinArgentina‑M.S.SANTINI,O.D.SALOMÓN,S.A.ACARDI,E.A.SANDOVAL&L.TARTAGLINO................................................................................................................................................187

Caninevisceralleishmaniasis:studyofmethodsforthedetectionofIgGinserumandeluatesamples‑F.B.FIGUEIREDO,M.F.MADEIRA,L.D.NASCIMENTO,T.R.ABRANTES,E.MOUTA‑CONFORT,S.R.L.PASSOS&T.M.P.SCHUBACH..............................................................193

MALARIAIn vitroantimalarialactivityandcytotoxicityofsomeselectedCubanmedicinalplants‑A.F.‑C.VALDÉS,J.M.MARTÍNEZ,R.S.LIZAMA,Y.G.GAITÉN,D.A.RODRÍGUEZ&J.A.PAYROL....................................................................................................................................................197

BACTERIOLOGYEvaluationofHelicobacterpyloricolonizationbyserologictest(IgG)anddyspepsiainvolunteersfromthecountrysideofMonteNegro,intheBrazilianWesternAmazonregion‑R.B.RIBEIRO,H.S.MARTINS,V.A.dosSANTOS,M.ELKHOURI,L.S.DUARTE,M.N.BURATTINI,Q.CORDEIRO,L.M.A.CAMARGO&C.E.P.CORBETT..........................................................................................................203

PARASITOLOGYMelanoides tuberculata(Mollusca:Thiaridae)asanintermediatehostofCentrocestus formosanus(Trematoda:Heterophyidae)inBrazil‑H.A.PINTO&A.L.deMELO.......................................................................................................................................................................................207

CurrentdistributionofAchatina fulica, intheStateofSãoPaulo,includingrecordsofAelurostrongylus abstrusus(Nematoda)larvaeinfestation‑F.P.OHLWEILER,M.C.A.GUIMARÃES,F.Y.TAKAHASHI&J.M.EDUARDO...................................................................................................211

ANIMALENVENOMATIONScorpionstingsinTurkey:epidemiologicalandclinicalaspectsbetweentheyears1995and2004‑Y.CESARETLI&O.OZKAN........................215

CASEREPORTHyperinfectionbyStrongyloides stercoralisprobablyassociatedwithrituximabinapatientwithmantlecelllymphomaandhypereosinophilia‑R.N.INCANI,M.HERNÁNDEZ&M.E.GONZÁLEZ...............................................................................................................................................221

SerologicalmonitoringofaToxoplasmainfectionafterhematopoieticstemcelltransplantation‑C.L.ROSSI,F.S.NASCIMENTO,S.deBARROS‑MAZON,D.F.DIAS,A.C.VIGORITO&C.A.deSOUZA................................................................................................................225

BOOKREVIEwAtlasdeneuroimágenesyneuropatologíadelaenfermedadHIV/SIDA‑M.E.CORTI,M.F.VILLAFAÑE&O.J.PALMIERI................................182

No.5‑September/October,2010EDITORIALArticlespublishedintheSeptember/October2010issueoftheRevistadoInstitutodeMedicinaTropicaldeSãoPaulo‑T.deBRITO,P.P.CHIEFFI&M.F.FRANCO.....................................................................................................................................................................................229

VIROLOGYPathogenicityofdifferentrabiesvirusisolatesandprotectiontestinvaccinatedmice‑E.M.S.CUNHA,A.F.C.NASSAR,M.C.C.S.H.LARA,E.C.M.VILLALOBOS,G.SATO,Y.KOBAYASHI,Y.SHOJI,T.ITOU,T.SAKAI&F.H.ITO................................................................................231

EPIDEMIOLOGYSyndromicsurveillance:etiologicstudyofacutefebrileillnessindenguesuspiciouscaseswithnegativeserology,Brazil,FederalDistrict,2008‑A.D.daSILVA&M.S.N.EVANGELISTA....................................................................................................................................................................237

TOxOCARIASISBehavioralchangesinRattus norvegicusexperimentallyinfectedbyToxocara canislarvae‑P.P.CHIEFFI,R.T.R.AQUINO,M.A.PASQUALOTTI,M.C.S.A.RIBEIRO&A.G.NASELLO...................................................................................................................................243

HEPATITISFrequencyofhepatitisBimmunityandoccupationalexposurestobodyfluidsamongBrazilianmedicalstudentsatapublicuniversity‑L.C.M.deOLIVEIRA&J.P.J.PONTES.......................................................................................................................................................................247

LEISHMANIASISHyponatremiainvisceralleishmaniasis‑F.A.LIMAVERDE,F.A.A.LIMAVERDE,F.J.V.VERONESE,A.S.NETO,G.FUC&E.M.LIMAVERDE.......................................................................................................................................................................................................253

VI

CaninevisceralleishmaniasisduetoLeishmania(L.)infantumchagasiinAmazonianBrazil:acomparisonoftheparasitedensityfromtheskin,lymphnodeandvisceraltissuesbetweensymptomaticandasymptomatic,seropositivedogs‑L.V.R.LIMA,L.A.CARNEIRO,M.B.CAMPOS,E.J.CHAGAS,M.D.LAURENTI,C.E.P.CORBETT,R.LAINSON&F.T.SILVEIRA..................................................................259

BRIEFCOMMUNICATIONSusceptibilityandmorbiditybetweenmaleandfemaleSwissmiceinfectedwithAngiostrongylus costaricensis - M.B.MENTZ,E.DALLEGRAVE&C.GRAEFF‑TEIXEIRA.............................................................................................................................................................267

EpidemiologicalinvestigationofanacutecaseofChagasdiseaseinanareaofactivetransmissioninPeruvianAmazonregion‑R.CABRERA,S.VEGA,A.G.CÁCERES,C.RAMALA.,C.ÁLVAREZ,P.LADERA,R.PINEDO&G.CHUQUIPIONDO.......................................................269

MYCOLOGYTenyearsexperiencewithJorgeLobo’sdiseaseintheStateofAcre,Amazonregion,Brazil‑W.J.WOODS,A.F.F.BELONE,L.B.CARNEIRO&P.S.ROSA......................................................................................................................................................................................273

CASEREPORTRupioidhistoplasmosis:firstcasereportedinanAIDSpatientinArgentina‑M.CORTI,M.F.VILLAFAÑE,O.PALMIERI&R.NEGRONI.......279

Themonitoringofhematopoieticstemcelltransplantdonorsandrecipientsfromendemicareasformalaria‑J.INOUE,C.M.MACHADO,G.F.M.C.LIMA,M.J.C.NASCIMENTO,V.R.COLTURATO&S.M.DISANTI.......................................................................................................281

RingenhancingintracraniallesionrespondingtoantituberculoustreatmentinanHIV‑infectedpatient‑D.PELLEGRINO,J.GERHARDT,F.M.V.PORFÍRIO,E.DEBORTHOLISANTOS,R.F.DAUAR,A.C.PENALVADEOLIVEIRA&J.E.VIDAL....................................................285

LETTERTOTHEEDITORAnalogiesinMedicine:fungusandliturgy‑J.S.ANDRADEFILHO&G.P.PENA....................................................................................................288

No.6‑November/December,2010EDITORIALArticlespublishedintheNovember/December2010issueofthe“RevistadoInstitutodeMedicinaTropicaldeSãoPaulo”‑T.deBRITO,P.P.CHIEFFI&M.F.FRANCO.....................................................................................................................................................................................289

TOxOPLASMOSISPreliminaryreportofHIVandToxoplasma gondiioccurrenceinpregnantwomenfromMozambique‑S.P.B.L.SITOE,B.RAFAEL,L.R.MEIRELES,H.F.ANDRADEJÚNIOR&R.THOMPSON.................................................................................................................................291

MICROBIOLOGYProfileofpatientswithBaggio‑Yoshinarisyndromeadmittedat“InstitutodeInfectologiaEmilioRibas”‑E.A.GOUVEIA,M.F.ALVES,E.MANTOVANI,L.K.OYAFUSO,V.L.N.BONOLDI&N.H.YOSHINARI.............................................................................................................297

AIDSNeurologiccytomegaloviruscomplicationsinpatientswithAIDS:retrospectivereviewof13casesandreviewoftheliterature‑C.ALMEIDASILVA,A.C.PENALVADEOLIVEIRA,L.VILAS‑BOAS,M.C.D.S.FINK,C.S.PANNUTI&J.E.VIDAL...................................305

LEPROSYLeprosycontrol:perspectives&epidemiologicalandoperationalaspects‑A.GONÇALVES,G.G.MANTELLINI&C.R.PADOVANI................311

VIROLOGYAdenovirusrespiratoryinfection:significantincreaseindiagnosisusingPCRcomparingwithantigendetectionandculturemethods‑E.STROPARO,C.R.CRUZ,M.C.DEBUR,L.R.VIDAL,M.B.NOGUEIRA,S.M.ALMEIDA,L.A.PEREIRA,I.ROTTA&S.M.RABONI.....317

PARASITOLOGYMelanoides tuberculataasintermediatehostofPhilophthalmus gralliinBrazil‑H.A.PINTO&A.L.deMELO.....................................................323

CASEREPORTTreatmentofseverechromoblastomycosiswithitraconazoleand5‑flucytosineassociation‑V.S.ANTONELLO,M.C.APPELDASILVA,E.CAMBRUZZI,D.A.KLIEMANN,B.R.SANTOS&F.QUEIROZ‑TELLES.........................................................................................................329

EnvenomationbyMicrurus coralsnakesinBrazilianAmazonregion:reportoftwocases‑P.P.O.PARDAL,J.S.O.PARDAL,M.A.C.GADELHA,L.S.RODRIGUES,D.T.FEITOSA,A.L.C.PRUDENTE&H.W.FAN.....................................................................................333

BRIEFCOMMUNICATIONEvaluationofEnterovirus71immunestatusinSãoPauloState,Brazil‑A.LUCHS,A.CILLI,D.H.RUSSO,F.F.COSTA,R.C.C.CARMONA&M.C.S.T.TIMENETSKY............................................................................................................................................................................................339

AUTHORINDEx.....................................................................................................................................................................................................343

SUBJECTINDEx.....................................................................................................................................................................................................345

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